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Development of new dairy ingredient with prebiotic and antioxidant properties through
the electro-activation processing of sweet cheese whey
Thèse
Ourdia Kareb
Doctorat en sciences et technologie des aliments Philosophiae Doctor (Ph. D.)
Québec, Canada
© Ourdia Kareb, 2018
Development of new dairy ingredient with prebiotic and antioxidant properties through
the electro-activation processing of sweet cheese whey
Thèse
Ourdia Kareb
Sous la direction de :
Mohammed Aider, directeur de recherche
Claude P. Champagne, codirecteur de recherche
Julie Jean, codirectrice de recherche
iii
RÉSUMÉ
Le but de ce projet était de développer la technologie d'électro-activation comme une
approche novatrice pour la valorisation intégrale des constituants du lactosérum. Ce
sous-produit est riche en composants de valeur avec des propriétés biologiques et
fonctionnelles prometteuses. La première étape de ce travail visait à déterminer les
conditions optimales pour la production du lactulose en utilisant le lactosérum comme
source de lactose. Un rendement de 35% a été obtenu avec une pureté élevée à des
températures de 0, 10 et 25°C et un court temps de réaction (≅ 40 minutes). En outre,
l'analyse protéomique a révélé l'hydrolyse des protéines de lactosérum et la formation
simultanée de produits de réaction Maillard aux propriétés antioxydantes élevées. La
deuxième étape visait à élucider les mécanismes antioxydants qui régissaient les
hydrolysats de protéines de lactosérum à réaction de Maillard (MRP-whey) et à
caractériser leurs structures. L'effet antioxydant a été attribué à de multiples propriétés
et pourrait être utilisé à la fois comme antioxydants primaires et secondaires. La
structure moléculaire du MRP-whey a été caractérisée comme étant des bases de Schiff.
De plus, des peptides bioactifs multifonctionnels, avec des propriétés biologiques ont
également été identifiés. Dans la troisième étape, l'efficacité du lactosérum électro-
activé (lactosérum-EA) possédant des propriétés prébiotique et antioxydante sur la
croissance des bactéries probiotiques testées a été démontrée. Le lactosérum-EA avait
comparativement un meilleur effet bifidogène que le lactulose. De plus, on a constaté
que le lactosérum-EA produisait un effet protecteur sur L. johnsonii La-1 durant sa
croissance en présence d'oxygène. Cet effet peut être lié, en partie, à la capacité du
lactosérum-EA à éliminer le peroxyde d'hydrogène et à prévenir son accumulation dans
le milieu de croissance. Les spectres FTIR ont montré que le lactosérum-EA empêchait
l'oxydation des lipides de la membrane cellulaire en limitant le changement dans l'ordre
structurel des acides gras. Dans l'ensemble, le lactosérum-EA comme prébiotique
possédant une capacité antioxydante a un grand potentiel d'application, non seulement
dans l'industrie laitière, mais aussi comme ingrédient fonctionnel avec diverses
bioactivités et fonctionnalités.
iv
ABSTRACT
The goal of this project was to develop the electro-activation technology as innovative
approach for an integral valorization of whey constituents. This by-product is rich in
valuable components with promising biological and functional properties. The first step
of this work aimed to determine the optimal conditions of lactulose production using
whey as a source of lactose and a yield of 35% was obtained with high purity under
temperatures of 0, 10 and 25°C and short reaction time (≅ 40 minutes). Moreover, the
proteomic analysis revealed the hydrolysis of whey proteins and simultaneous
formation of Maillard reaction products with high antioxidant properties. The second
step aimed to elucidate the antioxidant mechanisms that governed the Maillard-reacted-
whey protein hydrolysates (MRPs-whey) and to characterize their structures. The
antioxidant effect was ascribed to a multiple properties and could be used as both
primary and secondary antioxidants. The molecular structure of the MRPs-whey was
characterized to be Schiff base compounds. Additionally, multifunctional bioactive
peptides, with biological properties were also identified. In the third step, the efficacy
of electro-activated whey (EA-whey) as a combined prebiotic and antioxidant
component on the growth of tested probiotic bacteria was demonstrated. The EA-whey
was comparatively a better bifidogenic factor than lactulose. Additionally, EA-whey
was found to elicit a protective effect on L. johnsonii La-1 during its growth in the
presence of oxygen. This effect may be related, in part, to the ability of EA-whey to
scavenge hydrogen peroxide metabolites and prevent its accumulation in the growth
medium. FTIR spectra showed that EA-whey prevented the cell membrane lipids
oxidation by limiting the change in the structural order of fatty acids. Overall, EA-whey
as a prebiotic supplement possessing antioxidant capacity has great potential for
application not only in the dairy industry but also as a functional food ingredient with
potent bioactivities and functionalities.
v
TABLE OF CONTENTS
RÉSUMÉ .......................................................................................................... iii
ABSTRACT ..................................................................................................... iv
TABLE OF CONTENTS .................................................................................. v
LIST OF TABLES ......................................................................................... xiii
LIST OF FIGURES ........................................................................................ xiv
LIST OF ABBREVIATIONS ...................................................................... xviii
DEDICATIONS ............................................................................................. xix
ACKNOWLEDGMENTS ............................................................................... xx
FOREWORD ................................................................................................ xxii
INTRODUCTION ............................................................................................. 1
1. CHAPTER 1: Literature review ................................................................ 4
1.1 Functional foods .................................................................................. 4
1.1.1 Probiotics ....................................................................................... 6
1.1.1.1 Probiotics concept ................................................................... 6
1.1.1.2 Commercially used probiotic foods ........................................ 6
1.1.1.3 Selection of potential probiotics ............................................. 8
1.1.1.4 Mechanisms of action of probiotics ...................................... 10
1.1.1.5 Beneficial health effects of probiotics .................................. 12
1.1.1.6 Factors affecting the survival of probiotics in dairy products ..
.............................................................................................. 14
1.1.2 Prebiotics ..................................................................................... 16
1.1.2.1 Prebiotics concept ................................................................. 16
1.1.2.2 Criteria of prebiotics ............................................................. 17
1.1.2.3 Sources, production of prebiotics ......................................... 17
vi
1.1.2.4 Beneficial health effects of prebiotics .................................. 20
1.1.2.5 Prebiotics as functional food ingredients .............................. 21
1.1.2.6 Synbiotic concept ................................................................. 23
1.1.2.7 Synbiotic in dairy products ................................................... 24
1.2 Whey ................................................................................................. 26
1.2.1 Whey in environmental consideration ......................................... 26
1.2.2 Whey composition ....................................................................... 27
1.2.2.1 Lactose whey ........................................................................ 28
1.2.2.2 Lactose-derived lactulose ..................................................... 28
1.2.2.2.1 General properties of lactulose ...................................... 28
1.2.2.2.2 Chemistry of lactulose synthesis ................................... 29
1.2.3 Methods of lactulose production ................................................. 31
1.2.3.1 Chemical synthesis ............................................................... 31
1.2.3.2 Enzymatic synthesis ............................................................. 31
1.2.3.3 Electro-activation synthesis .................................................. 32
1.2.4 Lactulose applications ................................................................. 33
1.2.4.1 Pharmaceutical applications ................................................. 33
1.2.4.2 Food applications .................................................................. 34
1.2.5 Whey proteins .............................................................................. 35
1.2.5.1 Nutritional value of whey proteins ....................................... 36
1.2.5.2 Whey proteins in food as a functional ingredient ................. 37
1.2.5.3 Physiological importance of whey proteins .......................... 39
1.2.5.4 Bioactive peptides generated from whey .............................. 40
1.2.5.4.1 Processing of bioactive peptides in whey ...................... 41
1.2.5.4.2 Functionalities of bioactive peptides in whey ............... 42
vii
1.2.5.4.2.1 Antihypertensive peptides ...................................... 43
1.2.5.4.2.2 Antioxidative peptides ............................................ 44
1.2.5.4.2.3 Antimicrobial peptides ........................................... 45
1.2.5.4.2.4 Immunomodulatory peptides .................................. 45
1.2.5.4.2.5 Other bioactive peptides ......................................... 46
1.2.5.5 Maillard reaction products from whey ................................. 47
1.2.5.5.1 Maillard reaction stages ................................................. 48
1.2.5.5.2 Maillard reaction processing ......................................... 50
1.2.5.6 Some proprieties of Maillard reaction products ................... 51
1.2.5.7 Antioxidant properties of MRPs-whey ................................. 51
1.3 Electro-activation .............................................................................. 53
1.3.1 The concept of electro-activation and devolvement .................... 53
1.3.2 Principles of electro-activated aqueous solutions ........................ 53
1.3.3 Application of electro-activation solutions .................................. 56
1.3.4 Electro-activation technology for whey valorization .................. 57
2. CHAPTER 2: Problematic, Hypothesis and Objectives ......................... 59
2.1 Problematic ........................................................................................ 59
2.2 Hypothesis ......................................................................................... 59
2.3 Main objective ................................................................................... 60
2.4 Specific objectives ............................................................................. 60
3. CHAPTER 3: Contribution to the production of lactulose-rich whey by in
situ electro-isomerization of lactose and effect on whey proteins after electro-activation
as confirmed by MALDI-TOF MS spectrometry and SDS-PAGE gel electrophoresis .
................................................................................................................. 61
3.1 RÉSUMÉ ........................................................................................... 62
3.2 ABSTRACT ...................................................................................... 63
viii
3.3 INTRODUCTION ............................................................................. 64
3.4 MATERIALS AND METHODS ...................................................... 67
3.4.1 Chemicals .................................................................................... 67
3.4.2 Electro-activation reactor and configuration ............................... 67
3.4.3 Electro-activation reaction ........................................................... 69
3.4.4 HPLC analysis of the reaction products ...................................... 69
3.4.5 Total protein content .................................................................... 70
3.4.6 Gel Electrophoresis (SDS-PAGE) ............................................... 70
3.4.7 MALDI-TOF-MS assessment ..................................................... 71
3.4.8 Determination of free amino acids by UPLC-UV florescence
detection ..................................................................................................... 71
3.4.9 Oxygene radical capacity (ORAC) of electro-activated whey .... 72
3.4.10 Statistical Analysis ...................................................................... 72
3.5 RESULTS AND DISCUSSION ....................................................... 73
3.5.1 Process of whey solution EA: Evolution of whey solution alkalinity
..................................................................................................... 73
3.5.2 Assessment of lactulose formation during whey electro-activation
..................................................................................................... 76
3.5.3 HPLC analysis of lactulose and other reaction by-products ........ 78
3.5.4 Effect of temperature ................................................................... 81
3.5.5 Effect of the electric current intensity ......................................... 83
3.5.6 Effect of the volume reaction ...................................................... 85
3.5.7 Effect of the feed whey concentration ......................................... 87
3.5.8 Proteomic analysis of the EA-whey ............................................ 89
3.5.8.1 SDS-PAGE profiles of EA-whey proteins ........................... 89
3.5.8.2 MALDI-TOF-MS analysis of whey proteins after EA ......... 91
ix
3.5.9 Total antioxidant capacity of EA-whey proteins ......................... 92
3.6 CONCLUSION ................................................................................. 96
4. CHAPTER 4: Impact of electro-activation on the antioxidant properties of
defatted whey .............................................................................................................. 97
4.1 RÉSUMÉ ........................................................................................... 98
4.2 ABSTRACT ...................................................................................... 99
4.3 INTRODUCTION ........................................................................... 100
4.4 MATERIALS AND METHODS .................................................... 102
4.4.1 Chemicals, reagents, membranes and electrodes ....................... 102
4.4.2 Electro-activation of whey induced Maillard reaction products 104
4.4.3 Determination of antioxidant activity ........................................ 104
4.4.3.1 Determination of reducing power of EA-whey .................. 104
4.4.3.2 Determination of 2,2-diphenyl-1-picrylhydrazyl radical-
scavenging activity of EA-whey ................................................................... 105
4.4.3.3 Determination of 2,2-diphenyl-1-picrylhydrazyl radical-
scavenging activity of EA-whey ................................................................... 105
4.4.3.4 Determination of hydroxyl radical scavenging activity of EA-
whey ............................................................................................ 106
4.4.3.5 Determination of chelating activity on Fe2+ of EA-whey ... 106
4.4.4 Measurement of browning intensity and fluorescence of electro-
activated whey ................................................................................................... 107
4.4.5 Statistical analysis ...................................................................... 107
4.5 RESULTS AND DISCUSSION ..................................................... 108
4.5.1 Oxygen radical absorbance capacity ......................................... 108
4.5.2 Reducing power ......................................................................... 110
4.5.3 Metal-chelating activity ............................................................. 112
x
4.5.4 2,2-Diphenyl-1-picrylhydrazyl scavenging activity .................. 114
4.5.5 Hydroxyl radical scavenging activity ........................................ 116
4.5.6 Effect of pH on brown colour development of electro-activated
whey ................................................................................................... 119
4.6 CONCLUSION ............................................................................... 122
5. CHAPTER 5: Electro-activation of sweet defatted whey: Impact on the
induced Maillard reaction products and bioactive peptides ...................................... 123
5.1 RÉSUMÉ ......................................................................................... 124
5.2 ABSTRACT .................................................................................... 125
5.3 INTRODUCTION ........................................................................... 126
5.4 MATERIALS AND METHODS .................................................... 128
5.4.1 Chemicals and reagents ............................................................. 128
5.4.2 Maillard reaction products (MRPs) generation from EA-whey 128
5.4.3 Determination of free and sugar-bound amino acids ................. 129
5.4.4 Determination of reducing sugars in MRPs-whey .................... 130
5.4.5 Structure characterization of MRPs-whey induced by electro-
activation ................................................................................................... 130
5.4.5.1 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) ............................................................................................ 130
5.4.5.2 FT-IR measurements .......................................................... 130
5.4.5.3 LC/MS-MS analysis ........................................................... 131
5.4.5.3.1 Samples preparation .................................................... 131
5.4.5.3.2 Mass spectrometry ....................................................... 131
5.4.5.3.3 Database searching ...................................................... 132
5.4.5.3.4 Criteria for protein identification ................................. 132
5.5 Statistical analysis ........................................................................... 132
xi
5.6 RESULTS AND DISCUSSION ..................................................... 133
5.6.1 Changes in free amino acids and reducing sugars ..................... 133
5.6.2 FTIR spectroscopy analysis ....................................................... 138
5.6.3 Identification of bioactive peptides from electro-activated whey ...
................................................................................................... 140
5.7 CONCLUSION ............................................................................... 143
6. CHAPTER 6: Effect of electro-activated sweet whey on growth of
probiotic bacteria of Bifidobacterium, Lactobacillus and Streptococcus genera under
model growth conditions ........................................................................................... 144
6.1 RÉSUMÉ ......................................................................................... 145
6.2 ABSTARCT .................................................................................... 146
6.3 INTRODUCTION ........................................................................... 147
6.4 MATERIALS AND METHODS .................................................... 150
6.4.1 Chemicals and reagents ............................................................. 150
6.4.2 Preparation of electro-activated whey ....................................... 150
6.4.3 Microorganisms and culture conditions .................................... 152
6.4.4 Experimental design .................................................................. 153
6.4.5 Evaluation of growth performance ............................................ 153
6.4.5.1 Effect of MRS supplementation on the growth of probiotic
bacteria under anaerobic conditions ........................................................... 153
6.4.5.2 Effect of MRS supplementation on the growth of La under
aerobic conditions ....................................................................................... 154
6.4.5.3 Fourier transform infrared spectroscopy analysis ............ 154
6.5 Statistical analyses ......................................................................... 155
6.6 RESULTS AND DISCUSSION ..................................................... 156
6.6.1 RESULTS .................................................................................. 156
xii
6.6.1.1 Effect of MRS supplementation on the growth of probiotic
bacteria under anaerobic conditions ........................................................... 156
6.6.1.2 Effect of MRS supplementation by EA-whey on the growth of
L. johnsonii La-1 under aerobic conditions ............................................... 161
6.6.1.3 FTIR analysis ...................................................................... 163
6.6.2 DISCUSSION ............................................................................ 165
6.6.2.1 Justification of the used strategy ........................................ 165
6.6.2.2 Response of the bifidobacteria to EA-whey ....................... 166
6.6.2.3 Response of the lactobacillus to EA-whey ......................... 167
6.6.2.4 Response of the specific yogurt cultures to EA-whey ........ 168
6.6.2.5 FTIR analysis of L. johnsonii membrane lipids ................. 170
6.7 CONCLUSION ............................................................................... 173
CHAPTER 7: General conclusion and perspectives ..................................... 174
6.1 GENERAL CONCLUSION ............................................................ 174
7.2 PESPECTIVES ....................................................................................... 176
REFERENCES .............................................................................................. 178
xiii
LIST OF TABLES
Table 1.1: Main approaches used for the production of prebiotic carbohydrates
(Playne & Crittenden, 2009). ...................................................................................... 19
Table 1.2: Food applications of prebiotics (Wang, 2009). ............................. 22
Table 1.3: Proximate composition and pH of sweet and acid whey (Yadav et
al., 2015). .................................................................................................................... 27
Table 1.4: Protein composition and basic characteristics of the whey proteins
(Yadav et al., 2015). .................................................................................................... 36
Table 1.5: Examples of application of whey protein ingredients in certain foods
and their functional properties adapted from (Bansal & Bhandari, 2016). ................. 38
Table 3.1: Oxygen radical absorbance capacity of whey 7% (w/v) as a function
EA time at 10°C and 400 mA. .................................................................................... 94
Table 5.1: Comparison of amino acids composition of native whey and EA-
Whey at different concentrations. ............................................................................. 134
Table 5.2: List of the identified amino acid sequences in the electro-activated
whey (EA-Whey) and their potential biological activity. ......................................... 141
Table 6.1: Main proximate composition and antioxidant properties of whey and
EA-whey used in this study. ..................................................................................... 152
Table 6.2: ODmax of the bacterial strains in supplemented MRS medium as a
function of the concentration and type of carbon source. ......................................... 157
Table 6.3: µmax (h-1) of the bacterial strains in supplemented MRS medium as
a function of the concentration and type of carbon source. ...................................... 159
Table 6.4: ODmax and µmax (h-1) of L. johnsonii La-1 was cultured in MRS-
supplemented medium under anaerobic conditions. ................................................. 162
Table 6.5: ODmax and µmax (h-1) of L. johnsonii La-1 was cultured in MRS-
supplemented medium under aerobic conditions. ..................................................... 162
xiv
LIST OF FIGURES
Figure 1.1: Some most used probiotic species (Holzapfel et al., 2001; Kumar et
al., 2016). ...................................................................................................................... 7
Figure 1.2: Desirable criteria for the selection of probiotics in commercial
applications adopted from (Mortazavian et al., 2012; Tripathi & Giri, 2014). ............. 8
Figure 1.3: Schematic diagram illustrating potential or known mechanisms
whereby probiotic bacteria might affect the microbiota. These mechanisms include (1)
competition for dietary ingredients as growth substrates, (2) bioconversion of, for
example, sugars into fermentation products with inhibitory properties, (3) production
of growth substrates, for example, EPS or vitamins, for other bacteria, (4) direct
antagonism by bacteriocins, (5) competitive exclusion for binding sites, (6) improved
barrier function, (7) reduction of inflammation, thus altering intestinal properties for
colonization and persistence within, and (8) stimulation of innate immune response.
IEC: epithelial cells, DC: dendritic cells, T:T-cells (O'Toole & Cooney, 2008). ....... 12
Figure 1.4: Health benefits from probiotic consumption (Parvez et al., 2006).
..................................................................................................................................... 14
Figure 1.5: Schematic representation of the chemistry mechanism of lactulose
isomerization trough LA transformation, adapted from Aissa & Aïder (2013a). ....... 30
Figure 1.6: Mechanism of action of lactulose and significance of the bacterial
metabolism of lactulose (Panesar & Kumari, 2011). .................................................. 34
Figure 1.7: Alternative modes of bioactive peptide generation (Madureira et al.,
2010). .......................................................................................................................... 41
Figure 1.8: Scheme of the Maillard reaction adapted from (Hodge, 1953). ... 49
Figure 1.9: Schematic illustration of a basic water electrolysis system (Zeng &
Zhang, 2010). .............................................................................................................. 55
Figure 3.1: Schematic representation of the experimental set-up for lactose-
whey isomerization. .................................................................................................... 68
Figure 3.2: Profile of pH as a function of EA time in whey (Cwhey = 7% w/v), at
different experimental conditions (a) current intensities (400, 600, and 800 mA) at
10°C and 100 mL, (b) working temperatures (0, 10, and 25°C) at 400 mA and 100 mL,
and (c) volume conditions (100, 200, and 300 mL) at 10°C and 400 mA. ................. 73
xv
Figure 3.3: Profile of lactulose yield as a function of EA time using different
feed solutions. The experimental conditions were; whey (Cwhey = 7% w/v), lactose
(CLactose = 5% w/v) at 10°C, 400 mA and 100 mL conditions. Data represent the mean
± standard deviation of three experiments. ................................................................. 77
Figure 3.4: Chromatogram profiles of whey sugars after EA at 7% (w/v)
concentration, temperature T = 10°C, current intensity, I = 400 mA and V=100 mL: (a)
t = 0 min, (b) t = 10 min, (c) t = 20 min, (d) t = 30 min, (e) t = 40 min, (f) t = 50 min,
(g) t = 60 min. ............................................................................................................. 79
Figure 3.5: Mechanism of the electro-isomerization of lactose to lactulose in
whey and the possible galactose formation pathways under low temperature using EA
process. ........................................................................................................................ 80
Figure 3.6: Profiles of lactulose and galactose produced as a function of EA
time at different processing temperatures (0, 10, 25°C), whey concentration 7% (w/v),
temperature T = 10°C, current intensity I = 400 mA and V = 100 mL. ..................... 82
Figure 3.7: Profiles of lactulose yield as a function of EA time at different
current intensities (400, 600, and 800 mA). The concentration of feed whey and volume
were fixed at Cwhey = 7% (w/v) and V = 100 mL, respectively. The working
temperatures were varied, (a) at 25°C, (b) at 10°C and (c) at 0°C.............................. 84
Figure 3.8: Profile of lactulose yield produced as a function of EA time at
different volume conditions (100, 200 and 300 mL). The concentration of feed whey
and temperature were fixed at Cwhey = 7% (w/v) and T = 10°C, respectively. The current
intensities were varied, (a) at 400 mA, (b) at 600 mA and (c) at 800 mA. ................. 86
Figure 3.9: Profiles of lactulose yield produced as a function of EA time at
different whey concentration (7, 14 and 28% w/v). The temperature and volume
conditions were fixed at T = 10°C and V = 100 mL, respectively. The current intensities
were varied, (a) at 400 mA, (b) at 600 mA and (c) at 800 mA. .................................. 88
Figure 3.10: Electrophoretic pattern of EA whey proteins as a function of
reaction time under non-reducing conditions (a) and reducing conditions (b). The
temperature and current intensity were fixed at T = 10°C and I = 400 mA, respectively.
MW, molecular weight of protein standard; BSA, bovine serum albumin; β-LG, α-LA
and IGs. ....................................................................................................................... 90
xvi
Figure 3.11: MALDI mass spectrum acquired in the linear mode: (a) untreated
whey and (b) after 40 min of the EA. MALDI mass spectra acquired in the reflectron
mode: (c) untreated sample and (d) after 40 min of EA. ............................................ 93
Figure 4.1: Schematic representation of the reactor used for whey EA: AEM
and CEM indicate the anion and cation exchange membrane, respectively. ............ 103
Figure 4.2: Effect of EA time and current intensities on the ORAC activity of
whey at different concentrations: (a) at 7%, (b) at 14% and (c) at 21% (w/v). Error bars
show standard deviation (n = 3). ............................................................................... 109
Figure 4.3: Effect of EA time and current intensities on the reducing power
activity of whey at different concentrations: (a) at 7%, (b) at 14% and (c) at 21% (w/v).
Error bars show standard deviation (n = 3). .............................................................. 111
Figure 4.4: Effect of EA time and current intensities on the iron chelating ability
of whey at different concentrations: (a) at 7%, (b) at 14% and (c) at 21% (w/v). Error
bars show standard deviation (n = 3). ....................................................................... 113
Figure 4.5: Effect of EA time and current intensities on the DDPH radical
scavenging activity of whey at different concentrations; (a) at 7%, (b) at 14% and (c)
at 21% (w/v). Error bars show standard deviation (n = 3). ....................................... 115
Figure 4.6: Effect of EA time and current intensities on the hydroxyl radical
scavenging activity of whey at different concentrations: (a) at 7%, (b) at 14% and (c)
at 21% (w/v). Error bars show standard deviation (n = 3). ....................................... 117
Figure 4.7: Changes in pH (a), absorbance at 294 nm (b), browning intensity at
420 nm (c), and fluorescence intensity (d) of whey at different concentrations
(diamonds, 7%; squares, 14%; triangles, 21%) during electro-activation at 60 mA for
up to 45 min; error bars show standard deviation (n = 3). ........................................ 120
Figure 5.1: Schematic representation of the experimental set-up for the EA of
whey. ......................................................................................................................... 129
Figure 5.2: Sugar profiles of EA-whey at different concentrations up to 45 min
at 600 mA current intensity: (a) untreated sample, (b) 7% (w/v), (c) 14% (w/v) and (d)
21% (w/v). ................................................................................................................. 135
Figure 5.3: Electrophoretic pattern of EA-whey proteins under non-reducing
conditions (a) and reducing conditions (b)................................................................ 137
xvii
Figure.5.4: Infrared spectra of EA-whey at different concentrations after 45 min
of EA treatment. ........................................................................................................ 139
Figure 6.1: Schematic representation of the electro-activation reactor used for
production of EA-whey. ............................................................................................ 151
Figure 6.2: Normalized FTIR spectra (3000-2800 cm-1) of L. johnsonii La-1
cells grown under (a) anaerobic conditions and (b) aerobic conditions in control MRS
and BMRS culture medium supplemented with EA-whey at different concentrations.
................................................................................................................................... 164
xviii
LIST OF ABBREVIATIONS
ACE: Angiotensin-Converting Enzyme AEM: Anion Exchange Membrane ANOVA: Analysis Of the Variance ARP: Amadori rearrangement product BOD: Biological Oxygen Demand BSA: Bovine Serum Albumin CEM: Cation Exchange Membrane CFU: Colony Forming Units COD: Chemical Oxygen Demand DDPH: 2,2-Diphenyl-1-Picrylhydrazyl EA: Electro-Activation EAS: Electro-Activated Solutions FAO: Food and Agriculture Organization FOS: Fructooligofructose FTIR: Fourier Transform Infrared GOS: Galactooligosaccharide GRAS: Generally Recognized As Safe HPLC: High Performance Liquid Chromatography HRSA: Hydroxyl Radical Scavenging Activity Igs: Immunoglobulins LC-MS/MS: Liquid Chromatography tandem Mass Spectrometry LF: Lactoferrin LP: Lactoperoxidase MALDI-TOF: Matrix-Assisted Laser Desorption/Ionization-Time Of Flight MRPs: Maillard Reaction Products MW: Molecular Weight ORAC: Oxygen Radical Absorbance Capacity PP: Proteose-peptone ROS: Reactive Oxygen Species SCFAs: Short-Chain Fatty Acids SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis UPLC-UV: Ultra Performance Liquid Chromatography Ultraviolet/Visible WHO: World Health Organization α-La: α-Lactalbumin β-Lg: β-Lactoglobulin
xix
DEDICATIONS
To the memory of my loved dad,
My haven of peace
And my twin
You stay in me.
xx
ACKNOWLEDGMENTS
This work represents an achievement of 4 years of research that could not even
have been carried out without the support and understanding of many important people
that I shall therefore acknowledge.
I would like to extend the utmost gratitude to my research director Dr.
Mohammed Aider for welcoming me in his research team and trusting me with this
project. His availability, guidance, support, and advice were always greatly
appreciated. I would also like to express my gratitude to my co-directors Dr. Claude
P., Champagne and Dre. Julie Jean for their support and helpful advice. I am very
grateful to Dr. Ahmed Gooma for his valuable comments and constructive criticisms
in my articles.
My gratitude also extends to Amara Ait-Aissa, Pascal dubé, Veronique Richard,
Diane Gagnon and, Marie Michele, Marine Béguin, Pascal Lavoie and Mélanie
martineau for their help in the lab and for their incredible patience. I would like to thank
Diane Lajoie and Christin Dumas for their availability in the preparation and signature
of administrative documents.
To the many friends I made along the way your presence made this experience
truly wonderful. I would especially like to thank Pamela, Mayank, Emna, Agathe,
Mathilde, Abdel, Tahar, Lamia, Samia, Menel, Karima and Sadia. I also must
acknowledge the great support I had from my colleagues in the Department of Food
Sciences especially, Alseny, Abdoulaye and Omar. My deep appreciation goes to
Ammar for fraternity; I could not accomplish this work without your support. I would
like as well to acknowledge Mahdi, Thank you very much for great friendship and kind
support.
I would also like to show my appreciation for Agathe, Khadija and Yasmée for
the amazing atmosphere and love during the three years working together.
xxi
It is hard to accomplish anything in life without the care and support only your
loved ones can give you. I am very fortunate to have a wonderful family that give me
their immense love and support. I would especially like to extend my deep love and
gratitude to my mom, who has been the great strength and motivation during the entire
periods away from home. Thank you so much letting me go where I wanted to be. I
would also like to show my appreciation for my wonderful brothers; Sofiane, Salim
and Samir for their strong support and continuous encouragement, I express here my
unconditional love “bad boys”. Special acknowledgements for my cousins, Ouisa and
Hakim for their support, immense help, inspiration and insatiable encouragement they
gave me. My gratitude also goes to the other members of my family: to my amazing
grandparents, uncles, aunts and their families.
I would also like to show my appreciation for my family in law, especially
Kahina and Djimy for their support.
Last, but not least, I am extremely thankful to my life partner, Omrak for
constant support and love. I guess, there is no words to express my feelings to you.
Thanks for bearing the hard times and have incredible comprehensiveness and patience
during all these years spent far from you.
THANK YOU !!!
xxii
FOREWORD
The present thesis is submitted to the Faculty of Graduate and Postdoctoral
Studies of Laval University (Faculté des études supérieures et postdoctorales de
l'Université Laval) to meet the requirements for obtaining the Philosophiae Doctor es
Sciences (Ph. D) degree in Food Science and Technology at the Faculty of Agriculture
and Food Sciences (Faculté des sciences de l’agriculture et de l’alimentation).
This doctoral thesis is composed of seven chapters and the results are presented
in the form of scientific articles submitted or published in international journals with
refereed committee. The first chapter represents the literature review, which aims to
provide the essential elements for a good understanding of the problematic of this
doctorate.
The second chapter presents the problematic, hypothesis and objectives of the
present work. Most experimental works and results obtained have been published or
submitted for publication in appropriate scientific journals.
The third chapter presents the article entitled "Contribution to the production of
lactulose-rich whey by in situ electro-isomerization of lactose and effect on whey
proteins after EA as confirmed by MALDI-TOF spectrometry and SDS-PAGE
electrophoresis" published in "Journal of Dairy Science". Authors: Ourdia Kareb,
Claude P. Champagne and Mohammed Aider.
The fourth chapter presents the second article entitled "Impact of electro-
activation on antioxidant properties of defatted whey" published in the "International
Dairy Journal". Authors: Ourdia Kareb, Ahmed I. Gomaa, Claude P. Champagne, Julie
Jean and Mohammed Aider.
The fifth chapter presents the third article entitled "Electro-activation of sweet
defatted whey: Impact on the induced Maillard reaction products and bioactive
xxiii
peptides" published in Food Chemistry. Authors: Ourdia Kareb, Ahmed I. Gomaa,
Claude P. Champagne, Julie Jean and Mohammed Aider.
The sixth chapter presents the fourth article entitled "Effect of electro-activated
sweet whey on growth of probiotic bacteria of Bifidobacterium, Lactobacillus and
Streptococcus genera under model growth conditions" submitted to publication to
"International Dairy Journal". Authors: Ourdia Kareb, Ahmed I. Gomaa, Claude P.
Champagne, Julie Jean and Mohammed Aider.
In all articles presented above, Ourdia Kareb is the first author who was in
charge of the conception, experimental design and execution of experimental works,
result analysis and article writing. Dr Mohammed Aider (thesis director) was involved
in scientific supervision, experimental design, correction, revision and submission of
manuscripts. Dr Claude champagne and Julie Jean (thesis co-directors) were involved
in experimental design, correction and revision of manuscripts; Dr Ahmed Gomaa was
involved in correction and revision of manuscripts.
1
INTRODUCTION
‘‘Let food be thy medicine and medicine thy food’’. In North America, more
than 93% of the population adopt the idea of eating as a mean of prevention against
diseases (Champagne, 2009). This social tendency attracts a keen interest within the
food industry, which puts functional foods on the market with a benefit effect beyond
simple nutritional function (Betoret et al., 2011; Bigliardi & Galati, 2013; Costa &
Jongen, 2006). Many of these so called “healthy foods” contain functional compounds
like probiotics, prebiotics, bioactive peptides and antioxidant compounds, as well as
other nutrients such as vitamins and specific minerals (Grajek et al., 2005; Korhonen,
2009; Li-Chan, 2015; Lobo et al., 2010; Stanton et al., 2005; Watson & Preedy, 2015).
The probiotic containing food products have been estimated to account for 60-
70% of the total functional foods market (Tripathi & Giri, 2014). Probiotics are found
mostly in dairy products, whith yogurts and fermented milks representing the major
products sold worldwide (Granato et al., 2010a). In Canada, the regularly dose of a
probiotic is estimated to approximately 109 viable cells per day to reach the targeted
health effect (Champagne et al., 2011). However, many questions remain about the
effectiveness, especially their survival from design until their site of action. These
microorganisms are subjected to many stresses during the manufacturing and storage
of the product until the consumption due the changes in pH, total acidity, temperature,
oxygen variations and depletion of nutrients leading to low survival rates (Donkor et
al., 2006; Talwalkar & Kailasapathy, 2004; Tripathi & Giri, 2014). Moreover, it has
been shown that probiotics exhibit slow growth in fermented dairy products due their
low proteolytic activity (Marafon et al., 2011; Shihata & Shah, 2000).
One of the most important scientific challenges for the dairy industry is to
promote probiotics response capacity by developing effective compounds than can
enhance their growth and survival under different conditions as well as during the
transit within the gastrointestinal tract, which is known to be a harsh medium for
probiotics. At the other hand, prebiotics, described as non-digestible carbohydrates, are
able to selectively stimulate the growth of beneficial gut microbiota, and are by far the
most used ingredients which help in the growth of probiotic bacteria (Romano et al.,
2015). In this regards, lactulose as a prebiotic derived from lactose is successfully used
2
in the food industry due to the nutritional and technological properties it provides in
dairy products (Seki & Saito, 2012). In addition, it offers additional health benefits as
a smooth laxative and is used by the pharmaceutical industry in the treatment of hepatic
encephalopathy. The total annual global production of lactulose has been estimated
around 50000 tons in 2009 (Playne & Crittenden, 2009). Lactulose can be produced by
isomerization of lactose through chemical or enzymatic approaches. Recent works
introduce electro-activation (EA) as an interesting approach of lactulose production. In
contrast to both chemical and enzymatic methods, electro-activation is considered to
be an economical process, is safe and is reagentless technology for the synthesis of
lactulose from lactose by self-generating of high alkalinity in the reaction medium
(Aider & Gimenez-Vidal, 2012; Aissa & Aïder, 2013a, 2013b, 2014a).
Other challenges for the dairy industry is to added value to the huge quantities
of whey produced from cheese and casein manufacturing. About 180 million tonnes of
whey were produced in 2012, and around 30-50% of the total production are still not
utilized (Sitanggang et al., 2016). Despite its potential as pollutant, whey is regarded
as a valuable source of numerous nutritional, functional and bioactive compounds.
Whey presents an elevated content of lactose and proteins, which can be used to
produce versatile benefit ingredients. The production of lactulose from whey by using
electro-activation should be a commendable challenge; since it presents the both
economical and environmental interests. In addition, it appears that no other study has
considered the possible synergistic effect between lactose and whey protein submitted
to EA. Meanwhile, the behaviour of whey proteins have not been tested in EA during
lactulose synthesis. Thus, their structural and functional properties, as affected by EA,
are not well known so far. It is possible that the EA of whey as non-thermal technology
could allow the formation of other functional compounds of interest, mainly Maillard
reaction products (MRPs) described to have excellent antioxidant properties. A better
understanding of the relationship between the structure and characteristics of these
conjugates will allow their use to improve functional properties of food products.
Additionally, the hydrolysis of whey proteins can occur and can result in the generation
of bioactive peptides. The latter have been described to perform physiological effects
in vivo, such as antioxidant, antimicrobial, antihypertensive and anticancer activities.
3
The aim of this Ph. D project was to study the process of sweet whey electro-
activation for the production, in situ, of lactulose, as well as to determine the impact of
this process on whey proteins structure and functionality. Moreover, the antioxidant
activity of electro-activated whey was studied in order to understand the involved
compounds. Finally, the synergetic effect of electro-activated whey due its prebiotic
and antioxidant properties was tested for the growth of probiotic bacteria under model
growth conditions.
4
1. CHAPTER 1: Literature review
1.1 Functional foods
It is thought that factors such as stressful lifestyle and changing dietary patterns
cause the raise in the incidence of chronic diseases, and by the way, their prevention
and treatment are very costly (Hu et al., 2000; Willett, 2002). Recent studies have
clearly linked the balanced diet to improved health and to prevent or to reduce the risk
of some diseases (Amine et al., 2002; Boushey et al., 2001; Darnton-Hill et al., 2004;
Erdmann et al., 2008). These findings lead to a huge interest in the population to
consume foods that contain functional ingredients, thereby promoting their health and
well-being (Milner, 1999; Siro et al., 2008). Consequently, research studies in the food
industry have concentrated on identifying bioactive ingredients, which provide
effective physiological benefits over and above the nutritional value of traditional food
products (Bigliardi & Galati, 2013; Goldberg, 2012). These compounds can be added
to, or naturally enhanced or derived in variety of foods to provide advantageous health
benefits, thus forming the basis of new market food segment so-called “functional
foods” (Hasler, 1998; Roberfroid, 2000).
The functional food term was first introduced in Japan, in the 1980s, for food
product fortified with, bioactive compounds that possessing positive physiological
effects and reducing the risk of diseases, in addition to being nutritious (Doyon &
Labrecque, 2008; Hasler, 2002). The functional food can be a natural or processed
product that contain one or more biologically active compounds. These foods include:
(i) usual foods with naturally occurring bioactive substances (e.g., dietary fibre), (ii)
foods supplemented with bioactive substances (e.g., probiotics, antioxidants), and (iii)
derived food ingredients (e.g., prebiotics, bioactive peptides) (Roberfroid, 2000; Siro
et al., 2008). Thus, together with their acceptance as healthy and nutritious products,
functional foods have led to their extended popularity across the world, especially in
industrialized countries (Bigliardi & Galati, 2013; Siro et al., 2008). The global market
for functional foods has been expected to be worth $305.4 billion by 2020. The faster
growing sector within this area corresponds to the dairy products fortified with
probiotics and/or prebiotics (Stanton et al., 2005). Their success among functional
5
foods is mainly due to their healthful image among consumers (Granato et al., 2010a).
Other potentially functional ingredients such as bioactive peptides and antioxidant
compounds have been continuously raising the interest in functional foods (Grajek et
al., 2005; Li-Chan, 2015; López‐Barrios et al., 2014; Patel, 2015).
During the last decades, consumers increasingly believe that functional foods
significantly contribute to their health and are looking for the bioactive compounds
found in them. Indeed, they are demanding for novel, high added value and affordable
functional ingredients (Costa & Jongen, 2006). As consequence, the food industry has
been focused their efforts to obtain innovative functional products that satisfy this
demand (Juriaanse, 2006; Khan et al., 2013). Indeed, one of the promising opportunity
in this area lies in development of functional ingredients that offer multiple health
benefits in a single food (Teratanavat & Hooker, 2006). The successful innovation in
functional products is challenging and complex process highly related to the cost
reduction by the incorporation of cheaper raw materials and the use of efficient and
adaptive processing technologies (Betoret et al., 2011; Bigliardi & Galati, 2013).
Therefore, the cheese whey, a co-product of the dairy industry, potentially rich in
lactose, proteins and minerals, is gained increasing attention to be valorized into
functional ingredient, which in turn allows avoiding the related environmental
problems of whey. Moreover, it is necessary to develop efficient technologies able to
reduce the cost of the process, treatment time and the eliminating of the use of reagents,
especially when these are toxic. Recent advances in the EA as an emerging technology
offers attracting opportunity to promote the development of whey as a functional
ingredient in respect of the environmental friendly concept.
Thus, since this review is about the valorization of whey as a functional
ingredient with potential prebiotic activity, an overview about probiotics and prebiotics
is described. Subsequently, insight into nutritional and biological benefits of whey, the
derived bioactive ingredients with special emphasis on lactulose, bioactive peptides
and Maillard reaction products, their properties as well as potential uses is given.
6
1.1.1 Probiotics
1.1.1.1 Probiotics concept
Ilya Ilyich Mechnikov, Nobel Prize in Physiology or Medicine 1908, evoked
for the first time the concept of «Probiotics» and proposed that yoghurt had beneficial
health effects because of the lactic acid bacteria it contains and played a major role in
maintaining intestinal health and prolonging life expectancy of Balkans (Anukam &
Reid, 2007; Gibson, 1999). The term probiotic comes from the Greek words "pro bios"
meaning "for life", and was first proposed in 1965 by Lilly and Stilwell. Later, Fuller
and Gibson (1997) focused on viability of probiotics and introduced the idea that they
have a beneficial effects on the host (Guarner et al., 2005). The current definition is the
one adopted by experts of the commissions' Food and Agriculture Organization United
Nations (FAO) and the World Health Organization (WHO) “live microorganisms
which when administered in adequate amounts; exert a beneficial effect on the host's
health”(FAO/WHO, 2002). However, the inclusion of the word “live” in definition was
controverted, when positive effects were reported for the same dead cells (Adams,
2010). Recent advances also widened the probiotic to probioactive concept “probiotic-
derived bioactive”, there would be to the generation of new bioactive compounds and
no further need to have longer shelf-live bacteria. Given these prerequisites, the
beneficial health of probiotic bacteria can resulted from the generation of new bioactive
compounds derived from both bacterial metabolisms and/or hydrolysis of food matrix
(Farnworth & Champagne, 2010). In the International Scientific Association for
Probiotic and Prebiotic (ISAPP) consensus meeting stated in October 2013, the expert
panels continue to untimely restrict the selection of potential probiotic to viable state
and the necessary to obtain claims recognition (Hill et al., 2014; Kumar et al., 2015).
Thus, bioactive bacterial products or lysed cells that also reported to modulate the
immune response were eliminated from the probiotic concept.
1.1.1.2 Commercially used probiotic foods
Probiotic bacteria are available in the market and consumed as either food
products or dietary supplements in the form of tablets or capsules, and they represent
important segment in the modern functional foods market (Granato et al., 2010b;
7
Stanton et al., 2005). Probiotics dairy products, especially yogurts and cheese were
shown to be the most consumed, since they exhibit good delivery carriers for
maintaining the viability of these bacteria (da Cruz et al., 2009; El-Dieb et al., 2012).
Moreover, the market is diversified to non-dairy products such as cereals, chocolate
bars, biscuits, drinks and even chewing-gum added probiotics (Foligné et al., 2013).
The commonly probiotics are from bifidobacteria and lactobacilli genera that
considered to be safe based on their historical presence in human gut and foods (Figure
1.1). Other less commonly species belonging to the genera Streptococcus, Escherichia
coli, Lactococcus, Enterococcus and Saccharomyces yeast are also used as probiotics
due to their safety and great health potential (Holzapfel et al., 2001; Kumar et al., 2016).
Figure 1.1: Some most used probiotic species (Holzapfel et al., 2001; Kumar et al., 2016).
8
1.1.1.3 Selection of potential probiotics
The appropriate selection of probiotic strains is a curial step to ensure the
desirable health benefits and a list of potential criteria appears in Figure 1.2. It must
meet security, functionality, good technological properties and the claimed health
benefits (FAO/WHO, 2002). The initial screening of probiotics includes the safety of
the microorganisms. Lactobacillus and Bifidobacterium strains that generally have the
status of "GRAS" (Generally Recognised As Safe) are widely recognized as probiotics.
However, the presence of plasmids carrying antibiotic resistance genes and /or
virulence factors reduces the security aspects (Saarela et al., 2000; Vesterlund et al.,
2007; Zhou et al., 2005).
Figure 1.2: Desirable criteria for the selection of probiotics in commercial applications adopted from (Mortazavian et al., 2012; Tripathi & Giri, 2014).
9
The viability and metabolic activity are the main features to ensure the
functionality of probiotic strains and to finally colonize their specific locations
(Mortazavian et al., 2012). Health Canada recommends a daily dose of 109 colony-
forming units (CFU) at time of consumption (Champagne et al., 2011). The probiotic
orally ingested in foods must remain viable and resist to the harsh conditions
encountered during gastrointestinal digestion, in particular amylases, pH acidity of the
stomach, pancreatic enzymes and bile salts (Hernandez-Hernandez et al., 2012). The
ability of selected probiotic to survive to this stressed environment is species and
strains-dependant (Huang & Adams, 2004; Tamime et al., 2005). Charteris et al. (1998)
studied the sensitivity of several probiotic strains in the gastrointestinal simulator.
These authors found that the majority of strains, except Lactobacillus. fermentum KLD,
were susceptible to resist to the gastric environment. The strains of Bifidobacterium
and Lactobacillus are mostly resistant to enzymes encountered during intestinal transit.
Lactobacillus acidophilus La-5 as well as Lactobacillus. johnsonii La-1 were more
resistant than Lacobacillus. casei strain and Lactobacillus. rhamnosus GG to bile salts.
In addition, there is an induction of resistance of Lactobacillus. rhamnosus GG to the
gastrointestinal environment with certain ingredients of the food matrix (Sumeri et al.,
2008). The probiotics adhesion capacity to the intestinal mucosa is also required to
guarantee the survival of the probiotics into the gut and then to exert their beneficial
action (Ouwehand & Salminen, 2003). Some probiotic strains demonstrated to have
the ability to exclude the pathogenic microbiota through a competition for the same site
adhesion at the intestinal mucosa (Collado et al., 2007). Further functional properties
such as their ability to grab nutrients, lactic acid production, and secretion of
antimicrobial metabolites such as bacteriocins and organic acids are required to the
selection of suitable probiotic strains (Saad et al., 2013; Saarela et al., 2000).
Moreover, for their industrial applications, probiotics must demonstrate their
technological ability to grow at large scale, remain stable at high survival levels during
processing and storage (Champagne et al., 2011; Saarela et al., 2000). It is important
that the selected strains involved in improving the organoleptic and textural qualities
of the final probiotic product (Champagne et al., 2005; Granato et al., 2010a). The
acidifying activity of probiotics exerts a protective effect against the spoilage flora and
10
improves its sensory qualities (Donkor et al., 2006). The production of
exopolysaccharides by some probiotic strains improves the rheological properties of
yoghurt and reduces syneresis (Badel et al., 2011). The ability of probiotic bacteria to
release bioactive molecules such as conjugated linoleic acid (CLA), γ-aminobutyric
acid (GABA), bioactive peptides and other molecules is very suitable from the
industrial point of view (Coakley et al., 2003; Gobbetti et al., 2010; Stanton et al.,
2005). In the processing of fermented dairy products, selecting the most biocompatible
combination of starter and probiotic cultures has to be chosen in the regard of the strains
growth and the final quality of the product (Gardner-Fortier et al., 2013; Vinderola et
al., 2002).
1.1.1.4 Mechanisms of action of probiotics
Probiotics have demonstrated their potential as therapeutic agents for a variety
of illnesses but the mechanisms associated with the probiotic activity have not been
fully elucidated yet. Several operating mechanisms have been highlighted and are
schematically summarized in Figure 1.3 (O'Toole & Cooney, 2008). Probiotics mainly
act in the gut homeostasis by excluding the pathogens invasion by competition with
them for limiting nutritional resources, reduction luminal pH or production of
antimicrobial substances and receptor adhesion in mucosal and epithelial cells (Ng et
al., 2009; Sarkar & Mandal, 2016). It has been reported that one of the efficient
mechanisms of bifidobacteria in the gut is by sequestering iron, thus causing
deprivation for enteropathogenic bacteria involved in infectious diseases (Vazquez-
Gutierrez et al., 2015). Selective fermentation of dietary carbohydrates by intestinal
probiotics resulted in metabolites of physiological importance such as short chain fatty
acids (SCFAs), acids and carbon dioxides which play role in the reduction of luminal
pH, consequently inhibiting the growth of acid-sensitive pathogens (Fukuda et al.,
2011; Yasmin et al., 2015). Probiotics also release antibacterial products referred to as
bacteriocins and de-conjugated salts leading to intestinal homeostasis (Bermudez-Brito
et al., 2012; Ng et al., 2009). In addition to direct antibacterial mechanisms, probiotics
contribute to enhance the epithelial barrier by competitive exclusion of pathogens and
toxins trough expression of the analogue trans-membrane or establishing biofilms and
11
surfactants (Oelschlaeger, 2010; Sherman et al., 2009). Probiotics promote intestinal
epithelial cell survival and restore barrier integrity after damages by enhanced the
expression and the redistribution of tight junction proteins (Bermudez-Brito et al.,
2012). Probiotics can also mediate their positive immunomodulatory effects by
enhancing and modulating pathogen induced inflammation related in inflammatory
bowel diseases like ulcerative colitis and Crohn’s disease (Chong, 2014). Probiotics
exert immunomodulatory effects through the expression of signaling molecules, which
interact with the immune cells by pattern recognition receptors (PRRs) like the well-
known toll-like receptors (TLRs). Thus, immune response may be even achieved by
probiotics-derived signaling molecules such as exopolysaccharides, teichoic acids,
peptidoglycan fragments or DNA-like peptidoglycan, and (Oelschlaeger, 2010; Sarkar
& Mandal, 2016). Moreover, probiotics may regulate the immune responses by
enhancing the innate immune molecules including mucins, trefoil factors and defensins
(Sherman, Ossa et al. 2009). Current reach also suggests that bacteria in the gut could
have a direct effect on the central nervous system via the vagus nerve, even in the
absence of an immune response (Gayathri & Rashmi, 2017) . For example, in the
mouse, the consumption of Lactobacillus. rhamnosus JB-1 modulate the central GABA
receptors and reduced the anxiety (Bravo et al., 2011). Thus, the understand of the
mechanisms of action and the appropriate probiotics enhances their effectiveness
application for the prevention and treatment of a certain diseases (Bermudez-Brito et
al., 2012).
12
Figure 1.3: Schematic diagram illustrating potential or known mechanisms whereby probiotic bacteria might affect the microbiota. These mechanisms include (1) competition for dietary ingredients as growth substrates, (2) bioconversion of, for example, sugars into fermentation products with inhibitory properties, (3) production of growth substrates, for example, EPS or vitamins, for other bacteria, (4) direct antagonism by bacteriocins, (5) competitive exclusion for binding sites, (6) improved barrier function, (7) reduction of inflammation, thus altering intestinal properties for colonization and persistence within, and (8) stimulation of innate immune response. IEC: epithelial cells, DC: dendritic cells, T:T-cells (O'Toole & Cooney, 2008).
1.1.1.5 Beneficial health effects of probiotics
A wide array of health benefits have been attributed to the consumption of
probiotics in adequate amount, including improvement of intestinal disorders,
enhancement of the immune response, alleviation of lactose intolerance and reduction
of serum cholesterol as well as prevention of colon cancer (Agarwal et al., 2016; Gill
& Prasad, 2008; Kechagia et al., 2013). Figure 1.4 summarizes the multiple benefits
of probiotics on human health (Parvez et al., 2006). Health attributes of probiotics can
be exerted locally in the gastrointestinal gut or systemically thought out the body
(Kumar et al., 2016). There are a documented benefits of Lactobacillus. rhamnosus GG
13
in treating several forms of diarrhea, including rotavirus diarrhea, travelers’ diarrhea,
relapsing Clostridium difficile diarrhea as well as to prevent antibiotic associated
diarrhea in children (McFarland, 2006; Szajewska et al., 2013). Other probiotics such
as Lactobacillus. reuteri DSM 17938 and Saccharomyces boulardii have been shown
to be effective for the treatment of acute gastroenteritis (Guandalini, 2008). The
consumption of some probiotic yogurts was related to improve lactose digestion and to
lower serum cholesterol levels through de-conjugation of bile salts (Shah, 2007). There
are also agreements relating to the effectiveness of Lactobacillus. rhamnosus GG and
Bifidobacterium. animalis subsp lactis Bb12 to reduce the severity of atopic eczema
(Doege et al., 2012; Isolauri et al., 2000). While some of the health benefits are well
established, others require additional studies in human to substantiate these benefits
(Rijkers et al., 2011). Promising applications of probiotics include the prevention of
respiratory and urogenital infections, prevention of dental caries and treatment of
inflammatory bowel diseases (Anderson et al., 2017; Chapman et al., 2011; Gille et al.,
2016; Stamatova & Meurman, 2009). More studies are anticipated for future
applications of probiotics in the treatment of rheumatoid arthritis, management of
depression, prevention of cancer diseases, and treatment of diabetes as well as using as
vaccine adjuvants (Gayathri & Rashmi, 2017; Sun & Buys, 2016; Tonucci et al., 2017;
Zoumpopoulou et al., 2017). These health properties are highly species and strain
specific and impacted by the various mechanisms of action mentioned above (Hill et
al., 2014; Sánchez et al., 2016). The convincing dose therapeutic effect is not precisely
defined because the strains do not survive in the same level to different stresses
encountered during their manufacturing and through the gastrointestinal passage (Reid
2008). Many of these probiotics are being delivered through dairy products, of which
yogurt is the most deliverer carrier used (Marco et al., 2017; Pei et al., 2017; Reid,
2015).
14
Figure 1.4: Health benefits from probiotic consumption (Parvez et al., 2006).
1.1.1.6 Factors affecting the survival of probiotics in dairy products
Dairy products are considered the most suitable carriers for delivering probiotic
cultures to the human gastrointestinal tract (Granato et al., 2010a; Mortazavian et al.,
2012; Ranadheera et al., 2010). There are mainly incorporated into products such as
cheese, yogurt, beverage, ice cream, and other dairy desserts (Ranadheera et al., 2010).
The application of these cultures in dairy products represents a huge challenge.The
probiotics may lose their viability and metabolic activity due the unfavorable
environment conditions during production and over storage period (Champagne, 2009;
Ross et al., 2005). Thus, factors such as chemical composition of product where they
are added, milk solid content, addition of salts, sugars and sweeteners, properties of
strains used and their interactions with the starter cultures, moment and proportion of
inoculation, content and availability of nutrient, temperature and duration of
15
fermentation, redox potential and dissolved oxygen, pH and titrable acidity,
concentration of final metabolites as well as storage temperature might significantly
affect the viability of probiotic cultures (Dave & Shah, 1997b; Kneifel et al., 1993;
Lourens-Hattingh & Viljoen, 2001; Mattila-Sandholm et al., 2002; Mortazavian et al.,
2011; Shah, 2000; Talwalkar & Kailasapathy, 2004; Vinderola et al., 2002). Probiotic
cultures, in particular, L. rhamnosus and bifidobacteria usually exhibited weakly
growth in milk due to their slow proteolytic activity and lack of some vitamins, as well
as to their low lactase levels (Gaudreau et al., 2005; Roy, 2005). During fermentation
and storage of bio-yogurt, these microorganisms are considerably affected by the
decrease of pH which may reach values as low as 3.6 (Lankaputhra et al., 1996).
Lactobacilli are generally more acid tolerant than bifidobacteria since their growth is
inhibited below pH value of 4.6 (Lee & Salminen, 2009; Shah et al., 1995). Moreover,
during the refrigerated storage, uncontrolled growth of some L. delbrueckii ssp.
bulgaricus at low pH results in excessive production of organic acids termed post-
acidification that may further affect the viability of acid-sensitive probiotics (Donkor
et al., 2006; Shah & Ravula, 2000). Another main stress that probiotic encountered in
fermented dairy products is their exposition to oxygen toxicity (Dave & Shah, 1997b;
Talwalkar & Kailasapathy, 2004). It is well known that oxygen easily penetrates and
dissolves during production, in addition to oxygen permeation through the packaging
(Lourens-Hattingh & Viljoen, 2001; Shah, 2000). Lactobacilli are aerotolerant or
anaerobic, while bifidobacteria are strictly anaerobic, thus the oxygen content and the
redox potential have significant importance in maintaining their survival, when the
oxygen-scavenging system in these bacteria is either reduced or completely absent
(Holzapfel et al., 2001). Consequently, when water is present, oxygen is reduced to
form toxic oxygenic metabolites such as hydrogen peroxide (H2O2), superoxide anion
(O2-) and the highly toxic hydroxyl radical (OH-) which lead to the cell death
(Talwalkar & Kailasapathy, 2004). Moreover, in the presence of oxygen, some L.
delbrueckii ssp. bulgaricus can produce H2O2 and may indirectly affect the viability of
probiotic bacteria during storage (Shah, 2000). The conservation of probiotic dairy
products at low temperature is also a matter of concern. Cold temperatures reduce the
membrane fluidity as well as influence the strain multiply and increase sensitiveness
16
toward sodium chloride, which may cause perturbation in the membrane integrity
(Corcoran et al., 2008).
Thus, many alternatives have been adopted to enhance the viability of
probiotics, the most commonly being stress adaptation, two step-fermentation,
microencapsulation as well as fortification of milk with different growth and promoting
factors such as hydrolyzed protein, whey derivatives, amino acids, with recently
emphasis on prebiotics and antioxidant compounds (Champagne, 2009; Dave & Shah,
1998; Dave & Shah, 1997a; Gouin, 2004; Mattila-Sandholm et al., 2002; Mohammadi
& Mortazavian, 2011; Mortazavian et al., 2012; Romano et al., 2015).
1.1.2 Prebiotics
1.1.2.1 Prebiotics concept
Currently, there is a great deal of interest in the use of prebiotics as functional
food ingredients as they combine health benefits and interesting technological
properties (Al-Sheraji et al., 2013; Charalampopoulos & Rastall, 2012; Roberfroid,
2002). The concept of prebiotics was introduced at the end of the 20th century and
originally was defined as "selectively fermented ingredient that induces specific
changes in the composition and/or the intestinal flora, conferring benefits to health and
well-being of the host" (Gibson & Roberfroid, 1995). Based on this definition, the most
known prebiotics are carbohydrates such as fructooligofructoses (FOS), inulin,
galactooligosaccharide (GOS) and lactulose, which resist digestion and can,
metabolized by health promoting colonic microflora, especially by bifidobacteria and
lactobacilli (Fuller & Gibson, 1997; Gibson, 2004; Gibson et al., 2004). At the 6th
ISAPP Meeting in 2008, the more recently definition of “dietary prebiotics” was
updated as “a selectively fermented ingredient that results in specific changes in the
composition and/or activity of the gastrointestinal microbiota, thus conferring
benefit(s) upon host health”. The most important emphasis is toward the extension of
the physiological benefits dominated by the gastrointestinal health to more targeted
sites such as the oral cavity, skin and the urogenital tract (Gibson et al., 2010).
Moreover, in the light of the current knowledge on the ecological and the functional
features of microbiota, Bindels et al. (2015) widen the concept of prebiotics and
17
included more compounds as potential prebiotic candidates such as resistant starches,
pectin, arabinoxylan, whole grains and non-carbohydrate compounds such as
polyphenols and bioactive peptides.
1.1.2.2 Criteria of prebiotics
There are many candidates for prebiotic name, however it must fulfilled some
criteria: (i) Resistance to gastric activity and resistance to hydrolysis by mammalian
enzymes, (ii) No absorption in the upper gastrointestinal tract, (iii) Fermentation by the
intestinal microbiota and (iv) Selective stimulation of the growth and/or activity of
intestinal bacteria believed to be beneficial to the host (Gibson et al., 2004). The
selectivity is the most difficult criterion to demonstrate. It is generally accepted that
prebiotics have potentially selective effect on bifidobacteria and lactobacilli genus said
to be beneficial for health, whilst decreasing bacteroides, clostridia and fusobacteria
branded detrimental (Rastall & Gibson, 2015). However, the selectivity criterion is not
assumed as previously when strains belonging the pointing detrimental groups such as
Faecalibacterium prausnitzii and Akkermansia muciniphila have shown to be
beneficial and to metabolize specific prebiotics (Hutkins et al., 2016). Prebiotics are
also appreciated for their technological properties (Huebner et al., 2007; Wang, 2009).
Thus, their stability during the product processing and their contribution to improve
organoleptic and functional properties of the foods is another potential prebiotic
selection criterion (Al-Sheraji et al., 2013; Charalampopoulos & Rastall, 2012).
1.1.2.3 Sources, production of prebiotics
The most food-grade utilized prebiotics are carbohydrates with different degree
of polymerisation. These include (FOS), inulin type-fructans, (GOS) and lactulose
(Gibson, 2004). Moreover, these is an emerging list of a potential prebiotics including
lactosucrose, isomalto-oligosaccharides (IMO), xylo-oligosaccharides (XOS), resistant
starch and soybean oligosaccharides (SOS) (Charalampopoulos & Rastall, 2009).
Some prebiotics are naturally present in common foods including milk, vegetables and
fruits such as asparagus, chicory, leek, banana, wheat and soybean (Fu & Wang, 2013).
However, their content is generally low to engender the desirable bifidogenic effect
18
and need more subsequent manufacturing steps (Michalak et al., 2014). The usually
used methods for prebiotics production are illustrated in Table 1.1. For examples,
inulin and soybean oligosaccharides are produced by direct extraction in aqueous
media. The FOS are manufactured from the partial enzymatic hydrolysis of inulin or
synthesised from sucrose using a fructosyltransferase enzyme. Industrially, lactulose is
mainly manufactured from lactose trough chemical isomerisation using alkaline
catalysts. The GOS and lactosucrose are another prebiotic compounds produced from
lactose by transglycosylation with ß-galactosidase and ß-fructofuranosidase enzymes,
respectively. These prebiotics are available in the market with different purity degree
as powders or syrups and formulated as supplements or incorporated as a functional
ingredients in various food products (Charalampopoulos & Rastall, 2009).
19
Table 1.1: Main approaches used for the production of prebiotic carbohydrates (Playne
& Crittenden, 2009).
Approach Process Prebiotic examples
Direct extraction
Extraction from raw plant
materials
Soybean oligosaccharides from
soybean whey
Inulin from chicory
Resistant starch from inulin
Controlled
hydrolysis
Controlled enzymatic hydrolysis
of polysaccharides; may be
followed chromatography to
purify the prebiotics
Fructooligosaccharides from
inulin
Xylooligosaccharides from
Arabinoxylan
Transglycosylation
Enzymatic process to build up
oligosaccharides from
disaccharides; may be followed
by chromatography to purify the
prebiotics
Galactooligosaccharides from
lactose
Fructooligosaccharides from
sucrose
Lactosucrose from
Lactose + sucrose
Chemical
processes
Catalytic conversion of
carbohydrates
Lactulose from rom alkaline
isomerization of lactose
Lactitol from hydrogenation of
lactose
20
1.1.2.4 Beneficial health effects of prebiotics
The most health promoting effects of prebiotics are believed to come through
the modulation of the gut microbiota towards a more beneficial microbiota composition
and their fermentation metabolites (Verbeke et al., 2015; Verspreet et al., 2016). The
beneficial effects of prebiotics include an increase of good bacteria and a decrease of
detrimental bacteria in the gut, alleviation of constipation, treatment of hepatic
encephalopathy, prevention of infections, increased absorption of minerals, regulation
of blood lipids and reduced of cancer risk (Al-Sheraji et al., 2013; Roberfroid et al.,
2010; Slavin, 2013). FOS and inulin prebiotics have shown to stimulate bifidobacteria
growth and to reduce harmful bacteria in the human colon (Gibson & Roberfroid,
1995). Prebiotics have shown protective effects against infections by exerting anti-
adhesive activity and inhibit binding of pathogens to the intestinal cells (Rastall et al.,
2005). FOS can directly stimulate bacteriocin production from Lactobacillus and
Lactococcus strains (Chen et al., 2007). Indeed, the fermentation of prebiotics in colon
leads to the increase in the production of short-chain fatty acids (SCFAs) with various
physiological effects. They low the pH of the colon making the growth difficult for
some pathogenic bacteria (Wang & Gibson, 1993). In addition, the acidification of the
colon leads to increased mucin production and reinforced the mucosal barrier and in
this way decreasing translocation of pathogen bacteria. The low pH also reduces the
formation of toxic compounds such as ammonia, biogenic amines, and phenolic
compounds (Lomax & Calder, 2009). The SCFAs including acetate, propionate and
butyrate exert important physiological effects on colonic epithelium as a source of
energy for the colonocytes as well as for immune system cells (Bermudez‐Brito et al.,
2015). Prebiotics are also known to interact with carbohydrate receptors on immune
cells in Peyer’s patches, to increase the cytotoxicity of natural killer cells and to
increase the production of IgA and various interleukins (Tuohy, 2008). Increasing
SCFAs has been also associated with improving availability of minerals in the colon,
which can be beneficial in preventing osteoporosis and avoiding diet-related anaemia.
Calcium and magnesium absorption has been found to be increased following ingestion
of GOS (Chonan et al., 2001). Indeed, inulin has been proved to reduce the plasma
levels of cholesterol and triacylglycerols and different mechanisms on lipid metabolism
21
are speculated. This was thought to be due to the inhibition of a liver lipogenic activity
which may be a result of increased propionate produced from the fermentation of inulin
by gut bacteria (Beylot, 2005). In addition, current studies support the positive effect
of fructan prebiotics on appetite sensation through increased secretion of the satiety-
inducing hormone glucagon-like peptide 1 and peptide YY (Cani et al., 2009). Other
promising health benefits associated with prebiotics are the reduction of inflammatory
bowel disease by stimulation the growth of immunomodulatory bifidobacteria (Sartor,
2004). These properties collectively give prebiotics myriad potential applications into
a broader range of foods.
1.1.2.5 Prebiotics as functional food ingredients
The main applications of prebiotics include dairy products, frozen desserts,
baked goods, breakfast cereals, fillings, processed meat and baby food formulations
(Table 1.2). In addition to their nutritional advantages, prebiotics offer a versatile
functional proprieties when incorporated in foods as reduced calorie value, fat replacer,
textural modification, organoleptic improvement, dietary fiber and prebiotic (Fu &
Wang, 2013). As functional food ingredient, prebiotics must be chemically stable
during food processing, such as high temperature, low pH, and Maillard reaction
conditions (Huebner et al., 2007). Significant decreasing prebiotic activity was only
seen for FOS when heated at low pH, so no longer offering selective stimulation. Inulin
is often used as fat replacer or texture modifier in low fat dairy products, such as milk
drinks, fresh cheeses, yoghurts and dairy desserts. In cheese, inulin seems particularly
suitable for fat replacement as it may contribute to an improved mouthfeel. As fat
replacement, inulin can also be applied in meat products, sauces and soups.
Additionally, inulin are used as prebiotic ingredient and improved the viability of
probiotic cultures in dairy products during food processing and storage (Karimi et al.,
2015). FOS are approximately between 0.3 and 0.6 the sweetness than sucrose and have
similar technological properties to sucrose and glucose syrups. They are usually
applied as sugar replacements in variety of dairy products as well as in foods for
diabetics and lower calorie food products (Bali et al., 2015). The GOS are used to
improve the textural proprieties of yogurts. They are also suitable to retain moisture in
22
bread and baked products, owing to their high moisture retaining capacity, as well as
in fruit juices owing to their high acid stability (Torres et al., 2010). Addition of the
mixture of GOS and FOS to infant formulas stimulates the growth of bifidobacteria
(Bakker-Zierikzee et al., 2005). XOS are moderately sweet, stable over a wide range
of pH and temperatures and have organoleptic characteristics suitable for incorporation
into juices (Otieno & Ahring, 2012).
Table 1.2: Food applications of prebiotics (Wang, 2009).
Applications Functional properties
Yoghurts and desserts
Beverages and drinks
Breads and fillings
Meat products
Dietetic products
Cake and biscuits
Chocolate
Sugar confectionary
Soups and sauces
Baby food
Sugar replacement, texture and mouthfeel, fiber and prebiotics
Sugar replacement, mouthfeel, foam stabilization and prebiotics
Fat or sugar replacement, texture, fiber and prebiotics
Fat replacement, texture, stability and fiber
Fat or sugar replacement, fiber and prebiotics
Sugar replacement, moisture retention, fiber, and prebiotics
Sugar replacement, heat resistance and fiber
Sugar replacement and prebiotics
Sugar replacement and prebiotics
Texture, body and mouthfeel, fiber, stability and prebiotics
23
1.1.2.6 Synbiotic concept
Due the potential synergy between probiotics and prebiotics to work together,
synbiotic foods are recently emerged as new segment of functional foods to enhance
the gastrointestinal health (Siro et al., 2008; Ziemer & Gibson, 1998). Synbiotic
concept is defined as “a mixture of probiotics and prebiotics that beneficially affects
the host by improving the survival and implantation of live microbial dietary
supplements in the gastrointestinal tract, by selectively stimulating the growth and/or
by activating the metabolism of one or a limited number of health-promoting bacteria,
and thus improving host welfare” (Gibson & Roberfroid, 1995). The symbiotic effect
between probiotics and prebiotics is achieved as a complementary mixture of both
benefits entities or as a synergistic approach to insure a layer protection for probiotics
face a diverse stresses that can encountered (Bengmark, 2001; Iyer & Kailasapathy,
2005). In optimized synbiotic foods, prebiotics should serve as a substrate for
fermentation and stimulate the growth of probiotic microorganisms, without
compromising the technological proprieties or the consumer acceptability of the
product (Watson & Preedy, 2015). In addition, they also act to improve the viability
and the metabolic activity of probiotics during their transit through the gastrointestinal
tract and implantation in the colon, while also stimulating indigenous beneficial
bacteria (Collins & Gibson, 1999; Nazzaro et al., 2012). The success creation of
synbiotic is not a just mixture, its development depends on the compatibility between
the chosen probiotic and prebiotic compounds and requires a both in vitro and in vivo
screening processes for most efficacy and possibly to reduce the dose (Nazzaro et al.,
2012; Pandey et al., 2015). Thus, the synbiotic concept found applications in various
functional foods, particularly in dairy products, which enhance their therapeutic values.
It appears that in some cases, synbiotic products offer higher benefit effects than those
of products containing only probiotics or prebiotics (Bengmark, 2003; Kolida &
Gibson, 2011).
24
1.1.2.7 Synbiotic in dairy products
One desirable attribute of a synbiotic is to improve survival of the probiotic
bacteria under manufacturing and storage conditions as well as during the transit
through the gastrointestinal (Mohammadi & Mortazavian, 2011). The incorporation of
probiotics into foods is often limited by the non-optimal environments in certain food
matrices such as pH, H2O2, organic acids, oxygen, which claimed to affect their
survival, especially in dairy products as described below. A wide range of prebiotics
are supplemented into milk based foods as probiotic protectants (Romano et al., 2015).
Oliveira et al. (2009a) evaluated the prebiotic potential of inulin at 4% in skim milk
fermented by co-cultures and pure cultures of L. acidophilus, L. rhamnosus, L.
bulgaricus, and B. lactis with S. thermophilus. The addition of inulin to milk is highly
benefit when exerted a bifidogenic effect and preserved almost viable counts during 7
days storage at 4°C. Furthermore, addition of inulin to the fermented milk enhanced
the acidification kinetics, favored post-acidification and contributed to an increase the
apparent viscosity when compared to fermented milk without inulin (Oliveira et al.,
2009a). Similarly, the addition of lactulose improved the quality of fermented skim and
milk showed a bifidogenic effect, when B. lactis was exhibited the highest cell counts
than those of the other selected probiotics (De Souza Oliveira et al., 2011). The same
authors formulated synbiotic fermented milk combinations with different prebiotics
such as maltodextrin, oligofructose and polydextrose. These authors concluded that the
combination of suited prebiotics and probiotics can be resulted in increasing conjugated
linoleic acids levels during processing of fermented milk (Oliveira et al., 2009b).
Prebiotics and probiotics are increasingly been used to produce potentially synbiotic
yogurts. Donkor et al. (2007b) investigated the prebiotic effects of adding inulin and
Hi-maize on the growth of two probiotics, L. acidophilus and L. casei in set-type
yogurt. Inulin was found to be more effective on the viability retention of both stains
during storage and to increasing the apparent viscosity of the yogurt products than Hi-
maize. Similarly, yogurt supplemented with oat β-glucans at levels of 0.25-0.5% (w/v)
enhanced the viability of three probiotic L. plantarum strains during storage period of
21 days at 4°C (Kılıç & Akpınar, 2013). Özer et al. (2005) investigated the effect of
supplementation of lactulose and inulin on the growth of L. acidophilus and B. bifidum
25
in yogurt. The results showed that lactulose was found to be more effective on the
growth of both probiotic stains than inulin (Özer et al., 2005). On the contrary, the
addition of prebiotics soluble corn fiber, polydextrose, and inulin in fermented yogurt
drinks did not improve the viability of B. lactis and L. acidophilus during the storage
period and showed to alter the sensory properties of the yogurt drink (Allgeyer et al.,
2010). The efficiency of prebiotics on the retention of probiotics viability would
depend on other nutritional compounds and/or intrinsic factors related the fermented
products. It is recommended that additional ingredients or methods as micro-
encapsulation of probiotics be used for ensuring higher survival rate (Chen et al., 2005).
In other study, Cardarelli et al. (2008) reported that the addition of oligofructose and/or
inulin yielded in increasing the probiotic viable count and improving the sensory
quality of petit-suisse cheese containing B. lactis and L. acidophilus. Di Criscio et al.
(2010) produced a synbiotic ice cream by adding L. casei and L. rhamnosus and inulin
as prebiotic substrate. The best synbiotic ice cream formulation was obtained with L.
casei and 2.5 % inulin and showed good nutritional and sensory properties. Angiolillo
et al. (2014) developed a synbiotic Fiordilatte cheese with an edible sodium alginate
coating as carrier of L. rhamnosus and FOS prebiotics. The viability of L. rhamnosus
was maintained for the entire storage period. In addition, the coating has a slight
antimicrobial effect against Pseudomonas spp. and Enterobacteriaceae contributing to
improve the sensory properties and prolonging the shelf life of the product. Considering
the different studies, addition of suitable prebiotics to synbiotic dairy products can
significantly improve the viability of probiotic bacteria in different formulations and
storage conditions.
26
1.2 Whey
1.2.1 Whey in environmental consideration
Whey is the major by-product of the dairy industry removed from cheese and
casein manufacture after that milk has been coagulated. In this context, from 10 L of
milk utilised during the production of 1-2 Kg of cheese, approximately 8-9 L of whey
is produced. The current total worldwide production of whey is over 160 million tons
per year and showing around 2% annual growth rate (Smithers, 2008). Hence, whey
poses challenge to the dairy industry and can cause significant environmental problem
without an adequate treatments. In addition to the huge volume, whey exhibits a high
biological oxygen demand (BOD) which estimated to be >35,000 ppm and a chemical
oxygen demand (COD) >60,000 ppm (Prazeres et al., 2012). Lactose, the main
component of whey contributes highly to its organic load matter effect (Carvalho et al.,
2013). Therefore, different approaches were addressed for the valorization of whey. A
portion of whey is used for animal feed and in food application, which is only half of
the global whey production (Spălățelu, 2012). So, whey must be further processed to
maximise benefits and to limit its environmental pollution impact. Recently, the
perception of whey and its utilization has changed from being a by-product to co-
product resource of various value-added products. Lactose and proteins as principal
compounds of whey are recognized to exhibit a number of nutritional, functional and
physiological features that make them potentially useful for versatile applications.
Thus, one attractive approach for whey valorization is the conversion of its lactose
content to bioactive compounds such as GOS, lactosucrose and lactulose prebiotics
(Gänzle et al., 2008; Nath et al., 2016). On the other hand, whey proteins have been
attracted a great interest for valorization as a potential source of bioactive peptides with
a wide range health benefits (Brandelli et al., 2015; Madureira et al., 2007; Patel, 2015).
27
1.2.2 Whey composition
Whey is obtained after removing casein from milk and containing about 93%
of the water in milk and 50% of the total solids of milk. Two types of whey are
produced depending on the manufacturing process involved; sweet and acid whey.
Sweet whey is obtained from rennet coagulation of milk during the production of hard
and soft cheese like cheddar or Swiss cheese. Acid whey is the co-product of fresh
acid-coagulated milk involved in the production of some chesses such as cottage cheese
and Greek style-yogurt (Bansal & Bhandari, 2016). The most widely produced type of
whey is the sweet whey (Tunick, 2008). The composition and physicochemical
characteristics of the two types of whey are quite similar as shown in Table 1.3. In
general, on dry basis, whey is composed about 70% of lactose, whey proteins (~14%),
minerals and some fat. The major differences being in the acidity and the mineral
contents as well as some whey protein fractions. Acid whey contains higher
concentration of minerals than sweet whey due the dissolution of the colloidal calcium
phosphate component of casein micelles during acidification process (Tsakali et al.,
2010). Sweet whey contains the glycomacropeptide fraction produced by the enzymatic
hydrolysis of κ-casein. Moreover, the amount of amino acids can vary on a degree of
casein hydrolysis. Thus, free amino acids content in sweet whey is about 4 times higher,
and in acid whey even 10 times higher than in milk.
Table 1.3: Proximate composition and pH of sweet and acid whey (Yadav et al., 2015).
Constituents Sweet whey (g/L) Acid whey (g/L)
Total solids Lactose Protein Fat Lactate Ash Calcium Phosphate Chloride pH
63-70 46-52 6-10 0.5 2 5 0.4-0.6 1-3 1.1 5.9-6.4
63.70 44-46 6-8 0.4 6.4 8 1.2-1.6 2-4.5 1.1 4.6-4.7
28
1.2.2.1 Lactose whey
Lactose is the main product of whey processing industry. The approximate
lactose content in cheese whey is 4.8 g/100 mL. Lactose has its own physicochemical,
nutritional, and functional values. Lactose (4-O-β-galactopyranosyl-D-glucopyranose,
C12H22O11) is a disaccharide comprising one glucose molecule linked to a galactose
molecule. The solubility of lactose is low compared with other disaccharides and is
only about 10% of that of sucrose at ambient temperature (Gänzle et al., 2008). The
sweetness of lactose solutions is about 20-30% that of sucrose and for this reason that
lactose is a suitable carbohydrate in infant formulas. Lactose promotes the health gut
and enhances the absorption of calcium and magnesium (Schaafsma, 2008).
Application of lactose in food industry include baby foods, reconstituted dairy products
as well as confectionary and bakery products. Refined lactose is commonly used in the
pharmaceutical industry as an excipient of tablets and as a carrier agent for medicines
(Božanić et al., 2014). However, a significant number of the world’s population suffers
from partial or complete lactose intolerance due to a metabolic inefficiency to
hydrolyze lactose to glucose and galactose. This limits the use of lactose in a number
of nutritional supplements (Schaafsma, 2008). Although, the demand of the food and
pharmaceutical industries for lactose still insufficient to absorb all of the actual whey
lactose available. Currently, conversion of lactose into value-added products is
considered a commendable challenge. A large number of lactose-derived prebiotics
such as GOS, lactulose, lactitol, lactosucrose, lactobionic acid and tagatose, have been
synthesized by different operating technologies considering raw whey as feedstock
(Gänzle et al., 2008; Nath et al., 2016; Playne & Crittenden, 2009). Currently,
considerable attention has been given to the synthesis of lactulose due to its excellent
therapeutic and functional properties, being a highly valuable lactose whey derivative
(Olano & Corzo, 2009; Panesar & Kumari, 2011).
1.2.2.2 Lactose-derived lactulose
1.2.2.2.1 General properties of lactulose
Historically, lactulose was first synthesized in 1930 from lactose following an
alkaline isomerization reaction (Montgomery & Hudson, 1930). In the end of the 50th
29
of the 20th century, the bifidogenic effect of lactulose was reported for the first time
(Petuely, 1957). Lactulose (4-O-β-D-galactopyranosyl-D-fructose) is a synthetic
disaccharide composed from a galactose moiety linked to a fructose moiety by a β (1-
4) glyosidic linkage, which is not hydrolyzed by mammalian digestive enzymes. Thus,
the ingested lactulose passes through the stomach and small intestine without
degradation. In the large intestine, lactulose contributes to restore or maintain good gut
health through the promotion of growth of beneficial bacterial flora over pathogenic
bacteria. Lactulose has around 0.48 to 0.62 times the sweetness of sucrose and 1.5 times
than lactose (Playne and Crittenden 2009). Lactulose became known first as a medical
laxative for the treatment of patients with hepatic encephalopathy and in patients with
chronic constipation and complication of liver disease (Panesar & Kumari, 2011). It is
also used as sweetener substituent in pediatric and geriatric diets and as prebiotic
functional ingredient in dairy foods (Seki & Saito, 2012). Because of its many
applications, demand lactulose production is increasing. The annual production was
estimated at 45.000 to 50.0000 tons in 2009 (Panesar & Kumari, 2011). Solvay
Pharmaceuticals (Netherlands) and Morinaga Milk Industry (Japan) are the leading
producers of lactulose. Currently, the commercially available lactulose is made by
chemical synthesis. Alternatively, lactulose can be produced by enzymatic routes or by
new emerging processes like electro-activation technology (Aider & Gimenez-Vidal,
2012; Silvério et al., 2016).
1.2.2.2.2 Chemistry of lactulose synthesis
Lactulose is rarely present in nature but is found in small amounts in high heat-
treated milk (Adachi, 1958). Lactulose is mainly based on the molecular rearrangement
of lactose under alkaline conditions via a Lobry de Bruyn-Alberda van Ekenstein (LA)
transformation in which the glucose moiety is converted into fructose (Aider & de
Halleux, 2007). The LA transformation proceeds through an intermediate enolization
of an aldose sugar to the corresponding ketose sugar. The α-carbon in the glucose
moiety of lactose can be seen in Figure 1.5a. The C-H bond on the α-carbon breaks
easily in alkaline media leading to its isomerization to fructose. The enediol
intermediate occurs when a double bond forms between carbons 1 and 2 in the glucose
30
moiety as shown in Figure 1.5b. The enediol intermediate (Figure 1.5c), then
undergoes rearrangement (Figure 1.5d) to form lactulose (Figure 1.5e, f). The reaction
requires catalysis as proton acceptors during lactose isomerization and this is achieved
by having an alkaline medium. The cleavage of the α-carbon and hydrogen bond are
favored by heat and high pH values leading to high reaction yield. The synthesis of
lactulose can be also achieved by the reaction of lactose with free amino groups. In this
case, the formed lactosylamine undergoes an Amadori rearrangement to form
lactulosylamine and after hydrolysis, lactulose could be obtained (Hodge, 1955).
Besides the formation of lactulose, the reaction routes comprise a subsequent
degradation of lactulose to galactose and isosaccharid acids, as well as an isomerization
of lactose into small amount of epilactose (Olano & Calvo, 1989).
Figure 1.5: Schematic representation of the chemistry mechanism of lactulose isomerization trough LA transformation, adapted from Aissa & Aïder (2013a).
31
1.2.3 Methods of lactulose production
1.2.3.1 Chemical synthesis
The chemical synthesis of lactulose is carried out via LA rearrangement in basic
media and an important number of catalysts were used. Calcium hydroxide, sodium
hydroxide, potassium hydroxide and carbonate, magnesium oxide, tertiary amines and
sodium aluminate have been employed as homogenous catalyst (Aider & de Halleux,
2007; Panesar & Kumari, 2011; Zokaee et al., 2002). However, most of these processes
provided low yield of lactulose and accomplished with the subsequent formation of by-
products and a brownish color. Moreover, homogeneous catalysis is generally
characterized by a huge challenge for catalyst removal at the end of the reaction. The
presence of side products is especially undesirable for pharmaceutical and medical
applications. Moreover, to achieve high yields, a considerable amount of catalysts is
required leading to an extensive separation and purification steps which increase the
production cost (Zokaee et al., 2002). Other compounds named heterogeneous catalysts
such as zeolites, sepiolites, egg and oyster shell have been used to obtain lactulose
(Boro et al., 2012; Montilla et al., 2005; Shukla et al., 1985; Villamiel et al., 2002). The
main advantage associated with these catalysis’s is their easy removal by
centrifugation. However, they are also characterized by very low reaction yields.
1.2.3.2 Enzymatic synthesis
The enzymatic synthesis of lactulose emerges as a suitable alternative to
overcome the drawbacks associated with chemical synthesis. Contrary to chemical
processes, enzymatic synthesis of lactulose is usually carried out under mild conditions,
which greatly limited the formation of the side products. Consequently, it provide high-
purity final product and simply the purification step (Kim & Oh, 2012). Lactulose is
synthesized through transgalactosylation of lactose, using fructose as galactosyl
acceptor and β-galactosidase or β-glycosidase as a biocatalysts (Guerrero et al., 2011;
Mayer et al., 2010). One of the great advantage of these process is the possibility to use
of whey and whey permeate as a raw material, while chemical synthesis requires high-
purity lactose to reduce secondary reactions (Padilla et al., 2015; Song et al., 2013).
However, depending on the microbial source of enzyme catalyst, the key problems are
the low yield and the lack of reaction selectivity. More recently, lactulose synthesis by
32
direct epimerization of lactose with cellobiose 2-epimerase was described (Kim & Oh,
2012). The formation of epilactose is also reported as a by-product of this enzymatic
process. A new strategies are proposed to increase the yield, productivity and the
specificity of lactulose synthesis, such as molecular modification of enzymes, use of
immobilized enzyme and continuous mode of reaction (Silvério et al., 2016; Wang et
al., 2013a).
1.2.3.3 Electro-activation synthesis
Recently, lactulose was successfully synthesized by a reagentless process which
is the EA technology (Aider & Gimenez-Vidal, 2012). EA is an emerging technology
based on the electrolysis of aqueous solutions, which makes it highly reactive and
useful in physico-chemical and biological reactions (Aider et al., 2012). The synthesis
of lactulose under the EA reactor follows the LA transformation. The isomerization
reaction requires proton acceptors, and this can be achieved by having a high alkaline
medium, which generated following water electrolysis at the cathode interface under
the influence of applied external electric field. The synthesis of lactulose under the EA
reactor is controlled by using an appropriate cell configuration of the reactor and ion
exchange membranes (Aider & Gimenez-Vidal, 2012; Aissa & Aïder, 2013b). The
electro-isomerization of lactulose was operated under low temperatures (0 to 30°C) and
approximately 25% lactulose yield with a purity of 95% were achieved in shortness
reaction time (Aissa & Aïder, 2013a). ). This yield is quite higher than that obtained by
the chemical synthesis which is operated at higher temperatures and in the presence of
strong bases (Zokaee et al., 2002). These results confirmed that by using EA; the
activation energy required for the isomerization of lactulose is markedly lower than for
the chemical methods. The optimization of some processing parameters such as an
electric current intensity, reaction time, reactor configuration and electrolyte
concentration resulted in increased lactulose yield to 30% (Aissa & Aïder, 2014a). In
contrast to the production of lactulose by both chemical methods, which present many
problems of purification steps as well as increasing the cost and energy consumption,
and enzymatic method which is very expensive, EA has advantages as is considered to
be a safe, clean, ecofriendly and energy-saving approach (Aissa & Aïder, 2013a).
33
Unfortunately, the lactulose yield is not improved and remaining lower compared to
that of chemical isomerization with a complexing agent (Sitanggang et al., 2016).
1.2.4 Lactulose applications
1.2.4.1 Pharmaceutical applications
As a drug, lactulose is mainly used for the treatment of constipation, hepatic
encephalopathy, Salmonella carrier, complication of liver disease, inflammatory bowel
disease, cancer prevention and immunology, anti-endotoxin effects, maintain blood
glucose and insulin level (Aissa & Aïder, 2014b; Panesar & Kumari, 2011; Schuster-
Wolff-Bühring et al., 2010). Lactulose is conveyed into the digestive tract and arriving
intact in the colon where it is fermented selectively (Gibson et al., 2004). The main
mechanisms by which lactulose exhibited their therapeutic effects are described in
Figure 1.6. It was recognized that lactulose exerts bifidogenic effect which contributes
to a good gut health by increasing numbers of Bifidobacteria and decreasing those of
harmful bacteria (Kim et al., 2015). The physiological effects of lactulose are mainly
the result of its fermentative metabolism which leads to the production of SCFAs,
hydrogen (H2) and carbon dioxide (CO2) lowering the faecal pH, thereby converting
free ammonia to non-absorbable ammonium ion (Schumann, 2002). The colonic
acidification is behind its systemic effects in the treatment of chronic constipation,
hepatic encephalopathy and improving the absorption of calcium and magnesium.
Lactulose has also been used to reduce the rate of Salmonella carriage in chronic
carriers and the carrier rate of Shigella was reported to be reduced (Szilagyi, 2010).
Other physiological effects attributed to lactulose have been described in the treatment
and the prevention of intestinal disorders, the modulation of lipid metabolism and
reducing the risk of colorectal cancer (Panesar & Kumari, 2011). However,
administration of lactulose at high dosages increases the osmotic pressure and causes
diarrhea (Hammer et al., 1989).
34
Figure 1.6: Mechanism of action of lactulose and significance of the bacterial metabolism of lactulose (Panesar & Kumari, 2011).
1.2.4.2 Food applications
Petuely has introduced the use of lactulose as a feed additive in 1957. Today,
lactulose is increasingly used as prebiotic in functional foods as well as a sugar
substitute in a variety of dietary formulations (Panesar & Kumari, 2011). It is added in
confectionary products, beverages, infant milk powders, bakery products and dairy
products, especially in yogurts (Seki & Saito, 2012). Lactulose also has some desirable
properties such as an excellent solubility in water, stable to acid and heat, and
contributes to improving the taste of food products (Olano & Corzo, 2009). For
example, several functional properties have been improved with the use of lactulose in
the manufacture of various dairy products as it can improve their sensory qualities,
physico-chemical and microbiological properties. Özer et al. (2005) studied the effect
of lactulose and inulin as a prebiotics on the growth of L. acidophilus LA-5 and B.
bifidum BB-02 in yogurt. The growth of the both probiotic strains was found to be more
significant with lactulose than inulin. Tabatabaie & Mortazavi (2008) also studied the
effect of lactulose on improving the survival of L. rhamnosus and B. bifidum in yoghurt
35
stored at 4ºC for five weeks. Both strains were stable and their survival was slightly
improved. Shaghaghi et al. (2013) studied the physicochemical, sensory and
microbiological properties of synbiotic yogurts containing both L. rhamnosus and L.
reuteri in the presence of lactulose, oligofructose and inulin prebiotics. The highest
acidifying activity and the best smell have been attributed to lactulose-supplemented
yogurt (Shaghaghi et al., 2013). The addition of lactulose improved the proprieties of
skim milk fermented by L. acidophilus, L. rhamnosus, B. lactis and L. bulgaricus in
co-culture with S. thermophiles (De Souza Oliveira et al., 2011). The growth of the
available probiotic strains was improved with particular concern to B. lactis. Moreover,
the addition of lactulose has reduced the fermentation time and the post-acidification
pH during the five weeks of storage at 4°C. Thus, supplementation of the dairy products
with lactulose, as a prebiotic, enhances the already healthful attributes of yogurt and
can provide more benefits to the consumer.
1.2.5 Whey proteins
Whey proteins represent 15-20% of total milk proteins (Sindayikengera & Xia,
2006). The major components of whey proteins are β-lactoglobulin (β-LG), α-
lactalbumin (α-LA), bovine serum albumin (BSA) and immunoglobulins (IGs). Beside
these compounds, whey contains also a minor proteins including lactoferrin (LF),
lactoperoxidase (LP) and proteose-peptone (PP) (Mollea et al., 2013).
Glycomacropeptide is realised from enzymatic hydrolysis of ҡ-casein during cheese
making (Li & Mine, 2004). Whey proteins have a globular structure with high
solubility, denatured by heat, insensitive to Ca2+, and can form intramolecular bonds
via disulfide bridges between cystein-sulfhydryl groups (Tavares & Malcata, 2013).
Some characteristics of each individual protein are presented in Table 1.4. Whey
represents a rich mixture of proteins with an inherent excellent nutritional, functional
and biological properties enabling them to be used in various food applications (De
Wit, 1998; Madureira et al., 2007).
36
Table 1.4: Protein composition and basic characteristics of the whey proteins (Yadav
et al., 2015).
Whey protein Content
(% w/w)
Molecular mass
(kDa)
Isoelectric point
β-LG 40-50 18.3 5.35-5.49
α-LA 12-15 14.0 4.2-4.5
Immunoglobulins 8.0 150-1000 5.5-8.3
BSA 5.0 66.0 5.13
LF 1.0 76.5 9.5-10.0
LP 0.5 78.0 9.5
Glycomacropeptide 12.0 6.8 4.3-4.6
PP 0.19 4-22 -
1.2.5.1 Nutritional value of whey proteins
The nutritional value of whey proteins is much higher than egg proteins and
other commonly consumed proteins (Smithers, 2008). Whey proteins comprise a high
content on essential and branched amino acids, as well as a balanced source of the
sulfur-containing amino acids (De Wit, 1998). Nutritional researches have shown that
whey proteins have stronger satiating effect and are considered as a potential candidate
for body-weight control and obesity treatment (Luhovyy et al., 2007). The branched
amino acids including isoleucine, leucine and valine play important roles as a regulator
of different metabolic functions and blood glucose homeostasis (Joy et al., 2013). The
abundance of leucine in whey is of particular interest because it plays a distinct role in
protein metabolism and muscle protein synthesis (Ha & Zemel, 2003). Whey proteins
provide a source of sulphur amino acids which are thought to play a role as
antioxidants, as a precursors to the potent intracellular antioxidant glutathione (Micke
37
et al., 2002). Thus, they supply important nutritional benefits when used as a food
ingredient.
1.2.5.2 Whey proteins in food as a functional ingredient
Apart from nutritional aspect, whey proteins possess excellent functional
properties and desirable sensory characteristics. Functional properties of whey proteins
are governed by the composition and structure of the proteins and can also influenced
by external environmental factors related to the food systems and processing conditions
(De Wit, 1998; Kinsella & Whitehead, 1989). The most functional applications of whey
proteins include emulsification, gelation, foaming stability, viscosity and sensory
properties. For example, β-LG has excellent gelling, foaming and emulsifying
properties while α-La exhibits good emulsifying (Dissanayake & Vasiljevic, 2009).
Whey proteins are used in numerous food applications including sport beverages,
liquid meat replacements, baked products and processed meats, pasta, salad dressings,
spreads and dips, artificial coffee creams, soups, ice cream, confectionary, infant foods
and various other dairy products (Bansal & Bhandari, 2016; Mulvihill & Ennis, 2003).
Functional properties of various whey-based ingredient foods are shown in Table 1.5.
Numerous novel processing technologies are now developed for increasing the
functional properties of whey proteins (Abd El-Salam et al., 2009).
38
Table 1.5: Examples of application of whey protein ingredients in certain foods and
their functional properties adapted from (Bansal & Bhandari, 2016).
Food category
Uses
Functionality
Dairy products Yogurt, ricotta cheese, quarg
Nutritional, yield, consistency, curd cohesiveness
Cream cheeses, cream cheese spreads, sliceable/squeezable cheeses, cheese fillings and dips
Gelling, emulsifier, sensory properties
Bakery products Cakes, bread, muffins, croissants
Nutritional, emulsifier and egg replacer
Beverages Soft drinks, fruit juices, powdered or frozen beverages
Nutritional
Milk-based flavored beverages
Colloidal stability, viscosity
Desserts products Ice-cream, frozen juice bars, frozen dessert coatings
Whipping properties, skim milk solids replacement, emulsifier, body/texture
Confectionary Aerated candy mixes, sponge cakes, meringues
Emulsifier, whipping properties, egg white replacement, fat binding, foam stability
Meat products Luncheon meats, frankfurters
Gelation, pre-emulsion, water holding capacity, fat binding
Injection brine for fortification of whole meat products
Yield, gelation
Infant formula Pre-term formula, Term formula, follow-on formula
Nutritional
39
1.2.5.3 Physiological importance of whey proteins
Consumption of whey proteins brings a number of biological benefits including
stimulation of satiation control and weight management, prevention of muscular
atrophy, exercise performance, cardiovascular health, anti-cancer effects, and
management of infections as well as enhancement of the immune functions (Keri
Marshall, 2004). Each protein has known bioactivities and used in several commercial
products (Korhonen & Pihlanto, 2003). The β-LG is thought to exert its benefit effects
as a source of cystein, which is important for the synthesis of GSH, a potent
intracellular antioxidant. GSH is important in immune regulation and cancer
prevention, in the improvement of liver functions, and in helping overcome GSH-
deficiency in seropositive and Alzheimer’s disease patients (Madureira et al., 2007).
The β-LG structure comprises a ligand binding site tending to bind to hydrophobic
molecules such as fat-soluble vitamins as well as lipids, consequently it may play a role
in the metabolism of fatty acids (Chatterton et al., 2006). The α-LA has mostly used in
infant formula as it has the most structurally similar protein profile compared to the
breast milk (Heine et al., 1991). As a regard of it high content in tryptophane, α-LA
may help to improve mood, sleep and congestive performance. This protein also
demonstrated to possess anti-tumor activity as it can selectively induce apoptosis in
tumor cells (Svensson et al., 2003). The lactoferrin (LF) possesses a wide range of
biological functions, it can protect the mammary gland and gastro-intestinal tract
against infections, provides superior nutrition through increased bioavailability of iron
and rich source of amino acids and promotes division, differentiation and growth of
immune system and mucosa intestinal cells (Pihlanto & Korhonen, 2003;
Wakabayashi et al., 2006). Thanks to its iron-chelating ability, LF also appears to have
antiviral, antifungal and antibacterial properties (Farnaud & Evans, 2003; Pan et al.,
2006). Lactoperoxidase is pertaining due it antimicrobial action and being explored in
the inhibition of dental caries (Tenovuo, 2002). Immunoglobulins exert an important
immunological protection to the neonates, combat infections and enhance gut health
(Mehra et al., 2006). The beneficial effects of glycomacropeptide on satiety and
phenylketonuria management have been validated (Sharma et al., 2013). In addition,
these proteins can extend their functions through the release of bioactive peptides
40
during intestinal digestion displaying a wide range of biological activities and this has
become an interesting approach to add value to whey. Moreover, it is interesting to
note that whey protein hydrolysates have improved absorption in the gut and lower
allergenicity compared to intact proteins (Foegeding & Davis, 2011).
1.2.5.4 Bioactive peptides generated from whey
According to the current knowledge, whey proteins seem to be by far a rich
sources of bioactive peptides (Madureira et al., 2010; McCarthy et al., 2014). Bioactive
peptides are defined as “specific protein fragments that have a positive impact on body
functions or conditions and which ultimately may influence health” (Kitts & Weiler,
2003). They are often latent or encrypted within their parent proteins to be liberated e.g
during gastrointestinal digestion or during food processing trough the action of
proteolytic enzymes (Meisel & Bockelmann, 1999). The size of bioactive peptides is
usually 2-20 amino acids in length and their primary sequence defines their function
(Korhonen & Pihlanto-Leppälä, 2001). As name implies, bioactive peptides shown to
possess numerous biological activities depending on their amino acid composition and
sequence. The most common are antihypertensive, antioxidative, immunomodulatory,
opioid, antimicrobial, and mineral-carrying activities (Hartmann & Meisel, 2007;
Korhonen, 2009; Pihlanto-Leppälä, 2000). Beside the common ones, other novel
functionalities of these bioactive peptides are also inscribed such as anticancer, anti-
obesity and antidiabetic bioactivities (Gaudel et al., 2013; Jakubowicz & Froy, 2013;
Shahidi & Zhong, 2008). Moreover, some bioactive peptides may multifunctional
activities, where specific peptide sequences may possess more than one biological
activity (Meisel, 2004). Bioactive peptides can modulate physiological functionalities
through binding to specific receptors or action on specific sites on target cells in various
systems of the body such as the immune system, the nervous system, the cardiovascular
system and the gastrointestinal system (Madureira et al., 2010; Möller et al., 2008).
Thus, today bioactive peptides have gained great interest as components in functional
foods and nutraceuticals (Li-Chan, 2015).
41
1.2.5.4.1 Processing of bioactive peptides in whey
There are various processes available to produce peptides with different
biological activities as described in Figure 1.7 (Madureira et al., 2010). Enzymatic
hydrolysis is the most common route used to produce bioactive peptides from whey
proteins (Gauthier & Pouliot, 2003; Korhonen & Pihlanto, 2006). Many of the derived
bioactive peptides have been produced using gastrointestinal enzymes, usually pepsin
and trypsin. For example, digestion of α-LA with pepsin; whilst β-LG with combined
pepsin and then with trypsin yielded opioid peptides, α- and β-lactorphins (Pihlanto-
Leppälä et al., 1996). Furthermore, it was shown that digestion of β-LG with
chymotrypsin produced β-lactotensin (Pihlanto-Leppälä et al., 1997).
Figure 1.7: Alternative modes of bioactive peptide generation (Madureira et al., 2010).
42
Besides enzymatic hydrolysis, fermentation of whey has been also identified as
one the routes for the production of bioactive peptides from milk proteins (Korhonen
& Pihlanto, 2006). However, the production of bioactive peptides from whey by
fermentation has been in the doubt which can explained for part due the limitations of
substrate cannot assure the microbial growth to release peptides (Madureira et al.,
2010). Pihlanto-Leppälä et al. (1998) shown that the fermentation of whey proteins
with various commercial dairy starters did exhibit antihypertensive activity only after
their digestion with pepsin and trypsin. Thus, additional enzymes have been used as a
strategy to maximize the release of peptides (Ortiz-Chao et al., 2009; Sinha et al., 2007;
Tsai et al., 2008). Proteins can be produced trough acid and alkali hydrolysis; however,
these processes still difficult to control and tend to yield final products with reduced
nutritional values. Alternatively, strategies have been further including the synthesis of
peptides based on sequence similarities of peptides having known biological activity.
Recombinant DNA techniques have been successfully used for the synthesis of large
bioactive peptides (Gill et al., 1996). One of the challenge faced in bioactive peptide
production is to obtain high yield amount with potent activity. Thus, there is a need for
further applicable food-grade processing that can be conduced under moderate
controlled conditions are suitable. Application of pre-treatments such as thermal,
sonication and hydrostatic pressure treatments can enhance enzymatic hydrolysis,
possibly by changing the conformation of native proteins and as a results increasing
the bioavailability of hydrolysis sites (Nongonierma & FitzGerald, 2015).
1.2.5.4.2 Functionalities of bioactive peptides in whey
Bioactive peptides derived from whey proteins have open up a wide range of
possibilities within the market for functional foods; of special interest are those
exhibited antihypertensive, antioxidant, immunomodulatory and antimicrobial
peptides (Korhonen & Pihlanto, 2006; McCarthy et al., 2014). Moreover, many of the
bioactive peptides are multifunctional and can possess more than one activity.
43
1.2.5.4.2.1 Antihypertensive peptides
Hypertension or high blood pressure is a major health problem in many
industrialized countries, and is a major risk factor for coronary heart disease, congestive
heart failure, stroke and renal disease (Haque & Chand, 2008; Tavares & Malcata,
2013). Whey derived-proteins with antihypertensive activity are the most relevant and
studied bioactivity due to their significant role in blood pressure reduction (Brandelli
et al., 2015). Many of these peptides were done as angiotensin converting enzyme
(ACE) inhibitory peptides and block the conversion of angiotensin I to angiotensin II.
The ACE is responsible for the elevation of blood pressure by converting angiotensin-
I to the potent vasoconstrictor, angiotensin-II, and by degrading bradykinin, a
vasodilatory peptide, and enkephalins (Coates, 2003). Therefore, inhibition of ACE
results in an overall antihypertensive effect. The ACE inhibitory peptides usually
contain 2-12 amino acids and Trp, Tyr, Phe, and Pro are the most potent C-terminal
amino acids of these peptides that bind at the ACE active site and thereby inhibit their
activity (López-Fandiño et al., 2006). The ACE-inhibitors derived from milk proteins
are attributed to different fragments of casein named casokinins or whey proteins
named lactokinins (Park & Nam, 2015). The most potent inhibitors of ACE were
derived from αs1-casein, αs2-casein and β-casein, after trypsin hydrolysis or an
extracellular proteinase from Lb. helveticus (Pihlanto-Leppälä et al., 1998; Yamamoto
et al., 1994). The hydrolysis of whey proteins also resulted in peptides with high ACE-
inhibitory. Concerning the whey proteins, which are more compact than the caseins, it
has been shown that native β-LG, α-La and whey protein concentrate have very low
ACE-inhibitory bioactivity, but it enzymatic hydrolysis resulted in high levels of ACE
inhibition (73-90%) (Mullally et al., 1997). Moreover, Hernández-Ledesma et al.
(2006) demonstrated that hydrolysis of β-LG with thermolysin under heating
conditions results in potent ACE inhibitory peptides. So far, the most potent β-LG
derived ACE inhibitory peptide reported is β-LG f(32-46) which had an IC50 value 8
μm and produced using Protease N Amano, a food-grade commercial proteolytic
preparation (Ortiz-Chao et al., 2009). The primary sequences of some ACE-inhibitory
peptides derived from α-LA and β-LG are described, for a review see Nongonierma et
al. (2016).
44
1.2.5.4.2.2 Antioxidative peptides
Oxidative processes are normal cellular events, but uncontrolled oxidation
involving a high level of reactive oxygen species (ROS) can damage biological
molecules leading to the development of chronic diseases, such as cancer,
cardiovascular diseases, neurological disorders and (Ames et al., 1993). Furthermore,
it is well known that ROS can mediate lipid oxidation in foods and reduce the
nutritional value and safety of foods by producing undesirable flavors and toxic
substances (Johnson & Decker, 2015). Therefore, it is important to prevent and to retard
the formation of free radicals in foods containing lipids and/or fatty acids. Synthetic
antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene
(BHT) are commonly used to inhibit lipid oxidation in food and biological systems.
However, some adverse effects and dose limitations were stimulated their replacement
by natural antioxidant compounds, especially by bioactive peptides derived milk
caseins and whey proteins (Mohanty et al., 2016). Therefore, in addition to their
biological mechanisms (e.g. chelation of transition metals by lactoferrin and increase
the body’s levels of glutathione), whey proteins can also used as source of antioxidant
peptides (Hernández-Ledesma et al., 2008; Pihlanto, 2006). Antioxidant peptides
derived from milk proteins are usually consist of 5-11 amino acids and contain
hydrophobic residues (Pro, His, Try and Trp) (Brandelli et al., 2015). Different
mechanisms have been attributed to antioxidant peptides including their ability to
scavenge ROS, chelate prooxidative transition metals, inhibition/activation of key
metabolic enzymes involved in oxidative processes or combination of this mechanisms
(Elias et al., 2008). Hernández-Ledesma et al. (2005) investigated the antioxidant
activity of hydrolysates from whey proteins β-LG and α-LA, by commercial proteases
(pepsin, trypsin, chymotrypsin, thermolysin and corolase PP). The results indicated that
corolase PP was the most appropriate enzyme to produce β-LG derived peptide having
high oxygen radical scavenging activity than BHA. Peng et al. (2009) indicated that
antioxidant activity of whey protein hydrolysates depended on peptide molecular
weight, with peptides of 0.1–2.8 kDa possessing the strongest radical scavenging
activity. Although, whey protein hydrolysates have also been found to inhibit lipid
45
oxidation in liposomal system and cooked pork patties (Peña-Ramos & Xiong, 2001;
Peña-Ramos & Xiong, 2003).
1.2.5.4.2.3 Antimicrobial peptides
Among whey proteins, LF and lysozyme in its intact format have well
documented for their antimicrobial properties. Various milk protein-derived peptides
have also been described to display antimicrobial properties against Gram positive and
Gram negative bacteria, yeast and fungi (Madureira et al., 2010). The antimicrobial
peptides are usually of short molecular weights over than 10 kDa with amphipathic,
cationic and hydrophobic properties (Demers-Mathieu et al., 2013; Pellegrini et al.,
2001). Their mechanism of action is thought to bind with the anionic sites on cell
surfaces leading to the distrusting of normal membrane permeability (Epand & Vogel,
1999). Peptides derived from LF, αs1-, αs2- and κ-casein have been the most described.
Other whey-derived peptides have also been described for their antimicrobial
properties (Benkerroum, 2010). Lactoferricin is a potent antimicrobial peptide which
displays a wide range activity against Gram-positive and Gram-negative bacteria
(Tomita et al., 2002). Furthermore, lactoferrampin, also derived from LF, has shown
antibacterial activity against Bacillus subtilis, E. coli and Pseudomonas aeruginosa
(Lopez-Exposito & Recio, 2008). Both α-LA and α-β-LG hydrolysates have yielded
with potent antimicrobial activities (Pellegrini et al., 2001; Pellegrini et al., 1999). For
example, E. coli growth was altered in the presence of α-LA and β-LG hydrolysates,
while the intact proteins failed in the inhibition of E. coli (Pihlanto‐Leppälä et al.,
1999). Therefore, use antimicrobial whey-derived peptides as natural food-grade
preservatives constitute an opportunity of paramount importance for the food industry
(Demers-Mathieu et al., 2013).
1.2.5.4.2.4 Immunomodulatory peptides
Immunomodulatory activity is also a common function of bioactive peptides
associated with milk caseins and whey proteins (McCarthy et al., 2014; Park & Nam,
2015). Immunomodulatory peptides either supress or stimulate the immune system
involving lymphocyte proliferation, antibody synthesis, and cytokine regulation and to
46
increase bacterial resistance (Nagpal et al., 2011). Several peptides generated through
the fermentation with proteolytic bacteria or via the action of gastric enzymes of milk
caseins were reported to interact with the immune functions. Isracidin is the first
antimicrobial released from α-casein trough the action of chymosin. As well as
exhibiting antimicrobial activities, isracidin known to protect mice against lethal
infection by Staphylococcus aureus and stimulate phagocytosis and immune responses
in mice infected with Candida albicans (Lahov & Regelson, 1996). Many other
peptides derived from both β- and κ-caseins were demonstrated to have various
suppressive or stimulatory effects on mononuclear cell and Payer’s patch cells (Baldi
et al., 2005; Tellez et al., 2010). Indeed, immunomodulatory peptides derived from α-
LA and β-LG hydrolysates were reported to impact on many components of the
immune system (Gauthier et al., 2006; Mercier et al., 2004; Saint-Sauveur et al., 2008).
Immunomodulatory peptides released from the hydrolysis of β-LG from fermentation
with L. paracasei repressed the lymphocyte stimulation, up regulated IL-10 and down-
regulated spleenocyte proliferation and down-regulate INF-γ and IL-4 secretion in
murine models (Prioult et al., 2004). Moreover, α-LA hydrolysates were showed to
have immunomodulatory effects trough the modulation of splenocyte proliferation and
cytokine secretion (Jacquot et al., 2010). Lactoferricin, an antimicrobial pepsin derived
peptide of LF has also been shown to significantly modulate cytokine production in
bovine blood leukocytes and monocytes (Prgomet et al., 2006).
1.2.5.4.2.5 Other bioactive peptides
Other types of bioactive peptides have been derived from whey proteins
including, opioid, antithrombotic, antiviral, anticancer and mineral binding peptides
and other biological activities (Brandelli et al., 2015; Nongonierma et al., 2016). Casein
and whey proteins have also proven to be a rich source of opioid peptides, which exhibit
properties similar to morphine-like effects. The major opioid peptides are β-
casomorphins derived from casein α- and β-lactorphins derived from α-LA and β-LG,
respectively (Pihlanto-Leppälä, 2000; Silva & Malcata, 2005). The most potent food-
derived antithrombotic peptides have been released from κ-casein (Chabance et al.,
1995). Lactoferricin and other peptides liberated from LF are also effective against
47
viral infections (Pan et al., 2006). Mineral-binding peptides are mainly released from
hydrolysis of casein (Vegarud et al., 2000). Also, peptides released from heated whey
proteins have been shown to have a great iron-binding abilities and antigenic properties
(Kim et al., 2007b). At last, several milk protein derived peptides reveal multifunctional
bioactivities. For examples, lactoferricin exhibit other activities such as antitumor and
anti-inflammatory activities and β-casomorphin that exhibits antihypertensive,
immunomodulatory, opioid and cytomodulatory activities (Meisel, 2004).
1.2.5.5 Maillard reaction products from whey
In recent years, attempts have been made to increase the functionality of whey
derived-peptides through Maillard reaction (MR), which plays an important role in the
development of new value-added food ingredients (Arihara et al., 2017; Mohan et al.,
2015). Maillard reaction refers to a complex series of sequential and parallel reactions
that occur usually between reducing sugars and available amine groups such as amino
acids, peptides or proteins without requiring to the addition of chemical reagent
(Hodge, 1953). The MR is known to occur in heated, dried or stored foods (Jaeger et
al., 2010). This reaction is generally associated to the formation of color, aroma and
texture, and are the key factors for consumer acceptance in various food products
(Martins et al., 2000; Van Boekel, 2006). The MR proceeds via a complex steps and
can be affected by several factors including the nature and ratio of the reactants, their
molecular weight, pH, temperature, water activity and reaction time (Oliver et al.,
2006). Resulting Maillard reaction products (MRPs) present varying structures with
different physicochemical characteristics, techno-functional properties and
bioactivities (Jiang et al., 2013; Li et al., 2013; Liu et al., 2014). Recent research on
MRPs has focused on their bioactive properties, such as antioxidant capacity as well,
in addition to the antimicrobial, antihypertensive, prebiotic and antigenicity proprieties,
among others (Hauser et al., 2014; Nicoli et al., 1999; Oh et al., 2016; Seiquer et al.,
2014; Wang et al., 2013b). Moreover, MR has been possibility associated with the
formation of antinutritional and carcinogenic compounds, such as the acrylamide
(Mottram et al., 2002). Hence, to use the MRPs as food ingredients, the MR must be
48
elaborated under well-controlled technological conditions with selecting the
appropriate optimal process conditions (de Oliveira et al., 2016; Jaeger et al., 2010).
1.2.5.5.1 Maillard reaction stages
The MR, also known as non-enzymatic browning, was first reported by Louis-
Camille Maillard, in 1912. Hodge presented the first original comprehensive scheme
of the MR in 1953 where it was showed that the chemistry of MR is very complex
(Figure 1.8). The chemistry underlying the MR encompasses a series of reactions
divided into three main stages: early, advanced and final stages. The early stage of MR
involves the condensation reaction between the carbonyl group of a reducing sugar
with the available amine group, yielding the cyclic N-glycosylamine with the release
of one water molecule. The formed N-glycosylamine rearranges to form the Amadori
rearrangement product (ARP). The nutritional value may be reduced during the early
stages of the Maillard reaction because of the ɛ-amino group of lysine residue is most
likely to react. The advanced stage involves. The advanced stage involves the
degradation of the ARP and subsequent reactions depend on the pH of the system in
which the reactions occur. Under pH equal to or lower than 7, sugar dehydration occurs
with the formation of furfural in case of pentose or hydroxymethylfurfural when hexose
is involved. Under neutral or alkaline conditions, the degradation of ARP involves the
formation of various products such as a reductones and a multiple of fission products.
These products are highly reactive and enter in new reactions. The final stage of the
MR involves aldol condensations and an aldehydes-amine condensation reaction
leading to the formation of brown nitrogenous polymers known as melanoidins. The
composition of melanoidins is very complex and the structure of these compounds has
not been resolved completely(Wang et al., 2011). Despite the clear description of the
main routes of the MRPs, it is accepted that contrary to the early stage, the advanced
and final stages involve a wide range of interactions leading to the formation of either
desired or undesirable compounds (de Oliveira et al., 2016).
49
Figure 1.8: Scheme of the Maillard reaction adapted from (Hodge, 1953).
50
1.2.5.5.2 Maillard reaction processing
The products derived from Maillard reaction (MRPs) are commonly prepared
using high temperatures, either dry or in wet/aqueous solution. The first method
involves the heating of a dry dispersion of proteins and reducing sugars under
controlled temperature and relative humidity conditions. Disadvantages of this method
include the irregular contact between the reactants and the long reaction time up to
several days or weeks. The second method is accomplished in an aqueous solution,
which is heated at controlled temperatures. This method has the advantages of better
control and shorter reaction time. Its drawbacks include the removal of the water and
buffer when recovering the final products (de Oliveira et al., 2016). Nevertheless, under
heating processes, it is difficult to prevent the formation of undesirable color and flavor
changes (Oliver et al., 2006). Apart from the conventional thermal approaches,
innovative food processing technologies using non-thermal treatment such as
irradiation, high pressure, pulsed electric field and ultrasound were reported to promote
the formation of MRPs (Chawla et al., 2009; Guan et al., 2010; Wang & Huang, 2017;
Yu et al., 2016). These technologies improve the functionalities of MRPs with minimal
changes to color and flavor. It was found that high pressure enhanced the initial stage
of the MR, and decelerated the advanced and final stages, resulting in slight browning
color. Avila Ruiz et al. (2016) evaluated the effects of high-pressure high temperature
method on the MRPs from whey protein-sugar. They also found reduced browning and
inhibition of advanced stage products compared to high-temperature treatments. The
conjugates from whey protein isolate and dextran prepared using pulsed electric field
showed better solubility and emulsifying properties compared to whey protein isolate
(Sun et al., 2011). The gamma irradiation has been successfully used to produce
potential antioxidant MRPs in whey protein dispersion (Chawla et al., 2009).
Moreover, high intensity ultrasound assisted heating significantly improved the
functionalities such as solubility, antioxidant and antimicrobial properties of chitosan-
fructose MRPs (Zhang et al., 2015). Further research in this field could offer novel
opportunities for the development of emerging technologies, as an EA proposal.
51
1.2.5.6 Some proprieties of Maillard reaction products
Numerous reports demonstrated that the functionality of whey proteins can be
greatly improved trough the MR processing (de Oliveira et al., 2016). Some techno-
functional properties such as solubility, heat stability, emulsifying, foaming properties
and water binding capacity were significantly increased (Chevalier et al., 2001c; Chiu
et al., 2009; Dickinson & Izgi, 1996; Hattori et al., 1997; Medrano et al., 2009). In the
other hand, physiological properties such as antioxidant, antimicrobial,
antihypertensive and cytotoxic properties were also greatly enhanced via MR cross-
linking (Chevalier et al., 2001a; Hongsprabhas et al., 2011; Nooshkam & Madadlou,
2016a). In the recent years, increasing attention has been headed for the MRPs-whey
as naturally potential antioxidant additives for the replacement of the toxic synthetic
antioxidant (Chawla et al., 2009; Joubran et al., 2013; Liu et al., 2014; Wang et al.,
2013b).
1.2.5.7 Antioxidant properties of MRPs-whey
Antioxidant compounds can play an important role in both food systems as well
as in the human body to reduce oxidative alterations. In foods, MRPs like antioxidants
are useful in preventing or retarding lipid peroxidation leading to the deterioration of
quality attributes such as flavour and colour of the food product during storage
(Vhangani & Van Wyk, 2016; Yilmaz & Toledo, 2005). MRPs-whey demonstrated
potential antioxidant properties and may have favourable effects on human health
(Chevalier et al., 2001a; Seiquer et al., 2014; Thorpe & Baynes, 1996). Generally, the
antioxidant activity of MRPs-whey is mainly due to synergistic effects between the
ability to inactivate reactive oxygen species, chelate prooxidative transition metals,
scavenge free radicals, and reduction of hydroxyl radicals (Chawla et al., 2009;
Mastrocola & Munari, 2000; Wang et al., 2013b). Liu et al. (2014) reported that the
whey protein isolate conjugated with glucose under dry-heating conditions had
improved antioxidant properties and exhibited a strong reducing power and radical-
scavenging activity. Wang et al. (2013b) also noted that the water soluble MRPs
prepared from the interaction of whey protein isolate and different sugars with varying
pH have high antioxidant activity. Thus, the MRPs-whey prepared under alkaline pH
52
showed higher antioxidant proprieties. Sun et al. (2006) showed that MRPs prepared
from aqueous model mixtures of α-LA and β-LG with ribose under high temperature
and alkali pH conditions exhibited stronger radical scavenging activity. The MRPs
produced from whey protein-lactose under wet conditions by irradiation have been
associated with suitable antioxidant activity (Chawla et al., 2009). The reducing power
and iron-chelating ability of MRPs-whey were increased upon irradiation. These MRPs
were also able to scavenge hydroxyl and superoxide anion radicals and can useful to
inhibit lipid oxidation. Likewise, it was demonstrated that ultrasound technology was
efficient to produce glycated β-LG, which possessed increased iron-chelating and
reducing power capacities without promote the final stage of MR (Stanic-Vucinic et
al., 2013). Other study showed that combined hydrolysis of milk proteins and MR
resulted in significantly greater antioxidant activity than did the formed hydrolysates
or MRPs alone (Oh et al., 2013). These authors also showed that the antioxidant activity
was higher for the whey protein concentrate groups than for the sodium caseinate
groups and could be used as potential antioxidants in the pharmaceutical, food, and
dairy industries (Oh et al., 2013). The MRPs prepared from reacting hydrolysed β-LG
with glucose showed strongest antioxidant activity to inhibit lipid oxidation compared
the heated hydrolysed β-LG (Dong et al., 2012). Mohan et al. (2015) also found that
the MRPs-whey occurred with simultaneous enzymatic hydrolysis gained significantly
higher reducing capacity. Nevertheless, there is not much information about the
relationship between structural characteristics and the physiological activity of MRPs.
In some studies, the structure of MRPs with antioxidative activity were identified, such
as reductones, heterocyclic compounds, or high molecular melanoidins, but most of the
active antioxidant compounds in MR are still unknown (Eiserich & Shibamoto, 1994;
Wang et al., 2011).
53
1.3 Electro-activation
1.3.1 The concept of electro-activation and devolvement
Electro-activation (EA) is an emerging science, which studies activation
energy, thermodynamics and physicochemical properties of water or aqueous solutions
subjected to the action of external electric field (Aider et al., 2012). EA as a technology
itself account for more than a 100 years. Initial interest was when it is special physico-
chemical properties were detected by Russian scientists while working with drilling
mud (Prilutsky & Bakhir, 1997). In 1972, a Russian engineer Bakhir observed that
electro-activated solutions had properties different from chemically alkalized or
acidified analogues. However, he also observed that these anomalous properties tended
to decrease with the time and finally becoming equal to their chemical counterparts
(Pastukhov & Morozov, 2000). Due to these observations, such solutions were called
electro-activated solutions (EAS) (Gnatko et al., 2011). Its effects are a result of
physical energy, which is generated by the kinetic energy of the molecules in the
applied medium. Since that, it soon became apparent that EA is a growing field of
research, its powerful effect drawn the scientists to investigate on its applications in
many areas. Some of the examples of the efficient of EA in food science are microbial
inactivation and food safety, protein extraction, and enzyme inactivation (Aider et al.,
2012). One of the most exciting feature of EA technology is fact that is a reagentless
technology. More recently, a number of studies are opened a promising opportunities
for the development of EA technology as a safe and green technology (Aider &
Gimenez-Vidal, 2012; El Jaam et al., 2016; Gerzhova et al., 2015; Koffi et al., 2014;
Liato et al., 2015).
1.3.2 Principles of electro-activated aqueous solutions
The generation of EAS is based on the electrolysis phenomena, which occurs at
the electrodes/solutions interfaces (Shaposhnik & Kesore, 1997). These solutions find
themselves in the no equilibrium (metastable) thermodynamic state characterized by
abnormal physico-chemical activity decreasing with time (Prilutsky & Bakhir, 1997).
The application of external electric field supplies the energy required for the electro-
chemical reaction to launch and engenders migration of the charged particles (ions)
54
toward the electrode of opposite charge. Oxidation and reduction are the two main
processes, which take place during the electrolysis reaction (Figure 1.9). In water or
any aqueous solutions, a reduction reaction occurs at the negatively charged electrode,
which is called ''cathode''. The electrons (e-) from the cathode are given to the
positively charged ions such as hydrogen cations to form hydrogen gas (equation 1.1):
Reduction in the cathode: 2H+ + 2e- → H2(g) Eq. (1.1)
At oppositely charged electrode called an “anode”, an oxidation reaction takes place.
In this case, free electrons are given to the anode. The simple example for this reaction
will be the negatively charged oxygen, which migrates toward the anode. The migration
yields the generation of oxygen gas (O2) by giving electrons to the anode to complete
the reaction as described in equation 1.2:
Oxidation in the anode: 4OH-→ O2(g) + 2H2O + 4e-
Eq. (1.2)
Molecules of water also can be reduced and oxidized on the surface of cathode and
anode, respectively, according to the following equations1.3 and 1.4:
Cathode reduction: 2H2O + 2e- → H2(g) + 2OH- Eq. (1.3)
Anode oxidizing: 2H2O → O2(g) + 4H+ + 4e- Eq. (1.4)
55
Figure 1.9: Schematic illustration of a basic water electrolysis system (Zeng & Zhang, 2010).
As the result of the passage of electric current, the EA of water molecules
acquired a higher activity and modified structural and physico-chemical properties
such as pH, oxido-reduction potential and electro conductivity, etc. The
thermodynamics, which characterize the EA water generation, is still unknown (Aider
et al., 2012). Acid anolyte with pH in the range of 2.3 – 2.7, high positive oxidation -
reduction potential (ORP > ± 1000 mV), high concentration of dissolved oxygen is
produced from the anode section. Moreover, alkaline catholyte with high pH value
(10.0 – 11.5), high dissolved hydrogen, and negative oxidation-reduction potential (-
800 to -900 mV), is produced from the cathode section (Huang et al., 2008). In order
to obtain EAS with desired properties, the electrolytic cell is usually equipped with a
selective ion exchange membrane. As water conducts poorly the electric current, salts
like NaCl, KCl, Na2SO4, and NaHCO3 are usually added to facilitate the charge transfer
and to minimize the system resistance.
56
As was already mentioned, the EAS could temporarily exist in an active state
possessing anomalous physicochemical properties, which decrease during the
relaxation period. Gnatko et al. (2011) assigned some factors, which could explain the
distinct properties of EAS. The formation of intermediate highly active metastable
compounds, in particular hydrogen peroxide and free radicals, which are able to exist
in the medium for a long time due to the cyclic chain reactions. Other possible
explanation is related to the modification of structural and energetic characteristics of
water. Finally, the formation and the presence of gaseous microbubbles also favors the
anomalous properties of solutions in activated state (Sprinchan et al., 2011). Pastukhov
& Morozov (2000) demonstrated the existence of difference in the vibrational spectrum
of the EA water compared to its chemical acidified or alkalized analogs by Raman
spectroscopy. The spectra of EA water was similar to those of concentrated alkali and
acids in spite of the fact that they had low mineralization. This assumed that EA water
had properties similar to concentrated acids and alkalis. The substantial differences are
assigned in the spectral region between 700 and 2700 cm-1. A broad peak was obtained
for EA water in this region, which was not present for their chemical counterparts
confirmed the formation of intermediates compounds. The intensity of the peak also
decreased with the time indicating the relaxation period.
1.3.3 Application of electro-activation solutions
The EAS have been attracted considerable interest in many fields including
water preparation, chemical industry, agriculture, medical and food industry (Aider et
al., 2012; Gnatko et al., 2011; Huang et al., 2008). The distinct properties of EAS
described above characterize their utilization. It emerges as an anolyte, which can be
used as power cleaning and disinfecting agent or as a catholyte, which can act as a
powerful detergent as well as an extraction agent. In the food industry, the most
common techniques used to inactivate microorganisms are conventional thermal
pasteurization and sterilization. However, their effectiveness depend on treatment
temperature and time exposure leading to decrease the nutritional and organoleptic of
the products (Lee et al., 2016). The strong bactericidal activity of the electro-oxidized
water was found against numerous pathogenic bacteria (Klintham et al., 2017; Posada-
57
Izquierdo et al., 2014; Wang et al., 2014) as well as against fungi and viruses (Fang et
al., 2016). EAS have also been used in combination with moderate sterilization of
canned vegetables in order to decrease the sterilization temperatures (El Jaam et al.,
2016; Liato et al., 2015). The use of EAS significantly improves the extraction of plant
proteins (Ismond & Welsh, 1992). The extract activity of EAS was found to be
increased in comparison with conventional technology (Gerzhova et al., 2015). EAS
were applied to improve the bread baking leading in lower fermentation time and
higher bread volume (Nabok & Plutahin, 2009). Another example of EA application is
lactulose production as described in section. Among other applications are
improvement of the antioxidant capacity of enzymes, stabilization of the maple sap soft
drink, biofilms prevention or treatment and poultry spraying and chilling, etc (Aider et
al., 2012). In the medical field, electro-activated solutions were extremely useful for
the disinfection of medical and technological equipment and surfaces (Pintaric et al.,
2015). As an eco-environmentally technology, EA used for the neutralization of acid
generation sulfide mine drainage system and of water contamination risk (Kastyuchik
et al., 2016).
1.3.4 Electro-activation technology for whey valorization
In the present day, the dairy industry is focusing on exploring new technologies
to recover high-added-value components from cheese whey that can be potentially used
in functional food and pharmaceutical fields. Several technologies are available to
concentrate and fractionate cheese whey proteins, while most of it lactose content still
underutilized. New tendencies are oriented to maximize the valorization of cheese
whey compounds. Thus, search for economically viable and appropriate uses of whey
proteins and lactose content is of fundamental importance. In addition, the green
concept could be employed in this context. As a green, ecofriendly and low cost
technology, EA may be an interesting strategy for maximum valorization of cheese
whey based on adding value to the lactose together with protein compounds. The
exploitation of the alkaline and catalytic properties of EA could be attractive to enhance
the functional and biological proprieties of whey. In previous studies, the high pH
generated in the cathode was exploited for the isomerisation of lactose to lactulose
58
without the addition of reagents and lengthy processing conditions (Aider & Gimenez-
Vidal, 2012; Aissa & Aïder, 2013a, 2013b, 2014a). As a result of continued research
interest, one of the promising opportunity is the production of lactulose-rich whey.
Hence, whey can be used as a valuable and cost effectiveness raw material for lactulose
synthesis. Moreover, these is a lack of studies on the impact of EA process on the
structural characteristics and functional properties of whey proteins.
59
2. CHAPTER 2: Problematic, Hypothesis and Objectives
2.1 Problematic
The consumption of the dairy products containing prebiotics and probiotic
bacteria owning for the claimed health effects associated therewith gained great interest
from consumers. The growth and the viability of these beneficial bacteria are important
features at the time of consumption. To deliver the therapeutic effects, it is
recommended to maintain survival of the probiotic bacteria at suitable levels ranging
from 6-7 Log CFU/mL at the time of consumption. However, the scientific literature
showed that many factors can affect the viability of probiotics in food including the
probiotic stains used, the pH, the concentration of assimilate substrates, the presence
of hydrogen peroxide and dissolved oxygen, among others. To improve the growth and
the viability of these beneficial bacteria, the addition of functional ingredients such as
prebiotics and antioxidant compounds can be accomplished with proper selection of
probiotic strains. Sweet whey, a major co-product of dairy industry can be regarded as
a promising subtract to stimulate the growth of probiotic bacteria. Whey is a potential
source of lactose and soluble proteins and their values can be harnessed by inducing
their bioconversion into value-added product to use as a new multifunctional
ingredient. In this perspective, it is derisible to explore novel technologies for integral
valorisation of the full potential of whey compounds. In this context, electro-activation
(EA) as an environmentally eco-friendly technology holds strong promise for broader
functionality of whey by the in-situ conversion of lactose and proteins to corresponding
active compounds; lactulose (as prebiotic) and Maillard reaction products (as
antioxidant) as well as liberation of bioactive peptides, which can increase their degree
of applicability in diversified functional products.
2.2 Hypothesis
The valorisation of the full potential of the cheese whey components requires
new technologies. The present work hypothesizes that the electro-activation (EA)
technology is an innovative approach to produce in situ enriched whey with functional
60
and bioactive compounds and will allow its use as a potential prebiotic and antioxidant
ingredient to stimulate the growth of probiotic bacteria.
2.3 Main objective
The main objective of this doctoral thesis is to develop new dairy ingredient with
prebiotic and antioxidant properties through the electro-activation processing of sweet
cheese whey. This innovative approach was also aimed to improve the added-value of
cheese whey by using electro-activation in terms of optimizing the yield of lactose
electro-isomerization into lactulose, enhancement of it antioxidant and bioactive
proprieties and it potential applicability as prebiotic/antioxidant ingredients to
stimulate the growth of probiotic bacteria.
2.4 Specific objectives
To verify the stated hypothesis and to achieve the main objective, the following
specific objectives are highlighted:
1) To study the process of the in situ electro-isomerization of lactose into lactulose (as
prebiotic) by using electro-activation technology. The effects of different processing
parameters such as working temperature, current electric field intensity, whey volume
and initial whey concentration on lactulose yield were investigated.
2) To elucidate the antioxidant properties (mechanisms) of electro-activated whey (EA-
whey) produced under optimized conditions selected from the first specific objective.
3) To characterize the structural properties of Maillard reaction products and to identify
potential bioactive peptides formed in the EA-whey.
4) To assess the potential of EA-whey as a potential prebiotic ingredient to stimulate
the growth of chosen probiotic strains from Bifidobacterium and Lactobacillus species.
The protective effect of EA-whey as an antioxidant ingredient on the growth of L.
johnsonii grown in the presence of oxygen was also assessed.
61
3. CHAPTER 3: Contribution to the production of lactulose-rich
whey by in situ electro-isomerization of lactose and effect on whey
proteins after electro-activation as confirmed by MALDI-TOF MS
spectrometry and SDS-PAGE gel electrophoresis
This chapter is presented as an article entitled: “Contribution to the production
of lactulose-rich whey by in situ electro-isomerization of lactose and effect on whey
proteins after electro-activation as confirmed by MALDI-TOF MS spectrometry and
SDS-PAGE gel electrophoresis”.
The authors are: Ourdia Kareb (Ph. D. candidate: planning and realization of
the experiments, results analysis and manuscript writing), Claude P. Champagne
(Thesis co-director: scientific supervision, correction and revision of the manuscript),
and Mohammed Aïder (Thesis director: scientific supervision, article correction and
revision).
62
3.1 RÉSUMÉ
Le lactosérum, coproduit majeur de l'industrie laitière, a récemment fait l'objet
de nombreuses applications technologiques. Nous avons étudié la conversion du
lactosérum en produits à haute valeur ajoutée tels que le lactulose et les composés
antioxydants. Cet article examine l'efficacité de l'électro-activation comme une
technologie écologique pour une valorisation optimale du lactosérum. L'effet de quatre
paramètres expérimentaux tels que la température (0, 10 et 25°C), l’intensité du courant
(400, 600 et 800 mA), le volume de la réaction (100, 200 et 300 mL) et la concentration
en lactosérum [7, 14 et 28% (p/v)] sur l'électro-isomérisation du lactosérum a été
étudié. Les caractéristiques structurales des protéines de lactosérum et leurs capacité
antioxydante ont également été étudiées. Le rendement maximal de 35% de lactulose
a été atteint après 40 min d’électro-activation à une température de 10°C sous un champ
de courant électrique de 400 mA et un volume de réaction de 100 mL avec une
concentration de lactosérum de 7% (p/v). L'électro-isomérisation du lactose en
lactulose est accompagnée par la formation de galactose, mais seulement à très faible
quantité. De plus, le lactosérum électro-activé possèderait un pouvoir antioxydant
significativement plus élevé comparé au lactosérum non traité. L'augmentation de la
capacité antioxydante du lactosérum électro-activé résulterait de l'effet synergique de
l’hydrolyse partielle des protéines du lactosérum et la formation de composés
antioxydants capables de piéger les radicaux libres. En conclusion, les résultats de cette
étude révèlent que le lactosérum traité par la technologie d'électro-activation
possèderait à la fois des propriétés prébiotique (lactulose) et antioxydante et pourrait
avoir une application substantielle dans l’industrie pharmaceutiques et aliments
fonctionnels.
Mots-clés: Lactosérum prébiotique; Lactulose; Électro-activation; Isomérization;
Capacité antioxidante.
63
3.2 ABSTRACT
Cheese-whey, a major co-product of the dairy industry, has recently been the
subject of many technological applications. We studied the conversion of whey into
high value added products such as a potential lactulose prebiotic and compounds with
antioxidant properties. This paper examines efficiency, safety, and economics of
electro-activation (EA) as an eco-friendly technology for a maximum valorisation of
whey. The effect of four experimental parameters such as low working temperatures
(0, 10, and 25°C), current intensities (400, 600, and 800 mA), volume conditions (100,
200, and 300 mL), and feed concentrations [7, 14, and 28% (w/v)] on lactose-whey
electro-isomerization to lactulose were studied. Structural characteristics of whey
proteins and antioxidant functionality were also investigated. The maximum yield of
35% of lactulose was achieved after 40 min of reaction at the working temperature of
10°C under 400 mA electric current field and 100 mL volume conditions with using
feed solution at 7% (w/v). The isomerization of lactose to lactulose is accomplished by
subsequent degradation to galactose, but only at a very small amount. Additionally,
EA-whey showed significantly elevated antioxidant capacity compared with the
untreated samples. The enhancement of antioxidant functionality of EA-whey resulted
from the synergistic effect of its partial hydrolysis and the formation of antioxidant
components that could scavenge free radicals. In conclusion, the findings of this study
reveal that the whey treated by the safety EA technology has both lactulose-prebiotic
and antioxidant properties and could have a substantial application in the manufacture
of pharmaceutical and functional foods.
Keywords: Prebiotic whey; Lactulose; Electro-activation; Isomerization; Antioxidant
capacity.
64
3.3 INTRODUCTION
Whey is the major co-product of the dairy industry that is removed after the
coagulation of casein during cheese manufacturing (Siso, 1996; Zadow, 1994). The
global world production of whey was estimated to 160 million tons per year, showing
a constant annual growth of about 2%, whereas in North America, 3.6 million tons of
whey is obtained every year during by the cheese manufacturing industry (Guimarães
et al., 2010). Due to its high volume production and pollutant load impacts, cheese
whey represents a significant problem for the dairy industry (Janczukowicz et al.,
2008). Whey exhibits a biochemical oxygen demand (BOD) of 0.6-60 g/L and a
chemical oxygen demand (COD) of 0.8-100 g/L. Lactose, the major component of
whey contributes highly to its organic load matter effect (Carvalho et al., 2013;
Kosikowski, 1979). On the other hand, nowadays whey is not considered only as a
waste but also as a source of added value components (Corzo‐Martínez et al., 2013; de
Souza et al., 2010; Guimarães et al., 2010; Koutinas et al., 2009; Madureira et al., 2010;
Tavares et al., 2011). However, only half of the global whey processing is now
transformed into marketable products (Panesar et al., 2007; Siso, 1996; Spălățelu,
2012) and many of them are linked to the protein fraction (Karam et al., 2013). In this
respect, there is a need to find other innovative and complete approaches for the
valorization of whey based on adding value to the lactose together with the protein
components (Banaszewska et al., 2014; Smithers, 2008). Whey typically retains 55%
of milk nutrients, including lactose (4.5-5% w/v), soluble proteins (0.6-0.8% w/v),
lipids (0.4-0.5% w/v) and other trace elements such as B group vitamins and mineral
salts (Kosikowski & Wzorek, 1977; Marwaha & Kennedy, 1988; Prazeres et al., 2012).
The qualitative and quantitative contents of whey make it a good raw material for the
production of versatile valuable products (Mollea et al., 2013) such as bioactive
peptides with substantial functionalities for both human health and food technology
applications (Madureira et al., 2007; Pihlanto & Korhonen, 2015; Smithers, 2008).
Lactose is presently the most underutilized whey component, mainly because of the
huge quantities generated by the cheese/casein manufacturing industry and its
limitations in regards to functional foods applications (Guimarães et al., 2010; Paterson
65
& Kellam, 2009). However, it provides an attractive opportunity to produce different
chemicals, pharmaceutical and functional ingredients for food applications (Gänzle et
al., 2008). Growing attention has recently been paid to some prebiotics derived from
lactose, such as lactulose, lactilol, lactobionic acid and galacto-oligosaccharides. They
are considered as substrates with high promoting effect on the growth of beneficial
bacteria such (probiotics) and improve the overall gut health (Cardelle-Cobas et al.,
2011; Gutiérrez et al., 2012; Saarela et al., 2003; Schaafsma, 2008; Seki & Saito, 2012).
Among these sugars derived from lactose, lactulose is considered as the most promising
one in regards to the prebiotic effect.
Lactulose (4-O-β-D-galactopyranosyl-D-fructose) is a disaccharide composed
of a moiety of galactose linked to a moiety of fructose by a β (1-4) glycosidic linkage
(Panesar & Kumari, 2011). It is recognized as a potential prebiotic, because it
beneficially affects the growth of health-promoting bacteria in the gastro-intestinal
tract (Roberfroid, 2007). In the pharmaceutical field, lactulose is mainly used as a
gentle laxative (Schuster-Wolff-Bühring et al., 2010). For food considerations, is of
both technological and nutritional interest since it has higher sweetening power than
lactose and is more soluble (Aissa & Aïder, 2014b; Huebner et al., 2007; Olano &
Corzo, 2009). The sweetness power of lactulose is 48-62% of that of sucrose that is
referred as the standard in measuring the sweetness. The sweetness of lactose versus
sucrose is about 17-20 % only. It has been introduced in various dairy and
confectionery products and sweetener for diabetics (Aider & de Halleux, 2007; Panesar
& Kumari, 2011; Schuster-Wolff-Bühring et al., 2010; Seki & Saito, 2012).
Industrial production of lactulose is commonly performed by three methods: (1)
chemical catalysts in alkaline media via a Lobry de Bruyn-Alberda van Ekenstein
rearrangement in which the glucose of lactose isomerizes to fructose (Martinez-Castro
et al., 1986; Montgomery & Hudson, 1930), (2) Amadori rearrangement in which the
carbonyl group of reducing lactose interacts with free amino groups from proteins or
amino acids (Adachi & Patton, 1961; Andrews, 1986; Bologa et al., 2009) and (3)
biocatalysis with β-galactosidase and glycosidase enzymes (Adamczak et al., 2009;
Gänzle, 2012). However, the aforementioned processes present some drawbacks,
which include the low catalysis efficiency, cost effectiveness, low yield of lactulose
66
and complexity of purification steps (Aissa & Aïder, 2013b). Currently, lactulose was
successfully synthesized by using a safe and eco-efficient process, which is the electro-
activation (EA) technology (Aider & Gimenez-Vidal, 2012; Aissa & Aïder, 2013b,
2014a). EA is an emerging technology based on electrolysis of aqueous solutions
(Bologa et al., 2009). EA has been used for enhancing the antioxidant activity of
enzymes, maple beverage stabilization and starch hydrolysis (Aider et al., 2012). In
regards to whey valorization, one of the most promising way consists of converting
part of lactose into lactulose in situ by submitting whey to EA process. Consequently,
a new product, lactulose enriched whey, can be produced and can be used as a valuable
ingredient with proven prebiotic effect. This approach will enhance the use and
valorization of whey in a complex form without need for its fractionation. Furthermore,
whey proteins could be subjected to the Maillard reaction with reducing sugars and
partial hydrolysis under the conditions occurred using the EA and improved the
antioxidant capacity of the final product (Oh et al., 2013).
In the present work, the first objective was to study the process of lactose
conversion into lactulose in situ of sweet cheese whey by using the EA technology.
The effect of four processing parameters such as the working temperature (0, 10 and
25°C), current electric field intensity (400, 600 and 800 mA), whey volume (100, 200
and 300 mL), as well as initial whey concentration (7, 14 and 28%, w/v) on lactose
electro-isomerization yield into lactulose was studied. The second objective of this
study was to investigate the structural changes of whey proteins after EA treatment in
the cathodic side of the reactor by SDS-PAGE and MALDI-TOF analysis and to
evaluate the antioxidant capacity of the product by the ORAC assay in order to target
find dual functionality such as potential prebiotic and natural antioxidant effect.
67
3.4 MATERIALS AND METHODS
3.4.1 Chemicals
All chemicals used in this study were of analytical grade. Whey powder was
obtained from Agropur (Agropur Cooperative, St-Hubert, Canada). The composition
of the whey (on a wet basis) is as follows: water content: 4.28%, lactose: 75.50%, total
proteins: 12.15%, ash 7.41%, and residual fat 0.85%. Lactose monohydrate was
purchased from Avantor Performance Materials (Center Valley, PA). Standard sugars
of lactulose, glucose, galactose, and fructose (HPLC grade) were purchased from
Sigma-Aldrich (St. Louis, MO). Sodium sulfate (Na2SO4) was purchased from
Anachemia (Montreal, Canada). All solutions were prepared in deionized water.
3.4.2 Electro-activation reactor and configuration
The EA apparatus used in this work was designed as shown in Figure 3.1. The
EA cell was immersed in the refrigerated heat exchanger bath equipped with circulating
system (Isotemp 1016S, Fisher Scientific, Ottawa, Canada) to keep the working
temperature at the desired values as specified for this parameter (0, 10, and 25°C). The
bath was filled with Polycool MIX-25 silicone heat fluid (PolyScience, Niles, IL) to
maintain the required temperature. The EA cell is composed of three compartments:
the anodic, central, and cathodic. The central compartment was connected to the anodic
and cathodic ones by anion and cation exchange membranes, respectively as described
in Figure 3.1. The used anion exchange membrane was the AMI-7001S and the cation
exchange membrane was the CMI-7000S. Both the membranes were purchased from
Membranes International Inc. (Ringwood, NJ). This configuration was chosen to
maintain acidic conditions in the anodic side while the alkaline conditions were kept in
the cathodic compartment in which the lactulose formation was targeted. To ensure the
passage of external electric current, 2 food-grade stainless-304 electrodes were
introduced in the anodic and cathodic compartments and connected to the positive and
negative poles of a DC-electric generator, respectively. The cathodic compartment was
initially filled with whey solution, whereas the central and anodic compartments were
filled with 0.5 M of Na2SO4 solution.
68
Figure 3.1: Schematic representation of the experimental set-up for lactose-whey isomerization.
69
3.4.3 Electro-activation reaction
The experiments were carried out by modifying the following parameters:
operating temperatures (0, 10, 25°C), current electric field intensity (400, 600, 800
mA), whey volume (100, 200, 300 mL), and whey concentrations [7, 14, and 28%
(w/v)]. For each experiment, freshly prepared solutions were placed into the cathodic
compartment in which the targeted reaction occurred. The solution was gently stirred
by using an agitator RW 20 DS1 (IKA, Wilmington, NC) at 200 rpm. Samples from
the cathodic compartment were collected every 10 min until the end of the EA process,
which was set for a total of 60 min. For each of these samples, the values of the
corresponding voltage and pH were measured by using a digital multimeter (Keithley,
Inerga Series, Cleveland, OH) and a pH meter (model Oakton pH 700, Eutech
Instruments, Cole-Parmer, Montreal, Canada). All samples were kept under
refrigerated conditions until analysis. All experiments were done in duplicate.
3.4.4 HPLC analysis of the reaction products
Sugar contents were determined in all samples before and after the EA
treatment by Agilent HPLC system (Agilent Technology, Millipore Corp., Milford,
MA) equipped with a refractive index detector. The column chosen was 300×6.5 mm
Carbohydrate Analysis (Waters Co., Milford, MA). Before introduction into the HPLC
system, the samples were centrifuged at 10.000 × g for 5 min at ambient temperature
(21 ± 1°C). The supernatant was diluted 10-fold, and an aliquot of 25 µL was injected.
The running time was set at 30 min per sample. Calibration curves were prepared using
each sugar standard in a range of 5 to 20% (w/v) concentration. Once the separation of
sugars was achieved, peaks were identified by comparing their retention time with the
standard sugars.
The yield of reacted (converted) lactose, which is the fraction of the initial
lactose transformed during the reaction, was calculated by the following equation (3.1):
Lactose yield(%) =�������� ����������������
���������� ������� × 100 Eq. (3.1)
70
Where A Lactose is the peak area of the lactose at time τ, corresponding to the
time at which the treated solution was sampled, and A Lactose initial is the peak area of the
standard of lactose samples.
Lactulose yield, which is the fraction of lactulose formed in the total
carbohydrates in cheese whey, was calculated by the following equation (3.2):
Lactulose yield (%) = A Lactulose / A Lactulose initial ×100 Eq. (3.2)
Where A Lactulose is the peak area of lactulose at time τ and A Lactulose initial is the
peak area of the standard of lactulose samples given by the HPLC.
Galactose yield, which is the amount of galactose, formed in the in the total
carbohydrates was calculated by the following Equation (3.3):
Galactose yield (%) = A Galactose / A Galactose initial ×100 Eq. (3.3)
Where A Galactose is the peak area of galactose at time τ and A Galactose initial is the
peak area (Given by HPLC) of the standard of galactose samples.
3.4.5 Total protein content
The total nitrogen of whey samples was measured by using a Leco analyzer
(Truspec, Leco, St. Joseph, MI, USA) according to Dumas nitrogen method (Bremner
& Mulvaney, 1982). The corresponding total protein was calculated by multiplying the
nitrogen content by a factor 6.38 (Finete et al., 2013).
3.4.6 Gel Electrophoresis (SDS-PAGE)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with
and without β-mercaptoethanol (β-ME) of whey proteins submitted to EA was
performed using mini-protein electrophoresis system (Bio-Rad Laboratories, Hercules,
Canada) according to the method of Laemmeli (1970). Briefly, the 1% (w/v) protein
solutions were diluted 1:1 ratio with an SDS-PAGE sample buffer and then heated at
95°C for 5 min. A 10-µL aliquot of each sample was loaded into the gels.
Electrophoresis running was conducted at constant electric current of 30 mA. After
running, the proteins bands were stained with Coomassie Brilliant Blue R- 250 (0.2%)
in 40% methanol and 10% acetic acid for 2 h and destained overnight in 10% methanol
and 10% acetic acid.
71
3.4.7 MALDI-TOF-MS assessment
To achieve a better resolution and to get a more detailed insight of EA whey
proteins, the protein profiles were analyzed by MALDI-TOF-MS. Prior to analysis, a
10 µL aliquot of each protein sample was diluted 50-fold in 0.1% trifluoroacetic acid.
The matrix mixture was prepared by combining α-cyano-4-hydroxycinnamic acid and
2,5-dihydroxybenzoic acid solutions in 1:1 volume ratio as described previously by
Fenaille et al. (2006). A 1-µL aliquot of a protein sample was mixed with the same
volume of the matrix mixture, and 1 µL of the resulting solution was then deposited on
a MALDI target and analyzed using a MALDI-TOF mass spectrometer instrument
(4800 MALDI-TOF/TOF, AbSciex, Concord, Canada) equipped with the nitrogen
laser 335 nm. The laser-desorbed positive ions were analyzed after acceleration by 20
kV under linear mass range of 4000 to 30000 Thomson (Th) for the proteins and under
the reflector mode in the mass range of 650 to 4000 Thomson (Th) for the peptides.
Mass calibration in linear mode was achieved using the proteins mixture composed of
insulin, thioredoxin, and apomyoglobin. The external calibration in reflector mode was
performed using the peptide mixture composed of the adrenocorticotropic hormone
fragments (1–17, 18–39, and 7–38) and angiotensin I, provided by AbSciex (Concord,
Canada). A total of 2000 individual shots of each spot samples were added to produce
a MALDI mass spectrum.
3.4.8 Determination of free amino acids by UPLC-UV florescence detection
The free AA composition of whey after EA was determined after derivatization
with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using the Waters Acquity
UPLC system (Milford, MA) equipped with UPLC AccQ·Tag Ultra Column (2.1 mm
× 100 mm) and with an Acquity tunable UV and fluorescence detectors as described in
an AccQ·Tag Ultra kit (Waters, 2012). Briefly, whey samples were centrifuged for
5min at 4500× g at 4°C. The standards and samples derivatization of AA were prepared
by mixing 60 µL of AccQ·Tag borate buffer, 10 µL of samples, 10 µL of internal
standard (Norvaline at 100 µM), and 20 µL of AccQ·Tag. Ultra reagents were added
into a recovery vial subsequently. The mixture was vortexed immediately for 5 s and
72
allowed to rest for 1 min before heating at 55°C for 10 min. Five microliters of the
solution obtained was injected into the Waters HPLC system and monitored using both
UV and florescence set at 260 and 473 nm, respectively.
3.4.9 Oxygene radical capacity (ORAC) of electro-activated whey
The automated ORAC assay with fluorescein was carried out with the BMG
Fluostar Galaxy micro-plate reader (Durham, NC) equipped with fluorescence filters
for an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The
experiment was performed at 37°C and pH 7.0 using Trolox as the standard (a water-
soluble derivative of vitamin E). A mix of 200 µL of fluorescein solution (0.036 mg/L),
75 µL of a 2.2′-azobis-2-aminopropane dihydrochloride solution (8.6 mg/L), and 20
µL of the diluted samples were added to a 96-well plate in triplicate. The analyzer
recorded the florescence 35 times for 120 s. The ORAC values of EA-whey were
expressed as micromoles of Trolox equivalent per liter of whey.
3.4.10 Statistical Analysis
Statistical analysis was performed using a complete randomized factorial design
with repeated measurements. The factors considered were the electric current,
temperature, volume, concentration, and reaction time. The dependent variables
studied were the pH, lactulose yield obtained, as well as yield of by-products (glucose,
galactose, and fructose) that occurred in the cathodic compartment of the EA cell. Each
treatment was carried out in triplicate, and mean values ± standard deviation were used.
The antioxidant capacity was performed using the regression model. Statistical analysis
(ANOVA) of the data was performed using SAS software (V9.3, SAS Institute Inc.,
Cary, NC).
73
3.5 RESULTS AND DISCUSSION
3.5.1 Process of whey solution EA: Evolution of whey solution alkalinity
Figure 3.2: Profile of pH as a function of EA time in whey (Cwhey = 7% w/v), at different experimental conditions (a) current intensities (400, 600, and 800 mA) at 10°C and 100 mL, (b) working temperatures (0, 10, and 25°C) at 400 mA and 100 mL, and (c) volume conditions (100, 200, and 300 mL) at 10°C and 400 mA.
74
The evolution of the whey solution pH in the cathodic compartment in which
the electro-isomerization of lactose into lactulose is anticipated under varying
experimental conditions is shown in 3.2. The effect of three current intensities (400,
600, 800 mA) on the pH evolution of whey at 10°C and 100 mL conditions are
summarized in the Figure 3.2a. The results showed that EA is effective in terms of
enhancing the alkalinity of whey without adding alkalinizing reagents such as NaOH.
To achieve this pH increase up to 10 and more, the results showed that the current
intensity is an important parameter because it is necessary to achieve the
electrochemical metastable state of aqueous solutions and to make it able for electro-
catalysis (Bologa et al., 2008; Sprinchan et al., 2011). Indeed, the whey solution
possesses the necessary properties for the rapid and efficient accumulation of actively
charged particles (Bologa et al., 2008). Unlike the lactose solution which suffers from
lack of conductivity, whey contains sufficient amount of mineral salts that ensure the
passage of electric current (Aissa & Aïder, 2013a). Thus, under the influence this one
(external electric field), electrolytic dissociations of aqueous solutions giving charged
electrolytes in the anodic and cathodic compartments occur. Under EA, a water
oxidation takes place in the anodic compartment leading to the formation of H+ cations,
according to the reaction (Eq. 3.4):
Anode 2H2O → O2 (g) + 4H+ + 4e- (Eq. 3.4)
At the same time, in the cathodic compartment, a water reduction into hydrogen
with release of HO- ions takes place according to the following reaction (Eq. 5):
Cathode 2H2O + 2e- → H2 (g) + 2 OH- (Eq. 3.5)
The formed hydroxyl ions OH- increase the alkalinity of the media that is a key
parameter in the lactose electro-isomerization into lactulose. Indeed, the hydroxyl ions
formed as result of water dissociation at the solution/cathode interface play the role of
proton acceptors when the isomerization of lactose into lactulose occurs according to
the Lobry de Bruyn-Alberda van Ekenstein rearrangement. In order to maintain this
condition at the cathodic side, a negatively charged cation exchange membrane was
used to repulse the hydroxyl ions that can be transported by the current flow. In our
75
study, the results showed a similar tendency of the evolution of pH as a function of
time under the different currents applied (Figure 3.2a). The curves obtained can be
divided into two stages. During the first stage, typically in the first 20 min of EA, a
rapid increase of pH occurred in the solution due to the high dissociation of water with
the subsequent generation of high amount of hydroxyl ions OH-. In the second stage,
the pH solution continued to increase gradually but at a lower rate than in the first stage,
which means that the solution was highly charged by hydroxyl ions OH-. It can be also
seen that the average pH of solution increases sharply with increasing the current
intensity applied to the EA reactor. This behavior can be explained by the proportional
increase of water electrolysis due to the increase of the current intensity. The optimum
pH of lactose isomerization to lactulose under EA for this study was 11 ± 0.3. The
results published by previous authors using chemical catalysis such as boric acid and
sodium hydroxide are in good agreement with those here described (Hashemi &
Ashtiani, 2010; Zokaee et al., 2002). Some works reported the decline of pH solution
as the reaction of isomerization of lactose to lactulose proceeds at the end (Angel de la
Fuente et al., 1999; Montilla et al., 2005). According to the studies of Berg & van
Boekel (1994) and Olano & Martinez-Castro (1981) in heated milk, the decrease of pH
resulted from the hydrolysis of lactulose into galactose and acid compounds such as
isosaccharinic and formic acids. Contrary to these works, the pH profile of the whey
submitted to EA during 60 min did not show any decreasing phase in all experimental
conditions. Thus, it can be assumed that under the effect of EA, high alkaline conditions
were maintained until the end of the reaction process. Thus, subsequent degradation of
lactulose into organic acids did not occur in these conditions. This result is very
important in terms of the differentiation between the conventional chemical lactose
isomerization into lactulose and the process based on the reagentless EA technology.
Similar results were obtained when lactose aqueous solution was used to synthesize
lactulose by using an EA reactor (Aider & Gimenez-Vidal, 2012; Aissa & Aïder,
2013a). The alkalinity of whey solution under different temperatures was also studied
and the results are shown in Figure 3.2b. The variation of temperatures did not affect
the evolution of the whey pH submitted to EA at the cathodic compartment (Figure
3.2b). On the other hand, when the volume conditions at the cathodic compartment
76
were varied, one can observe a different behavior of pH evolution. As a function of
running time, the ANOVA analyses showed a high significance level (P < 0.0001) of
the volume variation on the evolution of pH solution at the cathodic compartment. As
showed in Figure 3.2c, the maximum alkaline conditions were obtained when a whey
volume of 100 mL was used. Further increase of the whey volume resulted in a slower
increase of pH solution. For example, after 20 min of EA, the pH of solution reached a
value of 10.65 ± 0.4, 9.10 ± 0.90 and 7.70 ± 0.57 when the whey volumes of 100, 200
and 300 mL were used, respectively. The difference of the alkalinity of whey solution
is related to the concentration hydroxyl ions formed as well as to the solution electric
conductivity and buffering capacity (Al-Dabbas et al., 2011; Hill et al., 1985).
3.5.2 Assessment of lactulose formation during whey electro-activation
Figure 3.3 showed the results obtained when whey and lactose, at similar
lactose content, were electro-activated under 400 mA current electric field at 10°C in
a 100 mL volume conditions. The obtained results showed that the conversion of
lactose to lactulose was faster by using lactose aqueous solution compared to whey
solution. The lower level of lactulose formed in whey in the early stage of reaction until
20 min is due to the retarding of pH raise caused by the buffering action of whey
proteins and other compounds such as citrate and phosphate (Paseephol et al., 2008).
Furthermore, lactulose was not detected in whey at earlier reaction when pH was in the
range of that of milk and it was formed only when alkaline conditions were reached in
the cathodic compartment. Thus, one can conclude that lactulose did not remove as a
result of lactulosyl-lysine hydrolysis. These results agreed with previous reports which
showed that protein did not interact to increase the lactulose concentration in heated
milk (Andrews & Prasad, 1987; Martinez-Castro et al., 1986). Thus, it can be
concluded that lactulose was formed exclusively via the (LA) rearrangement using
whey as lactose source under EA conditions.
It was reported that in milk or whey, lactulose is subsequently formed by
different reaction mechanisms in two main forms as free lactulose and covalently
bound to amino groups of proteins as lactulosyl-lysine (Adachi & Patton, 1961;
Andrews, 1986). Under alkaline conditions, the free lactulose was mostly formed via
77
the (LA) transformation, in which the glucose moiety of lactose is isomerized to
fructose via an intermediate enolization reaction (Andrews & Prasad, 1987; Martinez-
Castro et al., 1986). The rearrangement of lactose to lactulose requires proton acceptors
which are ensured by achieving high alkaline medium in the cathodic compartment of
EA cell (Aider & Gimenez-Vidal, 2012). Regarding the Lactulosyl-lysine, it is formed
by the Amadori rearrangement in which the carbonyl group of lactose condenses with
the free amino groups of the protein (Adachi & Patton, 1961). Some workers suggested
that in the second mechanism, free lactulose could be also removed because of the
hydrolysis of bound lactulosyl-lysine. The conversion of lactose to lactulose in milk
during heating at 110-150°C during 20 min was studied by Berg & van Boekel (1994)
and the authors concluded that the major lactulose formed followed the LA
rearrangement and only 20% was by the Amadori pathway.
Figure 3.3: Profile of lactulose yield as a function of EA time using different feed solutions. The experimental conditions were; whey (Cwhey = 7% w/v), lactose (CLactose
= 5% w/v) at 10°C, 400 mA and 100 mL conditions. Data represent the mean ± standard deviation of three experiments.
78
3.5.3 HPLC analysis of lactulose and other reaction by-products
The conversion of lactose into lactulose was usually followed by its degradation
into various by-products as was reported by Berg & van Boekel (1994) for milk and
Montilla et al. (2005) for whey. The isomerization of lactose to lactulose and formation
of other by-products such as glucose, galactose and fructose were investigated by
HPLC chromatograms as shown in Figure 3.4. In the present study, experiments
carried out with a 7% (w/v) whey solution treated by EA at 10°C in a 100 mL volume
under 400 mA electric current field showed that as the EA processed, the formation of
lactulose (lactose conversion yield) increased gradually with a slight decrease at the
end of the treatment. A maximum conversion of lactose into lactulose of 36 ± 0.2%
was achieved after 50 min of EA. However, this high conversion was followed by an
increase of the galactose formation which was more observed at the early stage of the
EA treatment. At the same time, analysis of the HPLC chromatograms did not show
any formation of glucose or fructose (few traces in some cases). Thus, these results
clearly indicate that galactose is the only by-product formed after EA of lactose. This
means that lactulose formation by EA is quite different from the other chemical ways
used for this purpose. Our results are in accordance with those previously reported by
Ait-Aissa & Aider (2013a) who used EA, but do not agree with the findings of Olano
& Martinez (1981) who used chemical conversion method and showed that lactulose
was more easily degraded than lactose in alkaline solution. Moreover, only minimal
traces in the round of 0.17 ± 0.02 % of glucose and fructose were detected at the
advanced stages of the EA reaction in strong alkaline media. Similar results were
reported by Aider & Vidal (2012) under other experimental conditions. In agreement
with our results, no glucose or fructose has been previously detected in heated milk or
whey (Montilla et al., 2005; Olano et al., 1989). Mendez & Olano (1979) reported that
since no fructose and glucose were detected, it can be assumed that the disaccharides
lactose and lactulose were degraded into galactose and acid compounds. However, our
results did not show any pH drop in all cases such as showed in Figure 3.2. Thus, it
could be suggested that under conditions occurred in EA, glucose and fructose could
be isomerized to galactose or take part in the formation of lactulose after the ring
opening of the lactose molecule (Figure 3.5).
79
Figure 3.4: Chromatogram profiles of whey sugars after EA at 7% (w/v) concentration, temperature T = 10°C, current intensity, I = 400 mA and V=100 mL: (a) t = 0 min, (b) t = 10 min, (c) t = 20 min, (d) t = 30 min, (e) t = 40 min, (f) t = 50 min, (g) t = 60 min.
80
Furthermore, brown color was not observed which suggest that the advanced
Maillard reactions were not formed at the low temperatures used in this study. Thus, the
main route of lactose conversion under EA was its isomerization into lactulose with
subsequent formation of some galactose as the sole by-product of the reaction.
Figure 3.5: Mechanism of the electro-isomerization of lactose to lactulose in whey and the possible galactose formation pathways under low temperature using EA process.
81
3.5.4 Effect of temperature
In contrast with the previous studies, the isomerization of lactose into lactulose
under refrigerated conditions until 0°C has been addressed in the study of (Aissa & Aïder,
2013a) for the first time. By using lactose aqueous solution, these authors investigated the
effect of six temperatures (0, 5, 10, 15, 20 and 30°C) as a function of time on lactulose
synthesis under EA porcess in the cathode section of the reactor. Then, it is worthwhile
noting that they obtained approximately 25 % lactulose yield with a purity of 95% at low
temperatures (0-10°C). Based on the above results, the low temperatures are great suited
for lactulose isomerization under EA reactor since it limited the formation of by-product
to only galactose in small amount, a fact which constitutes a real advancement in the
knowledge and technology of lactulose synthesis. Therefore, three temperatures (25, 10,
and 0°C) were selected for the present study. Figure 3.6 shows the effect of the different
temperatures in a volume of 100 mL of whey under 400 mA of electric field on lactose
conversion into lactulose and galactose formation as a by-product. The profiles of lactulose
yield obtained were similar at all the temperatures. A maximum yield of 34.57 ± 1.37%
with low galactose formation was obtained at the working temperature of 10°C after 40
min of EA treatment. The formation of galactose was slightly lower in the range at 0-10°C
than at 25°C. On the other hand, further decrease of temperature down to 0°C resulted in
moderate decline of lactulose formed (32.18 ± 0.61%). Therefore, the temperature of 10°C
can be considered as a key parameter for the optimal synthesis of lactulose by using the
EA process. These results were in good agreement with those reported by Ait-Aissa &
Aïder (2013a) using lactose aqueous solution. Since the synthesis reaction of lactulose is
kinetically controlled by temperature, these results suggest that using EA; the activation
energy required for lactulose isomerization is markedly lower than for the chemical and
enzymatic methods. Besides, values of lactulose yield obtained in these experimental
conditions are slightly higher within the ranges usually reported in the literature. Thus, the
present process of lactulose synthesis is significantly more sensitive, rapid and simple than
those routinary used.
82
Figure 3.6: Profiles of lactulose and galactose produced as a function of EA time at different processing temperatures (0, 10, 25°C), whey concentration 7% (w/v), temperature T = 10°C, current intensity I = 400 mA and V = 100 mL.
Based on the findings of several researchers, the alkaline isomerization of lactose
to lactulose via LA rearrangement was usually carried out at high temperatures in the range
of 50 to 130°C combined with different reaction times (Andrews, 1986; Andrews & Prasad,
1987; Geier & Klostermeyer, 1983; Martinez-Castro & Olano, 1980; Montilla et al., 2005;
Olano et al., 1992; Paseephol et al., 2008; Sakkas et al., 2014; Song et al., 2013; Zokaee et
al., 2002). Using egg shell, a food grade catalyst and cheese whey permeate, Corzo-
Martinez et al. (2013) obtained a yield of 16.1% of lactose conversion into lactulose by
heating at 98°C during 150 min of reaction time. A similar lactulose yield of 18% was
reached by using milk ultrafiltrate within 60 min (Montilla et al., 2005). The synthesis of
lactulose from milk permeate using calcium carbonate-based catalysts was also achieved.
The lactulose yield reached did not exceed 21% by a heat treatment at 96°C during 120
min running time (Paseephol et al., 2008). Recently, Seo et al. (2015) combined cheese
whey as lactose source and sodium carbonate (Na2CO3) as catalyst for lactulose synthesis.
The authors obtained only 3-4% of lactose to lactulose conversion yield at 60°C. When the
temperature was increased up to 90°C, the lactulose yield increased up to 29.6% after 20
min of reaction time. In fact, Andrews & Prasad (1987) showed that the increasing of
temperature from 90°C to 130°C results in shortness reaction time from 120 min to 4 min
with positive effect on lactulose yield conversion. In spite of that, the rapid degradation of
83
lactulose in favor of galactose by-product constituted a limit of the process. It was
concluded that the amount of galactose increases with elevated temperature due to the
greater activation energy formed and which pushed the reaction on the other side pathways
(Calvo & Olano, 1989; Hashemi & Ashtiani, 2010; Olano & Calvo, 1989; Troyano et al.,
1992).
3.5.5 Effect of the electric current intensity
The effect of different current intensities on lactulose synthesis was also studied as
shown in Figure 3.7. The statistical analysis of obtained data showed a high significance
level (P < 0.0001) of the electric field intensity on lactulose yield. As it can be seen in
Figure 3.7a, when the EA reaction was carried out at 25°C, the isomerization of lactose
(in the whey) into lactulose was accelerated at 600 mA and 800 mA compared to 400 mA
as a function of running time. After 10 min of EA, the lactulose yield achieved at 400 mA
was only 4.48 ± 0.6%. At the same time, mean values of 8.23 ± 0.76 and 16.35 ± 1.3%
were recorded at 600 and 800 mA, respectively. It can be noticed that lactulose yield
increased in a linear fashion when the current intensity was increased from 400 to 800 mA.
As the reaction time was increased and after 30 min of EA, lactulose yield in the cathodic
compartment still increased and reached the maximum value of 35.78 ± 0.8% at the current
intensity of 800 mA. In similar conditions and by using a low current intensity of 200 mA,
Aider & Vidal (2012) reported only a value of 5.71 ± 0.31% lactulose produced from whey
permeate as feed solution. Thus, these results are in agreement with previously reported
data on the synthesis of lactulose using EA process, where the increasing lactulose yield
was clearly related to the increase of the current intensity (Aider & Gimenez-Vidal, 2012;
Aissa & Aïder, 2014a; Sprinchan et al., 2011). Nevertheless, a gradual decrease of lactulose
yield that could be attributed to its degradation into galactose after 30 min of EA was
observed at elevated current intensities than at 400 mA. Regardless on pH solution, these
results are in good agreement with the fact that the increase of electric field intensity
implied the Joule heating effect. During the passage of electric current through whey
solution, some resistance occurred and the temperature increased in the range of 3 to 6°C
in the reactor. The heated energy dissipated is proportional with electric current applied
and described as Joule effect (Aissa & Aïder, 2014a).
84
Figure 3.7: Profiles of lactulose yield as a function of EA time at different current intensities (400, 600, and 800 mA). The concentration of feed whey and volume were fixed at Cwhey = 7% (w/v) and V = 100 mL, respectively. The working temperatures were varied, (a) at 25°C, (b) at 10°C and (c) at 0°C.
In the same conditions with the working temperature of 10°C as seen in Figure
3.7b, a high lactulose yield values were also achieved. As a function of the reaction time,
lactose conversion into lactulose still increased until 40 min of EA in all experimental
conditions. The maximum conversion yield of 33.65 ± 0.2% of lactose into lactulose was
obtained after 30 min under 800 mA current electric field. After 40 min of EA, one can
distinguish two distinct behaviors on lactulose evolution: at 400 mA, lactulose synthesis
still predominant and reached the value of 34.57 ± 0.79 % with significant high purity,
while at higher current intensities higher by-products, mainly galactose, decreased its
synthesis. However, it can be noted that the rate of lactulose yield decrease at 10°C
occurred more slowly and was less important than at 25°C, due the fact that Joule effect
85
was quite negligible. Moreover, depending on the pH value of the reaction medium and
after 40 min of EA, the pH reached the values of 11.15 ± 0.2, 11.85 ± 0.1 and 11.95 ± 0.05
under the effect of 400, 600 and 800 mA, respectively. In fact, the rate of water electrolysis
is directly proportional to the electric current applied. On the other hand, preliminary
results of pH effect on lactulose synthesis indicate that the pH 11 ± 0.3 was more suitable
for lactulose synthesis which is an accordance with the results obtained at 400 mA after 40
min of EA. The same effect was also reported by other authors (Hashemi & Ashtiani, 2010;
Zokaee et al., 2002). Figure 3.7c showed the profiles of lactulose yield obtained under
different electric current applied at 0°C. The reaction rate of isomerization of lactose into
lactulose was slightly lower. However, as expected, the drop of lactulose yield being
slowest and less significant under these conditions. When the desired temperature range is
determined, the corresponding parts of the current intensity-time profile can be used to
extract the best current intensity field for isomerization of lactose into lactulose by EA. In
these assays (experiments), since the compromise between the highest lactulose yield and
the lowest by-product was carried out with, the lowest electric field applied of 400 mA;
this parameter was chosen to ensure the optimal lactulose yield and to reduce the energy
expenditure of the process.
3.5.6 Effect of the volume reaction
The curves described in Figure 3.8 show the change of the lactulose yield values
as a function of the processing time with varying the volume of the reacted whey (100, 200
and 300 mL) in the cathodic compartment of the EA reactor. All of the experiments were
carried out at 10°C. Analysis of the obtained data showed a general trend towards an
increased lactulose yield with decreasing the volume of the EA-whey in all cases.
Moreover, when the current electric field was increased, an important increase in lactulose
yield was observed at 100 mL compared to the result obtained when the higher whey
volumes were used (200 and 300 mL). The ANOVA analysis showed that the effect of the
volume on lactulose yield as a function of the running time was highly significant (P <
0.0001).
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Figure 3.8: Profile of lactulose yield produced as a function of EA time at different volume conditions (100, 200 and 300 mL). The concentration of feed whey and temperature were fixed at Cwhey = 7% (w/v) and T = 10°C, respectively. The current intensities were varied, (a) at 400 mA, (b) at 600 mA and (c) at 800 mA.
As it can be seen in Figure 3.8a, when the cathodic compartment was filled with
100 mL of whey solution and submitted to 400 mA, the evolution of lactulose yield showed
two distinguished zones: in the first one, lactulose yield still increased until 40 min, and in
the second part, it was constant until the end of EA reaction. By using the volumes of 200
mL and 300 mL, the rate of the electro-isomerization reaction was more slowly and
lactulose yield obtained was less important compared to 100 mL. After 40 min of EA, the
lactulose yield reached the values of 34.57 ± 0.79, 20.43 ± 0.81 and 7.08 ± 1.07% by using
100, 200 and 300 mL whey, respectively. The difference observed can be explained by the
fact that at any given constant electric current, an increase of the treated volume results
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lower pH increase due to the similar amount of OH- ions generated by electrolysis of water
at the cathode/solution interface. Thus, the lactulose yield is directly related to the amount
of hydroxyl ions formed, which play the role of proton acceptors and ensure the optimum
pH for lactose isomerization into lactulose. On the other hand, according to the Nernst law,
an increase in current intensity is required for decreasing the minimum voltage of water
decomposition.
Figure 3.8b and c present the comparison of the values of lactulose yield with
varying the volume of the EA-whey at 600 and 800 mA, respectively. The results showed
a strong dependence on the applied current electric when the reacted volume was increased.
The increasing of current electric applied resulted in a faster decomposition of water which
ensures an increasing of required alkalinity for lactulose synthesis. Highest lactulose yield
was obtained when the target compartment was filled with 200 mL, reaching values of
34.85 ± 1.09% and 35.95 ± 0.7% at the end of reaction process under 600 and 800 mA,
respectively. However, lactulose yield and purity obtained with increasing the whey
volume and applied electric current did not differ noticeably from the corresponding values
of lactulose yield obtained in 100 mL (34.57 ± 0.79%) under 400 mA electric current
applied. Thus, the optimum volume in which high lactulose yield was recovered was 100
mL.
3.5.7 Effect of the feed whey concentration
To assess the potential of the EA process for eventual large scale production level,
synthesis of lactulose was studied as a function of the feed solution concentrations. Having
selected previously the optimum conditions for lactulose production, the effect of three
initial concentrations of the whey [7, 14 and 28% (w/v)] was studied. The results showed
that the conversion of lactose to lactulose is feasible using different feed concentrations
under the EA process. However, the lactulose yield did not increase with increasing the
substrate concentration in all the cases. As shown in Figure 3.9a, when 400 mA of electric
field was applied, the lactulose production in a 7% (w/v) whey solution reached the
maximum of 34.57 ± 0.79 % after shorter time compared to the case when higher
concentration of whey was used.
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Figure 3.9: Profiles of lactulose yield produced as a function of EA time at different whey concentration (7, 14 and 28% w/v). The temperature and volume conditions were fixed at T = 10°C and V = 100 mL, respectively. The current intensities were varied, (a) at 400 mA, (b) at 600 mA and (c) at 800 mA.
Under these conditions, it was necessary to increase the reaction time to reach the
maximum lactulose concentration due to an incomplete reaction. Figure 3.9b and c showed
that with increasing the current electric intensity, the both concentrations of 7 and 14%
were suitable for lactulose production. Thus, it was possible to maintain a high yield of
lactulose production in an average value of 36 ± 0.54%. These results agree with those
reported by Montilla et al. (2005) who obtained a similar percentages of conversion by
using whey ultrafiltrate concentrated up to 5.2 and 7.6 fold. Further increase of the feed
solution up to 28% (w/v) was not suitable for lactulose synthesis by using the EA process.
At this concentration, the rate of the reaction was the slowest despites increasing of the
applied current intensity, and the lactulose yield reached was lowest compared to the other
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concentrations. These results could be explained by the high buffering capacity of the whey
solution at this concentration. This was corroborated by the lowest pH increase in the
cathodic compartment when 28% whey solution was used compared to the other
concentrations. Thus, it can be concluded that the suitable whey concentrations for
lactulose production that can be used are 7 and 14% (w/v).
3.5.8 Proteomic analysis of the EA-whey
3.5.8.1 SDS-PAGE profiles of EA-whey proteins
The profile of whey proteins after EA could be changed as result of the variation of
pH and the compositions of aqueous medium in the cathodic compartment of the reactor.
Therefore, the changes in whey proteins during simultaneous isomerization of lactose into
lactulose treated at 10°C and 400 mA were investigated by SDS-PAGE analysis under the
both reducing and non-reducing conditions (Figure 3.10). The SDS-PAGE patterns of
whey proteins after EA revealed a decline tendency of the level and intensity of protein
bands as the reaction time progressed. The three prominent native whey proteins including
α-lactalbumin (α-LA, 14.4 kDa), β-lactoglobulin (β-LG, 18.3 kDa), bovine serum albumin
(BSA, 67 kDa) and a faint band of immunoglobulins (IGs) fraction in the upper part of the
separating gel were clearly appeared in the untreated samples. After 20 min of EA, the
band intensity of the β-LG fraction was significantly diminished whereas a new band with
molecular weight of approximately 34 kDa appeared. The appearance of high molecular
weight fractions in the protein patterns could be resulted from protein aggregates formed
through disulphide linkages (Lillard et al., 2009; Monahan et al., 1995) or a conjugated
protein-carbohydrate achieved by Maillard reaction products (MRPs) (Chevalier et al.,
2001b; Diftis & Kiosseoglou, 2006).
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Figure 3.10: Electrophoretic pattern of EA whey proteins as a function of reaction time under non-reducing conditions (a) and reducing conditions (b). The temperature and current intensity were fixed at T = 10°C and I = 400 mA, respectively. MW, molecular weight of protein standard; BSA, bovine serum albumin; β-LG, α-LA and IGs.
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To confirm whether whey proteins conjugated with the sugars present in the
cathodic compartment, the SDS-PAGE pattern was investigated under reducing conditions
(Figure 3.10b). A larger broader spectral smearing zone was still detected under the
reducing conditions, but of minor intensity, which indicates that the both covalently
disulphide bonds and protein cross-links with sugars occurred. The conjugation
demonstrated by SDS-PAGE was also supported by MALDI-TOF-MS analysis as
discussed below. After 40 min of EA, the characteristic bands were gradually disappeared
but some traces were still observed. Thus, the less perceptibility of the bands is interpreted
by the partial hydrolysis of whey proteins to smaller products than the limit detection of
the gel electrophoresis. The combined Maillard reaction products with whey proteins and
it hydrolysates has been previously reported (Oh et al., 2013) and the authors suggested
that these products can contribute to enhance the antioxidant capacity of the product.
Therefore, the changes in whey proteins after EA could result in improved antioxidant
functionality.
3.5.8.2 MALDI-TOF-MS analysis of whey proteins after EA
MALDI-TOF-MS analysis is widely used to monitor the main modifications
induced in whey proteins by thermal processing, including their glycation pattern in the
presence of lactose and sugars (Carulli et al., 2011; Meltretter et al., 2009). Furthermore,
no studies have been reported so far to applying MALDI-TOF-MS to investigate the
modifications in whey proteins submitted to EA process. In this study, MALDI-TOF-MS
analysis was performed for screening the structural modifications of whey proteins
submitted to EA treatment in the presence of a mixture of different sugars such as lactose,
lactulose and galactose. It is well known that the interaction of reducing sugars with
proteins might lead to the formation of protein bound carbonyls as early Maillard reaction
products. Covalent bounds of lactose and galactose mix with proteins whey resulted in 324
Da and 162 Da increments (Carulli et al., 2011). The results of MALDI spectrum of the
native whey proteins showed the main signals at m/z 14189 and 18362 Da, which
corresponded to the expected mass of α-LA and β-LG, respectively. Another peak signal
exhibiting mass values of 6784 Da, which could be attributed to proteose-peptone, was also
detected. The profile of EA-whey proteins during 40 min at temperature of 10°C and
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current electric field of 400 mA is shown in Figure 3.11b. The MALDI mass spectra were
characterized by a Gaussian peak shape including new peaks due the great heterogeneity
of the conjugated-proteins with sugars formed as described in other works (Corzo-Martínez
et al., 2008). A decrease of the signal intensity was also observed due the aggregation and
partial hydrolysis of the native proteins under the alkaline conditions occurred in the
cathodic compartment of the EA reactor. Furthermore, the native and EA-whey proteins in
the range of 650-4000 Da were also detected and analyzed (Figure 3.11c and d). Compared
to the native spectra, new polypeptide peaks were assigned by their mass, which resulted
from the partial proteins hydrolysis as observed previously by SDS-PAGE analysis.
3.5.9 Total antioxidant capacity of EA-whey proteins
The whey proteins and its hydrolysates have been shown to have antioxidant
activities which can act as free radical scavenging, metal ion chelation and inhibition of
lipid peroxidation (Hernández-Ledesma et al., 2005; Oh et al., 2013; Önay-Uçar et al.,
2014). Thus, in addition to the lactose electro-isomerized into lactulose, the total
antioxidant capacity of EA-whey proteins and their capacity to scavenge the peroxyl
radicals was measured by the ORAC assays. There was a significant difference in radical-
scavenging activity of whey as the EA-time increased (P < 0.0014). As shown in Table
3.1, the TAC of EA-whey increased gradually with increasing the reaction time, except at
20 min when a decline was observed. The decrease observed can be resulted from the
polymerization of whey proteins as confirmed by SDS-PAGE pattern (Figure 3.10). The
β-LG in a more folded conformation, which would have prevented the hydrophobic amino
acid residues from the free radicals, hence, reduced the ORAC values.
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Figure 3.11: MALDI mass spectrum acquired in the linear mode: (a) untreated whey and (b) after 40 min of the EA. MALDI mass spectra acquired in the reflectron mode: (c) untreated sample and (d) after 40 min of EA.
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Table 3.1: Oxygen radical absorbance capacity of whey 7% (w/v) as a function EA time at 10°C and 400 mA. Electro-activated whey time (min) ORAC (µmol equivalent Trolox/L)
Control
10
20
30
40
50
60
1383.50 ± 1.82
1729.90 ± 3.70
1392.70 ± 1.88
3055.20 ± 5.05
4487.60 ± 4.37
4783.90 ± 1.67
7160.80 ± 3.59
Data represent the mean ± standard deviation of tree replicates. Linear effect from regression model, R2 = (0.899).
Conway et al. (2013) also found a low capacity of whey proteins concentrate to
scavenge the free radicals resulted from thermal denaturation, which induced it,
polymerization. Compared to the control untreated whey (none EA), the whey proteins
after EA showed an increase of the TAC up to 324% and 571% after 40 and 60 min of EA,
respectively. It is recognized that the antioxidant properties of whey proteins can be
enhanced through its hydrolysis to peptides with relatively low molecular mass (< 6 kDa)
as well as an aromatic and hydrophobic free amino acids (Pihlanto, 2006). Thus, the potent
antioxidant capacity of whey may be partly due to in situ protein hydrolysis that occurred
during EA, which could have produced peptides derived products with greater exposed
ionizable and hydrophobic amino acid residues leading to act with the free radicals. The
present results were in agreement with previous reports where the hydrolysates from the α-
La and β-LG proteins obtained by enzymatic hydrolysis showed strong antioxidant
properties (Hernández-Ledesma et al., 2005). Additionally to the presence of protein
hydrolysates, other compounds such as reducing sugars and polymer protein-sugars formed
under Maillard reaction were also reported to improve the antioxidant action of whey
proteins, especially in alkaline pH conditions (Lertittikul et al., 2007; Morales & Jiménez-
Pérez, 2001). The hydroxyl groups of reducing sugar and Maillard reaction products were
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suggested to play an important role through the donation of hydrogen to form stable radical
species (Wang et al., 2013b). Although, Hernandez-Ledesma (2005), found that free amino
acids such as tryptophan followed by methionine and cysteine liberating from whey protein
hydrolysis have a great antioxidant properties because of their capacities to donate
hydrogen proton to free radicals. In our present study, the content of free amino acids in
the cathodic compartment was also investigated. It was found that glutamic acid content
was the major amino acid present followed by proline with fewer amounts of the other
amino acid residues (results not shown). Glutamic acid was reported to act as antioxidant
compound because of its capacity to chelate metal ions (Kim et al., 2007a; Salami et al.,
2010). Indeed, the reducing of the availability of the other amino acid residues is due to
their reaction with the carbonyl function of reducing sugars present in the reaction medium
(Guan et al., 2010). Moreover, it is worthwhile noting that some authors investigated the
radical scavenging activity of MRPs from model and food systems and they concluded that
the heat treatment resulted in the reducing of the antioxidant properties (Vhangani & Van
Wyk, 2013). Therefore, it could be concluded that the enhancement of the antioxidant
functionality of whey after EA is the synergistic effect of its partial hydrolysis and
generation of mixtures of bioactive compounds with a high ability to scavenge free radicals.
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3.6 CONCLUSION
The main purpose of this work was to evaluate the dual efficiency of EA technology
to enhance the lactulose yield and antioxidant properties of cheese whey. The results
demonstrated the usefulness of EA process for efficient lactose in situ isomerization of
whey at low temperatures and short reaction time. In contrast to enzymatic and chemical
methods, EA has been proved a simple, clean, safe and green technology, which provides
a higher lactulose yield with high purity. Whey, a low value by-product of cheese industry
was proven an excellent raw material to produce lactulose rich product with high
antioxidant capacity. The presence of proteins did not show an adverse effect on the
isomerization of lactose to lactulose and provide a great antioxidant capacity. The
combination of whey and EA technology reveals a deeper valorization for whey
components such as potential lactulose and natural antioxidant. One of the interesting
opportunities was to use whey rich on lactulose in-situ with great antioxidant capacity as a
functional ingredient to improve growth and survival of probiotics incorporated in several
dairy products. Thus, due to its expected dual prebiotic and antioxidant properties, EA-
whey could be constituted a promising functional ingredient in several food formulations.
In the future study, research on the identification of the antioxidant compounds in whey
submitted to EA is needed to elucidate this characteristic.
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4. CHAPTER 4: Impact of electro-activation on the antioxidant
properties of defatted whey
This chapter is presented as an article entitled “Impact of electro-activation on the
improvement of antioxidant properties of whey”.
The authors are: Ourdia Kareb (Ph. D. candidate: planning and realization of the
experiments, results analysis and manuscript writing), Ahmed Gooma, Claude P.
Champagne (Thesis co-director: scientific supervision, correction and revision of the
manuscript), Julie Jean (Thesis co-director: scientific supervision, correction and revision
of the manuscript) and Mohammed Aïder (Thesis director: scientific supervision, article
correction and revision).
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4.1 RÉSUMÉ
L'activité antioxydante du lactosérum électro-activé (lactosérum-EA) a été étudiée.
Les effets de l'intensité du courant électrique (400, 500 ou 600 mA) et la concentration en
lactosérum [7, 14 ou 21% (p / v)] en fonction du temps de la réaction (0, 15, 30 ou 45 min)
sur les propriétés antioxydantes du lactosérum dégraissé ont été évaluées. L'intensité du
courant et le temps de réaction sont les paramètres les plus significatifs (P < 0.001). La
capacité d'absorption des radicaux oxygénés, de piégeage des radicaux 2,2-diphényl-1-
picrylhydrazyle et hydroxyles du lactosérum-EA ont significativement augmentés suite à
la formation de produits de réaction de Maillard (PRMs). En outre, le lactosérum EA
posséderait un pouvoir réducteur et une forte capacité de chélation du Fe2+. Les PRMs
dérivés du lactosérum-EA à 14% (p/v) posséderaient la plus forte activité de piégeage des
radicaux hydroxyles. Les résultats démontrent le potentiel de la technologie d'électro-
activation pour améliorer les propriétés fonctionnelles du lactosérum; le lactosérum-EA
pourrait être utilisé dans l'industrie alimentaire comme un antioxydant naturel pour
remplacer les antioxydants synthétiques.
Mots-clés : Lactosérum ; Électro-activation ; Antioxydants.
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4.2 ABSTRACT
Antioxidant activity of electro-activated whey (EA-whey) was investigated. The
effects of electric current intensity (400, 500 or 600 mA) and whey concentration [7, 14 or
28% (w/v)] as a function of reaction time (0, 15, 30 or 45 min) on antioxidant properties
of defatted whey were studied. The extent of current intensity and reaction times were the
most significant parameters (P < 0.001). Oxygen radical absorbance capacity and 2,2-
diphenyl-1-picrylhydrazyl as well as hydroxyl radical scavenging activities of the EA-
whey were significantly increased by the formation of Maillard reaction products (MRPs).
Moreover, EA-whey possessed reducing power and strong Fe2+ chelating ability. The
MRPs derived from the EA-whey with 14% had the highest hydroxyl radical scavenging
activity. The findings demonstrate the potential of electro-activation technology to enhance
the functional proprieties of whey; EA-whey could be used in the food industry as a natural
antioxidant ingredient to replace synthetic antioxidants.
Keywords: Whey; Electro-activation; Antioxidant.
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4.3 INTRODUCTION
Whey is a by-product of dairy industry that retains approximately 55% of milk
nutrients (Siso, 1996; Smithers, 2008). It contains valuable proteins such as β-lactoglobulin
(β-LG) and α-lactalbumin (α-La) that have been widely recognised for their nutritional and
physiological health-promoting benefits (Korhonen & Pihlanto, 2006; Mohanty et al.,
2016; Möller et al., 2008; Patel, 2015). In the food industry, whey proteins are increasingly
used in food formulations for their techno-functional and sensory properties (Chatterton et
al., 2006; Korhonen, 2006). Currently, with the rapid growth in the nutraceutical food
sector, whey ingredients have found new applications as sources of bioactive compounds
to develop novel functional foods. Several recent studies have aimed to improve the
functionality of whey by converting it to whey-derived products rich in bioactive
compounds, with a focus on antioxidant activity (Brandelli et al., 2015; Elias et al., 2008).
In this regard, antioxidant compounds derived from whey have potential as natural
functional ingredients (de Castro & Sato, 2014; de Oliveira et al., 2016; Peng et al., 2010).
Recently, the Maillard reaction (MR), known as non-enzymatic browning, has been
reported to be a potential way to promote the antioxidant properties of whey (Oliveira et
al., 2014). Maillard reaction products (MRPs) occur naturally between the available ɛ-
amino groups in the proteins and the reducing-end carbonyl group of sugars (Hodge & Rist,
1953). MRPs from whey exhibited high antioxidant activities in different model and real
food systems (Dong et al., 2012; Jiang & Brodkorb, 2012; Wang et al., 2013b). These
activities include scavenging of reactive oxygen species, inactivation of free radical chain,
inhibition of lipid peroxidation, and chelation of transition metals (de Castro & Sato, 2014;
Liu et al., 2014; Oh et al., 2013; Stanic-Vucinic et al., 2013; Wang et al., 2013b). Moreover,
MRPs in whey present other interesting properties such as prebiotic, antimicrobial,
antihypertensive and anti-allergenic activities (Chevalier et al., 2001c; Corzo-Martínez et
al., 2013; Hwang et al., 2011). However, the major challenge in this area remains the
development of efficient technologies that can control the extent of the MRPs and confer
the desired characteristics without the need for harsh processing (Chawla et al., 2009;
Jaeger et al., 2010; Jiang & Brodkorb, 2012).
In this context, we have previously reported electro-activation (EA) as an efficient
emerging technology for whey valorisation without need for fractionation. Moreover, EA
101
showed the potential to increase the antioxidant activity of whey (Kareb et al., 2016). It has
been hypothesised that the enhanced antioxidant functionality of the EA-whey resulted
from the synergistic effect of protein hydrolysis, and glycation of amino acids with
different peptides, proteins and reducing sugars under reducing conditions, that are
favourable for promoting MRPs. Moreover, a conversion of lactose to lactulose (a
prebiotic) was also achieved concurrently under the same conditions. Therefore, EA-whey
could be considered as a complex mixture of bioactive compounds, in which several
conjugates and distinct antioxidant mechanisms are possibly acting.
In this study, the potential of EA technology to enhance the antioxidant properties
of whey was investigated. Therefore, the main objectives of this work were: (i) to
investigate the influence of three experimental conditions (electric current intensity: 400,
500 or 600 mA; whey total solids concentration: 7, 14 or 21% (w/v), and reaction time: 0,
15, 30 or 45 min) on the antioxidant activity of EA-whey and (ii) to elucidate the
mechanisms of antioxidant activity of the EA-whey.
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4.4 MATERIALS AND METHODS
4.4.1 Chemicals, reagents, membranes and electrodes
Food-grade whey powder (lactose 75%, protein 12%, moisture content < 5%) was
purchased from Agropur Production (St-Hubert, Canada). Sodium sulphate (Na2SO4) was
purchased from Anachemia (Montreal, Canada). All reagents and chemicals used for this
study were of analytical grade and were purchased from Sigma Chemical Co. (Oakville,
Canada). Reagents were prepared freshly and stored under conditions preventing
deterioration. All the solutions were prepared in deionised water and were filtered through
0.45 μm Millipore filters before use. The anion (AM-40) and cation (CM-40) exchange
membranes used for whey EA treatment were purchased from the Schekina-Azot
(Shchekina, Russian Federation). Before use, the membranes were wiped with ethanol-
containing pads and dipped in a saturated solution of NaCl for 24h. After that, they were
transferred to another NaCl solution diluted twice compared with the first one for another
24h. The same procedure was repeated in a dilute NaCl solution for 24h. Thereafter, the
membranes were used in the EA reactor after rinsing with distilled water. The anodic
electrode was made of corrosion stable ruthenium-iridium-coated titanium; in the cathodic
compartment, the electrode was dimensionally stable type 304 stainless steel.
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Figure 4.1: Schematic representation of the reactor used for whey EA: AEM and CEM indicate the anion and cation exchange membrane, respectively.
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4.4.2 Electro-activation of whey induced Maillard reaction products
Maillard reaction products (MRPs) derived from EA-whey were prepared under
alkaline conditions following the procedure described in our previously study (Kareb et al.,
2016). Briefly, whey powder was freshly reconstituted to the required total solids
concentrations (7, 14 or 21%, w/v) in deionised water and stored overnight at 4°C. Equal
volume (125 mL) of each preparation was kept in the cathodic compartment, while the
central and anodic compartments were filled with Na2SO4 solution at 0.5 m concentration
to ensure the passage of the electric current. Solutions were submitted to different current
intensities (400, 500 or 600 mA) and were gently stirred by using an agitator (Model: RW
20 DS1, IKA, Cole-Parmer, Montreal, Canada) at 200 rpm min−1. The EA treatment of
whey was carried at ambient temperature. Samples from the cathodic compartment were
collected every 15 min during the duration of the EA process of 45 min, and stored at 4°C
until analyses. All experiments were done in triplicate. The pH was measured by using a
digital multimeter (Keithley, Inerga Series, Cleveland, OH, USA) and a pH meter, model
Oakton pH 700 (Eutech Instruments, Cole-Parmer, Montreal, Canada) (Figure 4.1).
4.4.3 Determination of antioxidant activity
The antioxidant potential of EA-whey was assessed by different assays based on
various mechanisms including its capacity to prevent and scavenge radical formation, as
well as its ability to chelate transition metals.
4.4.3.1 Determination of reducing power of EA-whey
The reducing power of EA-whey was measured as described by Mohammadian &
Madadlou (2016). with some modifications. Briefly, 1 mL sample (10 mg mL−1) was
mixed with 2.5 mL of 0.2 m sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium
ferricyanide (VWR, Montreal, Canada). The mixture was incubated in water bath at 50°C
for 20 min followed by addition of 2.5 mL of 10% trichloroacetic acid (Fisher-Canada,
Ottawa, Canada), after cooling to room temperature. Thereafter, the mixture was
centrifuged at 5000 × g for 10 min and 2.5 mL of supernatant was mixed with 2.5 mL
distilled water and 0.5 mL of 0.1% ferric chloride (VWR, Montreal Canada). After 10 min
of incubation, the absorbance of the reaction mixture was measured at 700 nm with a
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UNICO UV-2100 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
The increase in absorbance was used to measure the increased reducing power.
4.4.3.2 Determination of 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity of
EA-whey
The oxygen radical absorbance capacity (ORAC) of EA-whey was tested as
described previously by Ou et al. (2001). A mixture of 200 μL of fluorescein solution
(0.036 mg L−1), 75 μL of a 2,2′-azobis-2-aminopropane dihydrochloride solution (AAPH,
8.6 mg L−1) (Sigma–Aldrich, Saint-Louis, MO, USA) and 20 μL of the appropriate diluted
samples were added to a 96-well plate. The experiment was performed at 37°C and pH 7.0
and the fluorescence emission was immediately read at 520 nm (λex: 485 nm) with a BMG
Fluostar Galaxy microplate reader (Durham, NC, USA). Trolox (Sigma–Aldrich, Saint-
Louis, MO, USA) was used as standard (a water-soluble derivative of vitamin E) and
ORAC values of EA-whey were expressed as μmoL of Trolox equivalent L−1 of whey.
4.4.3.3 Determination of 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity of
EA-whey
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the EA-
whey was estimated according to the procedure of Wang et al. (2013b). An aliquot of MPRs
(250 μL) was added to 1 mL of 0.1 mm DPPH solution prepared freshly in methanol. The
reaction solution was then mixed vigorously and allowed to stand at room temperature for
30 min in the dark. The mixture was centrifuged at 5000 × g for 10 min. The absorbance
of the supernatant was measured at 517 nm using a UNICO UV-2100 spectrophotometer
(Agilent Technologies, Palo Alto, CA, USA). The percentage of DPPH radical scavenging
activity was calculated using the following equation:
Radical scavenging activity (%) = 100 − [(Asample − Acontrol) / Ablank × 100] (Eq.4.1)
The control was prepared in the same manner, except that distilled water was used
instead of the MRP sample. For the blank, the assay was conducted in the same manner,
except that ethanol was added instead of DPPH solution.
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4.4.3.4 Determination of hydroxyl radical scavenging activity of EA-whey
The MRPs from EA-whey was also tested for their ability to scavenge the hydroxyl
radicals as described by Chawla et al. (2009). An aliquot of 100 μL of EA-whey was mixed
with 1 mL phosphate buffer (0.1 M pH 7.4) containing 1 mM ferric chloride (FeCl3), 1 mM
EDTA, 1 mM ascorbic acid, 30 mM deoxyribose, and 20 mM H2O2. The mixture was
incubated at 37°C for 90 min. Immediately, after that, 2 mL of 2.8% (w/v) TCA and 2 mL
of 1% (w/v) TBA were added and mixed vigorously. The absorbance of the mixture was
measured at 536 nm. The percentage of hydroxyl radical scavenging was calculated using
the following equation (Chawla et al., 2009), where HRSA is the Hydroxyl radical
scavenging activity.
HRSA (%) = [(Asample − Acontrol) / (Ablank − Acontrol) × 100] (Eq. 4.2)
Control experiments were carried out by the addition the same solutions except for
the sample that was substituted with deionized water. The blank was prepared in the same
manner, except that deionized water was used instead of H2O2.
4.4.3.5 Determination of chelating activity on Fe2+ of EA-whey
The ability of EA-whey to chelate ferrous (Fe2+) was assessed as described by
(Kosseva et al., 2009) with some modifications. Briefly, 250 µL of sample (2.5 mg mL-1)
was first diluted with 2.5 mL of deionized water. Then, the solution was mixed with 50 µL
of 2 mM FeCl2 and 100 µL of 5 mM Ferrozine. After 10 min of incubation at room
temperature, the mixture was centrifuged at 5000 × g for 5 min and the absorbance at 562
nm was measured. Deionized water was used in the control instead of the sample. Results
were the average of three measurements and expressed as chelating activity (%) as follows:
Fe2+ chelating ability (%) = [(Acontrol − Asample) / Acontrol] × 100 (Eq. 4.3)
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4.4.4 Measurement of browning intensity and fluorescence of electro-activated
whey
The amount of intermediate and final MRPs formed during the EA treatment of
whey was monitored by absorbance measurement at 294 and 420 nm, respectively. The
fluorescence intensity was measured at an excitation wavelength of 365 nm and an
emission wavelength of 440 nm (Agilent Technologies, Palo Alto, CA, USA). Samples at
initial concentration of 10-µg mL-1 were diluted 10-fold and 200-fold with 100 mM
phosphate buffer at pH 7.4 for analysis.
4.4.5 Statistical analysis
Three independent experimental trials were conducted for all experiments. The
results were expressed as mean values ± standard deviation. Data were analyzed for
statistical differences between treatments by one-way analysis of variance (ANOVA) using
the LSD multiple comparisons procedure of SAS 9.2 (SAS Institute Inc., Cary, NC, USA)
and differences at p value <0.05 were considered statistically significant.
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4.5 RESULTS AND DISCUSSION
The antioxidant potencies of the electro-activated whey (EA-whey) products can be
affected by the EA reaction conditions, such as, pH, current intensity, reactant
concentration and reaction time. Indeed, formation of MRPs by EA is a complex process
in which hydrolysis of whey proteins is accompanied by a simultaneous conversion of
lactose to other reducing sugars, particularly lactulose (a prebiotic). Accordingly, to
accurately measure the antioxidant activities, it is important to use different assays based
on different mechanisms to confirm and complement the results and elucidate the
mechanism of antioxidant activity.
4.5.1 Oxygen radical absorbance capacity
The ORAC assay was used in this study to estimate the peroxyl radical inhibition
capacity of whey submitted to EA process. The ORAC assay is based on the inhibition of
peroxyl radical-induced oxidations, through the donation of hydrogen atom and breaking
the radical chains (Prior et al., 2005). Figure 4.2a, b and c present the change in ORAC of
EA-whey as function of reaction time. The ORAC values of all the samples increased
significantly (P < 0.001) as a function of reaction time. The highest increase of ORAC
values was derived from 7% EA-whey that reached 6.7-fold, followed by 14% EA-whey
that was 5.3-fold higher than the control reaction after 45 min of EA treatment at 600 mA.
The ORAC values derived from EA-Whey with 21% total solids concentration was only
slightly increased by 2.1-fold. Thus, increasing of whey solids concentration did not
improve its antioxidant capacity; this could be an indication that the EA had a characteristic
reaction rate, resulting in less change in the conformation of the whey proteins at higher
concentration. Previously, we reported the same tendency when the focus was based on the
conversion of whey lactose to lactulose under EA (Kareb et al., 2016). Regardless of
reactant concentration and reaction time, the current intensity had a pronounced effect on
the improvement of antioxidant capacity values of all simples (P < 0.001). Indeed, the
increase in ORAC values was accomplished with increasing alkalinity in the target
compartment of the reactor (P < 0.001).
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Figure 4.2: Effect of EA time and current intensities on the ORAC activity of whey at different concentrations: (a) at 7%, (b) at 14% and (c) at 21% (w/v). Error bars show standard deviation (n = 3).
110
Consequently, more brown compounds were formed from the pools of products
generated in the reactor, as detected by browning intensity measurement, expressed as the
increased absorbance of the EA-whey (Figure 4.7b and c). These results agreed with the
results of Wang et al. (2013b) who reported improved antioxidant activities of MRPs
through the interactions of whey protein isolates and sugars in alkaline pH conditions.
Moreover, the present results were also in agreement with a previous study in which whey
protein hydrolysates prepared at different alkaline conditions showed a strongest ORAC
values at higher pH conditions (Athira et al., 2015). The positive effect of EA treatment on
ORAC values of whey could result from the simultaneous molecular rearrangements and
glycation process between compounds generated in situ, which lead to the formation of
amphoteric lower molecular compounds with more active and available hydrogen ions and
carboxylic acid groups. EA is a complex process, which could lead to peptide and free
amino acids generation, peptide cross-linking, amino acid loss and polymerisation. Thus,
the results indicated that EA-whey could be considered as a good chain-breaking
antioxidant.
4.5.2 Reducing power
The reducing power is the main antioxidant assay used to determine the ability of
antioxidant compounds to donate electrons and to reduce the ferric chloride ferricyanide
complex to its ferrous form (Canabady-Rochelle et al., 2015). This assay particularly
measures the reducing activity of hydroxyl groups from compounds (Gu et al., 2010;
Vhangani & Van Wyk, 2013; Yoshimura et al., 1997). A significant increase in the
reducing power values of whey subjected to EA was found as a function of combined
current intensity and reaction time as seen in Figure 4.3a, b and c (P < 0.001). However,
it was also shown that there was a decrease of reducing power values under some
conditions. The reducing power values were not improved with increasing the
concentration of whey total solids. The observed trends could be explained by the fact that
the functional groups are not available, due to the change in the conformation and
polymerisation of whey proteins. The EA of whey could have resulted in formation of
MRPs that improve the reducing power through the availability of hydroxyl and pyrrole
groups of advanced MRPs (Yanagimoto et al., 2002).
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Figure 4.3: Effect of EA time and current intensities on the reducing power activity of whey at different concentrations: (a) at 7%, (b) at 14% and (c) at 21% (w/v). Error bars show standard deviation (n = 3).
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The reductones, intermediate compounds of MRPs-whey, were reported to exhibit
breaking at radical chains by the donation of hydrogen atoms (Rao et al., 2011). Moreover,
the hydroxyl groups of MRPs played a relevant role in reducing activity. In the study of
Yoshimura et al. (1997), the bromination of the hydroxyl groups decreased the reducing
power of glucose–glycine model system. Thus, the extent of the reducing power of EA-
whey could be explained to not only the formation of MRPs-whey, but also the distinct
role of EA processing in the formation of reducing conditions due the dynamic electrolysis
of water and production of hydroxyl groups. Therefore, the mechanisms by which whey
protein/peptides and Maillard conjugates exert their reducing activity is complex and will
probably vary depending on the pH, reaction time, and the array of individual antioxidant
compounds present in the end-product.
4.5.3 Metal-chelating activity
Iron chelating ability is argued as the best assay to determine the ability of
antioxidant to chelate metals in food systems (Morales et al., 2005). Transient iron
chelation acts indirectly as an antioxidant mechanism since the Fenton reaction, responsible
for hydroxyl radical formation and inhibiting subsequent radical chain reaction (Jing &
Kitts, 2004). Figure 4.4a, b and c shows the Fe2+ chelating ability of aqueous EA-whey as
a function of the effect of whey concentration, current intensity and reaction time. As
expected, all the three tested concentrations of EA-Whey had a significantly higher ferrous
iron chelating activity when compared with the none EA-whey (P < 0.001) due to their
content of the protein, peptides and MRPs. With EA-whey having 7% total solids
concentration, the Fe2+ chelating activity increased sharply during the first 15 min
(P < 0.001) and reached about 70%, followed by a slow decrease over the remaining EA
reaction time. Similar results were observed with the EA-whey having 14% total solids
concentration with progressive chelating ability until the 30 min of EA treatment.
However, a linear relationship was found between the Fe2+ chelating activity and the EA-
whey having 21% total solids concentration as function of reaction time (P < 0.001). Also,
the current intensity showed a significant effect (P < 0.001) on increasing the chelating
irons. During the EA treatment, whey compounds undergo two chemical changes; on one
side, proteins degrade to smaller fractions and free amino acids.
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Figure 4.4: Effect of EA time and current intensities on the iron chelating ability of whey at different concentrations: (a) at 7%, (b) at 14% and (c) at 21% (w/v). Error bars show standard deviation (n = 3).
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On the other hand, these compounds can crosslink directly with the reducing sugars
to form MRPs-whey. Some authors have associated the metal chelating ability of whey
proteins with the derived peptides with lower molecular mass and free amino acids (Cruz-
Huerta et al., 2016; Kim et al., 2007b; Vavrusova et al., 2015). On the other hand, some
studies also reported that the peptides of higher molecular mass were better able to chelate
irons (Gu et al., 2010; Yoshimura et al., 1997). It is also interesting to notice that Liu et al.
(2012) reported a more effective Fe2+ chelating activity from the MRPs produced from soy
than soy protein hydrolysates. Furthermore, the negative charge, location of chelating site
and distribution of carboxyl groups of the amino acids seem to be directly related to the
extent of iron chelation ability (Caetano-Silva et al., 2015; Canabady-Rochelle et al., 2015;
Chaud et al., 2002). Besides the formation of versatile compounds leading to chelation of
iron, the EA processing induces dynamic water electrolysis in the cathodic compartment,
resulting in an increase of the negative charge concentration through the accumulation of
hydroxyl groups, which could play a role of proton acceptors, then chelate iron.
4.5.4 2,2-Diphenyl-1-picrylhydrazyl scavenging activity
The changes in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
activity of EA-whey at different concentrations are shown in Figure 4.5a, b and c. The
DPPH radical scavenging activity increased sharply in EA-whey of 7 and 14% total solids
concentration to reach approximately 60% after 15 min of EA treatment at 600 mA
(P < 0.001). At 45 min of EA, the radical scavenging activity reached 71% and 67% values
for EA-whey with 7 and 14% total solids, respectively. The DPPH radical scavenging
activity was significantly higher under high current intensity as 600 mA when compared
with the other current fields used (P < 0.001). Upon concentration of the whey to 21% total
solids, the DPPH radical scavenging activity increased at a slower rate compared with the
other two concentrations. These results are in agreement with previous studies in which
MRPs derived from sugar/protein interactions in aqueous model systems possessed a good
DPPH radical scavenging activity (Jiang & Brodkorb, 2012; Jiang et al., 2013; Stanic-
Vucinic et al., 2013). MRPs obtained by gamma radiation of a whey model system also
exhibited a strong DPPH scavenging activity (Chawla et al., 2009).
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Figure 4.5: Effect of EA time and current intensities on the DDPH radical scavenging activity of whey at different concentrations; (a) at 7%, (b) at 14% and (c) at 21% (w/v). Error bars show standard deviation (n = 3).
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In the model systems, only one reducing sugar and a protein were used. In the EA
reactor, the reactants exceed those of the model systems, resulting in the generation of
heterogeneous compounds with different structures, degrees of polymerisation and
antioxidant potencies. Thus, EA treatment generates exposed amino acids that donate
hydrogen and could react with the unstable DDPH, subsequently, converting them to more
stable DDPH-H molecules and terminating the oxidation chain reaction. Hernández-
Ledesma et al. (2005) showed that the peptides generated from the hydrolysis of β-
lactoglobulin with Corolase PP also exhibited antiradical activity. On the other hand, these
results were in agreement with those of Wang et al. (2013b), who also found that MRPs
derived from whey protein isolate conjugated to different sugars had DPPH radical
scavenging activity. During the MR, the intermediate and final brown polymer products
can act as hydrogen donors and contribute to the DPPH radical scavenging activity (Chawla
et al., 2009; Wang et al., 2011). Moreover, Benjakul et al. (2005) noted that sugar
caramelisation could also participate in the antiradical activity. The type of sugars reacted
with whey protein is another main factor, determining the positive antiradical activity of
the final products (Chevalier et al., 2001c). They reported that lactose is more reactive than
lactulose for Maillard conjugation with both whey protein isolates and its hydrolysates.
Our results indicated that the DPPH radical scavenging activity correlated well with ORAC
of EA-whey. This property confirms the primary antioxidant activity through the donation
of hydrogen atoms. Thus, EA-whey may have a potential use to prevent lipid oxidation in
dairy products.
4.5.5 Hydroxyl radical scavenging activity
Among the reactive oxygen species, the hydroxyl radicals (.OH) are the most known
toxic radical, as they can attack and damage biological macromolecules. Furthermore, they
can also accelerate the oxidation of lipid in foods. In the presence of hydrogen peroxide,
hydroxyl radicals are formed through the Fenton-type reaction (Özyürek et al., 2008).
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Figure 4.6: Effect of EA time and current intensities on the hydroxyl radical scavenging activity of whey at different concentrations: (a) at 7%, (b) at 14% and (c) at 21% (w/v). Error bars show standard deviation (n = 3).
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The deoxyribose degradation assay has been widely used to evaluate the hydroxyl
radical scavenging ability which produces malonaldehyde and then reacts with
thiobarbituric acid (TBA) to form a pink chromogen with maximum absorbance at λ532nm
(Halliwell et al., 1987). In the presence of antioxidant compounds, deoxyribose competes
with deoxyribose to react and scavenge the .OH radicals preventing the formation of
malonaldehyde. Hydroxyl radical scavenging activities of MRPs produced from EA of
whey at different concentrations are shown in Figure 4.6a, b and c. The results showed
that the EA treatment exerted a positive effect to scavenge the hydroxyl radicals for the
whey tested under certain conditions (p > 0.001). Regarding the reaction time, two
noticeable changes were observed. The first is a marked increase in .OH inhibition rate
after 30 min of EA treatment. The second is a decrease in .OH inhibition rate in the second
part of the process occurred, particularly at 600 mA. Also, the MRPs derived from EA-
whey with 7% total solids concentration yielded the most active .OH radical scavenging,
followed by those derived from EA-whey at 14% total solids concentration, with 70% and
60% scavenging ability, respectively. MRPs derived from EA-whey at 21% total solids
displayed a gradual and slower .OH inhibition rate as a function of reaction time, compared
to other whey concentrations. These results are in agreement with earlier reports that
reported hydroxyl radical inhibition in whey after irradiation or in a heated model systems
composed of glucose, fructose, and ribose/lysine due to the formation of MRPs (Chawla et
al., 2009; Wijewickreme et al., 1999). The results shown indicate that the antioxidant
proprieties of EA-whey increased with increasing reaction time. One exception was the
hydroxyl radical scavenging activity, which significantly decreased after 45 min in the case
of EA-whey at 600 mA. These findings are in agreement with those of (Yoshimura et al.,
1997) who observed that .OH inhibition rate of MRPs produced from a glucose-glycine
mixture decreased with increasing heating time. This observation can be possibly explained
by depression of Fenton reaction through the reduction of Fe+3 in favour of Fe+2 ions by the
reducing compounds formed under prolonged EA reaction time. Moreover, it can be
assumed that MRPs were unable to degrade the excessive hydrogen peroxide in the
presence of transition metal Fe+2 which can contribute to undesired Fenton reaction
products through the propagation of .OH and, since the concentration of reactive
antioxidant compounds was lowered, it will not able to scavenge the .OH generated. The
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results showed that the EA of whey acted simultaneously with different mechanisms of
action and could be potentially used to retard lipid peroxidation in food systems.
4.5.6 Effect of pH on brown colour development of electro-activated whey
We determined the impact of the EA treatment on the physiochemical and the
structural changes of whey solutions at different feed concentrations after having optimised
the current intensity at 600 mA. The existence of a relationship among the pH of the
medium, the development of brown colour, the molecular weight distribution, and the
extent of the antioxidant activities as examined. The changes in the pH of MRPs-derived
from EA-whey at different concentrations as a function of reaction time was shown in
Figure 4.7a. The pH of all systems increased significantly from their initial pH of 5.8 as
the EA reaction time increased up to 11.5 ± 0.25 at the end of the processing (P < 0.001).
Among the concentrations tested, 21% EA-whey showed a slower increase of the pH as a
function of reaction time by the action of buffering capacity of whey proteins (Paseephol
et al., 2008). According to these results, it is clear that the antioxidant proprieties of whey
were promoted when the alkaline conditions were established in the cathodic compartment.
Our results are in agreement with previous studies in which the higher pH influenced
significantly the glycation and the polymerisation of whey protein with reducing sugars,
forming compounds with stronger antioxidant characteristics (Chawla et al., 2009; Wang
et al., 2013b). In the MR, the open chain form of the reducing sugars and the charged amino
acid groups are quickly formed at alkaline conditions (Martins et al., 2000).
Some authors have pointed out that the pH of protein-sugar models decreased with
the prolongation of combined heating temperature-reaction time due the degradation of the
intermediate MRPs to organic acids (Benjakul et al., 2005; Gu et al., 2010; Jiang &
Brodkorb, 2012; Vhangani & Van Wyk, 2013).
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Figure 4.7: Changes in pH (a), absorbance at 294 nm (b), browning intensity at 420 nm (c), and fluorescence intensity (d) of whey at different concentrations (diamonds, 7%; squares, 14%; triangles, 21%) during electro-activation at 60 mA for up to 45 min; error bars show standard deviation (n = 3).
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Contrary to these studies, the enhancement of antioxidant capacities of EA-whey
was accomplished without a decrease of the pH of the solution and production of organic
acids. It is noticeable that a simultaneous conversion of lactose to lactulose is also promoted
at alkaline conditions (Kareb et al., 2016). Thus, this fact opens promising possibilities for
use of EA-whey as an antioxidant and prebiotic. In general, the development of brown
colour is related to the enhancement of antioxidant properties of protein-sugar systems in
which the non-browning enzymatic occurred (Anese & Fogliano, 2001; Oyaizu, 1986).
Depending on the reactivity of reactants and the reaction rate, MR is characterised by the
formation of intermediate and advanced brown compounds which can be measured by the
absorbance at 294 and 420 nm (Figure 4.7b, c and d), as well as fluorescent compounds
(Stanic-Vucinic et al., 2013). In the presence of reducing sugars, the levels of intermediate
MRPs that contribute to the formation of brown products increased with increasing reaction
time. The reaction rate was inversely proportional to whey solids concentration (P < 0.001).
A parallel increase in the brown colour intensity measurement was also observed as a
function of reaction time (P < 0.001).
The formation of MRPs derived from EA-whey with 7% total solids was higher
compared with EA-whey with 14 and 21% total solids, respectively. It is also noticeable
that the formation of intermediate compounds was higher than the formation of the brown
compounds, which revealed the dominance of early stage of MR under the EA treatment.
This provides an interesting approach for food industry, when the extent of browning is not
accepted for some foods such as dairy products (Morales & Jiménez-Pérez, 2001).
Development of MR has been accomplished with the formation of fluorescent compounds
(Jing & Kitts, 2002). The fluorescence intensity of EA-whey in the three tested
concentrations was promoted with prolonged reaction time (P < 0.001). Moreover, the
fluorescence of EA-whey at 7% total solids concentration was sharply increased within
30 min followed by a slight decrease until the end of reaction. Such decrease may be due
to the conversion of intermediate products to advanced MRPs. Under the EA reactor, the
conversion of lactose to a mixture of sugars was found to contribute to the browning
intensity through caramelisation at less significant level. It is worth noting that MRPs
coming from EA-whey at 14% total solids concentration displayed the more interesting
characteristics for an application in dairy industry due it higher hydroxyl radical scavenging
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activity and less developed of browning colour. Techno-functional properties of EA-whey
are worthy to be considered in broad-spectrum application, especially in dairy industry.
4.6 CONCLUSION
The results presented herein are the first that show the potential of electro-activation
as a non-thermal technology to enhance the formation of MRPs with potential antioxidant
activities. There was a marked increase in the reducing power of EA-whey that correlated
well with high ORAC and DPPH radical scavenging activities. EA-whey possessed
potential iron chelating ability, with some interference with its hydroxyl radical scavenging
activity to some extent. Overall, EA-whey could be suitable as a natural and effective
antioxidant for preventing or terminating the oxidation reaction chain of lipid peroxidation
in food processing. The increase of antioxidant capacity of EA was greatly affected by the
intrinsic conditions occurring in the reactor such as pH and the accumulation of negative
charges. To assist the development of EA technology in the dairy industry, future work on
more comprehensive models that could reflect the bioactive peptides profile of whey
protein during the EA is worth carrying out.
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5. CHAPTER 5: Electro-activation of sweet defatted whey: Impact on
the induced Maillard reaction products and bioactive peptides
This chapter is presented as an article entitled “Electro-activation of sweet defatted
whey: Impact on the induced Maillard reaction products and bioactive peptides”.
The authors are: Ourdia Kareb (Ph. D. candidate: planning and realization of the
experiments, results analysis and manuscript writing), Ahmed Gooma, Claude P.
Champagne (Thesis co-director: scientific supervision, correction and revision of the
manuscript), Julie Jean (Thesis co-director: scientific supervision, correction and revision
of the manuscript) and Mohammed Aïder (Thesis director: scientific supervision, article
correction and revision).
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5.1 RÉSUMÉ
L'électro-activation (EA) a été utilisée pour conférer de la valeur ajoutée au
lactosérum doux dégraissé. Cette étude visait à examiner et à caractériser les composés
bioactifs formés dans différentes conditions d'EA par approches moléculaire et
protéomique. Les effets de l'intensité du courant électrique (400, 500 ou 600 mA) et de la
concentration du lactosérum [7, 14 ou 28% (p/v)] en fonction du temps d'électro-activation
(0, 15, 30 ou 45 min) ont été évalués. Les variables dépendantes ciblées étaient la formation
des produits de la réaction de Maillard (PRMs) et la composition de l’hydrolysat des
protéines du lactosérum EA. Les profils SDS-PAGE indiquaient la formation d'hydrolysats
et de composés glycosylés avec différent poids moléculaire. L’analyse FTIR a indiqué la
prédominance de PRM intermédiaires, tels que les bases de Schiff. L'analyse LC-MS/MS
a permis l’identification de nombreuses séquences de peptides bioactifs multifonctionnels
résultant de l’hydrolyse des protéines du lactosérum.
Mots-clés : Lactosérum; Électro-activation; Peptides bioactifs; Réaction de Maillard;
Glycation
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5.2 ABSTRACT
Electro-activation (EA) was used to add value to sweet defatted whey. This study
aimed to investigate and to characterize the bioactive compounds formed under different
EA conditions by molecular and proteomic approaches. The effects of electric current
intensity (400, 500 or 600 mA) and whey concentration [7, 14 or 28% (w/v)] as a function
of the EA time (0, 15, 30 or 45 min) were evaluated. The targeted dependent variables were
the formation of Maillard reaction products (MRPs) and the whey protein hydrolysates.
SDS–PAGE analyses indicated the formation of hydrolysates and glycated compounds
with different molecular weight distributions. FTIR analysis indicated the predominance
of intermediate MRPs, such as the Schiff base compounds. LC-MS/MS proteomics
analysis showed the production of multi-functional bioactive peptides due to the hydrolysis
of whey proteins.
Keywords: Whey; Electro-activation; Bioactive peptides; Maillard reaction; Glycation
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5.3 INTRODUCTION
Whey is a dairy industry co-product that contains approximately 55% of milk
components (Smithers, 2008). It is rich in proteins such as β-lactoglobulin (β-LG) and α-
lactalbumin (α-LA) with high nutritional and physiological health-promoting effects
(Korhonen & Pihlanto, 2006; Mohanty et al., 2016; Möller et al., 2008; Patel, 2015).
Moreover, whey-derived ingredients are increasingly used in different food formulations
due to their technological, functional and organoleptic characteristics (Chatterton et al.,
2006). Furthermore, the growth of foods with nutraceutical properties involves intensive
use of whey-derived ingredients for their possible bioactivity and health promoting
benefits. To achieve this objective, different approaches were proposed to improve the
functionality of whey by producing whey-derived products containing bioactive
compounds with special focus on antioxidant activity (Brandelli et al., 2015; de Castro &
Sato, 2014; Oliveira et al., 2014; Peng et al., 2010). Enzymatic treatments are generally
used to improve the bioactive potential of whey through the formation of active peptides
(Hernández-Ledesma et al., 2005; Peng et al., 2010; Pihlanto, 2006; Pihlanto & Korhonen,
2015). Indeed, whey protein hydrolysates demonstrated high efficacy as natural
antioxidants by preventing lipid oxidation in different foods. Several antioxidant
hydrolysates from β-LG and α-La with high radical-scavenging activity were isolated
(Hernández-Ledesma et al., 2005). Unfortunately, this strategy suffered from serious
drawbacks, such as lack of specificity, high process cost, low catalysis efficiency and the
need for using huge volumes of chemicals and thermal treatments to stop the hydrolysis
process (Contreras et al., 2011; de Castro & Sato, 2014; Kosseva et al., 2009; Power et al.,
2013).
Recently, the Maillard reaction (MR), also known as non-enzymatic browning, was
pursued as a promising mean of enhancing the antioxidant activity of whey (Oliveira et al.,
2014). Maillard reaction products (MRPs) occur between available ɛ-amino groups in the
proteins, peptides, free amino acids and the reducing-end carbonyl group of sugars. This
reaction is highly enhanced by heating (Arena et al., 2017). Different studies have shown
that MRPs from whey exhibit high antioxidant activities in different model and real food
systems (Dong et al., 2012; Jiang & Brodkorb, 2012; Wang et al., 2013b). Various
mechanistic approaches have been suggested to explain the observed antioxidant activities
127
of MRPs-whey. Among these mechanisms, scavenging of reactive oxygen species,
inactivation of free radical chain, inhibition of lipid peroxidation and chelation of transition
metals were suggested (de Castro & Sato, 2014; Liu et al., 2014; Oh et al., 2013; Stanic-
Vucinic et al., 2013; Wang et al., 2013b). Moreover, MRPs-whey could present other
interesting properties such as a prebiotic effect, antimicrobial activity and antihypertensive
and anti-allergenic activities (Chevalier et al., 2001a; Corzo-Martínez et al., 2013; Hwang
et al., 2011). Nevertheless, the major challenge in this area remains the need to develop
efficient technologies that can control the extent of the MRPs and confer the desired end-
product characteristics without the need of energy-intensive high thermal and lengthy
processing conditions (Chawla et al., 2009; Jaeger et al., 2010; Jiang & Brodkorb, 2012).
In this context, we have previously reported the electro-activation (EA) as an
emerging technology to promote the antioxidant activity of water-soluble whey under wet
conditions (Kareb et al., 2016). The enhancement of the antioxidant properties of electro-
activated whey (EA-whey) resulted from the combined effect of hydrolysed
proteins/peptides and glycation of amino group containing molecules of whey with
reducing sugars under alkaline conditions, which are favorable for the formation of
Maillard reactions products (MRPs). Interestingly, a conversion of lactose to lactulose (a
prebiotic) was also achieved concurrently under the same conditions. Therefore, EA-whey
could be considered as a complex mixture of bioactive compounds in which several
conjugates and distinct antioxidant mechanisms are potentially acting. Furthermore, the
antioxidant activity of EA-whey can be influenced by many factors, such as time, pH,
concentration and type of reactants, in addition to the intrinsic reaction conditions related
to the EA processing.
In this study, we investigated the potential of the electro-activation (EA) as a
promising efficient technology to enhance the bioactive properties of whey under
controlled conditions. The main objective of this work was to investigate the effect of the
EA process under different experimental conditions (electric current intensity, whey total
solids concentration, and reaction time) on the induced Maillard reaction products (MRPs)
and bioactive peptides formation.
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5.4 MATERIALS AND METHODS
5.4.1 Chemicals and reagents
Food-grade whey powder (lactose 75%, proteins 12% and moisture content less
than 5%) was purchased from Agropur Cooperative (St-Hubert, Quebec, Canada). Sodium
sulfate (Na2SO4) was purchased from Anachemia (Montreal, Quebec, Canada). All
reagents and chemicals used for this study were of analytical grade and were purchased
from Sigma Chemical Co. (Oakville, Ontario, Canada). Reagents were freshly prepared
and stored under adequate conditions to prevent deterioration. All the solutions were
prepared in deionized water and were filtered through 0.45 µm Millipore filters before use.
5.4.2 Maillard reaction products (MRPs) generation from EA-whey
MRPs derived from EA-whey were prepared under alkaline conditions following a
previously described procedure (Kareb et al., 2016) (Figure 5.1). Briefly, whey powder
was freshly reconstituted to the required concentrations (7, 14 or 21% (w/v)) in deionized
water and stored overnight at 4°C. An equal volume (125 mL) of each preparation was kept
in the cathodic compartment, while the central and anodic compartments were filled with
Na2SO4 solution at 0.5 M concentration to ensure the passage of current, as shown in
Figure 5.1. Solutions were submitted to different current intensities (400, 500 or 600 mA)
and were gently stirred at 200 rpm min-1, corresponding to 0.54 RCF, using an agitator
Model: RW20DS1 (Coleparmer Canada, Montreal, Canada). Samples from the cathodic
compartment were collected at 15 min intervals during the 45 min EA process. The samples
were then stored at 4°C. All experiments were performed in triplicate. The pH was
measured by a digital multimeter (Keithley, Inerga Series, Cleveland, OH) equipped with
a pH-probe (model Oakton pH 700, Eutech Instruments, Cole-Parmer, Montreal, Canada).
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Figure 5.1: Schematic representation of the experimental set-up for the EA of whey.
5.4.3 Determination of free and sugar-bound amino acids
Free amino acid groups and glycation degree of the conjugates derived from EA-
whey after 45 min of treatment were analyzed using an EZ:faast kit according to the
manufacturer’s instructions (Torrance, CA, USA). The derivatized free amino acids were
separated, identified and quantified by gas chromatography (GC) equipped with an AOC-
20i auto-injector and a FID 2010 Plus (Fisons Instruments Plus) that is connected to GC
solution software (Mandel Scientific Inc., Guelph, ON, Canada). The data corresponded to
the concentration of free amino acid (µmol). The degree of glycation of EA-whey was
expressed as the glycation ratio of EA samples to the native sample (C0).
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5.4.4 Determination of reducing sugars in MRPs-whey
Reducing sugars such as lactose, lactulose, glucose, galactose and fructose in whey
subjected to EA for 45 min were quantified using an HPLC Agilent Technology (Millipore
Corp., Milford, MA, USA). A column 300×6.5 mm Carbohydrate Analysis (Waters Co.,
Milford, MA USA) and a refractive index detector (Waters, Model 410) were used. The
supernatant was diluted 100 times, and then, a 25-μL aliquot was injected. The running
time was set at 30 min per sample. Once the separation of sugars was achieved, peaks were
identified by comparing their retention time to that of the standard sugars.
5.4.5 Structure characterization of MRPs-whey induced by electro-activation
5.4.5.1 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with or
without β-mercaptoethanol (β-ME) of EA-whey proteins was performed according to a
previously described method (Laemmli, 1970). Briefly, the 1% (w/v) protein solutions
were diluted in a 1:1 ratio with an SDS-PAGE Laemmli sample buffer and then heated at
95°C for 5 min. A 10-µL aliquot of each sample was loaded into a 4-20% gradient of
polyacrylamide gel. The separation was performed at a constant voltage of 30 mA. After
the run, the protein bands were stained by Coomassie Brilliant Blue R-250 (0.2%) in 40%
methanol and 10% acetic acid for 2 h and de-stained overnight with 10% methanol and
10% glacial acetic acid.
5.4.5.2 FT-IR measurements
To monitor the structural changes of whey compounds and the formation of MRPs
after 45 min of EA treatment, Fourier transform infrared (FTIR) spectroscopy analysis was
performed according to a previously described method. The infrared spectra were measured
at room temperature with a Magna 560 Nicolet spectrometer with a liquid nitrogen cooled
narrow-band mercury cadmium telluride (MCT) detector and a Golden Gate diamond ATR
accessory (Specac Ltd., London, UK). Each spectrum was obtained from 256 scans at a
resolution of 4 cm−1 with a Happ-Genzel apodization. The scale of each spectrum was
normalized to minimize differences due to sample concentration, and the baseline was
corrected (Gomaa et al., 2013).
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5.4.5.3 LC/MS-MS analysis
The sample preparation and mass spectrometry experiments were performed by the
Proteomics platform of the Centre de Génomique de Quebec, CHU de Quebec-Laval
University.
5.4.5.3.1 Samples preparation
Samples without digestion: Proteins (100 µg) from control and 7% EA-whey were
washed with Amicon 10 kDa column. Protein fractions less than 10 kDa were acidified
with 5% formic acid (FA) and purified on the Hydrophilic-Lipophilic Balance (HLB)
column (Waters). After elution, samples were vacuum-dried and re-suspended in 0.1%
(v/v) formic acid prior to the analysis by mass spectrometry.
Samples digested with trypsin: A total of 25 µg of 7% EA-whey was solubilized in 25 µl
of 50 mM ammonium bicarbonate-1% sodium deoxycholate and heated at 95°C for 5 min.
Samples were reduced with DTT (1 µg) at 37°C for 30 min and alkylated iodoacetamide
(5 µg) at 37°C for 20 min. Trypsin (1 µg) was then added, and samples were incubated
overnight at 37°C. Trypsin hydrolysis was stopped by acidification with 3% acetonitrile-
1% TFA-0.5% acetic acid. Peptides were purified on Stage-tip (C18), vacuum dried and
then re-suspended in 0.1% formic acid for analysis by mass spectrometry.
5.4.5.3.2 Mass spectrometry
Peptide samples were separated by online reversed-phase (RP) nanoscale capillary
liquid chromatography (nanoLC) and analyzed by electrospray mass spectrometry (ES
MS/MS). The experiments were performed with a Ekspert NanoLC425 (Eksigent) coupled
to a 5600+ mass spectrometer (AB Sciex, Framingham, MA, USA) and equipped with a
nanoelectrospray ion source. Peptide separation took place on a nano cHiPLC column 3
µm, 120A C18, 15 cm × 0.075 mm internal diameter. Peptides were eluted with a linear
gradient from 5-35% solvent B (acetonitrile, 0.1% formic acid) for 35 minutes at 300
mL/min. Mass spectra were acquired by a data-dependent acquisition mode using Analyst
software version 1.7. Each full-scan mass spectrum (400 to 1250 m/z) was followed by
132
collision-induced dissociation of the twenty most intense ions. Dynamic exclusion was set
for a period of 12 s and a tolerance of 100 ppm.
5.4.5.3.3 Database searching
All MS/MS peak lists (MGF files) were generated using Protein Pilot (AB Sciex,
Framingham, MA, USA, Version 5.0) with the paragon algorithm. MGF sample files were
then analyzed using Mascot (Matrix Science, London, UK; version 2.5.1). Mascot was
designed to search the complete proteome Bos taurus database (release December 2014
32422 entries) assuming the tryptic digestion (or no enzyme for the non-digested trypsin).
Mascot was searched with a fragment ion mass tolerance of 0.10 Da and a parent ion
tolerance of 0.10 Da. Oxidation of methionine for the trypsin digested sample was specified
as a variable modification and carbamidomethylation (C) as a fixed modification. Two
missed cleavages were allowed.
5.4.5.3.4 Criteria for protein identification
Scaffold (version Scaffold 4.2.0, Proteome Software Inc., Portland, OR, USA) was
used to validate the MS/MS based peptide and protein identifications. Peptide
identifications were accepted if they could be established at greater than 85% probability
by the Peptide Prophet algorithm with Scaffold delta-mass correction. Similarly, protein
identifications were accepted if they could be established at greater than 85% probability
and contained at least one identified peptide. Protein probabilities were assigned by the
Protein Prophet algorithm. Proteins that contained similar peptides and thus could not be
differentiated based on MS/MS analysis were grouped (Keller et al., 2002).
5.5 Statistical analysis
Three independent experimental trials were conducted for all experiments. The
results were expressed as the mean values ± standard deviation. Data were analyzed for
significant differences between treatments by one-way analysis of variance (ANOVA)
using the LSD multiple comparisons procedure of SAS 9.2 (SAS Institute Inc., Cary, NC,
USA). Statistically significant differences were considered at p value < 0.05.
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5.6 RESULTS AND DISCUSSION
5.6.1 Changes in free amino acids and reducing sugars
As seen in Table 5.1, a significant decrease in free amino acid content of whey was
observed with extending the EA time to 45 min for all concentrations. A total of 66.9% of
the free amino acids were lost in 7% EA-whey solution compared to 40.8% and 48.2% for
14% and 21% EA-whey, respectively. This reduction could be explained by the attachment
of free amino acids to the reducing compound. From the above results, the amino acids
could be classified into three groups according to their relative reactivity rate and
availability. The first group corresponded to the highest reactive amino acids, including
alanine, leucine, isoleucine, glycine, histidine and methionine. The second group included
proline, glutamate and glutamic acid, which exhibited intermediate reactivity.
The third group included aspartic acid and tyrosine. Lysine was completely lost in
the 7% EA-whey solution in contrast to the significant 2.5- and 2-fold increase for 14%
and 21% EA-whey solutions, respectively. During the EA, the loss of amino groups may
be related to the formation of a Schiff-base and the rapid transformation of the
intermediates to brown compounds, while the release of free amino groups was attributed
to the extensive hydrolysis of whey proteins. The differing reactivity rate of reducing
sugars is another parameter that affects the extent of the MR formation (Morales &
Jiménez-Pérez, 2001). For instance, the glycation reaction rate of the reducing sugars
depends on the percentage of the acyclic formed and the electrophilicity of the carbonyl
groups (Naranjo et al., 1998). Under the same alkaline conditions, the EA of whey leads to
the isomerization of lactose to lactulose via the Lobry de Bruyn-van Ekenstein
transformation with subsequent degradation to galactose by-product (Kareb et al., 2016).
Taking into consideration the reactivity of sugars, monosaccharide has a higher reactivity
than disaccharides with respect to the extent of MR glycation and the development of
browning color (Laroque et al., 2008). As shown in Figure 5.2, lactose, lactulose or
galactose amounts in EA-whey varied as a function of whey concentration. Galactose
content was higher in 7% EA-whey than in 14% and 21% EA-whey. This difference is
correlated with the pH increase in 7% EA-whey compared to the other concentrations.
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Table 5.1: Comparison of amino acids composition of native whey and EA-Whey at different concentrations.
Name Concentration Amino acids Native
Whey-7% EA-Whey-7%
Native Whey-14%
EA-Whey 14% Native Whey-21%
EA-Whey 21%
ALA 57.0 4.1 87.1 12.7 82.3 21.7
GLY 30.0 5.1 78.8 19.8 173.8 56.7 VAL 74.1 7.0 68.7 3.9 37.6 5.2 LEU 16.7 0.7 14.6 2.6 9.0 4.4
ILE 6.7 <LOQ 6.8 0.6 <LOQ <LOQ THR 2.5 0.0 4.6 1.5 39.5 <LOQ
SER 10.8 3.0 16.2 12.1 13.4 14.5 PRO 62.5 37.6 100.6 81.1 231.7 141.6 ASP 15.7 20.3 28.8 26.7 24.1 33.6
MET 23.0 0.0 15.0 1.5 26 2.1 GLU 199.9 69.7 413.2 311.5 1018.4 509.4
PHE 4.3 3.0 0.0 13.1 0.0 23.5 GLN 4.2 1.7 6.6 2.9 9.2 3.8 LYS 3.0 0.0 1.3 3.2 12.6 25.5
HIS 3.6 0.3 3.2 1.1 21.2 6.2 TYR 3.6 18.7 25.4 20.9 10.7 36.8
Total (ppm) 517.6 171.2 870.9 515.2 1709.5 885
Glycation degree (%)
66.9
40.8
48.2
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The main contributing factor for this distinctive increase of the pH is the higher
buffering capacity of the whey at higher total solids concentration (14 and 21%). Therefore,
galactose content likely makes an important contribution to the glycation and the formation
of MRPs. Moreover, Nooshkam and Madadlou (2016) reported that lactose is more reactive
than lactulose for Maillard conjugation with both intact whey protein isolates and its
hydrolysates (Nooshkam & Madadlou, 2016b). These results also indicated that the
development of MRPs depended on the reactivity of the sugars involved. However, the role
of the reactivity rate of the different sugars formed during the EA in the enhancement of
the antioxidant potencies was not explored.
Figure 5.2: Sugar profiles of EA-whey at different concentrations up to 45 min at 600 mA current intensity: (a) untreated sample, (b) 7% (w/v), (c) 14% (w/v) and (d) 21% (w/v).
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5.6.2 Gel electrophoresis (SDS-PAGE)
Changes in EA-whey protein were investigated by SDS-PAGE under both reducing
and non-reducing conditions. Figure 5.3a and b shows the SDS-PAGE patterns of 7%,
14% or 21% EA-whey at 600 mA current intensity during the 45 min EA process. As
expected from our previous results, whey proteins undergo two main changes, including
polymerization and glycation. A significant decrease in the protein bands was observed as
a function of EA time. The banding patterns of 7% EA-whey tend to disappear completely
at the end of reaction, which is contrary to 14% and 21% EA-whey. Furthermore, at 30 min
of treatment, a new band with greater molecular weight of approximately 36 kDa appeared
in 14% and 21% EA-whey; however, this band became less perceptible at 45 min. The
formation of glycated compounds was confirmed by the smearing zones detected in the
presence of β-Mercaptoethanol (Figure 5.3b). Thus, the EA treatment of whey at different
total solids concentrations had a characteristic reaction rate and resulted in a wide range of
mass distributions of proteins/peptides. The formation of new peptide fractions could have
significant impact on antioxidant proprieties of EA-whey (Kareb et al., 2017b). The SDS-
PAGE patterns explained well the decline of the reducing power in whey prepared at 14%
and 21% (w/v) at 30 min of EA, i.e., the polymerization change that rendered the reducing
groups unavailable. Athira et al. (2015) reported that the hydrolysates of whey derived from
alcalases treatment possessed high antioxidant activities and that peptides with molecular
weights of 3 kDa exhibited the strongest radical scavenging activity (Athira et al., 2015).
The ORAC and DDPH values also seem to be favored when the hydrolysis of whey protein
was pronounced, as confirmed by the band profiles of 7% EA-whey.
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Figure 5.3: Electrophoretic pattern of EA-whey proteins under non-reducing conditions (a) and reducing conditions (b).
138
5.6.2 FTIR spectroscopy analysis
FTIR was used to monitor the changes in the molecular structure of MRPs derived
from EA-whey. Figure 5.4 shows the normalized spectra within the mid-infrared range
between 4000-400 cm−1 of non-treated whey and 7%, 14% or 21% EA-whey after 45 min
of EA processing. Functional groups, including NH2 from amino acids, may be lost, while
the amount of the compounds associated to MR, such as the Amadori compound (C=O),
Schiff base (C=N) and pyrazines (C–N) may be increased through the formation of MRPs.
The bands centered at 1642 cm−1, which corresponds to amide I (C=O stretching and C-N
bending), and at 1583 cm−1, which corresponds to amide II (N–H bending and C-N
stretching), are useful to provide information on the structural changes in the untreated and
EA-treated whey proteins. The areas under the peaks correspond to the concentration of
the functional groups eluted from the corresponding bands. The spectrum of untreated
whey protein showed strong amide I and II bands at 1633 cm-1 and 1540 cm-1, respectively.
FTIR spectrum of 21% EA-whey showed a significant decrease in both amide I and amide
II bands due to the protein hydrolysis. Fourier self-deconvoluted spectra showed two
distinctive peaks in the amide I region at 1640 cm-1 and 1652 cm-1 in addition to a peak in
the amide II region at 1545 cm-1. The peak at 1640 cm-1 indicates the modification of the
protein secondary structure from mainly β-sheet structures to helical and random structures
due to the hydrolysis of the protein. The peaks at 1652 and 1545 cm-1 indicate the formation
of Schiff base (C=N) between the reducing end of sugars and the amino groups of whey
proteins as intermediate Maillard components (Wnorowski & Yaylayan, 2003). Further
decrease in amide I was shown in the spectrum of 14% EA-whey accompanied by Schiff
base formation at 1648 cm-1 and an increase at 1545 cm-1, indicating even greater Schiff
base formation (C=N). Spectrum of 7% EA-whey showed the disappearance of amide I
with more formation of Schiff base evidenced by the two peaks at 1652 and 1545 cm-1.
These results corroborated with the SDS-PAGE profiles, which also showed the
disappearance of whey protein bands. A new strong peak at 1582 cm-1 that was attributed
to the glycation process between the protein and sugars appeared in the three EA samples.
139
Figure.5.4: Infrared spectra of EA-whey at different concentrations after 45 min of EA treatment.
140
It was worth noting that in 7% EA-whey, this peak is increased, and its absorption
is more intense, which is reflective of the highest ORAC and DDPH radical scavenging
activities for that concentration compared with the other concentrations (14 and 21% of
total solids). An increase in the region of 1050–950 cm-1 in 21% EA-whey corresponds to
the increase in the vibrations of side chain amino acids due to the protein hydrolysis.
Additionally, the many overlapping peaks in the region of 1180–953 cm-1 corresponds to
the lactose that results from stretching vibration modes of C–C and C–O and the bending
mode of C–H bonds (Nooshkam & Madadlou, 2016b). The significant intensity decrease
of this region within the 7% EA-whey is likely due to consumption of amino acid
sidechains and sugars during the formation of the Maillard products that appeared at 1545
and 1652 cm-1. Furthermore, the decreasing the ratio between 1020 and 1035 cm-1 is
indicative of the conversion of lactose to galactose and lactulose. A decrease in 1020 and
1069 cm-1 was observed due to the glycation of lactose and its participation in forming
Maillard components. Finally, FTIR spectra indicated that only intermediate products of
MR were formed (e.g., Schiff base); however, no Amadori products were formed though
it has been reported that Amadori products (C=O) give rise to peaks at 1660 and 1700 cm-
1 (Yaylayan & Locas, 2007).
5.6.3 Identification of bioactive peptides from electro-activated whey
Many studies have demonstrated that enzymatic hydrolysis of whey proteins can
yield peptides exhibiting various bioactivities. Thus, in addition to the formation of the
antioxidant MRPs, the EA treatment led to the hydrolysis of whey proteins. Moreover, it
was postulated that the final product could have other bioactive properties. Bioactive
peptides usually have a molecular weight lower than 10 kDa. In the SDS-PAGE profile of
7% EA-whey, a significant degradation of the intact proteins to small hydrolysates less
than 10 kDa was obtained after 45 min of EA. This sample was chosen for further
investigation of its peptide profiles with LC-MS-MS analysis.
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Table 5.2: List of the identified amino acid sequences in the electro-activated whey (EA-Whey) and their potential biological activity.
Protein source AA sequences Identify Activity References
β-casein
TQSLVYPFPGPIPN
QTQSLVYP ACE-inhibitory Kohmura et al., 1990
TQSLVYP ACE-inhibitory
QSLVYP ACE-inhibitory
SLVYP ACE-inhibitory
VYPFPGPI Antiamnestic ACE-inhibitory
Van der Ven et al., 2002
VYPFPG ACE-inhibitory Abubakar et al., 1998
YPF Opioid Brantl et al., 1979
YPFP Opioid
YPFPGP Opioid agonist Meisel, 1998
YPFPGPI
Opioid agonist ACE-inhibitory
Meisel, 1998; Meisel et al, 2006
YPFPGPIPN ACE-inhibitory FitzGerald et al., 2004
TPVVVPPFLQP ACE-inhibitory Abubakar et al., 1998
FLQP Dipeptidyl peptidase IV inhibitor
Nongonierma and FitzGerald, 2013
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TPVVVPPFLQPEVMGVS
LQP
ACE-inhibitory Dipeptidyl peptidase IV
inhibitor
Nongonierma and FitzGerald, 2013
LTLTDVE ACE-inhibitory Hayes et al., 2007
SLTLTLTDVENL QSWMHQPHQ ACE-inhibitory Phelan et al., 2014
MHQPHQPLPPT YPQRDMPIQ ACE-inhibitory Hayes et al., 2007
QRDMPIQAFLL
RDMPIQ Antioxidative De Gobba et al., 2014
AFLL ACE-inhibitory De Gobba et al., 2014
VLNENLLR Antibacterial Hayes et al., 2006
α–s1-casein
QGLPQEVLNENLL VAP ACE-inhibitory Maruyama et al., 1987
FFVAPFPEVFGK
FVAP ACE-inhibitory Maruyama et al., 1985
FFVAP ACE-inhibitory Maruyama et al., 1985
FFVAPFPEVFGK
ACE-inhibitory Anticancer
Juillerat-Jeanneret
VTSTAV
ACE-inhibitory FitzGerald et al., 2004
κκκκ-casein VIESPPEINTVQVTSTAV
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Whey proteins include the major β-LG, α-LA, immunoglobulins, BSA, in addition
to other minor proteinaceous components, such casein fragments that result from enzymatic
cheese making. In 7% EA-whey, 27 peptide sequences were identified. All the bioactive
peptides were derived from α, β and γ casein. The peptide FFVAPFPEVFGK extracted
from α–s1-casein was previously reported as a bioactive peptide possessing an Angiotensin
Converting Enzyme (ACE) inhibition and anticancer activity. Other peptides with multi-
functionality could promote various bioactivities such as an ACE and dipeptidyl-peptidase
IV (DPP-IV) inhibition, opioid agonist, anti-amnestic properties, as well as anti-oxidative
and antimicrobial activities (Table 5.2). Thus, EA can be used to target a simultaneous
production of the MRPs and enriched bioactive peptides from whey protein with desirable
health benefits. The peptides derived from 7% EA-whey contained only one sequence
fraction with anti-oxidative properties. Indeed, almost all the anti-oxidative activity of the
EA-whey resulted from the MR. Tryptic digestion of 7% EA-whey did not resulted in any
further hydrolysis or production of peptides. This lack of hydrolysis and peptide production
is likely caused by glycation in MRPs, thereby rendering the trypsin cleavage sites
(arginine and lysine) unavailable. Further model system studies with whey protein isolates
are required to demonstrate the efficacy of the EA technology in the production of bioactive
peptides.
5.7 CONCLUSION
This study contributed to understand the potential of the EA to enhance the
biological activity of whey without need of fractionation. This work permitted the synthesis
of whey enriched, in situ, by Maillard reaction products (MRPs) and bioactive peptides by
EA technology. Moreover, the identification of structural characteristics of Maillard
reaction products and bioactive peptides derived from EA-whey was carried out. It has
been demonstrated by detailed characterization that EA-whey could be a source of Maillard
reaction products and bioactive peptides. The molecular structure of MRPs-whey was
characterized as Schiff base compounds. Finally, this study highlighted the potential of as
the EA as a non-thermal technology to enhance the formation of Maillard reaction products
(MRPs) with potential biological activity that can be used in functional food formulations.
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6. CHAPTER 6: Effect of electro-activated sweet whey on growth of
probiotic bacteria of Bifidobacterium, Lactobacillus and Streptococcus
genera under model growth conditions
This chapter is presented as an article entitled “Effect of electro-activated sweet
whey on growth of probiotic bacteria of Bifidobacterium, Lactobacillus and Streptococcus
genera under model growth conditions”.
The authors are: Ourdia Kareb (Ph. D. candidate: planning and realization of the
experiments, results analysis and manuscript writing), Ahmed Gooma, Claude P.
Champagne (Thesis co-director: scientific supervision, correction and revision of the
manuscript), Julie Jean (Thesis co-director: scientific supervision, correction and revision
of the manuscript) and Mohammed Aïder (Thesis director: scientific supervision, article
correction and revision).
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6.1 RÉSUMÉ
Récemment, nous avons démontré l'efficacité de l'électro-activation à améliorer les
propriétés fonctionnelles du lactosérum qui pourrait être utilisé comme prébiotique et
antioxydant en raison in situ de son enrichissement en lactulose et des produits de la
réaction de Maillard ayant un potentiel antioxydant. L'objectif de la présente étude était
d'évaluer l'effet du lactosérum électro-activé (lactosérum-EA) sur la croissance de
probiotiques des genres de Bifidobacterium, Lactobacillus et Streptococcus dans des
cultures pures en comparaison au lactosérum non EA, Lactulose, lactose, saccharose,
glucose et galactose à différentes concentrations (1,25, 2,5 et 5%). La cinétique de
croissance bactérienne en fonction de la densité optique maximale (ODmax) et du taux
maximal de croissance (μmax) a été étudié. En outre, l’effet du lactosérum-EA sur la
croissance de L. johnsonii La-1 en présence d'oxygène a été évalué. L’analyse FTIR a aussi
été utilisée pour évaluer l’impact de l’ajout du lactosérum EA sur la structure de la
membrane bactérienne. Les résultats obtenus ont montré que le lactosérum-EA améliorerait
la croissance de toutes les souches étudiées. Il ressort aussi des résultats que le lactosérum-
EA ait un effet bifidogénique significatif en comparaison au lactulose. La croissance de la
souche L. johnsonii La-1 en présence d’oxygène a été grandement améliorée lorsque le
milieu de culture a été supplémenté avec le lactosérum-EA. Cet effet stimulant pourrait en
partie été lié au pouvoir du lactosérum-EA à prévenir l'accumulation du peroxyde
d'hydrogène. Les spectres FTIR ont montré que le lait d'EA-EA agit comme antioxydant
en ce qui concerne l'oxydation des lipides de la membrane cellulaire en limitant la
modification de l'ordre structurel des acides gras. Ainsi, le lactosérum-EA ayant un
potentiel prébiotique et antioxydant combiné, pourrait être utilisé comme ingrédient actif
dans la fabrication de produits laitiers fermentés fonctionnels.
Mots-clés: Électro-activation; Lactosérum; Lactulose; Prébiotique; Antioxydant;
Probiotique
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6.2 ABSTARCT
Recently, we demonstrated the efficacy of electro-activation to improve the
functional proprieties of whey, which can be used as prebiotic and antioxidant agent
through lactulose and Maillard reaction products formation. The aim of the present study
was to evaluate the effect of electro-activated sweet whey (EA-whey) on growth of
probiotics of Bifidobacterium, Lactobacillus and Streptococcus genera in pure cultures and
to compare EA-whey with non-electro-activated whey, lactulose, lactose, sucrose, glucose
and galactose at different concentrations (1.25, 2.5 and 5%). The bacterial growth was
monitored through maximum optical density (ODmax) and maximum growth rate (µmax)
measurements. Moreover, the effects of EA-whey on the growth of L. johnsonii La-1 in the
presence of oxygen was assessed. FTIR spectroscopy analyses of the bacterial membrane
structure was monitored. The results showed that EA-whey enhanced the growth of all the
test bacteria. They clearly demonstrated a promoting bifidogenic effect of EA-whey in
comparison to lactulose. The growth of L. johnsonii La-1 was greatly enhanced in the
presence of oxygen when the culture medium was supplemented with EA-whey. This
growth promoting effect could be linked, for part, to EA-whey to prevent the accumulation
of hydrogen peroxide metabolites in the growth medium. FTIR spectra showed that EA-
whey acts as antioxidant in regards to cell membrane lipids oxidation by limiting the
change in the structural order of fatty acids. Thus, EA-whey, a potential prebiotic and
antioxidant, could be used as active ingredient in manufacturing functional fermented dairy
products.
Keywords: Electro-activation; Whey; Lactulose; Prebiotic; Antioxidant; Probiotic
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6.3 INTRODUCTION
Nowadays, the functional foods are recognized to offer an excellent opportunity to
maintain and improve consumer’s health (Bogue et al., 2017; de Boer et al., 2016; Pastrana
et al., 2017; Siro et al., 2008). The dairy products fortified with appropriate probiotics,
prebiotics or their synbiotic combinations are the mostly popular functional foods with
proven success to produce beneficial synergistic effects on human’s well-being (Dwivedi
et al., 2016; Hailu et al., 2009; Illanes & Guerrero, 2016; Parvez et al., 2006).
Probiotic bacteria, live microorganisms with convincing positive health effects,
belonging to the genera Bifidobacterium and Lactobacillus species are successfully
incorporated in functional dairy products (Ranadheera et al., 2010; Vasiljevic & Shah,
2008). These bacteria play important role in maintaining consumer’s health by improving
the intestinal microbial balance, besides producing bioactive compounds (Dwivedi et al.,
2016; Kumar et al., 2016). It is recognised that a daily dose of 109 viable cells/ml or g of
product are required at the point of consumption to unsure their health benefits on the host
(Champagne et al., 2011). However, these microorganisms encountered various stresses
during the manufacturing and the storage of product until the consumption due the changes
in pH, oxygen variations, acidity, and the depletion of nutrients which arguably leads to
low survival rates (Champagne et al., 2005; Donkor et al., 2007b; Hernandez-Hernandez
et al., 2012; Jokinen et al., 2011). Furthermore, probiotics have low proteolytic activities,
hence their slow growth in dairy products (Donkor et al., 2007a; Shihata & Shah, 2000).
Thus, researches are currently focusing on the elaboration of multifunctional bioactive
compounds that can better promote the growth of probiotic bacteria and then, developing
new functional products.
Prebiotics are described as “a selectively fermented ingredient that allows specific
changes, both in the composition and/or activity in the gastrointestinal microbiote that
confers benefits upon host well-being and health” (Gibson et al., 2004; Glenn &
Roberfroid, 1995). The most used prebiotics are inulin, fructo-oligosaccharides, galacto-
oligosaccharides and lactulose (Roberfroid, 2007). Lactulose has received much attention
from the dairy industry due its prebiotic functionality and interesting technological
properties (Al-Sheraji et al., 2013; Huebner et al., 2008). For its prebiotic effect, lactulose
was reported to promote the growth of Bifidobacteria and Lactobacilli bacteria (Ballongue
148
et al., 1997; Oliveira et al., 2011; Özer et al., 2005; Saarela et al., 2003; Watson et al.,
2013). Furthermore, lactulose is also used as a gentle laxative to improve the chronic
constipation and is used as pharmaceutical agent in the treatment of the hepatic
encephalopathy (Schuster-Wolff-Bühring et al., 2010). Lactulose is mainly produced
following chemical and enzymatic isomerisation of lactose (Aider & de Halleux, 2007;
Wang et al., 2013a).
Recently, electro-activation (EA) was successfully used for the isomerisation of
lactose into lactulose without alkalinizing chemicals added. The process was based on a
self-generation of the needed alkaline conditions following redox reaction at the
solution/cathode interface. Thus, considering these facts, this innovative technology can be
seen as an eco-friendly and reagentless approach that holds a promising issue for useful
production of lactulose without chemical reagents (Aissa & Aïder, 2013a, 2013b; Kareb et
al., 2016). Applied to sweet whey, these previous studies motivated the authors of the
present work to postulate a new concept of prebiotic with improved functionalities through
compositional modifications with a final aim to find complementary alternative for a better
stimulation of the growth of probiotic bacteria. Effectivity, whey can serve as a precursor
of lactulose synthesis through in situ electro-isomerization of lactose by EA processing
(Kareb et al., 2016). Moreover, simultaneous hydrolysis of whey proteins and glycation
with the reducing sugars occurred under EA conditions, giving rise to Maillard reaction
products (MRPs) that greatly improve the antioxidant capacity of electro-activated whey
(EA-whey) (Kareb et al., 2017b) . Indeed, the MRPs are frequently used to improve the
technological and biological proprieties of foods, and could be particularly interesting as
potential prebiotic because they beneficially affect the growth of health-promoting bacteria
in the gastrointestinal tract as reported in some recent studies (Corzo-Martínez et al., 2012;
Corzo-Martínez et al., 2013; Seiquer et al., 2014). Although some studies have been
demonstrated the positive effect of adding antioxidant compounds to stimulate the growth
of probiotic bacteria when oxygen sensitive species are used, there is not published reports
on the effect of MRPs-rich EA-whey on the growth of probiotic bacteria, since research is
focused principally on phenolics such as green tea extract rich in polyphenols (de Lacey et
al., 2014; Gaudreau et al., 2013; López de Lacey et al., 2014; Muniandy et al., 2016; Zhao
& Shah, 2014). Moreover, EA-whey also contains other interesting compounds, which can
149
stimulate the growth of these beneficial bacteria like small peptides, free amino acids,
vitamins, carbohydrates and minerals. Other characteristics of EA-whey, which could be
interesting, are the improved buffering capacity and the potential redox. In addition, this
product contains lactose, glucose, and galactose, without prebiotic properties, which
increase carbohydrates availability and thus, providing on optimum growth medium for
probiotic bacteria. In addition, the inclusion of functional and nutritive whey in dairy
products rather than milk can ultimately reduce the production cost (Almeida et al., 2009).
Therefore, it is of great importance to introduce new prebiotic that may result in
better growth of probiotic bacteria. Thus, the aim of the present study was to investigate
the ability of EA-whey as a prebiotic of new generation to stimulate the growth of potential
probiotic strains from Bifidobacterium and Lactobacillus species. The effect of EA-whey
as an antioxidant compounds was also monitored. To the best of our knowledge, nobody
focused on this issue in the past; therefore, this paper was aimed to study the combined
effects such as prebiotic and antioxidant of EA-whey on the growth of the chosen probiotic
bacteria.
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6.4 MATERIALS AND METHODS
6.4.1 Chemicals and reagents
Food grade whey powder (75% lactose, 12% total proteins, 7% ash and moisture
content less than 5%) was obtained from Agropur Cooperative (St-Hubert, Quebec,
Canada). Sodium sulphate (Na2SO4) was purchased from Anachemia (Montreal, Quebec,
Canada). Analytical grade carbohydrates such as fructose, glucose, galactose, sucrose,
lactulose and lactose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bacterial
media like MRS and M17 broths were provided from Fisher Scientific (Fisher Scientific
Company, Ottawa, Canada). All other reagents and chemicals used in this study were of
analytical grade and were purchased from Sigma-Aldrich (Oakville, Ontario, Canada).
6.4.2 Preparation of electro-activated whey
EA-whey was prepared under alkaline conditions following the same procedure
described in our previous study (Kareb et al., 2016). The configuration of the electro-
activation reactor as well as the reaction conditions used are shown in Figure 6.1. Briefly,
whey powder at the concentration of 7% (w/v) was reconstituted in deionized water and
stored overnight at 4°C before treatment to allow full dissolution. The solution was then
submitted to EA treatment in the cathodic compartment of the used reactor at 600 mA
nominal electric current intensity. The electro-activated whey solution was gently stirred
by using an agitator (Model: RW 20 DS1, IKA) fixed at 200 rpm.min-1 during 45 min until
the optimum pH of 11± 0.3 was reached and which is required for the optimal conversion
of lactose to lactulose (Kareb et al., 2016). The EA-whey was lyophilised and stirred to
obtain homogenised powder. Sugars content such as lactose, lactulose, glucose, galactose
and fructose were quantified by high performance liquid chromatography by using an
HPLC Agilent Technology system (Millipore Corp., Milford, MA, USA) (Kareb et al.,
2016). The degree of protein hydrolysis after EA treatment was also analysed by using
EZfaast kit according to the manufacturer’s instructions (Phenomenex, Torrance, CA,
USA).
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Figure 6.1: Schematic representation of the electro-activation reactor used for production of EA-whey.
The oxygen radical absorbance capacity (ORAC) and the reducing power of EA-
whey were measured as described in our previous study (Kareb et al., 2017b) . The pH was
monitored by using a pH meter (model Oakton pH 700, Eutech Instruments, Cole-Parmer,
Montreal, Canada). The oxido-reduction potential (ORP) values of the EA-whey was
estimated by using a VWR Symphony platinum electrode (VWR Scientific, West Chester,
PA, USA). The composition and the physiochemical characteristics of whey as a control
and those of the EA-whey are presented in Table 1.
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Table 6.1: Main proximate composition and antioxidant properties of whey and EA-whey used in this study.
Compositions Whey EA-whey
Carbohydrates (%)
• Lactose
• Lactulose
• Galactose
Proteins hydrolysis
Antioxidant properties
• ORAC
• Reducing power
pH
Oxidation-reduction potential, mV
100 %
-
-
-
1236 ± 153
0.45 ± 0.05
5.85 ± 0.15
245 ± 25
62 %
28.6 %
7.85 %
0.5 %
7674 ± 145
1.3 ± 0.1
10.95 ± 0.2
-124 ± 15
(−): Absence of ingredient
6.4.3 Microorganisms and culture conditions
Four commercial yogurt probiotic strains of Lactobacillus and Bifidobacterium
species, specifically: Lactobacillus johnsonii La1 (Nesté); Lactobacillus rhamnosus GG
(Chr. Hansen, Hoersholm, Denmark); Bifidobacterium animalis subsp. lactis Bb12 (Chr.
Hansen); and Bifidobacterium longum subsp. bifidum R0175 from here onward called
(Lallemand Health Solutions, Montréal, QC, Canada) and the yogurt starter culture
Streptococcus thermophilus R0292 (Lallemand) and Lactobacillus bulgaricus R5083
(Lallemand). The probiotic strains were selected to be used in the present study on the basis
of their different abilities to assimilate lactose and lactulose as a carbon source. Moreover,
La1 was chosen as a control positive because it grows well in whey and produces
extracellular toxic H2O2 in relatively high amount to test the effect of EA-whey
supplementation on the culture medium as an antioxidant under aerobic conditions. The
stock cultures were prepared by mixing a fresh MRS-grown culture with sterile 20%
glycerol and sterile 20% (w/v) reconstituted skim milk in a 2:5:5 ratios. All stains were
subcultured in appropriate medium at least twice prior to the experiments. The incubation
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period was variable so that each culture was collected once it had reached the beginning of
the stationary growth phase. The purity of stains tested was routinely monitored.
6.4.4 Experimental design
To study the ability of the chosen probiotic bacterial stains to use different
carbohydrate sources, basal MRS free carbohydrates (BMRS) medium was prepared
following the study of Saarela et al. (2003). The composition of the BMRS was as follows
peptone from casein (10.0 g.L-1), yeast nitrogen (5.0 g.L-1), K2HPO4 (5.0 g.L-1), K2HPO4
(5.0 g.L-1), MgSO4 (0.2 g.L-1), MnSO4 (0.05 g.L-1) and Tween 80 (1 mL). The pH was
adjusted to 6.5 and the medium was sterilised at 121°C for 15 min. The sources of carbon
(carbohydrates) tested were as follows: glucose, galactose, sucrose, lactose, lactulose,
commercial sweet whey and EA-whey. Freshly prepared BMRS was supplemented with
three final concentrations of each individual carbohydrate tested at final concentration of
1.25, 2.5 and 5% (w/v) and then sterilized by passing through 0.45 µm membrane filters.
Commercial whey and EA-whey were sterilized by irradiation and added to the BMRS to
obtain the similar concentrations as for the other carbohydrates. The BMRS without any
added carbohydrates and BMRS supplemented with glucose were used as a negative and
positive control, respectively.
6.4.5 Evaluation of growth performance
6.4.5.1 Effect of MRS supplementation on the growth of probiotic bacteria under
anaerobic conditions
The growth of the starter culture and the probiotic strains was studied by using the
PowerwaveTM Microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT,
USA) in conjunction with Gen5TM Microplate software for the data acquisition. Two wells
of the microplate were filled with 200 μL of each BMRS at different sugar concentrations
(1.25, 2.5 and 5%) and supplemented with 10 μL of fresh culture of each individual
bacterial strain. Unless otherwise stated, to ensure the anaerobic conditions, 50 μL of sterile
mineral oil was added to the wells on the surface of the inoculated media as described in
the study of Gaudreau et al. (2013). The plates were incubated for 24 h at 37oC in anaerobic
conditions by using anaerobic jars containing an Anaerogen sachet (Oxoid, Nepean,
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Ontario, Canada). The optical density at a wavelength of 600 nm (OD600nm) readings were
taken every hour, and each read was preceded by a 30 seconds moderate shaking step.
Three independent assays were carried-out, and the resulting growth data were expressed
as the mean of these replicates. The growth rate was calculated for strain-carbohydrate
source-concentration combinations per the formula µmax = (Inx2 − Inx1) \ (t2 − t1), where x1
and x2 were absorbance values measured within the exponential phase of growth at times
t1 and t2, respectively.
6.4.5.2 Effect of MRS supplementation on the growth of La under aerobic conditions
To evaluate the hypothesis that EA-whey plays a role in the protection against
oxygen stress, this study investigated the role of EA-whey as antioxidant during the growth
of La in presence of oxygen. Indeed, La was shown because it produces radical oxygen
species like H2O2 in high amount during growth (Hougaard et al., 2016). To carry out this
experiment, fresh culture of La was prepared as described previously in anaerobic
conditions. The growth curves of La in the presence of oxygen during 24 hours were
monitored. BMRS supplemented with commercial whey and EA-whey at different
concentrations (1.25, 2.5 and 5%) was used as above. The plates were incubated for 24 h
at 37oC in aerobic conditions. The OD600nm readings were taken every hour, and each read
was preceded by a 30 seconds moderate shaking step. Three independent replicates were
carried out for each condition and the data presented are the average of the three assays.
6.4.5.3 Fourier transform infrared spectroscopy analysis
The effect of EA-whey supplementation of the medium culture on the membrane
structure of La was monitored by using Fourier transform infrared (FTIR) spectroscopy. A
1% inoculum of a fresh culture of L. johnsonii La-1 was grown at 37°C in 50 mL test tubes
in BMRS broth supplemented with EA-whey at different concentrations (1.25, 2.5 and 5%
w/v) in presence of oxygen under quickly continuous agitation. For the anaerobic
conditions, the tubes were incubated in anaerobic jars containing an Anaerogen sachet
(Oxoid, Nepean, Ontario, Canada). Bacteria cells were collected when the growth reached
the beginning of the stationary phase and were harvested by centrifugation at 9800 rpm for
5 min at 4oC and washed twice with deionized water. The pellet was placed on a CaF2
155
window and dried 15 min at 50oC as described by Alvarez-Ordóñez & Prieto (2010). The
infrared spectra were measured at room temperature with a Magna 560 Nicolet
spectrometer with a liquid nitrogen cooled narrow-band mercury cadmium telluride (MCT)
detector and a Golden Gate diamond ATR accessory (Specac Ltd., London, UK). Each
spectrum was obtained from accumulation of 128 scans at a resolution of 2 cm−1 from 4000
to 900 cm-1 with a Happ-Genzel apodization. Spectral processing was performed using
Omnic software (version 3.1, Thermo Electron Corporation, Beverly, MA, USA). The
scale of each spectrum was normalized to minimize differences due to sample amount and
the baseline was corrected in the spectral region 3000-2800 cm-1 to correct spectra.
6.5 Statistical analyses
Three independent replicates of each above-mentioned experiment were performed
and mean of the obtained results was reported. Statistical analyses were performed using
SAS software (SAS software, Inc., San Jose, CA, USA) on the results obtained for the
strains growth on various carbon sources. A one-way analysis of variance (ANOVA) at a
significance level of 95% (p < 0.05) was used for the statistical evaluation of resulted
derived from the measurement of the ODmax and µmax for each bacterial strain with each
substrate studied.
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6.6 RESULTS AND DISCUSSION
6.6.1 RESULTS
6.6.1.1 Effect of MRS supplementation on the growth of probiotic bacteria under
anaerobic conditions
To understand the effects of electro-activated whey (EA-whey) on growth of the
selected probiotic bacteria, a concentration-dependent analysis was carried out. Growth
curves under anaerobic conditions of the selected strains of the genera Lactobacillus,
Bifidobacterium and Streptococcus under different growth conditions by using different
carbon sources (glucose, galactose, sucrose, lactose, lactulose, whey and EA-whey) are
shown in Tables 6.2-6.3 as two main parameters reflecting the growth performance of the
tested bacteria: the maximum optical density (ODmax) and the maximum growth rate (µmax)
which were used to compare the substrates preferences. No growth was observed for any
of the tested strains in the control (basic) medium without carbon source added (data not
shown). As it can be observed, the probiotic strains studied showed different growth
properties for the same substrate, and also showed distinguishing reactions to the different
carbon sources. This indicates that this parameter (type of added sugar) has a significant
effect regarding to the growth intensity of each bacterial strain. In general, the growth levels
obtained on glucose and galactose were high for all the studied probiotic strains. Lactulose
was markedly utilized only by the Bifidobacteria strains, whereas the electro-activated
whey (EA-whey) was intensively utilized by all the tested strains. Furthermore, an
examination of the data in Tables 6.2-6.3 showed that the growth parameters of the
probiotic strains in the media containing EA-whey was often higher than in whey, and
comparable to those obtained with glucose. This indicated a high level of EA-whey
carbohydrate assimilation.
The growth profiles of B. lactis Bb12 indicated that this strain grew to high cell
densities in the media supplemented with all the tested carbohydrates, except for sucrose.
The growth rate of B. lactis Bb12 was greatly enhanced by supplementing the growth
medium with 5% (w/v) of EA-whey.
157
Table 6.2: ODmax of the bacterial strains in supplemented MRS medium as a function of
the concentration and type of carbon source.
Strain Medium
ODmax
Concentration, %
1.25 2.5 5
Bb12
Glucose 1.85 ± 0.11bc 1.85 ± 0.10bc 1.84 ± 0.09bc
Galactose 1.90 ± 0.11ab 1.88 ± 0.10b 1.87 ± 0.10b
Sucrose 1.12 ± 0.12d 0.74 ± 0.04d 0.72 ± 0.06d
Lactose 1.87 ± 0.12ab 1.89 ± 0.09b 1.81 ±0.03bc
Lactulose 1.82 ± 0.12bc 1.77 ± 0.10c 1.74 ± 0.11c
EA-whey 1.96 ± 0.13a 2.02 ± 0.10a 2.17 ± 0.10a
Whey 1.75 ± 0.10c 1.83 ± 0.11bc 1.83 ± 0.09bc
GG
Glucose 1.84 ± 0.11a 1.86 ± 0.09a 1.85 ± 0.10a
Galactose 1.85 ± 0.10a 1.87 ± 0.10a 1.87 ± 0.10a
Sucrose 0.61 ± 0.03d 0.58 ± 0.03d 0.56 ± 0.02d
Lactose 0.59 ± 0.04d 0.53 ± 0.03d 0.51 ± 0.02d
Lactulose 0.56 ± 0.03d 0.54 ± 0.02d 0.51 ± 0.01d
EA-whey 1.07 ± 0.01b 1.22 ± 0.02b 1.45 ± 0.01b
Whey 0.69 ± 0.06c 0.77 ± 0.04c 1.03 ± 0.06c
R0175
Glucose 1.85 ± 0.10ab 1.76 ± 0.08c 1.63 ± 0.03bc
Galactose 1.90 ± 0.10a 1.88 ± 0.10b 1.73 ± 0.11b
Sucrose 1.69 ± 0.07c 1.62 ± 0.07d 1.61 ± 0.10c
Lactose 1.85 ± 0.10ab 1.81 ± 0.10bc 1.62 ± 0.08bc
Lactulose 1.77 ± 0.11bc 1.72 ± 0.12cd 1.58 ± 0.11c
EA-whey 1.92 ± 0.11a 2.00 ± 0.10a 2.10 ± 0.09a
Whey 1.73 ± 0.08c 1.82 ± 0.10bc 1.68 ± 0.09bc
R0292 Glucose 1.83 ± 0.10b 1.86 ± 0.10b 1.84 ± 0.09b
158
Galactose 1.85 ± 0.12b 1.87 ± 0.11b 1.86 ± 0.10b
Sucrose 1.69 ± 0.10c 1.61 ± 0.10c 1.64 ± 0.10c
Lactose 1.88 ± 0.14ab 1.87 ± 0.10b 1.62 ± 0.04c
Lactulose 1.84 ± 0.11b 1.79 ± 0.09b 1.54 ± 0.08c
EA-whey 1.97 ± 0.11a 2.04 ± 0.11a 2.11 ± 0.10a
Whey 1.84 ± 0.12b 1.88 ± 0.11b 1.88 ± 0.09b
R5083
Glucose 1.80 ± 0.10b 1.86 ± 0.10b 1.85 ± 0.10c
Galactose 1.84 ± 0.09ab 1.87 ± 0.10b 1.86 ± 0.10c
Sucrose 0.60 ± 0.03c 0.57 ± 0.02c 0.56 ± 0.03d
Lactose 1.83 ± 0.09ab 1.82 ± 0.10b 1.84 ± 0.12c
Lactulose NG NG NG
EA-whey 1.92 ± 0.10a 2.01 ± 0.11a 2.26 ± 0.07a
Whey 1.83 ± 0.11ab 1.92 ± 0.12ab 2.07 ± 0.11b
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Table 6.3: µmax (h-1) of the bacterial strains in supplemented MRS medium as a function
of the concentration and type of carbon source.
Strain Medium
µmax (h-1)
Concentration, %
1.25 2.5 5
Bb12
Glucose 0.57 ± 0.02a 0.47 ± 0.03b 0.45 ± 0.03b
Galactose 0.47 ± 0.02b 0.42 ± 0.02c 0.42 ± 0.02bc
Sucrose 0.13 ± 0.01c 0.20 ± 0.02e 0.20 ± 0.02e
Lactose 0.42 ± 0.01c 0.38 ± 0.01d 0.37 ±0.01d
Lactulose 0.42 ± 0.01c 0.40 ± 0.01cd 0.40 ± 0.01c
EA-whey 0.55 ± 0.04a 0.58 ± 0.02a 0.53 ± 0.04a
Whey 0.54 ± 0.01a 0.46 ± 0.03b 0.43 ± 0.01bc
GG
Glucose 0.72 ± 0.01a 0.58 ± 0.02a 0.54 ± 0.01a
Galactose 0.46 ± 0.01c 0.46 ± 0.01b 0.45 ± 0.01c
Sucrose 0.36 ± 0.01e 0.38 ± 0.02c 0.35 ± 0.01e
Lactose 0.40 ± 0.03d 0.48 ± 0.03b 0.38 ± 0.03d
Lactulose 0.39 ± 0.02d 0.48 ± 0.03b 0.37 ± 0.04de
EA-whey 0.52 ± 0.01b 0.47 ± 0.01b 0.49 ± 0.01b
Whey 0.48 ± 0.02c 0.48 ± 0.02b 0.47 ± 0.02bc
R0175
Glucose 0.56 ± 0.02ab 0.50 ± 0.02b 0.50 ± 0.04c
Galactose 0.44 ± 0.01c 0.39 ± 0.02d 0.30 ± 0.02e
Sucrose 0.42 ± 0.07c 0.49 ± 0.04b 0.59 ± 0.03a
Lactose 0.43 ± 0.01c 0.39 ± 0.02d 0.38 ± 0.01d
Lactulose 0.43 ± 0.01c 0.42 ± 0.01c 0.37 ± 0.05d
EA-whey 0.57 ± 0.05a 0.65 ± 0.02a 0.55 ± 0.01b
Whey 0.53 ± 0.02b 0.51 ± 0.03b 0.57 ± 0.02ab
R0292 Glucose 0.66 ± 0.01a 0.56 ± 0.01a 0.54 ± 0.01a
160
Galactose 0.47 ± 0.01b 0.46 ± 0.01b 0.44 ±0.01b
Sucrose 0.25 ± 0.02e 0.28 ±0.03e 0.39 ± 0.02c
Lactose 0.35 ± 0.02d 0.26 ± 0.02ef 0.21 ± 0.01e
Lactulose 0.35 ± 0.02d 0.25 ± 0.02f 0.21 ± 0.01e
EA-whey 0.43 ± 0.01c 0.35 ± 0.02c 0.33 ± 0.03d
Whey 0.42 ± 0.03c 0.32 ± 0.02d 0.37 ± 0.04c
R5083
Glucose 0.71 ± 0.02a 0.57 ± 0.02a 0.53 ± 0.01a
Galactose 0.45 ± 0.01b 0.45 ± 0.01b 0.44 ± 0.01b
Sucrose 0.34 ± 0.03d 0.36 ± 0.02d 0.36 ± 0.03c
Lactose 0.35 ± 0.03d 0.33 ± 0.02e 0.28 ± 0.02e
Lactulose NG NG NG
EA-whey 0.34 ± 0.02d 0.33 ± 0.02e 0.24 ± 0.02f
Whey 0.39 ± 0.01c 0.40 ± 0.04c 0.33 ± 0.01d
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The other Bifidobacterium strain tested, B. longum R0175, was able to grow in all
the media with the highest growth occurring in the medium supplemented with EA-whey.
Moreover, results showed that both B. lactis Bb12 and B. longum R0175 strains grew quite
well in the media supplemented with lactulose and Whey (non-electro-activated). In the
1.25 to 5% range, the concentration of the supplement had little influence on the growth of
these two bifidobacteria.
The strain Lactobacillus rhamnosus GG exhibited varying growth levels, as a
function of substrate and concentration. At the added sugar concentration of 1.25%, L.
rhamnosus GG reached the highest biomass levels with glucose and galactose as carbon
source. When using EA-whey in the growing medium, L. rhamnosus GG ODmax values
were higher than in Whey, lactose, lactulose and sucrose. It was noteworthy that ODmax
values of L. rhamnosus GG increased as a function of the Whey and EA-whey
concentrations (p > 0.001), but µmax data were not affected by concentration.
The growth of S. thermophilus R2092 was similar to that of L. bulgaricus R5083
when glucose, galactose, lactose and Whey were used to supplement the MRS-base growth
medium. However, data showed that S. thermophilus R2092 utilised well lactulose and
sucrose, whereas these carbon sources were minimally assimilated by L. bulgaricus R5083.
Furthermore, ODmax values were highest of strains R2092 and R5083 were highest when
electro-activated whey (EA-whey) was the supplement. However, this did not extend to
corresponding μmax values, which were highest on glucose and galactose. These data show
that biomass levels (ODmax) and growth rates (µmax) are not similarly affected by the various
supplements.
6.6.1.2 Effect of MRS supplementation by EA-whey on the growth of L. johnsonii La-
1 under aerobic conditions
Data in Tables 6.4-6.5 were obtained under anaerobic and aerobic conditions in
low concentration of oxygen since oil was added at the surface of the media in the wells to
avoid diffusion of oxygen in the media during the incubation. To examine the potency of
EA-whey as prebiotic and antioxidant compound, it was used to supplement the growth
medium of L. johnsonii La-1 which is a probiotic strain well-known to produce high
162
amount of a toxic hydrogen peroxide (H2O2) when cultivated under aerobic conditions
(Pridmore et al., 2008).
Table 6.4: ODmax and µmax (h-1) of L. johnsonii La-1 was cultured in MRS-supplemented
medium under anaerobic conditions.
Medium Concentration,
%
Anaerobic conditions (oil at
surface)
ODmax µmax(h-1)
EA-whey
1.5 1.76 ± 0.03e 0.54 ± 0.11ab
2.5 2.20 ± 0.04b 0.52 ± 0.01ab
5 2.27 ± 0.02a 0.41 ± 0.01c
Glucose
(positive control)
1.5 1.74 ± 0.02e 0.59 ± 0.01a
*NG: when the ODmax < 0.5 a,b,c,d,e,f : Values which are followed by the same superscript letter are not
significantly different (p > 0.05)
Table 6.5: ODmax and µmax (h-1) of L. johnsonii La-1 was cultured in MRS-supplemented
medium under aerobic conditions.
Medium Concentration,
%
Aerobic conditions
ODmax µmax(h-1)
EA-whey
1.5 1.55 ± 0.01f 0.57 ± 0.01a
2.5 1.89 ± 0.01d 0.53 ± 0.01ab
5 1.97 ± 0.03c 0.50 ± 0.01b
Glucose
(positive control)
1.5 NG NG
*NG: when the ODmax < 0.5 a,b,c,d,e,f : Values which are followed by the same superscript letter are not
significantly different (p > 0.05)
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In the present study, L. johnsonii La-1 was cultured under aerobic and anaerobic
conditions (with or without oil at the surface of the medium) and the results illustrating the
growth performances are summarized in Tables 6.4-6.5. Supplementation of the culture
medium by EA-whey greatly enhanced the growth of L. johnsonii La-1 under all the tested
conditions. At the supplementation concentration of 1.25%, L. johnsonii La-1 reached
similar ODmax values as the values obtained when it was grown in glucose and EA-whey
under anaerobic conditions. Furthermore, L. johnsonii La-1 was characterized by high µmax
value when cultured under aerobic conditions. By increasing the EA-whey concentration
in the growth medium up to 2.5% and 5%, the growth level (ODmax) of L. johnsonii La-1
was enhanced under both anaerobic and aerobic conditions (p < 0.001). However, µmax
values with EA-whey were not improved over those in glucose. These results clearly
demonstrate that electro-activated whey possesses the two expected properties, which are
a prebiotic effect and antioxidant activity.
6.6.1.3 FTIR analysis
FTIR spectra in acyl chain regions of the membrane of L. johnsonii La-1 cells
grown under both aerobic and anaerobic conditions in glucose-supplemented MRS (with
2% glucose) and in basic MRS media supplemented with 1.25 to 5% (w/v) EA-whey are
shown in Figure 6.2. The spectral region (3000-2800 cm-1) is dominated by stretching
vibrations of the CH3, CH2 and CH groups presented in the hydrocarbon chains of fatty
acid of the various bacterial membrane (Helm et al., 1991). The bands with frequencies
around 2960, 2930, 2875 and 2855 cm-1 have been assigned to asymmetric stretching
vibration of CH3 (νasCH3) and CH2 (νasCH2) groups and symmetric stretching vibrations
of CH3 (νsCH3) and CH2 (νsCH2), respectively (Davis & Mauer, 2010). These bands are
useful in the study of the physical properties of phospholipids (Mantsch & McElhaney,
1991). Moreover, the area under each band gives information about the amount of each
corresponding group, whereas wavenumber position provides information about structural
conformation of the lipid acyl chains. The bands corresponding to νasCH2 and νsCH2 are
conformation-sensitive and a shift to higher wavenumbers of those bands implies
introduction of disorder in the lipid acyl chains (Kóta et al., 1999).
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Figure 6.2: Normalized FTIR spectra (3000-2800 cm-1) of L. johnsonii La-1 cells grown under (a) anaerobic conditions and (b) aerobic conditions in control MRS and BMRS culture medium supplemented with EA-whey at different concentrations.
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Differences in the intensity of FTIR spectra for cells grown in the absence or
presence of oxygen can be observed (Figure 6.2a and b). Higher absorbance intensity of
the corresponding peaks was obtained for cells grown under both aeration conditions in
culture medium supplemented with 1.25 to 5% (w/v) of EA-whey in comparison with cells
grown in MRS. The wavenumber of the peak corresponding to νasCH2 stretching
vibrations was higher for cells grown under aerobic condition in the MRS in comparison
with cells grown in culture medium enriched with 5% EA-whey (from 2930 to 2928 cm-1).
6.6.2 DISCUSSION
6.6.2.1 Justification of the used strategy
It is well known that many probiotic bacteria show poor growth in milk. This can
be principally linked to three problems: limited lactose assimilation, proteolytic activity
and redox level of the milk. Thus, several substances have been studied for their potential
use as growth-promoting agents with the aim of finding suitable supplements to be
incorporated into milk-based products containing probiotic bacteria (Corcoran et al., 2004;
Dave & Shah, 1998; Gomes et al., 1998; Janer et al., 2004; Ravula & Shah, 1998). Among
them, different carbohydrates having potential prebiotic effects were increasingly studied
and added to probiotic-containing preparations to enhance their growth and ability to
compete with starter cultures when added in fermented dairy products (Gibson &
Roberfroid, 1995; Mäkeläinen et al., 2010; McComas & Gilliland, 2003; Oliveira et al.,
2011; Oliveira et al., 2009b). Recently, much attention has been paid to galacto-
oligosaccharides (GOS) derived from lactose and lactulose (Cardelle-Cobas et al., 2011;
García-Cayuela et al., 2014; Golowczyc et al., 2013). Moreover, the growth of some
probiotic strains was improved in milk supplemented with whey proteins and casein
hydrolysates (Bury et al., 1998; Petschow & Talbott, 1990). Thus, with the aim of
developing synbiotic combinations, the growth of representative probiotic strains
belonging to Bifidobacterium, Lactobacillus and Streptococcus genera was evaluated as
response to electro-activated whey (EA-whey) added to basic growth medium known to be
non-favourable for probiotics. In the present study, the used in vitro screening test
evaluated the combined prebiotic and antioxidant potential of EA-whey used to stimulate
the growth of the selected probiotic strains under model conditions to exclude the inference
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with some constituents of the matrix such as lactose and proteins if milk is used. The
targeted objective is that the most promising synbiotic will be further tested for their
prebiotic and antioxidant properties to be used in the development of fermented synbiotic
(with enhanced functionality) products.
6.6.2.2 Response of the bifidobacteria to EA-whey
The results of growth under anaerobic conditions showed different growth profiles
reflecting the ability of the used strains to metabolise different carbohydrates. The results
confirmed a species and strain-dependant behaviour, which is in agreement with the
reported information in the scientific literature (Gopal et al., 2001; Hopkins et al., 1998;
Huebner et al., 2007; Saarela et al., 2003). Among the tested bifidobacteria, two strains;
namely B. lactis Bb12 and B. longum R0175 were able to effectively use lactulose, whey
and EA-whey, which is rich of lactulose. The growth of these two Bifidobacterium strains
was stimulated proportionally with increasing EA-whey concentration in the growth
medium. Likewise, supplementation of the culture medium with lactulose and whey also
promoted the growth of these bifidobacteria strains but at less extend compared with EA-
whey, especially at 5% supplementation level. The used bifidobacteria strains were able to
assimilate a variety of carbon sources ascribed to their ability to produce enzyme β-
galactosidase, which is able to hydrolyse the β (1-4) link in different disaccharides such as
lactose and lactulose (Alander et al., 2001; Gopal et al., 2001; Smart et al., 1993). In the
same way, lactulose is a well-known commercially available prebiotic that has been already
recognized as a bifidogenic factor (Kneifel, 2000). Thus, in good agreement with these
findings, the enhanced growth of bifidobacteria strains grown in media supplemented with
EA-whey could be partially explained by its lactulose (prebiotic) content, lactose and
galactose which are well assimilated by B. lactis Bb12 and B. bifidum R0175. Indeed and
as shown in Table 6.1, EA-whey contains predominantly β (l-4) disaccharides: about 62%
lactose, 28% lactulose and about 8% galactose. Bifidobacteria can utilise galactose because
monosaccharides are assimilated faster, followed by lactulose and leave lactose for later
use. Beitane & Ciprovica (2011) showed that bifidobacteria poorly assimilate lactose in the
presence of lactulose in milk. On the other hand, the slow growth of bifidobacteria in milk
is also partially due to the lack of small peptides and free amino acids, which are essential
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as nitrogen source. The addition of whey proteins and acid casein hydrolases has been
previously reported to promote the growth of bifidobacteria (Gomes et al., 1998; Petschow
& Talbott, 1990). Dave & Shah (1998) reported the enhancement of growth and viability
of some bifidobacteria strains in yogurt supplemented with whey proteins. It is worthy to
highlight that all these factors are potentially present in the electro-activated whey (EA-
whey) used in the present study. Moreover, the presence of some conjugated Maillard
reaction products can also promote the growth of some bifidobacteria strains (Corzo-
Martínez et al., 2013). In our previous study, we demonstrated that EA-whey contains
glycoconjugated compounds with different hydrolysis and glycation degrees such as the
early Amadori compounds (Kareb et al., 2017a). In addition to the bifidogenic activity of
EA-whey through it lactulose content, some bifidogenic effects through its Maillard
reaction products content could be also expectable. Our results are in line with those
reported by Corzo-Martínez et al. (2012) who showed that the β-lactoglobulin-lactose
conjugates led to highest bifidogenic effects than lactulose alone. Thus, as EA-whey
contains a variety of compounds, it is difficult to ascertain which of these compounds
enhance preferentially the growth of B. lactis Bb12 and B. longum R0175 and the rate at
which a single compound is assimilated. Growth-promoting factors other than carbon and
azote sources could be also related to the growth-promoting properties of EA-whey.
Indeed, it could be also linked to the lowering redox potential of EA-whey, which was
characterized, by high reductive potential (antioxidative medium). Similarly, Bolduc et al.
(2006) have demonstrated that low redox potential values were appropriate for the growth
of bifidobacteria during storage.
6.6.2.3 Response of the lactobacillus to EA-whey
At the other hand, admitting that most bifidobacteria strains can use galacto-
oligosaccharides, only few lactobacilli strains possess this ability (Amaretti et al., 2007;
Ignatova et al., 2009). L. rhamnosus GG preferred glucose and galactose
(monosaccharides) in comparison to lactose, sucrose and lactulose (disaccharides). L.
rhamnosus GG was ascribed to have low ability to utilize lactose which is related to the
non-functionality of transporter machinery of lactose (Douillard et al., 2013). The
comparative genomic analysis of L. rhamnosus GG reveals that it is unable to hydrolyse
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casein, the major protein in milk (Kankainen et al., 2009). The growth parameters values
obtained in EA-whey were higher than those obtained with native whey. Moreover,
supplementation of the growth medium with of 5% EA-whey greatly enhanced the growth
performance of L. rhamnosus GG as shown by the µmax values obtained, which were
comparable to those obtained with galactose, control medium, and higher than those
obtained in the medium supplemented with native whey. The growth promoting effects of
EA-whey for this strain could be explained as follows: L. rhamnosus GG at first used the
readily fermented galactose, only after that, it utilised less easily a portion of lactose and
lactulose in the culture media supplemented with EA-whey. After the exhaustion of the
preferentially carbon sources, additive promoting effects could be present in EA-whey. No
information has been published regarding the Maillard reaction products (MRPs) as
promoting growth factor of L. rhamnosus GG. However, consistent with our results,
Kankainen et al. (2009) also reported that L. rhamnosus GG has a limited capacity to
synthesize amino acids; therefore, it requires exogenous amino acids and peptides for
growth. The electro-activation (EA) processing of whey, as presented in our previous
works, resulted in the partial hydrolysis of whey proteins to small molecular weight
peptides and rendered it available for direct L. rhamnosus GG uptake and requiring only
minimal energy to liberate the free amino acids when compared to intact proteins in native
whey. One of the interesting results of this study is that EA-whey may benefit to enhance
the growth of lactobacilli (probiotics), specially targeting those did not metabolise
prebiotics having β (1-4) linkages and which possess low proteolytic activity. Since the
BMRS has three sources of peptides, it is unlikely that the free amino acids and peptides
of EA-whey had a major effect on growth under our experimental conditions. However, it
can be expected that supplementation of milk itself would better allow the expression of
the growth-enhancing peptides of EA-whey. Further studies are warranted on this aspect.
6.6.2.4 Response of the specific yogurt cultures to EA-whey
One of the objective of the present study is to identify promising synbiotic
candidates for functional yogurt manufacture. The specific yogurt cultures, S. thermophilus
R2092 and L. bulgaricus R5083, significantly grew in EA-whey in comparison to all the
tested substrates indicating a stimulating (prebiotic) effect of EA-whey. Moreover,
169
supplementation of the medium with whey also greatly enhanced its growth. The efficiency
of EA-whey on S. thermophilus R2092 could be related to the accessibility of the required
free amino acids or bioactive peptides as easy nitrogen source. Our results are, to some
extent, similar to those reported by Bury et al. (1998), who indicated that whey protein
fractions (α-LA and β-LG) enhanced the growth of S. thermophilus ST20 and L. bulgaricus
11842 used in yogurt manufacturing. Here we saw that besides being fermented by the
selected probiotic strains, EA-whey was well utilised by the yogurt cultures, suggesting the
importance of careful selection of suitable starter culture for further maximizing the growth
of probiotic bacteria and development synbiotic products. The results of this preliminary
screening study indicate that EA-whey could present real potential in the development of
new functional products as it promoted the growth of pure probiotic cultures belonging to
Bifidobacterium and Lactobacillus. The results also established the prebiotic and
bifidogenic potential of EA-whey through its easy accessibility as carbon and nitrogen
sources and opens perspectives for the developing prebiotic of novel generation which may
have better functionality than those shared in the market.
6.6.2.5 Response of the L. johnsonii La-1 to EA-whey
The most sensitive strain used in the present study, L. johnsonii La-1, had the
highest growth in the medium supplemented with EA-whey under both anaerobic and
aerobic conditions. This strain was described to be highly sensitive to oxidative stress and
when incubated in the presence of oxygen, it is characterized by evident lower growth and
stagnation at the early stage (Hertzberger et al., 2013). Hertzberger et al. (2014) identified
a conserved NADH-dependent Flavin reductase that is prominently involved in H2O2
production by L. johnsonii La-1. The ODmax value obtained under aerobic conditions in
MRS was under 0.7, which confirmed the oxygen sensitivity of this strain. Moreover, the
µmax value obtained in medium supplemented with 1.25% of EA-whey under aerobic
condition was similar to those obtained in the control medium. In general, the L.
acidophilus species does not possess the enzyme catalase, which can scavenge the H2O2
(Gilliland & Speck, 1977). Without sufficient scavenging mechanisms or/and oxygen
scavenger substances, the accumulation of H2O2 can eventually lead to viability loss of
probiotics, especially L. johnsonii La-1. Dave & Shah (1997a) reported that the viability of
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L. acidophilus was affected by the elevated level of H2O2 produced by L. bulgaricus in
yogurts. Corroborating with these findings, Dave & Shah (1997a) also demonstrated the
effectiveness of ascorbic acid as oxygen scavenger to improve at great extend the viability
of probiotics in yogurt prepared with starter culture. Thus, the good growth of L. johnsonii
La-1 under aerobic conditions could be explained by the strong ability of EA-whey to
scavenge oxygen and oxygen reactive species, and to prevent the accumulation of H2O2
produced during growth of L. johnsonii La-1. In this context, we already demonstrated that
the antioxidant activity of the EA-whey is based on its ability to scavenge oxygen radicals,
to have reducing power and to trap positively charged electrophilic species or carry out
metal chelation to form inactive complexes (Kareb et al., 2016; Kareb et al., 2017b). The
enhancement of L. johnsonii La-1 growth could also be explained fort part by the negative
redox potential of EA-whey, which could result through the availability of reducing
compounds. Similar results were reported by (Gaudreau et al., 2013) who showed the
antioxidant potential of green tea extract and protective effect on the growth of L. helveticus
R0052 grown under aerobic conditions. These authors also showed the role of green tea
extract to modulate the structure of lipid membrane of L. helveticus R0052 (Gaudreau et
al., 2016). Thus, to gain insight into the possible protective effect of EA-whey on the
membrane structure of L. johnsonii La-1, FTIR analysis was employed to monitor the effect
of EA-whey at different concentrations (1.25 to 5%) on the structure and order of
membrane lipids of L. johnsonii La-1. The results clearly showed the protective effect of
EA-whey against cell membrane lipids oxidation.
6.6.2.5 FTIR analysis of L. johnsonii membrane lipids
L. johnsonii La-1 had optimal growth in the medium supplemented with EA-whey
under both anaerobic and aerobic conditions. The lower biomass level (ODmax < 0.7) that
was obtained in non-supplemented MRS medium for cells grown under aerobic conditions
than those grown under anaerobic conditions confirmed the oxygen sensitivity of L.
johnsonii La-1 (Tables 6.4-6.5). This strain was previously described to be sensitive to
oxidative stress and when incubated in the presence of oxygen, low growth and stagnation
at the early stage was observed (Hertzberger et al., 2013). Moreover, Herzberger et al.
(2014) identified a conserved NADH-dependent Flavin reductase that is prominently
171
involved in H2O2 production by L. johnsonii La-1. In general, the L. acidophilus species
does not possess catalase enzyme, which can decompose (scavenge) the H2O2 (Gilliland &
Speck, 1977). Without sufficient scavenging mechanisms or/and oxygen scavenger
substances, the accumulation of H2O2 can eventually lead to viability loss of L. johnsonii
La-1. Dave & Shah (1997b) reported that the viability of L. acidophilus was affected by
the elevated level of H2O2 produced by L. bulgaricus in yogurts. Corroborating with these
findings, Dave & Shah (1997a) also demonstrated the effectiveness of ascorbic acid to
scavenge H2O2 and to improve the growth of H2O2-sensitive probiotic bacteria. Thus, the
good growth of L. johnsonii La-1 under aerobic conditions could be explained by the strong
ability of EA-whey to scavenge and prevent the accumulation of H2O2 produced during
cell growth. In our previous study, we reported that EA-whey exerted antioxidant
properties based on its ability to scavenge oxygen radicals, to have reducing power and to
trap positively charged electrophilic species or carry out metal chelation to form inactive
complexes (Kareb et al., 2017b). The enhanced growth of L. johnsonii La-1 in EA-Whey
supplemented MRS medium could be explained for part by the reducing (negative) redox
potential of EA-whey which could create an adequate environmental growth conditions
(Bolduc et al., 2006). Similarly, Gaudreau et al. (2013) showed that addition of green tea
extract (GTE) as a complex of antioxidant compounds resulted in enhancement growth of
L. helveticus R0052 grown under aerobic condition trough the reduction of redox potential.
These authors also showed the role of GTE to modulate the structure of lipid membrane of
L. helveticus R0052 (Gaudreau et al., 2016). Effectively, exposition of probiotics to oxygen
can damage (oxidize) their lipids leading to increasing the membrane permeability due the
higher synthesis of unsaturated fatty acids (Talwalkar & Kailasapathy, 2004). Thus, to gain
insight into the possible protective effect of EA-whey on the membrane structure of L.
johnsonii La-1, FTIR spectroscopy analysis was monitored in this study. The differences
observed in the intensity bands corresponding to the functional lipid groups reflect the
effects of presence or absence of oxygen exposure to bacterial membrane. The shift to a
higher wavenumber of vasCH2 groups stretching bands for L. johnosnii cells grown under
aerobic conditions in non-supplemented MRS suggests a decrease in the acyl chain of
membrane lipids. Moreover, supplementation of the growth medium with 5% (w/v) EA-
whey resulted in lower wavenumbers of the νasCH2 stretching bands confirming the
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positive effect of EA-whey leading to the increase of density of the acyl chains on lipids of
the bacterial cells. Thus, supplementation of the growth medium with EA-whey resulted in
the limitation of oxidative damage, rendered L. johnosnii La-1 strain more resistant by
either prevention of H2O2 accumulation, and subsequently limited the alteration of the
phospholipids bilayer. Our results are in agreement with those of Gaudreau et al. (2016)
who reported a positive (protective) effect green tea extract (GTE) added at 2000 μg/mL
GTE on L. helveticus cells by changing their lipid composition in response to oxygen
exposure. Thus, in addition to their prebiotic properties, EA-whey could be used as natural
antioxidant to unsure additional protection for the growth of oxygen-sensitive probiotic
bacteria during manufacturing and storage of dairy products.
In summary, EA-whey can address the three main problems associated with growth
of probiotics in milk through a variety of carbohydrates, growth-promoting peptides and
antioxidant activity.
A word should finally be said on the interest of this EA-whey product from the
perspective of a “durable” agri-food sector. The advantage of the EA-whey process is that
no dairy by-product is generated. From an ecological standpoint, this is desirable.
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6.7 CONCLUSION
As far as we know, this is the first study on the effect of developed EA-whey as
potential prebiotic and antioxidant on pure cultures growth. EA-whey contains versatile
compounds, which allow extending the proteolytic and glycolytic properties of probiotic
bacteria. However, functional efficiency of EA-whey is not governed only nitrogen and
carbon compositions but also by the several other factors that should be further
investigated. These results can be used for selecting probiotic strains to design synbiotic
formulations and further essays will be conducted on the stability of probiotic bacteria
during storage in food matrices containing EA-whey and during gastrointestinal transitions.
EA-whey could be an interesting ingredient for commercial producers of probiotics
because this ingredient is natural and relatively inexpensive and its addition to culture
media is very simple and could allow growth and the viability of probiotic strains during
product conception and storage. In addition to the demonstrated prebiotic and the
antioxidant effects of EA-whey, subsequent investigations connected some health
promoting consequences could be provide new perspectives for the EA-whey application
in pharmaceutical fields. The EA-whey process is in line with current priorities of the sector
towards clean technologies for a durable agri-food sector.
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CHAPTER 7: General conclusion and perspectives
6.1 GENERAL CONCLUSION
The research undertaken in this doctoral project successfully achieved the following
objectives:
(i) Optimisation of key parameters for lactulose production from whey
using electro-activation,
(ii) Enhancement of antioxidant and other biological proprieties of electro-
activated whey and structural characterization of relevant molecules.
(iii) Utilizing electro-activated whey as functional ingredient for improving
the growth of probiotic bacteria.
This research project broadly enhanced our understanding of electro-activation and more
importantly yielded valuable evidence supporting the technological feasibility and
potential of electro-activation as a tool for adding value to whey, an abundant by-product
of the dairy industry.
In a previous study, electro-activation (EA) was successfully used as a reagent less
and energy frugal technology for electro-isomerization of lactose into lactulose. The study
although provided key insights into EA as a technology for electro-isomerization of lactose
into lactulose using milk lactose, it did not expand into other sources such as whey. The
present study tried to expand the application of EA with sweet whey as lactose source. The
production of lactulose from cheese whey with the help of EA has not been previously
attempted. Thus, the first objective of this thesis was focused on the optimization of some
key experimental parameters on lactulose yield such as the electric current intensity,
reaction time, temperature, whey volume, as well as whey concentration. The maximum
yield (35% of lactulose) with high purity was achieved with 100 mL volume at a working
temperature of 10 °C after 40 min of electro-activation (reaction) under a 400 mA electric
current intensity by using a 7% feed whey solution. Interestingly, it was observed that the
electro-isomerization of lactose in situ of whey to lactulose was accomplished with a
simultaneous enhancement in the antioxidant activity of whey. Although the reason for the
175
increase in the antioxidant capacity of electro-activated whey (EA-whey) can be attributed
to the formation of Maillard reaction products (MRPs), the mechanism(s) of the antioxidant
activity of EA-whey has not yet been elucidated. One of the challenges in understanding
MRPs is the lack of efficient non-thermal technologies that can control the extent of
Maillard reaction (MR), while attaining the desirable functional characteristics. Therefore,
based on the work presented here, we proposed EA as an innovative technology to prepare
MRPs-whey. Furthermore, in order to identify the possible antioxidant mechanism(s) of
EA-whey, the impact of varying experimental conditions (current intensity, and feed
concentration as a function of the reaction time) on the antioxidant activity was also
investigated. The antioxidant activity of the Maillard-reacted-whey protein hydrolysates
was ascribed to a cooperative effect of multiple reactions; including their ability to break
or terminate oxidative chains, transferring electrons, donating hydrogen atoms, and to
sequestering pro-oxidative metal ions. Thus, it could be said that MRPs derived from EA-
whey may act as both primary and secondary antioxidants.
There is a dearth of information relating the antioxidant activity of MRPs-whey to
their structural characteristics and, therefore, structural characteristics of MRPs-whey were
also studied. It was found that electro-activation promoted the intermediate stage of MR,
and decaled the final stage, resulting in slight browning. The molecular structure of the
MRPs-whey was characterized to be Schiff base compounds that have been reported in the
scientific literature to be safe for human health.
It was observed that in addition to the formation of MRPs, EA also led to the
hydrolysis of whey proteins. Thus, it was postulated that the final products could have other
bioactive properties. Based on the SDS-PAGE profile, 7% whey was chosen for further
investigations by using LC-MS-MS technique. The peptide profiling showed 27 peptides,
all derived from α, β and κ caseins. The peptide FFVAPFPEVFGK derived from α-casein
was previously reported to exhibit ACE inhibition and anticancer activities. Other
multifunctional bioactive peptides, with biological properties (ACE-inhibition,
antimicrobial activity, opioid agonist, and antioxidant activity) were also identified.
One other interesting opportunity which this work presented was to use EA-whey
as a functional ingredient along with prebiotics possessing antioxidant properties which
could potentially promote the growth of probiotic bacteria. The efficacy of EA-whey on
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the growth of probiotics of Bifidobacterium, Lactobacillus and Streptococcus genera in
pure cultures was demonstrated in this study. The results not only showed that all probiotic
strains grew to the highest culture optical density using EA-whey as sole carbon source but
also showed that EA-whey was comparatively a better bifidogenic factor than lactulose.
Additionally, EA-whey was found to elicit a protective effect on L. johnsonii La-1 during
growth in the presence of oxygen. This effect may be related to the ability of EA-whey to
scavenge hydrogen peroxide and prevent its accumulation. The FTIR analysis of the cells
growing with EA-whey and without it showed that EA-whey changed the lipid cell
membrane structure order of the cells growing in EA-whey enriched medium.
In conclusion, all these results confirmed the starting hypothesis. The outcome of this
work validates the efficacy of EA approach as a green, simple, safe and rapid technology
for the production of in situ enriched whey with versatile functionalities and bioactivities.
This study optimized the key parameters and resulted in high lactulose yield with high
purity at low temperatures and short reaction time. The EA is an emerging technology, that
can achieve the formation of functional MRPs-whey under controlled processing
conditions and meet consumer requirements for safer products. Additionally, the biological
properties of EA-whey were enhanced through the generation of new bioactive peptides.
Overall, EA-whey as a prebiotic supplement possessing antioxidant capacity has immense
potential for application not only in the dairy industry but also as a functional food
ingredient with potent bioactivities and designer functionalities.
7.2 PESPECTIVES
The results obtained in this research not only provide valuable new information
pertaining to EA, it also opens new doors for further and in-depth study. It will be of interest
to study the technological feasibility of EA to generate bioactive peptides in simplified
protein model systems. The whole spectrum of bioactivity of MRPs-whey can be studied
to identify other functionalities (anti-inflammatory, antihypertensive and antimicrobial
properties). Additionally, further research on the techno functional properties of MRPs-
whey needs to be done as MR has been reported to be an effective approach to improve the
technofunctional properties of whey proteins. This may help in broadening the potential
application base of MRPs-whey in the food industry. Another exciting avenue of study is
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encapsulation technology with respect to MRPs-whey; MRPs formed from whey proteins
conjugated to isomaltooligosaccharide has been reported to be an excellent carrier agent
for the encapsulation of probiotics in comparison to whey protein and
isomaltooligosaccharide co-components. Based on these findings, EA-whey enriched with
lactulose and MRPs could be a promising encapsulating material for maintaining the
viability of probiotics during exposure to gastrointestinal environmental conditions.
Finally, for further extending the application in dairy products, EA-whey should be
incorporated in probiotic yogurts to stimulate probiotics’ growth and survival during
manufacturing process and storage period.
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REFERENCES
Abd El-Salam, M., El-Shibiny, S., & Salem, A. (2009). Factors affecting the functional
properties of whey protein products: a review. Food Reviews International, 25(3),
251-270.
Adachi, S. (1958). Formation of lactulose and tagatose from lactose in strongly heated
milk. nature, 181, 840-841.
Adachi, S., & Patton, S. (1961). Presence and Significance of Lactulose in Milk Products:
A Review. Journal of dairy science, 44(8), 1375-1393.
doi:http://dx.doi.org/10.3168/jds.S0022-0302(61)89899-8
Adamczak, M., Charubin, D., & Bednarski, W. (2009). Influence of reaction medium
composition on enzymatic synthesis of galactooligosaccharides and lactulose from
lactose concentrates prepared from whey permeate. Chemical Papers, 63(2), 111-
116.
Adams, C. A. (2010). The probiotic paradox: live and dead cells are biological response
modifiers. Nutrition research reviews, 23(01), 37-46.
Agarwal, S., Misra, R., Vishvkarma, P., & Saxena, A. (2016). PROBIOTICS-A NOVEL
APPROACH FOR HEALTH BENEFITS.
Aider, M., & de Halleux, D. (2007). Isomerization of lactose and lactulose production.
Trends in Food Science & Technology, 18(7), 356-364.
Aider, M., & Gimenez-Vidal, M. (2012). Lactulose synthesis by electro-isomerization of
lactose: Effect of lactose concentration and electric current density. Innovative
Food Science & Emerging Technologies, 16, 163-170.
Aider, M., Gnatko, E., Benali, M., Plutakhin, G., & Kastyuchik, A. (2012). Electro-
activated aqueous solutions: theory and application in the food industry and
biotechnology. Innovative Food Science & Emerging Technologies, 15, 38-49.
Aissa, A. A., & Aïder, M. (2013a). Ion exchange membranes controlled electro-catalytic
synthesis of lactulose from lactose under refrigerated conditions. Innovative Food
Science & Emerging Technologies, 20, 299-309.
doi:http://dx.doi.org/10.1016/j.ifset.2013.09.013
Aissa, A. A., & Aïder, M. (2013b). Lactose isomerization into lactulose in an electro-
activation reactor and high-performance liquid chromatography (HPLC)
179
monitoring of the process. Journal of Food Engineering, 119(1), 115-124.
doi:http://dx.doi.org/10.1016/j.jfoodeng.2013.05.011
Aissa, A. A., & Aïder, M. (2014a). Electro-catalytic isomerization of lactose into lactulose:
The impact of the electric current, temperature and reactor configuration.
International Dairy Journal, 34(2), 213-219.
doi:http://dx.doi.org/10.1016/j.idairyj.2013.08.010
Aissa, A. A., & Aïder, M. (2014b). Lactulose: production and use in functional food,
medical and pharmaceutical applications. Practical and critical review.
International journal of food science & technology, 49(5), 1245-1253.
Al-Dabbas, M. M., Al-Ismail, K., & Al-Abdullah, B. M. (2011). Effect of Chemical
Composition on the Buffering Capacity of Selected Dairy Products Jordan Journal
of Agricultural Sciences, 7(4), 690-700.
Al-Sheraji, S. H., Ismail, A., Manap, M. Y., Mustafa, S., Yusof, R. M., & Hassan, F. A.
(2013). Prebiotics as functional foods: A review. Journal of Functional Foods, 5(4),
1542-1553.
Alander, M., Mättö, J., Kneifel, W., Johansson, M., Kögler, B., Crittenden, R., . . . Saarela,
M. (2001). Effect of galacto-oligosaccharide supplementation on human faecal
microflora and on survival and persistence of Bifidobacterium lactis Bb-12 in the
gastrointestinal tract. International Dairy Journal, 11(10), 817-825.
Allgeyer, L. C., Miller, M. J., & Lee, S. Y. (2010). Sensory and microbiological quality of
yogurt drinks with prebiotics and probiotics. Journal of dairy science, 93(10),
4471-4479. doi:https://doi.org/10.3168/jds.2009-2582
Alvarez-Ordóñez, A., & Prieto, M. (2010). Changes in ultrastructure and Fourier transform
infrared spectrum of Salmonella enterica serovar Typhimurium cells after exposure
to stress conditions. Applied and environmental microbiology, 76(22), 7598-7607.
Amaretti, A., Bernardi, T., Tamburini, E., Zanoni, S., Lomma, M., Matteuzzi, D., & Rossi,
M. (2007). Kinetics and metabolism of Bifidobacterium adolescentis MB 239
growing on glucose, galactose, lactose, and galactooligosaccharides. Applied and
environmental microbiology, 73(11), 3637-3644.
180
Ames, B. N., Shigenaga, M. K., & Hagen, T. M. (1993). Oxidants, antioxidants, and the
degenerative diseases of aging. Proceedings of the National Academy of Sciences,
90(17), 7915-7922.
Amine, E., Baba, N., Belhadj, M., Deurenbery-Yap, M., Djazayery, A., Forrester, T., . . .
MBuyamba, J. (2002). Diet, nutrition and the prevention of chronic diseases:
report of a Joint WHO/FAO Expert Consultation: World Health Organization.
Anderson, J. L., Miles, C., & Tierney, A. C. (2017). Effect of probiotics on respiratory,
gastrointestinal and nutritional outcomes in patients with cystic fibrosis: A
systematic review. Journal of Cystic Fibrosis, 16(2), 186-197.
doi:http://doi.org/10.1016/j.jcf.2016.09.004
Andrews, G. R. (1986). Formation and occurrence of lactulose in heated milk. Journal of
Dairy Research, 53(04), 665-680. doi:doi:10.1017/S0022029900033197
Andrews, G. R., & Prasad, S. K. (1987). Effect of the protein, citrate and phosphate content
of milk on formation of lactulose during heat treatment. Journal of Dairy Research,
54(02), 207-218.
Anese, M., & Fogliano, V. (2001). The Maillard reaction in foods. Chemical-technological
and nutritional aspects. Industrie Alimentari (Italy).
Angel de la Fuente, M., Juárez, M., de Rafael, D., Villamiel, M., & Olano, A. n. (1999).
Isomerization of lactose catalyzed by alkaline-substituted sepiolites. Food
Chemistry, 66(3), 301-306.
Angiolillo, L., Conte, A., Faccia, M., Zambrini, A. V., & Del Nobile, M. A. (2014). A new
method to produce synbiotic Fiordilatte cheese. Innovative Food Science &
Emerging Technologies, 22, 180-187.
doi:https://doi.org/10.1016/j.ifset.2013.09.010
Anukam, K. C., & Reid, G. (2007). Probiotics: 100 years (1907-2007) after Elie
Metchnikoff’s observation. Communicating current research and educational
topics and trends in applied microbiology, 1, 466-474.
Arena, S., Renzone, G., D’Ambrosio, C., Salzano, A. M., & Scaloni, A. (2017). Dairy
products and the Maillard reaction: A promising future for extensive food
characterization by integrated proteomics studies. Food Chemistry, 219, 477-489.
doi:http://dx.doi.org/10.1016/j.foodchem.2016.09.165
181
Arihara, K., Zhou, L., & Ohata, M. (2017). Bioactive Properties of Maillard Reaction
Products Generated From Food Protein-derived Peptides. Advances in Food and
Nutrition Research, 81, 161-185.
doi:http://dx.doi.org/10.1016/bs.afnr.2016.11.005
Athira, S., Mann, B., Saini, P., Sharma, R., Kumar, R., & Singh, A. K. (2015). Production
and characterisation of whey protein hydrolysate having antioxidant activity from
cheese whey. Journal of the Science of Food and Agriculture, 95(14), 2908-2915.
Avila Ruiz, G., Xi, B., Minor, M., Sala, G., van Boekel, M., Fogliano, V., & Stieger, M.
(2016). High-pressure–high-temperature processing reduces Maillard reaction and
viscosity in whey protein–sugar solutions. Journal of Agricultural and Food
Chemistry, 64(38), 7208-7215.
Badel, S., Bernardi, T., & Michaud, P. (2011). New perspectives for Lactobacilli
exopolysaccharides. Biotechnology advances, 29(1), 54-66.
Bakker-Zierikzee, A. M., Alles, M. S., Knol, J., Kok, F. J., Tolboom, J. J., & Bindels, J. G.
(2005). Effects of infant formula containing a mixture of galacto-and fructo-
oligosaccharides or viable Bifidobacterium animalis on the intestinal microflora
during the first 4 months of life. British Journal of Nutrition, 94(05), 783-790.
Baldi, A., Ioannis, P., Chiara, P., Eleonora, F., & Roubini, C. (2005). Biological effects of
milk proteins and their peptides with emphasis on those related to the
gastrointestinal ecosystem. Journal of Dairy Research, 72(S1), 66-72.
Bali, V., Panesar, P. S., Bera, M. B., & Panesar, R. (2015). Fructo-oligosaccharides:
production, purification and potential applications. Critical reviews in food science
and nutrition, 55(11), 1475-1490.
Ballongue, J., Schumann, C., & Quignon, P. (1997). Effects of lactulose and lactitol on
colonic microflora and enzymatic activity. Scandinavian Journal of
Gastroenterology, 32(sup222), 41-44.
Banaszewska, A., Cruijssen, F., Claassen, G. D. H., & van der Vorst, J. G. A. J. (2014).
Effect and key factors of byproducts valorization: The case of dairy industry.
Journal of dairy science, 97(4), 1893-1908. doi:http://dx.doi.org/10.3168/jds.2013-
7283
182
Bansal, N., & Bhandari, B. (2016). Functional Milk Proteins: Production and Utilization—
Whey-Based Ingredients Advanced Dairy Chemistry (pp. 67-98): Springer.
Beitane, I., & Ciprovica, I. (2011). The study of carbohydrates fermentation ability of B.
lactis in milk. Paper presented at the Conference Proceedings of the 6th Baltic
Conference on Food Science and Technology FOODBALT-2011, Jelgava, Latvia,
5-6 May, 2011. Innovations for food science and production.
Bengmark, S. (2001). Pre-, pro-and synbiotics. Current Opinion in Clinical Nutrition &
Metabolic Care, 4(6), 571-579.
Bengmark, S. (2003). Use of some pre-, pro-and synbiotics in critically ill patients. Best
Practice & Research Clinical Gastroenterology, 17(5), 833-848.
Benjakul, S., Lertittikul, W., & Bauer, F. (2005). Antioxidant activity of Maillard reaction
products from a porcine plasma protein–sugar model system. Food Chemistry,
93(2), 189-196.
Benkerroum, N. (2010). Antimicrobial peptides generated from milk proteins: a survey and
prospects for application in the food industry. A review. International Journal of
Dairy Technology, 63(3), 320-338.
Berg, H., & Van Boekel, M. (1994). Degradation of lactose during heating of milk. 1.
Reaction pathways. Netherlands Milk and Dairy Journal, 48, 157-175.
Bermudez-Brito, M., Plaza-Díaz, J., Muñoz-Quezada, S., Gómez-Llorente, C., & Gil, A.
(2012). Probiotic mechanisms of action. Annals of Nutrition and Metabolism,
61(2), 160-174.
Bermudez‐Brito, M., Sahasrabudhe, N. M., Rösch, C., Schols, H. A., Faas, M. M., & Vos,
P. (2015). The impact of dietary fibers on dendritic cell responses in vitro is
dependent on the differential effects of the fibers on intestinal epithelial cells.
Molecular nutrition & food research, 59(4), 698-710.
Betoret, E., Betoret, N., Vidal, D., & Fito, P. (2011). Functional foods development: trends
and technologies. Trends in Food Science & Technology, 22(9), 498-508.
Beylot, M. (2005). Effects of inulin-type fructans on lipid metabolism in man and in animal
models. British Journal of Nutrition, 93(S1), S163-S168.
Bigliardi, B., & Galati, F. (2013). Innovation trends in the food industry: the case of
functional foods. Trends in Food Science & Technology, 31(2), 118-129.
183
Bindels, L. B., Delzenne, N. M., Cani, P. D., & Walter, J. (2015). Towards a more
comprehensive concept for prebiotics. Nature reviews Gastroenterology &
hepatology, 12(5), 303-310.
Bogue, J., Collins, O., & Troy, A. J. (2017). Chapter 2 - Market analysis and concept
development of functional foods A2 - Bagchi, Debasis. In S. Nair (Ed.), Developing
New Functional Food and Nutraceutical Products (pp. 29-45). San Diego:
Academic Press.
Bolduc, M.-P., Raymond, Y., Fustier, P., Champagne, C. P., & Vuillemard, J.-C. (2006).
Sensitivity of bifidobacteria to oxygen and redox potential in non-fermented
pasteurized milk. International Dairy Journal, 16(9), 1038-1048.
Bologa, M., Stepurina, T., Bologa, A., Polikarpov, A., & Sprinchan, E. (2009).
Optimization of lactose isomerization into lactulose by the electrophysical method.
Surface Engineering and Applied Electrochemistry, 45(5), 415-419.
Bologa, M. K., Sprinchan, E. G., & Bologa, A. M. (2008). Recovery of lactulose products
and protein-mineral concentrate. Surface Engineering and Applied
Electrochemistry, 44(5), 410-414. doi:10.3103/s1068375508050128
Boro, J., Deka, D., & Thakur, A. J. (2012). A review on solid oxide derived from waste
shells as catalyst for biodiesel production. Renewable and Sustainable Energy
Reviews, 16(1), 904-910.
Boushey, C. J., Coulston, A. M., Rock, C. L., & Monsen, E. (2001). Nutrition in the
Prevention and Treatment of Disease: Academic Press.
Božanić, R., Barukčić, I., & Lisak, K. (2014). Possibilities of whey utilisation. Austin J.
Nutr. Food Sci., 2(7), 1036.
Brandelli, A., Daroit, D. J., & Corrêa, A. P. F. (2015). Whey as a source of peptides with
remarkable biological activities. Food Research International, 73, 149-161.
doi:http://dx.doi.org/10.1016/j.foodres.2015.01.016
Bravo, J. A., Forsythe, P., Chew, M. V., Escaravage, E., Savignac, H. M., Dinan, T. G., . .
. Cryan, J. F. (2011). Ingestion of Lactobacillus strain regulates emotional behavior
and central GABA receptor expression in a mouse via the vagus nerve. Proceedings
of the National Academy of Sciences, 108(38), 16050-16055.
184
Bremner, J. M., & Mulvaney, C. (1982). Nitrogen—total. Methods of soil analysis. Part 2.
Chemical and microbiological properties(methodsofsoilan2), 595-624.
Bury, D., Jelen, P., & Kimura, K. (1998). Whey protein concentrate as a nutrient
supplement for lactic acid bacteria. International Dairy Journal, 8(2), 149-151.
Caetano-Silva, M. E., Bertoldo-Pacheco, M. T., Paes-Leme, A. F., & Netto, F. M. (2015).
Iron-binding peptides from whey protein hydrolysates: Evaluation, isolation and
sequencing by LC–MS/MS. Food Research International, 71, 132-139.
doi:http://dx.doi.org/10.1016/j.foodres.2015.01.008
Calvo, M. M., & Olano, A. (1989). Formation of galactose during heat treatment of milk
and model systems. Journal of Dairy Research, 56(05), 737-740.
doi:doi:10.1017/S0022029900029307
Canabady-Rochelle, L. L. S., Harscoat-Schiavo, C., Kessler, V., Aymes, A., Fournier, F.,
& Girardet, J.-M. (2015). Determination of reducing power and metal chelating
ability of antioxidant peptides: Revisited methods. Food Chemistry, 183, 129-135.
doi:http://dx.doi.org/10.1016/j.foodchem.2015.02.147
Cani, P. D., Lecourt, E., Dewulf, E. M., Sohet, F. M., Pachikian, B. D., Naslain, D., . . .
Delzenne, N. M. (2009). Gut microbiota fermentation of prebiotics increases
satietogenic and incretin gut peptide production with consequences for appetite
sensation and glucose response after a meal. The American journal of clinical
nutrition, 90(5), 1236-1243.
Cardarelli, H. R., Buriti, F. C. A., Castro, I. A., & Saad, S. M. I. (2008). Inulin and
oligofructose improve sensory quality and increase the probiotic viable count in
potentially synbiotic petit-suisse cheese. LWT - Food Science and Technology,
41(6), 1037-1046. doi:https://doi.org/10.1016/j.lwt.2007.07.001
Cardelle-Cobas, A., Corzo, N., Olano, A., Peláez, C., Requena, T., & Ávila, M. (2011).
Galactooligosaccharides derived from lactose and lactulose: influence of structure
on Lactobacillus, Streptococcus and Bifidobacterium growth. International journal
of food microbiology, 149(1), 81-87.
Carulli, S., Calvano, C. D., Palmisano, F., & Pischetsrieder, M. (2011). MALDI-TOF MS
characterization of glycation products of whey proteins in a glucose/galactose
185
model system and lactose-free milk. Journal of agricultural and food chemistry,
59(5), 1793-1803.
Carvalho, F., Prazeres, A. R., & Rivas, J. (2013). Cheese whey wastewater:
characterization and treatment. Science of the total environment, 445, 385-396.
Chabance, B., Jollès, P., Izquierdo, C., Mazoyer, E., Francoual, C., Drouet, L., & Fiat, A.-
M. (1995). Characterization of an antithrombotic peptide from α-casein in newborn
plasma after milk ingestion. British Journal of Nutrition, 73(4), 583-590.
Champagne, C. P. (2009). Some technological challenges in the addition of probiotic
bacteria to foods Prebiotics and probiotics science and technology (pp. 761-804):
Springer.
Champagne, C. P., Gardner, N. J., & Roy, D. (2005). Challenges in the addition of probiotic
cultures to foods. Critical reviews in food science and nutrition, 45(1), 61-84.
Champagne, C. P., Ross, R. P., Saarela, M., Hansen, K. F., & Charalampopoulos, D.
(2011). Recommendations for the viability assessment of probiotics as concentrated
cultures and in food matrices. International journal of food microbiology, 149(3),
185-193.
Chapman, C., Gibson, G. R., & Rowland, I. (2011). Health benefits of probiotics: are
mixtures more effective than single strains? European journal of nutrition, 50(1),
1-17.
Charalampopoulos, D., & Rastall, R. A. (2009). Prebiotics and probiotics science and
technology (Vol. 1): Springer.
Charalampopoulos, D., & Rastall, R. A. (2012). Prebiotics in foods. Current Opinion in
Biotechnology, 23(2), 187-191. doi:https://doi.org/10.1016/j.copbio.2011.12.028
Charteris, W., Kelly, P., Morelli, L., & Collins, J. (1998). Development and application of
an in vitro methodology to determine the transit tolerance of potentially probiotic
Lactobacillus and Bifidobacterium species in the upper human gastrointestinal
tract. Journal of applied microbiology, 84(5), 759-768.
Chatterton, D. E. W., Smithers, G., Roupas, P., & Brodkorb, A. (2006). Bioactivity of β-
lactoglobulin and α-lactalbumin—Technological implications for processing.
International Dairy Journal, 16(11), 1229-1240.
doi:http://dx.doi.org/10.1016/j.idairyj.2006.06.001
186
Chaud, M. V., Izumi, C., Nahaal, Z., Shuhama, T., Bianchi, M. d. L. P., & Freitas, O. d.
(2002). Iron derivatives from casein hydrolysates as a potential source in the
treatment of iron deficiency. Journal of Agricultural and Food Chemistry, 50(4),
871-877.
Chawla, S. P., Chander, R., & Sharma, A. (2009). Antioxidant properties of Maillard
reaction products obtained by gamma-irradiation of whey proteins. Food
Chemistry, 116(1), 122-128.
doi:http://dx.doi.org/10.1016/j.foodchem.2009.01.097
Chen, K. N., Chen, M. J., Liu, J. R., Lin, C. W., & Chiu, H. Y. (2005). Optimization of
incorporated prebiotics as coating materials for probiotic microencapsulation.
Journal of food science, 70(5).
Chen, Y. S., Srionnual, S., Onda, T., & Yanagida, F. (2007). Effects of prebiotic
oligosaccharides and trehalose on growth and production of bacteriocins by lactic
acid bacteria. Letters in applied microbiology, 45(2), 190-193.
Chevalier, F., Chobert, J.-M., Genot, C., & Haertlé, T. (2001a). Scavenging of free radicals,
antimicrobial, and cytotoxic activities of the Maillard reaction products of β-
lactoglobulin glycated with several sugars. Journal of Agricultural and Food
Chemistry, 49(10), 5031-5038.
Chevalier, F., Chobert, J.-M., Mollé, D., & Haertlé, T. (2001b). Maillard glycation of $\
beta $-lactoglobulin with several sugars: comparative study of the properties of the
obtained polymers and of the substituted sites. Le lait, 81(5), 651-666.
Chevalier, F., Chobert, J.-M., Popineau, Y., Nicolas, M. G., & Haertlé, T. (2001c).
Improvement of functional properties of β-lactoglobulin glycated through the
Maillard reaction is related to the nature of the sugar. International Dairy Journal,
11(3), 145-152.
Chiu, T.-h., Chen, M.-l., & Chang, H.-c. (2009). Comparisons of emulsifying properties of
Maillard reaction products conjugated by green, red seaweeds and various
commercial proteins. Food Hydrocolloids, 23(8), 2270-2277.
Chonan, O., Takahashi, R., & Watanuki, M. (2001). Role of activity of gastrointestinal
microflora in absorption of calcium and magnesium in rats fed β1-4 linked
187
galactooligosaccharides. Bioscience, biotechnology, and biochemistry, 65(8),
1872-1875.
Chong, E. S. L. (2014). A potential role of probiotics in colorectal cancer prevention:
review of possible mechanisms of action. World Journal of Microbiology and
Biotechnology, 30(2), 351-374.
Coakley, M., Ross, R., Nordgren, M., Fitzgerald, G., Devery, R., & Stanton, C. (2003).
Conjugated linoleic acid biosynthesis by human‐derived Bifidobacterium species.
Journal of Applied Microbiology, 94(1), 138-145.
Coates, D. (2003). The angiotensin converting enzyme (ACE). The international journal
of biochemistry & cell biology, 35(6), 769-773.
Collado, M. C., Meriluoto, J., & Salminen, S. (2007). In vitro analysis of probiotic strain
combinations to inhibit pathogen adhesion to human intestinal mucus. Food
Research International, 40(5), 629-636.
Collins, M. D., & Gibson, G. R. (1999). Probiotics, prebiotics, and synbiotics: approaches
for modulating the microbial ecology of the gut. The American Journal of Clinical
Nutrition, 69(5), 1052s-1057s.
Contreras, M. d. M., Hernández-Ledesma, B., Amigo, L., Martín-Álvarez, P. J., & Recio,
I. (2011). Production of antioxidant hydrolyzates from a whey protein concentrate
with thermolysin: Optimization by response surface methodology. LWT - Food
Science and Technology, 44(1), 9-15.
doi:http://dx.doi.org/10.1016/j.lwt.2010.06.017
Conway, V., Gauthier, S. F., & Pouliot, Y. (2013). Antioxidant activities of buttermilk
proteins, whey proteins, and their enzymatic hydrolysates. Journal of agricultural
and food chemistry, 61(2), 364. doi:10.1021/jf304309g
Corcoran, B., Ross, R., Fitzgerald, G., & Stanton, C. (2004). Comparative survival of
probiotic lactobacilli spray‐dried in the presence of prebiotic substances. Journal
of Applied Microbiology, 96(5), 1024-1039.
Corcoran, B., Stanton, C., Fitzgerald, G., & Ross, R. (2008). Life under stress: the probiotic
stress response and how it may be manipulated. Current pharmaceutical design,
14(14), 1382-1399.
188
Corzo-Martínez, M., Ávila, M., Moreno, F. J., Requena, T., & Villamiel, M. (2012). Effect
of milk protein glycation and gastrointestinal digestion on the growth of
bifidobacteria and lactic acid bacteria. International journal of food microbiology,
153(3), 420-427.
Corzo-Martínez, M., Hernandez-Hernandez, O., Villamiel, M., Rastall, R. A., & Moreno,
F. J. (2013). In vitro bifidogenic effect of Maillard-type milk protein–galactose
conjugates on the human intestinal microbiota. International Dairy Journal, 31(2),
127-131. doi:http://dx.doi.org/10.1016/j.idairyj.2013.01.004
Corzo-Martínez, M., Moreno, F. J., Olano, A., & Villamiel, M. (2008). Structural
characterization of bovine β-lactoglobulin− galactose/tagatose Maillard complexes
by electrophoretic, chromatographic, and spectroscopic methods. Journal of
agricultural and food chemistry, 56(11), 4244-4252.
Corzo‐Martínez, M., Copoví, P., Olano, A., Moreno, F. J., & Montilla, A. (2013). Synthesis
of prebiotic carbohydrates derived from cheese whey permeate by a combined
process of isomerisation and transgalactosylation. Journal of the Science of Food
and Agriculture, 93(7), 1591-1597.
Costa, A. I., & Jongen, W. (2006). New insights into consumer-led food product
development. Trends in Food Science & Technology, 17(8), 457-465.
Cruz-Huerta, E., Maqueda, D. M., de la Hoz, L., Nunes da Silva, V. S., Pacheco, M. T. B.,
Amigo, L., & Recio, I. (2016). Short communication: Identification of iron-binding
peptides from whey protein hydrolysates using iron (III)-immobilized metal ion
affinity chromatographyand reversed phase-HPLC-tandem mass spectrometry.
Journal of Dairy Science, 99(1), 77-82. doi:http://dx.doi.org/10.3168/jds.2015-
9839
da Cruz, A. G., Buriti, F. C. A., de Souza, C. H. B., Faria, J. A. F., & Saad, S. M. I. (2009).
Probiotic cheese: health benefits, technological and stability aspects. Trends in
Food Science & Technology, 20(8), 344-354.
Darnton-Hill, I., Nishida, C., & James, W. (2004). A life course approach to diet, nutrition
and the prevention of chronic diseases. Public health nutrition, 7(1a), 101-121.
Dave, R., & Shah, N. (1998). Ingredient supplementation effects on viability of probiotic
bacteria in yogurt. Journal of Dairy Science, 81(11), 2804-2816.
189
Dave, R. I., & Shah, N. P. (1997a). Effectiveness of ascorbic acid as an oxygen scavenger
in improving viability of probiotic bacteria in yoghurts made with commercial
starter cultures. International Dairy Journal, 7(6-7), 435-443.
Dave, R. I., & Shah, N. P. (1997b). Viability of yoghurt and probiotic bacteria in yoghurts
made from commercial starter cultures. International Dairy Journal, 7(1), 31-41.
Davis, R., & Mauer, L. (2010). Fourier transform infrared (FT-IR) spectroscopy: a rapid
tool for detection and analysis of foodborne pathogenic bacteria. Current research,
technology and education topics in applied microbiology and microbial
biotechnology, 2, 1582-1594.
de Boer, A., Urlings, M. J. E., & Bast, A. (2016). Active ingredients leading in health
claims on functional foods. Journal of Functional Foods, 20, 587-593.
doi:10.1016/j.jff.2015.11.025
de Castro, R. J. S., & Sato, H. H. (2014). Advantages of an acid protease from Aspergillus
oryzae over commercial preparations for production of whey protein hydrolysates
with antioxidant activities. Biocatalysis and Agricultural Biotechnology, 3(3), 58-
65.
de Lacey, A. L., Pérez-Santín, E., López-Caballero, M., & Montero, P. (2014). Survival
and metabolic activity of probiotic bacteria in green tea. LWT-Food Science and
Technology, 55(1), 314-322.
de Oliveira, F. C., Coimbra, J. S. d. R., de Oliveira, E. B., Zuñiga, A. D. G., & Rojas, E. E.
G. (2016). Food protein-polysaccharide conjugates obtained via the maillard
reaction: A review. Critical reviews in food science and nutrition, 56(7), 1108-
1125.
De Souza Oliveira, R. P., Rodrigues Florence, A. C., Perego, P., De Oliveira, M. N., &
Converti, A. (2011). Use of lactulose as prebiotic and its influence on the growth,
acidification profile and viable counts of different probiotics in fermented skim
milk. International Journal of Food Microbiology, 145(1), 22-27.
doi:http://dx.doi.org/10.1016/j.ijfoodmicro.2010.11.011
de Souza, R. R., Bergamasco, R., da Costa, S. C., Feng, X., Faria, S. H. B., & Gimenes, M.
L. (2010). Recovery and purification of lactose from whey. Chemical Engineering
190
and Processing: Process Intensification, 49(11), 1137-1143.
doi:http://dx.doi.org/10.1016/j.cep.2010.08.015
De Wit, J. (1998). Nutritional and functional characteristics of whey proteins in food
products. Journal of dairy science, 81(3), 597-608.
Demers-Mathieu, V., Gauthier, S. F., Britten, M., Fliss, I., Robitaille, G., & Jean, J. (2013).
Antibacterial activity of peptides extracted from tryptic hydrolyzate of whey
protein by nanofiltration. International Dairy Journal, 28(2), 94-101.
doi:http://dx.doi.org/10.1016/j.idairyj.2012.09.003
Di Criscio, T., Fratianni, A., Mignogna, R., Cinquanta, L., Coppola, R., Sorrentino, E., &
Panfili, G. (2010). Production of functional probiotic, prebiotic, and synbiotic ice
creams. Journal of Dairy Science, 93(10), 4555-4564.
doi:https://doi.org/10.3168/jds.2010-3355
Dickinson, E., & Izgi, E. (1996). Foam stabilization by protein-polysaccharide complexes.
Colloids and Surfaces A: Physicochemical and Engineering Aspects, 113(1-2),
191-201.
Diftis, N., & Kiosseoglou, V. (2006). Physicochemical properties of dry-heated soy protein
isolate–dextran mixtures. Food Chemistry, 96(2), 228-233.
Dissanayake, M., & Vasiljevic, T. (2009). Functional properties of whey proteins affected
by heat treatment and hydrodynamic high-pressure shearing. Journal of dairy
science, 92(4), 1387-1397.
Doege, K., Grajecki, D., Zyriax, B.-C., Detinkina, E., zu Eulenburg, C., & Buhling, K. J.
(2012). Impact of maternal supplementation with probiotics during pregnancy on
atopic eczema in childhood–a meta-analysis. British journal of nutrition, 107(01),
1-6.
Dong, S., Panya, A., Zeng, M., Chen, B., McClements, D. J., & Decker, E. A. (2012).
Characteristics and antioxidant activity of hydrolyzed β-lactoglobulin–glucose
Maillard reaction products. Food Research International, 46(1), 55-61.
doi:http://dx.doi.org/10.1016/j.foodres.2011.11.022
Donkor, O. N., Henriksson, A., Vasiljevic, T., & Shah, N. P. (2006). Effect of acidification
on the activity of probiotics in yoghurt during cold storage. International Dairy
Journal, 16(10), 1181-1189. doi:http://doi.org/10.1016/j.idairyj.2005.10.008
191
Donkor, O. N., Henriksson, A., Vasiljevic, T., & Shah, N. P. (2007a). Proteolytic activity
of dairy lactic acid bacteria and probiotics as determinant of growth and in vitro
angiotensin-converting enzyme inhibitory activity in fermented milk. Le Lait,
87(1), 21-38.
Donkor, O. N., Nilmini, S. L. I., Stolic, P., Vasiljevic, T., & Shah, N. P. (2007b). Survival
and activity of selected probiotic organisms in set-type yoghurt during cold storage.
International Dairy Journal, 17(6), 657-665.
doi:https://doi.org/10.1016/j.idairyj.2006.08.006
Douillard, F. P., Ribbera, A., Järvinen, H. M., Kant, R., Pietilä, T. E., Randazzo, C., . . .
von Ossowski, I. (2013). Comparative genomic and functional analysis of
Lactobacillus casei and Lactobacillus rhamnosus strains marketed as probiotics.
Applied and environmental microbiology, 79(6), 1923-1933.
Doyon, M., & Labrecque, J. (2008). Functional foods: a conceptual definition. British Food
Journal, 110(11), 1133-1149.
Dwivedi, S. L., Puppala, N., & Ortiz, R. (2016). Microbiome, Prebiotics, and Human
Health Reference Module in Food Science: Elsevier.
Eiserich, J. P., & Shibamoto, T. (1994). Antioxidative activity of volatile heterocyclic
compounds. Journal of Agricultural and Food Chemistry, 42(5), 1060-1063.
El Jaam, O., Fliss, I., & Aider, M. (2016). Application of electro-activated potassium
acetate and potassium citrate solutions combined with moderate heat treatment on
the inactivation of Clostridium sporogenes PA 3679 spores. Innovative Food
Science & Emerging Technologies, 33, 483-488.
El-Dieb, S., Rabo, F. A., Badran, S., El-Fattah, A. A., & Elshaghabee, F. (2012). The
growth behaviour and enhancement of probiotic viability in bioyoghurt.
International Dairy Journal, 22(1), 44-47.
Elias, R. J., Kellerby, S. S., & Decker, E. A. (2008). Antioxidant activity of proteins and
peptides. Critical reviews in food science and nutrition, 48(5), 430-441.
Epand, R. M., & Vogel, H. J. (1999). Diversity of antimicrobial peptides and their
mechanisms of action. Biochimica et Biophysica Acta (BBA)-Biomembranes,
1462(1), 11-28.
192
Erdmann, K., Cheung, B. W., & Schröder, H. (2008). The possible roles of food-derived
bioactive peptides in reducing the risk of cardiovascular disease. The Journal of
nutritional biochemistry, 19(10), 643-654.
Fang, J., Cannon, J. L., & Hung, Y.-C. (2016). The efficacy of EO waters on inactivating
norovirus and hepatitis A virus in the presence of organic matter. Food Control, 61,
13-19.
FAO/WHO. (2002). WHO working group report on drafting guidelines for the evaluation
of probiotics in food. London, Ontario, Canada, 30.
Farnaud, S., & Evans, R. W. (2003). Lactoferrin—a multifunctional protein with
antimicrobial properties. Molecular immunology, 40(7), 395-405.
Farnworth, E. R., & Champagne, C. (2010). Production of probiotic cultures and their
incorporation into foods. Bioactive foods in promoting health. Academic, London,
3-17.
Finete, V. d. L. M., Gouvêa, M. M., Marques, F. F. d. C., & Netto, A. D. P. (2013). Is it
possible to screen for milk or whey protein adulteration with melamine, urea and
ammonium sulphate, combining Kjeldahl and classical spectrophotometric
methods? Food Chemistry, 141(4), 3649-3655.
doi:http://dx.doi.org/10.1016/j.foodchem.2013.06.046
Foegeding, E. A., & Davis, J. P. (2011). Food protein functionality: a comprehensive
approach. Food Hydrocolloids, 25(8), 1853-1864.
Foligné, B., Daniel, C., & Pot, B. (2013). Probiotics from research to market: the
possibilities, risks and challenges. Current opinion in microbiology, 16(3), 284-
292.
Fu, L., & Wang, Y. (2013). Sources and Production of Prebiotics. Probiotics and Prebiotics
in Food, Nutrition and Health, 50.
Fukuda, S., Toh, H., Hase, K., Oshima, K., Nakanishi, Y., Yoshimura, K., . . . Ohno, H.
(2011). Bifidobacteria can protect from enteropathogenic infection through
production of acetate. nature, 469(7331), 543-547.
doi:http://www.nature.com/nature/journal/v469/n7331/abs/10.1038-nature09646-
unlocked.html - supplementary-information
193
Fuller, R., & Gibson, G. R. (1997). Modification of the intestinal microflora using
probiotics and prebiotics. Scandinavian Journal of Gastroenterology, 32(sup222),
28-31.
Gänzle, M. G. (2012). Enzymatic synthesis of galacto-oligosaccharides and other lactose
derivatives (hetero-oligosaccharides) from lactose. International Dairy Journal,
22(2), 116-122. doi:http://dx.doi.org/10.1016/j.idairyj.2011.06.010
Gänzle, M. G., Haase, G., & Jelen, P. (2008). Lactose: crystallization, hydrolysis and value-
added derivatives. International Dairy Journal, 18(7), 685-694.
García-Cayuela, T., Díez-Municio, M., Herrero, M., Martínez-Cuesta, M. C., Peláez, C.,
Requena, T., & Moreno, F. J. (2014). Selective fermentation of potential prebiotic
lactose-derived oligosaccharides by probiotic bacteria. International Dairy
Journal, 38(1), 11-15.
Gardner-Fortier, C., St-Gelais, D., Champagne, C. P., & Vuillemard, J.-C. (2013).
Determination of optimal conditions for γ-aminobutyric acid production by
Lactococcus lactis ssp. lactis. International Dairy Journal, 32(2), 136-143.
Gaudel, C., Nongonierma, A. B., Maher, S., Flynn, S., Krause, M., Murray, B. A., . . .
Newsholme, P. (2013). A whey protein hydrolysate promotes insulinotropic
activity in a clonal pancreatic β-cell line and enhances glycemic function in ob/ob
mice. The Journal of nutrition, 143(7), 1109-1114.
Gaudreau, H., Champagne, C., Remondetto, G., Bazinet, L., & Subirade, M. (2013). Effect
of catechins on the growth of oxygen-sensitive probiotic bacteria. Food research
international, 53(2), 751-757.
Gaudreau, H., Champagne, C. P., & Jelen, P. (2005). The use of crude cellular extracts of
Lactobacillus delbrueckii ssp. bulgaricus 11842 to stimulate growth of a probiotic
Lactobacillus rhamnosus culture in milk. Enzyme and Microbial Technology, 36(1),
83-90. doi:http://doi.org/10.1016/j.enzmictec.2004.06.006
Gaudreau, H., Champagne, C. P., Remondetto, G. E., Alvarez, P. A., Gomaa, A., &
Subirade, M. (2016). Tea extract render probiotic Lactobacillus helveticus more
resistant to oxygen exposure through lipid modification mechanism. Food
Research International, 81, 141-148.
doi:http://dx.doi.org/10.1016/j.foodres.2015.12.031
194
Gauthier, S., & Pouliot, Y. (2003). Functional and biological properties of peptides
obtained by enzymatic hydrolysis of whey proteins. Journal of Dairy Science, 86,
E78-E87.
Gauthier, S. F., Pouliot, Y., & Saint-Sauveur, D. (2006). Immunomodulatory peptides
obtained by the enzymatic hydrolysis of whey proteins. International Dairy
Journal, 16(11), 1315-1323. doi:http://dx.doi.org/10.1016/j.idairyj.2006.06.014
Gayathri, D., & Rashmi, B. S. (2017). Mechanism of development of depression and
probiotics as adjuvant therapy for its prevention and management. Mental Health
& Prevention, 5, 40-51. doi:http://doi.org/10.1016/j.mhp.2017.01.003
Geier, H., & Klostermeyer, H. (1983). Formation of lactulose during heat treatment of milk.
Milchwissenschaft.
Gerzhova, A., Mondor, M., Benali, M., & Aider, M. (2015). A comparative study between
the electro-activation technique and conventional extraction method on the
extractability, composition and physicochemical properties of canola protein
concentrates and isolates. Food Bioscience, 11, 56-71.
Gibson, G. R. (1999). Dietary modulation of the human gut microflora using the prebiotics
oligofructose and inulin. The Journal of nutrition, 129(7), 1438S-1441s.
Gibson, G. R. (2004). Fibre and effects on probiotics (the prebiotic concept). Clinical
Nutrition Supplements, 1(2), 25-31. doi:https://doi.org/10.1016/j.clnu.2004.09.005
Gibson, G. R., Probert, H. M., Van Loo, J., Rastall, R. A., & Roberfroid, M. B. (2004).
Dietary modulation of the human colonic microbiota: updating the concept of
prebiotics. Nutrition research reviews, 17(02), 259-275.
Gibson, G. R., & Roberfroid, M. B. (1995). Dietary modulation of the human colonic
microbiota: introducing the concept of prebiotics. The Journal of nutrition, 125(6),
1401.
Gibson, G. R., Scott, K. P., Rastall, R. A., Tuohy, K. M., Hotchkiss, A., Dubert-Ferrandon,
A., . . . Loh, G. (2010). Dietary prebiotics: current status and new definition. Food
Sci Technol Bull Funct Foods, 7, 1-19.
Gill, H., & Prasad, J. (2008). Probiotics, immunomodulation, and health benefits Bioactive
Components of Milk (pp. 423-454): Springer.
195
Gill, I., López-Fandiño, R., Jorba, X., & Vulfson, E. N. (1996). Biologically active peptides
and enzymatic approaches to their production. Enzyme and Microbial Technology,
18(3), 162-183.
Gille, C., Böer, B., Marschal, M., Urschitz, M. S., Heinecke, V., Hund, V., . . . Poets, C. F.
(2016). Effect of probiotics on vaginal health in pregnancy. EFFPRO, a randomized
controlled trial. American Journal of Obstetrics and Gynecology, 215(5), 608.e601-
608.e607. doi:http://doi.org/10.1016/j.ajog.2016.06.021
Gilliland, S., & Speck, M. (1977). Instability of Lactobacillus acidophilus in Yogurt1.
Journal of Dairy Science, 60(9), 1394-1398.
Glenn, G., & Roberfroid, M. (1995). Dietary modulation of the human colonic microbiota:
introducing the concept of prebiotics. J. nutr, 125, 1401-1412.
Gnatko, E. N., Kravets, V. I., Leschenko, E. V., & Omelchenko, A. (2011). Emergence of
the science and technology of electroactivated aqueous solutions: applications for
environmental and food safety Environmental security and ecoterrorism (pp. 101-
116): Springer.
Gobbetti, M., Cagno, R. D., & De Angelis, M. (2010). Functional microorganisms for
functional food quality. Critical reviews in food science and nutrition, 50(8), 716-
727.
Goldberg, I. (2012). Functional foods: designer foods, pharmafoods, nutraceuticals:
Springer Science & Business Media.
Golowczyc, M., Vera, C., Santos, M., Guerrero, C., Carasi, P., Illanes, A., . . . Tymczyszyn,
E. (2013). Use of whey permeate containing in situ synthesised galacto-
oligosaccharides for the growth and preservation of Lactobacillus plantarum.
Journal of dairy research, 80(03), 374-381.
Gomaa, A., Sedman, J., & Ismail, A. (2013). An investigation of the effect of microwave
treatment on the structure and unfolding pathways of β-lactoglobulin using FTIR
spectroscopy with the application of two-dimensional correlation spectroscopy
(2D-COS). Vibrational Spectroscopy, 65, 101-109.
Gomes, A. M., Malcata, F. X., & Klaver, F. A. (1998). Growth enhancement of
Bifidobacterium lactis Bo and Lactobacillus acidophilus Ki by milk hydrolyzates.
Journal of Dairy Science, 81(11), 2817-2825.
196
Gopal, P. K., Sullivan, P. A., & Smart, J. B. (2001). Utilisation of galacto-oligosaccharides
as selective substrates for growth by lactic acid bacteria including Bifidobacterium
lactis DR10 and Lactobacillus rhamnosus DR20. International Dairy Journal,
11(1), 19-25.
Gouin, S. (2004). Microencapsulation: industrial appraisal of existing technologies and
trends. Trends in Food Science & Technology, 15(7), 330-347.
Grajek, W., Olejnik, A., & Sip, A. (2005). Probiotics, prebiotics and antioxidants as
functional foods. ACTA BIOCHIMICA POLONICA-ENGLISH EDITION-, 52(3),
665.
Granato, D., Branco, G. F., Cruz, A. G., Faria, J. d. A. F., & Shah, N. P. (2010a). Probiotic
dairy products as functional foods. Comprehensive Reviews in Food Science and
Food Safety, 9(5), 455-470.
Granato, D., Branco, G. F., Nazzaro, F., Cruz, A. G., & Faria, J. A. (2010b). Functional
foods and nondairy probiotic food development: trends, concepts, and products.
Comprehensive reviews in food science and food safety, 9(3), 292-302.
Gu, F.-L., Kim, J. M., Abbas, S., Zhang, X.-M., Xia, S.-Q., & Chen, Z.-X. (2010). Structure
and antioxidant activity of high molecular weight Maillard reaction products from
casein–glucose. Food Chemistry, 120(2), 505-511.
Guan, Y.-G., Lin, H., Han, Z., Wang, J., Yu, S.-J., Zeng, X.-A., . . . Sun, W.-W. (2010).
Effects of pulsed electric field treatment on a bovine serum albumin–dextran model
system, a means of promoting the Maillard reaction. Food Chemistry, 123(2), 275-
280.
Guandalini, S. (2008). Probiotics for children with diarrhea: an update. Journal of clinical
gastroenterology, 42, S53-S57.
Guarner, F., Perdigon, G., Corthier, G., Salminen, S., Koletzko, B., & Morelli, L. (2005).
Should yoghurt cultures be considered probiotic? British Journal of Nutrition,
93(06), 783-786.
Guerrero, C., Vera, C., Plou, F., & Illanes, A. (2011). Influence of reaction conditions on
the selectivity of the synthesis of lactulose with microbial β-galactosidases. Journal
of Molecular Catalysis B: Enzymatic, 72(3), 206-212.
197
Guimarães, P. M. R., Teixeira, J. A., & Domingues, L. (2010). Fermentation of lactose to
bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese
whey. Biotechnology advances, 28(3), 375-384.
doi:http://dx.doi.org/10.1016/j.biotechadv.2010.02.002
Gutiérrez, L.-F., Hamoudi, S., & Belkacemi, K. (2012). Lactobionic acid: A high value-
added lactose derivative for food and pharmaceutical applications. International
Dairy Journal, 26(2), 103-111. doi:http://dx.doi.org/10.1016/j.idairyj.2012.05.003
Ha, E., & Zemel, M. B. (2003). Functional properties of whey, whey components, and
essential amino acids: mechanisms underlying health benefits for active people
(review). The Journal of nutritional biochemistry, 14(5), 251-258.
doi:http://dx.doi.org/10.1016/S0955-2863(03)00030-5
Hailu, G., Boecker, A., Henson, S., & Cranfield, J. (2009). Consumer valuation of
functional foods and nutraceuticals in Canada. A conjoint study using probiotics.
Appetite, 52(2), 257-265. doi:http://dx.doi.org/10.1016/j.appet.2008.10.002
Halliwell, B., Gutteridge, J. M., & Aruoma, O. I. (1987). The deoxyribose method: a simple
“test-tube” assay for determination of rate constants for reactions of hydroxyl
radicals. Analytical biochemistry, 165(1), 215-219.
Hammer, H. F., Santa Ana, C. A., Schiller, L. R., & Fordtran, J. S. (1989). Studies of
osmotic diarrhea induced in normal subjects by ingestion of polyethylene glycol
and lactulose. Journal of Clinical Investigation, 84(4), 1056.
Haque, E., & Chand, R. (2008). Antihypertensive and antimicrobial bioactive peptides
from milk proteins. European Food Research and Technology, 227(1), 7-15.
Hartmann, R., & Meisel, H. (2007). Food-derived peptides with biological activity: from
research to food applications. Current opinion in biotechnology, 18(2), 163-169.
Hashemi, S. A., & Ashtiani, F. Z. (2010). The isomerization kinetics of lactose to lactulose
in the presence of sodium hydroxide at constant and variable pH. Food and
Bioproducts Processing, 88(2–3), 181-187.
doi:http://dx.doi.org/10.1016/j.fbp.2009.11.001
Hasler, C. M. (1998). Functional foods: their role in disease prevention and health
promotion. FOOD TECHNOLOGY-CHAMPAIGN THEN CHICAGO-, 52, 63-147.
198
Hasler, C. M. (2002). Functional foods: benefits, concerns and challenges—a position
paper from the American Council on Science and Health. The Journal of nutrition,
132(12), 3772-3781.
Hattori, M., Ogino, A., Nakai, H., & Takahashi, K. (1997). Functional improvement of β-
lactoglobulin by conjugating with alginate lyase-lysate. Journal of Agricultural and
Food Chemistry, 45(3), 703-708.
Hauser, C., Müller, U., Sauer, T., Augner, K., & Pischetsrieder, M. (2014). Maillard
reaction products as antimicrobial components for packaging films. Food
Chemistry, 145, 608-613.
Heine, W. E., Klein, P. D., & Reeds, P. J. (1991). The importance of alpha-lactalbumin in
infant nutrition. The Journal of nutrition, 121(3), 277-283.
Helm, D., Labischinski, H., Schallehn, G., & Naumann, D. (1991). Classification and
identification of bacteria by Fourier-transform infrared spectroscopy.
Microbiology, 137(1), 69-79.
Hernandez-Hernandez, O., Muthaiyan, A., Moreno, F. J., Montilla, A., Sanz, M. L., &
Ricke, S. (2012). Effect of prebiotic carbohydrates on the growth and tolerance of
Lactobacillus. Food microbiology, 30(2), 355-361.
Hernández-Ledesma, B., Dávalos, A., Bartolomé, B., & Amigo, L. (2005). Preparation of
antioxidant enzymatic hydrolysates from α-lactalbumin and β-lactoglobulin.
Identification of active peptides by HPLC-MS/MS. Journal of Agricultural and
Food Chemistry, 53(3), 588-593.
Hernández-Ledesma, B., Ramos, M., Recio, I., & Amigo, L. (2006). Effect of β-
lactoglobulin hydrolysis with thermolysin under denaturing temperatures on the
release of bioactive peptides. Journal of Chromatography A, 1116(1), 31-37.
Hernández-Ledesma, B., Recio, I., & Amigo, L. (2008). β-Lactoglobulin as source of
bioactive peptides. Amino Acids, 35(2), 257-265.
Hertzberger, R., Arents, J., Dekker, H. L., Pridmore, R. D., Gysler, C., Kleerebezem, M.,
& de Mattos, M. J. T. (2014). H2O2 production in species of the Lactobacillus
acidophilus group: a central role for a novel NADH-dependent flavin reductase.
Applied and environmental microbiology, 80(7), 2229-2239.
199
Hertzberger, R. Y., Pridmore, R. D., Gysler, C., Kleerebezem, M., & de Mattos, M. J. T.
(2013). Oxygen relieves the CO 2 and acetate dependency of Lactobacillus
johnsonii NCC 533. PloS one, 8(2), e57235.
Hill, A. R., Irvine, D. M., & Bullock, D. H. (1985). Buffer Capacity of Cheese Wheys.
Journal of food science, 50(3), 733-738. doi:10.1111/j.1365-2621.1985.tb13784.x
Hill, C., Guarner, F., Reid, G., Gibson, G. R., Merenstein, D. J., Pot, B., . . . Salminen, S.
(2014). Expert consensus document: The International Scientific Association for
Probiotics and Prebiotics consensus statement on the scope and appropriate use of
the term probiotic. Nature reviews Gastroenterology & hepatology, 11(8), 506-514.
Hodge, J. E. (1953). Dehydrated foods, chemistry of browning reactions in model systems.
Journal of Agricultural and Food Chemistry, 1(15), 928-943.
Hodge, J. E. (1955). The amadori rearrangement. Advances in carbohydrate chemistry, 10,
169-205.
Hodge, J. E., & Rist, C. E. (1953). The Amadori Rearrangement under New Conditions
and its Significance for Non-enzymatic Browning Reactions2. Journal of the
American Chemical Society, 75(2), 316-322.
Holzapfel, W. H., Haberer, P., Geisen, R., Björkroth, J., & Schillinger, U. (2001).
Taxonomy and important features of probiotic microorganisms in food and
nutrition. The American journal of clinical nutrition, 73(2), 365s-373s.
Hongsprabhas, P., Kerdchouay, P., & Sakulsom, P. (2011). Lowering the Maillard reaction
products (MRPs) in heated whey protein products and their cytotoxicity in human
cell models by whey protein hydrolysate. Food Research International, 44(3), 748-
754.
Hopkins, M., Cummings, J., & Macfarlane, G. (1998). Inter-species differences in
maximum specific growth rates and cell yields of bifidobacteria cultured on
oligosaccharides and other simple carbohydrate sources. Journal of Applied
Microbiology, 85(2), 381-386.
Hougaard, A. B., Pindstrup, H., Arneborg, N., Andersen, M. L., & Skibsted, L. H. (2016).
Free radical formation by Lactobacillus acidophilus NCFM is enhanced by
antioxidants and decreased by catalase. Food Research International, 79, 81-87.
200
Hu, F. B., Stampfer, M. J., Manson, J. E., Grodstein, F., Colditz, G. A., Speizer, F. E., &
Willett, W. C. (2000). Trends in the incidence of coronary heart disease and
changes in diet and lifestyle in women. New England Journal of Medicine, 343(8),
530-537.
Huang, Y., & Adams, M. C. (2004). In vitro assessment of the upper gastrointestinal
tolerance of potential probiotic dairy propionibacteria. International Journal of
Food Microbiology, 91(3), 253-260.
Huang, Y.-R., Hung, Y.-C., Hsu, S.-Y., Huang, Y.-W., & Hwang, D.-F. (2008).
Application of electrolyzed water in the food industry. Food control, 19(4), 329-
345.
Huebner, J., Wehling, R., & Hutkins, R. W. (2007). Functional activity of commercial
prebiotics. International Dairy Journal, 17(7), 770-775.
Huebner, J., Wehling, R., Parkhurst, A., & Hutkins, R. W. (2008). Effect of processing
conditions on the prebiotic activity of commercial prebiotics. International Dairy
Journal, 18(3), 287-293.
Hutkins, R. W., Krumbeck, J. A., Bindels, L. B., Cani, P. D., Fahey, G., Goh, Y. J., . . .
Rastal, R. A. (2016). Prebiotics: why definitions matter. Current opinion in
biotechnology, 37, 1-7.
Hwang, I. G., Kim, H. Y., Woo, K. S., Lee, J., & Jeong, H. S. (2011). Biological activities
of Maillard reaction products (MRPs) in a sugar–amino acid model system. Food
Chemistry, 126(1), 221-227.
doi:http://dx.doi.org/10.1016/j.foodchem.2010.10.103
Ignatova, T., Iliev, I., Kirilov, N., Vassileva, T., Dalgalarrondo, M. l., Haertlé, T., . . .
Ivanova, I. (2009). Effect of oligosaccharides on the growth of Lactobacillus
delbrueckii subsp. bulgaricus strains isolated from dairy products. Journal of
agricultural and food chemistry, 57(20), 9496-9502.
Illanes, A., & Guerrero, C. (2016). Chapter 2 - Functional Foods and Feeds: Probiotics,
Prebiotics, and Synbiotics Lactose-Derived Prebiotics (pp. 35-86). San Diego:
Academic Press.
Ismond, M., & Welsh, W. (1992). Application of new methodology to canola protein
isolation. Food Chemistry, 45(2), 125-127.
201
Isolauri, E., Arvola, T., Sütas, Y., Moilanen, E., & Salminen, S. (2000). Probiotics in the
management of atopic eczema. Clinical & Experimental Allergy, 30(11), 1605-
1610.
Iyer, C., & Kailasapathy, K. (2005). Effect of co‐encapsulation of probiotics with prebiotics
on increasing the viability of encapsulated bacteria under in vitro acidic and bile
salt conditions and in yogurt. Journal of Food Science, 70(1).
Jacquot, A., Gauthier, S. F., Drouin, R., & Boutin, Y. (2010). Proliferative effects of
synthetic peptides from β-lactoglobulin and α-lactalbumin on murine splenocytes.
International Dairy Journal, 20(8), 514-521.
doi:http://dx.doi.org/10.1016/j.idairyj.2010.02.013
Jaeger, H., Janositz, A., & Knorr, D. (2010). The Maillard reaction and its control during
food processing. The potential of emerging technologies. Pathologie Biologie,
58(3), 207-213.
Jakubowicz, D., & Froy, O. (2013). Biochemical and metabolic mechanisms by which
dietary whey protein may combat obesity and Type 2 diabetes. The Journal of
nutritional biochemistry, 24(1), 1-5.
Janczukowicz, W., Zieliński, M., & Dębowski, M. (2008). Biodegradability evaluation of
dairy effluents originated in selected sections of dairy production. Bioresource
technology, 99(10), 4199-4205.
doi:http://dx.doi.org/10.1016/j.biortech.2007.08.077
Janer, C., Peláez, C., & Requena, T. (2004). Caseinomacropeptide and whey protein
concentrate enhance Bifidobacterium lactis growth in milk. Food Chemistry, 86(2),
263-267.
Jiang, Z., & Brodkorb, A. (2012). Structure and antioxidant activity of Maillard reaction
products from α-lactalbumin and β-lactoglobulin with ribose in an aqueous model
system. Food Chemistry, 133(3), 960-968.
Jiang, Z., Wang, L., Wu, W., & Wang, Y. (2013). Biological activities and
physicochemical properties of Maillard reaction products in sugar–bovine casein
peptide model systems. Food Chemistry, 141(4), 3837-3845.
Jing, H., & Kitts, D. (2002). Chemical and biochemical properties of casein–sugar Maillard
reaction products. Food and Chemical Toxicology, 40(7), 1007-1015.
202
Jing, H., & Kitts, D. D. (2004). Antioxidant activity of sugar–lysine Maillard reaction
products in cell free and cell culture systems. Archives of biochemistry and
biophysics, 429(2), 154-163.
Johnson, D. R., & Decker, E. A. (2015). The role of oxygen in lipid oxidation reactions: a
review. Annual review of food science and technology, 6, 171-190.
Jokinen, H., Gouw, A., Madureira, S., Ylikoski, R., Van Straaten, E., Van Der Flier, W., .
. . Schmidt, R. (2011). Incident lacunes influence cognitive decline The LADIS
study. Neurology, 76(22), 1872-1878.
Joubran, Y., Mackie, A., & Lesmes, U. (2013). Impact of the Maillard reaction on the
antioxidant capacity of bovine lactoferrin. Food Chemistry, 141(4), 3796-3802.
Joy, J. M., Lowery, R. P., Wilson, J. M., Purpura, M., De Souza, E. O., Wilson, S. M., . . .
Jäger, R. (2013). The effects of 8 weeks of whey or rice protein supplementation
on body composition and exercise performance. Nutrition journal, 12(1), 86.
Juriaanse, A. (2006). Challenges ahead for food science. International journal of dairy
technology, 59(2), 55-57.
Kankainen, M., Paulin, L., Tynkkynen, S., von Ossowski, I., Reunanen, J., Partanen, P., . .
. Lebeer, S. (2009). Comparative genomic analysis of Lactobacillus rhamnosus GG
reveals pili containing a human-mucus binding protein. Proceedings of the National
Academy of Sciences, 106(40), 17193-17198.
Karam, M. C., Gaiani, C., Hosri, C., Burgain, J., & Scher, J. (2013). Effect of dairy powders
fortification on yogurt textural and sensorial properties: a review. Journal of Dairy
Research, 80(04), 400-409.
Kareb, O., Champagne, C. P., & Aïder, M. (2016). Contribution to the production of
lactulose-rich whey by in situ electro-isomerization of lactose and effect on whey
proteins after electro-activation as confirmed by matrix-assisted laser
desorption/ionization time-of-flight-mass spectrometry and sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Journal of Dairy Science.
Kareb, O., Gomaa, A., Champagne, C. P., Jean, J., & Aïder, M. (2017a). Electro-activation
of sweet defatted whey: Impact on the induced Maillard reaction products and
bioactive peptides. Food Chemistry, 221, 590-598.
203
Kareb, O., Gomaa, A. I., Champagne, C. P., Jean, J., & Aïder, M. (2017b). Impact of
electro-activation on antioxidant properties of defatted whey. International dairy
journal. doi:http://dx.doi.org/10.1016/j.idairyj.2016.09.009
Karimi, R., Azizi, M. H., Ghasemlou, M., & Vaziri, M. (2015). Application of inulin in
cheese as prebiotic, fat replacer and texturizer: A review. Carbohydrate Polymers,
119, 85-100.
Kastyuchik, A., Karam, A., & Aïder, M. (2016). Effectiveness of alkaline amendments in
acid mine drainage remediation. Environmental Technology & Innovation, 6, 49-
59.
Kechagia, M., Basoulis, D., Konstantopoulou, S., Dimitriadi, D., Gyftopoulou, K.,
Skarmoutsou, N., & Fakiri, E. M. (2013). Health benefits of probiotics: a review.
ISRN nutrition, 2013.
Keller, A., Nesvizhskii, A. I., Kolker, E., & Aebersold, R. (2002). Empirical Statistical
Model To Estimate the Accuracy of Peptide Identifications Made by MS/MS and
Database Search. Analytical Chemistry, 74(20), 5383-5392.
doi:10.1021/ac025747h
Keri Marshall, N. (2004). Therapeutic applications of whey protein. Alternative Medicine
Review, 9(2), 136-156.
Khan, R. S., Grigor, J., Winger, R., & Win, A. (2013). Functional food product
development – Opportunities and challenges for food manufacturers. Trends in
Food Science & Technology, 30(1), 27-37.
doi:http://dx.doi.org/10.1016/j.tifs.2012.11.004
Kılıç, G. B., & Akpınar, D. (2013). The effects of different levels of β-glucan on yoghurt
manufactured with Lactobacillus plantarum strains as adjunct culture. Journal of
Food, Agriculture & Environment, 11(1), 281-287.
Kim, G.-N., Jang, H.-D., & Kim, C.-I. (2007a). Antioxidant capacity of
caseinophosphopeptides prepared from sodium caseinate using Alcalase. Food
Chemistry, 104(4), 1359-1365.
Kim, K. M., Han, K., Kim, J. H., Suh, H. J., & Park, Y. (2015). Supplementation of
Lactulose Modulates Intestinal Environment. The FASEB Journal, 29(1
Supplement), LB344.
204
Kim, S. B., Seo, I. S., Khan, M. A., Ki, K. S., Lee, W. S., Lee, H. J., . . . Kim, H. S. (2007b).
Enzymatic Hydrolysis of Heated Whey: Iron-Binding Ability of Peptides and
Antigenic Protein Fractions. Journal of Dairy Science, 90(9), 4033-4042.
doi:http://dx.doi.org/10.3168/jds.2007-0228
Kim, Y.-S., & Oh, D.-K. (2012). Lactulose production from lactose as a single substrate
by a thermostable cellobiose 2-epimerase from Caldicellulosiruptor
saccharolyticus. Bioresource Technology, 104, 668-672.
Kinsella, J. E., & Whitehead, D. M. (1989). Proteins in Whey: Chemical, Physical, and
Functional Properties. Advances in Food and Nutrition Research, 33, 343-438.
doi:http://dx.doi.org/10.1016/S1043-4526(08)60130-8
Kitts, D. D., & Weiler, K. (2003). Bioactive proteins and peptides from food sources.
Applications of bioprocesses used in isolation and recovery. Current
pharmaceutical design, 9(16), 1309-1323.
Klintham, P., Tongchitpakdee, S., Chinsirikul, W., & Mahakarnchanakul, W. (2017).
Combination of microbubbles with oxidizing sanitizers to eliminate Escherichia
coli and Salmonella Typhimurium on Thai leafy vegetables. Food Control, 77, 260-
269.
Kneifel, W. (2000). In vitro growth behaviour of probiotic bacteria in culture media with
carbohydrates of prebiotic importance. Microbial Ecology in Health and Disease,
12(1), 27-34.
Kneifel, W., Jaros, D., & Erhard, F. (1993). Microflora and acidification properties of
yogurt and yogurt-related products fermented with commercially available starter
cultures. International Journal of Food Microbiology, 18(3), 179-189.
Koffi, K., Labrie, S., Genois, A., Aissa, A. A., & Aïder, M. (2014). Contribution to the
development of a method of maple sap soft drink stabilization by electro-activation
technology. LWT-Food Science and Technology, 59(1), 138-147.
Kolida, S., & Gibson, G. R. (2011). Synbiotics in health and disease. Annual review of food
science and technology, 2, 373-393.
Korhonen, H. (2009). Milk-derived bioactive peptides: From science to applications.
Journal of Functional Foods, 1(2), 177-187.
205
Korhonen, H., & Pihlanto, A. (2003). Food-derived bioactive peptides-opportunities for
designing future foods. Current pharmaceutical design, 9(16), 1297-1308.
Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: production and functionality.
International Dairy Journal, 16(9), 945-960.
Korhonen, H., & Pihlanto-Leppälä, A. (2001). Milk protein-derived bioactive peptides:
novel opportunities for health promotion: dairy nutrition for a healthy future.
Bulletin-International Dairy Federation(363), 17-26.
Korhonen, H. J. (2006). Technological and health aspects of bioactive components of milk.
International Dairy Journal, 16(11), 1227-1228.
doi:http://dx.doi.org/10.1016/j.idairyj.2006.07.001
Kosikowski, F. V. (1979). Whey utilization and whey products. Journal of dairy science,
62(7), 1149-1160.
Kosikowski, F. V., & Wzorek, W. (1977). Whey Wine from Concentrates of Reconstituted
Acid Whey Powder. Journal of dairy science, 60(12), 1982-1986.
doi:http://dx.doi.org/10.3168/jds.S0022-0302(77)84132-5
Kosseva, M. R., Panesar, P. S., Kaur, G., & Kennedy, J. F. (2009). Use of immobilised
biocatalysts in the processing of cheese whey. International journal of biological
macromolecules, 45(5), 437-447.
Kóta, Z., Debreczeny, M., & Szalontai, B. (1999). Separable contributions of ordered and
disordered lipid fatty acyl chain segments to νCH2 bands in model and biological
membranes: A Fourier transform infrared spectroscopic study. Biospectroscopy,
5(3), 169-178.
Koutinas, A. A., Papapostolou, H., Dimitrellou, D., Kopsahelis, N., Katechaki, E.,
Bekatorou, A., & Bosnea, L. A. (2009). Whey valorisation: A complete and novel
technology development for dairy industry starter culture production. Bioresource
technology, 100(15), 3734-3739.
doi:http://dx.doi.org/10.1016/j.biortech.2009.01.058
Kumar, H., Salminen, S., Verhagen, H., Rowland, I., Heimbach, J., Bañares, S., . . .
Lalonde, M. (2015). Novel probiotics and prebiotics: road to the market. Current
Opinion in Biotechnology, 32, 99-103.
doi:http://dx.doi.org/10.1016/j.copbio.2014.11.021
206
Kumar, M., Nagpal, R., Hemalatha, R., Yadav, H., & Marotta, F. (2016). Probiotics and
prebiotics for promoting health: through gut microbiota. Probiotics, Prebiotics, and
Synbiotics: Bioactive Foods in Health Promotion, 938.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. nature, 227, 680-685.
Lahov, E., & Regelson, W. (1996). Antibacterial and immunostimulating casein-derived
substances from milk: Casecidin, isracidin peptides. Food and Chemical
Toxicology, 34(1), 131-145. doi:http://dx.doi.org/10.1016/0278-6915(95)00097-6
Lankaputhra, W., Shah, N., & Britz, M. (1996). Evaluation of media for selective
enumeration of Lactobacillus acidophilus and Bifidobacterium species. Food
Australia.
Laroque, D., Inisan, C., Berger, C., Vouland, É., Dufossé, L., & Guérard, F. (2008). Kinetic
study on the Maillard reaction. Consideration of sugar reactivity. Food Chemistry,
111(4), 1032-1042. doi:http://dx.doi.org/10.1016/j.foodchem.2008.05.033
Lee, S. H., Choi, W., & Jun, S. (2016). Conventional and Emerging Combination
Technologies for Food Processing. Food Engineering Reviews, 8(4), 414-434.
Lee, Y. K., & Salminen, S. (2009). Handbook of probiotics and prebiotics: John Wiley &
Sons.
Lertittikul, W., Benjakul, S., & Tanaka, M. (2007). Characteristics and antioxidative
activity of Maillard reaction products from a porcine plasma protein–glucose model
system as influenced by pH. Food Chemistry, 100(2), 669-677.
Li, E. W., & Mine, Y. (2004). Immunoenhancing effects of bovine glycomacropeptide and
its derivatives on the proliferative response and phagocytic activities of human
macrophagelike cells, U937. Journal of agricultural and food chemistry, 52(9),
2704-2708.
Li, Y., Zhong, F., Ji, W., Yokoyama, W., Shoemaker, C. F., Zhu, S., & Xia, W. (2013).
Functional properties of Maillard reaction products of rice protein hydrolysates
with mono-, oligo-and polysaccharides. Food Hydrocolloids, 30(1), 53-60.
Li-Chan, E. C. (2015). Bioactive peptides and protein hydrolysates: research trends and
challenges for application as nutraceuticals and functional food ingredients.
Current Opinion in Food Science, 1, 28-37.
207
Liato, V., Labrie, S., Viel, C., Benali, M., & Aïder, M. (2015). Study of the combined effect
of electro-activated solutions and heat treatment on the destruction of spores of
Clostridium sporogenes and Geobacillus stearothermophilus in model solution and
vegetable puree. Anaerobe, 35, 11-21.
Lillard, J., Clare, D., & Daubert, C. (2009). Glycosylation and expanded utility of a
modified whey protein ingredient via carbohydrate conjugation at low pH. Journal
of dairy science, 92(1), 35-48.
Liu, P., Huang, M., Song, S., Hayat, K., Zhang, X., Xia, S., & Jia, C. (2012). Sensory
characteristics and antioxidant activities of Maillard reaction products from soy
protein hydrolysates with different molecular weight distribution. Food and
Bioprocess Technology, 5(5), 1775-1789.
Liu, Q., Kong, B., Han, J., Sun, C., & Li, P. (2014). Structure and antioxidant activity of
whey protein isolate conjugated with glucose via the Maillard reaction under dry-
heating conditions. Food Structure, 1(2), 145-154.
doi:http://dx.doi.org/10.1016/j.foostr.2013.11.004
Lobo, V., Patil, A., Phatak, A., & Chandra, N. (2010). Free radicals, antioxidants and
functional foods: Impact on human health. Pharmacognosy reviews, 4(8), 118.
Lomax, A., & Calder, P. (2009). Probiotics, immune function, infection and inflammation:
a review of the evidence from studies conducted in humans. Current
pharmaceutical design, 15(13), 1428-1518.
López de Lacey, A. M., Pérez-Santín, E., López-Caballero, M. E., & Montero, P. (2014).
Survival and metabolic activity of probiotic bacteria in green tea. LWT - Food
Science and Technology, 55(1), 314-322.
doi:http://dx.doi.org/10.1016/j.lwt.2013.08.021
Lopez-Exposito, I., & Recio, I. (2008). Protective effect of milk peptides: antibacterial and
antitumor properties Bioactive Components of Milk (pp. 271-294): Springer.
López-Fandiño, R., Otte, J., & van Camp, J. (2006). Physiological, chemical and
technological aspects of milk-protein-derived peptides with antihypertensive and
ACE-inhibitory activity. International Dairy Journal, 16(11), 1277-1293.
doi:http://dx.doi.org/10.1016/j.idairyj.2006.06.004
208
López‐Barrios, L., Gutiérrez‐Uribe, J. A., & Serna‐Saldívar, S. O. (2014). Bioactive
peptides and hydrolysates from pulses and their potential use as functional
ingredients. Journal of Food Science, 79(3).
Lourens-Hattingh, A., & Viljoen, B. C. (2001). Yogurt as probiotic carrier food.
International Dairy Journal, 11(1), 1-17.
Luhovyy, B. L., Akhavan, T., & Anderson, G. H. (2007). Whey proteins in the regulation
of food intake and satiety. Journal of the American College of Nutrition, 26(6),
704S-712S.
Madureira, A., Tavares, T., Gomes, A. M. P., Pintado, M., & Malcata, F. X. (2010). Invited
review: physiological properties of bioactive peptides obtained from whey proteins.
Journal of Dairy Science, 93(2), 437-455.
Madureira, A. R., Pereira, C. I., Gomes, A. M., Pintado, M. E., & Malcata, F. X. (2007).
Bovine whey proteins–Overview on their main biological properties. Food
Research International, 40(10), 1197-1211.
Mäkeläinen, H., Saarinen, M., Stowell, J., Rautonen, N., & Ouwehand, A. (2010). Xylo-
oligosaccharides and lactitol promote the growth of Bifidobacterium lactis and
Lactobacillus species in pure cultures. Beneficial microbes, 1(2), 139-148.
Mantsch, H. H., & McElhaney, R. N. (1991). Phospholipid phase transitions in model and
biological membranes as studied by infrared spectroscopy. Chemistry and Physics
of Lipids, 57(2), 213-226. doi:http://dx.doi.org/10.1016/0009-3084(91)90077-O
Marafon, A., Sumi, A., Granato, D., Alcântara, M., Tamime, A., & de Oliveira, M. N.
(2011). Effects of partially replacing skimmed milk powder with dairy ingredients
on rheology, sensory profiling, and microstructure of probiotic stirred-type yogurt
during cold storage. Journal of Dairy Science, 94(11), 5330-5340.
Marco, M. L., Heeney, D., Binda, S., Cifelli, C. J., Cotter, P. D., Foligné, B., . . . Pihlanto,
A. (2017). Health benefits of fermented foods: microbiota and beyond. Current
Opinion in Biotechnology, 44, 94-102.
Martinez-Castro, I., & Olano, A. (1980). Influence of thermal processing on carbohydrate
composition of milk. Formation of epilactose. Milchwissenschaft, 35(1), 5-8.
209
Martinez-Castro, I., Olano, A., & Corzo, N. (1986). Modifications and interactions of
lactose with mineral components of milk during heating processes. Food
Chemistry, 21(3), 211-221.
Martins, S. I. F. S., Jongen, W. M. F., & van Boekel, M. A. J. S. (2000). A review of
Maillard reaction in food and implications to kinetic modelling. Trends in Food
Science & Technology, 11(9), 364-373. doi:http://dx.doi.org/10.1016/S0924-
2244(01)00022-X
Marwaha, S., & Kennedy, J. (1988). Whey—pollution problem and potential utilization.
International journal of food science & technology, 23(4), 323-336.
Mastrocola, D., & Munari, M. (2000). Progress of the Maillard reaction and antioxidant
action of Maillard reaction products in preheated model systems during storage.
Journal of Agricultural and Food Chemistry, 48(8), 3555-3559.
Mattila-Sandholm, T., Myllärinen, P., Crittenden, R., Mogensen, G., Fondén, R., &
Saarela, M. (2002). Technological challenges for future probiotic foods.
International Dairy Journal, 12(2), 173-182.
Mayer, J., Kranz, B., & Fischer, L. (2010). Continuous production of lactulose by
immobilized thermostable β-glycosidase from Pyrococcus furiosus. Journal of
biotechnology, 145(4), 387-393.
McCarthy, R., Mills, S., Ross, R., Fitzgerald, G., & Stanton, C. (2014). Bioactive peptides
from casein and whey proteins. Milk and Dairy Products as Functional Foods, 23-
54.
McComas, K., & Gilliland, S. (2003). Growth of probiotic and traditional yogurt cultures
in milk supplemented with whey protein hydrolysate. Journal of food science,
68(6), 2090-2095.
McFarland, L. V. (2006). Meta-analysis of probiotics for the prevention of antibiotic
associated diarrhea and the treatment of Clostridium difficile disease. The American
journal of gastroenterology, 101(4), 812-822.
Medrano, A., Abirached, C., Panizzolo, L., Moyna, P., & Añón, M. C. (2009). The effect
of glycation on foam and structural properties of β-lactoglobulin. Food Chemistry,
113(1), 127-133. doi:http://dx.doi.org/10.1016/j.foodchem.2008.07.036
210
Mehra, R., Marnila, P., & Korhonen, H. (2006). Milk immunoglobulins for health
promotion. International Dairy Journal, 16(11), 1262-1271.
Meisel, H. (2004). Multifunctional peptides encrypted in milk proteins. Biofactors, 21(1-
4), 55-61.
Meisel, H., & Bockelmann, W. (1999). Bioactive peptides encrypted in milk proteins:
proteolytic activation and thropho-functional properties. Antonie van Leeuwenhoek,
76(1-4), 207-215.
Meltretter, J., Birlouez‐Aragon, I., Becker, C. M., & Pischetsrieder, M. (2009). Assessment
of heat treatment of dairy products by MALDI‐TOF‐MS. Molecular Nutrition &
Food Research, 53(12), 1487-1495.
Mendez, A., & Olano, A. (1979). Lactulose. A review of some chemical properties and
applications in infant nutrition and medicine. Paper presented at the Dairy Science
Abstracts.
Mercier, A., Gauthier, S. F., & Fliss, I. l. (2004). Immunomodulating effects of whey
proteins and their enzymatic digests. International Dairy Journal, 14(3), 175-183.
Michalak, M., Mikkelsen, J. D., Jonsson, G. E., & Pinelo, M. (2014). Enzymatic production
and purification of prebiotic oligosaccharides by chromatography and membrane
systems. Technical University of DenmarkDanmarks Tekniske Universitet,
Department of Systems BiologyInstitut for Systembiologi.
Micke, P., Beeh, K., & Buhl, R. (2002). Effects of long-term supplementation with whey
proteins on plasma glutathione levels of HIV-infected patients. European journal
of nutrition, 41(1), 12-18.
Milner, J. (1999). Functional foods and health promotion. The Journal of nutrition, 129(7),
1395S-1397s.
Mohammadi, R., & Mortazavian, A. (2011). Review article: technological aspects of
prebiotics in probiotic fermented milks. Food Reviews International, 27(2), 192-
212.
Mohammadian, M., & Madadlou, A. (2016). Characterization of fibrillated antioxidant
whey protein hydrolysate and comparison with fibrillated protein solution. Food
Hydrocolloids, 52, 221-230.
211
Mohan, A., Udechukwu, M. C., Rajendran, S. R., & Udenigwe, C. C. (2015). Modification
of peptide functionality during enzymatic hydrolysis of whey proteins. RSC
Advances, 5(118), 97400-97407.
Mohanty, D., Mohapatra, S., Misra, S., & Sahu, P. (2016). Milk derived bioactive peptides
and their impact on human health–A review. Saudi Journal of Biological Sciences,
23(5), 577-583.
Mollea, C., Marmo, L., & Bosco, F. (2013). Valorisation of cheese whey, a by-product
from the dairy industry Food Industry: InTech.
Möller, N. P., Scholz-Ahrens, K. E., Roos, N., & Schrezenmeir, J. (2008). Bioactive
peptides and proteins from foods: indication for health effects. European journal of
nutrition, 47(4), 171-182.
Monahan, F. J., German, J. B., & Kinsella, J. E. (1995). Effect of pH and temperature on
protein unfolding and thiol/disulfide interchange reactions during heat-induced
gelation of whey proteins. Journal of agricultural and food chemistry, 43(1), 46-
52.
Montgomery, E. M., & Hudson, C. (1930). Relations between rotatory power and structure
in the sugar group. XXVII. Synthesis of a new disaccharide ketose (lactulose) from
lactose1. Journal of the American Chemical Society, 52(5), 2101-2106.
Montilla, A., Del Castillo, M., Sanz, M., & Olano, A. (2005). Egg shell as catalyst of
lactose isomerisation to lactulose. Food Chemistry, 90(4), 883-890.
Morales, F. J., Fernández-Fraguas, C., & Jiménez-Pérez, S. (2005). Iron-binding ability of
melanoidins from food and model systems. Food Chemistry, 90(4), 821-827.
Morales, F. J., & Jiménez-Pérez, S. (2001). Free radical scavenging capacity of Maillard
reaction products as related to colour and fluorescence. Food Chemistry, 72(1),
119-125.
Mortazavian, A., Ghorbanipour, S., Mohammadifar, M., & Mohammadi, M. (2011).
Biochemical properties and viable probiotic population of yogurt at different
bacterial inoculation rates and incubation temperatures. Philipp Agric Scientist, 94,
111-116.
212
Mortazavian, A. M., Mohammadi, R., & Sohrabvandi, S. (2012). Delivery of probiotic
microorganisms into gastrointestinal tract by food products: INTECH Open
Access Publisher.
Mottram, D. S., Wedzicha, B. L., & Dodson, A. T. (2002). Food chemistry: acrylamide is
formed in the Maillard reaction. nature, 419(6906), 448-449.
Mullally, M. M., Meisel, H., & FitzGerald, R. J. (1997). Angiotensin-I-converting enzyme
inhibitory activities of gastric and pancreatic proteinase digests of whey proteins.
International Dairy Journal, 7(5), 299-303. doi:http://dx.doi.org/10.1016/S0958-
6946(97)00018-6
Mulvihill, D., & Ennis, M. (2003). Functional milk proteins: production and utilization
Advanced dairy chemistry—1 Proteins (pp. 1175-1228): Springer.
Muniandy, P., Shori, A. B., & Baba, A. S. (2016). Influence of green, white and black tea
addition on the antioxidant activity of probiotic yogurt during refrigerated storage.
Food Packaging and Shelf Life, 8, 1-8.
doi:http://dx.doi.org/10.1016/j.fpsl.2016.02.002
Nabok, M., & Plutahin, G. (2009). Baking wheat bread using electro-activated aqueous
solutions. Communications of the Kuban State Agrarian University, Krasnodar,
Russia.
Nagpal, R., Behare, P., Rana, R., Kumar, A., Kumar, M., Arora, S., . . . Yadav, H. (2011).
Bioactive peptides derived from milk proteins and their health beneficial potentials:
an update. Food & function, 2(1), 18-27.
Naranjo, G. B., Malec, L. S., & Vigo, M. (1998). Reducing sugars effect on available lysine
loss of casein by moderate heat treatment. Food Chemistry, 62(3), 309-313.
Nath, A., Verasztó, B., Basak, S., Koris, A., Kovács, Z., & Vatai, G. (2016). Synthesis of
lactose-derived nutraceuticals from dairy waste whey—a review. Food and
Bioprocess Technology, 9(1), 16-48.
Nazzaro, F., Fratianni, F., Nicolaus, B., Poli, A., & Orlando, P. (2012). The prebiotic source
influences the growth, biochemical features and survival under simulated
gastrointestinal conditions of the probiotic Lactobacillus acidophilus. Anaerobe,
18(3), 280-285.
213
Ng, S., Hart, A., Kamm, M., Stagg, A., & Knight, S. (2009). Mechanisms of action of
probiotics: recent advances. Inflammatory bowel diseases, 15(2), 300-310.
Nicoli, M., Anese, M., & Parpinel, M. (1999). Influence of processing on the antioxidant
properties of fruit and vegetables. Trends in Food Science & Technology, 10(3),
94-100.
Nongonierma, A., O’keeffe, M., & FitzGerald, R. (2016). Milk protein hydrolysates and
bioactive peptides Advanced Dairy Chemistry (pp. 417-482): Springer.
Nongonierma, A. B., & FitzGerald, R. J. (2015). Bioactive properties of milk proteins in
humans: A review. Peptides, 73, 20-34.
Nooshkam, M., & Madadlou, A. (2016a). Maillard conjugation of lactulose with
potentially bioactive peptides. Food Chemistry, 192, 831-836.
doi:http://dx.doi.org/10.1016/j.foodchem.2015.07.094
Nooshkam, M., & Madadlou, A. (2016b). Microwave-assisted isomerisation of lactose to
lactulose and Maillard conjugation of lactulose and lactose with whey proteins and
peptides. Food Chemistry, 200, 1-9.
doi:http://dx.doi.org/10.1016/j.foodchem.2015.12.094
O'Toole, P. W., & Cooney, J. C. (2008). Probiotic bacteria influence the composition and
function of the intestinal microbiota. Interdisciplinary perspectives on infectious
diseases, 2008.
Oelschlaeger, T. A. (2010). Mechanisms of probiotic actions–a review. International
Journal of Medical Microbiology, 300(1), 57-62.
Oh, N. S., Koh, J. H., Park, M. R., Kim, Y., & Kim, S. H. (2016). Hypolipidemic and
antiinflammatory effects of fermented Maillard reaction products by Lactobacillus
fermentum H9 in an animal model. Journal of Dairy Science, 99(12), 9415-9423.
Oh, N. S., Lee, H. A., Lee, J. Y., Joung, J. Y., Lee, K. B., Kim, Y., . . . Kim, S. H. (2013).
The dual effects of Maillard reaction and enzymatic hydrolysis on the antioxidant
activity of milk proteins. Journal of Dairy Science, 96(8), 4899-4911.
doi:http://dx.doi.org/10.3168/jds.2013-6613
Olano, A., Calvo, M., & Corzo, N. (1989). Changes in the carbohydrate fraction of milk
during heating processes. Food Chemistry, 31(4), 259-265.
214
Olano, A., & Calvo, M. M. (1989). Kinetics of lactulose, galactose and epilactose
formation during heat-treatment of milk. Food Chemistry, 34(4), 239-248.
doi:http://dx.doi.org/10.1016/0308-8146(89)90101-5
Olano, A., & Corzo, N. (2009). Lactulose as a food ingredient. Journal of the Science of
Food and Agriculture, 89(12), 1987-1990.
Olano, A., & Martinez-Castro, I. (1981). Formation of lactulose and epilactose from lactose
in basic media. A quantitative study. Milchwissenschaft (Germany, FR).
Olano, A., Santa-Maria, G., Corzo, N., Calvo, M., Martinez-Castro, I., & Jimeno, M.
(1992). Determination of free carbohydrates and Amadori compounds formed at
the early stages of non-enzymic browning. Food Chemistry, 43(5), 351-358.
Oliveira, F. C. d., Coimbra, J. S. d. R., de Oliveira, E. B., Zuñiga, A. D. G., & Rojas, E. E.
G. (2014). Food protein-polysaccharide conjugates obtained via the maillard
reaction: A review. Critical reviews in food science and nutrition(just-accepted),
00-00.
Oliveira, R. P. D. S., Florence, A. C. R., Perego, P., De Oliveira, M. N., & Converti, A.
(2011). Use of lactulose as prebiotic and its influence on the growth, acidification
profile and viable counts of different probiotics in fermented skim milk.
International journal of food microbiology, 145(1), 22-27.
Oliveira, R. P. D. S., Perego, P., Converti, A., & De Oliveira, M. N. (2009a). Effect of
inulin on growth and acidification performance of different probiotic bacteria in co-
cultures and mixed culture with Streptococcus thermophilus. Journal of Food
Engineering, 91(1), 133-139. doi:https://doi.org/10.1016/j.jfoodeng.2008.08.013
Oliveira, R. P. S., Florence, A. C. R., Silva, R. C., Perego, P., Converti, A., Gioielli, L. A.,
& Oliveira, M. N. (2009b). Effect of different prebiotics on the fermentation
kinetics, probiotic survival and fatty acids profiles in nonfat symbiotic fermented
milk. International journal of food microbiology, 128(3), 467-472.
doi:https://doi.org/10.1016/j.ijfoodmicro.2008.10.012
Oliver, C. M., Melton, L. D., & Stanley, R. A. (2006). Creating proteins with novel
functionality via the Maillard reaction: a review. Critical reviews in food science
and nutrition, 46(4), 337-350.
215
Önay-Uçar, E., Arda, N., Pekmez, M., Yılmaz, A. M., Böke-Sarıkahya, N., Kırmızıgül, S.,
& Yalçın, A. S. (2014). Comparison of antioxidant capacity, protein profile and
carbohydrate content of whey protein fractions. Food Chemistry, 150(0), 34-40.
doi:http://dx.doi.org/10.1016/j.foodchem.2013.10.119
Ortiz-Chao, P., Gómez-Ruiz, J. A., Rastall, R. A., Mills, D., Cramer, R., Pihlanto, A., . . .
Jauregi, P. (2009). Production of novel ACE inhibitory peptides from β-
lactoglobulin using Protease N Amano. International dairy journal, 19(2), 69-76.
doi:http://dx.doi.org/10.1016/j.idairyj.2008.07.011
Otieno, D. O., & Ahring, B. K. (2012). The potential for oligosaccharide production from
the hemicellulose fraction of biomasses through pretreatment processes:
xylooligosaccharides (XOS), arabinooligosaccharides (AOS), and
mannooligosaccharides (MOS). Carbohydrate Research, 360, 84-92.
Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an
improved oxygen radical absorbance capacity assay using fluorescein as the
fluorescent probe. Journal of Agricultural and Food Chemistry, 49(10), 4619-4626.
Ouwehand, A. C., & Salminen, S. (2003). In vitro adhesion assays for probiotics and their
in vivo relevance: a review. Microbial ecology in health and disease, 15(4), 175-
184.
Oyaizu, M. (1986). Studies on products of browning reaction--antioxidative activities of
products of browning reaction prepared from glucosamine. Eiyogaku zasshi=
Japanese journal of nutrition.
Özer, D., Akin, S., & Özer, B. (2005). Effect of Inulin and Lactulose on Survival of
Lactobacillus AcidophilusLA-5 and Bifidobacterium Bifidum BB-02 in
Acidophilus-Bifidus Yoghurt. Revista de Agaroquimica y Tecnologia de Alimentos,
11(1), 19-24.
Özyürek, M., Bektaşoğlu, B., Güçlü, K., & Apak, R. (2008). Hydroxyl radical scavenging
assay of phenolics and flavonoids with a modified cupric reducing antioxidant
capacity (CUPRAC) method using catalase for hydrogen peroxide degradation.
analytica chimica acta, 616(2), 196-206.
Padilla, B., Frau, F., Ruiz-Matute, A. I., Montilla, A., Belloch, C., Manzanares, P., &
Corzo, N. (2015). Production of lactulose oligosaccharides by isomerisation of
216
transgalactosylated cheese whey permeate obtained by β-galactosidases from dairy
Kluyveromyces. Journal of Dairy Research, 82(3), 356-364.
Pan, Y., Lee, A., Wan, J., Coventry, M. J., Michalski, W. P., Shiell, B., & Roginski, H.
(2006). Antiviral properties of milk proteins and peptides. International Dairy
Journal, 16(11), 1252-1261. doi:http://dx.doi.org/10.1016/j.idairyj.2006.06.010
Pandey, K. R., Naik, S. R., & Vakil, B. V. (2015). Probiotics, prebiotics and synbiotics-a
review. Journal of food science and technology, 52(12), 7577-7587.
Panesar, P. S., Kennedy, J. F., Gandhi, D. N., & Bunko, K. (2007). Bioutilisation of whey
for lactic acid production. Food Chemistry, 105(1), 1-14.
Panesar, P. S., & Kumari, S. (2011). Lactulose: production, purification and potential
applications. Biotechnology advances, 29(6), 940-948.
Park, Y. W., & Nam, M. S. (2015). Bioactive peptides in milk and dairy products: a review.
Korean journal for food science of animal resources, 35(6), 831.
Parvez, S., Malik, K. A., Ah Kang, S., & Kim, H. Y. (2006). Probiotics and their fermented
food products are beneficial for health. Journal of applied microbiology, 100(6),
1171-1185.
Paseephol, T., Small, D. M., & Sherkat, F. (2008). Lactulose production from milk
concentration permeate using calcium carbonate-based catalysts. Food Chemistry,
111(2), 283-290. doi:http://dx.doi.org/10.1016/j.foodchem.2008.03.051
Pastrana, L., González, R., Estévez, N., Pereira, L., Rodríguez Amado, I., Fuciños, P., . . .
Troncoso, R. (2017). 7 - Functional Foods Current Developments in Biotechnology
and Bioengineering (pp. 165-200): Elsevier.
Pastukhov, V., & Morozov, V. (2000). Raman scattering of light by the electroactivated
water. Optics and Spectroscopy, 88(1), 35-37.
Patel, S. (2015). Functional food relevance of whey protein: A review of recent findings
and scopes ahead. Journal of Functional Foods, 19, Part A, 308-319.
doi:http://dx.doi.org/10.1016/j.jff.2015.09.040
Paterson, A. H. J., & Kellam, S. J. (2009). 24 - Transformation of lactose for value-added
ingredients. In M. Corredig (Ed.), Dairy-Derived Ingredients (pp. 625-643):
Woodhead Publishing.
217
Pei, R., Martin, D. A., DiMarco, D. M., & Bolling, B. W. (2017). Evidence for the effects
of yogurt on gut health and obesity. Critical reviews in food science and nutrition,
57(8), 1569-1583.
Pellegrini, A., Dettling, C., Thomas, U., & Hunziker, P. (2001). Isolation and
characterization of four bactericidal domains in the bovine β-lactoglobulin.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1526(2), 131-140.
doi:http://dx.doi.org/10.1016/S0304-4165(01)00116-7
Pellegrini, A., Thomas, U., Bramaz, N., Hunziker, P., & von Fellenberg, R. (1999).
Isolation and identification of three bactericidal domains in the bovine α-
lactalbumin molecule. Biochimica et Biophysica Acta (BBA) - General Subjects,
1426(3), 439-448. doi:http://dx.doi.org/10.1016/S0304-4165(98)00165-2
Peña-Ramos, E. A., & Xiong, Y. L. (2001). Antioxidative Activity of Whey Protein
Hydrolysates in a Liposomal System1. Journal of Dairy Science, 84(12), 2577-
2583. doi:http://dx.doi.org/10.3168/jds.S0022-0302(01)74711-X
Peña-Ramos, E. A., & Xiong, Y. L. (2003). Whey and soy protein hydrolysates inhibit lipid
oxidation in cooked pork patties. Meat Science, 64(3), 259-263.
doi:http://dx.doi.org/10.1016/S0309-1740(02)00187-0
Peng, X., Kong, B., Xia, X., & Liu, Q. (2010). Reducing and radical-scavenging activities
of whey protein hydrolysates prepared with Alcalase. International Dairy Journal,
20(5), 360-365. doi:http://dx.doi.org/10.1016/j.idairyj.2009.11.019
Peng, X., Xiong, Y. L., & Kong, B. (2009). Antioxidant activity of peptide fractions from
whey protein hydrolysates as measured by electron spin resonance. Food
Chemistry, 113(1), 196-201.
doi:http://dx.doi.org/10.1016/j.foodchem.2008.07.068
Petschow, B. W., & Talbott, R. D. (1990). Growth promotion of Bifidobacterium species
by whey and casein fractions from human and bovine milk. Journal of Clinical
Microbiology, 28(2), 287-292.
Petuely, F. (1957). Lactobacillus bifidus flora produced in artificially-fed infants by
bifidogenic substances (bifidus factor). Zeitschrift fur Kinderheilkunde, 79(2), 174-
179.
218
Pihlanto, A. (2006). Antioxidative peptides derived from milk proteins. International
Dairy Journal, 16(11), 1306-1314.
doi:http://dx.doi.org/10.1016/j.idairyj.2006.06.005
Pihlanto, A., & Korhonen, H. (2003). Bioactive peptides and proteins. Advances in Food
and Nutrition Research, 47, 175-276.
Pihlanto, A., & Korhonen, H. (2015). 3 - Bioactive peptides from fermented foods and
health promotion A2 - Holzapfel, Wilhelm Advances in Fermented Foods and
Beverages (pp. 39-74): Woodhead Publishing.
Pihlanto-Leppälä, A. (2000). Bioactive peptides derived from bovine whey proteins: opioid
and ace-inhibitory peptides. Trends in Food Science & Technology, 11(9), 347-356.
Pihlanto-Leppälä, A., Koskinen, P., Paakkari, I., Tupasela, T., & Korhonen, H. J. (1996).
Opioid whey protein peptides obtained by membrane filtration.
Pihlanto-Leppälä, A., Paakkari, I., Rinta-Koski, M., & Antila, P. (1997). Bioactive peptide
derived from in vitro proteolysis of bovine β-lactoglobulin and its effect on smooth
muscle. Journal of Dairy Research, 64(01), 149-155.
Pihlanto-Leppälä, A., Rokka, T., & Korhonen, H. (1998). Angiotensin I Converting
Enzyme Inhibitory Peptides Derived from Bovine Milk Proteins. International
Dairy Journal, 8(4), 325-331. doi:http://dx.doi.org/10.1016/S0958-
6946(98)00048-X
Pihlanto‐Leppälä, A., Marnila, P., Hubert, L., Rokka, T., Korhonen, H., & Karp, M. (1999).
The effect of αα‐lactalbumin and ββ‐lactoglobulin hydrolysates on the metabolic
activity of Escherichia coli JM103. Journal of Applied Microbiology, 87(4), 540-
545.
Pintaric, R., Matela, J., & Pintaric, S. (2015). Suitability of electrolyzed oxidizing water
for the disinfection of hard surfaces and equipment in radiology. Journal of
Environmental Health Science and Engineering, 13(1), 6.
Playne, M., & Crittenden, R. (2009). Galacto-oligosaccharides and other products derived
from lactose Advanced dairy chemistry (pp. 121-201): Springer.
Posada-Izquierdo, G. D., Pérez-Rodríguez, F., López-Gálvez, F., Allende, A., Gil, M. I., &
Zurera, G. (2014). Modeling growth of Escherichia coli O157: H7 in fresh-cut
219
lettuce treated with neutral electrolyzed water and under modified atmosphere
packaging. International journal of food microbiology, 177, 1-8.
Power, O., Jakeman, P., & FitzGerald, R. (2013). Antioxidative peptides: enzymatic
production, in vitro and in vivo antioxidant activity and potential applications of
milk-derived antioxidative peptides. Amino Acids, 44(3), 797-820.
Prazeres, A. R., Carvalho, F., & Rivas, J. (2012). Cheese whey management: A review.
Journal of Environmental Management, 110, 48-68.
Prgomet, C., Peters, S., & Pfaffl, M. W. (2006). Influence of bovine lactoferrin and
lactoferricin on cytokine expression in LPS‐treated cultivated bovine blood cells.
Journal of the Science of Food and Agriculture, 86(4), 640-647.
Pridmore, R. D., Pittet, A.-C., Praplan, F., & Cavadini, C. (2008). Hydrogen peroxide
production by Lactobacillus johnsonii NCC 533 and its role in anti-Salmonella
activity. FEMS microbiology letters, 283(2), 210-215.
Prilutsky, V., & Bakhir, V. (1997). Electrochemically activated water: anomalous
properties, mechanism of biological action. M., VNIIIMT.
Prior, R. L., Wu, X., & Schaich, K. (2005). Standardized methods for the determination of
antioxidant capacity and phenolics in foods and dietary supplements. Journal of
Agricultural and Food Chemistry, 53(10), 4290-4302.
Prioult, G., Pecquet, S., & Fliss, I. (2004). Stimulation of interleukin-10 production by
acidic β-lactoglobulin-derived peptides hydrolyzed with Lactobacillus paracasei
NCC2461 peptidases. Clinical and diagnostic laboratory immunology, 11(2), 266-
271.
Ranadheera, R., Baines, S., & Adams, M. (2010). Importance of food in probiotic efficacy.
Food Research International, 43(1), 1-7.
Rao, M., Chawla, S., Chander, R., & Sharma, A. (2011). Antioxidant potential of Maillard
reaction products formed by irradiation of chitosan–glucose solution. Carbohydrate
Polymers, 83(2), 714-719.
Rastall, R. A., & Gibson, G. R. (2015). Recent developments in prebiotics to selectively
impact beneficial microbes and promote intestinal health. Current Opinion in
Biotechnology, 32, 42-46.
220
Rastall, R. A., Gibson, G. R., Gill, H. S., Guarner, F., Klaenhammer, T. R., Pot, B., . . .
Sanders, M. E. (2005). Modulation of the microbial ecology of the human colon by
probiotics, prebiotics and synbiotics to enhance human health: an overview of
enabling science and potential applications. FEMS microbiology ecology, 52(2),
145-152.
Ravula, R., & Shah, N. (1998). Effect of acid casein hydrolysate and cysteine on the
viability of yogurt and probiotic bacteria in fermented frozen dairy desserts.
Australian Journal of Dairy Technology, 53(3), 175.
Reid, G. (2015). The growth potential for dairy probiotics. International Dairy Journal,
49, 16-22. doi:http://doi.org/10.1016/j.idairyj.2015.04.004
Rijkers, G. T., De Vos, W. M., Brummer, R.-J., Morelli, L., Corthier, G., & Marteau, P.
(2011). Health benefits and health claims of probiotics: bridging science and
marketing. British Journal of Nutrition, 106(09), 1291-1296.
Roberfroid, M. (2002). Functional food concept and its application to prebiotics. Digestive
and Liver Disease, 34, S105-S110.
Roberfroid, M. (2007). Prebiotics: the concept revisited. The Journal of nutrition, 137(3),
830S-837S.
Roberfroid, M., Gibson, G. R., Hoyles, L., McCartney, A. L., Rastall, R., Rowland, I., . . .
Stahl, B. (2010). Prebiotic effects: metabolic and health benefits. British Journal of
Nutrition, 104(S2), S1-S63.
Roberfroid, M. B. (2000). Concepts and strategy of functional food science: the European
perspective. The American journal of clinical nutrition, 71(6), 1660s-1664s.
Romano, N., Tymczyszyn, E., Mobili, A., & Gómez-Zavaglia, A. (2015). Prebiotics as
protectants of lactic acid bacteria Bioactive foods in promoting health: Probiotics,
prebiotics, and synbiotics, Part 1: Prebiotics in health promotion (pp. 155-164):
Academic Press, Elsevier.
Ross, R., Desmond, C., Fitzgerald, G., & Stanton, C. (2005). Overcoming the technological
hurdles in the development of probiotic foods. Journal of Applied Microbiology,
98(6), 1410-1417.
Roy, D. (2005). Technological aspects related to the use of bifidobacteria in dairy products.
Le lait, 85(1-2), 39-56.
221
Saad, N., Delattre, C., Urdaci, M., Schmitter, J.-M., & Bressollier, P. (2013). An overview
of the last advances in probiotic and prebiotic field. LWT-Food Science and
Technology, 50(1), 1-16.
Saarela, M., Hallamaa, K., Mattila-Sandholm, T., & Mättö, J. (2003). The effect of lactose
derivatives lactulose, lactitol and lactobionic acid on the functional and
technological properties of potentially probiotic Lactobacillus strains. International
Dairy Journal, 13(4), 291-302.
Saarela, M., Mogensen, G., Fonden, R., Mättö, J., & Mattila-Sandholm, T. (2000).
Probiotic bacteria: safety, functional and technological properties. Journal of
biotechnology, 84(3), 197-215.
Saint-Sauveur, D., Gauthier, S. F., Boutin, Y., & Montoni, A. (2008). Immunomodulating
properties of a whey protein isolate, its enzymatic digest and peptide fractions.
International Dairy Journal, 18(3), 260-270.
Sakkas, L., Moutafi, A., Moschopoulou, E., & Moatsou, G. (2014). Assessment of heat
treatment of various types of milk. Food Chemistry, 159(0), 293-301.
doi:http://dx.doi.org/10.1016/j.foodchem.2014.03.020
Salami, M., Moosavi-Movahedi, A. A., Ehsani, M. R., Yousefi, R., Haertlé, T., Chobert,
J.-M., . . . Ebadi, S. A. (2010). Improvement of the antimicrobial and antioxidant
activities of camel and bovine whey proteins by limited proteolysis. Journal of
agricultural and food chemistry, 58(6), 3297-3302.
Sánchez, B., Delgado, S., Blanco‐Míguez, A., Lourenço, A., Gueimonde, M., & Margolles,
A. (2016). Probiotics, gut microbiota and their influence on host health and disease.
Molecular Nutrition & Food Research.
Sarkar, A., & Mandal, S. (2016). Bifidobacteria—Insight into clinical outcomes and
mechanisms of its probiotic action. Microbiological Research, 192, 159-171.
Sartor, R. B. (2004). Therapeutic manipulation of the enteric microflora in inflammatory
bowel diseases: antibiotics, probiotics, and prebiotics. Gastroenterology, 126(6),
1620-1633.
Schaafsma, G. (2008). Lactose and lactose derivatives as bioactive ingredients in human
nutrition. International Dairy Journal, 18(5), 458-465.
doi:http://dx.doi.org/10.1016/j.idairyj.2007.11.013
222
Schumann, C. (2002). Medical, nutritional and technological properties of lactulose. An
update. European journal of nutrition, 41.
Schuster-Wolff-Bühring, R., Fischer, L., & Hinrichs, J. (2010). Production and
physiological action of the disaccharide lactulose. International Dairy Journal,
20(11), 731-741.
Seiquer, I., Rubio, L. A., Peinado, M. J., Delgado‐Andrade, C., & Navarro, M. P. (2014).
Maillard reaction products modulate gut microbiota composition in adolescents.
Molecular nutrition & food research, 58(7), 1552-1560.
Seki, N., & Saito, H. (2012). Lactose as a source for lactulose and other functional lactose
derivatives. International Dairy Journal, 22(2), 110-115.
Seo, Y. H., Park, G. W., & Han, J.-I. (2015). Efficient lactulose production from cheese
whey using sodium carbonate. Food Chemistry, 173, 1167-1171.
Shaghaghi, M., Pourahmad, R., & Adeli, H. (2013). Synbiotic yogurt production by using
prebiotic compounds and probiotic lactobacilli. Int. Res. J. Appl. Basic Sci, 5, 839-
846.
Shah, N. (2000). Probiotic bacteria: selective enumeration and survival in dairy foods.
Journal of Dairy Science, 83(4), 894-907.
Shah, N., & Ravula, R. (2000). Influence of water activity on fermentation, organic acids
production and viability of yogurt and probiotic bacteria. Australian Journal of
Dairy Technology, 55(3), 127.
Shah, N. P. (2007). Functional cultures and health benefits. International Dairy Journal,
17(11), 1262-1277. doi:http://doi.org/10.1016/j.idairyj.2007.01.014
Shah, N. P., Lankaputhra, W. E., Britz, M. L., & Kyle, W. S. (1995). Survival of
Lactobacillus acidophilus and Bifidobacterium bifidum in commercial yoghurt
during refrigerated storage. International Dairy Journal, 5(5), 515-521.
Shahidi, F., & Zhong, Y. (2008). Bioactive peptides. Journal of AOAC International, 91(4),
914-931.
Shaposhnik, V., & Kesore, K. (1997). An early history of electrodialysis with
permselective membranes. Journal of Membrane Science, 136(1-2), 35-39.
223
Sharma, R., Rajput, Y. S., & Mann, B. (2013). Chemical and functional properties of
glycomacropeptide (GMP) and its role in the detection of cheese whey adulteration
in milk: a review. Dairy science & technology, 93(1), 21-43.
Sherman, P. M., Ossa, J. C., & Johnson-Henry, K. (2009). Unraveling mechanisms of
action of probiotics. Nutrition in Clinical Practice, 24(1), 10-14.
Shihata, A., & Shah, N. (2000). Proteolytic profiles of yogurt and probiotic bacteria.
International Dairy Journal, 10(5), 401-408.
Shukla, R., Verykios, X. E., & Mutharasan, R. (1985). Isomerization and hydrolysis
reactions of important disaccharides over inorganic heterogeneous catalysts.
Carbohydrate research, 143, 97-106.
Silva, S. V., & Malcata, F. X. (2005). Caseins as source of bioactive peptides. International
Dairy Journal, 15(1), 1-15.
Silvério, S. C., Macedo, E. A., Teixeira, J. A., & Rodrigues, L. R. (2016). Biocatalytic
Approaches Using Lactulose: End Product Compared with Substrate.
Comprehensive Reviews in Food Science and Food Safety, 15(5), 878-896.
Sindayikengera, S., & Xia, W.-s. (2006). Nutritional evaluation of caseins and whey
proteins and their hydrolysates from Protamex. Journal of Zhejiang University
Science B, 7(2), 90-98.
Sinha, R., Radha, C., Prakash, J., & Kaul, P. (2007). Whey protein hydrolysate: Functional
properties, nutritional quality and utilization in beverage formulation. Food
Chemistry, 101(4), 1484-1491.
doi:http://dx.doi.org/10.1016/j.foodchem.2006.04.021
Siro, I., Kápolna, E., Kápolna, B., & Lugasi, A. (2008). Functional food. Product
development, marketing and consumer acceptance—A review. Appetite, 51(3),
456-467.
Siso, M. I. G. (1996). The biotechnological utilization of cheese whey: A review.
Bioresource Technology, 57(1), 1-11. doi:http://dx.doi.org/10.1016/0960-
8524(96)00036-3
Sitanggang, A. B., Drews, A., & Kraume, M. (2016). Recent advances on prebiotic
lactulose production. World Journal of Microbiology and Biotechnology, 32(9),
154.
224
Slavin, J. (2013). Fiber and prebiotics: mechanisms and health benefits. Nutrients, 5(4),
1417-1435.
Smart, J. B., Pillidge, C. J., & Garman, J. H. (1993). Growth of lactic acid bacteria and
bifidobacteria on lactose and lactose-related mono-, di-and trisaccharides and
correlation with distribution of β–galactosidase and phospho-β–galactosidase.
Journal of dairy research, 60(04), 557-568.
Smithers, G. W. (2008). Whey and whey proteins—from ‘gutter-to-gold’. International
Dairy Journal, 18(7), 695-704.
Song, Y. S., Lee, H. U., Park, C., & Kim, S. W. (2013). Batch and continuous synthesis of
lactulose from whey lactose by immobilized β-galactosidase. Food Chemistry,
136(2), 689-694.
Spălățelu, C. (2012). Biotechnological valorisation of whey. Innovative Romanian Food
Biotechnology, 10, 1-8.
Sprinchan, E. G., Bologa, M. K., Stepurina, T. G., Bologa, A. M., & Polikarpov, A. A.
(2011). Peculiarities of the electric activation of whey. Surface Engineering and
Applied Electrochemistry, 47(1), 66-69. doi:10.3103/s1068375511010182
Stamatova, I., & Meurman, J. H. (2009). Probiotics: health benefits in the mouth. Am J
Dent, 22(6), 329-338.
Stanic-Vucinic, D., Prodic, I., Apostolovic, D., Nikolic, M., & Velickovic, T. C. (2013).
Structure and antioxidant activity of β-lactoglobulin-glycoconjugates obtained by
high-intensity-ultrasound-induced Maillard reaction in aqueous model systems
under neutral conditions. Food Chemistry, 138(1), 590-599.
Stanton, C., Ross, R. P., Fitzgerald, G. F., & Van Sinderen, D. (2005). Fermented
functional foods based on probiotics and their biogenic metabolites. Current
opinion in biotechnology, 16(2), 198-203.
Sumeri, I., Arike, L., Adamberg, K., & Paalme, T. (2008). Single bioreactor gastrointestinal
tract simulator for study of survival of probiotic bacteria. Applied microbiology and
biotechnology, 80(2), 317-324.
Sun, J., & Buys, N. J. (2016). Glucose-and glycaemic factor-lowering effects of probiotics
on diabetes: a meta-analysis of randomised placebo-controlled trials. British
Journal of Nutrition, 115(07), 1167-1177.
225
Sun, W.-W., Yu, S.-J., Zeng, X.-A., Yang, X.-Q., & Jia, X. (2011). Properties of whey
protein isolate–dextran conjugate prepared using pulsed electric field. Food
Research International, 44(4), 1052-1058.
doi:http://dx.doi.org/10.1016/j.foodres.2011.03.020
Sun, Y., Hayakawa, S., Puangmanee, S., & Izumori, K. (2006). Chemical properties and
antioxidative activity of glycated α-lactalbumin with a rare sugar, d-allose, by
Maillard reaction. Food Chemistry, 95(3), 509-517.
doi:http://dx.doi.org/10.1016/j.foodchem.2005.01.033
Svensson, M., Fast, J., Mossberg, A. K., Düringer, C., Gustafsson, L., Hallgren, O., . . .
Svanborg, C. (2003). α‐Lactalbumin unfolding is not sufficient to cause apoptosis,
but is required for the conversion to HAMLET (human α‐lactalbumin made lethal
to tumor cells). Protein Science, 12(12), 2794-2804.
Szajewska, H., Skorka, A., Ruszczyński, M., & Gieruszczak‐Białek, D. (2013). Meta‐
analysis: Lactobacillus GG for treating acute gastroenteritis in children–updated
analysis of randomised controlled trials. Alimentary pharmacology & therapeutics,
38(5), 467-476.
Szilagyi, A. (2010). Functional Disaccharides: Lactulose, Lactitol, and Lactose. Handbook
of prebiotics and probiotics ingredients. Health benefits and food applications (eds
SS Cho & ET Finocchiaro) CRC Press Taylor & Francis Group Boca Raton New
York, 95-122.
Tabatabaie, F., & Mortazavi, A. (2008). Influence of Lactulose on the survival of probiotic
strains in yoghurt.
Talwalkar, A., & Kailasapathy, K. (2004). A review of oxygen toxicity in probiotic
yogurts: influence on the survival of probiotic bacteria and protective techniques.
Comprehensive Reviews in Food Science and Food Safety, 3(3), 117-124.
Tamime, A., Saarela, M., Sondergaard, A. K., Mistry, V., & Shah, N. (2005). Production
and maintenance of viability of probiotic microorganisms in dairy products.
Probiotic dairy products, 39-72.
Tavares, T., Contreras, M. d. M., Amorim, M., Pintado, M., Recio, I., & Malcata, F. X.
(2011). Novel whey-derived peptides with inhibitory effect against angiotensin-
226
converting enzyme: In vitro effect and stability to gastrointestinal enzymes.
Peptides, 32(5), 1013-1019. doi:http://dx.doi.org/10.1016/j.peptides.2011.02.005
Tavares, T. G., & Malcata, F. X. (2013). Whey proteins as source of bioactive peptides
against hypertension: INTECH Open Access Publisher.
Tellez, A., Corredig, M., Brovko, L. Y., & Griffiths, M. W. (2010). Characterization of
immune-active peptides obtained from milk fermented by Lactobacillus helveticus.
Journal of Dairy Research, 77(2), 129-136.
Tenovuo, J. (2002). Clinical applications of antimicrobial host proteins lactoperoxidase,
lysozyme and lactoferrin in xerostomia: efficacy and safety. Oral diseases, 8(1),
23-29.
Teratanavat, R., & Hooker, N. H. (2006). Consumer valuations and preference
heterogeneity for a novel functional food. Journal of Food Science, 71(7).
Thorpe, S. R., & Baynes, J. W. (1996). Role of the Maillard reaction in diabetes mellitus
and diseases of aging. Drugs & aging, 9(2), 69-77.
Tomita, M., Wakabayashi, H., Yamauchi, K., Teraguchi, S., & Hayasawa, H. (2002).
Bovine lactoferrin and lactoferricin derived from milk: production and applications.
Biochemistry and Cell Biology, 80(1), 109-112.
Tonucci, L. B., Olbrich dos Santos, K. M., Licursi de Oliveira, L., Rocha Ribeiro, S. M.,
& Duarte Martino, H. S. (2017). Clinical application of probiotics in type 2 diabetes
mellitus: A randomized, double-blind, placebo-controlled study. Clinical Nutrition,
36(1), 85-92. doi:http://doi.org/10.1016/j.clnu.2015.11.011
Torres, D. P., Gonçalves, M. d. P. F., Teixeira, J. A., & Rodrigues, L. R. (2010). Galacto‐
oligosaccharides: Production, properties, applications, and significance as
prebiotics. Comprehensive Reviews in Food Science and Food Safety, 9(5), 438-
454.
Tripathi, M. K., & Giri, S. K. (2014). Probiotic functional foods: Survival of probiotics
during processing and storage. Journal of Functional Foods, 9, 225-241.
doi:http://doi.org/10.1016/j.jff.2014.04.030
Troyano, E., Martinez-Castro, I., & Olano, A. (1992). Kinetics of galactose and tagatose
formation during heat-treatment of milk. Food Chemistry, 45(1), 41-43.
doi:http://dx.doi.org/10.1016/0308-8146(92)90010-Y
227
Tsai, J.-S., Chen, T.-J., Pan, B. S., Gong, S.-D., & Chung, M.-Y. (2008). Antihypertensive
effect of bioactive peptides produced by protease-facilitated lactic acid
fermentation of milk. Food Chemistry, 106(2), 552-558.
Tsakali, E., Petrotos, K., D’Allessandro, A., & Goulas, P. (2010). A review on whey
composition and the methods used for its utilization for food and pharmaceutical
products. Paper presented at the 6th International Conference on Simulation and
Modelling in the Food and Bio-Industry (FOODSIM 2010).
Tunick, M. H. (2008). Whey protein production and utilization: a brief history. Whey
processing, functionality and health benefits, 1-13.
Tuohy, K. (2008). Commentary on ‘Prebiotics, immune function, infection and
inflammation: a review of the evidence’. British journal of nutrition, 101(5), 631-
632.
Van Boekel, M. (2006). Formation of flavour compounds in the Maillard reaction.
Biotechnology advances, 24(2), 230-233.
Vasiljevic, T., & Shah, N. P. (2008). Probiotics—from Metchnikoff to bioactives.
International Dairy Journal, 18(7), 714-728.
Vavrusova, M., Pindstrup, H., Johansen, L. B., Andersen, M. L., Andersen, H. J., &
Skibsted, L. H. (2015). Characterisation of a whey protein hydrolysate as
antioxidant. International Dairy Journal, 47, 86-93.
doi:http://dx.doi.org/10.1016/j.idairyj.2015.02.012
Vazquez-Gutierrez, P., Lacroix, C., Jaeggi, T., Zeder, C., Zimmerman, M. B., & Chassard,
C. (2015). Bifidobacteria strains isolated from stools of iron deficient infants can
efficiently sequester iron. BMC microbiology, 15(1), 3.
Vegarud, G. E., Langsrud, T., & Svenning, C. (2000). Mineral-binding milk proteins and
peptides; occurrence, biochemical and technological characteristics. British
Journal of Nutrition, 84(S1), 91-98.
Verbeke, K. A., Boobis, A. R., Chiodini, A., Edwards, C. A., Franck, A., Kleerebezem, M.,
. . . Tuohy, K. M. (2015). Towards microbial fermentation metabolites as markers
for health benefits of prebiotics. Nutrition research reviews, 28(01), 42-66.
228
Verspreet, J., Damen, B., Broekaert, W. F., Verbeke, K., Delcour, J. A., & Courtin, C. M.
(2016). A critical look at prebiotics within the dietary fiber concept. Annual review
of food science and technology, 7, 167-190.
Vesterlund, S., Vankerckhoven, V., Saxelin, M., Goossens, H., Salminen, S., & Ouwehand,
A. C. (2007). Safety assessment of Lactobacillus strains: presence of putative risk
factors in faecal, blood and probiotic isolates. International journal of food
microbiology, 116(3), 325-331.
Vhangani, L. N., & Van Wyk, J. (2013). Antioxidant activity of Maillard reaction products
(MRPs) derived from fructose–lysine and ribose–lysine model systems. Food
Chemistry, 137(1), 92-98.
Vhangani, L. N., & Van Wyk, J. (2016). Antioxidant activity of Maillard reaction products
(MRPs) in a lipid-rich model system. Food Chemistry, 208, 301-308.
Villamiel, M., Corzo, N., Foda, M. I., Montes, F., & Olano, A. n. (2002). Lactulose
formation catalysed by alkaline-substituted sepiolites in milk permeate. Food
Chemistry, 76(1), 7-11.
Vinderola, C., Mocchiutti, P., & Reinheimer, J. (2002). Interactions among lactic acid
starter and probiotic bacteria used for fermented dairy products. Journal of Dairy
Science, 85(4), 721-729.
Wakabayashi, H., Yamauchi, K., & Takase, M. (2006). Lactoferrin research, technology
and applications. International Dairy Journal, 16(11), 1241-1251.
doi:https://doi.org/10.1016/j.idairyj.2006.06.013
Wang, H., Yang, R., Hua, X., Zhao, W., & Zhang, W. (2013a). Enzymatic production of
lactulose and 1-lactulose: current state and perspectives. Applied microbiology and
biotechnology, 97(14), 6167.
Wang, H.-Y., Qian, H., & Yao, W.-R. (2011). Melanoidins produced by the Maillard
reaction: Structure and biological activity. Food Chemistry, 128(3), 573-584.
Wang, J. J., Zhang, Z. H., Li, J. B., Lin, T., Pan, Y. J., & Zhao, Y. (2014). Modeling Vibrio
parahaemolyticus inactivation by acidic electrolyzed water on cooked shrimp using
response surface methodology. Food Control, 36(1), 273-279.
229
Wang, W., & Huang, L. (2017). Ultrahigh pressure promotes colorless bovine serum
albumin-glucose conjugates generation. Innovative Food Science & Emerging
Technologies, 39, 137-140.
Wang, W.-q., Bao, Y.-h., & Chen, Y. (2013b). Characteristics and antioxidant activity of
water-soluble Maillard reaction products from interactions in a whey protein isolate
and sugars system. Food Chemistry, 139(1), 355-361.
Wang, X., & Gibson, G. (1993). Effects of the in vitro fermentation of oligofructose and
inulin by bacteria growing in the human large intestine. Journal of Applied
Bacteriology, 75(4), 373-380.
Wang, Y. (2009). Prebiotics: Present and future in food science and technology. Food
Research International, 42(1), 8-12.
Watson, D., O'Connell Motherway, M., Schoterman, M., Neerven, R., Nauta, A., &
Sinderen, D. (2013). Selective carbohydrate utilization by lactobacilli and
bifidobacteria. Journal of applied microbiology, 114(4), 1132-1146.
Watson, R. R., & Preedy, V. R. (2015). Probiotics, Prebiotics, and Synbiotics: Bioactive
Foods in Health Promotion: Academic Press.
Wijewickreme, A., Krejpcio, Z., & Kitts, D. (1999). Hydroxyl Scavenging Activity of
Glucose, Fructose, and Ribose‐Lysine Model Maillard Products. Journal of Food
Science, 64(3), 457-461.
Willett, W. C. (2002). Balancing life-style and genomics research for disease prevention.
Science, 296(5568), 695-698.
Wnorowski, A., & Yaylayan, V. A. (2003). Monitoring Carbonyl-Amine Reaction between
Pyruvic Acid and α-Amino Alcohols by FTIR SpectroscopyA Possible Route To
Amadori Products. Journal of Agricultural and Food Chemistry, 51(22), 6537-
6543. doi:10.1021/jf034581y
Yadav, J. S. S., Yan, S., Pilli, S., Kumar, L., Tyagi, R., & Surampalli, R. (2015). Cheese
whey: A potential resource to transform into bioprotein, functional/nutritional
proteins and bioactive peptides. Biotechnology advances, 33(6), 756-774.
Yamamoto, N., Akino, A., & Takano, T. (1994). Antihypertensive effect of the peptides
derived from casein by an extracellular proteinase from Lactobacillus helveticus
CP790. Journal of Dairy Science, 77(4), 917-922.
230
Yanagimoto, K., Lee, K.-G., Ochi, H., & Shibamoto, T. (2002). Antioxidative activity of
heterocyclic compounds found in coffee volatiles produced by Maillard reaction.
Journal of Agricultural and Food Chemistry, 50(19), 5480-5484.
Yasmin, A., Butt, M. S., Afzaal, M., van Baak, M., Nadeem, M. T., & Shahid, M. Z. (2015).
Prebiotics, gut microbiota and metabolic risks: Unveiling the relationship. Journal
of Functional Foods, 17, 189-201. doi:http://doi.org/10.1016/j.jff.2015.05.004
Yaylayan, V. A., & Locas, C. P. (2007). Vinylogous Amadori rearrangement: Implications
in food and biological systems. Molecular Nutrition & Food Research, 51(4), 437-
444. doi:10.1002/mnfr.200600126
Yilmaz, Y., & Toledo, R. (2005). Antioxidant activity of water-soluble Maillard reaction
products. Food Chemistry, 93(2), 273-278.
doi:http://dx.doi.org/10.1016/j.foodchem.2004.09.043
Yoshimura, Y., Iijima, T., Watanabe, T., & Nakazawa, H. (1997). Antioxidative effect of
Maillard reaction products using glucose-glycine model system. Journal of
Agricultural and Food Chemistry, 45(10), 4106-4109.
Yu, H., Seow, Y.-X., Ong, P. K. C., & Zhou, W. (2016). Generating Maillard reaction
products in a model system of d-glucose and l-serine by continuous high-intensity
ultrasonic processing. Innovative Food Science & Emerging Technologies, 36, 260-
268. doi:http://dx.doi.org/10.1016/j.ifset.2016.07.011
Zadow, J. (1994). Utilisation of milk components: Whey Robinson: Modern Dairy
Technology (pp. 313-373): Springer.
Zeng, K., & Zhang, D. (2010). Recent progress in alkaline water electrolysis for hydrogen
production and applications. Progress in Energy and Combustion Science, 36(3),
307-326. doi:http://dx.doi.org/10.1016/j.pecs.2009.11.002
Zhang, H., Yang, J., & Zhao, Y. (2015). High intensity ultrasound assisted heating to
improve solubility, antioxidant and antibacterial properties of chitosan-fructose
Maillard reaction products. LWT - Food Science and Technology, 60(1), 253-262.
doi:http://dx.doi.org/10.1016/j.lwt.2014.07.050
Zhao, D., & Shah, N. P. (2014). Influence of tea extract supplementation on bifidobacteria
during soymilk fermentation. International journal of food microbiology, 188, 36-
44.
231
Zhou, J., Pillidge, C., Gopal, P., & Gill, H. (2005). Antibiotic susceptibility profiles of new
probiotic Lactobacillus and Bifidobacterium strains. International journal of food
microbiology, 98(2), 211-217.
Ziemer, C. J., & Gibson, G. R. (1998). An overview of probiotics, prebiotics and synbiotics
in the functional food concept: perspectives and future strategies. International
Dairy Journal, 8(5-6), 473-479.
Zokaee, F., Kaghazchi, T., Zare, A., & Soleimani, M. (2002). Isomerization of lactose to
lactulose—study and comparison of three catalytic systems. Process Biochemistry,
37(6), 629-635. doi:http://dx.doi.org/10.1016/S0032-9592(01)00251-5
Zoumpopoulou, G., Pot, B., Tsakalidou, E., & Papadimitriou, K. (2017). Dairy probiotics:
Beyond the role of promoting gut and immune health. International Dairy Journal,
67, 46-60. doi:http://doi.org/10.1016/j.idairyj.2016.09.010
