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PLH McSweeneyUniversity College Cork, Ireland
Mastering cheese texture and flavour during ripening
University College Cork (1845)
Food
Food
Food
Food
Food
Food
Food
Food
Food in…BEES/ERIHistory, GeographyFood Business and Development (CUBS)Management and Marketing (CUBS)APCMicrobiologyProcess and Chemical EngineeringMedicine
Food
Food: the strategic priority for UCC
Ireland: the dairy islandIrlanda: la isla lechera
University College Cork
The dairy industry in Ireland
Kerry Group. Market capitalisation US$18.5 bn
Irish owned multinationals
1.4 m dairy cows. Approx
16,500 dairy farmers in Ireland. Average herd size 80cows (and growing). Average cow produces 5,000 litres milk per annum. Average farm
output is ca. 11,000 L/Ha
7,300 m litres of milk
But we consume <10%. Export 80% of production. Growing!
205,000 tonnes of cheese(mainly Cheddar L)
Ingredients…
Make 11% of the world’s
trade in infant
formula
Product mix determined by seasonality of milk supply
Outline…
• Overview of cheese ripening, coagulants
• Calcium equilibrium and cheese texture
• Optical properties of low-fat cheese• Redox potential and cheese
Outline…
• Overview of cheese ripening, coagulants
• Calcium equilibrium and cheese texture
• Optical properties of low-fat cheese• Redox potential and cheese
University College Cork
Stages of manufacture
University College Cork
Metabolism of lactate…
Propionate, Acetate, CO2Propionibacterium freudenreichii
Oxidative metabolism
Oxidation toformate, ethanol
and acetateLAB?
Butyrate, CO2, H2Clostridium sp.
H
CCOOHH3C
HO
Ca lactate crystals
University College Cork
O
CO
O
CO
O
CO
OHC
O
O
O
O
H
CH3C
COOHOH
Chymosinand plasmin
Caseins
Intermediate-sized peptides
Proteinases fromLactococcus
Short peptides
Peptidases of Lactococcusand NSLAB
Amino acids
Triglyceride
Fatty acidL-Lactate D-Lactate
Lactose
Fermentationby starter
Racemizationby NSLAB
COOH
CH2
C COOHHO
H2C
COOH
H
CH3C
OHCOOH
Catabo
lism of
lacta
te
VOLATILE FLAVOUR COMPOUNDS
Citrate
Metabolism of citrate
Lipas
e
Fatty acid catabolismAmino acid catabolism
Interactions betweenproducts
Interactions betweenproducts
Other products
Lipolysis andmetabolism of fatty acids
University College Cork
O
CO
O
CO
O
CO
OHC
O
O
O
O
H
CH3C
COOHOH
Chymosinand plasmin
Caseins
Intermediate-sized peptides
Proteinases fromLactococcus
Short peptides
Peptidases of Lactococcusand NSLAB
Amino acids
Triglyceride
Fatty acidL-Lactate D-Lactate
Lactose
Fermentationby starter
Racemizationby NSLAB
COOH
CH2
C COOHHO
H2C
COOH
H
CH3C
OHCOOH
Catabo
lism of
lacta
te
VOLATILE FLAVOUR COMPOUNDS
Citrate
Metabolism of citrate
Lipas
e
Fatty acid catabolismAmino acid catabolism
Interactions betweenproducts
Interactions betweenproducts
Other products
Proteolysis
University College Cork
Origins of proteases in cheese
§ Coagulant (chymosin, others)§ Milk (plasmin, somatic cell proteinases)§ Starter bacteria§ Secondary starter§ Non-starter microflora
University College Cork
Coagulant§ Rennet coagulation of milk…§ Proportion of added rennet activity retained in curd§ Little hydrolysis of b-casein in cheese due to hydrophobic
interactions of hydrophobic C-terminal region § Principally responsible for primary hydrolysis of as1-casein
University College Cork
Coagulant§ Rennet coagulation of milk…§ Proportion of added rennet activity retained in curd§ Little hydrolysis of b-casein in cheese due to hydrophobic
interactions of hydrophobic C-terminal region§ Principally responsible for primary hydrolysis of as1-casein
Chymosin1 199
1 23 24 199
102 199
Chymosin
f1-9, f1-13
Lactocepin
Further hydrolysis
Phe23-Phe24
Leu101-Lys102
University College Cork
Coagulant§ Rennet coagulation of milk…§ Proportion of added rennet activity retained in curd§ Little hydrolysis of b-casein in cheese due to hydrophobic
interactions of hydrophobic C-terminal region § Principally responsible for primary hydrolysis of as1-casein§ Chymosin assay…
P T E F F R L
NO2
CHYMOSIN
Synthetic heptapeptide substrate
F R L
NO2
Product absorbsat 300 nm
University College Cork
pH4.5 5.0 5.5 6.0 6.5 7.0
Enzy
me
activ
ity u
nit (
mm
ol
pdt.
mg
dry
mat
ter-1
hr-1
)
0
2
4
6
8
10
12
14
% Casein1 2 3 4 5 6 7
Enzy
me
activ
ity u
nit (
mm
ol
pdt.
mg
dry
mat
ter-1
hr-1
)
0.0
0.5
1.0
1.5
2.0
2.5
Tota
l Enz
yme
activ
ity
(mm
ol p
dt. h
r-1)
0
50x103
100x103
150x103
200x103
250x103
Chymosin (IMCU/ml milk)0.05 0.10 0.15 0.20
Enzy
me
activ
ity u
nit (
mm
ol
pdt.
mg
dry
mat
ter-1
hr-1
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Ionic strength of milk (M)0.0 0.2 0.4 0.6 0.8
Casein micelle size (nm)100 150 200 250 300 350 400
Enzy
me
activ
ity u
nit (
mm
ol
pdt.m
g dr
y m
atte
r-1 h
-1)
0.0
0.2
0.4
0.6
0.8
● pH at whey drainage (chymosin)● pH of milk (chymosin)● pH of milk (C. parasitica proteinase)
University College Cork
0
10000
20000
30000
40000
50000
60000
70000
80000
Cast Feta Feta Parmesan Emmental Cheddar Camembert Gouda Port Salut BuffaloMozzarella
PizzaMozzarella
Cheese varieties
Peak
Are
a/m
g dr
y m
atte
r/h
University College Cork
A interesting coagulant…
University College Cork
Characteristics of good rennet substitutes
• High milk clotting : proteolytic ratio• Proper specificity• Good activity in milk• Easily denatured (in whey)• Few suitable enzymes…
Camel chymosinCHY-MAXTM M
??
• No significant differences in composition and pH; suggestion of increased yield
• Primary proteolysis was significantly lower in camel chymosin cheeses large quantitative differences between the peptide profiles of cheeses; however, the levels of amino acids were similar
• Flavour differences• Camel chymosin can be used
successfully to make Cheddar cheese with lower levels of proteolysis but with good flavour.
University College Cork
Inhibition of coagulant
β-CN
αs1-CN
β-CN (f 106-209)
β-CN (f 29-209)
β-CN (f 108-209)
αs1-CN (f 102-199)
αs1-CN (f 24-199)αs1-CN degradationproducts
β-CN (f1-189/192)
NaCN C 0.1 0.25 0.4 2 C 0.1 0.25 0.4 2 C 0.1 0.25 0.4 2
1 d 180 d90 d
β-CN
αs1-CN
β-CN (f 106-209)
β-CN (f 29-209)
β-CN (f 108-209)
αs1-CN (f 102-199)
αs1-CN (f 24-199)αs1-CN degradationproducts
β-CN (f1-189/192)
NaCN C 0.1 0.25 0.4 2 C 0.1 0.25 0.4 2 C 0.1 0.25 0.4 2
1 d 180 d90 d
Urea-polyacrylamide gel electrophoretograms (12.5% T, 4% C, pH 8.9) of bovine sodium caseinate (CN) and the Cheddar cheeses manufactured from milk containing 0 (C), 0.1, 0.25, 0.4 or 2% equine blood serum after 1, 90 and 180 d of ripening.
Bansal et al.
a2-Macroglobulin720 kDa
University College Cork
O
CO
O
CO
O
CO
OHC
O
O
O
O
H
CH3C
COOHOH
Chymosinand plasmin
Caseins
Intermediate-sized peptides
Proteinases fromLactococcus
Short peptides
Peptidases of Lactococcusand NSLAB
Amino acids
Triglyceride
Fatty acidL-Lactate D-Lactate
Lactose
Fermentationby starter
Racemizationby NSLAB
COOH
CH2
C COOHHO
H2C
COOH
H
CH3C
OHCOOH
Catabo
lism of
lacta
te
VOLATILE FLAVOUR COMPOUNDS
Citrate
Metabolism of citrate
Lipas
e
Fatty acid catabolismAmino acid catabolism
Interactions betweenproducts
Interactions betweenproducts
Other products
Amino acidcatabolism
University College Cork
Catabolism of amino acids
Met Tyr Trp
MGLCBLCGL
TPL TIL
Methanethiol
Phenol
Indole
Aromatic amino acidsBranched-chain amino acids
Methionine
ELIMINATION
a-Keto acid
Aldehyde
Alcohol
Ester Methyl thioester
Carboxylic acid
Acyl-CoA
Hydroxyacid
DimethyldisulphideDimethyltrisulphide
Benzaldehyde(Phe)
Hydroxybenzaldehyde(Tyr)
Indole-3-acetate(Trp)
Cresol, skatole(Tyr, Trp)
TRANSAMINATION
HA-DH
Oxidati
on
a-KADH
a-KADC
aldDHAlcohol DH
?
?
Aminotransferase
University College Cork
Degradation of a-keto acids
H2N CH C
CH2OH
O
OH
a-Keto acid(p-Hydroxy phenylpyruvate)
a-Amino acid(Tyrosine)
Hydroxyacid
AldehydeCarboxylic acid Alcohol
O C C
CH2OH
O
OH
Chemicaldegradation
C
OH
O H
p-Hydroxy-benzaldehyde
Outline…
• Overview of cheese ripening, coagulants
• Calcium equilibrium and cheese texture
• Optical properties of low-fat cheese• Redox potential and cheese
University College Cork
University College Cork
Ammonia produced at surfaceby proteolysis diffuses into cheese
Migration of soluble Ca, PO43- and lactate towards surface
Lactate metabolised at surfaceCa3(PO4)2 precipitates
Growth of Penicillium camemberti at surface
pH
High pH
Low pH
Dire
ctio
n of
mig
ratio
n
Con
cent
ratio
n gr
adie
nt
Cheese softens
fromsurface towards
core
Textural changes during ripening…• Role of calcium, pH in softening of Camembert-type cheeses is well
established…
• What is the role of calcium in softeningof Cheddar cheese?Biochemical and physicochemicalprocesses… Role of as1-casein?
50
60
70
80
90
100
0 30 60 90 120 150 180Ripening time (d)
Hard
ness
(% d
ecre
ase)
Softening of Cheddar
cheese
University College Cork
Role of chymosin in cheese texture development?§ Chymosin inhibited by pepstatin
(IsoVal-Val-Val-Stat-Ala-Stat)
Useful tool to study cheese texture
University College Cork
2D Graph 1
Ripening time (d)0 30 60 90 120 150 180
Har
dnes
s (N
)
80
120
160
200
240
Inso
lubl
e C
alci
um/T
otal
Cal
cium
(%)
50
55
60
65
70
75
80
Res
idua
l as1
-cas
ein
(%)
0
20
40
60
80
100
Time vs H ctrl Time vs Ca ctrl Time vs as1 ctrl
2D Graph 2
Ripening time (d)0 30 60 90 120 150 180
Har
dnes
s (N
)
80
120
160
200
240
Res
idua
l as1
-cas
ein
(%)
0
20
40
60
80
100
Inso
lubl
e C
alci
um/T
otal
Cal
cium
(%)
50
55
60
65
70
75
80
HardnessResidual as1-caseinInsoluble Calcium
2D Graph 2
Ripening time (d)0 30 60 90 120 150 180
Har
dnes
s (N
)
80
120
160
200
240
Res
idua
l as1
-cas
ein
(%)
0
20
40
60
80
100
Inso
lubl
e C
alci
um/T
otal
Cal
cium
(%)
50
55
60
65
70
75
80
HardnessResidual as1-caseinInsoluble Calcium
Control (100% residual chymosin activity)
Pepstatin (16% residual chymosin activity)
Hardness decreased significantly (p<0.001) irrespective of as1-casein hydrolysis
Calcium solubilization most closely related to initial softening
Hydrolysis of as1-casein plays a role but not as important as solubilization of Ca
University College Cork
G' @ 0.4 Hz
Temperature (oC)
10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0
G' (
Pa)
101
102
103
104
105
106
8.34 g/L6.95 g/L5.56 g/L2.78 g/L1.39 g/L
Loss tangent @ 0.4 Hz
Temperature (oC)
10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0
Loss
tang
ent
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
8.34 g/L6.95 g/L5.56 g/L2.78 g/L1.39 g/L
Storage modulus[CCP] had no significant effect on G´ at 20oCG´ at 70oC increased significantly with increasing [CCP]
Loss tangentLTmax increased significantly with decreasing [CCP]
[CCP] had no significant effect on temperature of LTmaxRheological properties of Cheddar cheese at 70oC as a
function of colloidal calcium phosphate content
Colloidal calcium phosphate content of cheese (g calcium/100 g protein)
1.40 1.60 1.80 2.00 2.20 2.40
Stor
age
and
Loss
mod
ulus
(Pa)
0
50
100
150
200
250
300
350
400
Loss
tang
ent
0.60
0.80
1.00
1.20
1.40
1.60
1.80
G'G"Tan d
Rheological properties of Cheddar cheese at 70oC as a function of colloidal calcium phosphate content
Colloidal calcium phosphate content of cheese (a.u./100 g protein)
0.80 0.90 1.00 1.10 1.20 1.30 1.40
Stor
age
(G')
and
Loss
(G")
mod
uli (
Pa)
0
50
100
150
200
250
300
350
400
Loss
Tan
gent
0.60
0.80
1.00
1.20
1.40
1.60
1.80
G'G"Tan d
G´´ > G´
G´ > G´´
Rheological properties at 70oC
Variation in CCP levels using synthetic Cheddar cheese aqueous phase
University College Cork
Influence of Ca equilibrium on melt
Ø Insoluble Ca decreases during ripening and is responsible for increased melt during early ripening
Ø Cheeses with lower insoluble Ca have increased meltability
Loss
tang
ent
Lowest Insoluble Ca
Highest Insoluble Ca
Cheeses with same pH
(Choi et al. 2008)
University College Cork
Varying the texture of Cheddar cheese by controlling levels of soluble Ca using trisodium citrate or CaCl2 added at salting
Five vats of Cheddar cheese manufactured –
Control (100% NaCl)Experimental 1 (90% NaCl, 10% TSC)Experimental 2 (70% NaCl, 30% TSC)Experimental 3 (90% NaCl, 10% CaCl2)
Experimental 4 (70% NaCl, 30% CaCl2)
Levels of NaCl were varied to maintain a constant ionic strength to avoid different moisture levels
6000
8000
10000
12000
14000
16000
18000
7 14 28 56 112 202Time (days)
Har
dn
ess
(G)
ControlTSC 10%TSC 30%CaCl2 10%CaCl2 30%
University College Cork 35
1. Manufacture of Gouda-type cheesescontaining a Ca-sequestering agent (EDTA) encapsulated in liposomes
2. Determine the effect of EDTA on the textural and rheological properties of Gouda-type cheeses without affecting composition
Liposomes
EDTA addition + mix
UT/Homogenisation/Microfluidisation
Microfiltration
EDTA suspended in buffer + liposome
Outline…
• Overview of cheese ripening, coagulants
• Calcium equilibrium and cheese texture
• Optical properties of low-fat cheese• Redox potential and cheese
University College Cork
Translucency in low-fat Cheddar Cheese
• Colour is considered the mostimportant attribute to foodappearance.
• Importance of fat content andtemperature in cheese colour:
– Fat increases light scatering,affecting whiteness. (L* values)
– Fat reduction on cheese increasestranslucency (decreases L*values) and hence acceptability
– High temperatures increaseswhiteness. However, it isInfluenced by fat content (meltingeffect)
• Other parameters that affectcolour in cheese:
– Salt content– Calcium content– Homogenisation of cheese-milk– Addition of annatto– Addition of titanium dioxide
Rodrigo A. Ibáñez (Chile)PhD. Student (April 2011 – March 2015)
University College Cork
Translucency of low-fat Cheddar Cheese• L* value used as an indicator of
translucency (the higher the value,the lower the translucency).
• The study of translucent materialshas also been studied by meansof the reflectance of a thin layer ofa sample under a black and awhite background.
• Based on this relation, theKubelka-Munk index (K/S) hasbeen developed (Judd andWyszecki, 1975)
R∞:reflectance of a layer of infinite thickness.R: reflectance of the sample layer above a whitebackground with a known reflectance RgR0: reflectance of the sample with an ideal blackbackground.
University College Cork
Effect of titanium dioxide, annatto and homogenisationon Kubelka-Munk index in half fat Cheddar Cheese
Titanium Dioxide
Homogenisation
Annatto
0, 20 and 40(g TiO2/100 kg cheesemilk)
0, 10 and 20(MPa)
0, 8.25 and 16.5(ml/100 kg cheesemilk)
Outline…
• Overview of cheese ripening, coagulants
• Calcium equilibrium and cheese texture
• Optical properties of low-fat cheese• Redox potential and cheese
University College Cork
Physicochemical parameters influencing cheese ripening…• pH, aw (≈[NaCl]), T well understood and controlled…• Oxidation-reduction potential largely ignored…
• ORP: measure of ability of chemical species to oxidize or reduce
• ORP of milk ≈ +250 to +350 mV, cheese varies (but very reducing)
• Affects microbial growth, production of aroma compounds by amino acid catabolism
University College Cork
Oxidation-reduction potential and cheese
• ORP affects the type of microorganisms that grow in and on
cheese…
• Evidence that ORP influences which species or strains of
NSLAB proliferate (Boucher et al., 2006).
• ORP contributes to the creation of conditions necessary for
balanced flavour development and influences compounds
formed…
University College Cork
University College Cork
Measurement of ORP in mature hard cheese
Filled with3 M KCl
Platinum Electrode
Reference Electrode
mV
20 mm
-450
-350
-250
-150
-50
50
150
250
350
0 250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250
Time (min)
OR
P (m
V)
University College Cork
Cheddar cheesemaking Redox Potential
pHTemperature
20
30
40
50
60
70
80
T (�
C)
-400
-320
-240
-160
-80
0
80
160
0 50 100
150
200
250
300
350
400
OR
P (m
V)
5.0
5.5
6.0
6.5
7.0
7.5
Time (min)
pH
Pasteurization Starter addition
CaCl2 & coagulant addition
Cutting & cooking Whey drainage Milling &salting
-400
-320
-240
-160
-80
0
80
160
410
510
610
710
810
910
1010
1110
1210 1310
1410
1510
ORP
(mV
)
9
Time (min)
-400
-320
-240
-160
-80
0
80
160
410
510
610
710
810
910
1010
1110
1210 1310
1410
1510
ORP
(mV
)
9
Time (min)
University College Cork
Changes during ripening
1 3 5 7 9 11 13 15 17 19 21 23 25 27
OR
P (m
V)
-400
-300
-200
-100
0
100
200
300
0 40 80
Time
Minutes Days
Reference electrode in agar containing saturated KCl gelled in plastic tube with holes
Miniature platinumelectrodes
University College Cork
Influence of microbial growth on ORP in Cheddar cheese extract during simulated cheese pressing
C C10 S2 T °C
-400
-300
-200
-100
0
100
200
300
400
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
OR
P (m
V)
0
5
10
15
20
25
30
35
40
Tem
pera
ture
(°C
)
Time (hour)
pH 5.4 add NaClStrain
L. l. lactis C10 L. l. lactis DL16 L. l. lactis ATCII454 L. l. lactis ML3 L. l. MM510 L. l. lactis ATC29146 L. l. lactis M70 L. l. Crem, Z8 L. l. Crem HP L. l. Crem 45P+ L. l. Crem SW224 L. l. Crem SK11 04 L. l. Crem UC317 L. l. Crem E L. l. Crem R L. l. Crem EB7 L. l. Crem D L. l. Crem W L. l. Crem G L. l. Crem S2 L. l. lactis C2 L. l. lactis 26-2 L. l. SCO213 L. l. Crem A L. l. Crem B L. l. Crem C L. l. Crem F L. l. Crem I L. l. Crem J L. l. Crem Q
University College Cork
ORP control during Cheddar ripening
• Cheddar is a dry salted variety• Redox agents added to the salted curd before
pressing
• Reducing agents:• Cysteine (2%)• Sodium hydrosulfite (0.05%,0.1%)• Oxidizing agent:• Potassium iodate (0.05%,0.1%,1%)
University College Cork
ORP and pH during ripening
5.10
5.30
5.50
5.70
5.90
0 20 40 60 80 100 120 140 160 180 200
pH
Ripening Time (days)
Control KIO3 1% KIO3 0.1% KIO3 0.05% Na2S2O4 0.1% Na2S2O4 0.05% Cys 2%
-0.400
-0.300
-0.200
-0.100
0.000
0.100
0.200
0.300
0 20 40 60 80 100 120 140 160 180 200
OR
P (
V)
Ripening time (days)
Control KIO3 1% KIO3 0.1% KIO3 0.05% Na2S2O4 0.1% Na2S2O4 0.05% Cys 2%
University College Cork
PCA of volatile compounds at 2 month ripening
University College CorkFood Institutional Research
Measure (FIRM)
Thank you!¡Gracias!