Post on 03-Oct-2021
transcript
University of VermontScholarWorks @ UVM
Graduate College Dissertations and Theses Dissertations and Theses
2015
Physicochemical Properties, Microstructure andProbiotic Survivability of Non-Fat Goat's MilkYogurt Using Heat Treated Whey ProteinConcentrate as a Fat ReplacerJames Thomas McCarthyUniversity of Vermont
Follow this and additional works at: https://scholarworks.uvm.edu/graddis
Part of the Food Science Commons, and the Nutrition Commons
This Thesis is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion inGraduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contactdonna.omalley@uvm.edu.
Recommended CitationMcCarthy, James Thomas, "Physicochemical Properties, Microstructure and Probiotic Survivability of Non-Fat Goat's Milk YogurtUsing Heat Treated Whey Protein Concentrate as a Fat Replacer" (2015). Graduate College Dissertations and Theses. 442.https://scholarworks.uvm.edu/graddis/442
PHYSICOCHEMICAL PROPERTIES, MICROSTRUCTURE AND PROBIOTIC
SURVIVABILITY OF NON-FAT GOAT’S MILK YOGURT USING HEAT
TREATED WHEY PROTEIN CONCENTRATE AS A FAT REPLACER
A Thesis Presented
by
James T. McCarthy
to
The Faculty of the Graduate College
of
The University of Vermont
In Partial Fulfillment of the Requirements
For the Degree of Master of Science
Specializing in Nutrition and Food Sciences
October, 2015
Defense date: May 5, 2015
Thesis Examination Committee
Mingruo Guo, Ph.D., Advisor
Jana Kraft, Ph.D., Chairperson
Stephen Pintauro, Ph.D.
Cynthia J. Forehand, Ph.D., Dean of the Graduate College
ABSTRACT
Probiotic dairy foods, especially non- and low-fat dairy products, are becoming
popular in the US. A non-fat goat’s milk yogurt containing probiotics (Lactobacillus
acidophilus and Bifidobacterium spp.) was developed using heat-treated whey protein
concentrate (HWPC) as a fat replacer and pectin as a thickening agent. Yogurts
containing non-heat treated whey protein concentrate (WPC) and pectin as well as one
with only pectin were also produced. A fat-free cow’s milk yogurt with pectin was also
used as a control yogurt. The yogurts were analyzed for chemical composition, water
holding capacity (syneresis), microstructure, changes in pH and viscosity, mold, yeast
and coliform counts, and probiotic survivability during storage at 4oC for 10 weeks. The
results showed that the non-fat goat’s milk yogurt made with 12% HWPC (12.5% WPC
solution heated at 85oC for 30 min at pH 8.5) and 0.35% pectin, had a significantly higher
viscosity (P<0.01) than any of the other yogurts and low syneresis than the goat’s yogurt with only pectin added (P<0.01). After 10 weeks in storage, viscosity and pH remained constant throughout all of the yogurts. Mold, yeast, and coliform counts were negative throughout the 10 week study. Bifidobacterium spp. remained stable and counts remained
above 106CFU g
-1 during the 10 week storage. However, the population of Lactobacillus
acidophilus dropped below 106CFU g
-1 after 2 weeks of storage. Microstructure analysis
of the non-fat goat’s milk yogurt determined by scanning electron microscopy revealed that HWPC interacted with casein micelles to form a more comprehensive network in the yogurt gel. The results indicate that HWPC could be used as a fat replacer to improve the consistency of non-fat goat’s milk yogurt and other products alike.
ii
ACKNOWLEDGEMENTS
I would like to thank the following:
Dr. Mingruo Guo, who has been an indispensable mentor on my journey to complete this
work. His patience with me over the years has certainly been great, but has always
managed to push my work further than I otherwise would have. His insights have been
invaluable and I certainly have enjoyed our many talks that always seemed to wander to
subjects outside of science.
Dr. Tiehua Zhang, who guided me through the research on this project.
Dr. Stephen Pintauro, who went to bat for me when dealing with the Graduate College
and was always ready to lend a friendly ear to listen to my frustrations.
Dr. Jana Kraft, the chair of my committee who was not afraid to criticize my work and
would help steer me in new directions for research.
Dr. Jane Ross, who, unbeknownst to her, was the reason I pursued this difficult path in
the first place.
And finally I need to thank Helen Walsh, who helped me and listened to many of my
petty problems over the years.
Thank you all.
iii
DEDICATION
I dedicate this work to my parents, Tom and Lois McCarthy, whose support has always
been a pillar of stability over the many years.
And to my loving fiancé, without whom I would never have completed this difficult task.
iv
TABLE OF CONTENTS
AKNOWLEDGMENTS………………………………………………………………………...…ii
DEDICATION…………………………………………………………………………………….iii
CHAPTER 1: COMPREHENSIVE LITERATURE REVIEW ....................................................... 1
1.1 Introduction ............................................................................................................................ 1
1.2 Goat Milk Chemistry ............................................................................................................. 1
1.2.1. Protein ............................................................................................................................ 1
1.2.2. Casein content ................................................................................................................ 2
1.2.3. Whey Proteins ................................................................................................................ 2
1.2.4. β-lactoglobulin ............................................................................................................... 3
1.2.5 α-lactalbumin .................................................................................................................. 3
1.2.6. Other Proteins ................................................................................................................ 4
1.2.7. Milk Fat Globule Membrane .......................................................................................... 5
1.2.8. Bioactive Peptides .......................................................................................................... 7
1.2.9. Non-Protein Nitrogen Compounds ................................................................................ 8
1.2.10. Goat Milk Protein Allergy ........................................................................................... 9
1.2.11. Carbohydrates ............................................................................................................ 10
1.2.12. Vitamins and Minerals ............................................................................................... 12
1.2.13. Fats ............................................................................................................................. 13
1.2.14. Medium Chained Fatty Acids .................................................................................... 14
1.2.15. Conjugated Linoleic Acid .......................................................................................... 15
1.2.16. Variation in Goat Milk ............................................................................................... 15
1.3 Yogurt Manufacturing ......................................................................................................... 17
1.3.1. Acid Induced Coagulation of Milk .............................................................................. 17
1.3.2. Goat Milk Yogurt ......................................................................................................... 18
1.3.3. Fat Replacers ................................................................................................................ 19
1.3.4. Heat-Treated Whey Protein ......................................................................................... 21
1.4. Probiotics ............................................................................................................................. 22
vii
1.4.1. Pathogen Inhibition ...................................................................................................... 23
1.4.2. Prevention of Diarrhea ................................................................................................. 25
1.4.3. Irritable Bowel Syndrome ............................................................................................ 28
1.4.4. Hepatic Disease ............................................................................................................ 30
1.4.5. Alcoholic Liver Disease ............................................................................................... 32
1.4.6. Genetic Engineering of Probiotics ............................................................................... 33
1.4.7. Oral Health ................................................................................................................... 33
CHAPTER 2: MANUSCRIPT....................................................................................................... 36
2.1 Introduction .......................................................................................................................... 36
2.2. Materials and Methods ........................................................................................................ 38
2.2.1. Materials ...................................................................................................................... 38
2.2.2. Preparation of Heat-Treated Whey Protein (HWPC) ................................................... 39
2.2.3. Preparation of Yogurt ................................................................................................... 39
2.2.4. Chemical Analysis ....................................................................................................... 40
2.2.5. Syneresis Testing ......................................................................................................... 40
2.2.6. Mold, Yeast and Coliform Counts and the Survivability of Probiotics ....................... 41
2.2.7. pH and Viscosity .......................................................................................................... 41
2.2.8. Microstructure Analysis by Scanning Electron Microscopy (SEM) ............................ 42
2.2.9. Statistical Analysis ....................................................................................................... 42
2.3 Results and Discussion ........................................................................................................ 42
2.3.1. Determination of Proper Amounts of HWPC and Pectin............................................. 42
2.3.2. Chemical Composition ................................................................................................. 44
2.3.3. Syneresis ...................................................................................................................... 45
2.3.4. Changes in pH During Storage .................................................................................... 46
2.3.5. Changes in Viscosity During Storage .......................................................................... 47
2.3.6. Probiotic Survivability During Storage ........................................................................ 48
2.3.7. Mold, Yeast, and Coliforms ......................................................................................... 50
2.3.8. Microstructure .............................................................................................................. 50
2.4. Conclusions ......................................................................................................................... 53
vii
2.5. Financial Acknowledgments ............................................................................................... 54
2.6. References ........................................................................................................................... 55
1
CHAPTER 1: COMPREHENSIVE LITERATURE REVIEW
1.1 Introduction
Goat milk is an important source of nutrition around the globe and has been
labeled as “the cow of the poor man” due to the prevalence of goat herds in less
industrialized countries (Haenlein, 1996). Goat milk is also on the rise in westernized
countries. This is due to its importance in treatment of allergies, medicinal properties, as
well as a growing demand upon gourmet food connoisseurs for various goat milk
products.
1.2 Goat Milk Chemistry
1.2.1. Protein
Goat milk, like cow milk, is a good source of high quality protein that meets or
exceeds the amounts of essential amino acids needed for human nutrition (Jenness, 1979).
In general, goat milk contains higher levels of protein ranging from 2.6g/L to 4.1g/L
(Raynal-Ljutovac et al., 2008). Having higher protein content offers one of the benefits
of drinking goat milk for people with acidity problems. Because of the elevated protein
content, goat’s milk has a higher buffering capacity. The different arrangements of
phosphates in those proteins also contribute to this (Jandal, 1996). Goat’s milk contains
several different classes of proteins including caseins and whey proteins some of which
are similar to those found in cow’s milk. However, profile of goat’s milk casein varies
greatly from cow milk, as due the type, size, and distribution of the casein micelles.
2
1.2.2. Casein content
Casein is the prominent non-soluble protein in goat milk. They consist of αs1-
casein, αs2-casein, β-casein, and κ-casein. Unlike cow’s milk where the major constituent
is α-casein, goat’s milk contains much more β-casein (Park & Hanlein, 2006). This
difference in caseins gives goat milk a much more friable curd upon coagulation. This is
not only important for processing but also in human nutrition. During processing a
weaker gel is formed, producing softer cheeses and watery fermented yogurts. In human
nutrition, this characteristic is important because the softer curd allows for better
digestibility. The soft curds allow for better mixing in the stomach so enzymes can
hydrolyze proteins and fat more easily. Another aspect of caseins in goat’s milk is that
they are also larger in size than in cow’s milk. This has important implications in goat
milk technology, but at this point the nutritional impact of this is unknown (Park, 2007).
1.2.3. Whey Proteins
Goat’s milk whey proteins consist of β-lactoglobulin, α-lactalbumin,
immunoglobulins and serum albumin. Serum albumins and immunoglobulins are
identical to those found in the blood and are not specific to the goat milk (Park et al,
2007). Whey proteins are broken down in the gut to amino acids and peptides, some of
which are biologically active and can have implications for human health. The
physiological effects of consuming whey protein that have been explored include an
increase in physical performance, prevention of muscle atrophy, recovery after exercise
for athletes, improved cardiovascular health, anti-cancer effects, management of
infections, and healthy aging (Smithers, 2008).
3
1.2.4. β-lactoglobulin
Goat milk β-lactoglobulin is a small, soluble globular protein which makes up the
largest fraction of non-casien protein. It consists of 162 amino acid residues, which
differs from bovine milk β-lactoglobulin at six residues which produces a protein that has
one more positively charged amino acid and three less negative groups at pH 5-9
(Jenness, 1980). This difference causes the goat milk β-lactoglobulin to have a slower
electrophoretic mobility at alkaline pH and a different titration curve than its cow
counterpart (Park & Haenlein, 2006). At pH 3-7, β-lactoglobulin is a dimer that has a
molecular mass of 36 kDa, while at pH <3 it exists as a monomer (Chatterton et al. 2006).
The functionality of β-lactoglobulin is attributed to its sulfur bridges between amino acid
residues Cys66-Cys160 and Cys106-Cys119 and the free thiol group, Cys121
(McKenzie, Ralston, and Shaw, 1972).
1.2.5 α-lactalbumin
Goat α-lactalbumin, like bovine α-lactalbumin, is a small, globular protein
containing 123 residues and plays an important role in lactose synthesis (Park et al.
2007). It differs from cow α-lactalbumin by 12 amino acid residues, most notably being
the absence of methionine (Park & Haenlein, 2006). α-lactalbumin owes its functionality
to five aspartate amino acids on residues 79-88 which gives it a high affinity for metal
ions, calcium in particular (Chatterton et al., 2006). During the biosynthesis of milk, α-
lactalbumin acts by changing the Km of glucose to make it a suitable substrate to accept
galactose from urinediphophogalactose with the aid of the Mn2+
complexed enzyme
galactosyltransferase (Ebner & Schanbacher, 1974).
4
1.2.6. Other Proteins
Lactoferrin is a protein found in the whey of goat’s milk. Lactoferrin is a
glycoprotein that can bind iron and has been shown to have a distinct bacteriostatic
potential on harmful microorganisms (Lee et al. 1997). Patton et al. (1997) observed the
presence of prosaposins in goat, cow, and human milk. Prosaposins are a neurotrophic
factor and are required for membrane renewal.
Folate-binding protein is another protein of importance as it is much higher in
goat’s milk than in cow’s milk. This protein binds folate and causes the observable folate
content to be much lower. This can lead to infants fed solely on goat’s milk to form what
is known as ‘goat’s milk anemia’ because folate is required for the formation of
hemoglobin (Jenness, 1979).
Cow’s milk also contains proteins known as agglutinating euglobulins which aid
in the clustering of milk fat globules and will lead to better creaming than observed in
goat’s milk. Jenness and Parkash (1971) showed that goat’s cream, when reconstituted
into whole milk with skim milk from cows, had similar creaming properties to that of
cows. They concluded that it was this protein, and not the size of the fat globules, that is
responsible for the better creaming rate of bovine milk. In 1984, Euber and Brunner
found the protein in cow’s milk responsible for creaming by adding various protein
fractions to heated milk and observing the separation of fat and identified the protein as
immunoglobulin M (Ig M). IgM absorbs onto a fat globule allowing it to cluster. They
postulated that antibodies specific to the milk fat globule membrane (MFGM) are
responsible for clustering. Patton et al. (1980) observed that when MFGM antiserums
5
where injected into the udder of a goat, the clustering of fat globules became so great that
they were not able to be re-dispursed even after violent shaking, furthering the antibody
clustering theory. Euber and Brunner (1984) further discuss that during homogenization,
complexes between IgM and κ-casein form. Because IgM is unavailable to interact with
MFGM, clustering does not occur and creaming is not observed in bovine milk.
1.2.7. Milk Fat Globule Membrane
The milk fat globule is made up of a thin, amphilic membrane which surrounds a
triglyceride core. The milk fat globule membrane (MFGM) is about 10-20 nm across and
keeps the globules from coalescing and protects from enzymatic degradation. It is made
up of many different components including glycoproteins, non-polar lipids,
phospholipids and sphingolipids which all contribute to the nutritional and technological
aspects of the MFGM (Dewettinck et al., 2008).
Isolates of MFGM are important in the food manufacturing industry for their use
as emulsifiers where they can be added to chocolate to prevent crystallization and
increase viscosity, prevent staling in baked products, improve rehydration in instant
products, and to stabilize margarine (Vamhoutte et al., 2004). MFGM phospholipids are
structurally different from soy and egg derived phospholipids. They can be used to create
lipid bilayers which surround an aqueous core in which hydrophobic molecules can be
trapped in the lipid portion and hydrophilic molecules can be inserted in the core. Their
unique composition enables them to be used for a range of applications in food such as
protecting sensitive ingredients, confining undesirable flavors, and increasing the
effectiveness of food additives (Singh, 2006). The sphingolipid portion of the MFGM has
6
a special use in the cosmetics industry where it is used as a precursor for the production
of ceramides which increase the water-holding properties of the epidermis and is added to
many skin care products (Zhang, Hellgren & Zu, 2006).
In its native state, the milk fat globule will act as filler in milk based gels.
However, most milk gels undergo some processing such as pasteurization and
homogenization which has an effect on the MFGM proteins. Processing of the milk
causes cross linking of proteins through the induction of disulfide bridges between β-
lactoglobulin and κ-casien which are then absorbed into the MFGM and increasing the
strength of the milk gel (Ye et al., 2004).
Dewettinch et al. (2008) explored the nutritional aspects of components of the
MFGM and found many claims. The researchers found that sphingolipids had a positive
effect on cancer and other bowel-related diseases, have a hypocholesterolemic effect, and
are bactericidal which can protect the human gut from infection. Phospholipids may
improve exercise performance by altering neuroendocrine function which leads to
lessened perceived muscle soreness. On the negative side, butyrophilin, a protein found
in the MFGM, may be one of the factors responsible for multiple sclerosis, an
autoimmune disease which affects the central nervous system.
It should be noted that much of the research on the properties of the MFGM are
from bovine milk. Cebo et al., (2009) set out to elucidate the proteins that are found in
the goat MFGM. They observed differences in the glycoprotein fraction, and found that
goat mucins are considerably larger than their cow counterparts. Caseins were also
7
present in the goat MFGM in contrast to none found in the cow MFGM. The researchers
hypothesized that this fact could support the theory that goat’s milk is formed from a
singular secretion mode known as an apocrine process. Other proteins of the goat
MFGM include mucin-1, mucin-X, fatty acid synthase, xanthine oxidase, butyrophilin,
lactadherin, and adipophilin.
1.2.8. Bioactive Peptides
Bioactive peptides are produced from the hydrolysis of proteins by enzymes in the
gut or microbiologically during fermentation. They are described as peptides which are
biologically active, having antihypertensive, antimicrobial, opiod, antioxidant,
immunomodulant, or mineral-binding properties in humans (Park et al. 2007).
Angiotensin converting enzyme (ACE) inhibitors are of special interest in the
treatment of high blood pressure. ACE is an enzyme which has the ability to regulate
blood pressure by producing vasopressor angiotensin II and turning off the vasodepressor
bradykinin. Inhibition of this enzyme is used to lower blood pressure in patients with
hypertension. While most studies are from peptides from cow’s milk, Park et al. (2007)
reported that ACE inhibitors can be produced from the β-lactoglobulin, κ-casein, and αs2-
casein found in goat’s milk.
Bioactive peptides have also been demonstrated to contribute to antibacterial
qualities in goat’s milk. Bactericidal properties have been observed in peptides that exert
great influence over both Gram-negative and Gram-positive bacteria (Wakabayashi et al.
8
2003). They have been found to be peptides from the hydrolysis of both whey and casein
fractions of goat’s milk.
Cardiovascular diseases are the leading cause of death in the modernized
countries (WHO, 2004). Thrombosis is the formation of a blood clot in the circulatory
system which can lead to tissue death, brain damage, or heart attack. Peptides from
caprine κ-casein were found to inhibit fibrinogen binding and therefore inhibit platelet
aggregation in the circulatory system (Manso et al, 2002).
Park et al. (2007) reviewed the literature and found several studies that indicated
the bioactive potential of milk proteins. Opioid behavior of certain hydrolysates of
bovine whey and casein was observed in one study. These peptides can affect social
behavior, lengthen gastric transit time by inhibiting peristalsis, modulate amino acid
uptake, and stimulate endocrine responses such as pancreatic release of insulin. Other
studies observed casein phosphopeptides forming soluble salts acting as carriers for
minerals as well as studies that described peptides having antioxidant properties. While
many of these studies were on bovine milk proteins, Park et al. (2007) suggests that due
to the great similarities of the sequence between proteins of different species, goat milk
peptides are predicted to have similar properties.
1.2.9. Non-Protein Nitrogen Compounds
Goat’s milk has a higher non-protein nitrogen (NPN) content than cow’s milk and
consists of ammonia, urea, creatinine and free amino acids (Park & Haenlein, 2006).
Two of the most significant NPN compounds found in goat’s milk are carnitine and
9
taurine. Carnitine is found in the whey fraction of goat’s milk. This compound has been
well studied and is most noted for its role in carrying fatty acids into the mitochondria for
oxidation and production of energy. Taurine plays a role in cardiovascular function and
development of skeletal muscle.
1.2.10. Goat Milk Protein Allergy
There has been much research in the area of replacing cow’s milk with goat’s
milk for people sensitive to the proteins in cow’s milk. Park (1994a) suggests that
anywhere from 40-100% of people who are allergic to cow’s milk may tolerate goat’s
milk. Symptoms of allergies include vomiting, diarrhea, colitis, epigastric distress,
malabsorption, eczema, urticarial, rhinitis, asthma, bronchitis, anaphylaxis, hyperactivity,
or migraines. Two kinds of allergic reactions exist. The first kind is reaginic (IgE
mediated) and is the most dangerous one and involves immediate hypersensitivity.
Histamine is released in tissues and causes vasodilation, smooth muscle contraction, and
secretion of mucus. A severe reaction can even lead to respiratory failure and ultimately
death. The second kind is nonreaginic and antibodies in milk react with antigen to form
complexes that can lead to inflammation and cytopathic effects.
αs1-Casein is the main constituent of cow’s milk and thought to be one of the
major proteins contributing to allergic reactions (β-lactoglobulin is the other major
culprit, also present in large amounts of caprine milk). The theory is that goat milk
lacking αs1-casein is therefore less allergenic (El-Agamy, 2007; Park 1994a). While
much anecdotal evidence exists that many people who cannot tolerate cow’s milk can
tolerate goat’s milk, there is much conflicting evidence. Restani (2004) suggests that the
10
low prevalence of people allergic to goat’s milk might be due to the fact that people with
cow’s milk allergy avoid milk altogether and is therefore underreported. The author
further points to a double-blind, placebo controlled study that used a skin prick test to test
for the allergenicity of goat’s milk vs cow’s milk. They showed that out of the 26
children who were sensitive to cow’s milk, 24 were also sensitive to goat’s milk.
However, the study did also find that the oral dose to elicit a response was much higher
for goat’s milk.
There is also evidence of people being allergic to goat’s milk but show signs of
tolerance for cow’s milk. Umpierrez et al. (1999) studied this phenomenon. They
observed one patient to have this specific affliction, but the patient was sensitive to the
casein component in the goat’s milk and cheese. In a 2006 study by Ah-Leung at al., 28
children were observed to have this condition. It was concluded that the characteristics of
the allergy are different than from cow’s milk allergy as it manifests itself later in a
child’s life. Moreover, all reactions to the goat milk protein were observed to be acute
and could be life threatening in all cases whereas cow’s milk allergy may be less severe.
1.2.11. Carbohydrates
Goat milk contains many carbohydrates, the largest fraction being the milk sugar
lactose which is a disaccharide containing one molecule of glucose and one molecule of
galactose attached by a β-1→4 glycosidic link (Park & Haenlein, 2006). It constitutes
about 44% of the total carbohydrates in goat milk and between 4.1% and 4.8% of the
weight of the whole milk (Park et al., 2007; Raynal-Ljutovac et al., 2008). Lactose is an
11
important sugar in human nutrition as it aids the intestinal assimilation of magnesium,
calcium, phosphorus, and vitamin D (Campbell & Marshall, 1975). Lactose also plays
an important role in the development of fermented milk products, as bacteria can use it as
a source of energy, hydrolyzing the sugar into smaller units consisting of lactic acid
which drops the pH of the milk and causes it to curdle.
Other carbohydrates in goat milk include oligosaccharides, glycoproteins,
glycopeptides, and nucleotide sugars (Park et al., 2007). Oligosaccharides are
carbohydrates containing 3-10 monosaccharie residues and are important in the growth of
bifidobacteria in the newborn gut, have strong antigenic properties, and may play a role
in brain development in the young (Gopal & Gill, 2000). Lara-Villoslada et al. (2006)
looked at the effect of goat milk oligosaccharides on induced rat colitis and found them to
have anti-inflammatory properties.
Goat milk contain a higher amount of nucleotide sugars (154µmol/100ml) than in
in cow milk (68µmol/100ml) (Park et al, 2008). These sugars are important in the
biosynthesis of milk as they act as glycosyl donors for glycosyltransferase in the
mammary gland and are precursors to glycoproteins, glycolipids, and oligosaccharides.
Glycoproteins and glycolipids are found in goat milk as well, many of which reside in the
milk fat globule membrane. Of particular note, ganglioside, which is a sialic acid that
contains glycosphingolipids, has been shown to protect against enterotoxins and
infections in the young (Puente et al. 1996).
12
1.2.12. Vitamins and Minerals
Park and Hanlein (2006), Park and Guo (2006), Park (1994b)and Raynal-Ljutovac
et al. (2008) examined the vitamin and mineral content of commercial goat milks. While
sometimes drastic variations between studies were observed, there is a general consensus
that goat’s milk is higher in calcium, phosphorus, potassium, magnesium and chlorine
while lower in both sodium and sulfur when compared to the cow’s milk counterpart. As
for vitamins, a serving of goat’s milk meets or exceeds the recommended daily amount of
thiamin, riboflavin, and pantothenate, has adequate amounts of vitamin A and niacin, and
is deficient in vitamin C, vitamin D, vitamin B12, pyridoxine, and folate. Folate
deficiency is due to the high levels of the folate-binding protein as mentioned earlier.
Aliaga et al. (2000) fed rats with a diet consisting of either goat’s milk, cow’s
milk or a standard diet. It was found that calcium and iron were better absorbed by rats
on the goat’s milk diet. Their hypothesis about why this was observed was that although
deficient, goat’s milk contains a higher level of vitamin D than cow’s milk which favors
the absorption of calcium. The higher level of cysteine in goat’s milk is thought to be the
reason for the increased uptake of iron. Another observed outcome of the study was that
the rats fed goat’s milk also ate less, thought to be due to the higher levels of medium-
chained fatty acids. These are absorbed at a faster rate and delivered directly to the liver.
Therefore, the rat is able to meet its energy needs sooner and becomes satiated faster.
This could have important implications in the study of weight management.
Many studies have been conducted on the effect of goat’s milk on the absorption
of various minerals. Barrionuevo et al. (2002) studied malabsorption syndrome induced
13
rats fed a diet of either goat’s or cow’s milk. They showed that rats fed goat’s milk
absorbed and utilized iron and copper better than the group fed cow’s milk. In a study of
anemic rats, Park et al. (1994a) investigated the effect of goat milk on the bioavailability
of iron. Because of the low levels of iron present in milk, both groups of rats were
supplemented with ferrous sulfate. They showed that rats fed with goat’s milk showed
more iron gain in their hemoglobin than rats fed the cow’s milk diet.
1.2.13. Fats
Fats in goat milk differ from cow’s milk in that the milk fat globules are smaller
and they have a higher level of medium-chain fatty acids (MCFA) than cow’s milk fat.
Goat’s milk fat globules range in size from 0.73-8.58 μm in diameter with an average size
of 2.76 μm compared to that of bovine fat globules with an average size of 3.51 μm
(Attaie, 2000). However, the smaller globules in goat’s milk make it less susceptible to
creaming and is therefore naturally homogenized. In human nutrition this is important
because the smaller globules have a greater total surface
area (21,778 cm2/mL vs 17,117 cm
2/mL) and are more easily hydrolyzed by pancreatic
lipase (Park & Haenlein, 2006). Meena, Rajput & Sharma (2014) compared the
digestibility of cow’s, goat’s, camel’s, and buffalo’s milk by subjecting each to
pancreatic lipase for a five hour period after each of the milks was adjusted to 3% fat.
They found that the amount of free fatty acids present in the milk was inversely
proportional to the size of the milk fat globule with the camels’ and the goat’s milk being
the most highly digested.
14
The lipid profile of goat’s milk is much different from that of cow’s milk. Goat’s
milk has higher levels of caproic (C6:0), caprylic (C8:0), capric (C10:0), lauric (C12:0),
branched-chain fatty acids, and total saturated fatty acids than cow’s milk with lower
levels of cholesterol and conjugated linoleic acid (Alonso et al. 1999). Branched chained
fatty acids and MCFAs mayplay a role in the flavor profile of goat milk.
1.2.14. Medium Chained Fatty Acids
The high levels of MCFA in goat’s milk have important influence on energy
utilization and production. They are also of value in the medical field treating patients
with various ailments. As the MCFA are hydrolyzed in the gastrointestinal tract, they are
quickly absorbed by the intestinal cells without the need for esterification and carried by
the hepatic portal vein to the liver where they can be oxidized for quick energy. This
quick metabolism produces a thermal expenditure which may play an important role in
weight reduction as well as reducing circulating cholesterol, especially low density
lipoproteins (Chillard, 2007; Raynal-Ljutovac, 2008).
The high digestibility of MCFA also has implications on nutrient absorption.
Tantibhedhyangkul and Hashim (1975) studied premature infants fed various diets
consisting of different levels of MCFA in addition to their normal diets. The results
showed that the infants fed the highest amount of MCFAs had better fat and nitrogen
absorption, but also increased calcium and magnesium absorption.
Another study by Alferez (2001) showed that rats with induced malabsorption
syndrome fed a diet of either cow’s milk or goat’s milk were able to better digest the
15
goat’s milk fat more easily and closely resembled the digestibility of the control of olive
oil. They also noted that rats fed on the goat’s milk diet had lower circulating levels of
cholesterol.
1.2.15. Conjugated Linoleic Acid
Conjugated linoleic acids (CLA) have gained much attention in recent years due
to their implication in human health. CLA may have beneficial health benefits including
suppression of cancer cells, anti-obesity, modulation of the immune system, reduction of
atherosclerosis, and diabetes. However, goat’s milk does not contain levels of CLA as
cow’s milk (Park, 2007).
Ledoux et al. (2002) also observed a different fatty acid profile for goats fed on
different diets. Goats were fed a diet of either alfalfa hay or Rumiluz (dehydrated alfalfa
from France) plus forage. Goats fed the Rumiluz diet showed an increase in the
production of trans-11-C18:1 fatty acid, or vaccenic acid. Because vaccenic acid is a
precursor of CLA, Park (2007) suggests that higher levels of vaccenic acid in goat’s milk
could lead to higher levels in humans.
1.2.16. Variations in Goat Milk
Levels of nutrients can vary greatly in goat’s milk due to varying production
systems, stage of lactation, season, climate, milking frequency, breed, goat hygiene
and/or feed (Goetsch, Zeng, and Gipson, 2011; Raynal-Ljutovac et al., 2008; Park et al.,
2007). This varies from cow’s milk in that variability is not readily observed because of
extensive co-mingling of milk from several different herds that produce milk year round.
16
It would seem likely that cow’s milk from small farmsteads would be affected by the
same variables as goat’s milk.
Guo et al. (2001) studied the seasonal changes in the properties of goat’s milk
over a one year period from co-mingled milk from 5 different breeds of goat from 12
different herds around New England and found that the protein levels were highest in
summer. The higher protein content of summer milk is superior for making certain types
of cheese because of a higher yield. Winter milk contained higher levels of fat, ash, and
calcium.
The influence on feeding systems was investigated by Morand-Fehr et al. (2007).
They found that goats fed primarily on pasture had lower cholesterol than the silage fed
counterparts. In addition, they also found that retinol and α-tocopherol was higher in the
goats fed primarily on pasture. Tsiplakou (2006) studied seasonal variations in CLA
levels in several herds of goat and sheep. While a dramatic increase was seen during the
early summer months for sheep’s milk, goat’s milk remained constant throughout the
summer. This is due to the sheep eating the rapidly growing green grass while the goat’s
diet consists mainly of shrubs. As expected, the levels of CLA in sheep’s milk were
observed to be higher, however the levels of CLAs in both sheep and goat’s milk was
higher than the milk taken from the non-grazing animals kept indoors year round.
The protein profile also can vary greatly between breeds of goat. In general, goat
milk contains a greater amount of αs2-casein than cow’s milk where αs1-casein is the
predominant protein. However Ambrololi et al. (1988) looked at milk from two different
17
breeds of goat, Alpine and Saanen, and analyzed their milk for protein type and curd
formation. It was found that the Alpine goats produced a higher level of αs1-casein,
having the ability to form a firmer curd more closely resembling cow’s milk cheeses.
1.3 Yogurt Manufacturing
1.3.1. Acid Induced Coagulation of Milk
Coagulation of most common yogurts involves bacterial fermentation from
Streptococcus thermophilus and Lactobacillus delbruekii spp. bulgaricus through the
production of lactic acid from the milk sugar lactose. The gradual drop in pH during
incubation induces many changes in the milk and its proteins, causing a gel to form.
Phadungath (2005) reviewed the acidification process, and describes the following
mechanisms for coagulation. At normal milk pH, which is slightly above 7.0, the caseins
in milk have a net charge of negative and are therefore repelled from each other and stay
dispersed throughout the liquid. As the pH of the milk drops between 6.7 – 6.0, the
charge on the micelles is lessened which leads to lowering of electrostatic repulsions.
Between 6.0 – 5.0, charged hairs on the surface of casein micelles shrink and, as these
hairs are responsible for the stability of the micelles, leads to a lessening of electrostatic
repulsion and increase in stearic de-stabilization. These shorter hairs on the micelle
surface increase the propensity for aggregation. Colloidal calcium phosphate (CCP) also
increases in solubility and is transferred out of the micelles into the aqueous phase of the
milk. As the milk reaches its isoelectric point of 4.6, micellar sub-units are formed and
the net negative charge is neutralized leading to reduced amphiphilic properties of the
caseins while increasing hydrophobic interactions. These hydrophobic interactions
18
causes the caseins to aggregate and form chains to link together in a three dimentional
network, effectively trapping water, fat and soluble compounds to increase the viscosity
of the milk and forming what we know as yogurt.
1.3.2. Goat Milk Yogurt
As discussed previously, goat’s milk contains different types of caseins than that
of its bovine counterpart. Specifically, it contains little to no αs1-casein which is a major
protein found in cow’s milk and is a major component for the structural integrity of milk
gels. The lack of this protein leads to slower coagulation time and weaker curd formation
(Ambrosoli, Di Stasio and Mazzocco, 1988) which results in a weak and watery curd
leading to runny yogurt that has a high degree of separation. Different strategies have
been deployed for overcoming this hurdle; the most common and oldest method being
increasing the solids through evaporation and more recently with the addition of dried
milk powder. Modern approaches have also been devised with much success. Vargas et
al. (2008) produced an acceptable yogurt through the mixing of both cow’s milk and
goat’s milk in a ratio of 1:1. Farnsworth et al. (2005) used a pre-incubation period with
microbial transglutaminase to effectively cross-link proteins in goat’s milk. They were
successful in using the enzyme-treated milk to make yogurt that was more viscous and
had less syneresis than untreated milk. It was also a more effective way to increase the
viscosity in yogurt when compared to the traditional method of increasing solids. Li and
Guo (2006) also explored a novel approach to the production of goat’s milk yogurt.
Using a heat treated whey protein isolate, they were able to increase the gelation
properties of goat’s milk. Upon the addition to milk and subsequent acidification
19
by microbial means, they produced a product that was viscous providing an acceptable
amount of separation.
1.3.3. Fat Replacers
Fat globules in yogurt play an important role in the structural stability of the
protein network of a milk gel, and the absence of which will decrease the viscosity of a
yogurt. Yogurts without fat, and subsequently a reduction in total solids, exhibit weak
body, whey separation, and poor texture (Mistry & Hassan, 1992). Lobato-Calleros et al.
(2014) suggests that the solution to this problem is to provide structural elements to the
continuous phase of the yogurt which will substitute for the functionality of the fat. This
can be accomplished with the addition of protein, carbohydrates, or a combination of the
two.
Lobato-Calleros et al. (2004) used whey protein concentrate (WPC) and
microparticulated whey protein to replace fat and found that they had flow and creep
properties similar to the full-fat yogurt. A similar study showed that 4.5% WPC added to
fat-free milk contributed to the gel matrix, and had rheological properties similar to a
yogurt containing 3.5% fat (Faria et al. 2001). Skim milk powder has also been used as
an alternative to fat. Mistry and Hassan (1992) used skim milk powder to produce
yogurts with 5.3-11.6% protein and showed that an acceptable yogurt could be produced
by the addition of 5.6% protein. Any higher than this, however, the yogurts became firm
and had an astringent flavor. The yogurts were also analyzed by scanning electron
20
microscopy and found that as the protein content increased, the porosity of the yogurt
decreased.
Many different carbohydrate fat replacers have been studied. Crispin-Isidro et al.
(2015) replaced some of the fat in yogurt with either agave fructans or inulin at levels of
20, 40, and 60g L-1
. The results indicated that the reduced fat yogurt with the addition of
40g L-1
inulin or 60 g L-1
agave fructans had superior sensory characteristics than that of
the control. Microstructure examination of the yogurts indicated that the thickening of
the two yogurts were functionally different. The agave fructans covered the casein
micelle and acted as cementing material while the inulin formed long gel structures that
absorbed onto the protein network. Aryana et al. (2007) studied the addition of inulins of
various lengths to produce an acceptable fat-free yogurt. The addition of long-chained
inulins produced a yogurt that had better body and texture than the yogurts with short-
chained inulins and had less syneresis that the full-fat control yogurt. The short-chain
inulin produced a yogurt with a lower pH and better flavor. This is due to the short-
chained inulins being a better substrate for bacterial fermentation than the longer chained
carbohydrates. Lobato-Calleros et al. (2014) examined the effect of adding starches to
milk to produce a low-fat yogurt. Native corn starch, chemically modified corn starch,
and tapioca starch were compared to a full-fat control. The yogurts with added starch
showed less syneresis than the control with the yogurt containing tapioca starch having
the least amount of syneresis. The yogurts with added starch as also exhibited increased
acidity due to the bacteria being able to convert some of the starch to lactic acid. The
control yogurt however, did have a lower viscosity-shear rate. Gum tragacanth, an
21
anionic polysaccharide obtained from the shrubs of Astragalus in Asia, was used in an
attempt to produce a fat-free yogurt (Aziznia, 2008). The attempt was not a success, and
they found that increasing amounts of gum tragacanth negatively correlated with firmness
and positively correlated with the amount of syneresis. The scanning electron
microscopy showed the presence of a very porous and open arrangement of the protein
network. This was due to the polysaccharide adsorbing to the casein micelles, and as the
pH dropped the gum tragacanth underwent changes producing loops and tails which
reduced the interaction of casein micelles leading to weak structure.
1.3.4. Heat-Treated Whey Protein
β-Lactoglobulin a major protein in whey and is largely responsible for the
functional properties of both whey protein concentrate (WPC) and isolate. These
functional properties are attributed to the fact that β-lactoglobulin contains two disulfide
bonds between cysteine residues, Cys66-Cys160 and Cys106-Cys119, and a free thiol
group, Cys121 (Sakurai and Goto, 2006). Upon the raising of the pH, these bridges may
be broken and then reformed with other cysteine residues in redox reactions, also known
as the Tanford Transition (Sakurai and Goto, 2006). When these reactions occur between
other molecules of β-lactoglobulin, polymerization occurs. However, because the
residues are mainly located on the inside of the molecule these disulfide bridges mainly
reform within the original protein. With heating above 70⁰C, the protein becomes
unfolded and exposes the cysteine residues to cysteine residues on neighboring proteins
and a network forms (Bryant and McClemments, 1998). Vardhanabhuti and Foegending
(1999) describe the process of network formation in two phases, the first being just
22
described as proteins interacting to form covalent bonds between each other to form
primary polymers. The extent and size of the molecules is dependent mostly upon the
initial concentration of the protein. Phase two consists of length of heating time where
the primary polymers aggregate through noncovalent bonds. Larger aggregates are
formed with longer heating times, increasing the strength of the final gel.
In order for the gel to form, the pH must be lowered below that of the isoelectric
point of β-lactoglobulin, pH 5.2 (Alting, et al. 2003). When the pH is high, electrostatic
repulsions between molecules keep them from interacting with each other. As the pH
drops, these repulsions are decreased and the aggregation between molecules is
promoted resulting in a network of protein to form, trapping water and any other soluble
substances in the milk (Bryant and McClemments, 1998).
1.4. Probiotics
The use of fermented milk as a medicine goes back thousands of years. In the
Old Testament (Genesis 18:8) states “Abraham owed his longevity to the consumption of
sour milk” and in 76 BC Plinius, the Roman historian, recommended fermented milk for
the treatment of gastroenteritis (Schrezenmeir and de Vrese, 2001). However it was not
until the Nobel prize winner Elie Metchnikoff (1907) surmised that fermented milks may
have consequences for health because of their ability to positively shift the microbial
balance in the intestine. It is now known that the bacteria in the milk confer the
beneficial effects and these bacteria have become known as probiotics. Many definitions
of probiotics have been put forth over the years, but the prevailing one states that “live
23
microorganisms that when administered in adequate amounts confer a health benefit to
the host” (Vasiljevic and Shah, 2008). The health benefits that have been scientifically
established include the ability to help with lactose intolerance by delivering β-
galactosidase to the gastrointestinal tract and prevention and reduction of symptoms of
antibiotic-associated diarrhea and rotavirus due to competitive exclusion and improved
immune response. Other potential benefits of probiotics include reduction of risk of
mutagenicity and carcinogenicity, prevention of inflammatory bowel disease, stimulation
of immune system, hypocholesterolemic effects, prevention or reduction of eczema and
allergies, inhibition of Helicobacter pylori and other intestinal pathogens, alleviation of
constipation, improvement of quality of life of patients with irritable bowel syndrome,
improvement of symptoms of rheumatoid arthritis, lowering of blood pressure in patients
with hypertension, urinary tract infections, hepatic diseases, weight control, influence of
oxalate degradation and kidney stone formation, and improvement in oral health
(Ouwehand, 2002; Saarela et al. 2002; Sanders, 2003; Mckinley, 2005;Vasiljevid and
Shah, 2008).
1.4.1. Pathogen Inhibition
Certain bacteria including Shigella, Salmonella, Klebsiella, Proteus, Escherichia
coli, Pseudomonas, Staphylococcus aureus, and Vibrio cholera can be harmful or even
fatal to humans (Cross, 2002). Probiotics can inhibit these pathogens by producing
antimicrobial substances or through direct competition for site colonization in the gut
(Saarela et al., 2000). Mechanisms for bacteriocidal effects of probiotics include
inhibition through production of bacteriocins or phages such as Acidolin, Acidophilin,
24
Bifilong, and Lactocidin; the production of organic acids such as lactic acid and hydrogen
peroxide, or by lowering the pH of the gut through the production of short-chain fatty
acids which inhibit pathogens from multiplying (Shah, 2007).
A recent study by Coman et al. (2014) studied the pathogen antagonistic
characteristics of two probiotic strains, Lactobacillus rhamnosus and Lactobacillus
paracasei. They tested them against an array of Gram-positive and Gram-negative
bacteria, as well as yeasts using several methods of in vitro evaluation including the
modified cross streak, radial streak, agar well diffusion, and liquid coculture assay
methods. They found that these probiotics had good antimicrobial properties against
many of the Gram-positive bacteria and yeasts, but had lesser effect on the Gram-
negative. The authors concluded that it is necessary to test probiotics by various methods
of in vitro screening in order to obtain significant results.
In a search for new strains of probiotic bacteria, Shokryazdan et al. (2014)
collected 140 bacterial isolates from human milk, infant feces, fermented grapes, and
fermented dates. The bacteria were then subjected to environment that imitates the
human gut: 0.3% bile salt for 4 hours, 1.9 mg/mL pancreatic enzymes for 3 hours, and pH
3.0 for 3 hours. Nine of the isolates were able to withstand all three of the tests and they
were all found to be gram positive and catalase negative indicating that they were
probably lactic acid bacteria. Further identification by 16S rRNA gene sequencing
showed that the nine bacteria were closely related to L. acidophilus, L. fermentum, L.
buchneri, and L. casei and their antimicrobial properties were compared to the known
probiotic L. casei Shirota against a wide array of pathogens. All of the newly discovered
25
lactic acid bacteria displayed probiotic properties, especially the strains related to L.
buchneri and L. casei which showed exceptional antimicrobial activity towards
Helicobacter pylori, Listeria monocytogenes, and Staphylococcus aureus.
1.4.2. Prevention of Diarrhea
Diarrhea can have many underlying causes but is usually caused by an acute
infection by bacteria in the gut. There is an estimated four billion cases of diarrhea per
year, causing 2.2 million deaths making it the fifth highest cause of death of all ages
(WHO, 2004). Probiotics have been shown to alleviate some of the negative symptoms
of acute enteric infections including those found in antibiotic-associated diarrhea (AAD),
rotavirus infection, as well as traveler’s diarrhea (Saad et al. 2013).
AAD occurs between 2-8 weeks after treatment with antibiotics and has two
underlying causes. Bacteria in the gut play a vital role in protection against pathogens
and during treatment the normal gut microflora is substantially diminished making the
host suseptable to infections, especially those of Clostridium difficile, Candida albicans,
Salmonella spp., and Klebsiellia caytoca. Moreover, because of the decreased activity of
bacteria in the gut, complex carbohydrates are not completely broken down leading to
osmotic water secretion (Iannitti and Palmieri, 2010). De Vrese et al. (2011) investigated
the use of Lactobacillus acidophilus and Bifidobacterium lactis to treat AAD over an 8-
week period using questionnaires to rate pain frequency, pain intensity, and duration of
pain. They found that the patients given the probiotics had an improvement in
gastrointestinal pain and length of AAD. However, there is conflicting evidence for the
benefits of using probiotics to treat AAD. In a randomized clinical trial of two groups of
26
kids ranging in age from 6 months to 14 years with respiratory tract infection being
treated with antibiotics, one group was given a placebo while the other received a
symbiotic containing a mixture of probiotics and fructooligosaccharide (Jafari et al.
2014). While the stool frequency and duration of diarrhea were slightly lessened, there
was no statistically significant difference between the two groups. Another randomized,
double blind study found similar results among a greater number of patients with the rate
of occurrence not changing between the groups (Song et al. (2010). Their results showed
that the symptoms of AAD (lower frequency and less watery stool) were lessened among
the group given the Lactobacillus probiotics. The authors admitted however, that the
period of observation was only 2 weeks although AAD can occur up to 8 weeks after the
administration of the antibiotics. To analyze the many conflicting results, a group of
researchers conducted a meta-analysis of existing randomized controlled studies.
Hempel et al. (2012) looked at 63 trials consisting of 11,811 patients being administered
antibiotics for various illnesses. A majority of the trials reviewed used interventions of
Lactobacillus rhamnosus and Lactobacillus casei, however they did include trials
conducted with Enterococcus, Bacillus, Streptococcus and the yeast Saccharomyces
boulardii. They concluded that the results suggest that there is some benefit to using
probiotics to treat AAD, however, they also suggested that further research is needed to
identify the probiotics with the greatest efficacy towards different types of antibiotics.
Rotavirus infections were discovered in 1970 and have been recognized as the
most common cause of diarrhea in children worldwide and it is estimated that almost
every child has been infected by rotavirus by the time they are 3-5 years old (Dalgic et al.
27
2011). According to the WHO, children with severe diarrhea should recieve rehydration
therapy and zinc to relieve symptoms of rotavirus. Dalic et al. (2011) set out to study
whether or not the administration of a probiotic Saccharomyces boulardii could help
lessen the symptoms of this disease as well. They also tested it against treatment with a
lactose-free formula. They found that the duration of diarrhea was significantly shorter in
the groups given either only the zinc or the probiotic plus zinc complex when compared
to the control. Another possibility of treating the virus infection is the use of
nitazoxanide which is a nitrothiazole benzamide compound approved by the Federal
Drug Administration for the treatment of diarrhea. A study by Teran, Teran-Escalera &
Villarroel in 2009 sought to challenge the drug with probiotics for the treatment of
gastrointestinal distress. The researchers studied 147 children between the ages of 28
days to 24 months diagnosed with rotavirus infection who were assigned to one of three
groups: one given nitazoxanide drug, one administered a probiotic solution containing
Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and
Saccharomyces boulardii, and a third control group given only a rehydration solution.
There were no significant differences between the two interventions, however, they found
that both the nitazoxanide and probiotic mixture shortened the duration of diarrhea to 54
hours and 48 hours, respectively, and were both statistically significantly less than the
control group which experienced diarrhea for an average of 79 hours. The two groups
also shortened the hospital stay of the infants, lessening the cost burden of treating
rotavirus.
28
Traveler’s diarrhea affects many internationals from industrial countries who
travel to developing countries. The most common cause traveler’s diarrhea is an
infection by Escherichia coli, Campylobacter, Shigella, and Salmonella. The usual
treatment is an the administration of an oral antibiotic. However, because of developing
information on the dangers of widespread antibiotic use, interest in other treatments has
been growing. Researchers in France tested a probiotic against a placebo on Wistar rats
infected with Escherichia coli (Bisson, et al. 2010). The probiotic, called Protecflor™, is
a combination of Lactobacillus rosell 11, L. rosell 52, Bifidobacterium rosell 175, and
Saccharomyces boulardii. They found that the rats given the probiotics displayed less
pronounced symptoms and showed an increase in anti-inflammatory cytokines
(interleukin(IL)-4 and IL-10) and a decrease in pro-inflammatory cytokines (IL-1, IL-6,
and tumor necrosis factor-α). It should be noted, however, that the company funding this
research, Lallemand SAS France, is also the maker of Protecflor™ which could have had
an impact on the findings.
1.4.3. Irritable Bowel Syndrome
Irritable bowel syndrome (IBS) is a common disorder of the gastrointestinal tract
affecting as much as 10% of the population and has symptoms that manifest themselves
as abdominal pain, discomfort, and frequent loose stools which can lead to impaired
social functions and a reduced quality of life (Saarela et al. 2002). The underlying cause
is unclear, but it has been hypothesized that it could be caused by heightened function of
the intestine due to an imbalance in neurotransmitters, visceral hypersensitivity, or
29
disturbed intestinal microbiota (Saarela et al. 2002). A randomized, double-blind,
placebo controlled trial was conducted on 50 individuals with IBS in 2012 by Cha et al.
to determine if a probiotic mixture containing Lactobacillus acidophilus, Lactobacillus
plantarum, Lactobacillus rhamnosus, Bifidobacterium breve, Bifidobacterium lactis,
Bifidobacterium longum,, and Streptococcus thermophilus had any effect on this
disorder(Cha et al., 2012). Their primary goal was to determine if adequate relief, which
was defined as relief from symptoms for at least half of the 10 week trial, stool quality,
and the quality of life as determined by a questionnaire, could be increased. At the end of
the 10 weeks, they found significant improvements in all three areas. Another group of
researchers, Lorenzo-Zuniga et al. (2014), conducted a similar study on individuals with
IBS using a mixture of Lactobacillus plantarum and Pediococcus acidilactici and looked
at the outcomes of health-related quality of life, symptom relief, and gastrointestinal
anxiety using the Viceral Sensitivity Index scale. They also assessed dose-dependent
outcomes by assigning patients to a placebo group, a high-dose group (1-3 x 1010
cfu/dose), and a low-dose group (3-6 x 109
cfu/dose). After 6 weeks of treatment both
quality of life and gastrointestinal anxiety increased in all groups including the placebo,
however there was a significant increase in both of the probiotic groups compared to the
placebo group. There was no difference between the two dose groups however. Another
study would be needed to investigate whether the dose could be lessened even further.
Recently a group of researchers conducted a meta-analysis of outcomes of probiotic
effectiveness in treating IBS (Didari et al. 2015). They analyzed the results of 24 clinical
trials, 15 of which were determined to be quality studies and included in the analysis.
Quality was determined by the Jadad score, which rates trials on a scale of 1-5 in the
30
areas of randomization, blinding, and dropout rate. Trials that were scored a 2 or below
were not included. With the 15 remaining studies, a total of 1793 patients with IBS were
included. They found that with probiotic treatment a alleviation of abdominal pain, lower
incidence of flatulence, and a higher quality of life could be achieved.
1.4.4. Hepatic Disease
Non-alcoholic fatty liver disease (NAFLD) can be described as an accumulation
of fat in the liver ranging from steatosis to steatohepatitis which may eventually progress
to cirrhosis (Buss et al., 2014). There is no formal treatment for NAFLD, however, it has
been recommended that losing weight, lowering cholesterol, and modification of diet
may be beneficial to slowing or stopping the progression of this disease. Recent studies
have started investigating at the relationship between the makeup of the intestinal
microbiota and NAFLD, specifically the increased prevalence of small intestinal bacterial
overgrowth (SIBO), the increase of ratio of Firmicutes to Bacteroidetes in the gut, and
increased permeability of the intestine. Paolella et al. (2014) have reviewed many studies
and suggested several mechanisms in which the makeup of gut bacteria may play a role
in the progression of this disease. The increased bacteria load in the small intestine will
lead to an increased absorption of bacterial products such as pathogen-associated
molecular patterns which have a tendency to activate toll-like receptor 4 leading to
systemic inflammation and fibrogenesis in the liver. The gut microbiota profile in
patients with NAFLD also reduces the expression of fasting-induced adipose factor
leading to increased lipogenesis, in free fatty acid uptake, and fat accumulation. The
31
increased bacterial growth in the small intestine causes the utilization of more dietary
polysaccharides, leading to an increase in short chain fatty acids and monoglycerides
which can be used in gluconeogenesis and lipogenesis. Furthermore, the bacteria
associated with NAFLD may convert choline into methylamines which induces insulin
resistance and further fat accumulation.
Ritze et al. (2014) studied the effects of a probiotic, Lactobacillus rhamnosus GG
(LGG), on NAFLD in mice. Mice were fed a diet that was high in fructose which does
not increase the body weight of the mice but induces hepatic steatosis. They showed that
LGG lowered fat accumulation in the liver, normalized the expression of genes
associated with lipid metabolism, reduced liver inflammation, as well as improved
intestinal barrier function.
A double-blind study was performed in human patients by Nabavi et al. (2014) to
determine the effects of a probiotic yogurt in individuals with NAFLD and its effects on
metabolic risk factors. The individuals were assigned to two groups, one that consumed
only yogurt fermented with Lactobacillus Bulgaricus and Streptocuccus thermophilus
and one that ate yogurt with the addition of Bifidibacterium lactis Bb12 and
Lactobacillus acidophilus La5. Metabolic factors that were examined were levels of
alanine aminotransferase (ALA), aspartate transferase (AST), glucose, total cholesterol,
triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL). The 8-
week intervention showed that yogurt fermented with probiotics had no effect on
triglycerides, HDL, or serum glucose but reduced levels of ALA, AST, LDL and total
cholesterol.
32
1.4.5. Alcoholic Liver Disease
Abuse of alcohol can damage the liver and is the leading cause of liver disease in
industrial countries (Vassallo et al., 2015). The mechanisms in alcoholic liver disease
(ALD) are similar to that of NAFLD as alcohol induces a change in the microbiome of
the intestine leading to increase of bacterial translocation due to bacterial overgrowth and
increased permeability of the gut (Yan, 2012). The one main difference is that alcohol
alone can cause steatosis of the liver.
Shi et al. (2015) studied the effects of Lactobacillus rhamnosus GG (LGG) in
mice who were fed a diet containing 35% ethanol for 4 weeks to induce the symptoms of
ALD before introducing a probiotic. They analyzed the metabolites in the liver and the
feces. The changes in the gut of the LGG fed mice were an increase in the production of
hepatadecanoic acid, a long-chain fatty acid produced by bacteria, which reduces
endotoxemia and decreases gut permeability. They also found that in the alcohol-fed
mice a reduction of various different amino acids (isoleucine, proline, threonine,
phenylalanine, and valine) was observed but that in mice fed LGG these levels were
normalized. All of these amino acids play an important role in liver function, and the
deficiency of them may lead to further fat accumulation in the liver.
The effects of probiotics on increased oxidative stress on the liver from ALD
through the increased inflammatory mediators which include tumor necrosis factor-α,
interleukin-1β, and interleukin-6 were studied. Arora et al. (2014) found that the liver of
rats with induced ALD could be positively affected by microencapsulated Lactobacillus
plantarum. The rats were chronically exposed for 12 weeks to alcohol with the final 8
33
weeks having L. plantarum added to their diet. They found that the levels of the
inflammatory markers as well as levels of endotoxemia were reduced in the rats fed the
microencapsulated probiotics. However, the same effect was not observed in the un-
encapsulated L. plantarum, likely due to its inability to survive in the harsh environments
in the stomach and gut.
1.4.6. Genetic Engineering of Probiotics
Genetic modification of certain bacteria is a new and exciting field in the area of
probiotics, and while controversial and may not be yet acceptable to consumers, is worth
discussing. In terms of nutritional enhancement, vitamin production may be enhanced as
researchers have accomplished by enhancing a riboflavin synthase beta subunit in
Lactobacillus lactis to produce more riboflavin upon fermentation (Arena et al. 2014).
However far more interest has been in genetically modifying probiotics for oral
vaccinations against enteric bacteria. In 2010, Yamamoto et al. genetically modified
Bifidobacterium longum to display Salmonella-antigens on its surface and fed it to mice
for two weeks. The vaccine was then challenged by administering the Salmonella typhi
to the mice at a lethal rate of 1.0 x 107
cfu/mouse to induce typhoid fever. 12 out of 14
mice that were not given the vaccine died, while in the group receiving the altered
Bifidobacterium only 2 of the 14 tested died. This could certainly have important uses in
human health and should be developed further.
1.4.7. Oral Health
Another area where new research has been emerging is in the application of
probiotics in regards to oral health. A greater number of bacteria may be candidates for
34
use in oral health as they do not need to survive the harsh environments of the stomach
and upper GI to confer protection to the host. They do, however, need to have similar
characteristics in their mode of action. Agrawal, Kapoor, and Shah (2012) defined
mechanisms necessary for a bacterium to be an oral probiotic as the ability to produce
antimicrobial substances, bind in the oral cavity, modulate immune responses in the cell,
and the ability to modify oral conditions including the production of biofilms. A study by
Bosch et al. (2012) investigated over 100 strains of bacteria from the mouth and feces of
heathy children and analyzed them for possible use as probiotics. Out of the 100 strains,
46 were identified for use as probiotics with Lactobadillus being the largest identified
genus followed by Lactococcus, Pediococcus, and Leuconostoc. They were then
evaluated in regards to their ability to aggregate, their production of malodour, their
adhesion to pig tongue cells, acid production, and their antagonistic effect on the oral
pathogens Porpyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola,
Prevotella denticola,and Streptococcus mutans. Their findings suggest that many of the
isolated strains are suitable candidates for oral probiotics, with Lactobacillus casei,
Lactobacillus brevis, and Pediococcus pentosaceus isolated from saliva scoring
particularly well and being suitable for follow up studies. A more recent in vitro study
examined strains of Bifidobacterium breve, Lactobacillus rhamnosus, and Lactobacillus
plantarum and their possible use to protect gingival fibroblasts from the oxidant activity
of hydrogen peroxide (Mendi and Ashm, 2014). Oxidative stress is one of the main
causes of periodontal tissue damage and the use of antioxidants in the form of vitamins,
plant polyphenols, and medicinal plants has been explored. The strains were tested on
their ability to produce exopolysaccharides, scavenge the α,α-diphenyl-1-picryl hydrazyl
35
radical, chelate iron ions, and to inhibit plasma lipid peroxidation. Out of the probiotics
tested, the Bifidobacterium brevis scored exceptionally high in all of these areas and may
be a good candidate to protect the mouth from periodontitis. This new research could
have many intriguing commercial uses; there are very few products in production
promoting themselves as a probiotic toothpaste.
36
CHAPTER 2: MANUSCRIPT
2.1 Introduction
Goat milk is an important source of nutrition for people across the globe,
nourishing more people in underdeveloped countries than bovine milk (Haenlein, 2004).
Goat milk products are gaining in popularity in developed countries in part due to people
becoming increasingly sensitive to cow’s milk (Park, 1994a), it is higher in many
minerals as well as having a greater abundance of certain vitamins (Ljutovac et al., 2008;
Park, 1994b), and its rising appeal to food enthusiasts (Haenlein, 1996).However goat’s
milk forms a weak curd in yogurt due to its lack of αs-1 casein (Guo, 2003). Fat,
especially saturated fat, has been increasingly criticized for its contributions to certain
health issues in developed countries, and the demand for low fat dairy products is on the
rise (Sandoval-Castilla et al., 2004).
The most common methods for overcoming the weak texture of goat’s milk
yogurt involve increasing content of total solids of the milk. This can be achieved by
boiling the milk, evaporation, addition of skim milk proteins, milk proteins, or plant-
based carbohydrate compounds (Li and Guo, 2006). Newer methods include treatment of
milk with microbial transglutaminase to form an acceptable yogurt (Farnsworth et al.
2006).
Whey proteins have many functional properties in food systems including
gelation, foaming, and water-holding capacity (Bryant and McClements, 1998), and heat
treatment with high pH increasing these properties. Wang et al. (2011) investigated used
cow’s milk whey protein isolate (WPI) to produce a full-fat goat’s milk yogurt of
37
acceptable quality. When heated above its isoelectric point of 5.2 and held for a period of
time, the proteins unfold and become susceptible to increased electrostatic interactions.
In addition, when β-lactoglobulin unfolds more thiol containing amino acids are exposed
leading to an increased reaction among the cysteine residues to form a network which
further increases the strength of the gel and increased aggregate size (Roefs and de Kruif,
1994). After the protein is denatured, it is added to the goat’s milk and fermented. When
the pH is lowered below 4.6, decreasing the electrostatic repulsions of the proteins allow
a complex network of whey proteins and casein to form, strengthening the gel upon
cooling (Bryant and McClements, 1998). Pectin is used as a thickening agent to further
increase the overall consistency of the yogurt (Vardhanabhuti and Foegeding, 1999), as
well as to improve the mouth feel and to prevent whey separation (Lucey et al., 1999).
Fat plays an important role in the structural integrity and mouth feel of yogurt, in
large part because it interacts with casein micelles to form a copolymer (Everett and
Rosiland, 2004). Removal of the fat can lead to increased syneresis, unfavorable texture,
and weak body (Mistry and Hassan, 1992). Many different types of fat replacers have
been explored in bovine yogurts including the addition of inulin (Aryana et al., 2007), β-
glucan (Sahan, Yasar & Hayaloglu, 2008), gum tragacanth (Aziznia et al., 2007), high
milk protein powder (Mistry and Hassan, 1992), and WPC (Calleros-Lobato et al., 2004).
Gelling properties of whey protein concentrate (WPC) and various plant-based
hydrocolloids have been investigated extensively (Beaulieu, Turgeon, and Doublier,
2001; Mishra, Mann &Joshi, 2000). The use of WPC as a fat replacer in bovine yogurts
has been explored (Guzman-González et al., 1999; Sandoval-Castillaet al., 2004),
38
while Zhang et al. (2015) examined its use for goat’s milk yogurt. WPC contains
35%-80% proteinand therefore less expensive than WPI, however, the main
constituent, β- lactoglobulin, is the same.
The health benefits and safety of probiotics have been well established (Saad et
al., 2013; Salminen et al., 1998; Vasiljevic and Shah, 2008), and the use of yogurt as a
delivery system for the microorganisms has been widely accepted (Lourens-Hattingh and
Vijoen, 2001). Lactobacillus acidophilus and Bifidobacteria have been well studied and
it has been suggested that a viable cell count of at least 106cfu g
-1 is needed for beneficial
effects (Guo, 2007). With rising amounts of consumers looking for low- fat, quality
sources of protein containing live culture probiotics, there is a need to develop a non-fat
goat’s milk yogurt. The objective of this study was to investigate the efficacy of a new
technique for developing a non-fat goat’s milk yogurt by using heat-treated
whey protein concentrate, in conjunction with pectin as a fat replacer produce a yogurt
with good consistency and low syneresis while remaining a good delivery system for
probiotics.
2.2. Materials and Methods
2.2.1. Materials
Fat-free goat’s milk was obtained from Oak Knoll Dairy in Winsor, VT. Fat-free
cow’s milk was purchased from a local grocery store. The yogurt starter culture, Yo-Fast
100, was purchased from Chr. Hansen (Milwaukee, WI) which contained a combination
of the microorganisms Streptococcus thermophilus, Lactobacillus delbruecki issp.
Bulgaricus, Lactobacillus acidophilus, and Bifidobacterium spp. Low-methoxy pectin,
39
GENU® texturizer type YA-100, was received from CP Kelco (Atlanta, GA). Whey
protein concentrate (WPC 80% protein) was acquired from Davisco Foods International
Inc. (Le Sueur, MN).
2.2.2. Preparation of Heat-Treated Whey Protein (HWPC)
WPC powder (125g) was blended and dissolved in purified water and held
overnight at 4˚C to fully hydrate proteins. It was then brought to 1000mL total volume to
produce a 12.5% (w/v) solution. The pH of the solution was then adjusted to 8.5 using
3M NaOH. The WPC solution was then heated to 85˚C and held for 30 minutes in a
water bath. It was cooled rapidly in an ice bath to bring it to room temperature. A non-
heat-treated whey protein (WPC) solution was . made by blending and refrigerating the
WPC powder and adjusted to 12.5% (w/v) without the addition of NaOH or heating.
2.2.3. Preparation of Yogurt
GENU® pectin was added to the goat’s milk at a concentration of 0.35% (w/v)
and hand blended for 1 minute. It was then heated to 82˚C to dissolve the pectin and to
pasteurize the solution. The milk was then cooled in a water bath to 40˚C. The prepared
HWPC was mixed with the milk at a concentration of 12% (v/v). A 10% (w/v) starter
culture slurry was prepared according to directions provided by Chr. Hansen and added at
a rate of 0.4% (v/v). The mixture was then incubated at 43˚C for 4.5 hours, after which it
was cooled in a refrigerator and held there for analysis. Another yogurt was produced
using the above mentioned technique only using the WPC as a substitute for the HWPC.
A third yogurt was made with pectin and starter but without the addition of either HWPC
or WPC. A cow’s milk yogurt was also produced using only pectin and starter as a
49
control. A total of three trials were prepared for each of the yogurts on separate days and
held at 4˚C for 10 weeks to analyze chemical composition, probiotic survivability, pH,
viscosity, and syneresis of each of the samples.
2.2.4. Chemical Analysis
The yogurt samples were analyzed for total solids, ash, protein, fat, and
carbohydrate content as described in the Association of Official Analytical Chemist
procedures (AOAC, 2002). Total solid content was determined by air-drying samples in
an oven for 24 hours. Ash was then assessed by taking the dried samples and using a
muffle furnace to burn off all non-mineral matter.
Protein content was analyzed using the Kjeldahl digestion method and a nitrogen
conversion factor of 6.38 (Kosikowski, 1977). Fat was examined in the yogurt using the
Mojonnier method for fat analysis of dairy products. Carbohydrate content was then
obtained by determining the variance in total solids to the other solid components. Three
trials from each of the formulations were examined for chemical content.
2.2.5. Syneresis Testing
The water-holding properties of the yogurts were examined by centrifugation as
described by Li and Guo (2006). A portion of each of the formulations of yogurt (Y)
were prepared and were weighed before incubation. The yogurt was then centrifuged at
4˚C for 10 minutes at 2500 RPMs (640 x g). The supernatant (S) layer was poured off
and weighed. Three trials of each were conducted. Syneresis was determined by using
the following formulation:
41
Syneresis (%) = (S/Y) X 100%
2.2.6. Mold, Yeast and Coliform Counts and the Survivability of Probiotics
Probiotic enumeration was performed on each of the four yogurts once a week for
a 10-week period according to the procedures of Chr. Hansen (2005). L. acidophilus was
grown on Difco™ Lacobacilli MRS Agar and anaerobically incubated at 37˚Cfor 3 days.
Bifidobacterium spp. were selectively cultivated on the same media with the addition of
L-cysteine hydrochloride, lithium chloride, and Dicloxacilin and anaerobically incubated
at 37˚C for 3 days. Mold and yeast counts were determined every two weeks by using
Yeast and Mold Petrifilm (3M™ Petrifilm™, St Paul, MN) and stored at room
temperature (21±2˚C) for 5 days. Coliform counts were determined checked every two
weeks by using ColiformPetrifilm (3M™ Petrifilm™, St Paul, MN) and stored at room
temperature (21±2˚C) for 3 days. Three trials of each were performed.
2.2.7. pH and Viscosity
The pH and viscosity of each of the formulations of yogurt was determined once a
week for a 10-week period. Three trials of each were tested. Each of these tests was
performed using yogurt that was brought back up to room temperature (21±2˚C) for one
hour before analysis. Viscosity was measured using a Brookfield viscometer (Brookfield
Engineering Laboratories Inc., Middleboro, MN) and stated in mPa·s. A reading was
taken after a 30 second period at 100 rpm. pH of the yogurts was determined using a pH
meter (IQ Scientific Instruments Inc., San Diego, MA)
42
2.2.8. Microstructure Analysis by Scanning Electron Microscopy (SEM)
SEM was carried out on all four of the yogurt formulations as described by Walsh
et al. (2010). Cubes of agar were set in 2.5% glutaraldehyde in 0.1M sodium cacodylate
buffer (pH 7.2) and allowed to set at 4˚C for 12 hours. The cubes were then washed in
triplicate for 10 minutes each in the buffer and post fixed in 1.0% osmium tetroxide. This
was followed by an additional three rinses in a diluted (50 mM) cacodylate buffer (pH
7.2). Using a series of ethanol to 100%, the cubes of agar were dehydrated and frozen in
liquid nitrogen and fractured. The pieces were then thawed in ethanol (100%) and dried
under CO2. After being mounted on aluminum SEM stubs, the cubes were then sputter
coated with 3 nm of Au/Pd and analyzed via a FEI Quanta 200F scanning electron
microscope at 5kV. Several micrographs were taken at various magnifications (500x &
2500x).
2.2.9. Statistical Analysis
The syneresis data and chemical composition were analyzed using 1-way
ANOVA with a Bonferroni adjustment to determine statistical differences among
formulations. The data collected over the 10-week period was analyzed using a 2-way
ANOVA with a Bonferroni adjustment post-test to compare statistical differences for
individual weeks.
2.3 Results and Discussion
2.3.1. Determination of Proper Amounts of HWPC and Pectin
The denaturing processes of the WPC are affected by both the initial pH and the
heating time. Li and Guo (2006) investigated the use of polymerized whey proteins to
43
improve the texture and water holding properties of fermented goat’s milk yogurt using a
cold-set gel using whey protein isolate (WPI). Because WPC is less pure than WPI, a
greater degree of denaturation must be obtained to see the same effects. This can be
achieved through an increase in the total pH as well as a longer heating time. A pH of
8.5 and a heating time of 30 minutes produced a gel that did not fully set at room
temperature. This property allowed it to be easily mixed with the goat milk during yogurt
production.
The level of HWPC to be added to skim milk was optimized by using either only
HWPC or pectin alone to produce goat’s milk yogurt. The results of Table 1 show that
the yogurt with 1.2% and 1.4% HWPC is more viscous and forms a stronger gel than that
of the goat’s milk yogurt with pectin. The level of HWPC that could be used in
conjunction with pectin was explored. Table 1 shows that a viscosity of approximately
1650mPas was achieved with two different combinations of HWPC and pectin. A level
of 1.2% HWPC and 0.25% pectin was chosen because the HWPC is more expensive than
pectin, and therefore, the yogurt with the lower level of whey protein is more cost
effective while still producing a product with a smooth and creamy texture.
44
Table 1. Effects of addition of HWPC, pectin, or both on the characteristics of fat-free goat’s milk yogurt
Addition to goat’s milk Viscosity (mPas) pH
1.4%HWPC 831 + 35d 4.21 ± 0.01
i
1.2%HWPC 809 + 12d 4.21 ± 0.01
i
1.0%HWPC 695 + 22c 4.22 ± 0.01
i
0.8%HWPC 575 + 11b 4.21 ± 0.01
i
0.45%pectin 718 + 24c 4.33 ± 0.00
k
0.35%pectin 731 + 13c 4.29 ± 0.01
j
0.25%pectin 732 + 23c 4.39 ± 0.02
k
0.15%pectin 673 + 33c 4.34 ± 0.02
k
Non-fat goat’s milk yogurt 235 + 28a 4.47 ± 0.01
h
1.4%HWPC+0.15%pectin 1434 + 22f 4.27 ± 0.01
j
1.4%HWPC+0.25%pectin 1649 + 12g 4.28 ± 0.01
j
1.2%HWPC+0.15%pectin 1433 + 19f 4.25 ± 0.01
i
1.2%HWPC+0.15%pectin 1651 + 40g 4.27 ± 0.02
j
1.0%HWPC+0.15%pectin 1385 + 33f 4.25 ± 0.02
i
1.0%HWPC+0.15%pectin 1305 + 34e 4.30 ± 0.02
j
0.8%HWPC+0.15%pectin 1241 + 17e 4.29 ± 0.01
j
Values with different superscript letters incicate significant difference (P<0.05) relative to non-fat goat’s
milk yogurt
2.3.2. Chemical Composition
Chemical composition of the various formulations of yogurt is summarized in
Table 2. There were significantly differences in both total solids (P<0.05) and ash
(P<0.05) in the cow’s milk in comparison to the goat’s milk yogurt with only pectin
added, however, there was no difference between total solids or ash content between any
of the other formulations. There was a significantly higher carbohydrate (P<0.01)
content in the cow’s milk yogurt in comparison to the HWPC goat’s yogurt but no
difference between any of the other yogurts. There was no difference in the fat content
between any of the yogurts as they are all made from non-fat milk. The protein content
45
was significantly higher in the HWPC goat’s milk yogurt when compared with the goat’s
milk yogurt with only pectin added (P<0.01) and the cow’s milk yogurt (P<0.01). This is
most likely due to the addition of the extra whey protein during manufacturing the goat’s
milk yogurt.
Table 2. Chemical composition of the non-fat goat’s milk yogurt and cow’s milk yogurt (mean ± standard
deviation, n=3)
% HWPC+ Pectin WPC + Pectin Pectin Cow's Milk +
Pectin
Total Solids 8.47±0.59a 8.54±0.22
a 8.29±0.45
b 8.78±0.32
b
Protein 3.79±0.71c 3.50±0.47
c 2.85±0.08
d 3.11±0.15
d
Carbohydrate 3.58±1.10e 3.98±0.49
e 4.34±0.35
f 4.63±0.45
f
Fat 0.34±0.05g 0.33±0.04
g 0.34±0.09
g 0.31±0.07
g
Ash 0.76±0.05h 0.73±0.04
h 0.77±0.04
h 0.72±0.01
i
*Values with different superscripts incicate significant difference (P<0.05) relative to nonfat cow’s milk
plus pectin
2.3.3. Syneresis
There was no difference in regards to syneresis between the HWPC goat’s milk
yogurt, the WPC goat’s milk yogurt, and the cow’s milk yogurt (Fig. 1). However,
without the addition of the whey protein to the milk before processing, the water holding
capacity of the goat’s milk yogurt was significantly lower (P<0.01) than any of the other
yogurts, likely due to the decrease in total protein content (Table 2).
46
Figure 1. Effects of HWPC and pectin on syneresis of goat’s and cow’s milk yogurt
2.3.4. Changes in pH During Storage
There are no significant changes in pH over a 10 week period for any of the
formulations of yogurt and there was no significant difference of pH in the HWPC goat’s
milk yogurt (Fig. 2) during storage at 4˚C over a 10-week period when compared to the
other yogurts. These data conflict with the findings of Wang et al (2012) which reported
a decrease in the pH over the course of 10 weeks.The pH of the WPC goat’s milk yogurt
was significantly lower than the goat’s milk yogurt with pectin (P<0.05) and the cow’s
milk yogurt (P<0.05).
24
22
20
18
16
14
12
10
8
6
4
2
0
1 - HTWP + Pectin 2 - WPC + Pectin 3 - Pectin 4 - Cow's Milk + Pectin
% S
yner
esis
47
5
4.8
4.6
4.4
4.2
4
3.8
3.6
3.4
3.2
3
HWPC + Pectin
WPC + Pectin
Pectin
Cow's Milk + Pectin
1 2 3 4 5 6 7 8 9 10
Week
Figure 2. Changes in pH of non-fat goat’s milk yogurts and non-fat cow’s milk yogurt during storage at 4˚C
2.3.5. Changes in Viscosity During Storage
The viscosities of the yogurts did not change over the 10-week period (Fig. 3).
The HWPC goat’s milk yogurt’s viscosity was significantly higher than the WPC goat’s
milk (P<0.01), the goat’s milk yogurt + pectin (P<0.01), and the cow’s milk yogurt
(P<0.01). This could be due to the denaturization that occurs during heat treatment of the
WPC. When denatured WPC form aggregates which will lead to molecules that are
larger in size, less symmetric in shape, and have a increased volume fraction than the
native molecules. This leads to an increased viscocity of the yogurt (Bryant and
McClements, 1998; Vardhanabhuti and Foegeding, 1999).
The viscosity of goat’s milk yogurt + pectin was significantly lower than either
the WPC goat’s milk yogurt (P<0.05) or the cow’s milk yogurt (P<0.01). The viscosity
of the WPC goat’s milk yogurt and the cow’s milk yogurt were not significantly
different.
pH
48
Figure 3. Changes in viscosity of non-fat goat’s milk yogurts and non-fat cow’s milk yogurt during storage
at 4˚C
2.3.6. Probiotic Survivability During Storage
The microbial counts for L. acidophilus were only above 106cfu*g
-1 for the first
two weeks of storage for all of the formulations of yogurt except the goat’s milk yogurt +
pectin, which fell below 106cfu*g
-1 after the first week (Fig. 4). Results show a gradual
decline in microbial populations during storage, however the HTWP goat’s milk yogurt
showed the steepest decline in populations between weeks 1 through 6. This could
account for the fact that probiotic levels ended up significantly lower at the end of the
trial when compared to the WPC goat’s milk yogurt (P<0.01), the goat’s milk yogurt +
pectin (P<0.01), and the cow’s milk yogurt (P<0.01). The steep decline in L.
acidophilus populations was most likely due to the production of hydrogen peroxide by
L. Bulgaricus during storage which inhibits L. acidophilus (Gilland and Speck, 1977).
2500
2250
2000
1750
1500
1250
1000
750
500
250
0
HWPC + Pectin
WPC + Pectin
Pectin
Cow's Milk + Pectin
1 2 3 4 5 6 7 8 9 10
Week
mP
as
49
9
8
7
6
5
4
3
2
HWPC + Pectin
WPC + Pectin
Pectin
Cow's Milk + Pectin
1
0
1 2 3 4 5 6 7 8 9 10
Week
Figure 4. Survivability of L. acidophilous during storage at 4˚C.
Counts of the Bifidobacterium spp. declined gradually for all formulations,
however, they remained above 106
cfu-1
for all 10 weeks of the trial for all of the
formulations of yogurt (Fig. 5). Lourens-Hattingh and Viljoen (2001) suggested that S.
thermophilus in the yogurt may act to reduce the oxygen present, creating an anaerobic
environment favorable to Bifidobacterium spp. The only significant difference in
formulations was between the WPC goat’s milk yogurt and the cow’s milk yogurt
(P<0.01).
Log
CFU
g-1
50
10
9
8
7
6
5
4
3
2
1
0
HWPC + Pectin
WPC + Pectin
Pectin
Cow's Milk + Pectin
1 2 3 4 5 6 7 8 9 10
Week
Figure 5. Survivability of Bifidobacterium spp. during storage at 4˚C.
2.3.7. Mold, Yeast, and Coliforms
There was no mold, yeast, or coliform detected in any of the samples during
storage. The absence of these organisms suggests that the yogurts were safe to eat after
storage at 4˚C for 10 weeks.
2.3.8. Microstructure
Figure 6 shows a scanning electron microscopy (SEM) photograph of the four
prepared yogurts at different magnifications: (a) goat’s milk yogurt with HWPC and
pectin, (b) goat’s milk yogurt with WPC and pectin, (c) goat’s milk yogurt with only
pectin, and (d) cow’s milk yogurt with pectin. It can be seen in Fig. 6a that the dispersion
of the HWPC was much more even due to the absence of large dark voids. The voids
seen in Fig. 6b show a less even distribution of the WPC-casein matrix and more
prominent dark voids. These voids represent areas filled by the serum phase, an excess
Log
CFU
g-1
51
which leads to an open or loose texture (Sandoval-Castilla et al., 2004) which can lead to
the observed lower viscosity of the non-heat treated WPC yogurt. Fig. 6c represents the
goat’s milk yogurt with only pectin added. Because of the inherent weak structure of
goat’s milk as a result of the lack of αs1-casein, the sample most likely settled out during
the process of creating the SEM photographs which is why the observed picture is so
compact and grainy.
Figure 6. Scanning electron microscopy (SEM) photographs (x2000 & x500) of non-fat goat’s
milk yogurt with HTWP (a), non-fat goat’s milk yogurt with WPC (b), non-fat goat’s milk yogurt with
pectin only (c), and non-fat cow’s milk yogurt with pectin (d).
52
The αs1-casein in cow’s milk produces a protein-based network capable of
binding water and increasing viscosity in a bacterially acidified yogurt. As the pH is
decreased, the electrostatic repulsions between the normally negative casein molecules
are lowered. Hydrophobic interactions will increase leading to the formation of a three
dimensional protein network comprised of casein strands. Below pH 5.0, colloidal
calcium phosphate leaks into the serum from casein further strengthen electrostatic
interactions, weakening electrostatic repulsions, and leading to a depolymerization of αs1-
casein. This allows for an increased interaction between hydrophobic portions of the
casein (Phadungath, 2005). The goat’s milk yogurt with its absence of αs1-casein does not
form this network, leading to a weaker gel (Park and Guo, 2006).
The use of heat treated WPI has previously been shown to increase the water
holding capacity, total solids, and texture in goat’s milk yogurt (Li and Guo, 2006; Wang
et al., 2011). The use of WPC in goat’s milk yogurt requires that the proteins undergo
extensive denaturation through the raising of the pH and heating. β-Lactoglobulin is the
primary protein in WPC. In its native form, it contains one thiol-containing cysteine
residue as well as two disulfide bridges, the more active residing nearest the N-terminal.
Upon heating at high pH, the disulfide bridges can be broken resulting in an increase in
reactive thiol groups. These residues can then interact with each other, forming large
aggregates (Alting et al., 2000; Nicolai, Britton, and Schmitt, 2011; Roefs and de Kruif,
1994). When added to the milk system and then inoculated with a starter culture the pH
begins to drop, the HWPC interacts with the casein molecules and forms aggregated
particulates in the presence of calcium salts. Calcium, which is a divalent cation, binds
53
the negative carboxylic groups on nearby protein molecules (Bryant and McClements,
1998).
LM pectin is an anionic hydrocolloid, capable of interacting with the positive
charges on casein molecules. The pectin will stabilize the casein micelles, leading to a
decrease in viscosity and an increase in water holding capacity (Everett and Rosiland,
2004). Pectin plays a role in binding calcium as well. If the casein micelles bond too
strongly, water holding capacity will decrease. Beauleiu et al (2001) suggests that the
pectin should bind enough calcium so that gelation can occur without the presence of
much aggregation.
2.4. Conclusions
The use of HTWP solution, along with pectin, appears to be an appropriate
substitute for fat in the production of goat’s milk yogurt when used at a level of 12% and
0.35% respectively. Microstructure analysis showed that HTWP formed large aggregates
and interacted with casein to create a large protein network in the goat’s milk yogurt.
Pectin is used to further stabilize the gel and improve mouth feel. While survival rates of
L. acidophilous were not optimal, levels of Bifidobacteria spp. remained viable
throughout the 10 week storage period at 4˚C. This study has found that HTWP in
conjunction with pectin can be used as a fat replacer in goat’s milk yogurt and may have
uses in other fat- free dairy products.
54
2.5. Acknowledgments
Financial support for this project was provided in part by a USDA-NIFA Hatch
Grant (UVM project number 027094-2012/13) and Oak Knoll Dairy in Winsor, VT.
55
2.6. References
Agrawal, V., Kapoor, S., Shah, N. (2012). Role of ‘Live Microorganisms’ (probiotics) in
prevention of caries: going on the natural way towards oral health. Indian Journal of
Multidisciplinary Dentistry, 2(3), 491-496.
Ah-Leung, S., Bernard, H., Bidat, E., Paty, E., Rance, F., Scheinmann, P., Wal, J.M.
(2006). Allergy to goat and sheep milk without allergy to cow’s milk. Allergy, 61, 1358-
1365.
Alferez, M., Barrionuevo, M., Aliaga, I., Sanz-Sampelayo, M.R., Lisbona, F., Robles,
J.C., Campos, M.S. (2001). Digestive utilization of goat and cow milk fat in
malabsorption syndrome. Journal of Dairy Research, 68, 451-461.
Aliaga, I., Alferez, M., Barrionuevo, F., Lisbona, F., and Campos, M.S. (2000). Influence
of goat and cow milk on the digestive and metabolic utilization of calcium and iron.
Journal of Physiological Biochemistry, 56, 201-208.
Alonso, L., Fontecha, J., Lozada, L., Fraga, M., Juarez, M. (1999). Fatty acid
composition of caprine milk: major, branched-chain, and trans fatty acids. Journal of
Dairy Science, 82, 878-884.
Alting, A.C., Hamer, R.J., de Kruif, C.G., Visschers, R.W. (2000). Formation of disulfide
bonds in acid induced gels of preheated whey protein isolate. Journal of Agriculture and
Food Chemistry, 48, 5001-5007.
Alting, A., Hamer, R., De Kruif, C., Visschers, R. (2003). Cold-set globular protein gels:
Interactions, structure, and rheology as a function of protein concentration. Journal of
Agriculture and Food Chemistry, 51, 3150-3156.
Ambrosoli, R., Di Stasio, L., Mazzocco, P. (1988). Content of αs1-casien and coagulation
properties in goat milk. Journal of Dairy Science, 71, 24-28.
AOAC (2002). Association of official analytical chemists: official methods of analysis,
Vol. II (17 ed.), Gaithersburg, MD, USA.
Arena, M., Fiocco, D., Massa, S., Vittorio, C., Russo, P., Spano, G. (2014). Lactobacillus
plantarum as a strategy for an In situ production of vitamin B2. Journal of Food and
Nutritional Disorders, S1.
Arora, S., Kaur, I., Chopra, K., Rishi, P. (2014). Efficiency of double layered
microencapsulated probiotic to modulate proinflammatory molecular markers for the
management of alcoholic liver disease. Mediators of Inflammation, Article ID 715130.
Aryana, K.J., Plauche, S., Rao, R.M., McGrew, P., Shah, N.P. (2007). Fat-free plain
yogurt manufactured with inulins of various chain lenth and Lactobacillus acidophilous.
Journal of Food Science, 72, M79-M84.
56
Attaie, R., Richter, R.L. (2000). Size distribution of fat globules in goat milk. Journal of
Dairy Science, 83, 940-944.
Azinia, S., Khosrowshahi, A., Madadlou, A., Rahimi, J. (2007). Whey protein
concentrate and gum tragacanth as fat replacers in nonfat yogurt: Chemical, physical and
microstructural properties. Journal of Dairy Science, 91, 2545-2552.
Barrionuevo, M., Alferez, M., Lopez-Aliaga, I., Sampelayo, M.R., Campos, M.S. (2002).
Beneficial effect of goat milk on nutritive utilization of iron and copper in malabsorption
syndrome. Journal of Dairy Science, 85, 657-664.
Beaulieu, M., Turgeon, S.L., Doublier, J.L. (2001). Rheology, texture, and microstructure
of whey proteins/low methoxylpectins mixed with gels with added calcium. International
Dairy Journal, 11, 961-967.
Bisson, J., Hidalgo, S., Rozan, P., Messaoudi, M. (2010). Preventive effects of different
probiotic formulations on travelers’ diarrhea model in Wistar rats. Disgestive Diseases
and Sciences, 55, 911-919.
Bosch, M., Nart, J., Audivert, S., Bonachera, M., Alemany, A., Fuentes, M., Cune, J.
(2012). Isolation and characterization of probiotic strains for improving oral health.
Archives of Oral Biology, 57, 539-549.
Bryant, C.M., McClements, D.J. (1998). Molecular basis of protein functionality with
special consideration of cold-set gels derived from heat-denatured whey. Trends in Food
Science and Technology, 9, 143-151.
Buss, C., Valle-Tovo, C., Miozzo, S., de Mattos, A.A., (2014). Probiotics and synbiotics
may improve liver aminotransferase levels in non-alcoholic fatty liver disease patients.
Annals of Hepatology, 13, 482-488.
Calleros-Lobato, C., Martinez-Torrijos, O., Sandoval-Castilla, O., Perez-Orozco, .P.,
Vernon-Carter, E.J. (2004). Flow and creep compliance properties of reduced-fat
yoghurts containing protein-based fat replacers. International Dairy Journal, 14, 777-
782.
Campbell, J.R., Marshall, R.T. (1975). The Science of Providing Milk for Man. McGraw-
Hill Book Co., New York, NY, 801 p.
Cebo, C., Caillat, H., Bouvier, F., Martin, P. (2009). Major proteins of the goat milk fat
globule membrane. Journal of Dairy Science, 93, 868-876.
Cha, B., Jung, S., Choi, C., Song, I., Lee, H., Kim, H., Do, J., Chang, S., Kim, K., Chung,
W., Seo, J. (2012). The effect of a multispecies probiotic mixture on the symptoms and
fecal mirobiota in diarrhea-dominant irritable bowel syndrome. Journal of Clinical
Gastroenterology, 46, 220-227.
57
Chatterton, D., Smithers, G., Roupas, P., Brodkorb, A. (2006). Bioactivity of β-
lactoglobulin and α-lactalbumin- Technological implications for processing.
International Dairy Journal, 16, 1229-1240.
Chillard, Y., Glasser, F., Ferlay, A., Bernard, L., Rouel, J., Doreau, M. (2007). Diet,
rumen biohydrogenation and nutritional quality of cow and goat milk fat. European
Journal of Lipid Science and Technology, 109, 828-855.
Crispin-Isidro, G., Lobato-Calleros, C., Espinosa-Andrews, H., Alvarez-Ramirez, J.
(2015). Effect of inulin and agave fructans addition on the rheological, microstructural
and sensory properties of reduced-fat stirred yogurt. Food Science and Technology, 62,
438-444.
Chr. Hansen (2005). Lactobacillus acidophilus, Lactobacillus casei, andBifidobacteria in
fermented milk products – guidelines.Methods for counting probiotic bacteria. Technical
Bulletin, F-6, 1-8.
Cross, M. (2002). Microbes versus microbes: Immune signals generated by probiotic
lactobacilli and their role in protection against microbial pathogens. FEMS Immunology
and Medical Microbiology, 34, 245-253.
Coman, M., Verdenelli, M., Cecchini, C., Silva, S.,Orpianesi, C., Boyko, N., Cresci, A.
(2014). In vitro evaluation of antimicrobial activity of Lactobacillus rhamnosus IMC
501, Lactobacillus paracasei IMC 502, and SYNBIO against pathogens. Journal of
Applied Microbiology, 117, 518-527.
Dalgic, N., Sancar, M., Bayraktar, B., Pullu, M., Hasim, O. (2011). Probiotic, zinc, and
lactose-free formula in children with rotavirus diarrhea: Are they effective? Pediatrics
International, 53, 677-682.
Didari, T., Mozaffari, S., Nikfar, S., Abdollahi, M. (2015). Effectiveness of probiotics in
irritable bowel syndrome: Updated systematic review with meta-analysis. World Journal
of Gastroenterology, 21(10), 3072-3084.
De Vrese, M., Kristen, H., Rautenberg, P., Laue, C., Schrezenmeir, J. (2011). Probiotic
lactobacilli and bifidobacteria in a fermented milk product with added fruit preparation
reduce antibiotic associated diarrhea and Helicobacter pylori activity. Journal of Dairy
Research, 78, 396-403.
Dewettinck, K., Rombaut, R., Thienpon, N., Le, T., Messens, K., Camp, J. (2008).
Nutritional and technological aspects of milk fat globule membrane material.
International Dairy Journal, 18, 436-457.
Ebner, K., Schanbacher, F. (1974). Biochemistry of lactose and related carbohydrates. Pg
77. In: Lactation: A Comprehensive Treatise. Vol 2. B.L. Larson and V.R. Smith, ed.
Academic Press, New York, NY.
58
El-Agamy, E.I. (2006). The challenge of the cow milk protein allergy. Small Ruminant
Research, 86, 64-72.
Euber, J.R., Brunner, J.R. (1984). Reexamination of fat globule clustering and creaming
in cow milk. Journal of Dairy Science, 67, 2821-2832.
Everett, D.W., Rosiland, E.M. (2004). Interactions of polysaccharide stabilizers with
casein aggregates in stirred skim-milk yogurt.International Dairy Journal,15, 1175-1183.
Faria, J., Vidigal, M., Loures, M., Minim, V., Minim, L. (2001). The effect of whey
protein concentrate as a fat replacer on the rheological characteristics of yogurt. Federal
Univeristy of Vicosa, Brasil.
Farnsworth, J.P., Li, J., Hendricks, G.M., Guo, M.R. (2006). Effects of transglutaminase
on functional properties and probiotic culture survivability of goat milk yogurt. Small
Ruminant Research, 65, 113-121.
Gilland, S.E., Speck, M.L. (1977). Instability of Lactobacillus acidophilus in yogurt.
Journal of Dairy Science, 60, 1394-1398.
Goetsch, A., Zeng, S., Gipson, T. (2011). Factors affecting goat milk production and
quality. Small Ruminant Research, 101, 55-63.
Gopal, P.K., Gill, H.S. (2000). Oligosaccharides and glycoconjugates in bovine milk and
colostrums. British Journal of Nutrition, 84, S69-S74.
Guo, M. R. (2003). Goat milk. In B. Caballero, L. Trugo, & P. Finglas (Eds.),
Encyclopedia of food sciences and nutrition (pp. 2944-2949). London, England:
Academic Press.
Guo, M.R. (2007). Functional Foods: Principles and Technology. Timonium, MD, USA:
CTI Publications.
Guo, M., Dixon, P., Park, Y., Gilmore, J., Kindstedt, P. (2001). Seasonal changes in the
chemical composition of commingled goat milk. Journal of Dairy Science, 84, 79-83.
Guzman-Gonzalez, M., Morais, F., Ramos, M., Amigo, L. (1999). Influence of skimmed
milk concentrate replacement by dry dairy products in a low fat set-type yoghurt model
system: Use of whey protein concentrates, milk protein concentrates, and skimmed milk
powder. Journal of the Science of Food and Agriculture, 79, 1117-1122.
Haenlein, G.F. (1996). Status and prospects of the dairy goat industry in the United
States. Journal of Animal Science, 74, 117-1181.
Haenlein, G.F.W. (2004). Goat milk in human nutrition. Small Ruminant Research,
51, 155-163.
59
Hempel, S., Newberry, S., Maher, A., Wang, Z., Miles, J., Shanman, R., Johnsen, B.,
Shekelle, P. (2012). Probiotics for the prevention and treatment of antibiotic-associated
diarrhea. Journal of the American Medical Association, 307, 1959-1969.
Iannitti, T., Palmieri, B. (2010). Therapeutical use of probiotic formulations in clinical
practice. Clinical Nutrition, 29, 701-725.
Jafari, S., Ahanchian, H., Kiani, M., Khakshour, A., Noorbakhsh, Z., Zamani, E., Ansari,
E., Kianifar, H. (2014). Synbiotic for prevention of antibiotic-associated diarrhea in
children: a randomized clinical trial. International Journal of Pediatrics, 2, 57-62.
Jandal, J.M. (1996). Comparative aspects of goat and sheep milk. Small Ruminant
Research, 22, 177-185.
Jenness, R. (1980). Composition and characteristics of goat milk: review 1968-1979.
Journal of Dairy Science, 63, 1605-1630.
Jenness, R., Parkash, S. (1971). Lack of a fat globule clustering agent in goats’ milk.
Journal of Dairy Science,54, 123-126.
Kosikowski, F. (1977). Cheese and Fermented Food. p. 573. Kosikowski and
Associates, Brooktondale, NY, USA.
Lara-Villoslada, F., Debras, E., Nieto, A., Concha, A., Galvez, J., Lopez-Huertas, E.,
Boza, J., Obled, C., Xaus, J. (2006). Oligosaccharides isolated from goat milk reduce
intestinal inflammation in a rat model of dextran sodium sulfate-induced colitis. Clinical
Nutrition, 25 (3), 477-488.
LeDoux, M., Rouzeau, A., Bas, P., Sauvant, D. (2002). Occurrence of trans-C18:1 fatty
acid isomers in goat milk: Effect of two dietary regimens. Journal of Dairy Science, 85,
190-197.
Lee, T.H., Shimazaki, K., Yo, S.L., Nam, M.S., Kim, S.J., Lee, K.K., Yu, D.Y. (1997).
Polymorphic sequence of Korean native goat lactoferrin exhibiting greater antibacterial
activity. Animal Genetics, 28, 367-369.
Li, J., Guo, M. (2006). Effects of polymerized whey proteins on consistency and water-
holding properties of goat’s milk yogurt. Journal of Food Science, 71, C34-C38.
Ljutovac, K., Lagriffoul, G., Paccard, P., Guillet, I., Chilliard, Y. (2008). Composition of
goat and sheep milk products: An update. Small Ruminant Research, 79, 57-72.
Lobato-Calleros, C., Martinez-Torrijos, O., Sandoval-Casilla, O., Perez-Orozco, J.,
Vernon-Carter, E. (2004). Flow and creep compliance properties of reduced-fat yoghurts
containing protein-based fat replacers. International Dairy Journal, 14, 777-782.
60
Lobato-Calleros, C., Ramirez-Santiago, C., Vernon-Carter, E.J., Alvarez-Ramirez, J.
(2014). Impact of native and chemically modified starches addition as fat replacers in the
viscoelasticity of reduced-fat stirred yogurt. Journal of Food Engineering, 131, 110-115.
Lourens-Hattingh, A., Viljoen, B.C. (2001).Yogurt as a probiotic carrier
food. International Dairy Journal, 11, 1-17.
Lorenzo-Zuniga, V., Llop, E., Suarez, C., Alvarez, B., Abreu, L., Espadaler, J., Serra, J.
(2014). I.31, a new combination of probiotics, improves irritable bowel syndrome-related
quality of life. World Journal of Gastroenteraology, 20(26), 8709-8716.
Lucey, J.A., Tamehana, M., Singh, H., Munro, P.A. (1999). Stability of model acid milk
beverage: effect of pectin concentration, storage temperature and milk heat treatment.
Journal of Texture Studies, 30, 305-318.
Manso, M.A., Escudero, C., Aliho, M., Lopez- Fandino, R. (2002). Platelet aggregation
inhibitory activity of bovine, ovine, and caprine κ-casien macropeptides and their tryptic
hydrolysates. Journal of Food Protection, 65, 1992-1996.
McKenzie, H., Ralston, G., Shaw, D. (1972). Location of sulphydryl and disulfide groups
in bovine β-lactoglobulin and effect of urea. Biochemistry, 11:4539.
McKinley, M. (2005). The nutrition and health benefits of yogurt. International Journal
of Dairy Technology, 58, 1-12.
Meena, S., Rajput, Y., Sharma, R. (2014). Comparative fat digestibility of goat, camel,
cow and buffalo milk. International Dairy Journal, 35, 153-156.
Metchnikoff, E. (1907). The prolongation of life. Optimistic studies. London:
Butterworth-Heinemann.
Mendi, A., Ashm, B. (2014). Antioxidant Lactobacilli could protect gingival fibroblasts
against hydrogen peroxide: a preliminary in vitro study. Probiotics and Antimicrobial
Protection, 6, 157-164.
Mishra, S., Mann, B., Joshi, V.K. (2000). Functional improvement of whey protein
concentrate on interaction with pectin. Food Hydrocolloids, 15, 9-15.
Mistry, V.V., Hassan, H.N. (1992). Manufacture of nonfat yogurt from a high milk
protein powder. Journal of Dairy Science, 75, 947-957.
Morand-Fehr, P., Fedele, V., Decandia, M., Frileux, Y. (2007). Influence of farming and
feeding systems on composition and quality of goat and sheep milk. Small Ruminant
Research, 68, 20-34.
Nabavi, S., Rafraf, M., Somi, M.H., Homayouni-Rad, A., Asghari-Jafarabadis, M.
(2014). Effects of probiotic yogurt consumption on metabolic factors in individuals with
nonalcoholic fatty liver disease. Journal of Dairy Science, 97, 7386-7393.
61
Nicolai, T., Britten, M., Schmitt, C. (2011). Β-lactoglobulin and WPI aggregates:
formation, structure, and applications. Food Hydrocolloids, 25, 1945-1962.
Ouwehand, A., Salminen, S., Isolauri, E. (2002). Probiotics: and overview of beneficial
effects. Antonie van Leeuwenhowk, 82, 279-289.
Paolella, G., Mandato, C., Pierri, L., Poeta, M., Di Stasi, M., Vajro, P. (2014). Gut-liver
axis and probiotics: Their role in non-alcoholic fatty liver disease. World Journal of
Gastroenterology, 20(42), 15518-15531.
Park, Y.W. (1994a). Hypo-allergenic and therapeutic significance of goat milk. Small
Ruminant Research, 14, 151-159.
Park, Y.W. (1994b). Nutrient and mineral composition of commercial US goat milk
yogurts. Small Ruminant Research, 13, 63-70.
Park, Y.W. (2000). Comparison of mineral and cholesterol composition of different
commercial goat milk products manufactured in USA. Small Ruminant Research, 37,
115-124.
Park, Y.W. and Guo, M.R. (2006). Goat Milk Products: Types of products,
manufacturing technology, chemical composition, and marketing. In Park, Y.W. and
Haenlein, F.W. (Eds.), Handbook of Milk of Non-Bovine Mammal (pp. 59-106). Ames,
IA, USA: Blackwell Publishing.
Park, Y.W., Haenlein, F.W. (2006). Handbook of Milk of Non-Bovine Mammals,
Blackwell Publishing: Ames, IA, USA.
Park, Y.W., Juarez, M., Ramos, M., Haenlein, G.F.W. (2007). Physico-chemical
characteristics of goat and sheep milk. Small Ruminant Research, 68, 88-113.
Phadungath, C. (2005). The mechanism and properties of acid-coagulated milk
gels. Journal of Science and Technology, 2005, 433-448.
Patton, S., Bogus, E.R., Sternberger, B.H., Trams, E.G. (1980). Antiserum to the milk fat
globule membrane preparation and capacity to suppress milk secretion. Biochimica et
Biophysica Acta, 597, 216-223.
Patton, S., Carson, G.S., Hiraiwa, M., O’Brian, J.S., Sano, A. (1997). Prosaposin, a
neutropic factor: presence and properties in milk. Journal of Dairy Science, 80, 264-272.
Puente, R., Garcia-Pardo, L.A., Rueda, R., Gil. A., Hueso, P. (1996). Seasonal variations
in the concentration of gangliosides and sialic acids in milk from different mammalian
species. International Dairy Journal, 6, 315-322.
Raynal-Ljutovac, K., Lagriffoul, G., Paccard, P., Guillet, I., Chillard, Y. (2008).
Composition of goat and sheep milk products: and update. Small Ruminant Research, 79,
57-72.
62
Restani, P. (2004). Goat milk allerginicity. Journal of Pediatric Gastroenterology and
Nutrition, 39, 323-324.
Ritze, Y., Bardos, B., Claus, A., Ehrmann, V., Bergheim, I., Schwiertz, A., Bischoff, S.
(2014). Lactobacillus rhamnosus GG protects against non-alcoholic fatty liver disease in
mice. Plos One 9(1): e80169.
Roeffs, P.F.M., de Kruif, K.G. (1994). A model for the denaturation and aggregation of
β-lactoglobulin.European Journal of Biochemistry, 226, 883-889.
Saad, N., Delattre, C., Urdaci, M., Schmitter, J.M., Bressollier, P. (2013).An overview of
the last advances in probiotic and probiotic field.Food Science and Technology, 50, 1-16.
Saarela, M., Lahteenmaki, L., Crittenden, R., Salminen, S., Mattila-Sandholm, T. (2002).
Gut bacteria and health foods- the European perspective. International Journal of Food
Microbiology, 78, 99-117.
Saarela, M., Mogensen, G., Fonden,R., Matto, J., Mattilla-Sandholm, T. (2000). Probiotic
bacteria: safety, runctional and technological properties. Journal of Biotechnology, 84,
197-215.
Sahan, N., Yasar, K., Hayaloglu, A.A. (2008). Physical, chemical and flavor quality of
non-fat yogurt as affected by a β-glucanhydrocolloidal composite during storage.Food
Hydrocolloids, 22, 1291-1297.
Sakurai, K., Goto, Y. (2006). Dynamics and mechanism of the Tanford transition of
bovine β-lactoglobulin studied using heteronuclear NMR spectroscopy. The Journal of
Molecular Biology, 356, 483-496.
Salminen, S., von Wright, A., Morelli, L., Marteau, P., Brassart, D., de Vos, W.M.,
Fonden, R., Saxelin, M., Collins, K., Mogensen, G., Birkeland, S., Mattila-Sandholm, T.
(1998).Demonstration of safety of probiotics – a review. International Journal of Food
Microbiology, 44, 93-106.
Sanders, M.E. (2003). Probiotics: Considerations for human health. Nutrition Reviews,
61, 91-99.
Sandoval-Castilla, O., Lobato-Calleros, C., Aguirre-Mandujano, E., Vernon-Carter, E.J.
(2004).Microsctructure and texture of yogurt as influenced by fat replacers. International
Dairy Journal, 14, 151-159.
Schrezenmeir, J., de Vrese, M. (2001). Probiotics, prebiotics and synbiotics –
approaching a definition. American Journal of Clinical Nutrition, 73, 361-364.
Shah, N.P. (2007). Functional cultures and health benefits. International Dairy Journal,
17, 1262-1277.
63
Shi, X., Wei, X., Yin, X., Wang, Y., Zhang, M., Zhao, C., Zhao, H., McClain, C., Feng,
W., Zhang, X. (2015). Hepatic and fecal metabolomics analysis of the effects of
Lactobacillus rhamnosus GG on alcoholic fatty liver disease in mice. Journal of
Proteome Research, 14, 174-1182.
Shokryazdan, P., Sieo, C.C., Kalavathy, R., Liang, J.B., Alitheen, N., Jahromi, M., Ho.,
Y.W. (2014). Probiotic potential of Lactobacillus strains with antimicrobial activity
against some human pathogenic strains. BioMed Research International, Article ID
927268, 16 pages.
Singh, H. (2006). The milk fat globule membrane – A biophysical system for food
applications. Current Opinions in Colloid and Interface Science, 11, 154-163.
Song, J., Kim, J., Jung, S., Kim, S., Park, H., Jeong, Y., Hong, S., Cheon, J., Kim, W.,
Kim, H., Ye, B., Yang, S., Kim, S., Shin, S., Kim, H., Sung, J., Kim, E. (2010). Effect of
probiotic Lactobacillus (Lacidofil Cap) for the prevention of antibiotic-associated
diarrhea: A prospective, randomized, double-blind, milticenter study. Journal of Korean
Medical Science, 25, 1784-1791.
Tantibhedhyangkul, P., Hashim, S. (1975). Medium-chain triglyceride feeding in
premature infants: Effects of fat and nitrogen absorption. Pediatrics, 61, 537.
Teran, C., Teran-Exalera, C., Villarroel, P. (2009). Nitazoxanide vs. probiotics for the
treatment of acute rotavirus diarrhea in children: a randomized single-blind controlled
trial in Bolivian children. International Journal of Infectious Diseases, 13, 518-523.
Tsiplakou, E., Mountzouris, K.C., Zervas, G. (2006). Concentration of conjugated
linoleic acid in grazing sheep and goat milk fat. Livestock Science, 103, 74-84.
Umpierrz, A., Quirce, S., Maranon, F., Cuesta, J., Garcia-Villamuza, Y., Lahoz, C.,
Sastre, J. (1999). Allergy to goat and sheep cheese with good tolerance to cow cheese.
Clinical and Experimental Allergy, 29, 1064-1068.
Vanhoutte, B., Rombaut, R., Dewettinck, K., Van der Meeren, P. (2004). Phospholipids.
In L.M.L Nollet, Food Analysis (pp. 349-382). New York, USA
Vasiljevic, T., Shah, N.P. (2008). Probiotics – from Metchnikoff to bioactives.
International Dairy Journal, 18, 714-728.
Vassallo, G., Mirijello, A., Ferulli, A., Antonelli, M., Landolfi, A., Gasbarrini, A.,
Addolorato, G. (2015). Review article: alcohol and gut microbiota- the possible role of
gut microbiota modulation in the treatment of alcoholic liver disease. Alimentary
Pharmacology and Therapeutics, 1-11.
Vargas, M., Chafer, M., Albors, A., Chiralt, A., Gonzalez-Martinez, C. (2008).
Physicochemical and sensory characteristics of yoghurt produced from mixtures of cows’
and goats’ milk. International Dairy Journal, 18, 1146-1152.
64
Varhanabhuti, B., Foegeding, E.A. (1999). Rheological properties and characterization of
polymerized whey protein isolates. Journal of Agriculture and Food Chemistry, 47,
3649- 3655.
Wakabayashi, H., Takase, M., Tomita, M. (2003). Lactoferricin derived from milk
protein lactoferrin. Current Pharmicilogical Discussions, 9, 1277-1287.
Walsh, H., Ross, J., Hendricks, G., Guo, M. (2010).Physico-chemical properties,
probiotic survivability, microstructure, and acceptability of a yogurt-like symbiotic oats-
based product using pre-polymerized whey protein as a gelation agent. Journal of Food
Science, 75, M327-337.
Wang, W., Bao, Y., Hendricks, G.M., Guo, M. (2011).Consistancy, microstructure, and
probiotic survivability of goats’ milk yoghurt using polymerized whey protein as a co-
thickening agent. International Dairy Journal, 24, 113-119.
WHO- The World Health Organisation. (2004). Website
[http://www.who.int/healthinfo/global_burden_disease/GBD_report_2004update_part2.p
df].
Yamamoto, S., Wada, J., Katayama, T., Jikimoto, T., Makamura, M., Kinoshita, S., Lee,
K., Kawabata, M., Shirakawa, T. (2010). Genetically modified Bifidobacterium
displaying Salmonella-antigen protects mice from lethal challenge of Salmonella
Typhimurium in a murine typhoid fever model. Vaccine, 28, 6684-6691.
Yan, A.W., Schnabl, B. (2012). Bacterial translocation and changes in the intestinal
microbiome associated with alcoholic liver disease. World Journal of Hepatology, 4(4):
110-118.
Ye, A., Singh, H., Taylor, M., Anema, S. (2004). Interactions of whey proteins with milk
fat globule membrane proteins during heat treatment of whole milk. Lait, 84, 269-283.
Zhang, L., Hellgren, L., Xu, X. (2006). Enzymatic production of ceramide from
sphingomyelin. Journal of Biotechnology, 123, 93-105.