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Studies on stability and efficacy of
microencapsulated folic acid in Cheddar cheese and
in methionine-induced hyperhomocysteinemia in
mice
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
i
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
ii
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
412 Folic acid measurement 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
55 Materials and methods 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
iii
Chapter 6 - EFFECT OF ENCAPSULATED FOLIC ACID DIETARY
SUPPLEMENTATION ON METHIONINE-INDUCED
HYPERHOMOCYSTEINEMIA IN MICE
61 Abstract 171
62 Introduction 172
63 Materials and methods 175
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
iv
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
13th
International Workshop on Bioencapsulation June 24th
ndash 26th
2005 pg 33-34
Kingston Ontario Canada
v
Publications and disseminations
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
Madziva H Kailasapathy K Phillips (2004) Alginate-pectin microcapsules as a
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
Madziva H Kailasapathy K Phillips (2004) Alginate-pectin microcapsules as a
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
vi
Publications and disseminations
Posters
Madziva H Kailasapathy K Phillips (2006) Effect of encapsulated folic acid
dietary supplementation on methionine-induced hyperhomocysteinemia in mice 39th
Annual Australian Institute of Food Science amp Technology Convention 9-12th
July
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
Spain
Endeavour International Postgraduate Research Scholarship recipient (2002-
2006) for PhD studies at University of Western Sydney
vii
List of abbreviations
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
xi
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
xii
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
xiii
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
xiv
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)
1
Chapter 1 Introduction
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
2
Chapter 1 Introduction
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)
3
Chapter 1 Introduction
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)
4
Chapter 1 Introduction
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
and
bull not interact with the fortificant or undergo changes to taste colour or
appearance as a result of fortification
5
Chapter 1 Introduction
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
6
Chapter 1 Introduction
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
7
Chapter 1 Introduction
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
8
Chapter 1 Introduction
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
9
Chapter 1 Introduction
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
10
Chapter 1 Introduction
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
11
Chapter 1 Introduction
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
12
Chapter 1 Introduction
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
13
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
14
Chapter 2 Literature Review
Fig 21 Chemical structure of folic acid (Source Hawkes amp Villota 1989a)
15
Chapter 2 Literature Review
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
16
Chapter 2 Literature Review
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
17
Chapter 2 Literature Review
Zhang et al 1999) and unexplained recurrent early pregnancy loss (Rajkovic et al
1997)
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
18
Chapter 2 Literature Review
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
19
Chapter 2 Literature Review
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
20
Chapter 2 Literature Review
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
21
Chapter 2 Literature Review
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
2004)
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)
22
Chapter 2 Literature Review
Fig 22 Homocysteine metabolism (Source Verhoeff et al 1998)
23
Chapter 2 Literature Review
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
child-bearing age consuming only about half the amount recommended while adult
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)
24
Chapter 2 Literature Review
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
25
Chapter 2 Literature Review
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)
26
Chapter 2 Literature Review
Table 2 2Countries with mandatory folic acid fortification
Country Year
mandatory folic
acid
fortification
introduced
Foods fortified with folic acid Level of
fortification
mg kg
Africa
Malawi South Africa Zambia
2002
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
15
North America Canada
November 1998
flour (white enriched enriched white) enriched bread enriched pasta enriched pre-cooked rice
15
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
14
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
20
Adapted from (Canadian Government 1998 Roche Vitamins Europe 2003 USFDA 1996)
27
Chapter 2 Literature Review
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
food
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
28
Chapter 2 Literature Review
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
29
Chapter 2 Literature Review
food is well below the recommended intake during pregnancy of 400 μg per
day
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
30
Chapter 2 Literature Review
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)
31
Chapter 2 Literature Review
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)
66 28
Christchurch (Schader and Corwin 1999 in NZMoH 2003)
63 17
Dunedin (Ferguson et al 2000)
11
Adapted from (Abraham amp Webb 2001 NZMoH 2004) Women who had been pregnant in the last 5 years
32
Chapter 2 Literature Review
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
33
Chapter 2 Literature Review
Fig 23 Structure of the human gut (a) and folate absorption in the mucosal cells (b) (Source Selhub et al 1983)
34
Chapter 2 Literature Review
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
35
Chapter 2 Literature Review
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
36
Chapter 2 Literature Review
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
37
Chapter 2 Literature Review
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
38
Chapter 2 Literature Review
Fig24a) Pteridine (highly soluble in water b) Pterin (low solubility in water and nonpolar solvents) and nonpolar solvents)
Source Hawkes and Villota 1989 a
39
Chapter 2 Literature Review
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)
40
Chapter 2 Literature Review
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
75b
30b
46b
34b
8b
19b
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
41
Chapter 2 Literature Review
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
42
Chapter 2 Literature Review
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
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
43
Chapter 2 Literature Review
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
44
Chapter 2 Literature Review
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
45
Chapter 2 Literature Review
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
46
Chapter 2 Literature Review
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
RDI
47
Chapter 2 Literature Review
(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
48
Chapter 2 Literature Review
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
49
Chapter 2 Literature Review
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
50
Chapter 2 Literature Review
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 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
51
Chapter 2 Literature Review
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
and
bull not interact with the fortificant or undergo changes to taste colour or
appearance as a result of fortification
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)
52
Chapter 2 Literature Review
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
53
Chapter 2 Literature Review
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
54
Chapter 2 Literature Review
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)
A number of countries have introduced mandatory requirements for folic acid
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)
55
Chapter 2 Literature Review
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
56
Chapter 2 Literature Review
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)
57
Chapter 2 Literature Review
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
58
Chapter 2 Literature Review
(A) (B)
Core material
Core particles
Wall material
Fig25 Schematic diagram of two representative types of microcapsules
59
Chapter 2 Literature Review
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)
60
Chapter 2 Literature Review
(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)
61
Chapter 2 Literature Review
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
62
Chapter 2 Literature Review
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
63
Chapter 2 Literature Review
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
64
Chapter 2 Literature Review
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
65
Chapter 2 Literature Review
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
66
Chapter 2 Literature Review
Fig 26 Gel formation via G blocks egg box model (Source Rees 1969)
67
Chapter 2 Literature Review
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
68
Chapter 2 Literature Review
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
1990)
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
69
Chapter 2 Literature Review
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)
70
Chapter 2 Literature Review
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
71
Chapter 2 Literature Review
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
72
Chapter 2 Literature Review
Fig 27 Advanced Spray-drying process (Source Sloten 2006)
73
Chapter 2 Literature Review
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
74
Chapter 2 Literature Review
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
75
Chapter 2 Literature Review
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
76
Chapter 2 Literature Review
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
77
Chapter 2 Literature Review
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
78
Chapter 2 Literature Review
Fig 28 Schematic diagram of spray-cooling
79
Chapter 2 Literature Review
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
80
Chapter 2 Literature Review
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
81
Chapter 2 Literature Review
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
82
Chapter 2 Literature Review
Fig 29 Wurster fluidized bed for microparticle coating (Source Alexandridou et
al 1999)
83
Chapter 2 Literature Review
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)
84
Chapter 2 Literature Review
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
85
Chapter 2 Literature Review
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
86
Chapter 2 Literature Review
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
87
Chapter 2 Literature Review
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
88
Chapter 2 Literature Review
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
89
Chapter 2 Literature Review
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)
90
Chapter 2 Literature Review
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
91
Chapter 2 Literature Review
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
92
Chapter 2 Literature Review
(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
93
Chapter 2 Literature Review
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
94
Chapter 2 Literature Review
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
95
Chapter 2 Literature Review
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
96
Chapter 2 Literature Review
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
97
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
98
Chapter 3 Materials and methods
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
99
Chapter 3 Materials and methods
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
32 Effect of time and calcium chloride concentration on cross-linking
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)
100
Chapter 3 Materials and methods
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
101
Chapter 3 Materials and methods
300 microm nozzle
Product
bottle
Reaction vessel with calcium chloride
Fig 32 Inotech Encapsulator reg (Inotech AG Dottikon Switzerland) used in this
study
102
Chapter 3 Materials and methods
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
103
Chapter 3 Materials and methods
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
104
Chapter 3 Materials and methods
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
105
Chapter 3 Materials and methods
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)
106
Chapter 3 Materials and methods
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)
107
Chapter 3 Materials and methods
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
108
Chapter 3 Materials and methods
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
39 Effect of simulated cheese press pressures on mechanical stability of
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
109
Chapter 3 Materials and methods
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
110
Chapter 3 Materials and methods
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
Chapter 3 Materials and methods
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
112
Chapter 3 Materials and methods
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
mean)
Statistical difference is indicated as follows
pgt005 = no statistical difference
plt005 = statistically significant
113
Chapter 3 Materials and methods
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
encapsulated folic acid
Cheese making and
capsules stability during
cheese ripening
Porcine ex-vivo
release of folic
acid
Fig 35 Diagrammatic summary of the major steps in this study
114
Chapter 4 Selection and optimisation of encapsulation parameters
115
4 Screening of food grade polymers and
optimisation of folic acid encapsulation parameters
to increase encapsulation efficiency and stability
41 Abstract
Most naturally occurring folate derivatives in foods are highly sensitive to
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
Chapter 4 Selection and optimisation of encapsulation parameters
116
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)
Chapter 4 Selection and optimisation of encapsulation parameters
117
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
Chapter 4 Selection and optimisation of encapsulation parameters
118
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
Chapter 4 Selection and optimisation of encapsulation parameters
119
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
Chapter 4 Selection and optimisation of encapsulation parameters
120
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
Chapter 4 Selection and optimisation of encapsulation parameters
121
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
encapsulated folic acid
3 The effect of various buffers to release the encapsulated folic acid in
simulated gastric conditions
Chapter 4 Selection and optimisation of encapsulation parameters
122
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
Chapter 4 Selection and optimisation of encapsulation parameters
123
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)
Chapter 4 Selection and optimisation of encapsulation parameters
124
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
Chapter 4 Selection and optimisation of encapsulation parameters
125
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
Chapter 4 Selection and optimisation of encapsulation parameters
126
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
Chapter 4 Selection and optimisation of encapsulation parameters
127
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
Chapter 4 Selection and optimisation of encapsulation parameters
128
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
Chapter 4 Selection and optimisation of encapsulation parameters
129
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
Chapter 4 Selection and optimisation of encapsulation parameters
130
0
100
200
300
400
500
600
700
800
20 40 60 80 100 120 140
Time (min)
Cap
sula
r d
iam
eter
(m
icro
met
ers)
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))
Chapter 4 Selection and optimisation of encapsulation parameters
131
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 ()
Shape
Alginatepectin (ww) a
742plusmn092
irregular
sphere
6040
886plusmn015
irregular
sphere
7030
782plusmn019
regular
sphere
8020
548plusmn02
regular
sphere
1000
Chapter 4 Selection and optimisation of encapsulation parameters
132
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100 120 140 160 180 200 220 240Time (min)
Folic
aci
d rel
ease
d (
)
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100 120 140 160 180 200 220 240Time (min)
Folic
acid
rel
ease
d (
)
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100 120 140 160 180 200 220 240
Time (min)
Folic
acid
rel
ease
d (
)
b)
c)
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
a)
Chapter 4 Selection and optimisation of encapsulation parameters
133
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100 120 140
Time (min)
F
olic
aci
d r
elea
se
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)
Chapter 4 Selection and optimisation of encapsulation parameters
134
0
20
40
60
80
100
0 20 40 60 80 100 120 140
Time (min)
Rel
ease
(
)
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)
Chapter 4 Selection and optimisation of encapsulation parameters
135
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8 9 10 11
Time (weeks)
R
eten
tio
n f
oli
c ac
id
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
Chapter 4 Selection and optimisation of encapsulation parameters
136
0
4
8
12
16
20
24
28
32
36
40
0 30 60 90 120 150 180 210 240
Time (min)
ug
ml
Fo
lic
acid
rel
ease
d
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
Chapter 4 Selection and optimisation of encapsulation parameters
137
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
Chapter 4 Selection and optimisation of encapsulation parameters
138
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
Chapter 4 Selection and optimisation of encapsulation parameters
139
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
Chapter 4 Selection and optimisation of encapsulation parameters
140
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
Chapter 4 Selection and optimisation of encapsulation parameters
141
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
Chapter 4 Selection and optimisation of encapsulation parameters
142
solution the capsule starts to form instantaneously and grows along the flux direction
of the Ca2+
ions
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
Chapter 4 Selection and optimisation of encapsulation parameters
143
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
Chapter 4 Selection and optimisation of encapsulation parameters
144
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
Chapter 4 Selection and optimisation of encapsulation parameters
145
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
Chapter 4 Selection and optimisation of encapsulation parameters
146
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
Chapter 4 Selection and optimisation of encapsulation parameters
147
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
Chapter 4 Selection and optimisation of encapsulation parameters
148
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
Chapter 4 Selection and optimisation of encapsulation parameters
149
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
Chapter 4 Selection and optimisation of encapsulation parameters
150
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
152
Chapter 5 Cheddar cheese and folic acid delivery
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
153
Chapter 5 Cheddar cheese and folic acid delivery
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
154
Chapter 5 Cheddar cheese and folic acid delivery
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
155
Chapter 5 Cheddar cheese and folic acid delivery
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
156
Chapter 5 Cheddar cheese and folic acid delivery
55 Materials and methods
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
59 Distribution of gel capsules in Cheddar cheese incorporated with three
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
157
Chapter 5 Cheddar cheese and folic acid delivery
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
158
Chapter 5 Cheddar cheese and folic acid delivery
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)
159
Chapter 5 Cheddar cheese and folic acid delivery
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150 180 210 240
Time (min)
F
oli
c a
cid
rete
nti
on
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
Sec
0 7200 14 400 21 600 28 800
10
08
06
04
New
ton
s c
m2
160
Chapter 5 Cheddar cheese and folic acid delivery
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150 180 210 240
Time (min)
F
oli
c a
cid
re
ten
tio
n
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)
161
Chapter 5 Cheddar cheese and folic acid delivery
a)
b)
c)
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
162
Chapter 5 Cheddar cheese and folic acid delivery
Table 51 Comparative distribution of capsules added in milk at 3 levels in Cheddar cheese
slices
Cheese section Distribution in pixels
2 a
Top 1
2
3
Middle 1
2
3
Bottom 1
2
3
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
163
Chapter 5 Cheddar cheese and folic acid delivery
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
Milk
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)
164
Chapter 5 Cheddar cheese and folic acid delivery
0
40
80
120
160
200
240
280
320
360
0 05 1 15 2 25 3
Time (months)
Fo
lic a
cid
rete
nti
on
(u
g1
00 g
)
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
165
Chapter 5 Cheddar cheese and folic acid delivery
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
166
Chapter 5 Cheddar cheese and folic acid delivery
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
1978)
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
167
Chapter 5 Cheddar cheese and folic acid delivery
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
168
Chapter 5 Cheddar cheese and folic acid delivery
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
169
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
171
Chapter 6 Bioactivity of encapsulated folic acid
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
172
Chapter 6 Bioactivity of encapsulated folic acid
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
173
Chapter 6 Bioactivity of encapsulated folic acid
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
174
Chapter 6 Bioactivity of encapsulated folic acid
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
63 Materials and methods
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
175
Chapter 6 Bioactivity of encapsulated folic acid
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)
176
Chapter 6 Bioactivity of encapsulated folic acid
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)
177
Chapter 6 Bioactivity of encapsulated folic acid
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
178
Chapter 6 Bioactivity of encapsulated folic acid
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
food
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)
179
Chapter 6 Bioactivity of encapsulated folic acid
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
180
Chapter 6 Bioactivity of encapsulated folic acid
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
ngml
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
20 gkg L-Met + 2 mg free folic acid
20 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
181
Chapter 6 Bioactivity of encapsulated folic acid
a)
182
Chapter 6 Bioactivity of encapsulated folic acid
b)
183
Chapter 6 Bioactivity of encapsulated folic acid
c)
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
184
Chapter 6 Bioactivity of encapsulated folic acid
ba
c d
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
185
Chapter 6 Bioactivity of encapsulated folic acid
a) b)
c)
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)
186
Chapter 6 Bioactivity of encapsulated folic acid
0
5000
10000
15000
20000
25000
a b c d e fDiet
Lesio
n a
rea
sq
uare
mic
ron
+ S
E
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
187
Chapter 6 Bioactivity of encapsulated folic acid
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-
T
c
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
61)
188
Chapter 6 Bioactivity of encapsulated folic acid
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
992)
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
The
yperhomocysteinemia induced by 20 gkg L-Met was partially counteracted by the
1
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
s
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
h
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
189
Chapter 6 Bioactivity of encapsulated folic acid
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
ve
e
is
h
s the
onocytemacrophage adhesion to the aortic endothelium can be significantly
n and
on
o
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
m
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)
190
Chapter 6 Bioactivity of encapsulated folic acid
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
mice
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
th
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
(F
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)
191
Chapter 6 Bioactivity of encapsulated folic acid
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
n
s encapsulated form than free as seen by a reduction in homocysteine levels and
ents Alginate ndash pectin encapsulation of folic acid enhanced its
g
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
it
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
192
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
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
193
Chapter 7 Conclusion
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
194
Chapter 7 Conclusion
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
NTDs
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
195
Chapter 7 Conclusion
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
196
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
197
Chapter 8 Future directions
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
198
Chapter 9 References
9 Literature cited
Abraham B WebbKL (2001) Interim evaluation of the voluntary folate fortification
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
11-15
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
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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
199
Chapter 9 References
Arcot J Shrestha AK Gusanov U (2002) Validation of a competitive protein binding
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252
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
200
Chapter 9 References
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
201
Chapter 9 References
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-
104
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
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Chapter 9 References
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-
184
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
Predictions J Nutr 132 2792-2798
Chourasia M K Jain S K (2003)Pharmaceutical approaches to colon targeted drug
delivery systems J Pharm Pharmaceut Sci 6(1) 33-66
203
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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
Clydesdale FM (1997) A proposal for the establishment of scientific criteria for health
claims for functional foods Nutr Rev 55(12) 413-422
Codex Alimentarius Commission (1991) General principles for the addition of essential
nutrients to foods CACGL 09-1987 (amended 1989 1991)
wwwcodexalimentariusnetsearchindexdoc Accessed January 2006
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
EM Merz RD Forrester MB Williamson RA Krishnamurti DS
Stevenson RE Dean JH (1995) Surveillance for anencephaly and spina bifida
and the impact of prenatal diagnosis--United States 1985-1994 MMWR CDC
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
serine and methionine kinetics Am J Physiol Endocrinol Metab 281 E1182-E1190
Daly D Mills JL Molloy AM Conley Lee YJ Kirke PN Weir DG Scott JM
(1997) Minimum effective dose of folic acid for food fortification to prevent neural
tube defects Lancet 350 1666-1669
204
Chapter 9 References
Daly LE Kirke PN Molloy AM Weir DG Scott JM (1995) Folate levels and
neural tube defects Implications for prevention JAMA 274 1698-1702
Darnton-Hill I (1998) Rationale and elements of a successful food-fortification program
ScrimshawNS 2 (19) Tokyo United Nations University Press Food and nutrition
bulletin httpwwwunueduunupressfoodV192ebeginhtm Accessed January
2006
Datamonitor (2004) World cheese consumptions patterns
httpwwwdatamonitorcom~99f16a6f99eb4d8687a6839536225437~ Accessed
May 2005
Day BP Gregory JF 3rd (1983) Thermal stability of folic acid and 5-tetrahydrofolic
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
Department of Health ndash UK (2000) Folic acid and the prevention of disease 2000 Report
on Health and Social Subjects no 50 London The Stationary Office
Department of Health and Human Services Public Health Service Food and Drug
Administration (DHHSPHS) (1992) Food standards amendment of the standards of
identity for enriched cereal-grain products to require the addition of folic acid
proposed rule USA Fed Regist 58 53305-53312
Deplancke B A Rex GH (2002) Redox control of the transsulfuration and glutathione
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
205
Chapter 9 References
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
DeZarn TJ (1995) Food ingredient encapsulation In Encapsulation and Controlled
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
206
Chapter 9 References
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-
237
Food Standards Australia New Zealand
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
Friggi A Bodard H Berenger FP Esclapez F Rolland PH (1989) Effects of SIN-1
on peripheral hemodynamics and viscoelastic properties of aorta in anaesthetised
rabbits Cardiovasc Drugs Ther 3 445-448
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
81
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
1221
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
total plasma Clin Chem 43 687-688
207
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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
eds Functional Foods II--Claims and Evidence Cambridge England Royal Society
of Chemistry Press pp82-90
Giovannucci E Rimm EB Ascherio A Stampfer ME Colditz GA Willett WC
(1995) Alcohol low-methionine-low-folate diets and risk of colon cancer in men
JNCI 87(4) 265-273
Glickman M (1969) Cellulose gums In Gum technology in the Food Industry Academic
Press Inc New York NY pp 398-471
Godfrey PSA Toone BK Carney MWP (1990) Enhancement of recovery from
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
trends Trends Food Sci Technol 15 330ndash347
Grant GT Morris ER Rees DA Smith PJC Thom D (1973) Biological
interactions between polysaccharides and divalent cations the egg-box model FEBS
Lett 32 195-198
Green JT Skeaff MC Rockell JEP Venn BJ (2005) Folic acid fortified milk
increases blood folate and lowers homocysteine concentration in women of child
bearing age Asia Pac J Clin Nutr 14 (2) 173-178
Greener IK Fennema O (1989) Evaluation of edible bilayer films for use as moisture
barriers for food J Food Sci 54 1400ndash1403
Gregoriadis G (1984) In Liposome Technology Vol 1ndash3 CRC Press Boca RatonFL
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Gregory JF 3rd (1989) Chemical and nutritional aspects of folate research analytical
procedures methods of folate synthesis stability and bioavailability of dietary
folates Adv Food Nutr Res 33 1-101
Gregory JF 3rd (1997) Bioavailability of folate Eur J Clin Nutr 51 Suppl 1S54-59
Han YH Yon M Hyun TH (2005) Folate intake with updated database and its
association to blood folate and homocysteine in Korean college students Euro J Clin
Nutr 59246-254
Harker LA Ross R Slichter SJ (1976) Homocysteine-induced arteriosclerosis The
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731-741
Hawkes JG Villota R (1989) a Folates in foods reactivity stability during processing
and nutritional implications Crit Rev Food Sci Nutr 28 (6) 439-539
Hawkes JG Villota R (1989) b Prediction of folic acid retention during spray
dehydration J Food Eng 10 (4) 287-317
Hertzberg S Kvittingen L Anthonsen T Skjadegk-Braeligk G (1990) Alginate as
immobilization material for biocatalysts in organic solvents Ann NY Acad
Sci 615 511-516
Hirsch S de la Maza P Barrera G Gattaacutes V Petermann M Bunout D (2002) The
Chilean Flour Folic Acid Fortification Program Reduces Serum Homocysteine
Levels and Masks Vitamin B-12 Deficiency in Elderly People J Nutr 132289-291
Homocysteine Studies Collaboration (2002) J Am Med Assoc 288 2015-2022
Honein MA Paulozzi LJ Mathews TJ Erickson JD Wong L-Y C (2001) Impact
of folic acid fortification of the US food supply on the occurrence of neural tube
defects JAMA 285 2981- 2986
Hoppner K Lampi B (1990) Total folate pantothenic acid and biotin content of yogurt
products Can Inst Food Sci Technol J 23 (4-5) 223-225
209
Chapter 9 References
Hugenholtz J Sybesma W Boels I Starrenburg MJC Chaves AC Mertens A
Kleerebezem M (2000) Metabolic engineering of lactic acid bacteria for the
improvement of fermented dairy products Proceedings of BTK2000 Animating the
cellular map (Hofmeyr Rohwer Snoep eds) p 285-290
Iyer C Kailasapathy K Peiris P (2004) Evaluation of survival and release of
encapsulated bacteria in ex vivo porcine gastrointestinal contents using a green
fluorescent protein gene-labelled E coli Food Sci TechnolLebensm-Wiss
Technol 37(6) 639-642
Jackson LS Lee K (1991) Microencapsulation and encapsulated ingredients Lebens
Wisst Und Technol 24 289ndash297
Janaswamy S Chandrasekaran R (2001) Three-dimensional structure of the sodium salt
of iota-carrageenan Carb Res 335 181-194
Jarvis M C (1984) Structure and properties of pectin gels in plant cell walls Plant cell
environ 7(3) 153-164
Jozwiaskowski MJ Jones D Franz RM (1990) Characterisation of a hot melt fluid bed
coating process from fine granules Pharm Res 7 3-10
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
Kang SS Wong PW Malinow MR (1992) Hyperhomocyst(e)inemia as a risk factor
for occlusive vascular disease Annu Rev Nutr 12 279ndash298
Kapusta L Haagmans MLM Steegers PAE Cuypers MHM Blom HJ Eskes
TKAB (1999) Congenital heart defects and maternal derangement of
homocysteine metabolism J Pediatr 135 773-774
210
Chapter 9 References
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
release kinetics J Pharm Sci 86(3) 323 ndash 328
Kim HHY Baianu IC (1991) Novel liposome microencapsulation techniques for food
applications Trends Food Sci Technol 2 55ndash60
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
high encapsulation efficiency under mild conditions In Liposome Technology Vol
1 Gregoriadis G Ed CRC Press Boca Raton FL
Kneifel W Kaufmann M Fleischer A Ulberth F (1992) Screening of Commercially
Available Mesophilic Dairy Starter Cultures Biochemical Sensory and
Microbiological Properties J Dairy Sci 75 3158-3166
Kneifel W Mayer H K (1991) Vitamin profiles of kefirs made from milks of different
species Int J Food Sci Technol 26 423-428
Koga T Claycombe K Meydani M (2002) Homocysteine increases monocyte and T-
cell adhesion to human aortic endothelial cells Atheroscler 161(2) 365-374
Kopelman IJ Meydav S Wwilmersdorf P (1977) Storage studies of freeze dried lemon
crystals J Food Technol 12 65ndash69
Kourkoutas Y Bosnea L Taboukos S Baras C Lambrou D Kanellaki M (2006)
Probiotic Cheese Production Using Lactobacillus casei Cells Immobilized on Fruit
Pieces J Dairy Sci 89 1439-1451
211
Chapter 9 References
Kumar M N V R Kumar N Domb A J Arora M (2002) A review of pharmaceutical
polymeric controlled drug delivery systems Adv Poly Sci 160 45 ndash 117
Lamb R (1987) Spray chilling Food Flav Ingredients Packag Process 9 (12) 39ndash42
LancasterP HurstT (2001) Trends in neural tube defects in Australia Canberra
Australian Food and Nutrition Monitoring Unit Commonwealth of Australia
Lane HW Nillen JL and Kloeris VL (1995) Folic acid content in thermostabilised and
freeze-dried space shuttle foods J Food Sci 30 538-540
Leboeuf R (2003) Homocysteine and Alzheimerrsquos disease J Am Diet Assoc 103304-307
Leichter J (1980) Folate content in the solid and liquid portions of canned vegetables Can
Inst Food Sci Technol 13 33ndash34
Lewis CJ Crane NT Wilson DB Yatley EA (1999) Estimated folate intake data
updated to reflect food fortification increased bioavailability and dietary
supplement use Am J Clin Nutr 70 198-207
Life Sciences Research Office (1992)Safety of amino acids used as dietary supplements
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controlled release formulation J Pharm Pharmacol 51 141-149
212
Chapter 9 References
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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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Trondhein Norway
213
Chapter 9 References
Maruyama T Shiota T Krumdieck CL (1978) The oxidative cleavage of folates ndash a
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Mason JB (1990) Intestinal transport of monoglutamyl folates in mammalian systems In
Folic Acid Metabolism in Health and Disease 1st Ed pp 47ndash64
Matsuda S Hatano H Kuramoto K Tsutsumi A (2001) Fluidization of ultrafine
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Mayer EL Jacobsen DW Robinson K (1996) Homocysteine and coronary
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McDonnell R Johnson Z Doyle A Sayers G (1999) Determinants of folic acid
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McKinley MC McNulty H McPartlin J Strain JJ Pentieva K Ward M Weir
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Clin Nutr 73 759-764
McLachlan J (1985) Macroalgae (seaweeds) industrial resources and their utilization
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McNulty H Cuskelly JG Ward M (2000) Response of red blood cell folate to
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Millqvist-Fureby A Malmsten M Bergenstahl B (2000) An aqueous polymer two-phase
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54ndash61
214
Chapter 9 References
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Levnedsmiddelkemi og Ernaeligring Levnedsmiddelstyrelsen Sundhedsministeriet
Denmark
Mnkeni AP Beveridge T (1983) Thermal destruction of 5-methyltetrahydrofolic acid in
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Muller H Diehl JF (1995) Effect of ionisation radiation on folates in food LWT Food
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Munjeri O Collet JH Fell JT (1997) Hydrogel beads based on amidated pectins for
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215
Chapter 9 References
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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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OrsquoConnor DL (1994) Folate status during pregnancy and lactation Adv Exp Med Biol 252
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Chapter 9 References
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375 ndash 380
Petri M Roubenoff R Dallal GE Nadeau MR Selhub J Rosenberg IH(1996)
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Pfeiffer CM Caudiall SP Gunter EW Bowman BA Jacques PF Selhub J
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2854
217
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Polk AE Amsden B Scarratt DJ Gonzal A Okhamafe AOGoosen MFA (1994)
Oral delivery in aquaculture controlled release of proteins from chitosan-alginate
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Poncelet D Lencki R Beaulieu C Halle J P Neufeld R J Fournier A (1992)
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Prevost H Divies C Rousseau E (1988) Continuous production with Lactobacillus
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Queensland Health (2002) An overview of indicators of nutritional status of Queensland
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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
218
Chapter 9 References
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Quinlivan EP Gregory JF III (2003) Effect of food fortification on folic acid intake in
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Rader JI Weaver CM Angyal G (2000) Total folate in enriched cereal-grain products
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1174
Rao DR Shahani KM(1987) Vitamin content of cultured milk products Cultur Dairy
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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
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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
801
Rees DA (1969) Structure conformation and mechanism in the formation of
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Reeves PG Nielsen FH Fahey GC (1993) AIN purified diets for laboratory rodents
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Refsum H Ueland PM Nygaringrd O Vollset SE (1998) Homocysteine and
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219
Chapter 9 References
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Reineccius GA (1991) Carbohydrates for flavour encapsulation Food Technol 46 (3)
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Renner E (1983) Milk and dairy products in human nutrition VV GmbH
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Roberfroid MB (1998) Prebiotics and synbiotics concepts and nutritional properties Br J
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Rumpler K Jacob M (1998) Continuous coating in fluidised bed Food Market Technol
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Sairam M Babu VR Rao KSVK Naidu BVK Hosamani KM Aminabhavi TM
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220
Chapter 9 References
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Sandberg A Andersson H Hallgren B Hasselblad K Isaksson B Hulteacuten L (1981)
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Schader I Corwin P (1999) How many pregnant women in Christchurch are using folic
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SchollTO JohnsonWG (2000) Folic acidinfluence on the outcome of pregnancy Am J
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Schorah CJ Devitt H Lucock MD Dowell AC (1998) The responsiveness of plasma
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Scott J Rebeille F Fletcher J (2000) Review ndash Folic acid and folates the feasibility for
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Seiss W Divies C (1975) Microencapsulation Angewandte Chemie Int Ed 14 539ndash550
Selhub J Dhar GJ Rosenberg IH (1983) Gastrointestinal absorption of folates and
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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
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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
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Shrestha KA Jayashree A Paterson LJ (2003) Edible coating materials ndash their
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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
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Slattery ML Potter DJ Samowitz W Schaffer D Leppert M (1999)
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Smidsrod O Skjak-Braek G (1990) Alginate as immobilization matrix for cells Trends
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SPSS (2002) SPSS 1150 for Windows Standard version SPSS Inc
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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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Tabata Y Ikada Y (1998) Protein release from gelatin matrices Adv Drug Del Rev
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Takahashi M Ikeda U Masuyama J Kitagawa S Kasahara T Saito M Kano S
Shimada K (1994) Involvement of adhesion molecules in human monocyte
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Takka S Ocak OH Acarturk F (1998) Formulation and investigation of nicardipine
HCI-alginate gel beads with factorial design-based studies Euro J Pharm Sci 6
241-246
Taylor AH (1983) Encapsulation systems and their applications in the flavour industry
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Thies C (1987) Microencapsulation In Encyclopedia of Polymer Science and Engineering
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editors] New York John Wiley amp Sons
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
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Ueland PM Refsum H Beresford SA Vollset SE (2000) Am J Clin Nutr 72 324-
332
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Chapter 9 References
Ueland PM Refsum H Stabler SP Malinow MR Andersson A Allen RH (1993)
Total homocysteine in plasma or serum methods and clinical applications Clin
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Vahteristo L Lehikoinen K Ollilainen V Koivistoinen PEVARO P (1998)
Ovenbaking and frozen storage affect folate vitamer retention Lebensm-Wiss u-
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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
homocysteine folate vitamin B12 and on the risk of premature atherosclerosis
Atheroscl 141 (1) 161-166
Vitamin Study (1991) Prevention of neural tube defects results of the medical research
council vitamin study Lancet 338 (8760) 131-137
Vollset EM Refsum H Tverdal A Nygaringrd O Nordrehaug JE Tell GS Ueland
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Voorn JTG Overbeek MJ (1957) Phase separation in polyelectrolyte solutions Theory
of complex coacervation J Cellul Comp Physiol 49 Suppl 1 7ndash26
225
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Wagner C (1985) Folate-binding proteins Nutr Rev 43 293ndash299
Wald DS Law M Morris JK (2002) Homocysteine and cardiovascular disease
evidence on causality from a meta-analysis Br Med J 325 1202-1208
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-
134
Wenstrom KD Johanning LG Johnston KE Dubard M (2001) Association of the
C677T methylenetetrahydrofolate reductase mutation and elevated homocysteine
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815
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
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Sci Nutr 47 315-322
Wigertz K Svensson UK Jagerstad M (1996) Folate and folate binding protein content
in dairy products J Dairy Res 64 239-252
226
Chapter 9 References
Williams PG Ross H Miller B (1995) Ascorbic acid and 5-methyltetrahydrofolate
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Sci 60 541-546
Wills L (1931) Treatment of pernicious anaemia of pregnancy and tropical anaemia
with special reference to yeast extract as a curative agent Ind J Med Res 17 777-
779
Witthoft CM Forssen K Johannesson L Jagerstad M (1999) Folates-food sources
analyses retention and bioavailability Scandanav J Nutr 43 138-146
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
227
Chapter 9 References
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
228
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