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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

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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.

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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.

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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).

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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

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