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Author: Krienke, Emily, C

Title: Determining Differences in Protein Hydrolysis in Fermented Food Products Analyzed

using Mass Spectrometry The accompanying research report is submitted to the University of Wisconsin-Stout, Graduate School in partial completion of the requirements for the

Graduate Degree/ Major: MS Food and Nutritional Sciences

Research Adviser: Dr. Marcia Miller-Rodeberg

Submission Term/Year: Fall, 2012

Number of Pages: 30

Style Manual Used: American Psychological Association, 6th

edition

I understand that this research report must be officially approved by the Graduate School and

that an electronic copy of the approved version will be made available through the University

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I attest that the research report is my original work (that any copyrightable materials have been

used with the permission of the original authors), and as such, it is automatically protected by the

laws, rules, and regulations of the U.S. Copyright Office.

STUDENT’S NAME: Emily Krienke

STUDENT’S SIGNATURE: _Emily Krienke_____________________________________ DATE: 12/14/12

ADVISER’S NAME (Committee Chair if MS Plan A or EdS Thesis or Field Project/Problem):

ADVISER’S SIGNATURE: ____________DATE: 12/14/2012

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Krienke, Emily, C. Determining Differences in Protein Hydrolysis in Fermented Food

Products Analyzed using Mass Spectrometry

Abstract

Yogurt is one of the most universally consumed and healthful dairy products in the world

due to its physical and chemical characteristics. The objective of this study was to examine

differences in protein fragmentation between yogurts made with various starter cultures. Yogurt

samples were made using three commercially sold yogurt starter cultures (Activia, Fage, and Old

Home) with a basic recipe. Casein was extracted at weekly intervals using standard methods and

dissolved in a phosphate buffer until use; a control sample was extracted using the same method

from milk. Fragmentation was first examined using gel electrophoresis. Protein concentrations

were obtained and samples were desalted before they were investigated on a Matrix-Assisted

Laser Desorption Ionizing Time-of-Flight (MALDI-TOF) Spectrometer.

Results showed distinct cleavage sites of the protein with differences between starter

cultures. All peptides originated from bovine casein Alpha S2 and were sliced from the middle of

the protein sequence. While results were consistent with preliminary data, samples were not run

in triplicate and thus not statistically significant. More experimentation should be conducted to

determine significance and explore sensory attributes. The ultimate goal will be able to explain

the effect of using different microorganisms on protein fragmentation and sensory

characteristics.

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Acknowledgements

I would like to give the most thanks to my advisor, Dr. Miller-Rodeberg; without whom

this project may never have been started or completed. A special thanks to Rebecca Hoeft as well

as Dr. Grant and her students for their help and patience. To my family and especially my father,

thank you for the constant support and praise, now and throughout my life; you have always

supported me and without you and your sacrifices, this degree would’ve never been possible, I

love you. And to my biggest pillar of support, my fiancé Sean Nelson, while you still probably

have not comprehended what the specifics of my thesis are, you never let me quit and always

gave me the confidence to continue pursuing my dreams.

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Table of Contents

…………………………………………………………………………………………………Page

Abstract …………………………………………………………………………………………...2

Acknowledgements ……………………………………………………………………………….3

List of Tables ……………………………………………………………………………………..6

List of Figures …………………………………………………………………………………….7

Chapter I: Introduction ……………………………………………………………………………8

Statement of the Problem ………………………………………………………………..12

Purpose of the Study …………………………………………………………………….12

Hypothesis ……………………………………………………………………………….12

Definition of Terms ……………………………………………………………………..12

Assumptions of the Study ……………………………………………………………….13

Limitations of the Study …………………………………………………………………13

Chapter II: Literature Review …………………………………………………………………...14

Chapter III: Methodology ……………………………………………………………………….19

Introduction ………….………………………………………………………………….19

Sample Selection ………………………………………………………………………..19

Instrumentation ………………………………………………………………………….19

Data Collection ………………………………………………………………………….19

Data Analysis ……………………………………………………………………………21

Limitations ………………………………………………………………………………21

Chapter IV: Results ……………………………………………………………………………...22

Item Analysis ……………………………………………………………………….......22

Chapter V: Discussion …………………………………………………………………………..27

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Limitations ………………………………………………………………………………27

Conclusions ……………………………………………………………………………...27

Recommendations …………………………………………………………………........27

References …………………………………………………………………………………........29

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List of Tables

Table 1: Original sample protein concentration data as determined by the Bradford assay……..23

Table 2: MALDI-TOF results of peptide fragment peak identity………………………………..25

Table 3: Peptide peaks, residue numbers, phosphorylation, and sequences……………………..26

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List of Figures

Figure 1: Bovine milk casein micelle structure………………………………………………….10

Figure 2: Hydrolysis reaction of peptides……………………………………………………….15

Figure 3: Preliminary gel electrophoresis data using the standard BSA. A) Activia, Fage, and Old Home commercially produced yogurts. B) Milk and Yogurt in 1x and 10x concentrations ……………………………………………………………………………………………22

Figure 4: Protein concentration standard curve using Bradford reagent and bovine serum albumin as the protein standard…………………………………………………………………...23

Figure 5: Bovine casein cleavage spectrums: control (milk), Activia first casein extraction (A1), Fage first casein extraction (F1), and Old Home first casein extraction (OH1)…………24

Figure 6: Bovine casein amino acid sequences…………………………………………………..26

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Chapter I: Introduction

Yogurt is one of the most universally popular dairy foods because of its versatility. It can

be consumed as a snack, as part of a meal, an ingredient, or a dessert. Historians generally agree

that yogurt was discovered by accidentally storing milk using primitive methods at warm

temperatures. Yogurt can be traced back as far as 6000 B.C to the Neolithic peoples of Central

Asia. It is believed that herdsmen started the practice of milking their animals and storing the

milk in containers made out of animal stomachs, which subsequently curdled the milk, making

yogurt. It was first industrially produced in Barcelona, Spain in the 1700s at a family-run

company called “Danone,” which would later become General Mills’ brand “Dannon” (Dairy

Goodness, 2011).

Yogurt is made by using bacteria to ferment milk. The bacteria used are known as starter

cultures and the two starter cultures required by law to be used in the yogurt making process are

Lactobacillus bulgaricus, and Streptococcus thermophilus, though other cultures may be added.

The cultures work to ferment lactose (milk sugar) and in the process produce lactic acid. The

increase in lactic acid causes the pH to drop, resulting in the milk proteins to clot and form the

soft gel characteristic of yogurt. The production of lactic acid affects the protein in yogurt, which

in turn affects the consistency and flavor properties (Cornell University, 2006). The flavor

components most commonly associated with plain yogurt are sour, tangy, and tart due to the

lactic acid content.

The Food and Drug Administration (FDA, 2011) described yogurt as food produced by

culturing one or more of the optional dairy ingredients (cream, milk, partially skimmed milk, or

skim milk, used alone or in combination) with a characterizing bacterial culture that contains the

lactic acid-producing bacteria, Lactobacillus bulgaricus and Streptococcus thermophilus (FDA,

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2011). According to the United States Department of Agriculture (USDA) standards, plain

yogurt must have no less than 3.25% fat and no less than 8.25% milk non-fat solids; low-fat and

nonfat yogurts have their specifications, respectively. Milk used to make yogurt must be Grade A

and pasteurized (USDA, 2001).

The health benefits of yogurt appear to be never ending. Yogurt contains vitamins B5 and

12, potassium, riboflavin, iodine, zinc, and phosphorus, as well as any additionally added

vitamins and minerals. Yogurt is also growing in popularity due to its ability to aid in digestion

because it contains probiotics. Probiotics are defined as microorganisms that positively

contribute to the body’s overall health and are used to regulate and restore balance to the normal

microflora of the digestive track (Probiotics, 2009).

Of all the vitamins in milk, vitamins D and B2 (referred to as riboflavin from here on) are

two of the most sensitive to degradation. Vitamin D is a fat soluble vitamin and is essential in

creating and maintaining healthy bones and calcium absorption. While it can be obtained through

spending 15 minutes in the sun, or from eating a variety of foods, Vitamin D fortified foods are

the best sources. Vitamin D is naturally found in fish, meat, and eggs. Deficiency in vitamin D

can lead to terrible disorders such as Rickets and Osteoporosis (Vitamin D Deficiency Guide,

2011). Riboflavin is a water soluble vitamin required for processing dietary fats, carbohydrates,

and proteins and converting them into energy. While riboflavin is not easily destroyed through

cooking, it is very light sensitive and thus it can be found in greater abundance in cartons of milk

rather than clear plastic or bottle containers (Faqs.org, 2011).

Milk products such as yogurt have two major groupings of protein: caseins and whey.

Casein proteins are composed of several similar proteins which form a multi-molecular, granular

structure called a casein micelle which contains water and salt ions, specifically calcium and

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phosphate, Figure 1. The casein structure in milk is an important part of the manner of digestion

of milk in the stomach and intestine, as well as it is the basis for humans’ ability to easily

separate some proteins and other components from cow’s milk. Individual molecules of casein

alone are not exceptionally soluble in milk; however the casein micelle granules are able to be

maintained as a colloidal suspension in milk. If this structure is disrupted, the micelles may be

dispersed and the casein may fall out of solution. This process forms a gelatinous material known

as the ‘curd,’ which is part of the yogurt making process. The major soluble whey proteins in

milk are ß-lactoglobulin and α-lactalbumin. Other whey proteins include immunoglobulins and

serum albumin (Milk Composition and Synthesis Resource Library, 2011).

Figure 1 Bovine milk casein micelle structure.

Milk proteins can be separated and identified based on their molecular weight by means

of gel electrophoresis. Through this method, the proteins can be separated by molecular weight

with the larger and smaller proteins migrating at different rates on the gel which are all compared

to a molecular weight standard (Milk Composition and Synthesis Library, 2011).

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Mass Spectrometry is another method used to separate matter based on its charge to mass

ratio. This method has previously been most commonly been used by chemists to run unknown

samples and determine their composition based on their molecular weight. Some of the best

examples of the versatility of Mass Spectrometry include identifying and quantifying pesticides

in water samples, determining steroid use in athletes, carbon dating ancient artifacts, as well as

looking for life on Mars (Van Bramer, 1998). In the realm of food science, mass spectrometry is

being used for pesticide analysis, antibiotics analysis, toxin analysis, and allergen testing

(Integrated ULF Biosystems, 2011).

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Statement of the Problem

It has not been published as to how yogurt starter cultures produce differences in protein

fragmentation. Additionally, no work has been done using the Mass Spectrometer to measure

protein size in fermented foods. Experimentation will be conducted using the MALDI-TOF mass

spectrometer to determine how using different starter cultures affect protein fragmentation in

milk during the yogurt-making process.

Purpose of the Study

Preliminary results using Gel Electrophoresis showed differences in protein content of

several yogurts. The protein content of homemade yogurt using various starter cultures was

determined using Mass Spectrometry. Experiments were conducted in the spring and fall

semesters of the 2012 school year at the University of Wisconsin-Stout.

Hypothesis

Data collected by mass spectrometry will show differences in protein fragmentation

between yogurts made with various starter cultures.

Definition of Terms

These terms need to be defined because they are not basic knowledge to the general public.

Casein. The main protein in milk and milk products.

Denature. When the structure of a protein is lost due to unfolding of the peptide chain;

typically caused by heat or acid.

Gel electrophoresis. A method to separate proteins based on their size.

Hydrolysis. The cleavage of a chemical compound by addition of water.

Mass Spectrometry. A method for separating and analyzing matter based on its charge

to mass ratio.

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Peptide. A compound comprised of two or more amino acids that are linked via peptide

bonds into a polymer.

Assumptions

It is assumed that the differing patterns of hydrolysis observed by the gel electrophoresis

analysis in preliminary studies would be confirmed by a more sensitive technique.

Limitations

The starter cultures used in this study were not well characterized (e.g. identifying initial

cell count) and therefore, some observed differences may be due to the culture characteristics.

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Chapter II: Literature Review

This chapter will begin with how the fermentation process makes dairy foods healthier

and will conclude with current research on protein analysis using mass spectroscopy.

As most consumers know, fermented foods are formed from living microorganisms.

Fermented foods are the primary products that probiotic bacteria are found in with dairy foods

cornering the market. In commercially produced yogurt, starter culture strains are first chosen

based on their physical properties and then on their associated health benefits. Probiotics have

been shown to be beneficial by helping to regulate the digestive system by maintaining the

balance of healthful bacteria (Heller, 2001).

The cultures typically chosen in commercial yogurt come from the Lactobacillus and

Bifidobacterium genera. Within Lactobacillus, the genera from which probiotic strains have

been isolated include: Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus casei,

Lactobacillus rhamnosus, Lactobacillus gasseri, and Lactobacillus reuteri. The probiotic

Bifidobacterium species include: Bifidobacterium bifidum, Bifidobacterium longum, and

Bifidobacterium infantis (Heller, 2001).

Yogurt, in addition to other dairy products, is an optimum carrier of probiotics. The

environment provided by yogurt is very favorable to the growth and survival of fermentation

organisms. Live microorganisms interact strongly with their environment and can change the

properties of the medium they are in which plays a pivotal role in the composition of yogurt

(Heller, 2001).

Some of the essential variables that affect microbial activity in yogurt include the degree

of hydrolysis of milk proteins, because they affect availability of essential amino acids, as well

as the kind and amount of carbohydrates available, Figure 2. Conversely, proteolytic properties

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may be important for future degradation of proteins because they may have significant effects on

the taste and texture of yogurt. Other fermented foods such as sausages and sauerkraut are also

considered good carriers of probiotic organisms but few similar products can be seen on the

market (Heller, 2001).

Figure 2 Hydrolysis reaction of peptides.

A chief characteristic of the production of probiotic fermented yogurt is the associations

between probiotics and starter culture organisms. Little is known about the interactions between

probiotic bacteria and starter culture organisms but synergistic and antagonistic effects have been

observed and recorded. What is acknowledged about the connections between the different

bacteria is that they seemed to be based on the specific strains rather than the species (Heller,

2001).

The nature of the interactions between yogurt, probiotics, and starter cultures largely

depends on the stage in which the probiotics are added i.e. before or after fermentation occurs.

There is little to no interaction between the starter cultures and probiotics that are added to

yogurt after fermentation however, the same cannot be said for adding them in the beginning

(Heller, 2001).

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When probiotics actively partake in the fermentation process special care must be taken

to ascertain that they will not negatively interact with the starter cultures and retard the growth.

One such controllable factor is the production of lactic acid because it decreases the pH which

incapacitates functionality of the probiotics in use. Another factor to consider when adding

probiotics before the fermentation process is to ensure sufficient viable probiotic cells survive

not only the formulation but also the intestinal passage through digestion so as to maintain their

health benefits (Heller, 2001).

Not only does the fermentation process allow for increased use of probiotics in yogurt to

boost health, it also extends the shelf life, the original purpose of fermenting milk. Numerous

different types of cultured milk have progressed due to the fact that fresh milk rapidly

deteriorates whereas a controlled fermentation with lactic acid producing bacteria (LAB) such as

starter cultures gives yogurt an extended shelf life. Research has shown that fermentation can

inhibit pathogenic bacteria that otherwise could cause diarrhea; this research has global

implications because one out of ten children in developing countries dies due to dehydration

caused by diarrhea (Sahlin, 1999).

Toxins and anti-nutritive aspects can also be reduced while the nutritive value can be

improved. Every food item contains microorganisms of varying types and in different

quantities. The microorganisms that will dominate will be dependent on several factors, and

occasionally microorganisms initially present in very low numbers in the food, such as lactic

acid bacteria, will reproduce and outnumber the other negative organisms inhibiting their growth

(Sahlin, 1999).

Fermented milk products like yogurt also boast increased health benefits for those who

are lactose intolerant. As the name implies, those who are lactose intolerant have traditionally

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veered away from milk products due to the discomfort they feel when consuming them; however

yogurt is one dairy product that does not cause the affected any upset. The fermentation process

is based on the microorganisms feeding off the milk sugar and producing lactic acid, leaving less

lactose in the product so it is more digestible for anyone who is lactose intolerant (Versa, 2000).

Some research has been done looking into possible additives to make dairy products even

healthier. Ahmed et. al conducted a study to determine the ‘hydrolysis action of Solanum

dubium serine protease on both ovine and caprine caseins in order to address an increasing

worldwide demand for alternative dairy products with improved organoleptic nutritional and

health properties’ (2011). Researchers used sheep (ovine) and goat milk (caprine) rather than

cow’s milk (bovine) because the study was conducted in Sudan, where sheep and goat’s milk is

more popular and were used to investigate the hydrolysis pattern of their casein after the addition

of Solanum dubium (Ahmed, 2011).

Experimentation was done using SDS-polyacrylamide gel electrophoresis according to

standard methods. Gel electrophoresis and staining revealed:

“that they were both sensitive to the action of the enzyme. Ovine caseins were

hydrolyzed completely in 6 h, whereas caprine caseins showed slight hydrolysis in 24 h.

The enzyme showed high degree of hydrolysis on ovine casein, thus it may lead to the

production of soft cheese from ovine milk. On the other hand, the enzyme showed low

degree of hydrolysis on caprine casein, and hence it could be used for the production of

hard cheese from caprine milk”

(Ahmed, 2011).

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Mass spectroscopy has been utilized in the past primarily to determine the various

chemical compounds and components in a given substance but more recently has been used for

food applications. Celia Henry Arnaud wrote the cover story in the Chemical and Engineering

News volume 84, issue 40 regarding the various uses for mass spectroscopy including the

assessment of proteins. The journal commentary concluded that mass spectroscopy was

indispensable for the study of proteins. It was important to note that the distinguishable

modifications included proteomics, phosphorylation, and biological structure which are key

attributes when studying proteins. The study indicated that mass spectrometry has some

difficulties ascertaining all changes in proteins (Arnaud, 2006).

No research has been published regarding the hydrolysis of proteins from fermented milk

products using mass spectroscopy for evaluation. The following research project will examine

the fragmentation patterns of casein protein from homemade yogurt samples using MALDI-TOF

mass spectroscopy. Samples were generated using three commercially sold yogurts as the

various starter cultures.

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Chapter III: Methodology

This chapter will begin with a description of the sample selection and include the

instruments used as well as data collection methods. In addition, this chapter will report the data

analysis and conclude with the methodological limitations being identified.

Sample Selection

Three variations of yogurt were made using a basic, homemade yogurt recipe, with three

different starter culture combinations; Bifidobacteria (Activia), L. bulgaricus, S. thermophilus,

and L. acidophilus, (Fage), and L. acidophilis (Old Home). Yogurt samples were made by

heating 177ml skim milk to 85° C then cooling to 50° C. Then, 178g of one of the three fat free

starter culture yogurts was stirred into the warm milk mixture, poured into a sterilized jar, and

capped. The mixture was placed in a water bath set at 50° C and allowed to sit for 3 hours before

being moved to a refrigerator until use.

Instrumentation

The instruments to be used are the gel electrophoresis instrument (GE Healthcare,

PhastSystem), Bradford spectroscopy analyzer (Biotek, Epoch Spec), and the MALDI-TOF mass

spectrometer (Bruker, Microflex). The reasoning behind using this mass spectrophotometer came

from its versatility in uses, reputation for valid and reliable results, as well as the lack of research

done using it to determine protein fragmentation in fermented foods.

Data Collection

Once yogurts were made, casein was extracted from each sample once a week for 3

weeks using a standard method: each yogurt sample was diluted in water to 1:1 where the pH

was decreased to 4.25 by adding drops of 6M hydrochloric acid. The solution was then heated to

and held at 37° C for 30 minutes. The solution was then vacuum-filtered; the filtrate was washed

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once with reverse osmosis water and filtered again. The casein filtrate was suspended in a

100mM phosphate buffer and stored at -80° C until analysis could be performed. A control was

created by extracting casein from the milk used to create the yogurt samples using the same

methods as previously described.

Samples were first analyzed using gel electrophoresis on a PhastGel system to determine

whether visible differences existed between various yogurts and protein fragmentation. The first

extraction of casein from the three yogurt samples were chosen for preliminary data. 10 µL of

each sample and 10 µL of a sample buffer were added to 3 eppendorf tubes, respectively. 10 µL

of bovine serum albumin (BSA) was added to a separate tube to be used as the standard. All

samples were placed in boiling water for five minutes. Samples were then loaded onto the

PhastGel system using the standard method for 10-15% SDS gels.

A Bradford assay was conducted where all samples, the control, and BSA standards were

run in duplicate at 1, 5, and 10x dilutions and analyzed at 595nm according to standard methods

to determine proper dilutions before analysis on MALDI. It was determined that a 10x dilution

of samples resulted in the correct protein concentration for MALDI-TOF analysis. The samples

were then desalted using a standard Zip Tip protocol. To exchange solvents the pipette tip was

properly conditioned using 100% acetonitrile (ACN) and 0.2% trifluoroacetic acid (TFA) then

each sample underwent a 5x dilution with the TFA solution. Salt was removed from the samples

using the TFA solution and the sample was finally eluted with 50% ACN. Zip Tipped samples

were then kept at -80°C until use.

Samples were then run via the MALDI-TOF mass spectrometer using standard methods:

The target plate was obtained, washed with ethanol, and allowed to air dry. The matrix solution

was prepared with 70 µL ACN, 30 µL distilled water, 0.1 µL TFA, and a spatula tip of cyano-4

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then vortexed for 10 seconds. Next, a 0.5 µL sample was pipetted on to the sample target and

allowed to dry; 0.5 µL was pipetted on top of the sample and allowed to dry; finally 1.0 µL 0.1%

TFA was pipetted on top of sample, allowed to sit for 5 seconds, removed with a pipette, and the

plate was allowed to dry. This procedure was repeated for all samples.

Once the plate was dry, it was loaded into the chamber and locked in. The number of

shots was set to 5 while the desired laser power was set to 54%. Each sample had five shots

taken before the spectrum was created.

Data Analysis

Preliminary data of the gels was analyzed via the PhastGel gel electrophoresis system

while the final data was analyzed via MALDI-TOF mass spectrometry and Protein Prospector

software.

Limitations

The methodological limitations will be the ability to consistently make yogurt because

the activity of cultures is variable. Additionally, samples were not run in triplicate on MALDI-

TOF; thus, data is only preliminary and not statistically significant.

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Chapter IV: Results

Item Analysis

Before examining the mass spectrometry results, 1 of 3 outcomes were postulated to be

possible: 1) random peptide cleavages resulting in a multitude of unique peptides, 2) complete

hydrolysis with only single amino acids left and no peptides, or 3) distinct peptide cleavage

points resulting in specific peptides being formed. Preliminary results from the PhastGel

electrophoresis indicated that there were visible differences between the protein sizes of the

various, different yogurts, as seen in Figure 3. These positive results suggested specific cleavage

sites and prompted the further study on MALDI-TOF but prior to mass spectroscopy analysis

could be conducted, a Bradford analysis was conducted to determine the best concentration to

fun on MALDI-TOF, Figure 4 and Table 1.

Figure 3 Preliminary gel electrophoresis data using the standard BSA. A) Activia, Fage, and Old Home commercially produced yogurts. B) Milk and Yogurt in 1x and 10x concentrations.

BSA Activia Fage Old Home BSA Milk 1x 10x Yogurt 1x 10x

B A

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Figure 4 Protein concentration standard curve using Bradford reagent and bovine serum albumin as the protein standard.

Sample 10X

Absorbance (595nm)

mg/mL in original solution

Activia 1 0.7125 7.39 Activia 2 0.4655 3.52 Activia 3 0.401 2.51 Fage 1 0.6635 6.63 Fage 2 0.5695 5.15 Fage 3 0.4225 2.85 Old Home 1 0.4405 3.13 Old Home 2 0.594 5.54 Old Home 3 0.5805 5.33 Table 1 Original sample protein concentration data as determined by the Bradford assay.

24

Results of this study showed distinct cleavage points as shown in the random sampling of

spectrums, Figure 5. The collective cleavages of each sample were organized and a general

pattern emerged, as shown in Table 2. Results showed that the control sample varied from all of

the yogurt samples and each yogurt sample had similar cleavage points within its own type yet

showed differing cleavage within the sample type over time.

Figure 5 Bovine casein cleavage spectrums: control (milk), Activia first casein extraction (A1), Fage first casein extraction (F1), and Old Home first casein extraction (OH1).

25

Extraction

# Origin 876 1081 1881 1995 2109 2326 2766 2796

Control Milk x x

1 Activia (A1) x x x x

2 Activia (A2)

3 Activia (A3) x x x x x

1 Fage (F1) x x

2 Fage (F2) x x x

3 Fage (F3) x x x

1 Old Home (OH1)

x x x

2 Old Home (OH2)

x x x

3 Old Home (OH3)

x x x

Table 2 MALDI-TOF results of peptide fragment peak identity.

Through using the computer program Protein Prospector, the sample peptide cleavages

were further analyzed by determining which peaks correlated to specific peptide sequences, as

seen in Table 3. It was determined that all observed peptide sequences were from bovine casein

Alpha S2 rather than Beta or Alpha S1, as seen in Figure 6. All the observed C or N terminals

peptide sample sequences were also cut from the middle of the protein sequence rather than the

ends.

26

Peak Expected Peak

Residue #

Phosphorylation Peptide Sequence

1 876.65 875.04 478-485 None NMAINPSK

2 1881.20, 1881.21, 1881.41, 1881.50, 1881.70

1882.73 590-603 3 KTVDMESTEVFTKK

3 2765.11, 2766.31

2765.88 455-477 1 NTMEHVSSSEESIISQETYKQEK

4 2796.9 2796.2 568-590 3 RNAVPITPTLNREQLSTSEENSKK

Table 3 Peptide peaks, residue numbers, phosphorylation, and sequences.

Figure 6 Bovine casein amino acid sequences. Bovine Casein: Beta

MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDEL

QDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPK

HKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSL

SQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV

Bovine Casein: Alpha S1

MKLLILTCLVAVALARPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIG

SESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYK

VPQLEIVPNSAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWY

YVPLGTQYTDAPSFSDIPNPIGSENSEKTTMPLW

Bovine Casein: Alpha S2

MKFFIFTCLLAVALAKNTMEHVSSSEESIISQETYKQEKNMAINPSKENLCSTFCKEVVR

NANEEEYSIGSSSEESAEVATEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPIV

LNPWDQVKRNAVPITPTLNREQLSTSEENSKKTVDMESTEVFTKKTKLTEEEKNRLNFLK

KISQRYQKFALPQYLKTVYQHQKAMKPWIQPKTKVIPYVRYL

Red: Milk Orange: A, F, & OH Green: F Blue: A

27

Chapter V: Discussion

Limitations

The samples were not run in triplicate and thus no statistical analysis could be performed.

Conclusions

Hydrolysis patterns and definitive cleavage points in bovine casein were observed. While

the three most common forms of bovine caseins are Beta, Alpha S1, and Alpha S2, the only

peptide cleavages observed were within the Alpha S2 casein. The exact reason is unknown;

Alpha S1 and Beta may not have been hydrolyzed or the hydrolyzed peptides from these proteins

were too large to be observed. The mass spectrometer was not calibrated to observe large

peptides >2.5K molecular weight which therefore limited the conditional scope.

The results attained from this research further support evidence that MALDI-TOF is a

good technique for deciphering protein hydrolysis. With such observed results, yogurt appears to

be a good food to continue studying regarding the relationship between yogurt fermentation time

and cleavage patterns to look at changes in digestibility or physical properties. These findings

will open doors for new studies looking into lessening if not eliminating children’s allergies of

milk proteins; if enough peptides can be hydrolyzed, children may have an easier time digesting

milk proteins.

Recommendations

The parameters of the mass spectrometer should be changed to isolate and identify both

slightly larger and smaller peptide sequences. Without any statistical significance, the data

collected was preliminary and therefore this experiment must be repeated to determine

significance of these findings. Gel electrophoresis experimentation should be continued to

identify characteristics and quantify proteins that are not yet fragmented. Sensory testing should

28

also be done to determine textural attributes and acceptability of the product over time. This

study did not examine why different microorganisms in the starter cultures caused the

differences in protein hydrolysis therefore mores experiments are needed here as well. These

future tests could lead to an improved awareness of what role various microorganisms play in

allowing for better digestibility of dairy products.

29

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