Studies on stability and efficacy of mice

Studies on stability and efficacy of

microencapsulated folic acid in Cheddar cheese and

in methionine-induced hyperhomocysteinemia in

Honest Sindile MADZIVA (MSc Food Science amp Technology UWS)

August 2006

A thesis submitted to the University of Western

Sydney in fulfilment of the requirement for the

degree of Doctor of Philosophy

Acknowledgements

A journey is easier when you travel together Interdependence is certainly more

valuable than independence This thesis is the result of four years of work whereby I

have been accompanied and supported by many people It is a pleasant aspect that I

now have the opportunity to express my gratitude to all of them My most sincere

thanks and heartfelt gratitude go to my Supervisory Panel namely Kasipathy (Kaila)

Kailasapathy Michael Phillips and Geoff Skurray for their constant encouragement

and support in the development of this research particularly for the many stimulating

and instructive discussions we had Their exceptional enthusiasm and integral views

on research and their mission for providing only high-quality work and not less has

made a deep impression on me Michael and Kaila thank you for those many useful

comments during the preparation of manuscripts for publication in peer refereed

journals your constructive criticism emboldened me for the work thus completed

This research was supported and funded by the University of Western Sydney

through the Centre for Advanced Food Research and the Centre for Plant amp Food

Science Special mention goes to the then CAFR Director Jim Hourigan for his

outstanding support and inspiration I am grateful to my colleagues both past and

present the technical staff at UWS Hawkesbury (Rob Sturgess amp Liz Kabanoff)

UNSW (Maria Sares amp Gavin McKenzie) and Terry Evans (USyd) for their

assistance during the progression of this research

I am very grateful to my wife Tariro and daughter Buhlebenkosi ndash Catherine for

their consummate love and unequivocal patience during the period of my PhD study

Special thanks go to our families the Madzivas and Kandukas for their

encouragement and support I am also extremely grateful to my bigger church

family at Windsor Seventh Day Adventist church for their inspiration and profound

support during the course of my studies I want to thank in particular my ldquoadoptedrdquo

parents Wal and Anne Cram for making me part of the family The chain of my

gratitude would definitely be incomplete if I failed express my sincere thanks to the

first cause of this chain the Almighty God and our Lord Jesus Christ ldquoTrust in the

LORD with all thine heart and lean not unto thine own understanding In all thy

ways acknowledge him and he shall direct thy pathsrdquo Proverbs 3 5-6

Dedication

This thesis is dedicated to our unborn child due on this earth on

12 September 2006 our bundle of joy ndash Buhlebenkosi Catherine

and the love of my life wife Tariro

Statement of Authentication

The work presented in this thesis is to the best of knowledge and belief original

except as acknowledged in the text I hereby declare that I have not submitted this

material either in whole or in part for a degree at any other institution

Signedhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip

Datehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip

Table of Contents

Publications Disseminations and Awards arising from this thesis v

List of Tables viii

List of Figures ix

List of Abbreviations xi

Abstract xii

Chapter 1 ndash GENERAL INTRODUCTION 1

11 Aim of this study 10

12 Objectives 10

13 Constraints of the study 11

14 Thesis overview 13

Chapter 2 ndash LITERATURE REVIEW

21 Discovery and Chemistry of folates 14

22 The role of folic acid in health and disease 17

23 Neural tube defects 18

24 Occlusive vascular diseases associated with elevated homocysteine 20

25 Strategies to increase blood folate levels 24

26 Intestinal absorption of folates 33

27 Stability of folates 35

28 Strategies to raise folate levels in food products 47

29 Cheese as a food vehicle 51

210 Regulatory requirements for Cheddar cheese fortification 55

211 Microencapsulation 56

212 Encapsulating materials 62

213 Microencapsulation techniques 72

214 Future trends 95

215 Summary of Literature 96

Chapter 3 ndash MATERIALS AND METHODS

31 Folic acid encapsulation procedure 98

32 Effect of time and calcium chloride concentration on cross-linking

completion in alginate-pectin hydrogels 100

33 Effect of buffer type on folic acid release from alginate-pectin

hydrogels 103

34 Single and blended polymers effect on folic acid encapsulation

efficiency 103

35 Effect of different drying techniques on retention of encapsulated

folic acid 104

36 Folic acid leakage 104

37 Morphology of gel capsules 105

38 Folic acid measurement 105

39 Effect of simulated cheese press pressures on mechanical stability of

capsules 109

310 Cheddar cheese making 110

311 Stability of folic acid during cheese ripening 112

312 Total folates measurement 112

313 Statistical analyses 113

Chapter 4 - SCREENING OF FOOD GRADE POLYMERS AND OPTIMISATION OF

FOLIC ACID ENCAPSULATION PARAMETERS TO INCREASE

ENCAPSULATION EFFICIENCY AND STABILITY

41 Abstract 115

42 Introduction 116

43 Aim 121

44 Objectives 121

45 Materials and methods 122

46 Preparation of polymer solutions and encapsulation procedure 122

47 Optimisation of encapsulation parameters 122

48 Selection of buffer for the release of folic acid 122

49 Effect of different drying methods on stability of encapsulated folic acid 123

410 In vitro release of folic acid and swelling properties of alginate-pectin hydrogels 123

411 Release of encapsulated folic acid from alginate-pectin hydrogels in

ex-vivo porcine gastrointestinal contents 124

413 Determination of calcium content in alginate-pectin hydrogels 125

414 Results 126

415 Discussion 138

416 Conclusion 151

Chapter 5 - EVALUATION OF ALGINATE-PECTIN GEL CAPSULES IN CHEDDAR

CHEESE AS A FOOD-CARRIER FOR THE DELIVERY OF FOLIC ACID

51 Abstract 152

52 Introduction 153

53 Aim 156

54 Objectives 156

56 Folic acid capsules 156

57 Evaluation of gel capsules for mechanical strength 156

58 Stability of gel capsules in milk 157

59 Distribution of gel capsules in Cheddar cheese incorporated with three

different methods 157

510 Stability of encapsulated folic acid during cheese ripening 158

511 Results 159

512 Discussion 167

513 Conclusion 170

Chapter 6 - EFFECT OF ENCAPSULATED FOLIC ACID DIETARY

SUPPLEMENTATION ON METHIONINE-INDUCED

HYPERHOMOCYSTEINEMIA IN MICE

61 Abstract 171

62 Introduction 172

64 Animals diets and treatments 175

65 Blood collection and homocysteine measurement 175

66 Histologic analysis of the aortic arch 177

67 En Face Immunofluorescence staining 1 78

68 Results 179

69 Discussion 188

610 Conclusion 192

Chapter 7 - OVERALL CONCLUSIONS 193

Chapter 8 - FUTURE DIRECTIONS 197

Chapter 9 - Literature cited 199

Publications and disseminations

List of publications

Madziva HS Phillips MW Kailasapathy K Effect of encapsulated folic acid dietary

supplementation on methionine-induced hyperhomocysteinemia in mice (Submitted

060506 Journal of Nutrition article in review process

Madziva H Kailasapathy K Phillips M (2006) Evaluation of alginate-pectin capsules in

Cheddar cheese as a food carrier for the delivery of folic acid LWT Food Sci Technol 39

146-151

Madziva H Kailasapathy K Phillips M (2005) Alginate-pectin microcapsules as a

potential for folic acid delivery in foods J Microencap 22(4) 343-351

Conference presentations

Proceedings

Madziva H Kailasapathy K Phillips M (2005) Cheddar cheese a potential food

carrier for the delivery of folic acid (abstract of oral presentation) InProceedings of

the Nutrition Society of Australia Asi Pac J Clin Nutr 14(Suppl) S76

Madziva H Kailasapathy K Phillips M (2005) Cheddar cheese ndash a possible

vehicle for encapsulated folic acid delivery (oral presentation) In Proceedings of

International Workshop on Bioencapsulation June 24th

ndash 26th

2005 pg 33-34

Kingston Ontario Canada

Madziva H Kailasapathy K Phillips (2004) Alginate-pectin microcapsules as a

potential folic acid delivery mechanism In Proceedings of the 12th

International

Workshop on Bioencapsulation (full text for poster presentation) 24-26th

September

pg 359-362Vitoria-Gaistez Spain

potential folic acid controlled release formulation in food systems (full text for a

poster presentation) In Proceedings of the First International Conference on Folates

Analysis Bioavailability and Health 11-14 February pg 101-107 Warsaw Poland

Oral presentations

Madziva H Kailasapathy K Phillips M (2005) Cheddar cheese ndash a potential

food carrier for the delivery of encapsulated folic acid (abstract) 38th

Annual

Australian Institute of Food Science amp Technology Convention 10-13th

July pg 48

Sydney Convention amp Exhibition Centre Sydney Australia

potential folic acid controlled release formulation in food systems (abstract) 37th

Annual Australian Institute of Food Science amp Technology Convention 25-28th

July Brisbane Convention amp Exhibition Centre Brisbane Australia

Posters

Madziva H Kailasapathy K Phillips (2006) Effect of encapsulated folic acid

dietary supplementation on methionine-induced hyperhomocysteinemia in mice 39th

Adelaide Convention Centre Adelaide Australia

Awards

Centre for Plant amp Food Science Directorrsquos Prize for Excellence - University of

Western Sydneyrsquos 3rd

Postgraduate Innovation Conference 2006

Deanrsquos Prize for Excellence - University of Western Sydneyrsquos 2nd

Postgraduate

Innovation Conference 2005

Septimus Birrell Awards ndash Runner up May 2005 Dairy Industry Association of

Australia (Inc)

Travel Award ndash International Society of Bioencapsulation (2004) for a poster

presentation at the 12th

International Workshop on Bioencapsulation Vitoria-Gaistez

Endeavour International Postgraduate Research Scholarship recipient (2002-

2006) for PhD studies at University of Western Sydney

List of abbreviations

5 10 MTHFR 5 10 Methylenetetrahydrofolate reductase

Alg-pect Alginate-pectin

ATPSs Aqueous two-phase systems

CBS Cystathionine β-synthase

CV Covariance

DE Degree of esterification

DNA Deoxyribonucleic acid

EPBA Enzyme protein binding assay

FR Folate receptor

PteGlu Pteroylglutamic acid

GIT Gastrointestinal tract

Hcy Homocysteine

HM High methoxy

ICJ Ileo-caecal junction

IUPAC International Union of Pure and Applied Chemistry

LM Low methoxy

LUV Large unilamellar vesicles

MRPs Maillard reaction products

NO Nitric oxide

NTD Neural tube defect

OVD Occlusive vascular disease

NZFSA New Zealand Food Safety Authority

NHMRC National Health Medical Research Council

NZMoH New Zealand Ministry of Health

FSAI Food Safety Authority of Ireland

US-FDA United Statesndash Food and Drug Authority

pABA p-Amino benzoic acid

PEG Polyethylene glycol

PVP Polyvinylpyrrolidone

RBC Red blood cell

RDI Recommended daily intake

SAH S-adenosylyhomocysteine

SAM S-adenosylmethionine

SPSS Statistical Package for Social Sciences

Abstract

Most naturally occurring folate derivatives in foods are highly sensitive to

temperature oxygen light and their stability is affected by food processing

conditions Edible polysaccharides (hydrocolloids) were evaluated for folic acid

encapsulation both as single and mixed polymers as a way of increasing folic acid

stability Initially the polymers were evaluated for their encapsulation efficiency

capsule forming ability and retention of folic acid bioactivity during drying and

storage Alginate and pectin polymers produced the highest encapsulation

efficiencies of 54 and 49 respectively Upon being combined and the

encapsulation conditions optimised the alginate ndash pectin (alg-pect) polymer mixture

showed approximately 90 folic acid encapsulation efficiency The blended

alginate and pectin polymer mix increased folic acid encapsulation efficiency and

reduced leakage from capsules compared with the individual polymers After 11

weeks of storage at 4ordmC retention of encapsulated folic acid in freeze-dried capsules

was 100 compared with free folic acid that was absent from 9 weeks onwards The

alg-pect capsules were tested for their stability in milk where pH was adjusted from

67 to 45 over a 4 h period Folic acid retention under these conditions was 100

indicating their ability to remain stable in milk The ability of the capsules to

withstand mechanical stress was tested under simulated cheese press-pressures for 4

h until a force of 843 gcm2 was achieved Folic acid retention of up to 80 was

recorded Folic acid release was studied at two pH values pH 12 and pH 82

depicting the stomach and the intestinal pH respectively The result clearly

demonstrated that capsules remained intact in acidic conditions but dissolved in an

alkaline environment which would be similar to the conditions in the small intestines

where folic acid is absorbed The in vitro release of folic acid was gradual with 90

released in 120 min Ex-vivo porcine experimental results showed a similar pattern as

in the in vitro studies Folic acid release was greater in the small intestinal contents

compared to gastric and colonic contents Three stages in Cheddar cheese

manufacturing namely addition of capsules to the milk incorporation of capsules to

the milled curd and injection into the pressed block of raw cheese were then

compared for capsules distribution The former showed greater even distribution

while the latter two showed poor and irregular distribution of capsules Encapsulated

folic acid showed more stability (100 ) in Cheddar cheese over the 15 months

ripening period compared to free folic acid (28 ) The bioactivity of encapsulated

folic acid was further studied in male Balbc mice (6-8 weeks) by inducing different

levels of hyperhomocysteinemia by feeding 10 gkg or 20 gkg methionine over a 12

week period Cheddar cheese was used as a food vehicle for the delivery of folic

acid Mice were fed 6 experimental diets as follows (i) methionine only intake (10

gkg) (ii) methionine ( 20 gkg) with free folic acid (2 mgkg) (iii) methionine (20

gkg) with encapsulated folic acid (2 mgkg) (iv) methionine (10 gkg) with free

folic acid (v) methionine (10 gkg) with encapsulated folic acid The control group

(vi) was fed Cheddar cheese without added folic acid Supplementation of the diet

with 10 gkg and 20 gkg methionine increased plasma homocysteine to 18 and 61

times the control respectively The homocysteine (7 micromolL) resulting from the

dietary addition of 10 gkg methionine was completely counteracted by the

encapsulated folic acid while free folic acid showed homocysteine (11 micromolL)

almost twice as high Similarly encapsulated folic acid caused substantial reduction

in plasma homocysteine and arterial lesions in mice fed the diet supplemented with

20 gkg methionine compared to free folic acid Encapsulated folic acid incorporated

in Cheddar cheese showed lower plasma homocysteine regardless of methionine load

in the diet Endothelium-dependent relaxation of the mice aorta was impaired while

there was also a significant increase in the adhesion and binding of monocytes to the

endothelium of hyperhomocysteinemic mice Such an adhesion is a common feature

linking the inflammation reaction and the development of early atherosclerosis in

hyperhomocysteinemia In conclusion this study demonstrates that the alg-pect

polymer combination gave the highest folic acid encapsulation efficiency retained

folic acid during capsule making storage incorporation into milk during cheese

making as well as preserved folic stability during cheese ripening The alg-pect

polymer capsules also offered protection to folic acid from deteriorative conditions in

the gastric conditions while they dissolved in an alkaline environment depicting the

small intestines where folic acid is absorbed Results obtained from the present study

demonstrate for the first time that dietary incorporation of encapsulated folic acid

using Cheddar cheese as the delivery vehicle mitigates against

hyperhomocysteinemia and monocytemacrophage adhesion in mice

Chapter 1 Introduction

1 Introduction

Mankind has been relatively unsuccessful in the search for the ultimate panacea for

all ills however in the field of functional foods few nutritional components have so

many fundamental and diverse biological properties as folic acid and related B group

vitamins Moreover few nutrients can claim to modulate if not overtly benefit such

a wide array of clinical conditions

Around 2500 years ago Hippocrates first espoused the food as medicine

philosophy which fell into obscurity by the 19th century The first 50 years of the

20th century saw the discovery of the essential elements and vitamins particularly in

the context of deficiency diseases Indeed by 1912 Casimir Funk had put forward the

vitamine theory proposing four different vitamines that would cure scurvy

pellagra beri-beri and rickets During the 1970s the shift in emphasis from

undernutrition to overnutrition and disease led to a flood of public health guidelines

on optimising nutritional parameters By the 1990s with an ageing health-conscious

population scientists from academia and the commercial world coalesced their

thinking to create the trend we now know as functional foods

Enrichment of flour as a US government intervention programme to correct problems

with nutrient deficiency was probably the first modern attempt to design a food for

functional purposes related to nutritional outcome The first consequence of this was

the eradication of pellagra with niacin and a current programme among world

governments aims to do the same for neural tube defects through mandatory

fortification of grain and other foods with folate at source (Lewis et al 1999 USA

Food Standards 1996)

Since 1995 in Australia and 1996 in New Zealand certain foods have been able to be

fortified with folic acid Standard 132 of the Food Standards Australia New

Zealand Code (the Code) permits folic acid to be voluntarily added to a maximum

claim of 100 μg per reference quantity to a number of cereal-based foods fruit and

vegetables juices and drinks yeast and meat extracts

Clearly the use of folate fortification has immense potential benefit Interest in folate

over the past decade has rocketed in comparison with other nutrients largely because

scientists have recognised the importance of this vitamin in treating a broad range of

both developmental and degenerative disorders that are sensitive to even marginal

deficiencies in B vitamins (Fenech 2002)

Although Lucy Willss 1931 description of yeast extract being effective against the

tropical macrocytic anaemia of late pregnancy in India represents the first record of

folate being used for prevention of disease folate as the critical factor involved was

not isolated nor was its structure elucidated until later Furthermore it was not until

more than half a century later that the significance of folate in preventive medicine

was once again shown in a series of papers culminating in the one by the Medical

Research Council Vitamin Study Group in 1991 documenting how periconceptional

folate prevents spina bifida This discovery was followed by a meta-analysis

published in 1995 which presented data from 27 studies involving more than 4000

patients with occlusive vascular disease and a similar number of controls (Boushey et

al 1995) Data showed that homocysteine was an independent graded risk factor for

atherosclerotic disease in the coronary cerebral and peripheral vessels This was of

particular interest as dietary folate lowers homocysteine through de novo

biosynthesis of methionine (Schorah et al 1998) and it opened new avenues for

intervention with vitamins to prevent disease Several single nucleotide

polymorphisms that are related to folate and other B vitamins were also discovered in

1995 These affect the risk not only of birth defects and vascular disease but also of

several cancers

Much of the current interest in folate stems from the discovery of several single

nucleotide polymorphisms that modulate risk for a range of important diseases

associated with considerable morbidity and mortality (Lucock 2000) Of even

greater importance is the fact that dietary folate can interact with the proteins that are

encoded by these variant genes and ameliorate risk to the extent that an overt

protection against the disease is conferred (Slattery 1998)

Folate is of great interest and of great clinical value a veritable panacea among

functional foods Given the fundamental importance of B vitamin nutrigenomics and

the pace of development in molecular diagnostics it is not hard to envisage a new era

in preventive medicine that has even greater emphasis on diet as a means to a long

and healthy lifemdashindeed a return to Hippocrates famous Let food be thy medicine

and medicine be thy food philosophy

There is consensus that it is difficult to achieve the recommended intake of folate

through diet alone with Australian and New Zealand figures showing women of

child-bearing age consuming only about half the amount recommended while adult

population aged 20 to 65 years consuming approximately ~ 100-150 microgd Total

folate intakes in women of child-bearing age have not increased significantly and are

still well below recommended intakes despite the Standard 132 of the Australia

New Zealand Food Standards Code (the Code)

The Recommended Dietary Intake (RDI) for total folate for Australia and New

Zealand range from 50-75 μg per day for infants to 200 μg per day for the general

adult population However the RDIs for pregnant and lactating women are

considerably higher at 400 μg and 350 μg per day respectively (NHMRC 1991)

In Australia various education initiatives have been undertaken by a number of

jurisdictions to encourage women of child-bearing age to increase their dietary folate

andor take folic acid supplements Despite these campaigns current advice for

supplemental folic acid is not followed by a majority of women in the target group

Reasons for this include

bull a large percentage of pregnancies are unplanned

bull lack of knowledge among women about the benefits of folic acid

bull knowledge not always equating to behavioural change and

bull numerous barriers to supplement usage such as cost access and compliance

issues (Lancaster amp Hurst 2001)

A UK study modelling the effect of food fortification on the population found that

the maximal protective effect against neural tube defects (NTDs) would be gained if

a fortification program were chosen such that the entire target group received an

intake of 400 μg folic acid per day On average this would result in red blood cell

(RBC) folate levels above the 900 nmolL optimal level which would prevent at

least 60 of NTDs from occurring (Daly et al 1997) Red blood cell (RBC) folate

status is recognised as a more reliable indicator of long-term folate status as it is not

easily affected by daily fluctuations due to food consumption (Booth et al 1998) A

very low risk of NTDs has been associated with maternal RBC folate levels greater

than or equal to 900 nmolL (Daly etal 1995)

Information on the levels of RBC folate in Australia and New Zealand suggests that

the mean RBC folate concentrations vary from 486 nmolL to 791 nmolL (both

median values) (Booth et al 1998 Ferguson et al 2000 Queensland Health 2002)

Comparison of these data with international optimal references of 900 nmolL

suggests that there may be potential for an increase in folate status to further reduce

rates of NTDs and other folate deficiency diseases Fortification with the more stable

synthetic folic acid becomes a natural route to address this problem however there

are practical issues associated with this approach

The selection of appropriate food vehicle(s) for fortification is an important

consideration A number of organisations (Codex Alimentarius Commission 1991

Darton-Hill 1998 Nutrivit 2000) have published criteria for selecting appropriate

food vehicle(s) including the need for the selected vehicle to

bull be regularly consumed by the population at risk in stable predictable amounts

(upper and lower intake levels known)

bull be available to the target population regardless of socio-economic status

bull supply optimal amounts of micronutrient without risk of excessive

consumption or toxic effects

bull retain high level stability and bioavailability of the added micronutrient under

standard local conditions of storage and use

bull be economically feasible

bull be centrally processed so that quality control can be effectively implemented

bull not interact with the fortificant or undergo changes to taste colour or

appearance as a result of fortification

The majority of countries (most South American nations some African and Asian

countries Canada and the US) with mandatory folic acid fortification have selected

cereal foods as delivery vehicle In Australia and New Zealand the food standards

body ANZFA is reviewing Standard 132 which currently permits voluntary

fortification with the view of making fortification of cereal based foods mandatory

In line with the above food vehicle selection criteria Cheddar cheese was selected in

the current study since it meets the criteria and provides the following beneficial

health effects among others calcium which has been reported to decrease low

density lipoprotein (LDL) cholesterol (Reid et al 2002) and triacylglycerol

concentrations (Yacowitz et al 1965 as well as increase the conversion of

cholesterol to bile acids (Vaskon et al 2002) the presence of peptides from bovine

casein inhibit angiotensin-converting enzyme (ACE) which partly explains the

antihypertensive effect of fermented milks (Pfeuffer amp Schrezenmeir 2000) and

most hard cheeses among them Edam Gouda and Cheddar have been reported to

contain 20 to 40 microg of total folates100 g (Scott 1989)

Cheese consumption has been a major success for the Australian dairy industry with

consistently strong growth to a current 13 kilograms per capita or 30 g per day It

features among the four major consumer dairy products on the Australian dairy

market namely drinking milk (fresh and UHT white and flavoured) cheese (with

Cheddar or Cheddar type cheeses topping the list) butter and dairy blends and

yoghurt (Australian Bureau of Statistics 2005) therefore Cheddar cheese

fortification with folic acid was considered in the current study

Numerous objectives and factors must be taken into consideration to guarantee a

successful food-fortification intervention programme - for example legal issues and

how they affect programme outcomes Technological constraints are also faced by

food-fortification technology and in this instance ndash the stability of folates

microencapsulation conditions release and product applicability however the

current research has been conducted to find solutions to these problems The typical

constraints like nutrient and food constituents interaction which in the case of iron

for example may react with fatty acids in the fortified food forming free radicals

that induce oxidation in the product resulting in the colour taste odour and

appearance alterations resulting in problems with consumer acceptability of the

product has been avoided altogether through the use of microencapsulation

Folic acid or pteroylglutamic acid (PGA) is a yellow crystal with a molecular weight

of 4414 which obviously impart some colour if applied directly to a product

Encapsulation which is an inclusion technique for confining a substance into a

polymeric matrix was selected as a method of choice for two major reasons first

the encapsulated compound becomes more stable than its isolated and free form

(Arshady 1994 Dziezak 1988) since it is protected from deteriorative reactions and

adverse environmental conditions prior to release and secondly to mask the yellow

colour of folic acid Further to this the encapsulation materials are all food grade

polymers widely used as stabilisers in the Food industry ndash alginate pectin xanthan

gum gelatin and iota-carrageenan

The susceptibility of folic acid to cleavage under acidic conditions light and high

temperature has long been established (Stokstad et al 1947) The degree and rate of

destruction is largely influenced by the pH of the medium reducing agents in the

buffer folate derivatives type of buffer and the food system 5-methyl-

tetrahydrofolic is the predominant food folate (Stokstad amp Koch 1967) and is readily

oxidised to 5-methyl-5 6-dihydrofolate (Donaldson amp Keresztesy 1962) In this

oxidised form it may represent a substantial amount of the total food folate 5-

methyl-5 6-dihydrofolate is rapidly degraded under the mildly acid conditions which

prevail in the postprandial gastric environment Under the same conditions 5-

methyl-tetrahydrofolic acid is relatively stable Ascorbic acid is actively secreted into

the gastric lumen and may be a critical factor in salvaging acid labile 5-methyl-5 6-

dihydrofolate by reducing it back to acid stable 5-methyl-tetrahydrofolic While

Lucock et al (1995) have postulated that this might be useful in optimising the

bioavailability of food folate this work avoids this problem altogether by using a

mixture of alginate and pectin to make capsules that are pH sensitive to achieve

intestinal release in the jejuni where folic acid is absorbed

Alginate and pectin in combination or alone have been successfully used for the

gastrointestinal delivery of probiotics (Iyer et al 2004 Gill et al 2000) drug

delivery (Ashford et al 1994) among others but not folic acid Controlled release

has been known to be effective in its delivery as it enhances compliance and efficacy

Sustained plasma levels are typically preferred to the peak-and-trough plasma profile

normally associated with oral delivery The protective effect of the capsules on folic

acid and their applicability was also evaluated in Cheddar cheese Elevations of

plasma total homocysteine (tHcy) have been inversely correlated with blood folate

levels and taking folic acid either as a supplement or in food has been shown to

lower tHcy concentration Previous studies on folic acid supplementation and its

effect on tHcy and vascular diseases have focussed on folic acid incorporated only as

a supplement (Naurath et al 1995 Ambrosi et al 1999 Sarwar et al 2000 Han et

al 2005) or fortified form but not in its encapsulated form In the present study the

bioactivity of encapsulated folic acid delivered through Cheddar cheese was also

evaluated for its effect on homocysteine and vascular consequences of methionine

induced hyperhomosysteinemia in mice

11 Aim

The aim of this study was to develop a robust microencapsulation procedure using

edible polymers to increase folic acid stability retain its vitamer activity when

incorporated in Cheddar cheese and evaluate its bio-effect on methionine-induced

hyperhomocysteinemia in mice

12 Objectives

The principal objectives of this study were to

1 Assess food grade polymers for folic acid encapsulation

2 Optimise a number of encapsulation parameters to increase folic acid

encapsulation efficiency

3 Study the various alg-pect capsule storage conditions and their effect on the

activity of encapsulated folic acid

4 Assess the release of folic acid from alg-pect capsules under in vitro acidic

and alkaline conditions to mimic the gastrointestinal environment

5 Study the stability of alg-pect capsules in a milk system under simulated

cheese press pressures when to incorporate the capsules during cheese

making for even distribution and integrity of the capsules during cheese

ripening

6 Assess the release of folic acid from the alg-pect capsules in porcine intestinal

contents (ex-vivo)

7 Study the bioeffect of encapsulated folic acid on methionine-induced

hyperhomocysteinemia in mice using Cheddar cheese as a food delivery

vehicle

13 Constraints of the study

It is noteworthy that this study demonstrates that folic acid encapsulation is as

achievable as it is a controllable process however a number of constraints were

encountered The selection of appropriate food grade polymers (xanthan gum

gelatin iota-carrageenan low methoxy pectin and alginate) for encapsulation

presented a huge challenge in terms of gelling mechanism and encapsulation

efficiency The interaction of the various hydrogels with folic acid was little known

and in most cases was extrapolated However the hydrogels with the highest

encapsulation efficiency were selected at first instance and their properties studied

later due to the fact that no such work has been reported in literature to date The

polymers were initially selected for their widespread use within the food industry and

because they are cheap and easy to handle Alginate and pectin have been used in

microencapsulation before and were selected for this reason

The folic acid assay kit TECRAreg Enzyme Protein Binding Kit (EPBA) was selected

as a method of choice due to the fact that itrsquos a rapid method It gives results in just

under two hours as opposed to at least 24 hours for the HPLC or 48 hours for the

Microbiological assay (MA) besides method provides high specificity towards

folate isomers It shows high sensitivity too up to 1 ngml while HPLC and MA are

not as sensitive but the shelf-life of the EPBA is very short Once opened some

reagents in the kit have to be used within 30 days while others have a 60 day

lifespan This meant that kits were only used when a large volume of samples were

available for analysis which delayed progress whilst creating a huge workload at the

time of the analysis

At a cost of AUD $770 a kit for a full 96-well plate analysis the kits were expensive

and this was compounded by their short shelf-life

The protective effect of the alginate-pectin capsules on folic acid was studied by

incubating them at 37 degC in different sections of ex-vivo porcine intestinal contents

Although the result gave useful information its application to the human gut system

can only be probable because of the differences in functionality This also holds for

the murine model in terms of the homocysteine levels and vascular pathological

events reported in this study The data generated are very valuable but they only give

an indication of what might be in humans

It would have been ideal to house the mice in individual cages and have a larger

sample size Caging the mice individually would have allowed for the monitoring of

food intake which had a far reaching effect on the outcome of the study A larger

sample size would have allowed for a continuous study of homocysteine elevation in

relation to food intake and weight gain In the results presented only the end point

results are reported The results are still valuable as they are even though they donrsquot

show the information like the transit time of the capsules in the gastrointestinal tract

and the bioavailability of the folic acid under test conditions What can be deduced

with certainty though are lower lesions and homocysteine reported for encapsulated

folic acid than for the free folic acid It is reasonable to conclude that despite the

stated constraints microencapsulation of folic acid enhanced its stability and

therefore its bioactivity

14 Thesis overview

This thesis consists of an introduction a literature review and materials and methods

(Chapters 1-3) as well as three experimental chapters (Chapters 4-6) The literature

review presents an overview of folates their discovery history and nomenclature

importance in health and disease losses and instability during processing and

storage the various strategies to increase folates intake retention and elevate levels

in certain food products through judicious selection of known folate producing starter

cultures metabolic engineering plant gene manipulation and fortification

(mandatory and voluntary) Chapter 4 describes the selection of food grade polymers

for folic acid using the encapsulation efficiency as an initial selection tool before

optimising the various parameters to improve folic acid stability during storage and

bioactivity under in vitro gastric conditions Chapter 5 presents the applicability of

the alginate-pectin capsules in Cheddar cheese making their likely behaviour under

cheese press pressures and stability of the encapsulated folic acid during cheese

ripening Chapter 6 reports on the bioactivity of the released encapsulated folic acid

in the presence of methinonine-induced hyperhomocysteinemia in mice This chapter

focuses on mice weight gain homocysteine levels and vascular lesions in the mice

aorta arch during the 12 week study period It also closely compares the outcomes of

free and encapsulated folic acid to the above studied parameters Chapter 7 sums up

the overall conclusions of this study while Chapter 8 suggests future directions for

this research

Chapter 2 Literature Review

2 Literature Review 21 Discovery and chemistry of folates

In 1931 Lucy Wills demonstrated that yeast extract was effective against tropical

macrocystic anaemia often observed during late pregnancy in India Although as yet

undiscovered the critical nutrient factor involved was folic acid Several workers

contributed to the isolation of this vitamin and the elucidation of its structure (Angier

et al 1946 Mitchell et al 1941) The name folic acid is derived from the Latin ndash

folium (leaf)

Folacin refers to a group of heterocyclic derivatives with similar biological function

and common basic structure N-[4[(2-amino-1 4-dihydro-4-oxo-6-pteridinyl)-

methyl amino] benzoyl] glutamic acid with or without additional

L-glutamic acid residues conjugated via peptide linkages through the け-carboxyl

groups of succeeding glutamate molecules (Fig21) This compound was originally

given its common name folic acid by Mitchell et al (1941) upon extracting the

biologically active compound from spinach leaves

The basic structural unit (Fig 21) comprises three subunits from left to right are the

pteridine bicyclic ring structure ρ-aminobenzoic acid (ρ-ABA) and the L-glutamic

acid groups (Stokstad amp Koch 1967) The first two subunits are referred to as the

basic folate unit or pteroic acid Salts of this basic unit are called pteroates and the

acyl group termedrdquopteroylrdquo hence when this pteroic acid is conjugated with one or

more L-glutamic acid residues the entire formula as shown in Figure 21 is called

Fig 21 Chemical structure of folic acid (Source Hawkes amp Villota 1989a)

pteroylglutamic acid (PteGlu) aside from its International Union of Pure and Applied

Chemistry (IUPAC) name

Naturally occurring folates exist primarily as reduced one-carbon-substituted forms

of pteroylglutamates differing in substituent and number of glutamyl residues

attached to the pteroyl group Five different one- carbon units namely 5 6 7 8-

tetrahydro-pteroylpolyglutamates which contain glutamic molecules linked by け-

peptide bonds are known The nutritional activity of these reduced polyglutamates is

expressed as long as the essential subunit structure of folic acid remains largely

intact Additionally folates are usually C1 substituted at the N-5 (eg 5-methyl 5-

formyl) or N-10 (eg 10-formly) positions or have a single C bridge spanning these

positions (eg 510-methylene 510-methyl) Thus there are many chemical

derivatives of folic acid that exhibit a common vitamin activity as folates and these

have been well described (Scott 1989 Wagner 1985)

The following discusses the implications of folate deficiencies and attempts to

integrate the nutritional and physiological importance of folates with their chemical

stability as affected by storage and or processing variables encountered during

everyday standard food preparation manufacturing techniques either at home or on

an industrial scale It also explores the current strategies to increase food folates from

a regulatory viewpoint as well as research initiatives

22 The role of folic acid in health and disease

Interest in the health benefits of folic acid has increased considerably over the last 15

years This was initially because of its role in preventing neural tube defects (NTD)

like spina bifida (MRC Vitamin Study Group 1991)

There is evidence to support the protective role of folate against coronary heart

disease (Brouwer et al 1999) indications for positive effects of a good folate status

for cognitive functions (Seshadri et al 2002) and on prevention of certain forms of

cancer (Giovannucci et al 1995) The remethylation of homocysteine a

S-containing amino acid intimately involves t he metabolism of folate and other

B-vitamins notably vitamin B12 Elevated plasma homocysteine a consequence of

marginal folate deficiency is an emerging as independent risk factor for several

types of vascular stroke (Quere et al 2002 Vollset et al 2001) and neuro-psychiatric

disturbances including depression and dementia (Bottiglieri 1996) It has been

suggested that elevated homocysteine may have direct proatherogenic effects

mediated via cholesterol dysregulation and the enhancement of monocyte and T-cell

adhesion to human aortic endothelial cells (Koga et al 2002)

In fact the health benefits of folate nutrition extend well beyond these important

conditions The various disorders now thought to be under the influence of either

folate status andor allelic variation in genes coding for folate-dependent enzymes

include not only NTDs and occlusive vascular disease (OVD) but other midline

defects such as cleft palate (Mills et al 1999) affective disorders (Godfrey et al

1990) several cancers (cervical bronchial colon and breast) (Slattery et al 1999

Zhang et al 1999) and unexplained recurrent early pregnancy loss (Rajkovic et al

Most of these disorders can be explained within the context of folate dependent one

carbon transfer reactions involving methionine purine and pyrimidine biosynthesis

However the precise underlying cause is most probably linked to (a) one or more

common gene polymorphisms of the Hcy remethylation cycle that alters cellular

folate disposition (b) low intakes of dietary folate or (c) impaired DNA elaboration

andor gene expression linked to folate metabolism In fact it is highly likely that a

combination of these factors (and as yet undiscovered gene mutations) may come

into play and precipitate disease NTDs are the longest known folate deficiency

condition while homocysteine is an emerging risk factor for OVDs but has also been

implicated in NTDs

23 Neural tube defects (NTDs)

Birth defects are the leading cause of infant mortality and have been so for the past

25 years causing 22 of all infant deaths Approximately 3-4 of all live births

are affected by a birth defect the etiologies of most of them are known (Botto et al

1999 Cragan et al 1995) The relationship between serious birth defects and their

prevention by folic acid is well established Much of the birth defect data focus on

well substantiated relationship between folic acid and prevention of neural tube

defects (NTDs) (Smithells et al 1976) and this emphasis is reflected in this section

The neural tube is the embryonic structure that develops into the brain and spinal

cord This structure which starts out as a tiny ribbon-like tissue normally folds

inward to form a closed tube by the 28th day after conception NTDs occur when the

embryonic neural tube fails to completely close during development NTDs are

malformations of the developing brain and spine most commonly spina bifida and

anencephaly Spina bifida (ldquoopen spinerdquo) is a defect of the spine that can cause

paralysis and hydrocephalus Children with the severe form of spina bifida have

some degree of leg paralysis and impaired bladder and bowel control Anencephaly

is a fatal condition in which the baby is born with a severely underdeveloped brain

and skull Absence of the majority of the brain and surrounding tissue results in death

before or shortly after birth Anencephaly is responsible for about 30 of NTDs

(Cragan et al 1995)

In Australia the average NTD incidence rates reported for 1996-1997 were 115

births and terminations of pregnancy per 10000 total births comprising

bull 46 per 10000 total births for anencephaly

bull 57 per 10000 total births for spina bifida and

bull 12 per 10000 total births for encephalocoele (Lancaster and Hurst 2001)

Based on South Australian data accumulated over a number of decades and

generalised to the Australian population it appears that up to 500 pregnancies (births

and terminations) are affected by a NTD each year (Lancaster amp Hurst 2001)

In New Zealand approximately 30 live or stillbirths are affected by a NTD each year

(NZFSA NZMoH 2004) In 1999 the prevalence rate per 10000 was 91 total

births (including live births stillbirths and terminations) (NZMoH 2004)There is

considerable evidence showing that increased folate intakes can reduce the risk of

NTDs (NHMRC 1995) An inverse correlation exists between folate status and the

risk of NTDs with up to 70 of NTDs potentially preventable by increasing folate

status (FSAI Nutrition Sub-committee 2003) Seven percent of infant deaths from

birth defects are a result of NTDs

Because NTDs occur early in foetal development prevention would be most

effective at the earliest phase of pregnancy often before women know that they are

pregnant Hence the best public health interventions must target all fertile women

millions of women who are of child bearing age

24 Occlusive vascular disease associated with elevated homocysteine

Elevated plasma and urinary homocysteine (Hcy) levels result from several inherited

and nutritional diseases that affect Hcy remethylation and transsulphuration The

plasma Hcy range in normal subjects is quoted as 7-24 mmolL with urinary levels

in the same range Plasma Hcy exists in sulphudryl and mixed disulphide form

Homocystinuria as an inborn error of metabolism was first described by Carson et

al (1963) Mudd et al (1964) later showed a deficiency of cystathionine く-synthase

in liver biopsies taken from homocystinuric individuals

Human and animal studies clearly link plasma Hcy with vascular disease sustained

Hcy treatment in primates results in changes that mimic those observed in early

human arteriosclerosis (Harker et el 1976) Clinical studies support the

experimental data and are consistent in their findings which indicate patients with

OVD have a higher blood Hcy than individuals with no disease Despite this most

patients with vascular disease had values within what had been considered to be

normal range (Stampfer amp Malinow 1995 Ueland et al 1993) A profound

reciprocal relationship exists between blood Hcy and blood vitamins (particularly

folate) Because of this folate supplements especially when in combination with

vitamins B6 and B12 may offer a preventative measure against OVD (BrattstrOumlm amp

Wilcken 2000 Schorah et al 1998)

It has been calculated that 9 of male and 54 of female coronary artery deaths in

the United States for example could be prevented by mandatory fortification of grain

products with 350 microg folic acid100g food (Motulsky 1996) In 1996 the US-FDA

mandated that folic acid fortification of 140 microg folic acid 100 g grain product be

instituted This was estimated to increase folate intake by 70 ndash 120 microgday The

potential efficacy of further increasing this level of fortification is currently under

debate

A few studies failed to find an association between plasma Hcy and OVD (Alfthan et

al 1994 Verhoeff et al 1998) however sufficient evidence now exists to support

such an association (Perry et al 1995 Petri et al 1996) Even modest elevations in

plasma Hcy have a pathological effect on vascular endothelium Hmocysteine sits on

the intersection of two important pathways and is regulated by several enzymes (Fig

22) The partitioning of Hcy between de novo methionine biosynthesis and

transsulphuration to cystathionine is allosterically regulated by S-

adenosylmethionine (SAM) at the level of cystathionine-く-synthase (stimulates) and

5 10 methylenetetrahydrofolate reductase (5 10 MTHFR) (inhibits) (Selhub amp

Miller 1992)

In the Hcy remethylation cycle 5 10-methylene-H4PteGlu is reduced to 5-methyl-

H4PteGlu by the flavoprotein 510 MTHFR This is the only reaction capable of

producing 5-methyl-H4PteGlu and in vivo is irreversible During this process SAM

is converted to S-adenosylhomocysteine (SAH) which is then hydrolysed back to

Hcy to recommence a new remethylation cycle (Finkelstein 1990) This is the only

route for Hcy production in vertebrates The SAMSAH ratio concentration of the de

novo methyl group acceptor Hcy and specific dietary factors particularly folate and

methionine but also vitamins B12 and B6 are therefore all important determinants of

one-carbon metabolism and the metabolic balance between remethylation and

transsulphuration pathways (Deplancke amp Rex 2002)

In the liver the remethylation cycle serves to degrade methionine Methionine is an

essential amino acid in humans and is present in the diet of people in developed

countries at about 60 over that required for protein synthesis and other uses

(Shoveller et al 2004) The excess methionine is degraded via the methylation

cycle to homocysteine which can either be catabolised to sulfate and pyruvate (with

the latter being used for energy) or remethylated to methionine Folate deficiency

decreases flux through the methylation cycle The most obvious expression of the

decrease in the methylation cycle is an elevation of plasma Hcy Previously it was

thought that a rise in plasma Hcy was nothing more than a biochemical marker of

possible folate deficiency However there is increasing evidence that elevations in

plasma Hcy are implicated in the etiology of cardiovascular disease (Doshi et al

The prooxidant activity of this thiol may inhibit production of endothelin-derived

relaxation factor and activate quiescent vascular smooth muscle cells However at

physiological concentrations Hcy may inhibit the vascular endothelial cell cycle at

or before GI ndash S junction This inhibition seems to be mediated by a drop in carboxyl

methylation membrane association and activity of p21 ras a GI regulator (Amouzou

et al 2004)

Fig 22 Homocysteine metabolism (Source Verhoeff et al 1998)

25 Strategies to increase blood folate levels

The Medical Research Council (MRC) European trial concluded in 1991 that ldquofolic

acid supplementation starting before pregnancy can now be firmly recommended for

all women who have had an affected pregnancy and public health measures should

be taken to ensure that the diet of all women who may bear children contains and

adequate amount of folic acidrdquo These data led the National Health and Medical

Research Council (NHMRC) in Australia (1991) and the US Public Health Service

in 1992 among world governments to recommend that all fertile women of child-

bearing age consume 400 microg of folate daily to reduce the risk of NTDs and women

at increased risk ndash those with previous NTD pregnancies ndash should consult their

doctor before conception

In the results of a survey conducted in Australia (Table 21) Abraham and Webb

(2001) reported an increase of less than 12 dietary folate intake over a 3 year by

all age groups (15-49 y) of the women surveyed There is consensus that it is difficult

to achieve the recommended intake of folate through diet alone with women of

population aged 20 to 65 years consuming approximately ~ 100-150 microgd

It is evident that additional strategies are needed to increase dietary folate intakes to

ensure a more effective prevention of NTD cases as well as other folic acid

deficiency related diseases In instances where increases in folate levels have been

reported this is largely attributable to the ldquopassiverdquo approach to folate

supplementation namely folic acid fortification with some effects from folic acid

supplementation (Honein et al 2001)

Table 21 Median dietary folate intakes of women (15-45 years) in Australia before and after voluntary folic acid fortification (Nov 1998)

Age groups of

women (years)

Median folate intake pre

fortification 1 (1995)

(μgday)

Median folate intake

November 1998 (μgday)

15-19 195 229 20-24 216 240 25-29 219 239 30-34 206 235 35-39 210 227 40-44 217 231 45-49 221 247 15-49 213 235

Adapted from (Abraham and Webb 2001) 1 derived from unweighted survey data

A number of countries have introduced mandatory requirements for folic acid

fortification of foods in an effort to reduce the incidence of NTDs These include

Canada the USA Indonesia and a number of South American and African countries

(Table 22) Voluntary fortification only is also permitted for certain foods in a

number of European countries (including United Kingdom Ireland and Hungary)

and in a number of Middle Eastern and Asian countries The results have been quite

encouraging for example in the US overall studies found an average increase of

almost 200 μg of folate per day across all sectors of the community including the

target group of reproductive-age women (Choumenkovitch et al 2002 Quinlivan amp

Gregory 2003)

Australia and New Zealand have policies promoting the use of folic acid

supplements and have promoted the use of supplements in conjunction with

campaigns promoting the consumption of folate rich foods It is recognised that to

be effective sufficiently high dosage supplements must be taken consistently

during the peri-conceptional period However research suggests that only a small

proportion of women take the supplements during the recommended period although

evidence from New Zealand and Western Australia suggests that this proportion

increased following public health campaigns but not higher than approximately 40

(Bower et al 2002 Ferguson et al 2000) It is unclear whether this rate could be

further increased by additional promotional effort A significant issue in relation to

supplementation is the fact that approximately 45-50 of pregnancies in Australia

and New Zealand are unplanned and the neural tube develops before many women

know they are pregnant (Schader amp Corwin 1999)

Table 2 2Countries with mandatory folic acid fortification

Country Year

mandatory folic

fortification

introduced

Foods fortified with folic acid Level of

fortification

Africa

Malawi South Africa Zambia

Maize flour Maize meal wheat flour white brown bread white brown Enriched maize meal

206 189-194 136 124 074 24

Middle East

Saudi Arabia 2000

Enriched wheat enriched treated flour

North America Canada

November 1998

flour (white enriched enriched white) enriched bread enriched pasta enriched pre-cooked rice

USA Phased in between 1996 and January 1998

Enriched cereal grain products including enriched wheat flour enriched bread rolls amp buns enriched corn grits amp corn meal enriched farina enriched rice enriched macaroni products

South America

Argentina Bolivia Chile Colombia Costa Rica the Dominican Republic Ecuador El Salvador Guatemala Honduras Mexico Nicaragua Panama Paraguay

2002 1996 1997 1996 2002 2003 1996 2002 2002 2002 1998 2002 2002 1998

wheat flour wheat flour wheat flour wheat flour wheat flour corn flour rice milk wheat flour wheat flour wheat flour corn flour wheat flour corn flour wheat flour corn flour wheat flour corn flour wheat flour corn flour wheat flour corn flour wheat flour

22 15 20-24 154 18 13 1816 18 06 18 13 18 13 18 13 04-08 04-08 18 13 18 13 30

South East Asia

Indonesia unknown

Enriched wheat flour

Adapted from (Canadian Government 1998 Roche Vitamins Europe 2003 USFDA 1996)

Voluntary fortification of food with folic acid commenced in Australia in 1995 and

in New Zealand in 1996 Information from 1999 indicates that in Australia at that

time 104 folate-fortified products were available while information from New

Zealand indicates that at the end of 2001 there were 81 folate-fortified foods In

both countries breakfast cereals are the predominant folate fortified food There does

not appear to be more recent data available on the extent or type of folate-fortified

The impact of voluntary fortification on dietary intake of folate can be estimated

using modelling of consumption patterns although an accurate determination is

hampered by the lack of up-to-date information on the available fortified foods

However it has been estimated that voluntary fortification has lead to a small

improvement in mean dietary intakes of folate in women of child-bearing age in both

Australia and New Zealand with an 11 increase in Australia (from 213 μg to 235

μg) and a 13 increase in New Zealand (from 203 μg to 234 μg) (Abraham amp

Webb 2001 Newton et al 2001) However the mean intakes were significantly

lower than recommended intake levels (of 400 μg) in both countries The difficulty

of achieving the required folate in pregnancy through dietary modification has been

widely acknowledged Skeaff and Mann (1998) state that the folate-NTD

relationship is the first well documented public health situation where the amount of

nutrient required is more than that which can be practically eaten by choosing foods

wisely

The NHMRC Expert Panel on Folate Fortification noted that the likelihood of

achieving a reduction in NTDs through nutrition education alone was limited and

that the ability of nutrition education programs to reach those at most need was

questionable (NHMRC 1995) Likewise the New Zealand Ministry of Health

(NZMoH) (2004) stated that promotion of diets high in naturally-occurring folate is

not recommended on its own as a policy option to increase folate

It is difficult to identify the impact of campaigns specifically targeting increased

consumption of folate rich foods This is because most of the Australian campaigns

have targeted both increased consumption of folate rich foods and folic acid

supplementation However it is generally reported that public health campaigns

specifically targeted at increasing only naturally-occurring folate in the diet have not

produced significant dietary modification in the population (Bower et al 2002) and

have not been able to sufficiently increase RBC folate to the levels required to confer

a protective effect against NTDs (Chan et al 2001)

Reasons for this may include the following

bull Obtaining adequate folate from food sources in pregnancy is difficult because

commonly eaten foods such as wholegrain cereals fruit and vegetables are

only low to moderate sources of folate and some excellent sources of folate

such as liver are not recommended during pregnancy In addition morning

sickness may limit the amount of food women are able to eat early in

pregnancy and

bull Women who are not planning a pregnancy are unlikely to consume adequate

amounts of folate during the peri-conceptional period Data from National

Nutrition Surveys indicates that womenrsquos mean 24 hour intake of folate from

food is well below the recommended intake during pregnancy of 400 μg per

Health promotion initiatives to increase the use of folic acid supplements have been

implemented in a number of countries as a means to increase folic acid intake and

reduce the incidence of NTDs Both Australia and New Zealand have folic acid

supplement policies The promotion of supplements offers a number of advantages

(NZMoH 2004 Skeaff et al 2003) These include

bull being able to deliver the recommended amount of folic acid to the target

population (in one tablet)

bull causing no increase in the exposure and potential adverse effects in other

population subgroups

bull synthetic folic acid has a greater bioavailability than naturally-occurring

bull folate and

bull preservation of consumer choice

Supplementation is of most benefit to women planning a pregnancy but to be

effective supplements of sufficient dosage need to be taken consistently during the

peri-conceptional period It has not been recommended as a sole strategy to reduce

the incidence of NTDs due to its disadvantages which include the following

bull approximately 40-50 of pregnancies in Australia and New Zealand are

unplanned and the neural tube develops before many women know they are

pregnant (NZMoH 2004)

bull the policy relies upon the knowledge motivation and compliance of women

bull cost of supplements may be a barrier for some population groups

bull folic acid supplementation may be affected by socioeconomic factors such that

women of higher socio-economic status are more likely to take the

recommended folic acid supplements (de Walle et al 1999)

bull folic acid supplementation may also be affected by cultural factors such that

women of Culturally and Linguistically Diverse backgrounds have lower

uptake levels of folic acid supplement use (McDonnell et al 1999) and

bull use of folic acid supplements appears to be affected by age with women aged

15 to 24 years less likely to use supplements than women over 25 years of age

(Abraham amp Webb 2001)

Only a small proportion of women take folic acid supplements as shown in Table

23 In summary folic acid supplementation is highly effective in optimising folate

status (McNulty et al 2000) but supplementation is currently not an effective

strategy in primary prevention because of poor compliance

26 Intestinal absorption of folates

Dietary folates are a mixture of various mono- and pteroylpolyglutamates (with two

to seven glutamate moieties) Before absorption in the jejunum (Fig 23a) dietary

polyglutamyl folates must first be deconjugated by the enzyme pteroylpolyglutamate

hydrolase (folate conjugase) to a monoglutamyl form Before the fully-oxidised

monoglutamyl form of the vitamin folic acid enters the portal circulation through

the mucosal cells (Fig23b) of the jejunum it is reduced to tetrahydrofolate and is

either methylated or formylated (Clark 2000 Perry amp Chanarin 1970 Selhub et al

1983) However when a single dose of more than 250 mg folic acid is fed

unmetabolised folic acid has been shown to be present in serum (Kelly et al 1997)

Table 2 3 Use of folic acid supplements peri-conceptionally in Australia and New Zealand Survey Percentage of women

surveyed aware of

importance of folate in

the peri-conceptional

period

Percentage of women

taking folic acid

supplements during the

peri-conceptional period

WA Department of Health Folate Campaign evaluation (Bower et al 2002)

Christchurch (Schader and Corwin 1999 in NZMoH 2003)

Dunedin (Ferguson et al 2000)

Adapted from (Abraham amp Webb 2001 NZMoH 2004) Women who had been pregnant in the last 5 years

There are two different transport systems for the absorption of folates In the first

transport system folates are bound to membrane-associated folate-binding

proteins and transported across the brush-border membrane by a carrier-mediated

mechanism However at high intraluminal concentration of folate (gt10 mmoll) a

second non-saturable diffusion-mediated transport system plays a major role in folate

absorption The effect of the amount ingested is most likely to be of significance if

the saturable transport system is saturated At physiological concentrations (lt5

mmoll) of folate in the lumen transport occurs mainly via the saturable transport

system (Mason 1990) A level of intake that causes saturation of this transport

system is unlikely to be reached with normal intakes of natural folate from food but

could easily be reached with synthetic folic acid

Fig 23 Structure of the human gut (a) and folate absorption in the mucosal cells (b) (Source Selhub et al 1983)

27 Stability of folates

It is crucial to have appropriate information on the availability of nutrients in given

products and the effect of different processing variables on their retention To this

end research has been carried out to quantify folate losses and develop strategies to

reduce such losses within Food Industry The different forms of folate have been

reported to differ in their susceptibility to loss during storage processing and

cooking The chemical lability of all naturally-occurring folates results in a

significant loss of biological activity during harvesting storage processing and

preparation Half or even three quarters of initial folate activity may be lost during

these processes Although natural folates rapidly loose activity in foods over periods

of days or weeks the synthetic form of this vitamin folic acid (eg in fortified

foods) is almost completely stable for months or even years (Blakley 1969) In this

form the pterine ring is not reduced rendering it very resistant to chemical

oxidation

The bioavailability of natural folates is affected by the incomplete conjugation in the

brush boarder of the mucosal cells (Gregory 1997) thereby reducing their

bioavailability by as much as 25-50 In contrast synthetic folic acid appears to be

highly bioavailable ndash 85 or greater (Gregory 1997 Cuskelly et al 2001) The low

bioavailability and more importantly the poor chemical stability of the natural

folates have a profound influence on the development of nutrient recommendations

This is particularly true if some dietary intake is the more stable and bioavailable

synthetic form folic acid

Harvesting represents a more or less severe stress to the metabolic machinery in a

plant The metabolic processes of synthesis breakdown and interconversion between

different forms of folate are likely to continue immediately post-harvest and in the

early steps of processing before enzymes are inactivated Furthermore harvesting

may be associated with physical damage and exposure to adverse environmental

conditions such as oxygen heat and pressure There have been a few systematic

studies of the effects of harvest and post-harvest storage on folate levels Pandrangi

and Laborde (2004) reported that total folate was reduced by 7 when held for 10 h

at ambient temperature 26 when held for seven days at 4 ordmC and by 27 when

held for 10 weeks at ndash 22 ordmC compared with fresh spinach They concluded that

speed to market rapid sale and consumption would be obvious routes for improving

folate delivery in fresh produce It may be anticipated that transport and storage at

low temperatures and in atmospheres with low oxygen tension may reduce loss rates

Folate retention has also been studied using the sous vide system of processing In

this method vegetables are heated in a vacuum- packed container the lack of

exposure to exogenous water would be expected to prevent losses due to water

leaching Compared with fresh broccoli only 11 of total folate was lost using this

system whereas 26-40 was lost by steam blanching (Petersen 1993) Commercial

canning of vegetables and legumes results in greater losses of folate than commercial

freezing therefore consumption of folate from canned sources could be significantly

improved by recommending consumption of the accompanying canning medium

During processing and cooking minimising the use of water and time at high

temperatures has been shown to significantly improve folate retention

All folates are in danger of oxidative degradation enhanced by oxygen light

sunlight oxidising and reducing agents and heat resulting in a splitting of the

molecule into biologically inactive forms of which p-aminobenzylglutamate is one

major form There are considerable differences in stability between various reduced

folate forms the order of stability is

5-HCO-H4folate gt 5-CH3-H4folate gt 10-HCO-H4folate gt H4folate

(5formly tetrahydrofolate 5methyltetrahydrofolate 10 5678 formyl tetrahydrofolate tetrahydrofolate)

A great deal of the information on the chemical and physical properties of folates has

been based on the extensive work published on pterin chemistry (Pfleiderer 1985

Wolstenholme amp Cameron 1954) As previously mentioned folates are based on the

pteridine bicyclic structure (Figure 24a) to which there have been introduced various

electron releasing substituents conferring stability to an otherwise unstable ring The

instability of pteridine may be attributed to the loss of aromatic character with high

ratio of ring nitrogens to carbons resulting in a depletion of the stabilising layer of π-

electrons normally associated with aromatic compounds Pterins (Figure 24b) on the

other hand with their increased presence of substituents increase their ability to

resupply electrons to the depleted pteridine molecule (Figure 24a)

In the cases with hydrogenated pteridines or pterins although the number of

electron-releasing substituents attached also affects their behaviour the stability of

these reduced derivatives seems to decrease with the addition of substituent groups

(Hawkes amp Villota 1989a)

It has been demonstrated that the presence of antioxidants like ascorbate

mercaptoethanol and thiols in adequate amounts protect folates by delaying the

destruction of the methyl derivatives (Chen amp Cooper 1979) These authors studied

the stability of two folate derivatives tetrahydrofolate and 5-methyl-tetrahydrofolate

They reported that the presence of oxygen increases degradation of both derivatives

during heating at 100 degC and that ascorbic acid has a protective effect against

oxidation because of its action as a reducing agent The methyl derivative appeared

to follow first-order kinetics in the range of 65 to 100degC with activation energy (Ea)

calculated as 95 kcalmol Results from heating tetrahydrofolate derivative were

variable with an actual lower rate of degradation at 100degC than at lower

temperatures thus an Ea could not be calculated according to their data

Tetrahydrofolate appeared less stable than the methyl derivative

In general the rate of reaction for folate breakdown in the presence of oxygen

depends on the type of folate derivative and the nature of the food matrix in

particular with respect to pH buffer composition catalytic trace elements and

antioxidants (Gregory 1997 Hawkes amp Villota 1989a) Most foods in modern

nutrition are consumed after being processed by household or industrial procedures

This aims for microbiological safety convenience regarding storage and distribution

and optimal nutritional value and organoleptic appeal Most studies reflect negative

effects from processing causing increasing losses with increasing severity of

processing conditions in terms of heating temperature and time

Fig24a) Pteridine (highly soluble in water b) Pterin (low solubility in water and nonpolar solvents) and nonpolar solvents)

Source Hawkes and Villota 1989 a

Leakage and oxidative degradation are the major reasons for folate losses during

processing and storage After thermal processing of vegetables as depicted in Tables

24 and 25 both leakage and oxidative degradation can cause losses up to 70-80

In high temperature short-time processing such as pasteurisation or UHT treatment of

milk no leakage but oxidative degradation of folates occurs resulting in rather

modest losses between 0 and 20 (Wigertz et al 1996) Oxidation is also a major

cause of folate losses during steam flaking spray drying and extrusion cooking of

cereals when producing pre-cooked cereal products for gruel porridge biscuits etc

(Gregory 1989) Processing resulted in considerable losses of folates whereas losses

during storage appeared to be moderate (Table 26)

Table 24 Folate losses in foods subjected to thermal processing

Thermal

processing

Conditions

Food Sample

Folate

losses

Effecting factor Reference

water blanching steam blanching steaming (pressure) sous-vide processing in vacuum bags tinning tinning boiling or pressure cooking infra-red or convection heating warm holding autoclaving oven baking pasteurisation UHT

3-6 min 20-40 min 40 min 10-305 min 1 hr (72degC) 20 min 25-35min (200degC) 15 sec (74degC) 5 sec (140degC)

spinach broccoli broccoli broccoli spinach broccoli Brussels sprouts Brussels sprouts cooked vegetables cooked vegetables folate standards in food model rainbow trout pollack chicken breast fillet milk milk

42-83 70-91 24-41 11 50 30 0 4-24 14 0a

leakage oxidation leakage oxidation oxidation leakage oxidation leakage oxidation leakage oxidation oxidation oxidation oxidation oxidation oxidation

DeSouza amp Eitenmiller 1990 Petersen 1993 Petersen 1993

DeSouza amp Eitenmiller 1990 Malin 1977 Malin 1977 Williams et al 1995

Williams et al 1995 Ristow et al 1982 Vahteristo et al 1998 Wigertz et al 1997

a losses of folic acid

b losses of 5-methyltetrahydrofolate

Table 25 Folate losses from combined processing

Combined

processing

Conditions Food example Folate

losses

Effecting factors Reference

blanching + tinning quick soaking + cooking overnight soak + cooking blanching + freezing blanching + blast-freezing + storage freeze drying + rehydration ionised radiation ionised radiation cookchill + reheating cookhot-hold

1h+20-150 min 16h+20-150 min 14 ndash 180 days 10 kGy 25 5 10 kGy 3 days (3degC) 30 min (72degC) 2h (72degC)

spinach peas + lentils beans various pulses peas + lentils beans various pulses spinach Brussels sprouts space shuttle food various foods spinach white cabbage Brussels sprouts various vegetables various vegetables

84 55 ndash 79 55 ndash 81 34 ndash 69 40 ndash 71 31 ndash 40 34 ndash 69 87 0 36 ndash 71 5-30 10 ndash 30 10 ndash 60 10 ndash 40 26 19 32

leakage oxidation leakage oxidation leakage oxidation leakage oxidation leakage oxidation enzymes oxidation oxidation radiation leakage oxidation leakage oxidation

DeSouza amp Eitenmiller 1990 Hopper amp Lampi 1993 DeSouza amp Eitenmiller 1990 DeSouza amp Eitenmiller 1990 Malin 1977 Lane et al 1995 Muller amp Diehl 1995 Muller amp Diehl 1995 Williams et al 1995 Williams et al 1995

Table 26 Folate losses during storage of food

Storage Time Food example Folate

losses

() Effecting factors

Reference

frozen frozen frozen chilled (3degC) room temperature chilled

8 months 188 days 6-7 months I day 8 weeks 2 weeks

blanched spinach fresh Brussels sprouts beef liver

strawberries

cooked vegetables UHT- milk filjolk yoghurt

17 42 0 5 0a

oxidation enzymes oxidation enzymes oxidation oxidation enzymes

DeSouza amp Eitenmiller 1990 Malin 1977 Vahteristo et al 1998 Williams et al 1995 Wigertz et al 1997

a losses of 5-methyltetrahrdofolate

Information regarding actual kinetics of thermal destruction of specific folate

derivatives is limited and information pertaining to storage studies is almost

nonexistent General studies though have shown that losses of folates may occur as a

result of heat treatment temperature and oxygen source of heat (Chen amp Cooper

1978) light chemical environment (OrsquoBroin et al 1975) pH (Paine-Wilson 1979)

leaching (Leichter 1980) and catalytic oxidation by metal ion concentration

(Vonderschmitt amp Scrimgeneour 1967)

Ruddick et al (1980) investigated the kinetics of 5-methyltetrahydrofolate

degradation in pH 73-phosphate buffer when influenced by oxygen-concentration

changes Temperatures ranged from 40 to 100degC and oxygen concentrations used

were from 63 ppm to an unlimited oxygen supply A pseudo first-order reaction was

suggested in the presence of unlimited oxygen with an Ea of 71 kcalmol but with

limited oxygen supplied The reaction appeared to be second-order This however is

in contradiction to later work by Mnkeni and Beveridge (1983) working in a

temperature range of 100 to 140degC This variability may be due to simply the

differences in temperature levels at which the investigations were carried out

Day and Gregory (1983) also found that under limited oxygen concentrations the

degradation of 5-methyltetrahydrofolate or folic acid in phosphate buffer pH 70 was

second order The authors investigated the effects of fortification with ascorbic acid

or ferrous iron on stability of folic acid and 5-methyltetrahydrofolate when heated

from 100 to 140degC Folic acid showed highest stability in the presence of iron and

the least stability in the unfortified system Similar trends were observed with 5-

methyltetrahydrofolate but with even higher retentions than the folic acid The very

high stability is at variance with most literature however the authors concluded that

the sealed pouches in which samples were treated provided a limited oxygen supply

and thus provided lower rates of degradation

Barrett and Lund (1989) also studied the thermal degradation of

5-methyltetrahydrofolate in relation to oxygen They suggested that the discrepancies

among studies might be due to problems of oxygen diffusion They reportedly

eliminated oxygen mass-transfer limitations and calculated significantly higher

activation energies than those previous reported 163 and 233 kcalmol for 5-

methyltetrahydrofolate heated in excess oxygen or excess nitrogen respectively in a

temperature range from 40 to 92degC

The kinetics of thermal destruction of several folates as affected by pH and buffer

ions were studied by Paine-Wilson and Chen (1979) Four folates - folic acid 5-

formyltetrahydrofolate acid 5-methyltetrahydrofolate and tetrahydrofolate were

subjected to 100degC and followed first-order kinetics in a wide pH range (10 to 120)

Folic acid and 5-formyltetrahydrofolate were found to be stable up to 10 hours of

heating at pH 40 to 120 with rapidly decreasing stability with increasing alkalinity

or acidity In the case of tetrahydrofolate the reaction rate constant decreased with

increasing pH 40 to 120 The effect of ionic species present in the buffering

solutions did not seem to play a significant part in thermal stability of 5-formyl

tetrahydrofolate or folic acid but greatly altered rates of degradation of 5-

methyltetrahydrofolate and tetrahydrofolate

The lsquouniversalrsquo buffer was found to cause greater thermal degradation in both the 5-

methyltetrahydrofolate and tetrahydrofolate when compared with HCIKCI

citratephosphate or citrate buffers

Hawkes and Villota (1989b) studied the kinetics of degradation of various folates

including tetrahydrofolate 5-methytetrahydrofolate and folic acid as a function of

pH in aqueous solutions without the presence of buffer ions and as a function of

moisture content in microcrystalline cellulose (Avicel)glycerol (6040) solid model

systems Quantitation of the folates was carried out using HPLC The studies showed

that maximum stability occurred at neutral pH and that folic acid exhibited much

greater stability than either of the two derivatives in either aqueous or solid systems

All folates tested showed decreased stability with increasing moisture in solid

Avicelglycerol systems at 80degC Degradation of the tetrahydrofolate was extremely

rapid at 80degC even at 50degC data for tetrahydrofolate acid still showed the highest

rate of destruction when compared with folic acid or 5-methyltetrahydrofolate

exposed to 80degC

They concluded that folic acid is by far more stable than either of the two

derivatives 5-methyltetrahydrofolate or tetrahydrofolate with respect to pH

temperature and moisture content Highest stability for each of the folates studied

was at neutral pH After collecting data as a function of temperature and pH for the

degradation of 5-methyltetrahydrofolate tetrahydrofolate and folic acid they

reported that first-order reactions could be provisionally applied Overall higher

stabilities of the various folates were found in this investigation when compared with

values reported previously This could be due in part to the absence of buffer ions

commonly used in other investigations

In addition HPLC methodology allowed separation and monitoring of the

degradation compounds of interest without any interference from artefacts occurring

during microbiological assays In terms of characterising kinetic parameters for

folate degradation a reproducible methodology such as HPLC needs to be used

Studies investigating the effect of temperature on the stability of

5-methyltetrahydrofolate have indicated a possible change in mechanism depending

upon relative temperature range (Hawkes amp Villota 1989b Tripet amp Kesselring

1975) Folic acid however demonstrated considerably higher stability over the 5-

methyltetrahydrofolate containing systems reiterating folic acid as the logical choice

for fortification There remains however a great deal of work to calculate rate

constants when affected by moisture in different temperature ranges and to determine

if the degradation of various folates follows true first-order kinetics as affected by

different variables

28 Strategies to raise folate levels in food products

(i) Plant gene manipulation

Scott et al (2000) studied the regulatory points as well as the rate limiting steps of

folate synthesis in plants as a way of enhancing the over-expression of the enzymes

that are limiting steps for tetrahydrofolate biosnythesis Since tetrahydrofolate is a

rather unstable molecule the authors reported that the molecule metabolism could be

oriented toward a stable derivative through plant gene manipulation The authors

reported a six fold increase in folate using this technique which was only 39 of

(ii) Bio-fermentation

Dairy products represent one of the important dietary sources of folates Milk is not a

rich source of dietary folate however many dairy products are processed using

microbial fermentations in which folate can be synthesised Variations in the ability

of commercial yoghurt starter culture mixtures to produce or utilise folates have been

reported (Kneifel amp Mayer 1991 Kneifel et al 1992 Alm 1982) Hoppner amp

Lampi (1990) reported mean total folate levels that ranged from 37 to 139 microg100 g

Most of the yoghurts contained higher folate levels than those found in milk

(Crittenden et al 2002) indicating synthesis by the culturing bacteria and some

addition from ingredients

The natural diversity amongst yoghurt starter cultures with respect to their capacity

to (over)produce folate has been exploited to design new complex starters which

yield yoghurts with elevated folate levels Specific high level folate producing S

thermophilus strains and Lactobacillus strains which are prototrophic for folate were

selected from large culture collections (Crittenden et al 2002 Lin amp Young 2000)

It was demonstrated that the combinations of S thermophilus bifidobacteria and E

faecium S thermophilus elevated folate levels in skim milk from 115 ngg to

between 40-50 ngg Generally lactobacilli (L delbrueckii ssp bulgaricus L

helviticus biotype jugurti) depleted the available folate in the skim milk

Fermentations using a combination of Bifidobacterium animalis and S thermophilus

resulted in a 6-fold increase in folate concentration

By selecting high folic acid producing strains or by using relative high amounts of S

thermophilus compared to the traditional L delbrueckii ssp bulgaricus yoghurt with

increased folic acid content should in principle is possible

Daily consumption of 100 g of this yoghurt would contribute approximately 15 of

the minimum required folate intake but only 2 of recommended consumption for

women of childbearing age

(iii) Metabolic engineering

Hugenholtz et al (2000) reported that 6-7 enzymes are directly involved in the

complex biosynthesis pathway for folate in the cheese starter bacterium Lactococcus

lactis thereby providing an alternative technique for obtaining starters with

improved capacity for folate biosynthesis through metabolic engineering Their

ability to increase folate production was developed by means of classical

mutagenesis combined with high throughput screening technology as well as

optimisation of fermentation conditions which allowed for up-regulation of folate

levels in the end-product This technique like others yielded folate quantities well

below the RDI

(iv) Multi-faceted approach

Public Health experts including those with experience in behaviour-change-

strategies considered three possible strategies to increase folate intake prior to

mandatory fortification 1) behaviour change programs to increase sources of folate

in the diet such as fruits and vegetables 2) health education programs to promote the

use of folic acid containing dietary supplements and 3) passive programs to increase

folic acid in the general food supply As far as pregnancy goes it is important to note

that any successful intervention must take place before conception because most

women do not realise that they are in the very early stages of pregnancy when neural

tube occurs There are at least two major difficulties with ldquopreconceptionrdquo lifestyle

change strategies (eg efforts to increase dietary intake of folate or promote the use

of supplements)

First approximately 50 of all pregnancies are unplanned thus if an intervention

were targeted to women intending to conceive about half of all women and their

foetuses would be missed Second much research has shown that efforts to promote

the use of folic acid supplements or nutrition counselling to increase dietary sources

of folate have not been successful in increasing folic acid intake in the target

population during that critical period (Daly et al 1997 Centres for Disease Control

1992) Therefore public health experts vigorously pursued the third option of food

fortification with an initial goal of ensuring that women consume 400 microg of

synthetic folic acid daily before and during the first 12 wk of pregnancy

Even with this kind of approach it has been noted that high levels of total folate have

been reported in follow up population studies and were thought to be due to overages

used by manufacturers to ensure food products contained at least the amount of folic

acid specified on the label throughout shelf life (Rader et al 2000 Whittaker et al

2001) The lability of folic acid necessitated this move which creates another

problem Of primary concern with such levels is their possibility of masking B12

deficiency anaemia in older people a condition known to increase in prevalence with

age (Hirsch et al 2002)

Emphasis therefore should be laid on the research and assessment of new creative

public health strategies to improve folic acid supplementation to reduce andor

eliminate folic acid deficiency related diseases This will solve the more complex

problem of choice that has been raised with mandatory fortification where it has been

argued that everybody has to consume fortified products even if they prefer not to

meaning that a decision is made for an entire population without asking for

individual informed consent Food fortification is sometimes thought of as

patronising and is associated with vague fears of medicalisation of food not

believing in scientific evidence and not trusting scientists politicians and other

decision-makers

Although increased folate levels in yogurts and fermented milks are possible through

judicious selection of inoculum species the folate levels remain relatively low in

terms of RDI Even with an optimal combination of strains it still appears that

fortification is required before yoghurt could be claimed to be a good source of

dietary folate Most of these bacterial activities do not reach maximal functionality

during milk fermentation and are up for improvement The same can be said of the

alternative post-harvest storage cooking and processing techniques to minimise

folate losses

29 Cheese as a food vehicle

The four major consumer dairy products on the Australian market are drinking milk

(fresh and UHT white and flavoured) cheese butter and dairy blends and yogurt

(Table 27) The trends in per capita consumption occurring over the past two

decades vary quite significantly by individual product These reflect changes in

consumer tastes and preferences in response to a multitude of variables such as the

multicultural influences on the food we eat health perceptions of dairy products and

manufacturers responses (eg low-fat variants new product development flavour

and packaging innovations competitive category offerings distribution and

availability

Per capita consumption of milk remained around the 100 litres per head for many

years until a slow but gradual decline began in the mid-to-late 1990s to just over 97

litres now Cheese consumption has been a major success for the Australian dairy

industry with consistently strong growth to a current 12 kilograms per head Butter

consumption slowed in the 1970s and 1980s as people began to limit their intake of

saturated fats but the trend has flattened with the advent of dairy blends (Australian

Bureau of Statistics 2005)

Of course yogurt is the ultimate healthy snack for time-pressed consumers and

consequently has shown consistent growth over the period

Elsewhere the average cheese consumption in the USA nearly tripled between 1970

and 2003 from 55 kg per person to 155 kg In 2000 (the latest year for which

nutrient data are available) cheese contributed 26 percent of the calcium in the US

diet (up from 11 percent in 1970) 12 percent of the saturated fat (up from 5 percent

in 1970) and 16 percent of the sodium (up from 6 percent in 1970) (USFDA) World

cheese consumption and Cheddar in particular has grown by 15 since 1997 with

the forecast set to increase further during the next decade (USFDA 1998) This

makes Cheddar cheese an important food commodity and the subject of an

international trade of substantial value Dairy products are also becoming popular

with consumers in other parts of the world (Table 28)

Datamonitor (2004) reported that the global cheese market had a compound growth

rate of 46 in the period 1999-2003 The strongest growth was in 2000 when the

market grew by 55 Natural cheese dominates the global cheese market

accounting for 875 of the marketrsquos value In 2008 the global cheese market is

forecast to have a volume of 123 billion Kg an increase of 86 since 2003

Milk (Litres) Cheese (kg) ButterAMF (kg)

Yogurt (kg)

199899 1025 107 29 51

199900 1015 111 30 54

200001 996 113 33 53

200102 977 116 34 56

200203 974 120 34 58

200304 980 117 35 59

Year Kg billion Growth

1999 105

2000 108 260

2001 110 170

2002 112 180

2003 114 170

Table 28 Global cheese market volume 1999-2003

Source Australian Bureau of Statistics 2005

Table 27 Per capita consumption of major dairy products in Australia

Source Datamonitor 2004

210 Regulatory requirements for Cheddar cheese fortification

The Food Standards - Australia and New Zealand Standard 131 Schedule 2 allows

for the addition of alginate and pectin in both natural and processed cheeses as

additives In the USA as well as in the UK and Australia Cheddar cheese

fortification with folic acid is not permitted although in the US folates biosynthesis

using starter cultures with a final product concentration of up 700 microg100 g cheese is

allowed (USDA NDB 42258) A primary concern in the UK is the possibility of

masking B12 deficiency anaemia in older people a condition known to increase in

prevalence with age and to affect about 1271 000 000 people in the general UK

population (Department of Health 2000)

fortification of mainly breakfast cereals in an effort to reduce the incidence of NTDs

These include Canada the USA Indonesia and a number of South American and

African countries (Table 22) Voluntary fortification only is also permitted for

certain foods in a number of European countries (including United Kingdom Ireland

and Hungary) and in a number of Middle Eastern and Asian countries The results

have been quite encouraging for example in the US overall studies found an average

increase of almost 200 μg of folate per day across all sectors of the community

including the target group of reproductive-age women (Choumenkovitch et al

2002 Quinlivan amp Gregory 2003)

211 Microencapsulation

Currently there is a trend towards a healthier way of living which includes a

growing awareness by consumers of what they eat and what benefits certain

ingredients have in maintaining good health Preventing illness by diet is a unique

opportunity for innovative so-called functional foods

These products often present new challenges to the food industry Existing and new

ingredients need to be incorporated into food systems in which they slowly degrade

and lose their activity or become hazardous by oxidation reactions Ingredients can

also react with components present in the food system which may limit

bioavailability or change the colour or taste of a product In many cases

microencapsulation can be used to overcome these challenges

(i) Definition

Encapsulation is an inclusion technique for confining a substance into a polymeric

matrix coated by one or more semi-permeable polymers by virtue of which the

encapsulated compound becomes more stable than its isolated or free form (Arshady

1994 Dziezak 1988) Encapsulation of food additives can give the final food

product better technological properties and in addition controlled release of

encapsulated micro-constituents under specific conditions

The microencapsulation technology has been used by the food industry for more than

60 years In a broad sense encapsulation technology in food processing includes the

coating of minute particles of ingredients (eg acidulants fats and flavours) as well

as whole ingredients (eg raisins nuts and confectionary products) which may be

accomplished by microencapsulation and macro-coating techniques respectively

(Kirby 1991) More specifically the microcapsule has the ability to preserve a

substance in the finely divided state and to release it as occasion demands These

microcapsules may range from sub-micrometer to several millimeters in size and

have a multitude of different shapes depending on the materials and methods used to

prepare them The food industry applies microencapsulation process for a variety of

reasons

1 Encapsulationentrapment can protect the core material from degradation by

reducing its reactivity to its outside environment (eg heat moisture air and

light)

2 Evaporation or transfer rate of the core material to the outside environment is

decreased retarded

3 the physical characteristics of the original material can be modified and made

easier to handle

4 the product can be tailored to either release slowly over time or at a certain

point (ie to control the release of the core material to achieve the property

delay until the right stimulus)

5 the flavour of the core material can be masked

6 the core material can be diluted when only very small amounts are required

yet still achieve a uniform dispersion in the host material and

7 it can be employed to separate components within a mixture that would

otherwise react with one another (Dziezak 1988 Gibbs et al 1999)

Various properties of microcapsules that may be changed to suit specific ingredient

applications include composition mechanism of release particle size final physical

form and cost The architecture of microcapsules is generally divided into several

arbitrary and overlapping classifications (Fig 25) One such classification is known

matrix encapsulation This is the simplest structure in which a sphere is surrounded

by a wall or membrane of uniform thickness resembling that of a henrsquos egg In this

design the core material is buried to varying depths inside the shell This

microcapsule has been termed a single-particle structure (Fig 25A) It is also

possible to design microcapsules that have several distinct cores within the same

microcapsule or more commonly number numerous core particles embedded in a

continuous matrix of wall material This type of design is termed the aggregate

structure (Fig 25B)

In order to improve the properties of food ingredients immobilization of food

ingredients onto a suitable polymer or addition of antimicrobial agents are common

practices in the food industries (Cha et al 2003) For example an important bacteria

used in the food industry lactic acid bacteria was first immobilized in 1975 on Berl

saddles and Lactobacillus lactis was encapsulated in alginate gel beads years later

(Linko 1985) Seiss amp Divies (1975) suggested that immobilized lactic acid bacteria

could be used to continuously produce yogurt However the alginate gel beads

leaked large quantities of cells

(A) (B)

Core material

Core particles

Wall material

Fig25 Schematic diagram of two representative types of microcapsules

The use of microencapsulated food ingredients allows food ingredients to be

carefully tailored to the specific release site through the choice and

microencapsulation variables specifically the method and food ingredients-polymer

ratio (Kirby 1991) The total amount of ingestion and the kinetics of release are

variables that can be manipulated to achieve the desired result (Kirby 1991 Gibbs et

al 1999) Using innovative microencapsulation technologies and varying the

copolymer ratio molecular weight of the polymer etc microcapsules can be

developed into an optimal food ingredient device (Kirby 1991) Microcapsule-based

systems increase the life span of food ingredients and control the release of food

ingredients Various properties of microcapsules that may be changed to suit specific

ingredient applications include composition mechanism of release particle size

final physical form and cost Before considering the properties desired in

encapsulated products the purpose of encapsulation must be clear In designing the

encapsulation process the following questions are taken into consideration

1 What functionality should the encapsulated ingredients provide the final

product

2 What kind of coating material should be selected

3 What processing conditions must the encapsulated ingredient survive before

releasing its content

4 What is optimal concentration of the active ingredient in the microcapsule

5 By what mechanism will the ingredient be released from the microcapsules

6 What are the particle size density and stability requirements for the

encapsulated ingredient

7 What are the cost constraints of the encapsulated ingredient (Kirby 1991)

(ii) Controlled Release

Controlled release may be defined as a method by which one or more active agents

or ingredients are made available at a desired site and time and at a specific rate

(Pothakamury amp Barbosa-Canovas 1995) With the emergence of controlled-release

technology some heat- temperature- or pH-sensitive additives can be used very

conveniently in food systems Such additives are introduced into the food system

mostly in the form of microcapsules The additive present in the microcapsule is

released under the influence of a specific stimulus at a specified stage For example

flavours and nutrients may be released upon consumption whereas sweeteners that

are susceptible to heat may be released toward the end of baking thus preventing

undesirable caramelization in the baked product (Reineccius 1991 Greener amp

Fennema 1989 Kamper amp Fennema 1984) The release of an active compound

from a matrix-type delivery system may be controlled by diffusion erosion or a

combination of both Homogeneous and heterogeneous erosion are both detectable

Heterogeneous erosion occurs when degradation is confined to a thin layer at the

surface of the delivery system whereas homogenous erosion is a result of

degradation occurring at a uniform rate throughout the polymer matrix (Pothakamury

amp Barbosa-Canovas 1995)

The advantages of controlled release are

1 the active ingredients are released at controlled rates over prolonged periods

of time

2 loss of ingredients during processing and cooking can be avoided or reduced

3 reactive or incompatible components can be separated (Dziezak 1988

Brannon-Peppas 1993)

212 Encapsulating materials

Over the last few years medical and pharmaceutical industries have shown an

increased interest in biopolymers Polymers prepared from renewable natural

resources have become increasingly important because of their low cost ready

availability water-solubility biocompatibility biodegradability and gel forming

ability

Agar and agarose are used for solidification of biological media both in microbiology

and in new biotechnological applications The main disadvantages of agar are the

instability relating to supply and rising costs due to the limited resources of the

species of red algae from which it is isolated This factor has led to considerable

investigation in an attempt to find polymers and gelling systems which can act as

agar substitutes In the food industry this has led to the development of carrageenan

but numerous attempts have shown difficulties in replacing microbiological-grade

agar (McLachlan 1985) Some substitutes (Moslemy et al2002) have been developed

based on polysaccharides of non-algal origin

Carrageenan gels while rheologically stable require high potassium levels that are

often incompatible particularly in medical applications (Poncelet et al 1992)

Consequently alginate has become the most widely used encapsulation matrix for

biological materials including plant cells (Redenbaugh et al 1986) mammalian cells

(Lim amp Sun 1980) yeasts (Shiotani amp Yamane 1981) bacteria (Provost et al 1985)

insulin (Lim 1983) toners (Canon 1984) magnetite (Burns et al 1985) and food

products Alginate forms stable reversible gels in the presence of multivalent cations

under gentle formulation conditions at room temperature

Alginate polymer is inexpensive widely available as food or medical grade material

and biocompatible Alginate also has several unique properties that have enabled its

use as a matrix for entrapment andor delivery of a variety of proteins and cells (Lim

amp Sun 1980) Over the last two decades more suppliers of alginates are appearing in

the market place the quality of the polymer is improving and alginates are now

being sold partially or fully characterized in terms of its physicochemical properties

The selection of microencapsulation method and coating materials are

interdependent Based on the coating material or method applied the appropriate

method or coating material is selected Coating materials which are basically film-

forming materials can be selected from a wide variety of natural or synthetic

polymers depending on the material to be coated and characteristics desired in the

final microcapsules The composition of the coating material is the main determinant

of the functional properties of the microcapsule and of how it may be used to

improve the performance of a particular ingredient An ideal coating material should

exhibit the following characteristics

1 Good rheological properties at high concentration and easy workability

during encapsulation

2 The ability to disperse or emulsify the active material and stabilize the

emulsion produced

3 Non-reactivity with the material to be encapsulated both during processing

and on prolonged storage

4 The ability to seal and hold the active material within its structure during

processing or storage

5 The ability to completely release the solvent or other materials used during

the process of encapsulation under drying or other desolventization

conditions

6 The ability to provide maximum protection to the active material against

environmental conditions (eg oxygen heat light humidity)

7 Solubility in solvents acceptable in the food industry (eg water ethanol)

8 Chemical non-reactivity with the active core materials

9 Inexpensive food-grade status (Gouin 2004)

Because no single coating material can meet all of the criteria listed above in

practice either coating materials are employed in combinations or modifiers such as

oxygen scavengers antioxidants chelating agents or surfactants are added Some

commonly used biocompatible and food-grade coating materials are listed in Table

29 However chemical modifications of the existing coating materials to manipulate

their properties are also being considered Those modified coating materials exhibit

better physical and mechanical properties when compared to individual coating

materials

(i) Alginate

Alginate is a natural non-toxic biodegradable biocompatible polysaccharide found

in all species of brown algae (Aslani amp Kennedy 1996) Structurally alginates are

regarded as a family of unbranched polysaccharides consisting of (1 4) linked く-D-

Mannuronic (M) acid and ά-L-Guluronic (G) acid residues at different proportions

and with different sequential occurrence (Draget et al 2002)Chemically alginate is

a polysaccharide composed of two types of uronic acids The solubility of alginate in

water depends on the associated cations and pH

Table 29 Some coating materials for microencapsulation of functional food

additives

Category Coating materials Common method References

Carbohydrate starch maltodextrins spray amp freeze drying Reineccius 1991

chitosan spray amp freeze drying

modified starch inclusion complexation Godshall 1988

Gum alginate carrageenan spray drying syringe method Greener amp

Fennema 1989

Lipids wax paraffin oils fats emulsion liposomes Kim amp Baianu 1991

Protein casein gelatin peptides emulsion spray-drying Ono 1980

Sodium alginate is a water-soluble compound which gels in the presence of divalent

cations such as calcium and to a lesser extent in the presence of magnesium (Aslani

amp Kennedy 1996)

The early hypotheses for gel formation was that calcium ions displaced hydrogen

ions on the carboxylic acid groups of adjacent chains and formed simple ionic

bridges between the chains Rees (1969) argued why that was unlikely and later he

put forward the egg-box model (Grant et al 1973) now generally accepted This

requires the cooperative mechanism of binding of two or more chains shown in Fig

26 The buckled chain of guluronic acid units is shown as a two-dimensional

analogue of a corrugated egg-box with interstices in which the calcium ions may

pack and be coordinated The analogy is that the strength and selectivity of

cooperative binding is determined by the comfort with which eggs of the particular

size may pack in the box and with which the layers of the box pack with each other

around the eggs (Grant et al 1973) The model can be extended to be three-

dimensional While calcium helps to hold the molecules together their polymeric

nature and their aggregation bind the calcium more firmly this has been termed

cooperative binding The structure of the guluronic acid chains gives distances

between carboxyl and hydroxyl groups which allow a high degree of coordination of

the calcium

Ionically crosslinked alginate gels are extensively studied gel systems and well

known with respect to structurefunction relationships The chemical composition

and sequence to a large extent determine the elastic modulus of the resulting gels and

alginates with a high content of guluronate giving the strongest gels (Draget et al

Fig 26 Gel formation via G blocks egg box model (Source Rees 1969)

2006) The modulus increases when going down from approximately 35

guluronate suggesting that polymannuronic sequences are able to take part in

intermolecular junction zones but to a lower extent compared to polyguluronate

Viscosity typically changes in proportion to the G content During the alginate

gelling divalent cations bind preferentially to guluronic acid blocks in a highly co-

operative manner the size of the co-operative unit is reported to be more than 20

monomers (Walsh et al 1996) A high guluronic content and homopolymer blocks

lead to higher interaction between alginate and calcium which results in a stronger

and stable gel

However in the emulsification step high G gives premature gelation resulting in

larger beads with larger dispersions and more porous gels On the other hand high M

content produces more-elastic weaker gels with good freezendashthaw behaviour

However at low or very high Ca2+ concentrations high M alginates produce weaker

gels Relative to its concentration with sodium alginate concentration below 10

almost no spherical particles were formed probably due to the lack of enough

carboxyl groups for gelation When sodium alginate concentration was higher

increasing the viscosity of aqueous phase resulted in larger droplets with a wide

distribution (Liu amp Krishnan 1999) Thus for a given application the concentration

of alginate must be controlled in terms of the particle size shape and distribution

Type form and concentration of divalent cation vector

Alginate gels in the presence of divalent cations Such gels can be heat treated

without melting although they may eventually degrade Gelling depends on ion

binding (Mg2+ltCa2+ltZn2+ltSr2+ltBa2+) with the control of cation addition being

important for the production of homogeneous gels Even though alginate particles

have been produced using zinc ions calcium is the main cation used because it is

considered as clinically safe easily accessible and economical Exactly why poly-G

and poly-M are not equivalent in this context is not completely clear but it might be

argued that there should be a greater entropy loss when the more flexible mannuronic

acid blocks are lined up compared to the more rigid guluronic blocks Alginate gels

are widely used in foods but perhaps best known for immobilising living cells due to

the gentle conditions under which it can be performed (SmidrOslashd amp Skjak-Braek

For immobilization of living cells entrapment in calcium-alginate has become a

widely used technique for both research and commercial purposes Enzymes were

entrapped in a matrix of alginate by Martinsen (1990) with high encapsulation

efficiency but the capsules were rather leaky owing to the high porosity of the

alginate gel However Hertzberg et al (1990) found that the enzyme would be

retained within the alginate matrix if the environment was a hydrophobic solvent

such as hexane The use of an organic solvent for food applications is however

controversial The fact that alginate is not a natural constituent of milk and must

therefore be declared as an additive must be considered as a disadvantage However

alginate capsules may have some applications in products from which they can be

removed before consumption of the product thereby avoiding the need for

declaration For instance active microorganisms have been immobilized in Ca 2+

alginate beads for fermentation of milk or whey products and reutilized after

fermentation (Prevost amp Divies 1988 Champagne et al 1989) while Iyer et

al(2004) and Kailasapathy (2006) successfully encapsulated Ecoli and probiotic

bacteria respectively

(ii) Pectin

Pectins are primarily a polymer of D - galacturonic acid and rhamnogalacturonan

making it an α-D-galacturonan The most unique and outstanding property of pectins

is their ability to form gels in the presence of Ca2+ ions or sugar and acid It is this

property that makes them an important ingredient of many food products The

physical characterisations of gel are the consequence of the formation of a

continuous three-dimensional network of cross-linked polymer molecules On a

molecular level an aqueous gel consists of three elements as reported by Jarvis

(1984)

1 Junction zones where polymer molecules are joined

2 Interjunction segments of polymers that are relatively mobile

3 Water entrapped in the polymer network

A junction zone may involve a single covalent bond between two chains or a

combination of hydrogen bonds and hydrophobic interactions between two polymer

chains running side by side The size of the aggregate that forms junction zones

depends on how much calcium is available Under low calcium levels

polygalacturonate forms primary units of two chains in antiparallel configuration

with about 50 of the carboxyl groups neutralised with calcium In the presence of

excess calcium several primary units form sheet-like aggregates with excess

calcium being weakly bound These secondary aggregates have been suggested to

add only little strength to polygalacturonate gels (Axelos amp Thibault 1991)

Higher Ca2+ concentrations at pH 3 to 5 can destroy the gel by increasing the cross-

linking to such an extent that pectin is precipitated (Glickman 1969)

Depending on the degree of methoxylation pectins are classified into (1) low

methoxy (LM) 25 to 50 and (2) high methoxy (HM) 50 to 80 pectins and

form gels of two types They are called acid and calcium gels and are formed from

HM and LM pectins respectively For the purposes of the work reported LM pectin

was used In LM pectins gel is formed in the presence of Ca2+ which acts as a

bridge between pairs of carboxyl groups of pectin molecules The pH should be

higher in the gelation of LM pectin because only dissociated carboxylic groups take

part in the salt-like cross-linkages LM pectins are chemically more stable to

moisture and heat than are HM pectins because of the latterrsquos tendency to de-esterify

in a humid atmosphere (Yoo et al 2006)

Amidated pectins are LM pectins in which some of the carboxylic acid groups are

amidated and this has been reported to increase their gel-forming ability while gel

strength increases with decreasing degree of methoxylation (DM) (Phillips amp

Williams 2000) This increased strength of amidated pectin gels was reported to be

due to hydrogen bonding between amide groups They are more tolerant of pH

variations and calcium levels than conventional pectins

213 Microencapsulation techniques

(i) Spray-Drying

Spray-drying encapsulation has been used in the food industry since the late 1950s to

provide flavours oils with some protection against degradationoxidation and to

convert liquids to powders Spray-drying is the most widely used microencapsulation

technique in the food industry and is typically used for the preparation of dry stable

food additives and flavours (Fig 27) The process is economical flexible in that it

offers substantial variation in microencapsulation matrix adaptable to commonly

used processing equipment and produces particles of good quality In fact spray-

drying production costs are lower than those associated with most other methods of

encapsulation One limitation of the spray-drying technologies the limited number of

shell materials available

Since almost all spray-drying processes in the food industry are carried out from

aqueous feed formulations the shell material must be soluble in water at an

acceptable level Typical shell materials include gum acacia maltodextrins

hydrophobically modified starch and mixtures thereof Other polysaccharides

(alginate carboxymethylcellulose guar gum) and proteins (whey proteins soy

proteins sodium caseinate) can be used as the wall material in spray-drying but their

usage becomes very tedious and expensive because of their low solubility in water

the amount of water in the feed to be evaporated is much larger due to the lower dry

matter content and the amount of active ingredient in the feed must be reduced

accordingly

Fig 27 Advanced Spray-drying process (Source Sloten 2006)

In this method the material for encapsulation is homogenized with the carrier

material at a different ratio The mixture is then fed into a spray dryer and atomized

with a nozzle or spinning wheel Water is evaporated by the hot air contacting the

atomized material The microcapsules are then collected after they fall to the bottom

of the drier (Taylor 1983)

Rosenberg and Sheu (1996) demonstrated the use of whey protein isolate as a wall

material for encapsulation of volatiles They encapsulated ethyl butyrate and ethyl

caprylate in whey protein isolate and 11 mixture of whey protein isolate and lactose

Retention of volatiles was significantly affected by wall solids concentration (10ndash

30 ww) initial ester load (10ndash75 ww of wall solids) and by ester and wall

type Ester retention in whey protein isolatelactose was higher than in whey protein

isolate

Spray-drying is a food manufacturerndashfriendly technique because it allows the food

processor to manipulate the preparation process to improve the quality of the final

product Shiga et al (2001) prepared flavour inclusion powder by a spray-drying

technique using the combined encapsulation method of inclusion by く-cyclodextrin

and emulsified by gum arabic where d-limonene and ethyl n-hexanoate were used as

model flavours The effective film-forming property and inclusion complex were

achieved by applying high pressure to the mixture of flavours and く-cyclodextrin

slurry using a microfluidizer It is reported that flavour retention during spray-drying

increased due to blending of gum arabic and b-cyclodextrin in the feed liquid The

release rate of flavours was manipulated by the blending of maltodextrin in the feed

liquid

It is important to protect the flavour loss during drying because high-temperature air

is commonly used in spray-drying Generally the retention of flavour in

microcapsules is manipulated by varying the spray-drying conditions and

compositions of wall material

Liu et al (2001) adopted a new technique where they used emulsified liquid flavour

for spray-drying Nearly 100 of d-limonene was retained during spray-drying

independent of the composition of the feed liquid However the stability of emulsion

droplets markedly affected the retention of flavours d-Limonene emulsion was quite

stable independent of the emulsifier while the emulsion of ethyl butyrate was

unstable with gum arabic as the emulsifier The use of a mixture of gum arabic and

soluble soybean polysaccharide as the emulsifier improved oiliness and adjusting

density of ethyl butyrate and adding gelatin increased the retention of ethyl butyrate

during spray-drying

In recent years new wall materials for use in spray-drying microencapsulation have

not really emerged A few exceptions are noteworthy though The investigations of

other natural gums and their emulsification and shell properties have been reported

Mesquite gum for instance has been shown to give a better stability of the ow

emulsions and higher encapsulation efficiency compared to gum acacia (Beristain et

al 2001) Augustin et al (2001) proposed the use of Maillard reaction products

(MRPs) obtained by the reaction at high temperature between protein and

carbohydrate to encapsulate oxidation-sensitive nutrients such as fish oils The MRPs

are known to exhibit antioxidant properties and form a stable and robust shell around

the oil phase The stability of the oil against oxidation was greatly improved

compared to non-encapsulated spray-dried samples in ordinary shell material More

interesting is the recent development of complex shell formulations for spray-drying

encapsulation For instance aqueous two-phase systems (ATPSs) which result from

the phase separation of a mixture of soluble polymers in a common solvent due to the

low entropy of mixing (∆Smix) of polymer mixtures can be used to design double-

encapsulated ingredients in a single spray-drying step

Millqvist-Fureby et al (2000) encapsulated Enterococcus faeligcium in a mixture of

polyvinylpyrrolidone (PVP) and dextran While proteins exhibit partitioning between

the two phases whole cells tend to concentrate in one of the polymer phases which

make them ideal candidates for ATPS spray-drying The structure of the

microcapsule whether PVP is the outer layer and dextran the inner core or vice

versa can be controlled by adjusting the ratio and concentration of the two polymers

Encapsulated E faeligcium in spray dried ATPS showed a survival rate of up to 45

after 4 weeks at room temperature

Chitosan is a hydrophilic biocompatible and biodegradable polysaccharide of low

toxicity In recent years it has been used for development of oral controlled drug

delivery systems It is also a well-known dietary food additive Desai and Park

(2005) have reported using the cross-linked chitosan as a wall material for the

encapsulation of vitamin C by a spray-drying technique Vitamin C a representative

water-soluble vitamin has a variety of biological pharmaceutical and

dermatological functions Vitamin C is widely used in various types of foods as a

vitamin supplement and as an antioxidant Chitosan was cross-linked with non-toxic

cross-linking agent ie tripolyphosphate

Vitamin Cndashencapsulated chitosan microspheres of different size surface

morphology loading efficiency and zeta potential with controlled-release property

could be obtained by varying the manufacturing parameters (inlet temperature flow

rate) and using the different molecular weight and concentration of chitosan

Microencapsulation of vitamin C improves and broadens its applications in the food

industry

Numerous materials have been used as flavour-encapsulating agents using a spray-

drying technique These include proteins gums and modified starches (Chin-Cheng

et al 1995) An area of research of increasing interest is the development of

alternative and inexpensive polymers that may be considered natural like gum

arabic and that could encapsulate flavours with the same efficiency as gum arabic

(Re 1998) Beristain and Vernon-Carter (1995) noted that a blend of 60 gum

arabic and 40 mesquite gum encapsulated 935 of orange peel oil Cardamom-

based oil microcapsules were successfully produced by spray-drying using mesquite

gum (Beristain et al 2001) The stability against drop coalescence of the emulsions

was elevated for all the gum oil ratios studied High flavour retention (836) was

attained during microencapsulation by spray-drying when a proportion of 41 gum

oil was used This confirmed the interesting emulsifying properties and good flavour-

encapsulation ability that qualify mesquite gum as an important alternative

encapsulating medium The microcapsules can be readily used as a food ingredient

Recent developments have been in the use of new carrier materials and a newly

designed spray dryer Bhandari et al (1992 showed that a new type of dryer called

the Leaflish spray dryer which uses a high air velocity with a temperature of 300 to

400 degC was effective for encapsulating citral and linalyl acetate without degradation

A disadvantage is that a separate agglomeration step is required to prevent separation

or to render the obtained powder soluble A chief advantage is that this technique can

be used for heat-labile materials

(ii) Spray-Chilling or Spray-Cooling

In spray-chilling and spray-cooling the core and wall mixtures are atomized into the

cooled or chilled air which causes the wall to solidify around the core Unlike spray-

drying spray-chilling or spray-cooling does not involve evaporation of water In

spray-cooling the coating material is typically some form of vegetable oil or its

derivatives However a wide range of other encapsulating materials may be

employed These include fat and stearin with melting points of 45ndash122 degC as well as

hard mono- and diacylglycerols with melting points of 45ndash65 degC

In spray-chilling the coating material is typically a fractionated or hydrogenated

vegetable oil with a melting point in the range of 32ndash42 degC (Blenford 1986) In

spray-chilling there is no mass transfer (ie evaporation from the atomized

droplets) therefore these solidify into almost perfect spheres to give free-flowing

powders Atomization gives an enormous surface area and an immediate as well as

intimate mixing of these droplets with the cooling medium Microcapsules prepared

by spray-chilling and spray-cooling are insoluble in water due to the lipid coating

Fig 28 Schematic diagram of spray-cooling

Consequently these techniques tend to be utilized for encapsulating water-soluble

core materials such as minerals water-soluble vitamins enzymes acidulants and

some flavours (Lamb 1987)

(iii) Fluidized-Bed Coating

Originally developed as a pharmaceutical technique fluidized-bed coating

is now increasingly being applied in the food industry to fine-tune the effect of

functional ingredients and additives The main benefits of such miniature packages

called microcapsules include increased shelf life taste masking ease of handling

controlled release and improved aesthetics taste and colour

Fluidized-bed coating increasingly supplies the food industry with a wide variety of

encapsulated versions of food ingredients and additives (Shilton amp Niranjan 1993)

Compared to pharmaceutical fluidized-bed coating food industry fluidized-bed

coating is more obliged to cut production costs and therefore should adopt a

somewhat different approach to this rather expensive technology Solid particles are

suspended in a temperature and humidity-controlled chamber of high velocity air

where the coating material is atomized (Zhao et al 2004)

Typical food processing applications of fluidization include freezing and cooling

drying puffing freeze-drying spray-drying agglomeration and granulation

classification and blanching and cooking (Jackson amp Lee 1991) Great variations in

available wall materials exist Cellulose derivatives dextrins emulsifiers lipids

protein derivatives and starch derivatives are examples of typical coating systems

and they may be used in a molten state or dissolved in an evaporable solvent This

technique is applicable for hot-melt coatings such as hydrogenated vegetable oil

stearines fatty acids emulsifiers and waxes or solvent-based coatings such as

starches gums maltodextrins

For hot melts cool air is used to harden the carrier whereas for solvent-based

coatings hot air is used to evaporate the solvent Hot-melt ingredients release their

contents by increasing the temperature or physical breakage whereas water-soluble

coatings release their contents when water is added Fluidized-bed encapsulation can

be used to isolate iron from ascorbic acid in multivitamins and in small tablets such

as childrenrsquos vitamins Many fortified foods nutritional mixes and dry mixes

contain fluidized-bedndashencapsulated ingredients Citric acid lactic acid sorbic acid

vitamin C sodium bicarbonate in baked goods and salt added to pretzels and meats

are all encapsulated Nowadays the applicability and the utility of fluidized-bed

coating and other microencapsulation techniques in the food industry is well

recognized (Arshady 1993)

A fascinating advancement in fluidized-bed coating technique was reported by

Matsuda et al (2001) for the fluidization and coating of very fine particles In

conventional fluidized-bed coating whether it is top-spray Wurster or rotational

the basic concept of fluidization relies on the compensation of the gravitational force

experienced by the particles by an upward moving air flow which ensures complete

fluidization of the particles (Fig 29) Typical fluidized-bed apparatus can efficiently

process particles from 100 mm to a few millimeters However for very small

particles other forces such as electrostatic forces start to play a major role in the

movement of the particles in the fluidization chamber and prevent adequate

fluidization

Colloidal particles have been used with some success to reduce electrostatic force

but are not much help in the fluidization of very small (submicron) particles in a

conventional fluidized-bed apparatus In this innovative process however the

gravitational force is multiplied through the use of a rotating perforated drum that

contains the particle The air flow is then applied tangentially to the rotation of the

drum as compensation for the gravitational force now a multiple (up to 37 g) of the

normal gravitational force The conventional top-spray method remains unique and

widely used technique in food industry This is due to its high versatility relatively

high batch size and relative simplicity (Matsuda et al 2001)

A continuous fluidized-bed coaters has been developed (Rumpler amp Jacob 1998)

With such a continuous fluidized-bed coating process manufacturers can adapt the

system to their own specific requirements while maintaining the flexibility needed

for a large material throughput and wide product ranges and while providing the

coating quality demanded in the food industry The efficiency of fluidized-bed

techniques is governed by process variables ambient variables and thermodynamic

factors Appropriate modification or combinations of these variables will yield the

desired results The use of melted fats waxes or emulsifiers as shell materials is a

relatively new but very promising and interesting concept From an industrial point

of view the inherent advantage of hot-melt fluidized bed coating lies in the fact that

the coating formulation is concentrated (no solvent as in aqueous-based coating

formulation) which means dramatically shorter processing times The energy input

is also much lower than with aqueous-based formulation since no evaporation needs

to be done

Fig 29 Wurster fluidized bed for microparticle coating (Source Alexandridou et

al 1999)

Very few reports have been published on hot-melt coating by fluidized beds since

Jozwiakowsksi et al (1990) described the coating of sucrose particles with partially

hydrogenated cottonseed oil and analysed the optimal processing parameters by

modified central composite design A number of patent applications very similar in

processing designs have been published using fats and emulsifiers of various

melting points and have developed an innovative fluidized-bed process for coating

particles with fats and waxes using supercritical carbon dioxide as the solvent for the

coating formulation (Pacifico et al 2001 Wu et al 2002) Here again minimal

energy input is needed to evaporate the solvent and the process might lead to lower

cost-in-use encapsulated ingredients

(iv) Extrusion

Encapsulation of food ingredients by extrusion is a relatively new process compared

to spray-drying Extrusion used in this context is not same as extrusion used for

cooking and texturizing of cereal-based products Actually extrusion as applied to

flavour encapsulation is a relatively low temperature entrapping method which

involves forcing a core material in a molten carbohydrate mass through a series of

dies into a bath of dehydrating liquid The pressure and temperature employed are

typically lt100 psi and seldom 115degC (Reneccius 1991) The coating material

hardens on contacting the liquids forming an encapsulating matrix to entrap the core

material Then the extruded filaments are separated from the liquid bath dried and

sized (Shahidi amp Han 1993) The carrier used may be composed of more than one

ingredient such as sucrose maltodextrin glucose syrup glycerine and glucose

(Arshady 1993)

Swisher (1957) created a novel encapsulating processes that is similar to the one

currently used today in the flavour industry The primary benefit claimed was the

maintenance of fresh flavour in encapsulated citrus oils which otherwise would

readily oxidize and yield objectionable off-flavours during storage He conducted an

accelerated shelf life test on encapsulated orange peel oil that contained an

antioxidant and found that its shelf life was about one year The advantage of this

method is that the material is completely surrounded by the wall material (true

encapsulation) and any residual oil or core material is removed from the surface in

an alcohol bath (DeZarn 1995)

This technique can be classified as a glass encapsulation system or a controlled-

release system depending on the polymeric materials used The polymer matrices

and the plasticizers used can be modified to produce the capsules for controlled

release in food application (Ubbink amp Schooman 2003) However microcapsules

produced from this method are commonly designed to be soluble in water by the use

of high-molecular-weight hydrophilic polymer Thus this encapsulation technique is

considered unsuitable for subsequent extrusion processing because the water in the

extruder melt can dissolve the capsules (Yuliani et al 2004)

(v) Centrifugal Extrusion

Centrifugal extrusion is another encapsulation technique that has been investigated

and used by some manufacturers A number of food-approved coating systems have

been formulated to encapsulate products such as flavourings seasonings and

vitamins These wall materials include gelatin sodium alginate carrageenan

starches cellulose derivatives gum acacia fatsfatty acids waxes and polyethylene

glycol

Centrifugal extrusion is a liquid co-extrusion process utilizing nozzles consisting of

concentric orifice located on the outer circumference of a rotating cylinder (ie

head) The encapsulating cylinder or head consists of a concentric feed tube through

which coating and core materials are pumped separately to the many nozzles

mounted on the outer surface of the device While the core material passes through

the centre tube coating material flows through the outer tube The entire device is

attached to a rotating shaft such that the head rotates around its vertical axis As the

head rotates the core and coating materials are co-extruded through the concentric

orifices of the nozzles as a fluid rod of the core sheathed in coating material

Centrifugal force impels the rod outward causing it to break into tiny particles By

the action of surface tension the coating material envelops the core material thus

accomplishing encapsulation The microcapsules are collected on a moving bed of

fine-grained starch which cushions their impact and absorbs unwanted coating

moisture Particles produced by this method have diameter ranging from 150 to 2000

mm (Schlameus 1995)

(vi) Lyophilization

Lyophilization or freeze-drying is a process used for the dehydration of almost all

heat-sensitive materials and aromas It has been used to encapsulate water-soluble

essences and natural aromas as well as drugs Except for the long dehydration period

required (commonly 20 h) freeze-drying is a simple technique which is particularly

suitable for the encapsulation of aromatic materials

The retention of volatile compounds during the lyophilization is dependent upon the

chemical nature of the system (Kopelman et al 1977)

(vii) Coacervation

Coacervation involves the separation of a liquid phase of coating material from a

polymeric solution followed by the coating of that phase as a uniform layer around

suspended core particles The coating is then solidified In general the batch-type

coacervation processes consist of three steps and are carried out under continuous

agitation

1 Formation of a three-immiscible chemical phase

2 Deposition of the coating

3 Solidification of the coating

In the first step a three-phase system consisting of a liquid manufacturing vehicle

phase a core material phase and a coating material phase is formed by either a direct

addition or in situ separation technique In the direct addition approach the coating-

insoluble waxes immiscible solutions and insoluble liquid polymers are added

directly to the liquid-manufacturing vehicle provided that it is immiscible with the

other two phases and is capable of being liquefied In the in situ separation

technique a monomer is dissolved in the liquid vehicle and is then subsequently

polymerized at the interface Deposition of the liquid polymer coating around the

core material is accomplished by controlled physical mixing of the coating material

(while liquid) and the core material in the manufacturing vehicle in the liquid phase

this sorption phenomenon is a prerequisite to effective coating

Continued deposition of the coating is prompted by a reduction in the total free

interfacial energy of the system brought about by a decrease of the coating material

surface area during coalescence of the liquid polymer droplets

Finally solidification of the coating is achieved by thermal cross-linking or

desolventisation techniques and forms a self-sustaining microcapsule The

microcapsules are usually collected by filtration or centrifugation washed with an

appropriate solvent and subsequently dried by standard techniques such as spray- or

fluidized-bed drying to yield free-flowing discrete particles (Kirby 1991) A large

numbers of coating materials have been evaluated for coacervation

microencapsulation but the most studied and well understood coating system is

probably the gelatingum acacia system However other coating systems such as

gliadin heparingelatin carrageenan chitosan soy protein polyvinyl alcohol

gelatincarboxy methylcellulose く-lactoglobulingum acacia and guar gumdextran

are also studied (Gouin 2004)

In recent years modified coacervation processes have also been developed that can

overcome some of the problems encountered during a typical gelatingum acacia

complex coacervation process especially when dealing with food ingredients for

example a room-temperature process for the encapsulation of heat-sensitive

ingredients such as volatile flavour oils (Arneodo 1996) In this process the coating

materials are mixed and then phase separation (coacervation) is achieved by

adjusting the pH The newly formed coacervate phase is allowed to separate and

sediment most of the supernatant water is removed and the flavour oil is then added

to the mixture kept at 50degC and emulsified rapidly

The initial volume of water is restored with room temperature water causing a quick

drop in the temperature which means that the flavour oils experience a high

temperature for only a few minutes compared to several hours for a typical

coacervation process

(viii) Centrifugal Suspension Separation

Centrifugal suspension is more recent microencapsulation process The process in

principle involves mixing the core and wall materials and then adding to a rotating

disk The core materials then leave the disk with a coating of residual liquid The

microcapsules are then dried or chilled after removal from the disk The whole

process can take between a few seconds to minutes Solids liquids or suspensions of

30 mm to 2mm can be encapsulated in this manner Coatings can be 1ndash200 mm in

thickness and include fats polyethylene glycol (PEG) diglycerides and other

meltable substances

Since this is a continuous high-speed method that can coat particles it is highly

suitable for foods One application is to protect foods that are sensitive to or readily

absorb moisture such as aspartame vitamins or methionine (Sparks 1989)

(ix) Co-crystallization

Co- crystallization is a new encapsulation process utilizing sucrose as a matrix for

the incorporation of core materials The sucrose syrup is concentrated to the

supersaturated state and maintained at a temperature high enough to prevent

crystallization A predetermined amount of core material is then added to the

concentrated syrup with vigorous mechanical agitation thus providing nucleation for

the sucroseingredient mixture to crystallize As the syrup reaches the temperature at

which transformation and crystallization begin a substantial amount of heat is

emitted Agitation is continued in order to promote and extend

transformationcrystallization until the agglomerates are discharged from the vessel

The encapsulated products are then dried to the desired moisture (if necessary) and

screened to a uniform size It is very important to properly control the rates of

nucleation and crystallization as well as the thermal balance during the various

phases (Rizzuto et al 1984)

The advantages of this technique include

1 It can be employed to achieve particle drying By means of this process core

materials in a liquid form can be converted to a dry powdered form without

additional drying

2 Products offer direct tableting characteristics because of their agglomerated

structure and thus offer significant advantages to the candy and

pharmaceutical industries

(x) Liposome Entrapment

Liposomes consist of an aqueous phase that is completely surrounded by a

phospholipid-based membrane When phospholipids such as lecithin are dispersed

in an aqueous phase the liposomes form spontaneously One can have either aqueous

or lipid-soluble material enclosed in the liposome They have been used for delivery

of vaccines hormones enzymes and vitamins (Gregoriadis 1984)

They consist of one or more layers of lipids and thus are non-toxic and acceptable for

foods Permeability stability surface activity and affinity can be varied through size

and lipid composition variations They can range from 25 nm to several microns in

diameter are easy to make and can be stored by freeze-drying

Kirby and Gregoriadis (1984) devised a method to encapsulate at high efficiency

which is easy to scale-up and uses mild conditions appropriate for enzymes It is

important to reiterate that large unilamellar vesicles (LUV) are the most appropriate

liposomes for the food industry because of their high encapsulation efficiency their

simple production methods and their good stability over time The great advantage

of liposomes over other microencapsulation technologies is the stability liposomes

impart to water-soluble material in high water activity application spray-dryers

extruders and fluidized beds impart great stability to food ingredients in the dry state

but release their content readily in high water activity application giving up all

protection properties

Another unique property of liposomes is the targeted delivery of their content in

specific parts of the foodstuff For example it has been shown that liposome-

encapsulated enzymes concentrate preferably in the curd during cheese formation

whereas non-encapsulated enzymes are usually distributed evenly in the whole milk

mixture which leads to very low (2ndash4) retention of the flavour-producing enzymes

in the curd They have prepared bromelain loaded liposomes for use as meat-

tenderizer to improve stability of the enzyme during the processing of the food and

subsequently improve the availability of the enzyme Benech et al (2002) showed

that liposome-entrapped nisin retained higher activity against Listeria innocua and

had improved stability in cheese production proving a powerful tool to inhibit the

growth of L innocua in cheese while not preventing the detrimental effect of nisin on

the actual cheese-ripening process

The main issues in liposome encapsulation for the food industry are

1 the scaling up of the microencapsulation process at acceptable cost-in-use

levels and

2 the delivery form of the liposome encapsulated ingredients

The development of a cost-effective drying method for liposome microcapsules and

development of a dry liposome formulation that readily reconstitutes upon

rehydration would ensure a promising future to liposome encapsulation of food

ingredients The recent advances in liposome technology have most probably solved

the first issue microfluidization has been shown to be an effective cost-effective and

solvent-free continuous method for the production of liposomes with high

encapsulation efficiency The method can process a few hundred litres per hour of

aqueous liposomes on a continuous basis (Zheng et al 1999) The other issue

concerns the aqueous form in which the liposomes are usually delivered Most of the

time if not always liposome formulations are kept in relatively dilute aqueous

suspensions and this might be a very serious drawback for the large-scale production

storage and shipping of encapsulated food ingredients

(xi) Inclusion Complexation

Molecular inclusion is another means of achieving encapsulation Unlike other

processes discussed to this point this technique takes place at a molecular level b-

cyclodextrin is typically used as the encapsulating medium く-Cyclodextrin is a

cyclic derivative of starch made up of seven glucopyranose units They are prepared

from partially hydrolysed starch (maltodextrin) by an enzymatic process The

external part of the cyclodextrin molecule is hydrophilic whereas the internal part is

hydrophobic The guest molecules which are apolar can be entrapped into the apolar

internal cavity through a hydrophobic interaction (Pagington 1986) This internal

cavity of about 065nm diameter permits the inclusion of essential oil compounds

and can take up one or more flavour volatile molecules In this method the flavour

compounds are entrapped inside the hollow centre of a く-cyclodextrin molecule

く-Cyclodextrin molecules form inclusion complexes with compounds that can fit

dimensionally into their central cavity These complexes are formed in a reaction that

takes place only in the presence of water Molecules that are less polar than water

(ie most flavour substances) and have suitable molecular dimensions to fit inside

the cyclodextrin interior can be incorporated into the molecule There are three

methods to produce the flavour-く-cyclodextrin complex In the first method b-

cyclodextrin is dissolved in water to form an aqueous solution and the flavours are

added to form an inclusion complex in crystalline form The crystal obtained is then

separated and dried In the second method b-cyclodextrin is dissolved in a lesser

amount of water than in the first method to form a concentrated suspension and the

flavours are mixed to form an inclusion complex in crystalline form The complex

then must be separated and dried In the third method b-cyclodextrin is dissolved in

a much lower water content to form a paste and the flavours are mixed during

kneading to form an inclusion complex This method is superior to the former two

because it does not require further separation and drying (Pagington 1986)

Encapsulating flavours in this way can provide better protection from volatilization

during extrusion However the use of く-cyclodextrin for food application is very

limited possibly due to regulatory requirements in a number of countries

214 Future trends

The use of microencapsulated food ingredients for controlled-release applications is

a promising alternative to solve the major problem of food ingredients faced by food

industries The challenges are to select the appropriate microencapsulation technique

and encapsulating material Despite the wide range of encapsulated products that

have been developed manufactured and successfully marketed in the

pharmaceutical and cosmetic industries microencapsulation has found a

comparatively much smaller market in the food industry The technology is still far

from being fully developed and has yet to become a conventional tool in the food

technologistrsquos repertoire for several reasons

First of all the development time is rather long and requires multidisciplinary

cooperation Secondly the low margins typically achieved in food ingredients

and the relative inertia of well-established corporations are an effective deterrent to

the development and implementation of novel technologies that could result in truly

unique food products whether for more effective production food fortification

neutraceuticals improved organoleptic properties or development of novelty food

products However the most important aspect of RampD from the very first lab-bench

tests is an understanding of the industrial constraints and requirements to make a

microencapsulation process viable from the transition to full-scale production to the

marketing of the final product

215 Summary of Literature

It has been known for some time that optimising blood folate levels around the time

of conception and in the early weeks of pregnancy can significantly reduce the

chance of NTD-affected pregnancies Around the world governments have adopted

a number of approaches aimed at increasing the folate status of women Some have

taken the decision to recommend folic acid supplementation (typically 400 microgday)

for all women of child bearing age In some countries a higher amount (4 mgday)

has been recommended for women who have already experienced an NTD-affected

pregnancy Some have a gone further and supported voluntary and in a few cases

mandatory folic acid fortification programmes often focusing on bread and flour

and many have stressed the importance of eating folate-rich foods eg green

vegetables liver oranges and whole grain foods Numerous studies have also

demonstrated the importance of folic acid in protecting against cardiovascular

diseases Alzheimerrsquos dementia affective disorders and DNA replication in the

general population This further increased research and study of folic acid The

greatest challenge is probably the lability of all folates during harvest storage

processing and preparation of food sources of the vitamin It has been reported that

losses of up to 85 have been recorded in some studies done in fresh vegetables

when they were subjected to processing conditions like blanching canning and

boiling Its synthetic analogue folic acid has been used widely in supplementation

and fortification The problem with the natural folates is not just stability but also

bioavailability On a comparative basis natural folates are 25-50 bioavailable

while folic acid is gt85 This leaves folic acid as the only folate derivative that can

be used But the stability of folic acid like all folates affected by a change in pH

from neutral in either direction oxygen temperature and moisture So to be able to

deliver it in sufficient enough quantities and in a bioactive form other techniques of

delivery are required Microencapsulation has been applied in instances where the

free compound would otherwise be easily degraded or discolour the product due to

ingredients interaction It has also been successfully applied in the delivery of

probiotics and other functional foods where controlled release is achieved at

designated times Whilst both fat soluble vitamins (eg D E K A) and some water

soluble vitamins (eg vitamin C) have been encapsulated the controlled release of

folic acid has not been studied yet Study of the microencapsulation parameters of

this vitamin and its delivery through a food vehicle will not only enhance its stability

but also the applicability of this technology in other food products hitherto

unexplored Widening the base of folic acid rich foods will increase consumer choice

for such foods and in the process mitigate against known folic acid disorders

Chapter 3 Materials and methods

3 Materials and Methods

Some general material and methods used in the experiments of this study are

described in this chapter The specific experimental materials and methods for each

particular experiment are given in respective chapters

Sodium alginate (viscosity of 2 wv solution at 25degC 250 cps) potassium salt of

pectin (28 degree of esterification (DE) low methoxy (LM) and 20 amidated)

gelatine iota-carrageenan xanthan gum citric acid di-sodium hydrogen phosphate

potassium dihydrogen phosphate sodium hydroxide and calcium chloride were all

purchased from Sigma-Aldrich (Castle Hill Sydney Australia) Folic acid was

obtained from Roche Pty Ltd (Sydney Australia) Starch (Hi-Maize trade1043) was

purchased from National Starch and Chemical Company (New Jersey USA)

31 Folic acid encapsulation procedure

A modified single step process by Blandino et al (2001) was followed to produce

microcapsules using the Inotechreg encapsulator research IE-50R (Inotech Dottikon

Switzerland) fitted with a 300μm nozzle Single polymer solutions were first

prepared to evaluate folic acid encapsulation efficiency by dissolving sodium

alginate iota-carrageenan LMA pectin gelatin or xanthan gum wv in 100 ml Milli-

Q water (Millipore Massachusetts USA) until a viscosity of 225 cps was recorded

The quantity of polymer dissolved was recorded The solutions were left overnight to

fully hydrate at ambient temperature except for gelatin which was placed in a water

bath at 37-40 ordmC with constant stirring for a similar period The pH of the solutions

was recorded in the morning before the next step

Folic acid was dispersed in the polymer solutions to give a final concentration of

1ngml folic acid The mixture was pumped through the encapsulation nozzle with a

continuous flow of nitrogen into a gently agitated aqueous solution of calcium

chloride (005 01 05 and 10 M) at 25ordmC where discrete folic acid laden

microcapsules formed upon contact with calcium chloride solution

The various calcium chloride concentrations and different gelation times ranging

from 0 to180 min were tested to optimise folic acid loading efficiency The reaction

vessel with calcium chloride was constantly stirred to keep the polymer droplets

from clumping A dropping height of about 10 cm between the nozzle and the

reaction vessel was maintained to ensure that spherical droplets were formed The

flow rate was set at 1 mlsec to allow for the same residence time of the capsules in

the calcium chloride solution

The second set of polymer solutions to evaluate encapsulation efficiency based on

polymer combinations were prepared by mixing equal volumes of any two of the

above single polymers in a volume of 100 ml before repeating the above procedure

except that only 01 or 05 M calcium chloride solutions were used in the reaction

vessel

The third series of polymer solutions to study the combined effect of alginate and

pectin concentration on folic acid encapsulation efficiency were carried out as above

except that the calcium chloride solution was mantained at 01 M The two polymers

were mixed in a 100 ml cylinder as follows alginate-pectin (1000 8020 7030

6040 5050 0100 2080 3070 4060) All procedures with folic acid were

performed in a dark room because it degrades in both ultra-violet and visible light

(Hawkes amp Villota 1989a) The capsules were stored at -20 ordmC in aluminium foil

wrapped vials filled with Milli-Q water until required The foil was used to protect

the capsules from light Folic acid was quantified using the TECRA method as

mentioned in 38

completion in alginate-pectin hydrogels

Although the cross-linking process between alginate-pectin alginate or pectin with

calcium ions began almost instantaneously different cross-linking times ranging

from 0 to 180 min were studied for each of the calcium chloride concentrations

(005 01 or 10 M) and polymer mixtures to determine the completion of gelation by

sampling 1 g (wet weight) of hydrogels at 20 min intervals The hydrogels were

rinsed twice with 500ml Milli-Q water to remove loosely associated folic acid as

well as help terminate the cross-linking process and analysed immediately for folic

acid content Cross-linking was considered complete when folic acid content

between subsequent sampled hydrogels remained constant The crude surface

morphology of the hydrogels was examined using a Nikon microscope model

Labophot-2 (Nikon Corporation Kanagawa Japan)

Magnetic stirrer

300 microm nozzle

Food grade

nitrogen gas at 02

bar pressure

Alginate-pectin hydrogels

in 01 M calcium chloride

solution

100 ml alginate-pectin + folic acid 1ngml mix

Drain calcium

chloride solution

Capsules

Fig 31 A simplified schematic illustration of microencapsulation

300 microm nozzle

Product

bottle

Reaction vessel with calcium chloride

Fig 32 Inotech Encapsulator reg (Inotech AG Dottikon Switzerland) used in this

33 Effect of buffer type on folic acid release from alginate-pectin hydrogels

To select a suitable buffer for the release of folic acid from the alginate-pectin gel

capsules 1 g (wet weight) of the capsules was added to a 100 ml of each of the

following buffers 01M phosphate buffer citrate buffer and Buffer TS within pH

range 60 ndash 85 One ml aliquot was withdrawn and replaced with an equal volume of

fresh buffer in a time course experiment from 30 min to 180 min before storage at -

20 degC in aluminium foil wrapped vials until required The TECRA EPBA method

(see 311) was used to determine folic acid released as a function of time Phosphate

buffer (pH 82) produced the greatest capsule dissolution as indicated by folic acid

release and was therefore used as a buffer of choice Information about the

composition of the different buffers mentioned above is given in Appendix 1

34 Single and blended polymer effect on folic acid encapsulation efficiency

Encapsulation efficiency was determined using the following relationship adapted

from Pillay and Fassihi (1995)

Folic acid encapsulation efficiency () = (AQTQ) times 100

Where AQ is the actual quantity of the vitamin present in the capsule matrices and

TQ is the 100 theoretical quantity of folic acid

The alginate-pectin gel capsules (1000 8020 7030 6040 5050 0100 2080

3070 4060) were evaluated for folic acid encapsulation efficiency after

cross-linking in 01 M calcium chloride for 180 min One g of gel capsules from each

alginate-pectin mix was dissolved in 01 M phosphate buffer (pH 82) and assayed

for folic acid as described in 38

35 Effect of different drying techniques on retention of encapsulated folic acid

Five g (wet weight) of freshly made alginate-pectin (7030) gel capsules were

air-dried (ambient ~ 25degC) for 24 h with a similar quantity freeze-dried over a 6 h

period in an Alpha-1-4 freeze dryer with controller LDC-1M (CHRISTreg Osterode

am Harz Germany) while a third batch was vacuum-dried at 25degC for 6 h A control

with free folic acid was prepared To test the effect of combined drying 5 g of

alginate-pectin gel capsules were vacuum dried for 3 h at 25 degC followed by freeze

drying for 3 h The dried samples were stored in airtight foil covered glass vials at -

20 degC 4degC and ambient temperature for 11 weeks All the vials were flushed with

nitrogen immediately before use

36 Folic acid leakage

To assess folic acid diffusion out of the capsules made from each of the four

alginate-pectin (100 0 80 20 70 30 60 40) mixed polymer groups 50 capsules

were randomly picked and placed in a 100 ml Erlenmeyer flask (covered with

aluminium foil) with 50 ml of Milli-Q water (pH 70) The water in the flask was

agitated at 100 rpm with a Teflon bar on a magnetic stirrer for 24 h at 25ordmC with 1 ml

sample withdrawn at an hourly interval for the first 10 h and then four hourly for the

remaining time The 1 ml sample withdrawn was replaced with an equal volume of

Milli-Q water Folic acid leakage was quantified as given below after thawing the

previously frozen sample stored in aluminium foil covered vials

37 Morphology of gel capsules

The morphology of the gel capsules was studied by measuring by their thickness and

shape using an optical microscope (Nikon microscope model Labophot-2 Nikon

Corporation Kanagawa Japan) fitted with a stage Occulometer The thickness of the

capsules was further studied by cutting them in half and carrying out measurements

in at least four different locations on the capsule The image processing software

IMAGE-PRO was used to measure the capsule thickness on an image of each half

capsule The thickness and shape of capsules reported represent the average of

twenty capsules obtained under the same experimental conditions Alginate capsules

were used as a reference for both shape and thickness measurements

38 Folic acid measurement using the TECRAreg Quantitative Folate Assay

(QFA) Kit

Principle of the test

This technique is based on the interaction of folate immobilised on the surface of the

removawells and a folate receptor- enzyme conjugate mixture When this mixture is

added to the wells free folate and immobilised folate compete for binding to the

conjugate After incubation the wells are washed free of unbound material The

addition of the substrate to the wells causes any unbound conjugate on the surface of

the wells to produce a colour The limit of detection for the kit is 01ngmL

Folate( ) is immobilised on the surface

of Removawells

A Folate Receptor (FR) Ezyme

conjugase and a sample

containing free folate are added to the

Removawells and mixed

Unbound material is removed from the

Removawells by washing

A colourless substrate is added

In the presence of the Enzyme the colourless substrate

changes to green The amount of colour is inversely

proportional to the level of folate in the sample ie the

more folate present the less the colour produced

Substrate converted to green

Substrate

Enzyme conjugated Folate Receptor

Folic acid

Fig 33 Principle of folic acid measurement by enzyme protein binding assay

(illustration) (Arcot et al 2002)

Each kit comes with following

Reagent number Item

1 Wash concentrate

2 Folate standard solutions

i 270 ngml

ii 090 ngml

iii 030 ngml

iv 010 ngml

v 000 ngml

3 Conjugate

4 Substrate diluent

5 Substrate

6 Stop solution

7 Folate coated removable wells

Reagent preparation

The sample buffer was made by dissolving Tris base (145g) and sodium chloride

(700g) in 800 ml of Milli-Q water followed by pH adjustment to 79-82 using 10M

HCI before making up to 1L with water A 100 ml of the buffer was diluted with 900

ml of water and used fresh as the working strength buffer in sample preparation

Sample preparation

Capsules (1 g wet weight) were dissolved in phosphate buffer (pH 82) after which 1

ml of sample was diluted in 99 ml of sample buffer and heated in a boiling bath for

10 min This was followed by rapid cooling of a 1 ml aliquot in an ice bath for 10-15

min and centrifugation (Sigma 6K-15 Martin Christ Osterode am Harz Germany)

at 2000 x g for 15 min The sample buffer was used to further dilute the supernatant

to an approximate concentration of 1 ngmL where necessary using the following

dilution factors 125 150 1100 Sample dilutions were assayed for folic acid on

the day they were prepared or stored at - 20degC until required

Performing the Assay

All kit components and ancillary reagents were brought to room temperature (20-

25degC) prior to use On a 96-well plate 6 wells were used for each sample and 2 for

each folate standard (a total of ten wells for the five standards) Conjugate (50microL)

was added into every well followed by 150 microL of each sample dilution or standard

with gentle tapping against the holder to mix The holder was covered with a cling

wrap film to minimise heat loss and incubated at 35-37 degC for 1 hr in a shaking

incubator (New Brunswick Scientific classic series C24K Edison NJ USA)

Contents were emptied into the sink with the residual liquid removed by striking the

holder firmly several times face down on a thick pile of absorbent paper towels A

wide nozzle squeeze bottle held above the plate was used under a heavy stream of

wash solution to completely fill each well taking care not to trap air bubbles in the

bottom of the wells The wells were washed and completely emptied for a total of

four times by repeating the above

Substrate (200 microL) was added to each well followed by incubation at room

temperature (20-25 degC) for 15 min before reading the results at 414 plusmn10 nm on a

Multiskan Spectrum Microplate Spectrophotometer model 1500 (Thermo

Labystems Vantaa Finland) If the zero standard was greater or equal to 15 200

microL of the Stop solution was added to each well then read within one hour If the

absorbance did not reach 15 after 40 min the result could not be used For the

results to be valid the absorbance of the zero standard had be greater than 15 and

less than 25

The results were interpreted by the software set in the Multiskan Spectrophotometer

to interpolate unknown sample concentrations from a logarithmic standard curve For

the test to be valid the r and r2 values had to be greater than 095

capsules

Alginate-pectin gel capsules previously stored in 10 saline solution at 20 ndash 25 ordmC

for 24 h were evaluated for their mechanical strength under simulated cheese press

pressures using a texture analyser (model TA-XT2 Stable Micro Systems Surrey

UK) equipped with a 3 cm (diameter) plexiglass piston During the test the piston

was lowered at a rate of 01 mms on 30 g of alginate-pectin gel capsules until a force

resistance of 588 g (843 g cm2) was detected and maintained for 4 h after which the

probe automatically moved back to its rest position A 1 ml fluid aliquot was

withdrawn from gel capsules at 30 min intervals and replaced with an equal volume

A control with capsules stored in plain Milli-Q was also performed The aliquots

were immediately frozen until required for folic acid measurement as detailed in 38

The time versus folic acid measured from the aliquots characterises the capsules gel

strength Examination of gel capsules to determine rapture or deformity as a result of

the simulated cheese press pressures was carried out an optical microscope (Nikon

microscope model Labophot-2 Nikon Corporation Kanagawa Japan) Fresh gel

capsules were used as reference

310 Cheddar cheese making

Cheddar cheeses were made in 1 kg batches in a 10 L water-jacketed mini-vat

(Armfield Ringhood England) using 15 -20 ml of L lactis spp lactis LL50C direct

vat set (DSM Food Specialities Sydney Australia) as the starter culture and 25 ml

(110) calf rennet (Home Cheese making supplies Werribee Victoria Australia)

The 10 L of milk used per batch was obtained from the University of Western

Sydneyrsquos Dairy Plant After addition of rennet the vat was covered with foil paper

and heated for 35 min at 31 degC without agitation before checking the coagulum for

the right consistency

Wire knives were then used to cut the curd with titratable acidity ranging between

009 to 012 This was followed by heating to 38 degC over a 55 min period with

agitation at the rate of 1 degC for every 10 min up to 33 degC and then 1 degC for every 5

min from 33 to 38 degC When acidity level reached 016-017 the whey was

drained off A 100 microm mesh cheese cloth was fitted at the end of the whey outlet

tube to prevent loss of alginate-pectin capsules (450-550 microm) during whey-off The

retained capsules were put back in the vat With the curd banked up and cut into four

blocks turning of the blocks every 15-20 min called cheddaring followed When

titratable acidity rose to 05 milling followed and 20 min later salting (25 of

expected yield) then hooping before pressing on a cheese press at a force of 0814

Ncm2 overnight and then vacuum packaged (Model AV Australian Vacuum

Packing Machines Pvt Ltd Brisbane Australia) in sterile plastic bags the next

morning The cheese was ripened at 8-10 degC for at least 3 months before use Figure

35 summarises Cheddar cheese making by the way of a flow process chart

Rennet 25

ml 110

Starter culture

20 ml LL50 C

10 g alg-pect capsules

Pasteurised full

cream milk ndash 10 L

Add 10 g of capsules after milling

the curd and mix adequately

Inject 10 g of capsules into the

pressed curd using a hypodermic

syringe ldquo19 Gaugerdquo before slicing

and packing

Sanitise all equipment with hot water

Heat milk to 31 degC and hold add starter

and capsules Agitate continuously

Add rennet agitate for 1-2 sec

Cover vat with foil paper and heat for 35

min without agitation Check curd for

right consistency cut with wire knives if

its alright otherwise leave a bit longer

When acidity reaches 016-017

place a 100micro cloth at the end of the

outlet tube and drain all the whey Put

back the recovered capsules

ldquoCookrdquo from 31degC to 38 degC over 55

min starting with 1degC every 10 min

until 33 degC and then 1degC every 5

min thereafter

When the acidity reaches 05 mill

the curd and salt (25 of expected

yield)

Bank the curd and commence the

cheddaring process by turn the curd

every 20 min Maintain the temperature

at or above 31degC

Leave for 20 min to allow for the

absorption of salt

Hoop and press the curd overnight

at a pressure of 0814 Ncm2

Cut the raw cheese block into

30 g slices and vacuum pack

Transfer to a cheese

maturation room and store at

8-10degC until required 111

Fig 34 Flow process chart for Cheddar

cheese manufacture

311 Stability of folic acid during cheese ripening

Cheeses were made as above and 5 g alginate-pectin gel capsules incorporated in

milk to give a folic acid concentration of 1ngml For comparison free folic acid was

added after milling the curd Cheese slices weighing 30 g each were individually

vacuum packed (Model AV Australian Vacuum Packing Machines Pvt Ltd

Brisbane Australia) and ripened at 8 - 10 ordmC for at least 3 months and sampled

monthly for measurement of folic acid content using the TECRAreg enzyme protein

binding assay (Section 38)

312 Total folates measurement

Total folates in Cheddar cheese were measured as reported by Shrestha et al (2003)

Briefly a desiccated chicken pancreas conjugase (No 0459-12-2 Difco

Laboratories Detroit MI 48232-7058) solution of 5 mgml was prepared A 10 g

sample was homogenised in a Waring blender with 100 ml of extraction buffer (01

M potassium phosphate 1 ascorbic acid pH 72) The homogenate was autoclaved

at 121degC for 10 min immediately cooled and centrifuged at 1000 g for 15 min

Aliquots of supernatant (10 ml) were either analysed straightaway or stored at ndash

40degC until required Chicken pancreas (15 ml) was added to 1 ml of sample extract

and volume adjusted to 10 ml using the phosphate ascorbate buffer The mixture was

incubated at 37 degC for 3 h followed by heating at 100 degC to de-activate the enzyme

After preparation of the standard folic acid (F-7876 Sigma Chemical Co St Louis

MO 63178) solutions 50 microl of inoculum was added to the sample and standard tubes

and incubated at 37degC for 16-18 h Turbidity was measured as absorbance value in a

spectrophotometer (Helios Gamma Thermo Electron Corporation Finland) set at

540 nm

313 Statistical analyses

Statistical evaluation of data was performed using SPSS software (SPSS software

SPSS Inc 2002) For numerical data such as capsule thickness and shape

encapsulation efficiency folic acid retention total Hcy lesion area serum folate or

mice growth the differences between the means were analysed by analysis of

variance (ANOVA) The data were expressed as mean plusmn SD (standard deviation of

Statistical difference is indicated as follows

pgt005 = no statistical difference

plt005 = statistically significant

Evaluation of folic acid

encapsulation efficiency by

single or mixed polymers

Polymer selection

Calcium chloride

concentration

Alginate-pectin

combinations

Cross-linking time

Optimisation of polymers with

highest encapsulation efficiency

Measurement of

parameters

Capsules

mechanical

strength

Folic acid

leakage In-vitro

release

Storage

studies

Evaluate bioactivity of

encapsulated folic acid

using homocysteine and

aorta pathological events

Animal experiments to test

the bioactivity of

Cheese making and

capsules stability during

cheese ripening

Porcine ex-vivo

release of folic

Fig 35 Diagrammatic summary of the major steps in this study

Chapter 4 Selection and optimisation of encapsulation parameters

4 Screening of food grade polymers and

optimisation of folic acid encapsulation parameters

to increase encapsulation efficiency and stability

41 Abstract

temperature oxygen light and their stability is affected by processing conditions

Folic acid incorporated microcapsules using alginate and combinations of alginate

(alg) and pectin (pect) polymers were prepared to improve the stability of folic acid

The stability was evaluated with reference to encapsulation efficiency the cross-

linking of alg-pect during capsule making and folic acid retention during drying and

storage Use of alginate in combination with pectin produced capsules showing

greater encapsulation efficiency than the two polymers individually The

encapsulation efficiency ranged from 55 to 89 in the different combinations of

mixed polymers After 11 weeks of storage at 4ordmC folic acid retention in freeze-dried

alg-pect capsules was 100 (alg-pect 7030) The blended alginate and pectin

polymer matrix increased folic acid encapsulation efficiency and reduced the leakage

from the capsules compared to those made with alginate alone Folic acid retention

remained constant after freeze drying and storage Alg-pect capsules showed

minimal in-vitro release in acidic conditions while sustained release was observed in

alkaline conditions pH 82 The optimised microencapsulation parameters using alg-

pect capsules described in this chapter demonstrate the potential for this method to be

applied to protect folic acid against adverse conditions like in the stomach and be

released in the small intestines where folic acid is absorbed This chapter is based on the publication Madziva H Kailasapathy K Phillips M

(2005) Alginate-pectin microcapsules as a potential for folic acid delivery in foods J Microencap 22(4) 343-357

42 Introduction

Recent knowledge supports the hypothesis that beyond meeting nutritional needs

diet may modulate various functions in the body and may play detrimental or

beneficial roles in the aetiology of some diseases Concepts in nutrition are

expanding from the past emphasis on survival hunger satisfaction and preventing

adverse effects to an emphasis on the use of foods to promote a state of well-being

and better health and to help reduce the risk of diseases These concepts are

particularly important in the light of the increasing cost of health care the steady

increase in life expectancy and the desire of older people for improved quality of life

in their later years These changes of emphasis in nutrition have over the past 12-15

years justified the efforts of health authorities in many countries to stimulate and

support research on physiologic effects and health benefits of foods and food

components

Due to the complexity of food ingredients and bioactives to achieve the above

microencapsulation has been reinvented to meet such challenges In the past

microencapsulation has been used to mask the unpleasant taste of certain ingredients

and also to simply convert liquids to solids However in recent years the concept of

controlled release of the encapsulated ingredient at the right place and the right time

has been fundamental in the development of functional foods A food can be said to

be functional if it contains a component that benefits one or a limited number of

functions in the body in a targeted way that is relevant to either the state of well-

being and health or the reduction of risk of a disease (Bellisle etal 1998) or if it has

physiologic or psychologic effect beyond the traditional nutritional effect

(Clydesdale 1997)

The health benefit of a functional food would be limited if the food is not part of the

diet hence the development of probiotic dairy products like yoghurt (Kailasapathy

2006) dairy fruit drink (Shah 2000) and cheese (Kourkoutas et al 2006) among

others Besides milk or dairy products (eg cheese) already contain biologically

active peptides which are of particular interest in food science and nutrition because

they have shown to play physiological roles including opioid-like features as well

as immunostimulating and anti-hypertensive activities and ability to enhance

calcium absorption (Meisel amp Fitzgerald 2003) In essence milk and dairy products

are functional foods in their own right Both lipid-soluble (eg vitamin A b-carotene

vitamins D E and K) and water-soluble (eg ascorbic acid) vitamins can be

encapsulated using various technologies (Thies 1987 Kirby 1991) The most

common reason for encapsulating these ingredients is to extend the shelf-life either

by protecting them against oxidation or by preventing reactions with components in

the food system in which they are present

The most promising target for functional food science has been the gastrointestinal

tract (GIT) functions associated with a balanced colonic microflora mediated by the

endocrine activity of the GIT dependent on the tractrsquos immune activity and

modulators of cell proliferation (Roberfroid 1998) To achieve such delivery food

grade polymers like calcium alginate-starch (Sultana et al 2000) alginate-chitosan

(Iyer et al 2004) and せ-carrageenan and locust bean gum have been used among

others Most of these studies demonstrate the protective effect of microencapsulation

by the survival of the high number of cells reported as they transit through the GIT

particularly the acidic stomach and delivery to the lower part of the intestine in a

viable state to colonise and confer beneficial probiotic effects

In this study a similar approach was taken to screen food grade polymers for folic

acid encapsulation efficiency followed by optimisation of the encapsulation

parameters Among the polymers selected for screening alginate and pectin emerged

with the highest encapsulation efficiency

Shrestha et al (2003) reported using edible coating materials with alginate and pectin

films among them to coat rice with folic acid Low methoxy pectin retained more

folic acid than other edible coating polymers including alginate The drawback of

this approach is the spraying of folic acid onto the product In the case of Cheddar

cheese higher moisture levels would cause off-flavours during proteolysis at some

stage during aging (Saldo et al 2000) Direct addition to a product like what

happens during industrial fortification of breakfast cereals is suitable for such

products which can be subjected to high spray-drying temperatures and still retain

their integrity such temperatures would be detrimental for cheese quality Such high

temperatures could inhibit the action of residual starter cultures as well as the

proliferation of non-lactic acid bacteria during ripening

It is often difficult using only food-grade ingredients to achieve a barrier which is

good enough to prevent these water-soluble compounds like folic acid from leaking

away into the food system The most obvious way to encapsulate these compounds is

by spray-cooling and spray-chilling These techniques involve dispersing the water-

soluble ingredient in a molten fat or wax and spraying this dispersion through heated

nozzles into a chamber at ambient temperature (spray-cooling) or at refrigeration

temperatures (spray-chilling) If the chamber is at room temperature the

encapsulation material has a melting point between 45 and 122degC If the chamber is

cooled materials melting at 32ndash42 degC can be used (Thies 1987) The microcapsules

are insoluble in water and will release their contents when the temperature of the

food product is raised above the melting temperature of the fat or wax Neither of the

two techniques would be appropriate for Cheddar cheese because the cheese is

always consumed ambient temperature while the lower melting point range of 32-42

degC may not guarantee complete release in the GIT If fat is used instead of the wax

this would be contrary to current world trend of cutting back on fat intake due to an

increase in obesity and related illnesses

Folic acid is a member of the vitamin B family and essential for the healthy

functioning of a variety of physiological processes in humans Chemically the

folates are a group of heterocyclic compounds and have a common basic structure

N-4 [(2-amino-1 4-dihydro-4-oxo-6-pteridinyl)-methyl amino] benzoyl] glutamic

acid with or without additional L-glutamic acid residues conjugated via peptide

linkages (Hawkes and Villota 1989b) Folic acid also known as pteroylglutamic

acid is composed of a pteridine ring p-amino benzoic acid (pABA) and glutamate

moieties Separately the three moieties have no vitamin activity

Most naturally occurring folate derivatives in foods are highly sensitive to such

parameters as oxygen temperature pH and light and thus their stability is affected by

food processing conditions With an increasing proportion of the world food supply

being subjected to some sort of processing and storage conditions investigations

have shown the effect on folates stability (Witthoft et al 1999) Most studies

demonstrate negative effects on folates stability from both industrial processing and

household preparation causing increasing losses with increasing severity of heating

temperature and time (Williams et al 1995 Wigertz et al 1997 Vahteristo et al

1998) The overall effect when considering the chemistry of natural folates is that

they are all unstable to a varying degree (Scott et al 2000) Folic acid where the

pteridine ring is not reduced is the cofactor produced synthetically by commercial

companies and the form found in supplements (tablets) breakfast cereals and flours

There is a need for extensive studies especially to develop new techniques for

enhancing folate content stability and bioavailability in food products Encapsulation

is an inclusion technique for confining a substance into a polymeric matrix coated by

one or more semi-permeable polymers by virtue of which the encapsulated

compound becomes more stable than its isolated or free form (Dziezak 1988

Arshady 1994) It has been used extensively to entrap drugs and bioactive

compounds and control their release into the GIT (Murata et al 1993 Polk 1994)

Microencapsulation using blended alginate and pectin was applied in this study as a

way of protecting folic acid from deteriorative reactions and adverse environmental

conditions which lead to loss of bioactivity The capsules were evaluated for

controlled release of folic acid in simulated acidic and alkaline gastric conditions

stability of folic acid during storage prior and ex-vivo behaviour of alginate-pectin

capsules in terms of folic acid release in porcine intestinal contents Results from

these experiments gave a good indication of the potential release of folic acid from

the capsules in the mice experiments reported in Chapter 6 The microencapsulation

and controlled release of folic acid using polymeric matrices has not been reported in

the reported literature

43 Aim

The aim of this study was to screen food-grade polymers for folic acid encapsulation

to enhance its stability and optimising the conditions for its encapsulation and

release

44 Objectives

1 To study the effect of folic acid encapsulation efficiency as influenced by

type of polymer calcium chloride (cation for gelling) concentration shape

and size of hydrogels cross-linking time and calcium uptake

2 Investigate the effect of different drying methods on the stability of the

3 The effect of various buffers to release the encapsulated folic acid in

simulated gastric conditions

45 Materials and methods

46 Preparation of polymer solutions and encapsulation procedure

Five different polymers were used in this study The type and concentration of the

polymers and calcium chloride as well as folic acid is described in chapter 31

47 Optimisation of encapsulation parameters

Various encapsulation parameters such as cross-linking time of hydrogels (0-180

min) shape and diameter single or mixed polymer and their effect on encapsulation

efficiency reduced leakage of folic acid were studied and the details are given in

sections 32 34 and 36

48 Selection of buffer for the release of folic acid

Three common buffers namely phosphate citrate and buffer TS were studied for

folic acid release by adding 5 g of alg-pect hydrogels into 100 ml of each buffer with

constant stirring The pH of the dissolution buffer was maintained at either ambient

temperature 30 degC or 37 degC An aliquot of the release medium (1 ml) was sampled at

30 min intervals from the respective buffers up to 240 min An equivalent amount of

fresh buffer pre-warmed at respective temperatures was replaced to keep the buffer

levels constant Collected samples were then analysed for folic acid content which

was plotted as a function of time The buffer selection studies were performed in

triplicates (n = 3) in identical manner

49 Effect of different drying methods on stability of encapsulated folic acid

The alg-pect hydrogels were evaluated for their ability to protect folic acid after air

freeze and vacuum drying and frozen storage for 11 weeks The procedure is detailed

in section 35

410 In vitro release of folic acid and swelling properties of alginate-pectin

hydrogels

The release of encapsulated folic acid from alg-pect hydrogels (7030) was studied at

pH values 12 (01M HCI) and pH 82 (phosphate buffer) simulating the stomach

and upper small intestinal pH environment respectively (Iyer et al 2004) Both

solutions were de-aerated prior to use while the temperature was maintained at 37 plusmn

05 ordmC Agitation was kept to the barest minimum throughout the whole procedure

At predetermined intervals 1mL of the fluid was sampled together with 1 g of

capsules for folic acid determination and swelling studies An equal volume of buffer

and acid was replenished after sampling at each interval The amount of folic acid

released was quantified using the TECRA method as described in 38 All

experiments were performed in triplicate and repeated twice The mean of folic

acid released plusmn sd is reported The capsules were removed and weighed after

carefully drying the surface water with absorbent paper towel The ratio of water

uptake was calculated as

All mass measurements of the swollen beads were taken on single pan balance

(Mettler AE 240S Switzerland) having an accuracy up to fifth decimal

Ratio of water uptake = (wet weight ndash dry weight)(dry weight)

411 Release of encapsulated folic acid from alginate-pectin hydrogels in ex-vivo

porcine gastrointestinal contents

Porcine stomach and intestinal contents were obtained from four freshly sacrificed

pigs (10-12 months old) at Wilberforce meats (Wilberforce NSW Australia)

Different sections of the gastrointestinal tract from the start of the stomach to the end

of the ileum were secluded by tying with a draw string to keep the contents separate

and placed in ice bath before transportation to the lab The contents of the respective

sections were manually squeezed out and rinsed with PBS into pre-cooled glass jars

Thereafter 8 X 20 ml from each jar was aspirated and incubated anaerobically in

test tubes with 1 g encapsulated folic acid capsules or an equivalent of free folic acid

in a water bath at 37 degC for 4 h to evaluate folic acid release and stability as well as

the behaviour of the capsules under such conditions Over the 4 h experimental

period 1 ml triplicate aliquots of the test tube contents were sampled at 30 min

intervals and assayed for folic acid as described in section 38

412 Folic acid measurement

Reagents and sample preparation as well as performance of folic acid assay from alg-

pect hydrogels to evaluate encapsulation efficiency cross-linking time in-vitro and

ex-vivo folic acid release leakage and stability after various drying treatments was

carried out according to section 38

413 Determination of calcium content in alg-pect hydrogels

Standard solutions and reagents

All standards were prepared by dilution with 045 M nitric acid The nitric acid was

prepared from 65 nitric acid diluted with Milli-Q water (gt182 M cm) Calcium

standard solutions (1 2 3 and 5 ppm) were prepared from a 1000 mg Lndash1 Ca standard

by dilution with 045 M nitric acid A 1 mg Lndash1 standard solution was used for

instrument optimization Two reference samples (Lyphocheckreg 62081 and 62082

BIO-RAD Laboratories ECS Division Anaheim CA USA) were used to evaluate

the accuracy of the total measurement of calcium using the atomic absorption

spectroscopy (AA-240FS Atomic Absorption flame emission spectrophotometer

Varian California USA)

Samples and sample preparation

Multiple alg-pect capsules (5 g wet-weight) were sampled every 20 min between 0

and 180 min from the calcium chloride reaction vessel during polymerisation and

acidified with 1 ml of 65 nitric acid and kept frozen (ndash20 degC) until analysis The

sample preparation step was simple Upon thawing 1 g of each of the acidified

capsules were diluted 10 times with 045 M nitric acid followed by addition of 5 ml

of 10 000 ppm lanthanum oxide (releasing agent) before direct aspiration into the

AAS instrument

Measurements

Before a measurement sequence was started the instrument was carefully mass

calibrated and optimized using a 1 mg Lndash1 Ca standard solution At the beginning of

every measurement sequence two blanks and four calcium standards (1 2 3 and

5 mg Lndash1) were measured in order to determine the background level and the detector

dead time Any possible drift in background level was monitored by the analysis of a

1 mg Lndash1 Ca standard for every six samples All sample solutions were analysed three

times in order to improve the precision of the final result Between samples a 2 min

wash with 045 M nitric acid was applied

414 Results

All of the selected edible polymers have wide application in food industry as binders

fillers and components of protective coatings among other uses In the current study

they were evaluated for folic acid encapsulation efficiency which varied widely with

the polymers (Table 41) The order of encapsulation efficiency for single polymer

was found to be in the order of alginate gt pectin gt iota-carrageenangtxanthan gum

gtgelatin Alginate showed an efficiency of 50 followed by pectin at 48 while

the remaining polymers showed less than 30 encapsulation efficiency A

substantial rise in the efficiency was observed when polymers were combined the

results were as follows alg-pectgtalg-carrageenangtalg-gelatingtalg-xanthun

gumgtpect-carrageenan (Table 41) The blended alg-pect gel capsules gave by far

the highest folic acid encapsulation efficiency and were therefore selected and

optimised for use throughout this study

The thickness of the capsules increased rapidly during the first 40 min of cross-

linking after which it levelled off at its maximum value (Fig 41) The gel capsules

cross-linked in 005 M calcium chloride showed little increase in diameter during

contact time whereas higher calcium chloride concentrations (01 M and 10 M)

increased quite rapidly over the same time

Blending alginate with pectin led to increased folic acid encapsulation efficiency up

to a maximum of 886 thereafter it started to decrease (Table 42) Further to this

the sphericity of the gel capsules was regular up to 20 pectin but thereafter became

irregular (Fig 42) However folic acid entrapment still increased beyond this pectin

ratio although it started to decline at 30 pectin at which folic acid measurements

were as inconsistent as they were variable

With the increase of pectin as well came the increase in wrinkled surface

morphology which was depressed compared to the smooth surface observed in

alginate only hydrogels Any increase in the pectin ratio beyond 40 resulted in

complete loss of sphericity in the hydrogels

To study the effect of buffer composition and temperature on folic acid release from

the hydrogels three common buffers namely citrate buffer phosphate buffer and

Buffer TS all at pH 82 were used There was a significant difference (plt005) in the

folic acid released in the phosphate buffer at all temperatures (ambient 30 degC and 37

degC ) compared to citrate buffer and Buffer TS (Fig 43) A slight increase in folic

acid release between ambient and 30 degC is evident However the 37 degC temperature

showed the highest release in all three respective buffers The alg-pect hydrogels

showed remarkable stability in acidic conditions (Fig 45) while they completely

released folic acid in alkaline conditions (Fig 44) This in-vitro release step was

designed to simulate gastric and intestinal conditions in the human gut The effect of

various drying techniques on folic acid retention during storage is shown in Fig 46

Combining vacuum and freeze drying shows a slightly higher retention than the two

techniques separately Air-drying showed folic acid loss despite the encapsulation

which points to the fact that appropriate drying is also a factor in retaining folic acid

encapsulated in alginate-pectin hydrogels

The ex-vivo porcine studies (Fig 47) reflect an important phenomenon in targeted

delivery that the hydrogels remained largely intact in gastric conditions but released

folic acid in both the small intestine and colon Folic acid was protected from the

deteriorative gastric conditions as shown in Fig 47

Table 41 Folic acid encapsulation efficiency using single and mixed polymers Polymer (225 cps) Encapsulation efficiency ()a

Alginate

Pectin

Xanthan gum

Gelatin

Iota-carrageenan

Alginate ndash pectin

Alginate ndash gelatin

Alginate ndash iota carrageenan

Alginate ndash Xanthan gum

Pectin ndash iota carrageenan

541 plusmn 00

486 plusmn 02

194 plusmn 50

150 plusmn 21

222 plusmn 04

794 plusmn 01

589 plusmn 04

591 plusmn 08

553 plusmn 06

538 plusmn 20

viscosity measured in centipoise a mean plusmn SD (n = 4)

folic acid encapsulated within the capsules

20 40 60 80 100 120 140

Time (min)

Fig 41 Effect of cross-linking time and calcium chloride concentration on capsule diameter

(alginate-pectin concentration A70P30) Calcium chloride 005M 01M

10M (Values are mean plusmn sd (n = 12))

Table 42 Encapsulation efficiency and shape of capsules loaded with folic acid in a

co-gelled matrix of alginate-pectin

a Equivalent concentration ratio total polymer concentration was maintained constant at

18 (ww) (n =30)

Fig 42 Photomicrographs of a) alg (X 40 magnification) and b) alg-pect (SEM) capsules

Type of capsule Characteristic

A60P40 A70P30 A80P20 AOP0

Folic acid encapsulated efficiency ()

Alginatepectin (ww) a

742plusmn092

irregular

sphere

886plusmn015

irregular

sphere

782plusmn019

regular

sphere

548plusmn02

regular

sphere

0 20 40 60 80 100 120 140 160 180 200 220 240Time (min)

0 20 40 60 80 100 120 140 160 180 200 220 240

Time (min)

Fig 43 Folic acid release a) at ambient temperature b) at 30 degC c) at 37 degC in

diamsphosphate buffer citrate buffer and Buffer TS at pH 82

0 20 40 60 80 100 120 140

Time (min)

Fig 44 Effect of pectin ratio on the release of alg-pect hydrogels in 01 M phosphate buffer

pH 82 alginate pectin - A100P0 ndashA70P30 Values are mean plusmn sd (n = 9)

0 20 40 60 80 100 120 140

Time (min)

Fig 45 Effect of the pectin ratio on the release of alginate-pectin hydrogels in 01 M HCI

(pH 12) alg- pect A100P0 A70P30 Values are mean plusmn sd (n = 9)

0 1 2 3 4 5 6 7 8 9 10 11

Time (weeks)

Fig 46 Effect of different drying techniques on folic acid retention in alginate-

pectin gel capsules ndash vacuum dried and freeze-dried ∆ vacuum dried diams

freeze dried air-dried free folic acid

0 30 60 90 120 150 180 210 240

Time (min)

Fig 47 Release of encapsulated folic acid in ex-vivo porcine gastrointestinal contents The

error bars represent plusmnsd (n=3) Symbols - small intestine ndash colon diams -

stomach

415 Discussion

Gelatin with a high carboxyl groups which makes it negatively charged was tested

for the encapsulation of folic acid with the expectation that the resultant hydrogen

bonds formed between the two would increase the encapsulation efficiency

However the results (Table 41) show that gelatin had in fact the lowest folic acid

loading of all the five polymers studied Whilst this property of gelatin is ideal for

the sustained release of basic or acidic protein (Tabata amp Ikada 1998) it is less than

ideal for folic acid This was most likely due to molecular repulsion between folic

acid and gelatin The charge introduced through the Encapsulator to prevent capsules

from agglomerating after they are formed appeared to have failed to alter gelatinrsquos

net charge The reduced isoelectric point of gelatin consistent with the net negative

charge it carries was of little consequence even in the presence of positively charged

glutamic acid moiety of folic acid

Further the gelatin capsules alone or in combination would be of limited use at a

later stage of Cheddar cheese making since a cooking temperature of up to 38ordmC is

reached since gelatin melts at 35-40 ordmC At this temperature gelatin behaves as

random coils which would lead to the loss of encapsulated folic acid due to the

disintegration of the capsular structure Since the vitamin is water soluble at this

stage of cheese making there is ubiquitous presence of water and so would be lost

into the whey Among other properties it had been selected for gelatin contains lt 1

methionine an amino acid used later in excess quantities in dietary formulations

for mice experiments to induce hyperhomocysteinemia

Other gums lacked desirable properties since the capsules formed were either soft

(xanthan gum) brittle (iota-carrageenan) and gave low encapsulation efficiency The

porosity of these capsules could be responsible for the low encapsulation

efficiencies Moreover the encapsulation efficiencies of water soluble drugs are in

general lower than that for slightly or insoluble drugs (Aslani amp Kennedy 1996)

This appears to be true also with folic acid

Xanthan gum is less polydisperse than most hydrocolloids Its natural state has been

proposed to be bimolecular antiparallel double helices and this appears not to favour

folic acid encapsulation It may form a very stiff intramolecular (single molecule

hairpin) double stranded helical conformation by the annealing of the less stiff

natural denatured elongated single stranded chains The weakly-bound network

formed is highly pseudoplastic as well as viscosity reducing considerably with shear

increase The rationale for this behaviour is that the hydrogen-bonded and entangled

association between the side chains of the highly extended molecules resist

dissociation Shear thinning with greater strain is mainly due to the conformation of

the side chains flattening against the backbone under shear so reducing the

intermolecular interactions This may well have led to the escape of folic acid into

the polymerising medium resulting in low encapsulation efficiency (Table 41)

All carrageenans are highly flexible polyemers which at higher concentrations wind

around each other to form double-helical zones Gel formation in す-carrageenans

involves helix formation with gel-inducing and gel-strengthening Ca2+

cations

Piculell (1991) reported that す-carrageenan has less specific ionic binding but

increased ionic strength which allows helices to form junction zones in soft elastic

gels The bonds therefore arise only from electrostatic interactions hence the

possible effects of monovalent ions are attributable to the level of impurities in this

polymer The three-dimensional structure of the す-carrageenan double helix has been

determined (as forming a half-staggered parallel threefold right-handed double

helix stabilised by interchain O2-HmiddotmiddotmiddotO-5 and O6-HmiddotmiddotmiddotO-2 hydrogen bonds between

the β-D-galactopyranose-4-sulfate units (Janaswamy amp Chandrasekaran 2002)

Incomplete formation of 1C4 3 6-anhydro-links has been reported to reduce the

extent of helix formation as the unbridged α-linked galactose residues may flip to the

4C1 conformation This not only retards helix formation but subsequently destabilises

aggregating linkages between the helices which leads to the non- formation of the

junction zones It is more than likely that this is what occurred in this instance

leading to such low folic acid encapsulation efficiency

The synergistic effect of alginate with these gums is very evident as well as its high

cross-linking potential yielding high encapsulation efficiency Although the nature of

the synergistic interaction between pectin and alginate in mixed gels is not fully

known it appears to be a specific association between specific chain sequences of

two polymers alginate poly-L-guluronate ldquoblocksrdquo and pectin poly-D-galacturonate

sequences of low charge density rather than based on incompatibility or exclusion

effects (Walkenstrom et al 2003) The interaction between alginate and pectin is

enhanced as the proportion of these sequences is increased Although the

conformation of individual chains is the same as in homotypic calcium-mediated

junctions the geometry of the interaction is quite different and instead of leaving

cavities capable of accommodating metal ions the near-mirror-image chains form a

close-packed nested structure (Janaswamy amp Chandrasekaran 2002)

This results in favourable noncovalent interactions between methylester groups of

pectin and the H-1 and H-2 of the polyguluronate It becomes apparent why alg-pect

capsules had the highest folic acid encapsulation efficiency compared with other

polymers Even before being combined alginate and pectin still had the highest folic

acid 54 and 48 respectively Naturally this points to their gelling mechanisms as

the most probable cause for such

The capsules are formed because blocks of guluronic (alginate) and galacturonate

(pectin) residues bind to cations resulting in a three dimensional network of strands

held together with ionic interactions The resultant network is a function of the

frequency and length of contiguous guluronic and galacturonate residues as well as

the concentration of the cation There is also the added advantage of the low charge

density sequences in both alginate and pectin which invariably tends to increase folic

acid presence in their matrices compared to the other polymers Simpson et al (2004)

reported that changes in frequency and length of the contiguous units alters the

overall strength of the gel and so does the changes in cation concentration on the

number of strands held together in the ldquoegg-boxrdquo model and thus alter the strength of

the capsule network This was confirmed by 01 and 05 M CaCl2 which resulted in

highest folic acid encapsulation efficiencies and the shortest hardening times

respectively

Encapsulation efficiency was used as the basic selection criteria for any of the

polymers for further study consequently alginate and pectin were selected while

xanthan gum gelatine and す-carrageenan were studied no further To optimise the

parameters affecting the making of microcapsules various factors were evaluated

sodium alginate concentration calcium chloride concentration the contact time with

polymerising agent (calcium chloride) and incorporation of pectin The relationship

between cross-linking time and capsule diameter at different calcium chloride

concentrations is shown in Fig 41 Alginate pectin (7030) blend was tested in

concentrations of the cationic solution fixed at 005 01 and 10 M wv calcium

chloride

Generally the diameter of the capsule increased (300 to 650 μm) with increasing

cross-linking time and then remained constant irrespective of the concentration of the

alginate-pectin and calcium chloride In principle this is in agreement with the

results reported by Blandino et al (2001) despite the fact that they used sodium

alginate only Liu amp Krishnan (1999) also found a similar trend even though they

studied encapsulation of drugs The degree of cross-linking is dependent on both the

concentration of the calcium chloride and the contact time in this solution The

thickness of the capsules increases rapidly within the first 40 min of the process (Fig

41) After this stage the thickness of the capsules levels off at its maximum value

All these results can be explained by taking into consideration the capsule formation

process which is assumed to be controlled by the diffusion of the components

involved in it In this regard the fact that the metallic cation has a smaller size than

the two polymer molecules means that it is mainly the cation that diffuses between

the alginate and pectin chains binding to unoccupied binding sites on the polymers

first on the alginate and then pectin Thus once the polymer mix hits the cationic

solution the capsule starts to form instantaneously and grows along the flux direction

of the Ca2+

It is logical to assume that the gelling process starts on the outside of the capsule

towards the core since at first instance the nearest gelling zones are on the external

When the Ca2+

ions reach the core gelling zones and saturate them the gelation

process is completed In the initial moments of the capsule formation process all the

binding sites for Ca2+

ions present in the alginate and pectin chains are unoccupied

so cations can bind rapidly to the mixed polymers However when diffusing through

a gel that has already formed on the outside of the capsule where all the binding sites

are occupied there is no opportunity for Ca2+

ions to bind until it reaches available

sites further towards the core in the gelling zones Thus calcium ions must diffuse

through the gel to react with alginate and pectin during the formation of capsules

This means that the maximum growth of the capsule is realised within the first 40

min of the process where resistance to diffusion caused by the gel is not significant

Pectin added in experimental quantities had little impact on cross-linking time except

for a slight hardening delay probably caused by pectinrsquos slower gelling with calcium

compared to alginate (Bodmeier amp Paeratakul 1989) This delay seems to be useful

in the co-gelled matrix for folic acid retention since as shown later high pectin

levels are consistent with folic acid retention only up to a certain level after which it

becomes erratic due to irregular and inconsistent shape of the capsules The main

features of the gel capsules observed are summarised in Table 42 The shape was

greatly affected by the amount of pectin in the polymer matrix The capsules lost

their spherical shape as a consequence of increased proportion of pectin The high

alginate capsules showed a more regular spherical shape while those with more

pectin (A70P30 and A60P40) showed irregular shapes

The average of amount of encapsulated folic acid and loading efficiency after the

encapsulation process are shown in Table 42The effect of calcium chloride

concentration on capsule formation kinetics was studied by fixing the anionic

solution at 01 M On increasing calcium chloride concentration the thickness of the

capsules increased at a given gelation time This result can be explained by the fact

that an increase in the calcium ions results in a larger concentration gradient between

the outside solution and the core

The time required to obtain maximum capsule thickness is considerably longer when

calcium chloride concentration exceeds 01 M This result confirms that the capsule

thickness increases continuously until complete saturation of the gelling zones It is

noteworthy that neither the difference in capsule thickness nor the length of the

gelling time had any effect on the folic acid loading efficiency suggesting that the

initial process is very rapid Generally loading of any substance depends on its

solubility and the hardening time allowed Increasing calcium chloride concentration

from 005 to 01M increased folic acid loading from 25 to 53 in all the capsules

The initial loss of the vitamin loaded in all capsules could be due to high water

solubility and rapid diffusion of folic acid through the weakly cross-linked capsules

The entrapment efficiency did not increase with increasing cross-linking time (60 to

180 min) for any capsules including A70P30 and A60P40 which had the highest

efficiencies throughout It is clear that increasing calcium chloride concentration up

to 01 M produces capsules with higher levels of calcium ions and increased

entrapment efficiency Consequently the cross-linking of the polymer and

compactness of the formed insoluble matrices also increased

It was also found that further increase in the concentration of calcium chloride (up to

10M) did not enhance folic acid loading These results are in agreement with Takka

et al (1998) and Mirghani et al (2000) Greater loading efficiency was obtained as

the concentration of pectin increased up to 30 thereafter it dropped showing that

the optimum concentration for a robust capsule had been achieved at this

concentration

The cross-linking of both alginate and pectin with calcium ions is explained by the

ldquoegg boxrdquo gelation where calcium holds the hydrocolloids strands together The

release of folic acid from this matrix is based on the successful destabilisation of the

ldquoegg boxrdquo model It is the combined effect of alkaline conditions and presence of the

much more reactive phosphate ions that appears to cause the disintegration of the gel

capsules This can be seen in all three test temperatures where the phosphate buffer is

used Further confirmation is shown by the citrate buffer which has got almost three

times less phosphate (based on disodium hydrogen phosphate and potassium

dihydrogen phosphate used in buffer preparation) than the phosphate buffer A strong

correlation based on phosphate ions presence and release of folic acid is evident On

the other hand folic acid release using the Buffer TS was lowest at all three

temperatures but also no phosphate containing material makes up this buffer The

release of folic acid by the gel capsules at 37 degC and in an alkaline environment

makes them suitable for folic acid delivery into the small intestines

Free folic acid degraded rapidly followed by air dried but encapsulated folic acid

Inappropriate drying of the capsules may lead to loss of the vitamin However it

should be noted that even with just air drying encapsulation offered protection to the

vitamin The protection offered by encapsulation has been reported for probiotic

bacteria (Kailasapathy 2006) enzymes (Caruso et al 2000) and drugs (Sairam et al

2006) to mention just a few The traditional drying techniques for such sensitive

materials aided the alginate-pectin hydrogels in protecting folic acid from loss of

vitamer activity The combined vacuum and freeze drying offered minimal benefit

compared to either of the two techniques individually It should be noted that

appropriate drying of the alginate-pectin gel capsules is fundamental to the ultimate

effectiveness of encapsulation to protect the vitamin against degradation during

storage The stability improved dramatically compared to just air dried capsules

Overall after 11 weeks of storage at 4ordmC freeze-dried capsules retained greater

amounts of folic acid than air-dried capsules This is due to uniform vacuum drying

which maintains the integrity of the capsule throughout the freeze-drying process It

is reasonable to conclude that since air-drying was an uncontrolled process lower

folic acid retention was a direct result of this process

Although moisture levels were not determined freeze-drying reduces moisture

content much faster and to a greater extent than air-drying High moisture levels as

well as the longer air drying process may have had a negative effect on the capsules

ability to reduce oxygen permeation Alginate-pectin capsules with between 30 and

40 pectin (A70P30 and A60P40) subjected to freeze-drying enhanced folic acid

stability This was due to the rapid freeze drying process with little or no moisture

retained in the capsules after drying and possibly the exclusion of oxygen that is

known to contribute to folic acid instability among factors It shows that the capsules

can be very stable in food product application over a long period

The use of nitrogen during capsule formation had the added advantage of displacing

oxygen as well thereby augmenting the protective effect of the alginate-pectin

system

The folic acid release from capsules was studied at two pH values acidic [(pH 12)

(Fig 44)] simulating the gastric pH and pH 82 (Fig 45) simulating the intestinal

pH It is evident that release of folic acid from the capsules would be minimal in

acidic conditions such as in the stomach The alginate capsules showed faster release

than alginate-pectin capsules they were comparatively less dense (porous) This

porous structure (alginate hydrogels) is more degradable than the high density

structure (alginate-pectin hydrogels) therefore the release behaviour of folic acid

from the former was much faster than that of the latter due to the added pectin

The dissolution times for releasing 90 of folic acid from the capsules in 01 M

phosphate buffer pH 82 were in the range 80 to 120 min with increasing pectin in

the polymer group This is possibly due to the formation of a gel structure with a

greater number of cross-linkings Release in alkaline pH was gradual Alginate

capsules (A100) although relatively stable in acidic conditions were incapable of

preventing the release beyond 25 min at pH 82 The most notable difference was

obtained with alginate-pectin (A70) where the release was beyond 100 min this

could be attributed to the presence of pectin gel which is stronger and more stable

than alginate gel in acidic and alkaline conditions Because the capsules are used in

the swollen state the study of their swelling process is of utmost importance in order

to evaluate the suitability of encapsulation

It was observed that the microcapsules had swollen in phosphate buffer pH 82 more

than in 01N HCI The release would depend on the diffusion of folic acid through

the insoluble matrix of the polymer in 01N HCI Folic acid loss was greater in

alginate than alginate pectin at pH 12 There is a rapid increase in the weight of the

capsules in phosphate buffer (pH 82) up to a maximum value after which a slight

decrease in the water retention value is observed A swelling equilibrium value was

reached 120 min later after which erosion and breakdown of capsules occurred

Erosion could occur through degradation of the alginate and pectin backbones into

smaller molecular weight components In addition the ion exchange with phosphate

buffer causes erosion of the capsules which greatly increase the folic acid release

rate (Kumar et al 2002) These results suggest that dried capsules will swell slightly

in the stomach and as they are subsequently transferred to upper intestine the

particles will begin to swell more and behave as matrices for the sustained release of

the incorporated folic acid

Inconsistent results are reported in the literature with regard to the dissolution rate of

alginate capsules in acid medium Several investigators including Bodmeier amp

Paeratakul (1989) Liu amp Krishnan (1999) and among others have reported slower

release from calcium alginate capsules in acidic solution than in alkaline while

Oslashstberg etal (1994) have reported a faster release in acidic solution Our findings

were similar to results reported by the former (Liu and Krishnan 1999 and

Bodmeier and Paeratakul 1989) This could be attributed to the difference in the

chemical composition in the alginate used by the different research groups

Commercially available alginate comes in various grades that differ in the

guluronicmannuronic acid ratios and the level of free acid groups

The alginates used by us and the former (Liu and Krishnan 1999 and Bodmeier and

Paeratakul 1989) may have had a similar chemical composition and similar physical

properties Amidated pectin are more tolerant of pH variations and calcium levels

than conventional pectins making them useful in ileac or colonic delivery systems

(Munjeri et al 1997) In general the release mechanism from swellable hydrophilic

system containing different ratios of polymeric materials and a highly soluble drug

and in this instance vitamin is influenced by a number of parameters (Kim amp Fassihi

1997) These include the rate of fluid infusion into the matrix the rate of matrix

swelling and molecular diffusion of the vitamin through the swollen capsules

polymer relaxation and chain disentanglement non-homogenous gel microstructure

and dissolutionerosion

The jejunum is the site of maximum absorption of free folate where absorption

occurs by a pH-dependent carrier-mediated system (Mason 1990) It is

demonstrable from the in-vitro and ex-vivo porcine intestinal contents studies that the

alginate-pectin hydrogels released folic acid in alkaline conditions (Fig 44 and 47

respectively) For such a delivery to occur this suggests that the hydrogels have the

capacity to protect folic acid during their transit time through upper GIT and do

allow for its release where it is absorbed Considering the information available it is

reasonable to speculate that alginate-pectin gel capsules offer positive benefits for

targeted folic acid delivery Absorption of free folic acid in rats was observed to

occur more readily in the jejunum than in the ileum (Strum 1981) The disintegration

of gel capsules was not only due to a change in pH but also due to the presence of

the phosphate ions which destabilise the calcium ions previously holding the

polymers together It is reasonable to assume that the disintegration of the gel

capsules is multifactorial of which pH and phosphate ions play an important role

The folic acid release in the small intestinal contents as well as that of the colonic

contents follows almost a similar pattern except that the latter is greater (Fig 47)

These are two very different environments with perhaps a different effect on the

capsules despite a trend during release The presence of bacterial enzymes localised

in the colonic region may well have contributed to the disintegration of the gel

capsules leading to the release of folic acid Unlike in dairy mediums like yoghurt

and cheese the bacteria in this region of the gut do not utilise folic acid nor did it

lose its vitamer activity in the ensuing conditions The large intestine produces no

digestive enzymes mdash chemical digestion is completed in the small intestine before

the chyme reaches the large intestine The pH in the colon varies between 55 and 7

(slightly acidic to neutral)

This gives rise to the possibility that folic acid encapsulated in alginate-pectin gel

capsules can be used for the delivery of the vitamin in colonic cancer treatment

However further studies may be needed to carefully evaluate the actual transit times

passage across the ileo-caecal junction (ICJ) and factors affecting this transit Since

passage across the ICJ has been shown to be extremely variable (Chourasia amp Jain

2003) site specificity from a timed release dosage form would expected to be poor

The blending of alginate and pectin presents pectin which is refractory to host gastric

and intestinal enzymes (Sandberg et al 1981) but is almost completely degraded by

the colonic bacterial enzymes to produce a series of soluble oligogalacturonates

(Cummings amp Englyst 1987)

Controlled release of food ingredients at the right place and the right time is a key

functionality that can be provided by microencapsulation A timely and target release

improves the effectiveness of food additives broadens the application range of food

ingredients and ensures optimal dosage thereby improving the cost effectiveness for

the food manufacturer (Augustin et al 2001) Reactive sensitive or volatile

additives (vitamins cultures flavours etc) can be turned into stable ingredients

through microencapsulation With carefully fine-tuned controlled release properties

microencapsulation is no longer just an added value technique but the source of

totally new ingredients with matchless properties

416 Conclusion

The microencapsulation of the labile folic acid using a mixture of alginate and pectin

(A70P30) has the ability to protect it from adverse environmental factors that lead to

its degradation Overall alginate pectin combinations conferred greater folic acid

stability compared to free folic acid and alginate alone The capsules largely

remained intact in acidic environment but released completely in alkaline conditions

giving rise to the idea that they can withstand the stomach pH but release in the

alkaline small intestines where folic acid is absorbed Since all the parameters for the

capsule making process can be controlled this approach has the potential to become

an alternative method for folic acid delivery in foods and even the GIT

Chapter 5 Cheddar cheese and folic acid delivery

5 Evaluation of alginate-pectin gel capsules in Cheddar

cheese as a food-carrier for the delivery of folic acid

51 Abstract

Milk and especially fermented dairy products like yoghurt buttermilk and different

varieties of cheeses are already recognised as good dietary sources of folates

However the levels are affected by seasonal variation in milk appreciable losses

during processing and considerable reduction during storage This study was

undertaken to evaluate the applicability of encapsulated folic acid in the making of

Cheddar cheese as an alternative food vehicle for the delivery of folic acid

Alginate-pectin hydrogels were studied for their behaviour in a milk system with a

pH reduction from 67 to 45 over a 4 h period at 37 degC Stress tolerance of the

capsules was studied under simulated cheese press pressures for 4 h until a force of

0814 Ncm2 was detected Three stages in Cheddar cheese making were studied for

capsule distribution in cheese while folic acid stability was also evaluated during the

15 months ripening period Folic acid retention in alg-pect capsules was 100

indicating their ability to remain intact in a milk system while 80 retention was

recorded for the simulated cheese press pressures Encapsulated folic acid showed

more stability (100 ) in Cheddar cheese over the 15 months ripening than the free

folic acid (38 ) The alg-pect had notable stability in a milk system significantly

improved stress tolerance properties as seen by high folic acid retention during

cheese pressing and even distribution in a cheese matrix

This chapter is based on the publication Madziva H Kailasapathy K Phillips

M (2006) Evaluation of alginate-pectin capsules in Cheddar cheese as a food carrier

for the delivery of folic acid LWT Food Sci Technol 39 146-151

52 Introduction

Consuming milk and dairy products is a quick and convenient way of obtaining

significant amounts of protein and most micronutrients including calcium B-group

vitamins (particularly riboflavin and B12 but also thiamine niacin B6 and folate)

vitamin A iodine magnesium phosphorus potassium and zinc Unfortunately

instead of maintaining or increasing milk consumption there has been a shift away

from milk consumption in favour of carbonated beverages (Wells 2001)

Several reports on the nutritive value of cultured dairy products eg buttermilk and

yoghurt have reported that folate content of such milk products vary widely ranging

from 4 microg to 19 microg100g (Scott 1989 Renner 1983) Food composition tables based

on microbiological assays report total folate values of between 5 microg and 18 microg per

100 g for various fermented milk products (Swedish National Food Administration

1993 Moller 1996) Hopper and Lampi (1990) also reported that yoghurt and

buttermilk contain 47 microg and 97 microg of 5-methyl-THF100 g respectively The plain

yoghurt in their study consisted of a culture of Streptococcus salivarius ssp

thermophilus and Lactobacillus delbrueckii ssp bulgaricus which could

continuously alter the composition and concentration of folate Rao and Shahani

(1987) found that the total folate levels in skimmed milk fermented by L bulgaricus

decreased from 98 microg to 16 microg within 36 h of incubation while S thermophilus and

L acidophilus increased the total folate levels substantially to 19 microg100g Although

the concept of increasing folate levels in dairy products through ldquonaturalrdquo synthesis

using bacterial cultures has been discussed earlier it is worth mentioning that as late

as 2002 (Crittenden et al) only a six fold folate (72 ng g-1

) has been reported even

with a careful selection of microorganisms

This means that daily consumption of 100 g of such a product would contribute

approximately 15 of the minimum required folate intake but only 2 of

recommended consumption for women of child-bearing age

Reddy (1975) found that storage of yoghurt at 5 degC for 8 to 16 days resulted in

considerable reduction of the folate content Unripened soft cheeses for example

plain cottage cheese contain between 12 microg and 27 microg total folates per 100 g based

on microbiological assays and HPLC analyses Ripened soft cheeses like Brie and

Camembert have been reported to contain between 50 microg and 100 microg total folate

100 g probably due to the synthesis of folates by microorganisms during ripening

(Scott 1989) Most hard cheeses among them Edam Gouda and Cheddar have

been reported to contain 20 to 40 microg of total folates100 g Whey (liquid and cream)

have been reported to have between 2 microg and 12 microg total folate100 g

The manufacture of one kg of cheese requires 10 litres of milk During curdling the

water soluble material whey proteins and water soluble vitamins is separated from

the semi-solids of casein fats and salts The 5-methyl-THF concentration in whey

and cheese indicates that approximately 50 of the milk folates are lost in whey

Since milk folate binding protein (FBP) is also reduced by approximately half this

means that the 5-methyl-THF in the whey fraction could still be bound to FBP

Generally curdling due to rennet occurs at a pH of approximately 55 to 6 a

complete dissociation between FBP and bound folates occurs only below 35

(Wagner 1985) Variations of folate levels in cheese might be due to different cheese

starter cultures which either produce or utilise folates

Furthermore the higher concentration of 5-methyl-THF observed in cottage cheese

compared to hard cheese could also be explained by the addition of pasteurised

cream to the final product and the considerable amount of whey left in the product

after processing

Fortification with folic acid in one or more of the commonly consumed dietary items

is now regarded as the best method to ensure that increased folate intake reduces the

risks associated with folate deficiency Most of western countries including

Australia practice voluntary folic acid fortification Folic acid fortification of other

food products like juices and flours is being pursued elsewhere However no studies

have been reported yet regarding Cheddar cheese fortification Besides the careful

selection and combination of milk fermentation cultures and alternative food

preparation and storage as a way of increasing dietary folate only the study of FBP

has been suggested in literature examined so far There has been no mention of

microencapsulation at all and so this work seeks to expand that thinking to include

encapsulated folic acid in dairy products

In this study we investigated the possible application of alginate-pectin gel capsules

to encapsulate folic acid for use in Cheddar cheese making as an alternative medium

for delivery of the vitamin The microencapsulation technique was applied to protect

folic acid from deteriorative reactions and adverse environmental conditions thus

enhancing its stability which has hitherto contributed to low folate levels in dairy

products

53 Aim

The aim of this study was to investigate the ability of alginate-pectin hydrogels to

protect folic acid in a complex fermented food matrix like Cheddar cheese

54 Objectives

1) To study the behaviour of alginate-pectin hydrogels in a curdled milk system

in which pH is reduced to simulate pH reduction in cheese making

2) Study the distribution of hydrogels in Cheddar cheese when incorporated

at different times during cheese making

3) Study the effect of cheese ripening on the integrity of the hydrogels and

therefore the stability of the encapsulated folic acid

56 Folic acid capsules

The alginate-pectin gel capsules were prepared as described in 31 while folic acid

measurements followed the procedure in 38

57 Evaluation of gel capsules for mechanical strength

Previously made alginate-pectin gel capsules were studied for their mechanical

strength under simulated cheese press pressures as described in 39

58 Stability of gel capsules in milk

Capsules (1 g) were added to 100 ml of full cream milk previously flushed with

nitrogen to expel any oxygen The pH of the milk was adjusted from 67 to 45 over a

4 h period to simulate pH changes during Cheddar cheese making

The adjustment of pH was done using freshly prepared 01N HCI A free folic acid

control was also performed The temperature of the milk was maintained at 38 plusmn 05

ordmC Samples for folic acid measurement were taken every half hourly for the duration

of the experiment

different methods

In order to investigate the distribution of the gel capsules during cheese making 10 g

of alginate-pectin with 2 mgkg folic acid and 5 g of alginate-starch capsules were

incorporated at three different stages a) into the milk during cheese making b) after

milling the curd and c) injected into raw cheese block after overnight pressing

Cheese making was performed as described in 315 A hypodermic syringe fitted

with a ldquo19rdquo gauge needle was used to inject 05 ml of capsules at 05 cm intervals

until a depth of 55 cm was reached before turning the cheese block upside down and

repeating the procedure

The pressed curd was then sliced into 30 g horizontal slices and sprayed with iodine

solution The starch incorporated as a filler material during capsules preparation

stained blueblack The distribution of the stained capsules was visualised with an

Olympus-SZH-ILK fitted with a digital camera (Olympus Optical Co Tokyo Japan)

and a Fibreoptic Illuminator (model 15001 Fibreoptic Lightguides Melbourne

Australia)

510 Stability of encapsulated folic acid during cheese ripening

This procedure was carried out as detailed in section 312

511 Results

Compressive forces that simulate cheese press pressures were applied to the alginate-

pectin capsules to evaluate their stress tolerance properties using folic acid retention

as an indicator of leakage of folic acid from capsules (Fig51) Storing the hydrogels

in 10 (wv) sodium chloride solution for at least 24 h prior to applying cheese

press pressures showed a slightly higher folic acid retention compared to without

The difference in response to stress of the two treatments as measured by the Texture

analyser produced an identical mammography (Fig 52) Folic acid leakage was

rapid (1) within the first 7000 sec (2 h) before slowing down between 7200 and 21

000 sec No further increase was recorded thereafter

It is demonstrable that encapsulated folic acid has better stability in a milk system

than the free form (Fig 53) and the difference is significant (plt005) Encapsulated

folic acid was retained at 100 while 70 of the free vitamin was retained in the

240 min test period The reduction of pH from 67 to 49 resulted in the loss of

vitamer activity since any shift of pH in either direction from neutral has been

reported to cause loss of folic acid The stage at which the hydrogels are incorporated

during the cheese making process has a direct bearing on their distribution (Fig 54)

Incorporating the hydrogels in milk just before adding rennet ensured even

distribution with a cumulative mean of 15 062 plusmn 2 354 compared to 36 908 plusmn 29 395

for after milling or 98 233 plusmn 82 593 when injected (Table 6) Variation between the

top middle and bottom sections of the cheese in which hydrogels were incorporated

in the milk was not significant (Table 5)

0 30 60 90 120 150 180 210 240

Time (min)

Fig 51 Folic acid retention in alg-pect capsules during simulated cheese press pressures

with NaCl ordmwithout NaCl Values are mean plusmn sd (n = 9)

Fig 52 Mammography of the response of alginate-pectin microcapsules to simulated cheese

press pressures

0 7200 14 400 21 600 28 800

0 30 60 90 120 150 180 210 240

Time (min)

Fig 53 Stability of capsules in full cream milk with pH adjusted from 67 to 45 over 4 h

with 01N HCI alg-pect capsules frac14 free folic acid Values are mean plusmn sd (n=6)

Fig 54 Micrographs (Icirc 260 area ndash 782 336 pixels2) of capsules in Cheddar

cheese slices added in a) milk b) after milling the curd and c) injected after

overnight pressing of the curd

Table 51 Comparative distribution of capsules added in milk at 3 levels in Cheddar cheese

slices

Cheese section Distribution in pixels

Middle 1

Bottom 1

15 200 plusmn 2 014

15 055 plusmn 2 188

14 060 plusmn 2 361

15 711 plusmn 1 857

15 003 plusmn 2 330

15 052 plusmn 2 304

14 473 plusmn 2 695

15 380 plusmn 2 617

15 622 plusmn 2 821

a mean plusmn sd

Table 52 Comparative distribution of capsules incorporated at 3 different stages of Cheddar

cheese making

Stage of incorporation of capsules Cumulative mean

(sum of capsules in pixels 2)a

After milling the curd

Injected into curd after overnight

pressing

15 062 plusmn 2 354

36 908 plusmn 29 395

98 233 plusmn 82 593

a mean plusmn SD (n = 54)

0 05 1 15 2 25 3

Time (months)

Fig 55 Folic acid retention in alg-pect capsules () and free folic acid (frac14) during Cheddar

cheese ripening (n = 6) Values are mean plusmn sd

512 Discussion

Capsules stored in 1 sodium chloride prior to use retained 15 more folic acid

than those stored in Milli-Q water This difference is quite substantial in terms of

folic acid since microgram quantities are required for the physiological functions in

the human body De Boissenson et al (2004) reported that sodium chloride

strengthens intermolecular hydrophobic associations of the alkali chains which in

turn lead to a decrease in the swelling ratio as well as porosity and an increase in

elasticity in alginate capsules while Perez-Mateos and Montero (2002) reported that

it increases gel strength work of penetration and hardness This is over and above

the stability brought by ionic junctions These findings explain the higher folic acid

retention in capsules stored in sodium chloride

The alginate used in this study was high mannuronic which is known to be softer and

more elastic than the high guluronic one This allows for ease of integration of the

capsules into the soft curd during pressing which reduces folic acid leakage Whilst

the simulated pressures were applied directly onto the capsules during the test in

cheese making the curd absorbs most of the pressure which resulting in lesser force

reaching the capsules per unit area The result suggests that the capsules can

conveniently be applied in cheese making for folic acid delivery

The measurable free folic acid is continuously decreasing with an increase in time

and a drop in pH According to Lucey et al (1996) when milk is acidified from pH

67 to 20 the buffering capacity is maximal at about pH 50 At this pH colloidal

phosphate is totally solubilised and the ldquofreerdquo inorganic and organic phosphates can

be associated with H+ resulting in a buffering peak

This phenomenon may have caused the gradual degradation of the free folic acid

Oxygen is a factor known to enhance the degradation of folates we assume that it

contributed also to folic acid loss since it has been reported to yield a 6-methylpterin

under aerobic conditions that are acidic (Stokstad et al 1947 Maruyama et al

The pH reduction was carried out to test the integrity of the capsules in simulated

Cheddar cheese manufacturing conditions The alg-pect capsules retained their

integrity upon decreasing the pH conditions and therefore provided protection to

folic acid which would have been otherwise degraded by the acidic environment The

advantages of investigating the reaction in a milk system instead of a buffer system

is that it is more representative of the actual cheese making process and the results

can thus be explained in a more specific way

The distribution of capsules in the cheese was evaluated based on when the capsules

were incorporated during cheese preparation at the start of the process with other

ingredients after milling the curd or after overnight pressing Incorporation of

capsules in the milk resulted in even distribution per unit area of cheese (Fig 54a)

while poor distribution was recorded when incorporated after the milling stage

(Fig 54b) and injection into the pressed block of raw cheese (Fig 54c) Capsule

clusters were quite evident in the latter two tests due to the fact that very little mixing

(Milling ndash 5 min) or none at all (injection) was involved making them unsuitable for

cheese application where inert materials like capsules are used Some cheese slices

from the milling stage had no capsules on them whatsoever further illustrating how

irregular the distribution was

This is in contrast with 55 min of stirring when capsules were added to the milk All

the results (Table 51) indicate a very even distribution of capsules with no

significant difference at 95 confidence interval between the top middle and

bottom slices of the cheese where capsules were added in the milk

This is in contrast with the other two options where the mean and standard deviation

are so large indicating less than even distribution (Table 52) The capsule

distribution in milk (Fig 54a) was then used in the ongoing research to fortify

Cheddar cheese with folic acid Encapsulated folic acid showed more stability in

Cheddar cheese over the 3 months ripening than the free folic acid (Fig 54) The

retention is 100 for the capsules and only 38 for free folic acid Up to 37

folic acid was detected in the whey after overnight cheese pressing for free folic acid

while the drop in pH to 54 may have altered the folic acid structure thereby making

it undetectable by the TECRA enzyme protein binding protocol

This results in a biologically inactive form if any one of the three folic acid moieties

is lost Paine-Wilson and Chen (1979) have reported rapidly decreasing stability of

folic acid with increasing alkalinity or acidity The stability is pH dependent In

neutral solution is it quite stable but instability increases with a shift in pH in either

direction

513 Conclusion

The combination of alginate and pectin polymers resulted in capsules with high

encapsulation efficiency notable stability in a milk system significantly improved

stress tolerance properties as seen by high folic acid retention during cheese pressing

and even distribution in a cheese matrix Encapsulated folic acid has excellent

stability in capsules during cheese ripening than free folic acid These results suggest

that Cheddar cheese may be an effective medium for folic acid delivery particularly

if alg-pect capsules are used

Chapter 6 Bioactivity of encapsulated folic acid

6 Effect of encapsulated folic acid dietary supplementation on

methionine-induced hyperhomocysteinemia in mice

61 Abstract

Homocysteine is located at the fork of two pathways in the metabolism of

methionine which relies on coenzymes derived from vitamin B12 folate and vitamin

B6 Mildly elevated total plasma homocysteine is an independent risk factor for

cardiovascular diseases To quantify the maintenance of bioactivity of encapsulated

folic acid the gel capsules were incorporated into a diet with excess dietary

methionine using Cheddar cheese as the food carrier and fed to mice Male Balbc

mice were fed 6 experimental diets with varying methionine content for 12- weeks to

induce hyperhomocysteinemia Supplementation of the diet with 10 gkg and 20 gkg

methionine increased plasma homocysteine to 18 and 61 times the control

respectively The hyperhomocysteinemia caused by the dietary addition of 10 gkg

methionine was completely counteracted by the encapsulated folic acid while free

folic acid showed a result two times higher Encapsulated folic acid caused

substantial reduction in plasma homocysteine and arterial lesions in mice fed the diet

supplemented with 20 gkg methionine than free folic acid Folic acid in its

encapsulated form reduced homocysteine levels and aortic pathological events far

more than free folic acid Alg ndash pect capsules enhanced folic acid stability which was

bioactive in sufficient enough quantities to normalise plasma total homocysteine

levels when Cheddar cheese was used as the food carrier

This chapter is based on a manuscript submitted on 090606 Madziva HS Phillips M

Kailasapathy K Effect of encapsulated folic acid dietary supplementation on methionine-

induced hyperhomocysteinemia in mice Am J Clin Nutr corrected proof article in press

62 Introduction

Less than optimal folate nutrition has been implicated as a risk factor in a number of

negative health outcomes including congenital neural tube defects (NTD)

(Wenstrom et al 2001 Kapusta et al 1999) anaemia during pregnancy (Scholl amp

Johnson 2000) low infant birth weight (OrsquoConnor 1994) colorectal cancer and

cardiovascular disease (Kim 1999) In addition a growing body of sound

epidemiological evidence has shown a strong association of elevated homocysteine

with incidence of vascular disease (Bautista et al 2002 Homocysteine Studies

Collaboration 2002 Wald et al 2002) The association remains strong even after

adjustment for major determinants of homocysteine such as age and renal function

folate vitamin B12 and vitamin B6 status suggest that homocysteine is an

independent risk factor for occlusive vascular disease (Cleophas et al 2000 Ueland

et al 2000) Elevations of plasma total homocysteine (tHcy) may occur due to

genetic defects and or an inadequate status of folate and its cofactors The data

provide the basis for a compelling if still controversial hypothesis that elevated

blood homocysteine is a cause of vascular disease This hypothesis has engendered

great interest because of the possibility that lowering blood homocysteine through

nutritional interventions might prove to be a safe and effective means of reducing

associated risk of disease

Homocysteine (Hcy) is an amino acid intermediate formed during the metabolism of

methionine (Fig 22) Hcy can be metabolized via two major pathways namely

remethylation pathway and transsulfuration pathway In the remethylation pathway

Hcy can be converted to methionine catalyzed by methionine synthase with folate as

a co-substrate or catalysed by betaine-Hcy methyltransferase using betaine as a co-

substrate

In the transsulfuration pathway Hcy is irreversibly converted to cystathionine by

cystathionine β-synthase (CBS) Factors that perturb the steps in Hcy metabolic

pathways can cause an increase in cellular Hcy levels and lead to its elevation in the

blood (Refsum et al 1998 Kang et al 1992)

Hyperhomocysteinemia is regarded as an independent risk factor for cardiovascular

diseases It is defined as plasma or serum homocysteine (Hcy) levels higher than

15 たM (Refsum et al 1998) Abnormal elevations of plasma Hcy levels up to 100ndash

250 たM have been reported in patients with severe hyperhomocysteinemia due to

genetic defects of enzymes that are involved in Hcy metabolic pathways (Refsum et

al 1998) Recent evidence suggests that hyperhomocysteinemia is also associated

with diseases that involve other organs In an early study McCully (1969) observed

extensive arteriosclerosis in a paediatric patient with severe hyperhomocysteinemia

and proposed a pathogenic link between elevated blood Hcy levels and

atherogenesis The necropsy also revealed moderately fatty liver

Subsequent investigations demonstrated an association between

hyperhomocysteinemia and liver disease Plasma Hcy levels are often elevated in

patients with liver cirrhosis or chronic alcohol-induced liver injury due to impaired

Hcy metabolism (Garcia-Tevijano et al 2001 Lu et al 2002) A recent study by

Woo et al (2005) demonstrated an early sign of hepatic steatosis (fatty liver) in

hyperhomocysteinemic rats They also reported that hyperhomocysteinemia caused

an activation of several transcription factors in the liver leading to increased HMG-

CoA reductase and cholesterol biosynthesis As a consequence hepatic lipid

accumulation and hypercholesterolemia occurred

Oxidative stress due to excessive generation of reactive oxygen species (ROS) has

been suggested as one of the important mechanisms for Hcy-induced cardiovascular

injury (Au-Yeung et al 2004) It has also been reported that hyperhomocysteinemia

could induce excessive superoxide anion generation and expression of inflammatory

markers as well as impairment of endothelium-dependent vessel relaxation (Woo et

al 2005) Superoxide anion is a reactive oxygen free radical that can rapidly interact

with nitric oxide (NO) to form highly reactive peroxynitrite a potent oxidant that can

cause tissue damage

Folic acid is a synthetic form of folate that is a water soluble B vitamin The active

metabolite of folic acid is 5-methyltetrahydrofolate that facilitates the remethylation

of Hcy to methionine Oral folic acid supplementation has been shown to reduce

plasma Hcy levels as well as improve the endothelial function in individuals with

mild hyperhomocysteinemia (Woo et al 1999)

In earlier work we reported a microencapsulation method (Madziva et al 2005) for

folic acid using food grade polymers alginate and pectin to increase its stability

since like all folates it is in danger of oxidative degradation which is enhanced by

oxygen light heat and a shift in pH either way from neutral The protective effect of

the capsules and their applicability was evaluated in Cheddar cheese as a food carrier

for the delivery of folic acid (Madziva et al 2006) Homocysteine is inversely

correlated with blood folate levels and taking folic acid either as a supplement or in

fortified food has been shown to lower total plasma homocysteine (tHcy)

concentration

Previous studies on folic acid supplementation and its effect on tHcy and vascular

diseases have focussed on folic acid incorporated only as a supplement (Naurath et

al 1995 Ambrosi et al 1999 Han et al 2005 Sarwar et al 2000) or fortified free

form (Green et al 2005) but not in its encapsulated form

In the present work the activity of encapsulated folic acid in Cheddar cheese on

homocysteine and the vascular consequences of hyperhomocysteinemia in the mouse

model were evaluated

64 Animals diets and treatments

The University of Western Sydneyrsquos Animal Care and Ethics Committee approved

all procedures used in this study (see Appendix 2) Thirty 6-weeks old male BALBc

mice (mean weight 215 plusmn 09) were obtained from Animal Resources Centre

(ARC) Perth Australia Mice were acclimated on a standard rodent diet

recommended by the ARC and fed ad libitum for 1 week They were systematically

assigned to six groups of similar mean body weights and fed for 12 weeks with the

control and experimental diets formulated on the basis of Sarwar et al (2000) with

vitamin free ethanol precipitated casein and the appropriate vitamin mix The control

group was fed Cheddar cheese without supplemental folic acid or methionine Four

diets were formulated to induce hyperhomocysteinemia as follows 20 gkg L-

Methionine + 2 mgkg free folic acid 20 gkg L-Methionine + 2 mgkg encapsulated

folic acid 10 gkg L-Methionine + 2 mgkg free folic acid 10 gkg L-Met + 2 mg

kg encapsulated folic acid in Cheddar cheese as the food carrier

A sixth methionine only diet (10 gkg) was used to evaluate the effects of natural

folates in Cheddar cheese All diets contained 1 sulphathiazole (10 gkg diet) a

nonabsorbed sulphur drug that inhibits folate formation by gut bacteria to ensure that

the animalrsquos only source of available folate is from the diet Mice were housed in

groups provided with free access to water with a 12-h light-dark cycle

65 Blood collection and homocysteine measurement

Mice were euthanized under CO2 anaesthesia Blood was collected by puncture of

the abdominal aorta into heparinised tubes and kept for lt1 h until plasma fractions

were separated Plasma was stored at -80 degC until further analysis Plasma

homocysteine (tHcy) concentrations were analysed using the reverse-phase HPLC

method of Araki and Sako (1987) with modifications as suggested by Gilfix et al

(1997) Briefly plasma samples were incubated with tris-carboxyethylphosphine to

reduce protein bound and oxidised forms of homocysteine followed by

derivatization with 7-fluorobenzofuran-4-sulphonic acid ammonium salt (SBD-F)

The fluorescent thiol derivatives were separated on a Waters C-18 column (5

micromolL 45 x 250mm) using isocratic elution (98 01 molL acetate pH 55 2

methanol) by means of a Shimadzu HPLC system (Tokyo Japan model SIL-10A

XL) complete with auto injector and fluorescence detector ndash RF-10A (excitation そ =

385 nm emission そ = 515 nm) Concentrations of tHcy were determined using an

external standard curve (inter- and intra-assay CV lt002) This is a commonly used

and well-supported method of assaying tHcy concentration (Pfeiffer et al 2000)

66 Histologic analysis of the aortic arch

Histologic changes were evaluated in the aortic arch of all 30 mice After

exsanguination the aortic arch was excised and perfused for 2 min with PBS

containing nitro-glycerine followed by perfusion with PBS containing 4

formaldehyde (3 min) before being fixed in 1 buffered formaldehyde The aortic

arch was then embedded longitudinally and cut into approximately thirty 4 microm

sections Four sections (20 microm apart) of a series of 10 sections which represented the

central area of the arch with an intact morphology of the complete arch were

analysed for lesions Serial slides were obtained and alternately stained with

haematoxylin-eosin-safranine for general observation Mason trichrome for

connective tissue and nuclear compounds and orcein for elastic tissue

Computerised morphodensitometric analysis of orcein-stained pathologic slides of

aortic arch was performed to evaluate the elastin content with the media and to give a

quantitative characterisation of elastic structure After selection of the zone of

interest the image was digitised on a 640 x 480 pixel frame using a normalised 256-

gray-level The analysis was carried out on a manually defined standardised

rectangular field whose major axis was a radial segment and whose width was fixed

at 100 microm Stained elastic elements were selected onto the image by interactively

setting a grey-level threshold Assuming homogeneity at staining the mean thickness

of each object was calculated as proportional to its mean residual grey level after

subtracting the background Images were taken using an Olympus DP70 digital

camera linked to an Olympus BX51 microscope (Olympus NY USA)

67 En Face Immunofluorescence staining

To detect monocytesmacrophages bound to the aortic

endothelium in vivo

immunofluorescence staining was performed

to detect the presence of

monocytesmacrophages according to the method by Dietrich et al (2000) The

thoracic aorta was isolated cut longitudinally and mounted on a glass

slide with the

endothelial side up Rabbit monoclonal antibodies against mouse ED-1 on the surface

of monocytesmacrophages (1100 Serotec) were added and the incubation was

carried out for 30 minutes The secondary antibodies for immunostaining were

fluorescein-conjugated (FITC-labeled) goat anti-rat immunoglobulin

antibodies

(Calbiochem-Novabiochem Corp NY USA) Monocytesmacrophages bound to the

aortic endothelium were identified with the use

of a fluorescence microscope

(Olympus DP70 digital camera linked to an Olympus BX51 fluorescence microscope

(Olympus NY USA)The number of monocytesmacrophages bound to the

endothelium was counted from 9 equally distributed sites on each aortic

segment

68 Results

Weight gain in mice fed the L-Met only (10 gkg) and L-Met (20 gkg + 2 mgkg free

folic acid) diet was significantly (Plt005) less during the 12 weeks of feeding than in

mice fed the control diet and encapsulated folic acid (Table 61)These differences in

growth were observed even though all the mice consumed identical quantities of

Severe hyperhomocysteinemia was induced by 10 gkg L-Met only (316 micromolL

homocysteine) as well as 20 gkg L-Met diet (25 3 micromolL) with free folic acid In

contrast the 10 gkg L-Met supplemented diet with encapsulated folic acid did not

increase homocysteine (70 micromolL) compared with the control (52 micromolL) while

free folic acid in the 10 gkg L-Met diet yielded twice the homocysteine (113

micromolL) In all instances encapsulated folic acid diet had cumulatively lower

homocysteine levels than free folic acid Plt005) Table 61 illustrates the tHcy

serum folate levels and weight gain after 12 weeks on the experimental diets

HPLC chromatograms of 20 gkg L-Met only homocysteine middle standard (20

ppm) and 20 gkg L-Met + 2 mgkg encapsulated folic acid are shown in Fig 61 To

determine whether the number of monocytesmacrophages present in the aortic

endothelium was increased in hyperhomocysteinemic

mice en face

immunofluorescence staining was performed with antibodies against ED-1 in freshly

isolated aortic segments

Occasionally cells positively stained with antibodies

recognizing ED-1 were observed on the surface of the aortic endothelium

isolated

from mice fed the control diet (Fig 62a) The number of ED-1ndashpositive cells present

in the endothelium of the aortas isolated from hyperhomocysteinemic mice was

significantly

higher than the number present in the control mice (Fig 62b)

indicating an increase in monocytesmacrophages in the aortic

endothelium in

hyperhomocysteinemic mice The number of ED-1ndashpositive cells bound to the aortic

endothelium was significantly reduced in mice fed the 20 gkg L-Met + 2 mgkg

encapsulated folic acid diet (Fig 62c) compared with mice fed 20 gkg L-Met only

diet The non-specific IgG did not result in positive staining in the aortic segment

(Fig 62d)

Dietary methionine enrichment significantly attenuated the increase in splitting and

fragmentation of elastic laminae smooth muscle cell hypertrophy and thickened

subendothelial space of the aorta beyond the baseline vascular pathology of control

mice and those with encapsulated folic acid (Fig 63) The 20 gkg L-Met + free

folic acid diet as well as the methionine only (10 gkg) resulted in nearly 2-fold

increase in lesion area compared with the control (lesion area was 25 293 plusmn 1054

microm2 vs 13 278 microm2 plusmn 0956 respectively Plt005) (Fig64) The encapsulated folic

acid only partially mitigated this increase despite completely normalising

homocysteine levels especially in the 10 gkg methionine diet

Table 61 Effects of free and encapsulated folic acid in a methionine supplemented diet on

growth and levels of plasma total homocysteine (tHcy) in male BALBc mice

Diet tHcy a

micromolL

Folic acid a

Weight gain

g12 wk

Control

10 gkg L-Met

10 gkg L-Met + 2 mg free folic acid

10 gkg L-Met + 2 mg encapsulated folic acid

52 plusmn 07

316 plusmn 22

113 plusmn 08

70 plusmn 09

253 plusmn 17

203 plusmn 07

790 plusmn 13

63 plusmn 34

207 plusmn 21

664 plusmn 09

98 plusmn 12

362 plusmn 11

273 plusmn 03b

218 plusmn 06c

237 plusmn 19d

265 plusmn 08e

226 plusmn 09f

252 plusmn 07g

a Mean plusmn SEM (n = 5) 6 replicates of pooled blood samples Values of weight gain (g12 wk) are

significantly different at Plt005 by ANOVA

Fig 61 HPLC chromatograms of a) 20 gkg L-Met only b) homocysteine standard and c)

20 gkg L-Met + 2 mgkg encapsulated folic acid

Fig 62 En face immunofluorescence staining of monocytes bound to the endothelium of mice aorta

Thoracic aortas were isolated from mice fed Cheddar cheese only diet (control a) 20 gkg L-Met + 2

mgkg folic acid (b) and 20 gkg L-Met only (d) ED-1ndashpositive cells were identified by fluorescence

microscopy at a magnification of x 200 Non-specific IgG was used as a negative control (d)

Photomicrographs are representative of 3 separate experiments Arrowheads point to ED-1ndashpositive

cells Results are expressed as mean plusmn SD (error bar) Plt005 compared with control values

Plt005 compared with values obtained from mice fed the 20 gkg L-Met only diet

Fig 63 Photomicrographs show the typical histological appearance of the aorta from

hyperhomocysteinemic (a and b) and control (c) mice illustrating (a) disruption of

elastic laminae (b) smooth muscle cell changes and endothelial cell hypertrophy

and (c) providing evidence that the stacking of the elastic laminae was preserved and

cellular hyperplasia and reorientation were prevented (H amp E X 40)

a b c d e fDiet

Aortic arch lesion area

a = Control

b = 10 gkg L-Met

c = 10 gkg L-Met + 2 mgkg encapsulated folic acid

d = 10 gkg L-Met + 2 mgkg free folic acid

e = 20 gkg L-Met + 2 mgkg encapsulated folic acid

f = 20 gkg L-Met + 2 mgkg free folic acid

Values of the area are means plusmn SEM n = 5 Means with superscripts without a common number differ

significantly Plt005

Fig 64 The effect of the six dietary regimens on the aortic lesion area in mice The lesions

increased in mice fed methionine only as well as 20 gkg-L Met and 10

et with free folic acid in comparison with controls and encapsulated folic

significantly

gkg-L M

acid Error bars represent standard errors

69 Discussion

his study was conducted to evaluate the extent of spontaneous homocysteine

ounteraction by folic acid under five broad conditions (i) 10 gkg L-Met (ii) 10

gkg free folic acid (iii) 10 gkg L-Met + 2 mgkg encapsulated

folic acid (iv) 20 gkg L-Met + 2 mgkg free folic acid (v) 20 gkg L-Met + 2 mgkg

encapsulated folic acid (vi) natural folates from Cheddar cheese These conditions

for rodents growth

according to Reeves et al (1993) which was provided by the non-

gkg L-Met + 2 m

enabled us to evaluate the effect of encapsulated folic acid on induced

hyperhomocysteinemia and the consequent arterial lesions against those of free folic

acid in this model using Cheddar cheese as the food carrier

The control diet together with the 10 gkg supplemental dietary methionine +

encapsulated folic acid supported optimum growth of mice (Table 61) and produced

the lowest tHcy levels confirming the nutritional adequacy of both diets The

recommended level of sulphur amino acids (methionine + cystine)

is 92 gkg

methionine supplemented experimental diet This means that the 10 gkg L-Met and

the 20 gkg L-Met provided a total of 192 (about two times the requirement) and

292 (about three times the requirement) gkg of total sulphur amino acids

respectively Increasing the dietary methionine to two times the requirement (10 gkg

supplemental L-Met) had no effect on growth especially with encapsulated folic acid

but a further increment to three times the requirement (20 gkg supplemental L-Met)

caused significant reduction in mice growth even with encapsulated folic acid (Table

The significant negative effects on mice growth obtained by feeding 20 gkg

supplemental methionine observed in the study confirms earlier reports that suggest

that methionine is one of the most toxic amino acids (Life Sciences Research Office

ing less vitamer activity in the former Even though folic acid is the most

table of all folates just like all of them is in danger of oxidative degradation which

yperhomocysteinemia induced by 20 gkg L-Met was partially counteracted by the

Methionine supplementation of up to 10 kkg or two times the requirement resulted

in a twofold increase in plasma tHcy (Table 61) in free folic acid than encapsulated

suggest

is enhanced by oxygen light heat and a shift in pH either way from 76 This results

in the splitting of the molecules into biologically inactive forms (British

Pharmacopoeia) The pH of Cheddar cheese ranges between 54-57 making it less

than ideal for stability of folic acid Rao et al (1984) have demonstrated that lactic

acid cultures do not only synthesise but also utilise folic acid which is consistent with

what has been reported by Crittenden et al (2002) and Lin and Young (2000)

Microencapsulation of folic acid protected it from such deteriorative reactions and

environmental conditions thus enhancing its stability and availability in sufficient

enough quantities to counteract the presence of homocysteine

2 mgkg folic acid and again encapsulated folic acid proving more effective for the

decrease of plasma tHcy than the free This further demonstrates the protective effect

of encapsulation making a strong case for possible application of this technique in

food systems for folic acid delivery

An increased plasma tHcy concentration is a strong independent risk factor for

arterial sclerosis and cardiovascular diseases (Duell amp Malinow 1997 Mayer et al

1996) It has been shown that hyperhomocysteinemia is also related to the incidence

f some other diseases such as Alzheimer dementia and cancer (Leboeuf 2003

onocytemacrophage adhesion to the aortic endothelium can be significantly

Seshadri et al 2002) Supplementation with folic acid and vitamin B12 is effecti

for lowering plasma tHcy (McKinley et al 2001 Brouwer et al 1999) and is

thought to be useful in preventing these diseases On the other hand it is critically

important to prevent the occurrence of hyperhomocysteinemia The current study

potently indicates that encapsulated folic acid makes a substantial contribution to th

prevention of these diseases through the avoidance of plasma tHcy elevation It

important to recognise that the alginate-pectin capsules released folic acid in

sufficient enough to counteract plasma tHcy which suggests that encapsulation wit

the two polymers did not interfere with folic acid vitamer activity or its release

It has been reported that diet-induced elevation of plasma Hcy levels stimulate

expression of chemokine (MCP-1) and adhesion (VCAM-1 and E-selectin)

molecules in the aortic endothelium (Dietrich et al 2000) As a consequence

elevated This is observable in Fig 62 Endothelium-dependent relaxation of the

aorta was impaired while there was also a significant increase in the adhesio

binding of monocytes to the endothelium of hyperhomocysteinemic mice The

adhesion of leukocytes including monocytes to arterial endothelium is a comm

feature linking the inflammation reaction and the development of early

atherosclerosis (Valente et al 1992 Takahashi et al 1994)

Increased monocytemacrophage binding and adhesion to the vascular endothelium

may represent an early feature of atherosclerotic development in

hyperhomocysteinemia Results obtained from the present study demonstrate for the

mia and are

ought to contribute to their premature vascular disease (Malinow et al 1993) In

an edematous and fibrous subendothelial space

ig 63) Ambrosi et al (1999) showed that feeding pigs a methionine rich diet for 4

first time that dietary incorporation of encapsulated folic acid using Cheddar cheese

as the delivery vehicle mitigates against hyperhomocysteinemia in

The concentrations in plasma tHcy recorded here ranging from 52 to 316 micromolL

are of the same kind as levels in patients with mild hyperhomocysteine

addition hyperhomocysteinemia developed in mice without significant changes in

serum lipid profile reflecting hypercholesterolemia Our findings reproduce the

situation encountered in young homocysteic patients whose serum lipid profiles

usually do not reveal hypercholesterolemia (Malinow et al 1993) and in whom

arteriosclerotic lesions contain little or no evidence of accumulated lipid (Fig 63) in

foam cells or extracellularly (Friggi et al 1989) The present results in experimental

hyperhomocysteinemic mice therefore are in accordance with the conclusions that

homocysteinemia could represent a non-lipid model for the initiation of

arteriosclerosis (Friggi et al 1989)

In methionine only (10 gkg) and 20 gkg + free folic acid homocysteinemic mice

showed hypertrophic cells covering

months induced hyperhomocysteinemia and atherosclerosis By and large methionine

supplementation with free folic acid resulted in hyperhomocysteinemia and lesion

acceleration however encapsulated folic acid resulted in less lesion (Fig 64)

The mice fed the control diet developed spontaneous lesions as shown by the white

bar The lesion area increased significantly in mice fed 10 gkg L-Met only A further

significant difference is apparent between encapsulated and free folic acid

s encapsulated form than free as seen by a reduction in homocysteine levels and

ents Alginate ndash pectin encapsulation of folic acid enhanced its

irrespective of the methionine levels in the diets Encapsulation not only preserves

folic acid bioactivity but the targeted release of the alginate-pectin capsules is also a

factor in the reduced lesion area Supplementation of the methionine rich diet with

encapsulated folic acid in Cheddar cheese successfully normalised plasma levels

610 Conclusion

The present results confirm the preventive therapeutic effect of folic acid more so i

aortic pathological ev

stability and was bioavailable in sufficient enough quantities to normalise plasma

tHcy levels when Cheddar cheese was used as the food carrier This makes a stron

case for possible application of this technique in food systems for folic acid delivery

Chapter 7 Conclusion

7 Overall conclusions

In the introduction section mention was made of the fact that all folates are in danger

of oxidative degradation enhanced by oxygen light sunlight oxidising and reducing

agents and heat resulting in a splitting of the molecule into biologically inactive

forms of which p-aminobenzylglutamate is one major form Their stability is also

pH dependent with the reduced folates being most stable at pH gt 8 and pH lt 2 and

least stable between pH 4 ndash 6 Further to this alternative strategies to fortification to

increase folates through starter culture selection and combination metabolic

bioengineering as well as improved storage and preparation methods have failed to

substantially yield high enough dietary folates Hence this thesis by exploring the

possibilities of a robust microencapsulation procedure for folic acid using food grade

polymers evaluating its behaviour in buffering systems and Cheddar cheese making

and animal feeding trials to evaluate the persistence folic acid was an important

alternative of delivering higher folic acid levels in cheese than reported so far

Around 2500 years ago Hippocrates first espoused the food as

medicine

the context of deficiency diseases Indeed by 1912 Casimir Funk

had put forward the

pellagra beri-beri and

rickets During the 1970s the shift in emphasis from

undernutrition to overnutrition and disease led to a flood of public health

guidelines

on optimising nutritional parameters By the 1990s with an ageing health conscious

Enrichment of flour and other staple foods with vitamins including folic acid in some

developing countries the US Canada while most European countries so far have

decided not to do so except for Hungary and Ireland has been probably the first

modern attempt to design a food for functional purposes related to

nutritional

outcome

One of the reasons for the inertia on folic acid fortification is the fear for adverse

effects Although folate is safe and almost free of toxicity there is concern that folic

acid (the synthetic form) may mask symptoms of vitamin B12 deficiency primarily

in the elderly population and may lead to progression of neurological symptoms

Whether or not to fortify foods with folic acid is a decision that is often discussed

principally in the context of neural tube defects occurrence From a scientific point of

view this may be wise since the evidence for the protecting effect of folates on NTD

birth prevalence is undisputed From a normative point of view it is obvious that

public health authorities should do good and follow the beneficence principle but the

difficulty of policy makers is that they have to balance the certainty of benefits to an

as yet unknown amount of risk to harm Thus the maleficence principle leads to the

discretion to move forward The precautionary principle thus leads to opposite

consequences avoiding potential harm of a fortification or supplementation as

initiated by governments but also avoiding that many people ingest adequate

amounts of folic acid that many children are born healthy and many elderly have

reduced risks of cardiovascular disease and cancer Delivering folic acid through alg-

pect capsules is a controlled process in which precise quantities are added and remain

stable over long periods This eliminates the overages approach adopted by certain

food manufacturers to ensure that the right quantities of folic acid are available in the

product at the time of consumption

It has been argued that a government policy to fortify implies a decision taken for an

entire population without asking for individual decision making and informed

consent In order to maximise social utility preventive strategies need to be chosen

that are effect and efficient

This study offers a less controversial alternative to the ramifications of mass use of

folate as a functional food By increasing the pool of folic acid fortified food

products this gives the consumer the power of choice Cheddar cheese like other

dairy products has bioactive peptides beneficial for health and so incorporating folic

acid into such a product expands its health benefits It has been demonstrated that

folic acid encapsulation is possible and is a controlled process The encapsulated

folic acid is stable during storage and after application in Cheddar cheese but above

all that it retains its bioactivity in sufficient enough quantities to influence plasma

homocysteine status and thus the risk for vascular disease in addition to diminishing

The overall aim of this study was to develop a robust microencapsulation protocol to

enhance folic acid stability and evaluate its bioactivity using animal studies by

inducing a known folic acid deficiency disorder It is demonstrable that the alginate-

pectin hydrogels besides offering the highest encapsulation efficiencies also had the

added benefit of protecting folic acid from the deteriorative gastric conditions as

shown by the results from in vitro and porcine ex-vivo studies Blending alginate and

pectin to a maximum of 30 of the latter shows that varying pectin content can be

used to control folic acid load The release of folic acid in simulated in vitro alkaline

conditions points to the fact that when ingested the delivery in the GIT is as targeted

as it is controlled

The free form of the vitamin had poor stability during storage cheese ripening as

well as reduced vitamer activity to completely counteract the effect of induced

hyperhomocysteinemia in mice In contrast encapsulated folic acid performed much

better under all test conditions This clearly shows that encapsulation is an effective

way to protect folic acid from adverse conditions and can be delivered in an active

form in a food vehicle like Cheddar cheese to reverse homocysteine elevation

Chapter 8 Future directions

8 Future directions

Folic acid encapsulation developed in this study demonstrates that

microencapsulation using food grade polymers confers stability to otherwise unstable

folic acid It is possible that other polymers may deliver higher encapsulation

efficiencies or even confer greater stability and so more research into the polymers to

increase the application base and variety is recommended Whilst coating of the

alginate-pectin gel capsules with poly-L-lysine to minimise folic acid loss during

cross-linking was trialled no further work was done due to lack of time Further

studies in this area is a worthwhile venture as this can also alter the mechanical

properties of the hydrogels thus further enhancing folic acid retention within the

hydrogels

There is a good indication that the capsules can be further modified to achieve

targeted release not just in the whole length of the small intestine but specifically the

jejunum where folic acid absorption is maximal This can be done by studying the

transit time of the current capsules in the upper gastro-intestinal section until such

time when folic acid release starts Such a study can be aided by use of a fluorescent

analogue of the synthetic folic acid Polymer ratios can then be adjusted to either

quicken or delay the release

Whilst an attempt was made to study the well known phenomenon of pectin-casein

interaction in fluid milk systems serious operational problems with fluorescein

isothiocyanate (FITC) staining were encountered with cheese The staining was as

inconsistent as it was as irreproducible between and among samples in the same

cheese batch What was of interest was the possible interaction between pectin in the

gel capsule and casein in the cheese and how this would impact on the release of

folic acid The results from the preliminary studies were inconclusive due to the

irregularity in casein-pectin staining between the raw cheese and cheeses at various

stages of maturation

About 30 gkg of calcium in cheese is in unbound form more work needs to be done

to establish any further interaction between the polymer gel capsules and the calcium

ions during cheese ripening

The fate of the alginate-pectin hydrogels during cheese ripening is not known It

would be of value to study the structural changes that may occur and relate them to

folic acid bioactivity To the best of our knowledge no studies have reported the

utilisation of folic acid by non-lactic acid bacteria and so challenge studies of these

bacteria with folic acid would give a good indication of what happens to the vitamin

were it to leak in later stages of cheese ripening when the barrier that protects folic

acid weakens

Chapter 9 References

9 Literature cited

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program Canberra Australian Food and Nutrition Monitoring Unit

Commonwealth of Australia

Alexandridou S Bachtsi A Kiparissides C (1999) Synthesis and characterization of

polymeric nanoparticles and microgels for drug delivery Proceedings 4th

International Symposium on ldquoPolymers in Dispersed Mediardquo Lyon France April

Alfthan G Pekkanen J Jauhiainen M Pitkaniemi J Karvonen M Tuomilehto J

Salonen JT Ehnholm C (1994) Relation of serum homocysteine and lipoprotein

(a) concentrations to atherosclerotic disease in a prospective Finnish population

based study Atheroscl 106 (1) 9-19

Alm L (1982) Effect of fermentation on B-Vitamin content of milk in Sweden J Dairy

Sci 65(3) 353-359

Ambrosi P Rolland PH Barlatier A Charpiot P Guisgand G Friggi A

Ghiringhelli O Habib G Bouvenot G (1999) J Am Coll Cardiol 34 274-279

Amouzou EK Chabi WN Adjalla EC Rodriguez-Gueacuteant MR Feillet F Villaume

C Sanni A Gueacuteant LG (2004) High prevalence of hyperhomocysteinemia

related to folate deficiency and the 677C T mutation of the gene encoding

methylenetetrahydrofolate reductase in coastal West Africa Am J Clin Nutr 79 (4)

619-624

Angier R B Boothe J H Hutchings B LMowat J H Semb J Stok5tad E L R

Subbarow Y Waller C W Cosuuch D B Fahrenbach M J Hultquist M E

Kuh E Northey E H Seeger D R Sickells J P Smith J M (1946) The

structure and synthesis of the liver L casei factor Sci 103 667-669

Araki A Sako Y (1987) Determination of free and total homocysteine in human plasma

by HPLC with fluorescence detection J Chromatogr 442 43-52

Arcot J Shrestha AK Gusanov U (2002) Validation of a competitive protein binding

assay kit for the analysis of folic acid fortified cereal foods Food Control 13 245-

Arneodo CJF (1996) Microencapsulation by complex coacervation at ambient

temperature FR 2732 240 A1

Arshady R (1993) Microcapsules for food J Microencap 10 (4) 413ndash435

Arshady R (1994) Methodology and nomenclature in microencapsulation Polymer

Preprints 35 63-64

Ashford M Fell J Attwood D Sharma H Woodhead P (1994) Studies on pectin

formulations for colonic drug delivery J Control Rel 30 225-232

Aslani P Kennedy RA (1996) Studies on diffusion in alginate gels I Effect of cross-

linking with calcium or zinc ions on diffusion of acetaminophen J Control Rel

42(1) 75-82 88

Augustin MA Sanguansri L Margetts C Young B (2001) Microencapsulation of food

ingredients Food Australia 53 220ndash223

Australian Bureau of Statistics (2005) - Apparent Consumption of Selected Foodstuffs

Australia Preliminary 43150 httpabsgovauAUSSTATS Accessed June 2005

Au-Yeung KK Woo CW Sung FL Yip JC Siow YL (2004)

Hyperhomocysteinemia activates nuclear factor-kappa B in endothelial cells via

oxidative stress Circ Res 94 28ndash36

Axelos MAV Thibault JF (1991) The chemistry of low-methoxyl pectin gelation In

Walter RH (Ed) The chemistry and technology of pectin Academic Press Inc

New York pp 109ndash118

Barrett DM Lund DB (1989) Effect of oxygen on thermal degradation of 5-methyl-

5678-tetrahydrofolic acid J Food Sci 54(1) 146-151

Bautista LE Arenas IA Penuela A Martinez LX (2002) Total plasma homocysteine

level and risk of cardiovascular disease a meta-analysis of prospective cohort

studies J Clin Epidemiol 55 882-887

Bellisle F Blundell J E Dye L Fantino M Fern E Fletcher R J Lambert

J Roberfroid M Specter S Westenhoumlfer J Westerterp-Plantenga M S (1998)

Functional food science and behaviour and psychological functions B J Nutr 80

(Suppl 1) S173-S193

Benech RO Kheadr EE Laridi R Lacroix C Fliss I (2002) Inhibition of Listeria

innocua in cheddar cheese by addition of nisin Z in liposomes or by in situ

production in mixed culture Appl Env Microbiol 68 3683ndash3690

Beristain CI Garcia HS Vernon-Carter EJ (2001) Spray-dried encapsulation of

cardamom (Elettaria cardamomum) essential oil with mesquite (Prosopis juliflora)

gum Lebensm-Wiss U-Technol 34 398ndash401

Beristain CI Vernon-Carter EJ (1995) Studies on the interaction of Arabic (Acacia

Senegal) and mesquite (Prosopis juliora) gum as emulsion stabilizing agents for

spray dried encapsulated orange peel oil Drying Technol 29 645ndash667

Bhandari BR Dumoulin HMJ Richard HMJ (1992) Flavour encapsulation of spray

drying Application to citral and linalyl acetate J Food Sci 51 1301ndash1306

Blakley R L (1969) The biochemistry of folic acid and related pterins North Holland

Publishing Co Amsterdam p143

Blandino A Maciacuteas M Cantero D (2001) Immobilisation of glucose oxidase within

calcium alginate gel capsules Process Biochem 36 601-606

Blenford D (1986) Fully protected Food Flav Ingred Packag Process 8 (7) 43ndash45

Bodmeier R Paeratakul O (1989) Spherical agglomerates of water-insoluble drugs J

Pharm Sci 78 964-967

Booth C Clark T Fenn A (1998) Folic acid riboflavin thiamin and vitamin B-6 status

of a group of first-time blood donors Am J Clin Nutr 68 1075-1080

Bottiglieri T (1996) Folate vitamin B12 and neuropsychiatric disorders Nutr Rev 54(12)

382-390

Botto LD Moore CA Khoury MJ Erickson JD (1999) Medical Progress neural-tube

defects N Eng J Med 341(20)1512

Boushey CJ Beresford SA Omenn GS Motulsky AG (1995) A quantitative

assessment of plasma homocysteine as a risk factor for vascular disease probable

benefits of increasing folic acid intakes JAMA 274 1049-57

BowerC RyanA RudyE MillerM (2002) Trends in neural tube defects in Western

Australia Aust N Z J Public Health 26 150-151

Brannon-Peppas L (1993) Controlled release in food cosmetic industries In MA El-

Nokaly DM Piatt DA Charpentier (Eds) Polymeric delivery systems American

chemical society Washington DC (ECS Symp Ser Vol520)

Brattstroumlm L Wilcken DVL (2000) Homocysteine and cardiovascular disease cause or

effect Am J Clin Nutr 72 (2) 315-323

British Pharmacopoeia Her Majestyrsquos Stationary Office London (p 616)

Brouwer IA van Dusseldorp M Thomas CM Duran M Hautavast JG Eskes TK

Steegers-Theunissen RP (1999) Low-dose folic acid supplementation decreases

plasma homocysteine concentrations a randomised trial Am J Clin Nutr 69 99-

Burns MA Kvesitadze GI Graves DJ (1985) Dried calcium alginatemagnetite

spheres a new support for chromatographic separations and enzyme immobilization

Biotechnol Bioeng 27 137ndash145

Canadian Government (1998) Canada Gazette Part II 32(24)

Canon K (1984) Electrostatic image development toners Japanese patent no 59 170853

Carson NA Cusworth DC Dent CE Field CM Neill DW Westall RG (1963)

Homocystinuria A new inborn error of metabolism associated with mental

deficiency Arch Dis Child 38425-436

Caruso F Dieter T Mohwald H Renneberg R (2000) Ezyme encapsulation in layer-

by-layer engineered polymer multilayer capsules Langmuir 16 1485-1488

Centers for Disease Control and Prevention (1992) Recommendations for the use of folic

acid to reduce the number of cases of spina bifida and other neural tube defects

MMWR Recomm Rep 41 1-7

Cha DS Cooksey K Chinnan MS Park HJ (2003) Release of nisin from various

heat-pressed and cast films Lebensm-Wiss U-Technol 36 209ndash213

Champagne CP Baillargeon-Cote C Goulet J (1989) Whey fermentation by

immobilized cells of Propionibacterium shermanii J Appl Bacteriol 66(3) 175-

Chan A Pickering J Hann E A Netting M Buford A Johnson A Keane RJ

(2001) Folate before pregnancy the impact on women and health professionals of a

population-based health promotion campaign in South Australia Med J Austral 174

631-636

Chen TS Cooper RG (1979) Thermal destruction of folacin effect of ascorbic acid

oxygen and temperature J Food Sci 44(3) 713-716

Chin-Cheng L Shan-Yang L Sun-Hwang L (1995) Microencapsulation of squid oil

with hydrophilic macromolecules of oxidative and thermal stabilization J Food Sci

60 36ndash39

Choumenkovitch SF Selhub J Wilson P W F Rader JI Rosenberg HI Jacques

PF (2002) Folic Acid Intake from Fortification in United States Exceeds

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Clark ED (2000) Rapid calculation of polar molecular surface area and its application to

the prediction of transport phenomena 1 Prediction of intestinal absorption J

Pharm Sci 88 (8) 807 ndash 814

Cleophas TJ Hornstra N van Hoogstraten B van de Meulen J (2000) Am J Cardiol

86 1005-1009

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Cooper R G Chen T-S King M A (1979) Thermal destruction of folacin in microwave

and conventional heating J Am Diet Assoc 73 406-410

Cragan JD Roberts HE Edmonds LD Khoury MJ Kirby RS Shaw GM Velie

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Stevenson RE Dean JH (1995) Surveillance for anencephaly and spina bifida

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Surveill Summ 44(4) 1-13

Crittenden RG Martinez NR Playne MJ (2002) Synthesis and utilisation of folate by

yoghurt starter cultures and probiotic bacteria Intl J Food Microbiol 80 217-222

Cummings JH Englyst HN (1987) Fermentation in the human large intestine and the

available substrates Am J Clin Nutr 45(Suppl 5 ) 1243-55

Cuskelly GJ Stacpoole PW Williamson J Baumgartner TG Gregory JF III

(2001) Deficiencies of folate and vitamin B6 exert distinct effects on homocysteine

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Daly D Mills JL Molloy AM Conley Lee YJ Kirke PN Weir DG Scott JM

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neural tube defects Implications for prevention JAMA 274 1698-1702

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ScrimshawNS 2 (19) Tokyo United Nations University Press Food and nutrition

bulletin httpwwwunueduunupressfoodV192ebeginhtm Accessed January

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httpwwwdatamonitorcom~99f16a6f99eb4d8687a6839536225437~ Accessed

May 2005

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acid in liquid model systems J Food Sci 48(2) 581-585

De Boisseson MR Leonard M Hubert P Marchal P Stequert A Castel C Favre E

Dellacherie E (2004) Physical alginate hydrogels based on hydrophobic or dual

hydrophobicionic interactions Bead formation structure and stability J Colloid

Interf Sci 273(1) 131-139

de Walle H E van der Pal K M den Berg LTW Jeeninga W Schouten JSAG De

Rover CMBSE Cornel MC (1999) Effect of mass media campaign to reduce

socioeconomic differences in womens awareness and behaviour concerning use of

folic acid cross sectional study BrMed J 319 291-292

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on Health and Social Subjects no 50 London The Stationary Office

Department of Health and Human Services Public Health Service Food and Drug

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proposed rule USA Fed Regist 58 53305-53312

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biosynthesis pathways Current Opin Clin Nutr Metabol Care 5 (1) 85-92

Desai KGH Park HJ (2005) Encapsulation of vitamin C in tripolyphosphate crosslinked

chitosan microspheres by spray drying J Microencap 22 179ndash192

DeSouza S Eitenmiller R (1990) Effects of different enzyme treatments on extraction of

total folate from various foods prior to microbiological and radioassay J Micronutri

Anal 7 37-57

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Release of Food Ingredients American Chemical Society Symposium Series no

590 pp 113ndash131 [SJ Risch and GA Reineccius editors] Washington DC

American Chemical Society

Dietrich H Hu Y Zou Y Dirnhofer S Kleindienst R Wick G Xu Q (2000) Mouse model

of transplant arteriosclerosis role of intercellular adhesion molecule-1 Arterioscler

Thromb Vasc Biol 20 343ndash352

Donaldson KO Keresztesy JC (1962) Naturally occurring forms of folic acid

Characterisation and properties of 5-CH3-DHF an oxidation product of 5-CH3-THF

J Biol Chem 237 (12) 3815

Doshi SN Moat SJ Lewis MJ McDowell IF Giddings JC Goodfellow J (2004)

Short-term high-dose folic acid does not alter markers for endothelial cell damage in

patients with coronary heart disease Intl J Cardiol 94 204-207

Draget KI Skjaringk-Braeligk G Stokke BT (2006) Similarities and differences between

alginic acid gels and ionically crosslinked alginate gels Food Hydrocoll 20(2-3)

170-175

Draget KI Smidsroslashd O Skjaringk-Braek G (2002) Alginates from Algae In Steinbuumlchel

A De Baets S Vandamme EJ (eds) Biopolymers vol 6 Polysaccharides II Wiley-

VCH Weinheim pp 215ndash244

Duell PB Malinow MR (1997) Homocyst(e)ine an important risk factor for

atherosclerotic vascular disease Curr Opin Lipidol 8 28-34

Dziezak JD (1988) Microencapsulation and encapsulated ingredients Food Technol 42

(4) 136ndash151

Fenech M (2002) Micronutrients and genomic stability a new paradigm for recommended

dietary allowances (RDA) Food Chem Toxicol 40 1113-1117

Ferguson EL Skeaff CM Bourne DM Nixon N Parnell WR (2000) Folate status

of representative populations in Dunedin issues for folate fortification

Department of Human Nutrition and Department of Food Science University of

Otago New Zealand

Finkelstein JD (1990) Methionine metabolism in mammals J Nutr Biochem 1(5) 228-

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httpwwwfoodstandardsgovau_srcfilesStandard_1_3_1_Additives_Part_3_v85

doc Accessed May 2006

Food standards amendment of standards of identity for enriched grain products to require

addition of folic acid final rule (1996) Fed Regist 61 8781-97

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on peripheral hemodynamics and viscoelastic properties of aorta in anaesthetised

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FSAI Nutrition Sub-committee (2003) Report on the mandatory fortification of flour with

folic acid for the prevention of neural tube defects Dublin Food Safety Authority

of Ireland

Funk C (1912) The preparation from yeast and certain foodstuffs of the substance the

deficiency of which in diet occasions polyneuritis in birds J Physiol 45(1-2) 75ndash

Garcia-Tevijano ER Berasain C Rodriguez JA Corrales FJ Arias R Martin-Duce

A Caballeria J Mato JM Avila MA (2001) Hyperhomocysteinemia in liver

cirrhosis mechanisms and role in vascular and hepatic fibrosis Hyperten 38 1217ndash

Gibbs BF Kermasha S Alli I Mulligan CN (1999) Encapsulation in food industry A

review Int J Food Scie Nutr 50 213ndash234

Gilfix BM Blank DW Rosenblatt DS (1997) Novel reductant for determination of

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Gill HS Rutherford KJ Cross ML (2000) Bovine milk a unique source of

immunomodulatory ingredients for functional foods In Buttriss J Saltmarsh M

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of Chemistry Press pp82-90

Giovannucci E Rimm EB Ascherio A Stampfer ME Colditz GA Willett WC

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psychiatric illness by methyl folate Lancet 336 392-395

Godshall MA (1988) The role of carbohydrates in flavour development Food Technol 42

(11) 71ndash74

Gouin S (2004) Microencapsulation Industrial appraisal of existing technologies and

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Greener IK Fennema O (1989) Evaluation of edible bilayer films for use as moisture

barriers for food J Food Sci 54 1400ndash1403

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Hawkes JG Villota R (1989) a Folates in foods reactivity stability during processing

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Hertzberg S Kvittingen L Anthonsen T Skjadegk-Braeligk G (1990) Alginate as

immobilization material for biocatalysts in organic solvents Ann NY Acad

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Levels and Masks Vitamin B-12 Deficiency in Elderly People J Nutr 132289-291

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Hoppner K Lampi B (1990) Total folate pantothenic acid and biotin content of yogurt

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Iyer C Kailasapathy K Peiris P (2004) Evaluation of survival and release of

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Jackson LS Lee K (1991) Microencapsulation and encapsulated ingredients Lebens

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Janaswamy S Chandrasekaran R (2001) Three-dimensional structure of the sodium salt

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Jozwiaskowski MJ Jones D Franz RM (1990) Characterisation of a hot melt fluid bed

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Kailasapathy K (2006) Survival of free and encapsulated probiotic bacteria and their effect

on the sensory properties of yoghurt LWT - Food Sci Technol 39(10) 1221-1227

Kamper SL Fennema O (1984) Water vapour permeability of an edible fatty acid

bilayer film J Food Sci 49 1482ndash1485

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for occlusive vascular disease Annu Rev Nutr 12 279ndash298

Kapusta L Haagmans MLM Steegers PAE Cuypers MHM Blom HJ Eskes

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homocysteine metabolism J Pediatr 135 773-774

Kelly P McPartlin J Goggins M Weir DG Scott JM (1997) Unmetabolized folic

acid in serum acute studies in subjects consuming fortified food and supplements

Am J Clin Nutr 65 1790-1795

Kim H Fassihi R (1997) Application of binary polymer system in drug release rate

modulation 2 Influence of formulation variables and hydrodynamic conditions on

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Kim HHY Baianu IC (1991) Novel liposome microencapsulation techniques for food

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Kim YI (1999) Folate and cancer prevention a new medical application of folate beyond

hyperhomocysteinemia and neural tube defects Nutr Rev 57 (10) 314-321

Kirby CJ (1991) Microencapsulation and controlled delivery of food ingredients Food Sci

Technol Today 5 (2) 74ndash80

Kirby CJ Gregoriadis G (1984)A simple procedure for preparing liposomes capable of

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Microbiological Properties J Dairy Sci 75 3158-3166

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Kourkoutas Y Bosnea L Taboukos S Baras C Lambrou D Kanellaki M (2006)

Probiotic Cheese Production Using Lactobacillus casei Cells Immobilized on Fruit

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Lamb R (1987) Spray chilling Food Flav Ingredients Packag Process 9 (12) 39ndash42

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Lane HW Nillen JL and Kloeris VL (1995) Folic acid content in thermostabilised and

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Lewis CJ Crane NT Wilson DB Yatley EA (1999) Estimated folate intake data

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Liu XD Atarashi T Furuta T Yoshii H Aishima S Ohkawara M Linko P (2001)

Microencapsulation of emulsified hydrophobic flavors by spray drying Drying

Technol 19 (7) 1361ndash1374

Lu SC TsukamotoH JM Mato JM (2002) Role of abnormal methionine metabolism

in alcoholic liver injury Alcohol 27 155ndash162

Lucey J A Gorry C OrsquoKennedy B Kalab M Tan-Kinita R Fox P F (1996) Effect

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Lucock M (2000) Folic acid nutritional biochemistry molecular biology and role in

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Lucock MD Priestnall M Daskalakis I Shoran CJ Wild J Levene MI (1995)

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Madziva H Kailasapathy K Phillips M (2005) Alginatendashpectin microcapsules as a

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LWT - Food Sci Technol 39(2) 146-151

Malin JD (1977) Total folate activity in Brussels sprouts the effects of storage

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Martinsen A (1990) Alginates as Immobilization Materials-A Study of Some Molecular

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Matsuda S Hatano H Kuramoto K Tsutsumi A (2001) Fluidization of ultrafine

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McKinley MC McNulty H McPartlin J Strain JJ Pentieva K Ward M Weir

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McLachlan J (1985) Macroalgae (seaweeds) industrial resources and their utilization

Plant Soil 89(1-3) 137 ndash 157

McNulty H Cuskelly JG Ward M (2000) Response of red blood cell folate to

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Meisel H FitzGerald RJ (2003) Biofunctional Peptides from Milk Proteins Mineral

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Millqvist-Fureby A Malmsten M Bergenstahl B (2000) An aqueous polymer two-phase

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54ndash61

Mills JL Kirke PN Molloy MA Burke H Conley MR Lee LK Mayne PD

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Mirghani A Idkaidek NM Salem MS Najib NM (2000) Formulation and release

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Moslashller A (1996) The composition of foodsmdashfourth edition Instituttet for

Levnedsmiddelkemi og Ernaeligring Levnedsmiddelstyrelsen Sundhedsministeriet

Denmark

Mnkeni AP Beveridge T (1983) Thermal destruction of 5-methyltetrahydrofolic acid in

buffer model systems J Food Sci 48(2) 595-598

Moslemy P Neufeld RJ Guiot SR (2002) Biodegradation of gasoline by gellan gum-

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MRC Vitamin Study Group (1991) Prevention of neural tube defects results of the Medical

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Mudd SH Finkelstein JD Irreverre F Laster L (1964) Homocysturia an enzymic

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Muller H Diehl JF (1995) Effect of ionisation radiation on folates in food LWT Food

Sci 29 187-190

Munjeri O Collet JH Fell JT (1997) Hydrogel beads based on amidated pectins for

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Murata Y Maeda T Miyamoto E Kawashima S (1993) Preparation of chitosan-

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Naurath HJ Joosten E Riezler R Stabler SP Allen RH Lindebaum J (1995)

Effects of vitamin B12 folate and vitamin B6 supplements in elderly people with

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Newton R Green T Bourn D (2001) The effects of fortification of the New Zealand

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Nutrivit 2000 Fortification basics choosing a vehicle

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NZMoH (1999) Folate folic acid and health Wellington Ministry of Health

NZMoH (2004) Improving folate intake in New Zealand Wellington Ministry of Health

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naturally occurring monoglutamate derivatives of folic acid A J Clin Nutr 28 438-

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157-172

Ono F (1980) New encapsulation technique with protein-carbohydrate matrix J Japan

Food Sci Technol 27 529ndash535

Ostberg T Lund ME Graffner C (1994) Calcium alginate matrices for oral multiple

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Pacifico CJ Wu WH (2001) Fraley M Sensitive substance encapsulation US Patent

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Pagington JS (1986) β-Cyclodextrin and its uses in the flavour industry In Developments

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Paine-Wilson B Chen TS (1979) Thermal destruction of folacin effect of pH and buffer

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Pandrangi S Laborde LF (2004) Retention of folate carotenoids and other quality

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Peacuterez-Mateos M Montero P (2002) Effects of Na+ K+ and Ca2+ on gels formed from fish

mince containing a carrageenan or alginate Food Hydrocoll 16(4) 375-385

Perry IJ Refsum H Morris RW Ebrahim SB Ueland PM Shaper AG(1995)

Prospective study of serum total homocysteine concentration and risk of stroke in

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Perry J Chanarin I (1970) Intestinal absorption of reduced folate compounds in man Br J

Haematol18(3) 329-339

Petersen MA (1993) Influence of sous vide processing steaming and boiling on vitamin

retention and sensory quality in broccoli florets Eur Food Res Technol 197 (4)

375 ndash 380

Petri M Roubenoff R Dallal GE Nadeau MR Selhub J Rosenberg IH(1996)

Plasma homocysteine as a risk factor for atherothrombotic events in systemic lupus

erythematosus Lancet 348 (9035) 1120-4

Pfeiffer CM Caudiall SP Gunter EW Bowman BA Jacques PF Selhub J

Johnson CL Miller DT Sampson EJ (2000) Analysis of factors influencing

the comparison of homocysteine values between the Third National Health and

Nutrition Examination Study (NHANES) and NHANES 1999+ J Nutr 1302850-

Pfeuffer M Schrezenmeir J (2000) Bioactive substances in milk with properties

decreasing risk of cardiovascular diseases Br J Nutr 84(Suppl s1) 155-159

Pfleiderer W (1985) Chemistry of naturally occuring pterins in Folates and Pteridines

(Blakely R L amp Benkovic S J eds) vol 2 pp 43ndash113 Wiley-Interscience New

Phillips G O Williams P A (2000) Handbook of hydrocolloids Woodhead Publishing

Limited Cambridge

Piculell L (1991) Effects of ions on the disorder-order transitions of gel-forming

polysaccharides Food Hydrocoll 5 57-69

Pillay V Fassihi R (1995) In vitro release modulation from crosslinked pellets for site

specific drug delivery to the gastrointestinal tract I Comparison of pH sensitive

drug release and associated kinetics J Control Rel 59 229-242

Polk AE Amsden B Scarratt DJ Gonzal A Okhamafe AOGoosen MFA (1994)

Oral delivery in aquaculture controlled release of proteins from chitosan-alginate

microcapsules Aquaculture Engineer 13 311 ndash 323

Poncelet D Lencki R Beaulieu C Halle J P Neufeld R J Fournier A (1992)

Production of alginate beads by emulsificationinternal gelation Methodol Appl

Microbiol Biotechnol 38(1) 39 ndash 45

Pothakamury UR Barbosa-Caacutenovas GV (1995) Fundamental aspects of controlled

release in foods Trends Food Sci Technol 6 397ndash406

Prevost H Divies C Rousseau E (1988) Continuous production with Lactobacillus

bulgaricus and Streptococcus thermophilus entrapped in calcium alginate

Biotechnol Lett 7 247ndash252

Queensland Health (2002) An overview of indicators of nutritional status of Queensland

adults collected as part of the AusDiab study 9-21

Queacutereacute I Perneger T Zittoun J Bellet H Gris J Dauregraves J Schved J Mercier E

Laroche J Dauzat M (2002) Red blood cell methylfolate and plasma

homocysteine as risk factors for venous thromboembolism a matched case-control

study Lancet 359(9308) 747-752

Quinlivan EP Gregory JF III (2003) Effect of food fortification on folic acid intake in

the United States Am J Clin Nutr 77 (1) 221-225

Rader JI Weaver CM Angyal G (2000) Total folate in enriched cereal-grain products

in the United States following fortification Food Chem 70(3) 275-289

Rajkovic A CatalanoPM Malinow RM (1997) Elevated homocyst(e)ine levels with

preeclampsia Obstet Gynecol 90 168-171

Rao DR Reddy AV Pulusani SR Cornwell PE (1984) Biosynthesis and utilisation

of folic acid and vitamin B12 by lactic cultures in skim milk J Dairy Sci 67 1169-

Rao DR Shahani KM(1987) Vitamin content of cultured milk products Cultur Dairy

Prod J Feb6ndash10

Reacute MI (1998) Microencapsulation by spray drying Drying Technol 16 1195ndash1236

Reddy KS (1975) B-complex vitamins in cultured and acidified yogurt J Dairy Sci 59

191ndash195

Redenbaugh K Paasch BD Nichol JW Kossler ME Viss PR Walker KA (1986)

Somatic Seeds Encapsulation of Asexual Plant Embryos Biol Technol 4 797 ndash

Rees DA (1969) Structure conformation and mechanism in the formation of

polysaccharide gels and networks Adv Carbohydr Chem Biochem 24 303-304

Reeves PG Nielsen FH Fahey GC (1993) AIN purified diets for laboratory rodents

Final report of the American Institute of Nutrition ad hoc writing committee on the

reformulation of the AIN-67A rodent diet J Nutr 123 1939-1951

Refsum H Ueland PM Nygaringrd O Vollset SE (1998) Homocysteine and

cardiovascular disease Annu Rev Med 49 31ndash62

Reid IR Mason B Horne A Ames R Clearwater J Bava U Orr-Walker B Wu F

Evans MC Gamble GD (2002) Effects of calcium supplementation on serum

lipid concentrations in normal older women A randomized controlled trial Am J

Med 112(5) 343-347

Reineccius GA (1991) Carbohydrates for flavour encapsulation Food Technol 46 (3)

144ndash147

Renner E (1983) Milk and dairy products in human nutrition VV GmbH

Volkswirtschaftlicher Verlag Federal Republic of Germany

Ristow KA Gregory JF Damron BL (1982) Effects of dietary fibre on the

bioavailability of folic acid monoglutamate J Nutr 112 750-758

Rizzuto AB Chen AC Veiga MF (1984) Modification of the sucrose crystal structure

to enhance pharmaceutical properties of excipient and drug substances Pharm

Roberfroid MB (1998) Prebiotics and synbiotics concepts and nutritional properties Br J

Nutr 80 S197ndashS202

Roche Vitamins Europe (2003) Mandatory food enrichment Bolwley A Nutrivew Basel

Switzerland Roche Vitamins Europe Ltd

httpwwwnutrivitorgvicstapleindexhtm Accessed January 2006

Rosenberg M Sheu TY (1996) Microencapsulation of volatiles by spray drying in whey

protein based wall systems Int Dairy J 6 273ndash284

Ruddick JE Vanderstoep J Richards JF (1980) Kinetics of thermal degradation of 5-

CH3-THF J Food Sci 45 1019

Rumpler K Jacob M (1998) Continuous coating in fluidised bed Food Market Technol

12 41-43

Sairam M Babu VR Rao KSVK Naidu BVK Hosamani KM Aminabhavi TM

(2006) pH sensitive interpenetrating network microgels of sodium alginate-acrylic

acid for the controlled release of ibuprofen J Appl Polym Sci 99 2671ndash2678

Saldo J Sendra E Guamis B (2000) High hydrostatic pressure for accelerating ripening

of goatrsquos milk cheese proteolysis and texture J Food Sci 65(4) 636-640

Sandberg A Andersson H Hallgren B Hasselblad K Isaksson B Hulteacuten L (1981)

Experimental model for in vivo determination of dietary fibre and its effect on the

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dietary methionine with or without adequate dietary vitamins on

hyperhomocysteinemia in rats Nutr Res 20 (12) 1817-1827

Schader I Corwin P (1999) How many pregnant women in Christchurch are using folic

acid supplements in early pregnancy N Z Med J 10 112(1101) 463-465

Schlameus W (1995) Centrifugal extrusion encapsulation In Encapsulation and Controlled

Release of Food Ingredients Risch SJ Reineccius GA Eds American Chemical

Society Washington DC

SchollTO JohnsonWG (2000) Folic acidinfluence on the outcome of pregnancy Am J

Clin Nutr 71 (5) 1295S-1303

Schorah CJ Devitt H Lucock MD Dowell AC (1998) The responsiveness of plasma

homocysteine to small increases in dietary folic acid a primary care study Eur J

Clin Nutr52 407-11

Scott J Rebeille F Fletcher J (2000) Review ndash Folic acid and folates the feasibility for

nutritional enhancement in plant foods J Sci Food Agri 80 795-824

Scott KJ (1989) Micronutrients in dairy products In Renner E (ed) ldquoMicronutrients in

Milk and Milk-based productsrdquo London Elsevier Applied Science pp 71-123

Seiss W Divies C (1975) Microencapsulation Angewandte Chemie Int Ed 14 539ndash550

Selhub J Dhar GJ Rosenberg IH (1983) Gastrointestinal absorption of folates and

antifolates Pharmacol Ther 20(3) 397-418

Selhub J Miller JW (1992) The pathogenesis of homocysteinemia interruption of the

coordinate regulation by S-adenosylmethionine of the remethylation and trans-

sulfuration of homocysteine Am J Clin Nutr 55 131-138

Seshadri S Beiser A Selhub J Jacques PF Rosenberg IH DrsquoAgostino RB

Wilson PW Wolf PA (2002) Plasma homocysteine as a risk factor for dementia

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Shah PN (2000) Probiotic bacteria Selective enumeration and survival in dairy foods J

Dairy Sci 83 894-907

Shahidi F Han XQ (1993) Encapsulation of food ingredients Crit Rev Food Sci Nutr33

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Shiga H Yoshii H Nishiyima T Furuta T Forssele P Poutanen K Linko P (2001)

Flavour encapsulation and release characteristics of spray-dried powder blended

encapsulant of cyclodextrin and gum arabic Drying Technol 19(7) 1385-1395

Shilton NC Niranjan K (1993) Fluidization and its applications to food processing Food

Struct 12 199ndash215

Shiotani T Yamane T (1981) A horizontal packed-bed bioreactor to reduce carbon

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Shoveller KA House DJ Brunton JA Pencharz PB Ball PA (2004) The Balance

of Dietary Sulfur Amino Acids and the Route of Feeding Affect Plasma

Homocysteine Concentrations in Neonatal Piglets J Nutr 134 609-612

Shrestha KA Jayashree A Paterson LJ (2003) Edible coating materials ndash their

properties and the use in the fortification of rice with folic acid Food Res Intl 36

921-928

Simpson NE Stabler CL Simpson CB Sambanis A Constantinidis I (2004) The

role of the CaCl2ndashguluronic acid interaction on alginate encapsulated βTC3 cells

Biomat 25(13) 2603-2610

Skeaff CM Mann J (1998) Should folate be added to flour to prevent neural tube defects

N Z Med J 111 417-418

SkeaffM GreenT MannJ (2003) Mandatory fortification of flour Science not

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Slattery ML Potter DJ Samowitz W Schaffer D Leppert M (1999)

Methylenetetrahydrofolate reductase diet and risk of colon cancer Cancer Epidem

Biom Prev 8 513-518

Sloten BV (2006) Advanced spray drying process

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Smidsrod O Skjak-Braek G (1990) Alginate as immobilization matrix for cells Trends

Biotechnol 8 71ndash78

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SPSS (2002) SPSS 1150 for Windows Standard version SPSS Inc

Stampfer MJ Malinow MR (1995) Can lowering homocysteine levels reduce

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Stokstad ELR Koch J (1967) Folic acid metabolism Physiol Rev 47 83

Strum WB (1981) Characteristics of the transport of pteroylglutamate and amethopterin in

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Sultana K Godward G Reynolds N Arumugaswamy R Peiris P Kailasapathy K

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Takahashi M Ikeda U Masuyama J Kitagawa S Kasahara T Saito M Kano S

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241-246

Taylor AH (1983) Encapsulation systems and their applications in the flavour industry

Food Flav Ingre Packag Process 5 (9) 48ndash51

Thies C (1987) Microencapsulation In Encyclopedia of Polymer Science and Engineering

pp 724ndash745 [HF Mark NM Bikales CG Overberger G Menges and JI Kroschwitz

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Tripet FY Kesselring UW (1975) The stability of folic acid in solid the state as a

function of temperature and humidity Pharm Acta Helv 50(10) 318-322

Ubbink J Schoonman A (2003) Flavour delivery systems Kirk-Othmer encyclopedia of

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Ueland PM Refsum H Beresford SA Vollset SE (2000) Am J Clin Nutr 72 324-

Ueland PM Refsum H Stabler SP Malinow MR Andersson A Allen RH (1993)

Total homocysteine in plasma or serum methods and clinical applications Clin

Chem 39 1764-1779

USFDA (1998) Food standards amendment of standards of identity for enriched cereal

grain products to require the addition of folic acid (final rule) US Food and Drug

Administration Fed Register 61 (44) 8781-8797

Vahteristo L Lehikoinen K Ollilainen V Koivistoinen PEVARO P (1998)

Ovenbaking and frozen storage affect folate vitamer retention Lebensm-Wiss u-

Technol 31 329-333

Valente AJ Rozek MM Sprague EA Schwartz CJ (1992) Mechanisms in intimal

monocyte-macrophage recruitment a special role for monocyte chemotactic protein-

1 Circul 86 (suppl III) III-20ndashIII-25

Vaskonen T Mervaala E Sumuvuori V Seppaumlnen-Laakso T Karppanen H (2002)

Effects of calcium and plant sterols on serum lipids in obese Zucker rats on a low-fat

diet Br J Nutr 87(3) 239-245(7)

Verhoeff BJ Trip MD Prins MH Kastelein JJP Reitsma PH (1998) The effect

of a common methylenetetrahydrofolate reductase mutation on levels of

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Atheroscl 141 (1) 161-166

Vitamin Study (1991) Prevention of neural tube defects results of the medical research

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Vollset EM Refsum H Tverdal A Nygaringrd O Nordrehaug JE Tell GS Ueland

PM (2001) Plasma total homocysteine and cardiovascular and noncardiovascular

mortality the Hordaland Homocysteine Study Am J Clin Nutr74(1) 130-136

Vonderschmitt DJ Scrimgeneour KG (1967) Reaction of Cu2+ and Fe3+ with

tetrahydropterin Biochem Biophys Res Commun 28(3) 302-305

Voorn JTG Overbeek MJ (1957) Phase separation in polyelectrolyte solutions Theory

of complex coacervation J Cellul Comp Physiol 49 Suppl 1 7ndash26

Wagner C (1985) Folate-binding proteins Nutr Rev 43 293ndash299

Wald DS Law M Morris JK (2002) Homocysteine and cardiovascular disease

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Walkenstroumlm P Kidman S Hermansson A Rasmussen PB Hoegh L (2003)

Microstructure and rheological behaviour of alginatepectin mixed gels Food

Hydrocol 17(5) 593-603

Walsh PK Isdell FV Noone SM ODonovan MG Malone DM (1996) Growth

patterns of Saccharomyces cerevisiae microcolonies in alginate and carrageenan gel

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18(5) 366-372(7)

Wells A S (2001) The role of milk in the British diet Intl J Dairy Technol 54 (4) 130-

Wenstrom KD Johanning LG Johnston KE Dubard M (2001) Association of the

C677T methylenetetrahydrofolate reductase mutation and elevated homocysteine

levels with congenital cardiac malformations Am J Obstet Gynecol 185(5) 806-

Whittaker P Tufaro PR Rader JI (2001) Iron and Folate in Fortified Cereals J Am

Colleg Nutr 20(3) 247-254

Wigertz K Hansen SI Hoier-Madsen L Witthoft CM Holm J Jagerstad M (1997)

Effect of milk processing on the concentration of folate-binding protein (FBP) the

folate-binding capacity and the retention of 5-methyltetrahydrofolate Int J Food

Sci Nutr 47 315-322

Wigertz K Svensson UK Jagerstad M (1996) Folate and folate binding protein content

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Witthoft CM Forssen K Johannesson L Jagerstad M (1999) Folates-food sources

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Wolstenholme G E W Cameron MP (1954) Chemistry and biology of pterins Little

Brown Boston

Woo KS Chook P Lolin YI Sanderson JE Metreweli C Celermajer DS

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hyperhomocysteinemia J Am Coll Cardiol 34 2002ndash2006

Woo W Siow YL Pierce GN Choy PC Minuk GY Mymin D K O (2005)

Hyperhomocysteinemia induces hepatic cholesterol biosynthesis and lipid

accumulation via activation of transcription factors Am J Physiol Endocrinol

Metab 288 E1002ndashE1010

Wu WH Roe WS Gimino VG Seriburi V Martin DE Knapp SE (2002) Low

melt encapsulation PCT QO 0074499

Yacowitz H Fleischman A I Bierenbaum M L (1965) Effects of oral calcium upon

serum lipids in man Brit Med J 1 1352-1357

Yoo S-H Fishman ML Hotchkiss AT Hyeon G L (2006) Viscometric behavior of

high-methoxy and low-methoxy pectin solutions Food hydrocoll 20(1) 62-67

Yuliani S Bhandari B Rutgers R DrsquoArcy B (2004) Application of microencapsulated

flavour to extrusion product Food Rev Intl 20 (2) 163ndash185

Zhang S HunterDJ Hankinson ES Giovannucci LE Rosner AB Colditz AG

Speizer EF Willett CW (1999) A Prospective Study of Folate Intake and the

Risk of Breast Cancer JAMA 281(17) 1632-1637

Zhao L Pan Y Li J Chen G Mujumdar AS (2004) Drying of a dilute suspension in

a revolving flow fluidized bed of inert particles Drying Technol 22 (1-2) 363ndash376

Zheng S Alkan-Onyuksel H Beissinger RL Wasan DT (1999) Liposome

microencapsulation without using any organic solvent J Dispers Sci Technol 20

1189ndash1203

Appendix 1 Experimental Buffers

Buffer test solutions

(i) Phosphate buffer solution (pH 82)

Dissolve 537 g of disodium hydrogen phosphate in water to make 1000 ml

(Solution A) Dissolve 204 g of potassium dihydrogen phosphate in water to make

1000 ml (Solution B) Combine 21 volumes of Solution A and 4 volumes of

Solution B and adjust the pH to 75 with either Solution A or Solution B

(ii) Citrate acid buffer solution (pH 82)

Dissolve 21 g of citrate acid in water to make 1000 ml (Solution A) Dissolve 284 g

of disodium hydrogen phosphate in water to make 1000 ml (Solution B) Combine

11 volumes of Solution A and 389 volumes of Solution B

(iii) Buffer TS (pH 82)

Combine 50 ml of 02 M potassium dihydrogen phosphate and 152 ml of 02 M

sodium hydroxide and dilute to 200 ml with water

(iv) Hydrochloric acid (pH 12)

Dilute 815 ml of hydrochloric acid to 1L with water

Appendix 3 Stains

Verhoeff mdash Van Gieson (Elastin)

Solutions

5 hematoxylin solution - Hematoxylin 10g 100 ETOH 200ml

Dissolve hematoxylin in 58-60oC water bath Filter Solution is stable for several

months

10 aqueous ferric chloride (prepare fresh)

Weigertrsquos iodine solution

Potassium iodide 2 gm

Iodine 1 gm

ddH2O 100 ml

Verhoeffrsquos staining solution (prepare fresh)

5 alcoholic hematoxylin 20ml

10 ferric chloride 8ml

Weigert lsquos iodine solution 8ml

Mix well solution should be jet black Use immediately 2 aqueous ferric chloride

( prepare fresh ) 5 aqueous sodium thiosulfate

Van Giesonrsquos counterstain

1 aqueous acid fuchsin 5ml

Saturated aqueous picric acid 100ml

Appendix 3 Stains

Procedure

1 Hydrate slides to distilled water

2 Stain in Verhoffrsquos solution for 1 hour ( Save solution Saved solution may be

used for restain

3 Rinse in tap water with 2 or 3 changes

4 Differentiate in 2 aqueous ferric chloride and agitate slides gently

Stop differentiation with several changes of tap water and check

microscopically for black elastic fiber staining and gray background Repeat 2

ferric chloride treatment and tap water rinses as necessary If elastic fiber

staining is too pale restain in the saved verhoeffs solution

It is better to slightly underdifferentiate the tissue 3 min differentiation

was used for 10um paraffin sections

5 Wash slides in tap water

6 5 sodium thiosulfate 1 min Discard solution

7 Wash in running tap water for 5 min

8 Counterstain in Van Giesonrsquos solution for 3-5 min

9 Dehyddrate clear in xylene and cover slip

Results

Elastic fibers---blue-black to black

Nuclei---bluie to black

Collagen---red

Other tissue elements---yellow

Table of Contentspdf


414 Results 126

416 Conclusion 151

11 Aim


Food Sample

Effecting factors



Some general material and methods used in the experiments of this study are described in this chapter The specific experimental materials and methods for each particular experiment are given in respective chapters

Sodium alginate (viscosity of 2 wv solution at 25degC 250 cps) potassium salt of pectin (28 degree of esterification (DE) low methoxy (LM) and 20 amidated) gelatine iota-carrageenan xanthan gum citric acid di-sodium hydrogen phosphate potassium dihydrogen phosphate sodium hydroxide and calcium chloride were all purchased from Sigma-Aldrich (Castle Hill Sydney Australia) Folic acid was obtained from Roche Pty Ltd (Sydney Australia) Starch (Hi-Maize trade1043) was purchased from National Starch and Chemical Company (New Jersey USA)


32 Effect of time and calcium chloride concentration on cross-linking completion in alginate-pectin hydrogels




Choumenkovitch SF Selhub J Wilson P W F Rader JI Rosenberg HI Jacques PF (2002) Folic Acid Intake from Fortification in United States Exceeds Predictions J Nutr 132 2792-2798

Madziva H Kailasapathy K Phillips M (2005) Alginatendashpectin microcapsules as a potential for folic acid delivery in foods J Microencap 22(4) 343-351

Vollset EM Refsum H Tverdal A Nygaringrd O Nordrehaug JE Tell GS Ueland PM (2001) Plasma total homocysteine and cardiovascular and noncardiovascular mortality the Hordaland Homocysteine Study Am J Clin Nutr74(1) 130-136


