BIOCHEMICAL CHANGES IN PROTEIN AND LIPID FRACTIONS OF ULTRA-HIGH TEMPERATURE TREATED MILK AND DAIRY DRINKS AT DIFFERENT STAGES OF PROCESSING AND STORAGE

MUHAMMAD AJMAL2009-VA-495

A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

IN

DAIRY TECHNOLOGY

UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES LAHORE

2019

DEDICATION

Dedicated to

My Generous and Affectionate Parents

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ACKNOWLEDGEMENTS

I am very pleased to ALLAH Almighty who is Merciful and awarded me health, tolerance and

knowledge to complete this work. After the Allah Merciful I am Very thankful to Holy Prophet

(P.B.U.H.) for which whole universe made and is Rahmatul-Lil-Alameen.

I acknowledge, with deep gratitude and obligation, the motivation, help, valuable time and

guidance given to me by Dr. Muhammad Nadeem, who was my supervisor.

I also would like to express my thanks to Dr. Nabila Ghulzar, my co-supervisor, who always

believed in me and never hesitate to provide relentless support and motivation at all times.

I am very thankful to the Dr. Muhammad Tayyab, committee member who gave me useful

information’s and always helped me in my research work.

I would like to express my deepest gratitude to my Mother, Father, Brothers, Sister, Relatives, all

Students and their Parents, for their emotional and honorable support throughout my academic career

and also for their love, patience, encouragement and prayers.

Sincere thanks to all my friends especially Dr. Sohail Ahmad, Maryam Batool, Hafiz

Muhammad Khalid Mehmood and Imran Taj khan for their precious time and help regarding data

handling & encouraging all the time during my stay in university. Lastly, I wish to express my sincere

thanks to all those who have one way or another helped me in making this study a success.

Muhammad Ajmal

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CONTENTS

DEDICATION (i)ACKNOWLEDGEMENTS (ii)LIST OF TABLES (iv)LIST OF FIGURES (vi)LIST OF ABBREVIATIONS (vii)ABSTRACT (viii)

SR. NO. CHAPTERS PAGE NO.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 3

3 EXPERIMENT NO. 1 12

4 EXPERIMENT NO. 2 26

5 EXPERIMENT NO. 3 39

6 EXPERIMENT NO. 4 48

7 EXPERIMENT NO. 5 59

8 SUMMARY 71

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LIST OF TABLES

TABLE NO. TITLE PAGE NO.

3.1 Effect of Immediate and Delayed Chilling of Raw Milk on Chemical Composition of Pasteurized Milk

21

3.2 Effect of Immediate and Delayed Chilling of Raw Milk on Fatty Acid Profile of Pasteurized Milk

22

3.3 Effect of Immediate and Delayed Chilling of Raw Milk on Vitamins and Mineral Content of Pasteurized Milk

23

3.4 Effect of Immediate and Delayed Chilling of Raw Milk on Lipid Oxidation of Pasteurized Milk

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3.5 Effect of Immediate and Delayed Chilling of Raw Milk on Antioxidant Capacity of Pasteurized Milk

25

4.1 Effect of UHT Treatment and Storage on Chemical Composition of Milk

33

4.2 Impact of UHT Treatment and Storage on Fatty Acid Profile of Milk 33

4.3 Effect of UHT Treatment and Storage on Triglyceride Profile of Milk 34

4.4 Effect of UHT Treatment and Storage on Organic Acids in Milk 34

4.5 Effect of UHT Treatment and Storage on Lipid Oxidation of Milk 35

4.6 Effect of UHT Treatment and Storage on Sensory Characteristics of Milk

35

5.1 Chemical Composition of UHT Milk 45

5.2 Protein Profile of UHT Milk 45

5.3 Amino Acid Profile of UHT Milk 46

5.4 Non-Casein Nitrogen and Non-Protein Nitrogen Content of UHT Milk and Sedimentation

47

5.5 Plasmin Activity, Sedimentation and Viscosity of UHT Milk 47

6.1 Effect of Storage Duration on Chemical Composition of UHT Treated Tea Whitener, Milk and Dairy Drink

55

6.2 Antioxidant capacity of UHT Treated Products at different storage intervals

56

6.3 Vitamin A and E Contents of UHT Treated Tea Whitener, Milk and Dairy Drink at Different Stages of Storage

56

6.4 Fatty Acids Profile of UHT Treated Products 57

6.5 Induction period of UHT Treated Tea Whitener, Milk and Dairy Drink at Different Storage Intervals

58

7.1 Chemical Composition of Flavored UHT Milk Different Stages of Storage

68

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7.2 Transition in Protein Profile of UHT Flavored Milk Different Stages of Storage

68

7.3 Transition in Maillard Reaction Products at Different Stages of Storage 69

7.4 Fatty Acid Profile of Flavored UHT Milk at Different Stages of Storage 69

7.5 Oxidative Stability of Flavored UHT Milk 70

7.6 Sensory Characteristics of Flavored UHT Milk 70

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LIST OF FIGURESFIGURE NO. TITLE PAGE NO.

4.1 Effect of UHT Treatment and Storage on Free Fatty Acids 36

4.2 Effect of UHT Treatment and Storage on Peroxide Value 36

4.3 Effect of UHT Treatment and Storage on Anisidine Value 37

4.4 Effect of UHT Treatment and Storage on Conjugated Dienes 37

4.5 Induction Period of Raw, UHT Treated Milk in Storage 38

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LIST OF ABBREVIATION

ABBREVIATIONS CAPTION UHT Ultra-High TemperatureК-CN Kepa-caseinß-Lg Beta-lactoglobulinα-La Alpha-lactalbuminIg ImmunoglobulinsBSA Bovine serum albuminTAG TriacylglycerolFFA Free fatty acidsSCFA Short-chain fatty acidsMCFA Medium-chain fatty acidsUSFA Unsaturated fatty acidsLCFA Long-chain fatty acidsLCUSFA Long-chain unsaturated fatty acidsPV Peroxide valueAV Anisidine valueTBARS Thiobarbituric acid reactive substancesDPPH 1, 1-diphenyl-2-picrylhydrazylHPLC High performance liquid chromatographySNF Solid not fatGC-MS Gas chromatography mass spectrometryFAP Fatty acid profileTBA Thiobarbituric   acid NCN Non-casein nitrogen NPN Non-protein nitrogen CRD completely randomized designDMR Duncan multiple rangeSD Standard deviation TAC Total antioxidant capacityALA Antioxidant activity in linoleic AcidAOCS American Oil Chemists SocietyHMF Hydroxy Methyl FurfuralFUR Estimation of FurosineCML Nε-carboxymethyl lysineALS Auto liquid samplerFID Flame ionization detectorRP Reducing powerTFC Total flavonoid contentCN CaseinSDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresisAAP Amino acids profileIP Induction PeriodFRSA Free radical scavenging activity

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ABSTRACTThis study is comprised of five experiments, in the first experiment chemical changes taking place in lipid fraction of pasteurized milk was studied. Raw milk from the same source was at once cooled down to chilling temperature and in second instance it was put on the lab shelf for two hours at ambient temperature and then chilled to same temperature, followed by pasteurization and chilled storage. In the second experiment, samples of UHT milk were tested for chemical characteristics and lipid oxidation. In third experiment, impact of UHT treatment (142oC for 2 sec) and 90 days ambient storage (30-35oC) on protein and amino acid profile of UHT milk and sedimentation was investigated. In fourth experiment, samples of UHT Treated products were stored for 90 days. Transition in antioxidant capacity, vitamin A, E, fatty acid profile was studied. Transition in protein profile, Maillard reaction products and lipid oxidation of flavored UHT treated milk was investigated. In fifth experiment, Samples of mango flavored UHT milk 200 ml pack (475 Nos.) were purchased from the market and stored at ambient temperature for the duration of 90 days. Proteolysis, hydroxyl methyl furfural furosine, Nε-carboxymethyl lysine, FAP, FFA, PV and sensory characteristics were studied at 0, 45 and 90 days of storage. It was observed that chemical characteristics and storage stability of raw milk in the second instance was different from the milk cooled at once. Therefore, it is recommended that milk collection system in the developing countries should be improved to avoid any delay in between milking and chilling of raw milk. The results of the second experiment disclosed that fatty acid and triglyceride profile were not affected till 60 days with a steady increase in lipase activity. Strong correlations between peroxide value and induction period were established. For the anticipation of oxidative stability of UHT milk, induction period can be used. After UHT treatment, β-lactoglobulin, β-lactalbumin, immunoglobulin and serum albumin decreased by 12.1%, 15.4%, 10.6% and 10.2% with no effect on casein fractions. Sedimentation was comprised of 14.9% αs1-casein, 3.42% αs2-casein, 9.88% β-casein, 2.27% κ-casein, 4.36% β-Lactoglobulin, 1.88% β-lactalbumin, 1.84% immunoglobulin and 0.61% Serum Albumin. Out of 80% casein and 20% whey, 38.5% and 8.7% became the part of sedimentation. Tyrosine content of flavored UHT milk increased during the storage. Tyrosine content of flavored UHT milk at 0, 45 and 90 days of storage were 3.5, 6.9 and 15.2 µg tyrosine/ml. HMF content of UHT milk at 0, 45 and 90 days of storage were 1.56, 4.18 and 7.61 (µmol/L). Furosine content of flavored UHT milk at 0, 45 and 90 days of storage intervals were 278, 392 and 561 mg/100g protein.

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CHAPTER 1INTRODUCTION

In Ultra-High Temperature (UHT) operation, milk is thermally processed at 138-142°C/2-5 seconds, followed by rapid cooling (25°C) and filling in aseptic conditions. UHT products are commercially sterile which can be stored in ambient conditions for the longer durations. UHT treatment enhanced the storage stability of milk. UHT treated fluid milk can be kept for several months (Rauh et al. 2014).

Casein and whey proteins are present in milk; former represents about 80% and latter accounts for 20% in milk. Functionality and stability of UHT milk largely depends upon the milk proteins. During UHT treatment, exposure of milk to high temperature induces biochemical changes in casein and whey proteins, free peptides may be produced by the UHT treatment. Dephosphorylation and hydrolysis take place in casein (Singh and Waungana 2001; Chavan et al. 2011). UHT treatment can also induce inter and intra-molecular cross-linkage of caseins due to the high reactivity of lysine and tyrosine in the presence of milk sugar (O’Connell and Fox 2003).

Due to the globular structure, whey proteins are considerably unfolding of spherical structure leads to sedimentation. Among the whey proteins Ig and BSA have the minimum thermal stability, β-Lg and α-La have shown medium and high degree of resistance to the heat treatment. Structure and strength of intermolecular bonds are the key factors that determine milk protein sustainability. Due to the passage of time, sedimentation takes place in UHT milk by processing temperature (Anema, 2003).

UHT treatment affects the lipid fraction of milk, major changes take place around short-chain fatty acid, triacylglycerol (TAG) and fat-soluble vitamins. UHT treatment modifications in triglyceride molecules and produced free fatty acids (FFA). Objectionable flavors in UHT milk are usually associated with free fatty acids (Meshref and Rowaily 2008). In 1912 Louis-Camille Maillard first time introduced Maillard reaction. It is complex process of reducing sugar and protein in the presence in milk. It is naturally occurring reaction between reducing sugar and protein with amino group of protein and carbonyl group. These reactions undergo a reaction called Amadori arrangement to produce derivative of amino deoxyfructose. Maillard reaction also occurred at room temperature.

Plasmin, bacterial lipases and proteinases survive the UHT processing (Britz and Robinson 2008). Proteolytic and lipolytic enzymes are the major spoilage agents of UHT milk, plasmin is the most important proteolytic enzyme in UHT milk and plasmin causes the coagulation of milk by hydrolyzing α and β-caseins. Proteases produce aggregates in milk and dairy products during extended storage (Datta and Deeth 2001). Proteolytic and lipolytic activities may lead to the shorter shelf life of UHT treated dairy products (Omoarukhe et al. 2010). Hydrolysis of protein is due to action of enzymatic and plasmin activity which increased the sedimentation of UHT milk (Bhatt, 2014). UHT treated milk has own attribute of flavor. Heat treatment and plasmin activity at storage of milk produced undesirable changes (Clare et al. 2005). Plasmin, protease and lipase disturbed the force of attraction of protein to protein balance (Liang et al. 2013).

Shelf life of UHT milk was 6 months, however, due to several unassignable causes, it is dropped to 3 months and the decline in shelf life is not coming to an end. Shelf life and quality related issues are also associated with flavored milk and immitant dairy products, such as dairy drink. Although, milk processing sector of the country is well established and millions of liters of UHT dairy products is manufactured on daily basis, root cause of the problem is still mysterious.

Consumption of UHT treated flavored milk, tea whitener and dairy drink is mounting across the country. Flavored milk is prepared by mixing milk with sugar, stabilizer, color and flavor, while dairy drink is manufactured by blending demineralized whey powder/skim milk powder with milk fat, stabilizers and emulsifiers followed by UHT treatment and aseptic packaging.Flavored UHT has another advantage of longer storage life as compared to pasteurized versions. In the storage, cooked flavor is dominated by off flavor due to the generation of methyl ketones Vazques-Landaveerde et al. 2006). Alpha amino group of lysine residues react with lactose which lead to the formation of lactosyl lysine which is subsequently converted to α-N-deoxylactulosyl-l-lysine via Amadori rearrangements. Lactulosyl-lysine cannot be absorbed in the body due to it biological unavailability (Krause et al. 2003).

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Maillard reaction decreases the functional properties of protein by influencing the thermal stability, foaming, gelation, emulsification and textural properties of protein decreases (Jansson et al. 2014). Maillard

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INTRODUCTIONreaction is influenced by time, pH and water activity (Mayer et al. 2010). Rate of Maillard reaction is mainly governed by the magnitude of carbohydrates and proteins in the processing and storage of milk. In the storage period, carbohydrate type significantly affected the formation of furosine and 2-methylbutanal (Basto et al. 2012). For the characterization of early and terminal stages of Maillard reaction, Furosine and CML are used, respectively (Rhfian-Henares et al. 2017).

No study has been performed on the profiling of casein, whey proteins, lipid phase, lipolytic and proteolytic activities at different stages of processing and storages of UHT treated products to know which stage or stages are critical with respect to the problem of short shelf life.

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CHAPTER 2REVIEW OF LITERATURE

2.1. UHT Milk Long shelf life of milk can be attained through UHT treatment of milk for a few second 135-140 °C

with (Kelly et al. 2012; Fitria et al. 2015). Pathogenic and spoilage bacteria which are present in raw milk can be destroyed by UHT treatment (Rauh et al. 2014). UHT milk is quite popular in developing countries, where cold chain facilities are lacking (Chove et al. 2013; Chavan et al. 2011). The shelf life is highly dependent on time-temperature combination. The heating process is divided into two types (direct and indirect) and in the indirect UHT heat treatment the milk is not contact with heating media (steam or water) (Tetra Pak, 2003). The milk composition has been observed different between two types of UHT milk. 2.2. Effect of UHT Treatment on Milk Proteins

UHT treated milk was characterized by structural changes such as protein denaturation. Exposure of casein to UHT treatment leads to dephosphorylation, which affected the micelle structure (Belitz et al. 2004). UHT treatment can lead to the formation of peptides, αs1, αs2 and β-casein are susceptible to hydrolysis. Heat treatment can also induce inter and intra-molecular cross-linkage of caseins due to the high reactivity of certain amino acids, such as lysine and tyrosine with in presence of milk sugar. Heat treatment caused destabilization of milk proteins which led to promote higher sedimentation (Lewis et al. 2011). Lin et al. (2010) result showed that whey protein was decreased 23% and 85% in pasteurized and UHT heat treatment respectively.2.3. Biochemical Changes in UHT milk

Bacterial proteases and lipases are more heat resistant than indigenous enzymes of milk; these enzymes retain about 30 to 70% of their activity after UHT treatment (Datta and Deeth 2001). During the storage of UHT milk, proteases and lipases induced several undesirable flavors in UHT milk such as bitter, unclean, fruity, yeasty and metallic (Datta and Deeth 2003). Biochemical changes in UHT milk have negative impact on the nutritional characteristics, flavor, physical stability, pH and acidity (Hassan et al. 2009). Dupont et al. (2007) reported that biochemical reactions caused undesirable changing in UHT milk such as oxidation flavor, age gelation, bitterness and appearance defect. Tamime (2008) studied that plasmin, protease and lipases activity enhanced the biochemical changes in UHT treated milk such as sedimentation and fat separation. A disadvantage related to heating the milk at high temperatures is that the nutritional and sensory quality decreases due to denaturation of proteins, thermal degradations of lipids and reactions between sugar and proteins (Maillard reaction). Therefore, the combination of temperature and time of the heat treatment must be as low and short as possible (Elliott et al. 2003). 2.4. Proteolytic Activity

Raw milk naturally contains plasmin and plasminogen at the concentration of about 0.3g/L and 2.7g/L respectively. Protein hydrolysis was due to action of enzymatic activity in milk (Clare et al. 2005). Plasmin reduced life of UHT milk because they produced heat- stable enzymes and survived at UHT treatment (Datta and Deeth 2001). UHT treated milk had long shelf life when less microbial load in raw milk (Tamime, 2008). When raw milk stored at lower temperature then less amount of psychrotrophic bacteria were there and less biochemical reactions occurred. Vijayakumar (2012) reported that lipases and proteases deteriorated the quality of UHT milk. Pulkkinen (2014) studied that psychotropic bacteria in dairy industry have important role in UHT milk after processing. Samarzija et al. (2012) studied that the temperature between 20-30°C is known optimum temperature for growth of bacteria. Heat resistant enzymes inactivated during UHT treatment and after processing they activated; ultimately cause spoilage the milk at ambient temperature (Richards et al. 2014). Heat resistant enzymes were survived after UHT treatment (Topçu et al. 2006). Samet-Bali et al. (2013) results showed refrigeration of raw milk before processing is necessary in bulk tanks but even that temperature accelerated the growth of psychtrophic bacteria. The refrigeration temperature did not prevent the growth of psychrotrophic bacteria (O’Brien and Guinee 2011). Plasmin and

4

plasminogen are effective milk components, which lead to breakdown of milk protein, particularly caseins such as β-casein and ɑ-casein. Peptides, proteose-

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REVIEW OF LITERATUREpeptones were produced by the action of plasmin. Age gelation in UHT milk was due to activation of plasminogen and that released by plasmin during storage of milk at low temperature; as a result, proteolysis occurred in UHT milk (Cilliers, 2007). Plasmin attach all types of protein but favorite target is caseins. It cleaves β-casein, ɑs1-casein and ɑs2-casein at risk for proteolysis by plasmin in UHT milk. Plasmin activity can be reduced by UHT treatment but not complete inactivated. Plasmins have ability reversible activation phenomena, so it survived at UHT treatment of milk (Gazi et al. 2014). Plasmin has ability to induced proteolysis in UHT process of milk. It was responsible for releasing the βК-complex which was known preliminary step for age gelation and sedimentation (Bavarian et al. 2010). Chavan et al. (2011) results showed mastitis milk undergoes for UHT process, it showed short shelf life, fast age gelation and reason for that increased proteolytic activity due to high levels of plasmin. Plasminogen level in mastitis milk is more than normal milk so activity of protease was faster than normal UHT milk. Richards et al. (2014) studies that both indigenous milk proteases as well as heat-stable proteases produced by psychrotrophic bacteria were responsible for proteolysis in UHT milk. Whey proteins undergo different changes during processing temperature as well as by the action of bacteria. Gaucher et al. (2011) studied that caseins micelle degraded by proteolytic activity in UHT milk. Bavarian et al. (2010) studies that β-casein faster degradation than ɑs1-casein by action of proteases. Chove et al. (2013) reported that proteolysis at high temperature was increased in UHT treated milk during storage. Proteolytic enzymes were more problematic than indigenous enzymes (Forsbäck, 2010). Protease enzymes produced by psychrotrophic bacteria mostly attack on casein protein instead of whey protein. Caseins protein more sensitive of protease than the whey protein and colloid calcium phosphate were affected by action of protease enzymes (Samarzija et al. 2012). Bagliniere et al. (2013) studied that proteolysis destabilized milk protein by Pseudomonas flurescens which was produced at chilling temperature before processing of milk. 2.5. Lipase Activity

Free fatty acids concentration was increased in UHT milk by the lipolytic activity of lipases enzymes. Lipases origin from psychrotrophic bacteria and have ability disturb the native membrane structure of fat globule and ultimately result degradation of fat within milk (Samarzija et al. 2012). Lipases attack on first and third position in di and tri- monoglycerides of milk and development of hydrolytic rancidity in UHT milk (O Brien and Guinee 2011). Richards et al. (2014) reported that lipase enzymes partially inactivate by heat treatment and activated during storage temperature and start their hydrolysis of fat as result off-flavors, rancidity, oxidation, bitter tastes and formation of soapy flavors in UHT milk. Lipase caused serious defects in UHT milk such as changes the viscosity, thickness, off-flavors, breakdown of lipid and short shelf life of milk. During processing of UHT treatment some lipase enzymes survived (Hassan et al. 2009). Samarzija et al. (2012) results showed that heat stable enzymes break down the major components of UHT milk. 2.6. Age Gelation (AG)Gelation in UHT milk was due to whey protein denatured by activity of protease enzymes which decrease electrostatic repulsion with in protein and formation of gel (Tijssen et a. 2007). Age gelation and bitterness were defect of UHT milk. By the activity of enzymes casein micelle disperses and form βК-complexes and as a result viscosity of UHT milk is gradually increased with activity of proteolysis of proteins (Richards et al. 2014). In age gelation fluidity of milk is decrease and viscosity of milk increase and as a thick white gel can be observed within UHT milk container. Age gelation is big problem in dairy industry. Age gelation rate in UHT milk was greater at high temperature as compared to low temperature (Holland et al. 2011). Pulkkinen (2014) studied showed that age gelation mechanism involved major two steps in first phase some structural changes in proteins and second stage some physiochemical reaction takes place to decrease the stability of proteins within UHT milk. Milk contains more than 70 indigenous enzymes so it is very biological active product (Gazi et al. 2014). Samarzija et al. (2012) studied that UHT treatment promoted the age gelation due to break down of fatty acids as well as triacylglycerol of UHT milk at storage.2.7. Sedimentation

Datta et al. (2002) studies that sedimentation composition contains aggregates of denatured protein, fat, inorganic salt and lactose. The composition of sedimentation varies types of heat treatment. Sedimentation affected the acceptability of UHT treated milk. Schalk et al. (2013) reported that sedimentation is major problem as a result of indirect heat treatment as compared to direct heat treatment

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REVIEW OF LITERATUREprocess. Lewis et al. (2011) studied that sedimentation in UHT milk enhanced due to denaturization of protein. UHT heat treatment cause aggregation in proteins which promote the formation of emulsion, instability of protein and sedimentation (Liang et al. 2013). Hassan et al. (2009) studied that rejection of UHT milk due to development of sedimentation, bitter taste and increased in thickness. Samarzija et al. (2012) studied that protease attack on casein because casein locates on micelle surface as well as casein have more open structure than whey so more exposed to enzymatic cleavage. De Kort et al. (2012) studied that enzymes decreased the pH value of UHT milk and at low pH caseins micelles isolate from casein micelle and caused sedimentation. Lewis et al. (2011) result showed that ionic calcium level more than 2 mM caused significant sedimentation in UHT milk.2.8. Lipid Oxidation

FFA are the precursor flavors defect such as, oxidized, cardboard, bitter, rancid, soapy, unclean and metallic. Milk contains about 21-23% oleic acid (C18:1), which is susceptible to auto-oxidation, rate of autoxidation of C18:1 is fifteen times greater than stearic acid (C18:0). Concentration of fat protein, fat and lactose decreased in UHT treated milk, concentration of SCFA, MCFA and USFA were lower in UHT treated milk (Miguel et al. 2015). Recombined milk became more viscous than fresh UHT milk, extent of lipolysis was similar in both types milk (Hassan et al. 2009). Lipid oxidation in recombined UHT milk estimated using FAP, PV, AV and TBARS. Value of all these parameters intensified in the storage, major deviations in fatty acid profile were also recorded (Meshref and Rowaily 2008). Oxidation of lipids, primary the unsaturated fatty acids is catalyzed by heat (Costa et al. 2011), light, enzymes, transition metals and by microorganisms (Shahidi and Zhong 2010). The oxidation of the fatty acids generates primary oxidation products including lipid hydroperoxides. These peroxides are unstable and decompose to radicals which react with new lipid molecules and initiate a propagation process in the so-called autoxidation (Elias et al. 2008). Psychrotrophic bacteria are favored by cold storage and may contribute to lipid degradation by the formation of the heat stable extracellular enzymes such as lipases and proteases. However, for UHT treated milk, during the long-term storage at higher temperatures the bacterial lipases may contribute to the degradation of lipids. The lipids are included in the Maillard reaction by the amino group of the phospholipid and by the aldehydes and ketones formed from the fatty acid oxidation. The lipid oxidation may also generate dicarbonyls such as glyoxal and methylglyoxal, as well as radicals, highly reactive with proteins and Maillard reaction compounds. Edvaldo et al. (2011) result showed that fatty acids profile of raw milk was different than UHT milk. 2.9. Maillard Reaction in UHT Milk

In Maillard reaction, amino acid (lysine) react with reducing sugar and as a result decreased the nutritional value of food. Maillard reaction destabilized the milk protein heat treatment and caused defect in milk products in vivo and vitro studies (Gilani et al. 2012). When melanoidins polymerization was formed, this leads to the formation of browning, structural, compositional changes in sugars and protein. Maillard reaction significantly affected the color, taste and digestibility of food (Pischetsrieder and Henle 2014). Maillard reaction promotes bitter and cooked flavored in heat treated milk and food. Browning reaction was accelerated with heat treatment and storage temperature. When sugar and protein react to each other as a result Strecker aldehydes and off-flavored produced in food, beverage and UHT milk. Proteolysis was occurred in many foods either due to heat-processing or naturally and as a result increased the free amino acid in milk and food items (Jansson et al. 2014). Heat treatment hydrolyzed the lactose into glucose and galactose which were more reactive than lactose. Some lactase hydrolysis into lactose and protease, and react with amino acids as a result Strecker aldehydes were produced (Troise et al. 2016). When depletion of amino acids residues formed melanoidins, as a result browning, sensory defects in UHT during storage temperature (Meltretter et al. 2014). Maillard reaction changed the functionality properties of protein including stability, solubility, emulsifying, foaming and structural changes (Lee et al. 2017). Maillard reaction was promoted by many factors including; types of reactants, time temperature combination, pH and water activity (van Boekel, 2001). Martins et al. (2001) studied that Maillard reaction was influenced by temperature and metallic

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REVIEW OF LITERATUREcations. Formation of colored compound was due to chemical reaction of reducing sugars (glucose and fructose), protein and some water. Le et al. (2011) studied that decolorization on powder milk was due to Maillard reaction and caused deterioration in the functional properties such as foaming, solubility and emulsifying. 2.10. Flavored Milk

Consumption of flavored milk is increasing in Pakistan; usually manufactured by blending milk with sugar, stabilizer, color and flavor followed by UHT treatment and aseptic packaging. Flavored milk was formulated with milk fat, lactose, whey powder, mineral, flavor and emulsifiers (Yanes et al. 2002). Yeung et al. (2017) studied that flavored milk contains both sweetener (sucrose, glucose-fructose syrup) and non-calories sweetener which depend upon the type of flavored milk. In flavored milk addition of milk fat improved the creaminess in milk (Anonymous, 2000). Phillips et al. (2007) studied that addition of whey powder in pasteurized flavored milk to full fill solid not fat (SNF) for great nutritional, biological and functional properties. The protein industry has started to supply whey powder for milk base beverages (Bariatrix, 2008). Opawumi and White (2004) studied that ingredients of flavored milk (protein, flavor, colors, sweetener and carbohydrates) were played vital role for better nutritional value and better sensory attributes. Murphy et al. (2008) studied that pasteurized flavored milk, dairy drink which was formed by adding coloring agent’s (artificial or natural), flavor agents and sweetener in milk. Adding some sugar in flavored milk may help in improving the application of nutritious foods. Flavored milk was an excellent source to increase milk consumption among children and a better way to help children to make their diets more nutritious. Frary et al. (2004) reported that many authorities were encouraging for consumption of milk by children and also fever of flavored milk in children diets. Flavored milk can help to close the nutrition gap in children. Malnutrition is a condition which caused by lack of essential nutrition. Kumari et al. (2016) reported that fruit base flavored milk preparation was same as chocolate milk by adding flavor, color, sugar, fruit (orange, pineapple, strawberry) and consumer warmly accepted. Bhargav (2013) sterilized oat flavored milk with different concentration (1%, 2%, 3%) with 7% sugar and he concluded that 2% oat with 7 sugar flavored milk was more acceptable on the basis of color, mouth feel, aroma and overall acceptability. Chatterjee and Patel (2016) prepared the sterilized flavored milk by adding sugar, cocoa powder, carrageenan and oat in milk, he founded that addition of oat improves the viscosity and mouth feel was more acceptable by sensory panel. In UHT treated milk products, chemical and biological changes are accelerated by addition of reducing sugar. During heat processing lactose is degraded into lactulose and acids as result producing lysinoalanine. The Schifs is temporary stable product which was formed during Amodori rearrangements other products include fructoselysine and lactoselysine (Holland et al. 2011). Furosine is an indicator for thermal treatment in the milk. Furosine content in milk is directly proportion to severity of heat. The greater temperature produced high content of furosine in milk. Elliott et al. (2003) studied that furosine content was increased with elevation of storage temperature of UHT treated milk. It was not present in raw milk therefore production of lactulose in milk was indication of heat severity (ISO, 2004). Hydroxyl methyl furfural is primary indicator in heat treated milk.

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REVIEW OF LITERATURE

2.11. Statement of ProblemSeveral studies have been performed on various chemical aspects of UHT treated dairy products,

however, detailed investigation on protein and lipid fractions of UHT treated dairy products requires.Keeping in view the above-mentioned facts, a comprehensive study was planned with the following objectives

To study the biochemical changes in protein and lipid fractions of UHT treated milk at various stages of processing and storage

To study the protein and lipid profiling of UHT flavored milk, tea whitener and UHT dairy drink

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REVIEW OF LITERATURE

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Chatterjee B, Patel T. 2016. Increased sensory quality and consumer acceptability by fortification of chocolate flavored milk with oat B glucan. Int J Clin Biomed Res. 2:25-28.

Chavan RS, Chavan SR, Khedkar CD, Jana AH. 2011. UHT milk processing and effect of plasmin activity on shelf life: a review. Compr Rev Food Sci Food Saf. 10(5):251–268.

Chove LM, Zacharia AI, Grandison AS, Lewis MJ. 2013. Proteolysis of milk heated athigh temperatures by native enzymes analysed by trinitrobenzene sulphonic acid (TNBS) method. Afri J Food Sci. 7:232–237.

Cilliers FP. 2007. A biochemical study of tissue type plasminogen activator in bovine milk.(Masters Dissertation, University of Stellenbosch, Stellenbosch.) Retrieved from:http://scholar.sun.ac.za/handle/10019.1/44516

Clare DA, Bang WS, Cartwright G, Drake MA, Coronel P, Slmunovic J. 2005.Comparison of Sensory, Microbiological, and Biochemical Parameters of Microwave VersesIndirect UHT Fluid Skim Milk During Storage. J Dairy Sci. 88: 4172-4182.

Datta N, Deeth HC. 2001. Age Gelation of UHT Milk. Food and Bioproducts Processing. 79:197-210.Datta N, Deeth HC. 2003. Diagnosing the cause of proteolysis in UHT milk. Lebensm Wiss Tech. 36:173–

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heat coagulation and heat-induced changes of concentratedmicellar casein solutions: the role of calcium-ion activity and micellar integrity. Int Dairy J. 26(2):112-119.

Dupont D, Lugand D, Rolet-Repecaud O, Degelaen J. 2007. ELISA to detect proteolysis of ultrahigh-temperature milk upon storage. J Agric Food Chem. 17:6857–6862.

Edvaldo N, Costa, Ellen CQ, Lacerda, Suian MS, Santos, Carilan MS, Santos, Marcelo Franco, Robério R, Silva, Julliana I and Simionato. 2011. Action of Successive Heat Treatments in Bovine Milk Fatty Acids. J Braz Chem Soc.22(11):2115-2120

Elias RJ, Kellerby SS, Decker E. 2008. Antioxidant activity of proteins and peptides. Crit Rev Food Sci Nutr. 48:430–441.

Elliott AJ, Dhakal A, Datta N and Deeth HC. 2003. Heat-induced changes in UHT milks Part 1.Astralian J Dairy Technol. 58(1):3–10.

Fitria A, Buckow R, Singh T, Hemar Y, Kasapis S. 2015. Colour change and proteolysis of skim milk during high pressure thermal – processing. J Food Engg. 147:102–10.

Forsbäck L. 2010. Bovine udder quarter milk in relation to somatic cell count: Focus on milkcomposition and processing properties. Doctoral Thesis, Swedish University of Agricultural Sciences, Uppsala. Retrieved from: http://pub.epsilon.slu.se/2332/

Frary CD, Johnson RK, Wang MQ. 2004. Children and adolescents' choices of foods and beverages high in added sugars are associated with intakes of key nutrients and food groups. J Adol Health. 34(1):56-63.

Gaucher I, Tanguy G, Faunquant J, Jardin J, Rousseau F, Robert B, Madec MN,Gaucheron F. 2011. Proteolysis of casein micelles by Pseudomonas fluorescens CNRZ 798 contributes to the destabilisation of UHT milk during its storage. Dairy Sci and Technol. 91:413-429.

Gazi I, Vilalva IC, Huppertz T. 2014. Plasmin activity and proteolysis in milk protein ingredients. Int Dairy J. 38(2):208–212.

Gilani GS, Xiao CW, Cockell KA. 2012. Impact of antinutritional factors in food proteins on the digestibility of proteins and the bioavailability of amino acids and on protein quality. Br J Nutr. 108:315-332.

Hassan A, Amjad I, Mahmood S. 2009. Microbiological and physicochemical analysis of different UHT milks available in market. Afri J Food Sci.  3(4):100–106.

Holland JW, Gupta R, Deeth HC, Alewood PF. 2011. Proteomic analysis of temperature-dependent changes in stored UHT milk. J Agric Food Chem. 59:1837–1846.

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Jansson T, Clausen MR, Sundekilde UK, Eggers N, Nyegaard S, Larsen LB, Ray C, Sundgren A, Andersen HJ, Bertram HC. 2014. Lactose-hydrolyzed milk is more prone to chemical changes than conventional ultra-high-temperature (UHT) milk. J Agric Food Chem. 62:7886–7896.

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Lewis M, Grandison A, Lin MJ, Tsioulpas A. 2011. Ionic calcium and pH as predictors of stability of milk to UHT processing. Milchwissenschaft. 66(2):197-200.

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CHAPTER 3EXPERIMENT No. 1

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EXPERIMENT NO.1Background

In many developing countries, milk chilling facilities are not available on the farm where milk is produced; rather these are located at the distance of 10–12 km. After milking, it takes about 2–3 h to reach milk to the chilling facilities. The milk is then chilled and transported to the milk processing plants for thermal processing and value addition. In developing countries, shelf life of pasteurized milk is only 3  days, as compared to 7–10 days in developed countries. The factors which are responsible for the shorter shelf life of pasteurized milk should be discovered for the improvement of dairy sectors of these countries. The magnitude of chemical changes which takes place in un-chilled milk and their effect on fatty acids profile, antioxidant status and lipid oxidation is not previously studied.Methods: Raw milk samples of the same farm were either rapidly chilled to 4°C immediately for 2 h followed by rapid chilling to 4°C. Immediately and delayed chilled raw milk samples were stored at 4°C for 72h. Both milk samples were pasteurized at 65°C, filled in 250 ml transparent PET bottles and stored at 4°C for 6 days. Fatty acid profile, selenium, zinc, total antioxidant capacity, TFC and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, FFA, PV and AV were determined at different stages of the experiment. This experiment was repeated with milk of same farm for at least five times.Results: Storing raw milk at ambient temperature (35±2°C) significantly influenced the pH and lactose content of milk. The loss of SCFA in delayed chilled milk was 1.19%, 3.27% and 1.60%, as compared to immediately chilled raw milk. In delayed chilled milk, loss of C18:1 and C18:2 after 3 days of storage period was 6.67% and 1.22. In delayed chilled milk after 6 days of storage, loss of C18:1 and C18:2 was 7.7% and 1.39%, respectively. In immediately chilled milk loss of C18:1 and C18:2 after 3 days of storage were 3.48% and 0.64%. In immediately chilled milk loss of C18:1 and C18:2 after 6 days of storage were 4.57% and 0.9%. Almost 41% vitamin E was lost when raw milk was stored at ambient temperature for 2 hrs. About 21% and 7% vitamin E was lost in delayed and immediately chilled milk, when samples were analyzed immediately after pasteurization. Losses of selenium and zinc contents after 2 h of ambient storage of raw milk were 0.43 and 224 μg/100 g. Rise of 0.13 (MeqO2/kg) was recorded, when un-chilled raw milk was stored at ambient temperature for 2 h. After 3 and 6 days of storage, PV of pasteurized milk (delayed chilled) was 0.88 and 1.56 (MeqO2/kg). After 3 and 6 days of storage, peroxide value of pasteurized (immediately chilled) was 0.39 and 0.42(MeqO2/kg). After 2 hrs of ambient storage, 18.41% flavonoids were lost. After 2 hrs of ambient storage of raw milk, loss of TAC and DPPH free radical scavenging activity (FRSA) was 29.31% and 44.53%. After 6 days of pasteurization, loss of TAC and DPPH, FRSA in delayed chilled raw milk was 72.1% and 89.57%.Conclusions

The findings of this investigation showed that delayed chilling of raw milk leads to several undesirable chemical changes in lipid fraction of milk.Keywords: Raw milk, Delay chilling, Lipids characterization, Pasteurization, Storage

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EXPERIMENT NO.1

3.1 BackgroundIn many Asian and African countries, most of the milk is produced on small farms. On an average

basis, about 10–15L milk is produced on each farm. Chilling facilities are usually installed to cater the milk of about 15–25 villages. The distance between the place of milk production and milk chilling facilities is about 10–12 KM. On an average basis, it takes about 2 h to reach milk from the point of production to the chilling facility. During this period, several chemical changes take place in raw milk. Due to perishable nature of milk, immediate chilling of raw milk on the farm is recommended; however, due to several reasons in developing countries, practically it is impossible to chill the milk on the farm. To increase the shelf life of raw milk, below 4°C chilling is recommended. In Europe, milk processing companies collect chilled milk after every 3 days. Chilled milk may undergo several biochemical changes; certain species of the genus bacillus and pseudomonas fluorescenscan produce proteases and lipases, these enzymes have the capability of hydrolyzing fat and proteins of milk. Raw milk (RM) at lower temperature, change in microflora of milk takes place, when population of psychrophilic bacteria reaches about 107 to 108/ml (Datta and Deeth 2001). During the storage of heat-treated milk, proteases and lipases induces several undesirable flavors such as bitter, foreign, unclean, fruity, yeasty, metallic and bitter flavor (Datta and Deeth 2003). Indigenous and bacterial lipases may lead to the hydrolysis of fat globule; the degree of free fatty acid production in pasteurized milk depends upon psychrophilic count, storage temperature and metal ions etc. (Gillis, 2005). Objectionable flavors in raw milk may also be due to the generation of FFA (Singh and Creamer 1992) Low temperature/ chilling induced biochemical changes in raw milk during the 3 days of storage period should be studied and as this aspect is not previously investigated. Thermal treatment is mandatory for the manufacturing of fluid milk and the most commonly used thermal technique is pasteurization (Sakkas et al. 2014). Pasteurized milk requires refrigeration during distribution/ storage and offers wide range of products for the dairy industry (Scott, 2008). Pasteurization and subsequent storage of milk may induce some undesirable biochemical changes in lipid fraction of milk e.g. hydrolysis and auto-oxidation, these undesirable changes lead to the lower consumer acceptability and shorter shelf life (Valero et al. 2001). Heat resistant bacterial lipases are one of the most common cause spoilages of milk (Chen et al. 2003). Milk fat is considered as one of the most significant milk constituents with respect to the variety of fatty acids (Richmond, 2007). Thermal processing may influence the physical and chemical characteristics of milk and auto-oxidation. Auto-oxidation in milk fat consequences in the generation of low molecular weight aldehydes, ketone and lactones (Nadeem et al. 2017). These low molecular weight substances induce offensive odor and reduce the number of fat-soluble vitamins in milk (Al-Rowaily, 2008). Few oxidation products are connected with the damage of cellular membrane, ageing, cancer and heart diseases (Anwar et al. 2010; Perkins et al. 2005). Chemical perspectives of pasteurized milk have been studied in detail. However, the effect of delayed/ immediate chilling, 72 hours of chilling on biochemical changes in lipid fraction of pasteurized milk needs more detailed investigation. This study was planned with the objective to determine the effect of immediate/ delayed chilling, 72 h of chilling on fatty acid profile and lipid oxidation in pasteurized milk using conventional and advanced analytical techniques.3.2. Methods3.2.1 Materials and Experimental Plan

Raw milk was obtained from a farm and all the milk producing animals were in good health. For hygienic milk collection, sampler and transparent glass bottles were sterilized. Raw milk obtained from a farm was divided into two parts and packaged in sterilized transparent glass bottles. One part was immediately cooled down to 4°C using chilled water (2°C) designated as immediately chilled milk, second part was allowed (35°C) for 2 hrs, then it was also cooled down to 4°C using chilled water (2°C), designated as delayed chilled milk. Both type of milk was subjected to 72 h chilling at 4°C, followed by pasteurization at 65°C for 30 min, filled in 250 ml transparent sterilized bottles and stored at 4°C for 6 days.3.2.2. Chemical Composition of Milk

Chemical composition of milk samples was determined on a lactoscan. Fat, protein, lactose content and pH were determined.3.2.3. Estimation of Fatty Acids Profile

16

EXPERIMENT NO.1For the estimation of fatty acid profile of milk, first fat was extracted using diethyl ether, 50–60  mg

in test tube with the help of digital micropipette, followed by addition of 3  ml 2, 2, 4 trimethyl pentane then 0.5 N sodium methoxide solution prepared in HPLC grade methanol was added, samples were vortex for 3 min, followed by 15 min staying time. 1 μl sample was injected into GC-MS (7890-B) (Qian et al. 2003; IUPAC, 1987)3.2.4. Total Flavonoid Content (TFC)

Estimation of TFC in milk samples was performed by a spectrophotometric method using AlCl3 as derivatizing agent. For this test, Rutin was used as standard, milk sample 0.1 ml and 5% solution of NaNO3 (0.2 ml) were mixed together and incubated for 5 min, then 1M NaOH 1mL and 0.2 mL AlCl3 were added. For the measurement of absorbance, spectrophotometer was used (510 nm) (Nile and Khobragade 2010)3.2.5. Total Antioxidant Capacity (TAC)

TAC was performed in terms of Ascorbic Acid Equivalent/g (Nabasree and Bratati 2007). Absorbance of the sample, blank and series of standards was recorded at 695 nm. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. Sample (1 ml) was mixed with DPPH solution, which was prepared in in methanol, contents of the test tube were mixed by vertexing followed by incubation at 25 °C for 20 min. Double beam spectrometer was used for determined absorbance at 517 nm and results were reported in percent inhibition of free radicals (Adesegun et al. 2008).3.2.6. Estimation of Vitamins

For the extraction of fat from milk, standard method was adopted (AOAC, 1997). For the estimation tocopherols, 200 μg sample was homogenized with n-hexane (1 ml), sample was injected into HPLC. For the preparation of mobile phase, acetic acid and ethyl acetate (0.5% both) in n-hexane, flow rate was 1.5  ml/min, results of tocopherol were reported in μg/g (Jang and Xu, 2009). For the estimation of vitamin, A, milk sample (25 ml) was blended 20 ml each ammonia and ethanol ammonia (25% and 96%). BHT was added to the upper layer at the rate of 0.0025% as an antioxidant, followed by drying the sample at 35  °C using rotary evaporator, 30 ml KOH (5% in ethanol) was added, saponified at 60 °C for 30 min, extracted with n-hexane, evaporated on a rotary evaporator. Measurement was performed on HPLC (Pece et al. 2014). For the determination of vitamin C, each 300 μl milk and metaphosphoric (0.56%) were mixed together. Absorbance was measured at 254 nm against ascorbic acid standard (Romeu-nadal et al. 2006).3.2.7. Lipid Oxidation

For the measurement of lipid oxidation in milk at different stages of storage and processing, FFA, PV and AV were determined for 6 days at the frequency of 0, 3 and 6 days using standard methods (AOCS, 1995).3.2.8. Determination of Zinc and Selenium

Selenium and Zinc were performed by the standard methods (Larmond, 1987).3.2.9. Statistical Analysis

Experiment was performed in a CRD; each treatment was replicated three times and every sample was analyzed for three times. Data was analyzed using two-way analysis of variance technique. For the determination of significant difference, DMR Test was used SAS 9.1 software (Steel et al. 1997).3.3. Results and Discussion

Chemical composition of milk Table 1 describes the chemical composition of raw, chilled, pasteurized, 3 and 6 days old milk. Delayed chilling, 72 h of chilling and pasteurization treatment did not have any impact on fat content of milk, however, storage had a significant effect on fat content. After 6  days of storage, fat and protein content of delayed chilled raw milk were significantly less than their initial values (p < 0.05). Delayed chilling of raw milk also had a significant impact on lactose content and pH of milk. Hassan et al. (2009) studied the physical and chemical characteristics of heat-treated milk during the storage, fat, protein and SNF content while significant changes were reported in pH of heat-treated milk after 12 during storage. AlKanhal et al. (1994) studied the effect of storage on compositional attributes of milk, they recorded a decline in protein content with the progression of storage period.3.3.1. Fatty Acid Profile

17

EXPERIMENT NO.1Milk fat is a highly complex fat, more 200 types of fatty acids have been recognized in it, it is the

most common and abundant source of SCFA. Specific flavor and taste perspectives of dairy products are mainly due to the SCFA. In addition to that, milk fat is also a reasonable source of oleic acid, which is regarded beneficial fatty acid in prevention of dyslipidaemia. Milk fat also contains some concentration of linoleic acid (C18:2) (Nadeem et al. 2015). In current investigation, storage of un-chilled raw milk for 2h considerably influenced the FAP, major transition was recorded in SCFA, MCFA and LCFA. Concentrations of SCFA, MCFA and LCUSFA in immediately chilled raw milk were 10.4%, 44.97% and 26.42%. While the concentration of SCFA, MCFA and LCUSFA in delayed chilled (raw milk chilled after 2h of ambient storage) were 9.21%, 41.7% and 24.82%. The decline in the concentration of short-chain fatty acids in delayed chilled milk was 1.19%, 3.27% and 1.60%, as compared to immediately chilled raw milk (Table  2). Functional properties of milk fat are mainly due to the medium-chain fatty acids (Nadeem and Ullah 2016). In delayed chilled milk, loss of C18:1 and C18:2 after 3 days of storage period was 6.67% and 01.22. In delayed chilled milk after 6 days of storage, loss of C18:1 and C18:2 was 7.7% and 1.39%, respectively. In immediately chilled milk loss of C18:1 and C18:2 after 3 days of storage was 3.48% and 0.64%. In immediately chilled milk loss of C18:1 and C18:2after 6 days of storage was 4.57% and 0.9%. From the FAP of milk at different stages of storage and processing, it is evident that delayed chilling of raw milk is the major reason for undesirable changes in lipid fraction of pasteurized milk.3.3.2. Transition in Vitamins

Milk has two different types of antioxidant systems; these are categorized as fat soluble and water-soluble antioxidant system. Vitamin A, E and carotenoids constitute the fat-soluble antioxidant system of milk while, vitamin C, tyrosine, casein, whey proteins, zinc and selenium etc. constitute the water-soluble antioxidant system. This antioxidant defense system can prevent oxidative stresses in the body; they can also help to inhibit the lipid oxidation in milk (Nadeem and Ullah 2016). Estimation of variation in vitamin content of the immediately chilled raw milk, delayed chilled milk, raw milk chilled for 72h, freshly pasteurized milk, 3 and 6 days old pasteurized milk may provide useful evidence about the antioxidant behavior of milk at these stages, therefore, these parameters were studied in this study using HPLC. Vitamin A content of immediately chilled raw milk was 0.46 μg/100 g, about 41% vitamin E was lost when raw milk was stored at ambient temperature for 2 hrs. Vitamins A content of immediately and delayed chilled milk were not affected by the chilling of raw milk for 72h. About 21% and 7% vitamin E was lost in delayed and immediately chilled milk, when samples were analyzed immediately after pasteurization. After 3  days of storage of pasteurized milk, loss of vitamin E in immediately and delayed chilled milk was 17.39%, after 3 days of storage of pasteurized milk, loss of vitamin E in delayed and immediately chilled raw milk was 76.1% as compared to the vitamin A content of raw milk (immediately chilled after milking). After 6 days of storage of pasteurized milk, loss of vitamin E in delayed and immediately chilled raw milk was 91.3% and 36.9% (Table 3). Ohlsson and Bengtsson (2002) monitored the changes in vitamin A content, when milk was subjected to heat treatment. Saffert et al. (2002) recorded a slight decrease in the amount of vitamin A of heat-treated milk. Vitamin A content of the raw milk subjected to immediate chilling were 0.63 mg/100 g, while storing of milk at ambient temperature for 2 hrs resulted in decline of 41.2% vitamin E. Losses of folic acid may be 50% in sterilized milk (Forssein, 2000). Pasteurization had a varying degree of impact on vitamin E content of milk, loss of vitamin E immediately chilled milk was only 5% while, 12% vitamin E was lost in delayed chilled milk. Effect of storage period on vitamin E content of pasteurized milk was also different for immediately and delayed chilled raw milk. After 3 days of storage of pasteurized milk (immediately chilled milk), loss of vitamin E in pasteurized milk was 21%, from the initial value of immediately chilled raw milk. After 3 days of storage of pasteurized milk (delayed chilled milk), loss of vitamin E pasteurized was 52%, from the initial value of immediately chilled raw milk. After 6 days of storage of pasteurized milk (immediately chilled milk), loss of vitamin E in pasteurized milk was 30%, from the initial value of immediately chilled raw milk. After 6 days of storage of pasteurized milk (delayed chilled milk), loss of vitamin E in pasteurized milk was 90.4%, from the initial value of immediately chilled raw milk. Vitamin C content of raw milk was 0.57 mg/100 g, amount of vitamin C significantly decreased in 2

18

EXPERIMENT NO.1hrs stored raw milk at ambient temperature. Storing the raw milk at ambient temperature leads to several chemical and biochemical changes which leads to the reduction of vitamin in the subsequent stages of processing and storage.

3.3.3. Selenium and ZincSelenium and zinc are micro mineral which are naturally present in milk, these have antioxidant

activity in milk. Research work has shown that pasteurization usually have no effect on total mineral content of milk, however, information regarding the changes in content of selenium and zinc is scarce. This is the first investigation in which effect processing and storage is determined on selenium and zinc content of milk. Loss of selenium and zinc after 2 h of ambient storage of raw milk were 0.43 and 224 μg/100 g (Table 3). Effect of storage on selenium and zinc on both immediately and delayed chilled milk, loss of selenium and zinc in immediately and delayed chilled milk were 0.55 and 496 μg/100 g. Selenium is an essential mineral, as per dietary guidelines, 50 μg/day selenium should be consumed on daily basis to prevent cardiovascular diseases and cancer etc. (Tapiero et al. 2003) Zinc is a major antioxidant mineral of milk, it is an essential mineral, it should be consumed at the rate of 15 mg/day. Literature reported the use of selenium for the supplementation of dairy products (Gulbas and Saldamli 2005). Effect of different methods of thermal processing on selenium and zinc content were determined, boiling and pasteurization of milk had no effect on contents of selenium and zinc (Fennema, 1996). 3.3.4. Lipid Oxidation

For determination of lipid oxidation in raw and pasteurized milk, FFA, PV and AV were analyzed. Free fatty acids are the indicators of hydrolytic rancidity; higher concentration of FFA in milk shows the excessive lipase activity in milk. While, PV and AV quantify the peroxides and aldehydes especially 2-alkenals (Nadeem et al. 2014). Free fatty acids affect the fats and oils in two ways, firstly, they lead to the generation of odoriferous compounds. Secondly, they lead to the acceleration of auto-oxidation, resulting in shorter shelf lipid (Frage et al. 1999). Keeping in view the influence of free fatty acids on sensory and storage perspectives, edible oil manufacturer strives to a free fatty acid in the range of 0.08% to 0.11%. Free fatty acids of immediately chilled raw milk after 6 days of pasteurization were 0.11%. The rise of 0.14% FFA during 6 days of storage of delayed chilled raw milk was due to the production of more lipases during the 2 hrs. of ambient storage. In milk lipases may be originated from milk and bacteria, in 2 hrs time, total plate counts significantly increased that leads to excessive lipase activity. Lipases of bacterial origin are more resistant to heat treatment; they survive the pasteurization treatment and may cause / speed up the spoilage of pasteurized milk (Hayes et al. 2002). Rise of 0.13(MeqO2/kg) was recorded, when un-chilled raw milk was stored at ambient temperature for 2 h. Chilling (72h) and pasteurization did not impart any noticeable impact on peroxide value. Milk sample having peroxide value of 0.35 at the time of chilling and immediately after pasteurization 0.48 (MeqO2/kg) revealed poor storage stability as compared to the milk sample has 0.22 peroxide value at the time of chilling and 0.24 (MeqO2/kg) immediately after pasteurization. Anisidine value of delayed chilled raw milk was also higher than immediately chilled milk at all stages of processing and storage (Table 4).3.3.5. Storage Effect on Antioxidant Capacity of Pasteurized Milk

Effect of pasteurization on various chemical characteristics of milk is investigated in detail, however, transition in antioxidant capacity of raw milk, chilled milk, pasteurized milk and long-term storage is not previously investigated. Feed is the source of flavonoids, if the feed has a higher magnitude of flavonoids, milk will have higher concentration. In current investigation, flavonoids were estimated in terms of Rutin equivalent/ml. Flavonoids have antioxidant properties (Nadeem et al. 2013). Total flavonoid content of the raw milk was 2.39 Rutin equivalent/ml. After 2hrs of ambient storage, 18.41% flavonoids were lost. While the loss of total flavonoids in immediately and delayed chilled raw milk for 72h was 7.53% and 9.62%, with no effect of pasteurization. After 3 days of storage of pasteurized immediately and delayed chilled raw milk,

19

EXPERIMENT NO.1the loss of total flavonoids was 11.75% and 35.14% (Table 5). Pasteurized immediately and delayed chilled raw milk after 6 days the loss of total flavonoids was 18.41% and 46.86%. Peroxide values and total flavonoid contents were strongly correlated; determination intervals showed a lower amount of total flavonoid content revealed the higher peroxide value. Total antioxidant capacity denotes the antioxidant status of milk and it is an important indication of the response of the milk to hostage the free radicals, this can be used good indication of antioxidant status of biochemical fluids (Sies, 2007). In this investigation, TAC was measured to observe the antioxidant behavior of raw, chilled, pasteurized milk at different stages of storage. TAC and DPPH (FRSA) of raw milk were 49.8% and 25.6%. After 72h of chilling of immediately and delayed chilled raw milk, loss of total antioxidant capacity was 8.43% and 37.55%. After 72h of chilling of immediately and delayed chilled raw milk, loss of DPPH (FRSA) was 8.2% and 53.1%. Concentration of antioxidant substances was influenced by the grazing (Ruiz de Gordoa et al. 2011). Milk oligosaccharides also possess antioxidant activity (Roy and Deepak 2014). 3.4. Conclusions

lactose and pH of delayed chilled raw milk was less than immediately chilled milk while major changes were recorded in FAP of delayed chilled milk during the storage period of 6 days. Delayed chilled raw milk showed higher peroxide value and lower antioxidant capacity. In addition to several other factors, delayed chilling of raw milk may also be one of the most important reason for the shorter shelf life of pasteurized milk in developing countries. From this work, it was concluded that raw milk should be immediately cooled for better shelf life. Therefore, it is recommended that milk collection system in the developing countries should be improved to avoid any delay in between milking and chilling of raw milk.3.5. REFERENCES Adesegun SA, Elechi NA, Coker HAB. 2008. Antioxidant activities of methanolic extract of Sapium

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Technologie. 36:173–182.Fennema OR. 1996. Food chemistry. 3rd ed. New York: Marcel Dekker.Forssein KM. 2000. Folates and dairy products: a critical update. J Am College of Nutr. 19(2):100–110. Frage N, Mozzon M, Lercker G. 1999. Effects of free fatty acids on oxidative stability of vegetable oil.  J Am

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cheese. Interna J Food Sci Nutrin. 56(2):141–146.Hassan A, Amjad I, Mahmood S. 2009. Microbiological and physicochemical analysis of different UHT milks

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species. J Food Sci. 67:861–867.IUPAC. 1987. Standard methods for the analysis of oils, fats and derivatives.  7. Oxford: IUPAC, Applied

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21

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components during storage of whole and skimmed UHT milk. Food Chem. 72(1):51–58.

22

EXPERIMENT NO.1

23

EXPERIMENT NO.1Table 3.1: Effect of Immediate and Delayed Chilling of Raw Milk on Chemical Composition of Pasteurized Milk.Stage of Sampling Fat% Protein% Lactose% pH TS%Raw Milk (Immediately Chilled After Milking)

4.32±0.04a 3.22±0.08a 4.62±0.08a 6.71±0.20a 13.2±0.03a

Raw Milk Chilled After 2 Hrs of Storage (32±1oC)

4.29±0.03a 3.18±0.06a 4.55±0.06b 6.61±0.05b 13.1±0.11a

Raw Milk Chilled for 72 Hrs (Immediately Chilled Milk)

4.30±0.02a 3.21±04a 4.61±0.5a 6.69±0.03a 13.2±0.08a

Raw Milk Chilled for 72 Hrs (Delayed Chilled Milk)

4.28±0.05a 3.18±0.06a 4.55±0.03b 6.60±0.02b 13.1±0.09a

0 Day After Pasteurization (Immediately Chilled Milk)

4.27±0.06a 3.21±0.07a 4.60±0.06a 6.61±0.05b 13.2±0.07a

0 Day After Pasteurization Treatment (Delayed Chilled Milk)

4.28±0.04a 3.16±0.05b 4.54±0.05b 6.67±0.04a 13.1±0.05a

After 3 Days of Storage (Immediately Chilled Milk)

4.25±0.08a 3.19±0.03a 4.59±0.04a 6.66±0.03a 13.2±0.08a

After 3 Days of Storage (Delayed Chilled Milk)

4.24±0.7a 3.11±0.08b 4.51±0.02b 6.58±0.07b 13.1±0.06a

After 6 Days of Storage (Immediately Chilled Milk)

4.26±0.05a 3.16±0.06a 4.57±0.03a 6.62±0.05a 13.0±0.03a

After 6 Days of Storage (Delayed Chilled Milk)

4.20±0.09b 2.95±0.09c 4.52±0.06b 6.54±0.06b 12.7±0.02b

In a column, if a mean is expressed by dissimilar letter, these are statistically significant (p<0.05)

24

25

Table 3.2: Effect of Immediate and Delayed Chilling of Raw Milk on Fatty Acid Profile of Pasteurized MilkFatty Acid

Raw Milk (Immediatel

y Chilled After

Milking)

Raw Milk Chilled After

2 Hrs of Storage (32±1oC)

Raw Milk Chilled for

72 Hrs (Immediatel

y Chilled Milk)

Raw Milk Chilled for

72 Hrs (Delayed Chilled Milk)

0 Day After Pasteurizatio

n (Immediatel

y Chilled Milk)

0 Day After Pasteurization Treatment

(Delayed Chilled Milk)

After 3 Days of Storage

(Immediately Chilled

Milk)

After 3 Days of Storage (Delayed Chilled Milk)

After 6 Days of Storage

(Immediately Chilled

Milk)

After 6 Days of Storage (Delayed Chilled Milk)

C4:0 3.48±0.92d 3.11±0.09e 3.46±0.5d 3.39±0.08d 3.15±0.07e 3.72±0.08c 4.12±0.9a 3.51±0.25c 3.95±0.3b 3.11±0.09e

C6:0 2.38±0.37d 2.19±0.07e 2.35±0.3c 2.28±0.11d 2.27±0.11d 2.52±0.12b 3.08±0.16a 2.39±0.11c 2.31±0.02d 2.16±0.06e

C8:0 1.41±0.14c 1.24±0.01e 1.39±0.06c 1.31±0.05d 1.31±0.03d 1.66±0.07b 1.78±0.02a 1.42±0.06c 1.61±0.03b 1.22±0.06e

C10:0 3.13±0.24d 2.67±0.03f 3.11±0.04d 2.95±0.12e 2.91±0.08e 3.55±0.09b 3.62±0.08a 3.36±0.07c 3.53±0.05b 2.96±0.01e

C12:0 3.56±0.16 3.17±0.04 3.55±0.05 3.24±0.04 3.61±0.12 3.84±0.10b 4.10±0.15a 3.42±0.03 3.77±0.12 3.24±0.03

C14:0 11.63±0.46b 10.16±0.01c 11.59±0.3b 10.67±0.06c 11.25±0.14b 11.51±0.03b 12.17±0.43a 10.64±0.31c 11.38±0.5b 9.73±0.09d

C16:0 29.78±1.35b 28.37±0.14c 29.72±0.7b 28.75±0.37c 29.11±0.07b 31.35±0.08a 31.98±0.16a 30.15±0.58b 29.10±0.38b 28.43±0.88c

C18:0 13.77±0.80a 12.55±0.12b 13.66±0.4a 12.85±0.14b 12.78±0.05b 10.27±0.64d 11.22±0.31c 9.60±0.24e 8.85±0.22f 8.62±0.36f

C18:1 24.89±1.30a 23.61±0.06b 24.85±0.6a 23.57±0.25b 21.47±0.13c 19.76±0.49e 21.41±0.72c 18.22±0.45e 20.32±0.61d 17.19±0.20f

C18:2 1.53±0.19a 1.21±0.02c 1.49±0.04a 1.29±0.03b 0.88±0.04c 0.43±0.01d 0.84±0.02c 0.31±0.03f 0.63±0.02e 0.14±0.01g

C18:3 0.49±0.01a 0.27±0.01b 0.48±0.01a 0.26±0.01b 0.19±0.01c 0.12±0.02d 0.16±0.01c 0.08±0.01f 0.11±0.01e ND

In a row, if a mean is expressed by dissimilar letter, these are statistically significant (p<0.05)

EX

PER

IME

NT

NO

. 1

EXPERMENT NO. 1Table 3.3: Effect of Immediate and Delayed Chilling of Raw Milk on Vitamins and Mineral Content of Pasteurized MilkStage of Sampling Vitamin

Aμg/100 g

α-Tocopherol

mg/100g

Vitamin C

mg/100g

Seleniumμg/100 g

Zincμg/100 g

Raw Milk (Immediately Chilled After Milking)

0.46±0.02a 0.63±0.06a 0.57±0.05a 3.29±0.04a 4735±0.60a

Raw Milk Chilled After 2 Hrs of Storage (32±1oC)

0.27±0.01b 0.37±0.02d 0.26±0.03d 2.86±0.05c 4511±0.44c

Raw Milk Chilled for 72 Hrs (Immediately Chilled Milk)

0.44±0.05a 0.61±0.03a 0.54±0.06a 3.28±0.03a 4726±0.66a

Raw Milk Chilled for 72 Hrs (Delayed Chilled Milk)

0.24±0.04d 0.34±0.02d 0.23±0.01d 2.85±0.02c 4506±0.53c

0 Day After Pasteurization (Immediately Chilled Milk)

0.41±0.08a 0.51±0.06b 0.38±0.02b 3.25±0.05a 4722±0.64a

0 Day After Pasteurization Treatment (Delayed Chilled Milk)

0.19±0.03e 0.25±0.03c 0.13±0.04d 2.81±0.02c 4491±0.55d

After 3 Days of Storage (Immediately Chilled Milk)

0.38±0.07b 0.42±0.04c 0.31±0.01c 3.23±0.03a 4714±0.67a

After 3 Days of Storage (Delayed Chilled Milk)

0.11±0.02f 0.11±0.01f 0.05±0.02e 3.06±0.01b 4364±0.52e

After 6 Days of Storage (Immediately Chilled Milk)

0.29±0.03c 0.33±0.04e 0.20±0.01d 3.19±0.03a 4695±0.63b

After 6 Days of Storage (Delayed Chilled Milk)

0.04±0.01g 0.06±0.02g Not Detected

2.74±0.05c 4239±0.49f

In a column, if a mean is expressed by dissimilar letter, these are statistically significant (p<0.05)

26

EXPERMENT NO. 1Table 3.4: Effect of Immediate and Delayed Chilling of Raw Milk on Lipid Oxidation of Pasteurized MilkStage of Sampling FFA%

(Oleic Acid)

Peroxide Value

(MeqO2/kg)

Anisidine Value

Raw Milk (Immediately Chilled After Milking) 0.05±0.01e 0.22±0.03f 2.58±0.8e

Raw Milk Chilled After 2 Hrs of Storage (32±1oC) 0.08±0.02d 0.35±0.05d 3.64±0.09c

Raw Milk Chilled for 72 Hrs (Immediately Chilled Milk) 0.05±0.01e 0.24±0.03e 2.59±0.07e

Raw Milk Chilled for 72 Hrs (Delayed Chilled Milk) 0.11±0.06c 0.36±0.06d 3.66±0.10c

0 Day After Pasteurization (Immediately Chilled Milk) 0.08±0.03d 0.24±0.04e 2.65±0.04e

0 Day After Pasteurization Treatment (Delayed Chilled Milk)

0.12±0.07c 0.48±0.08c 3.72±0.07c

After 3 Days of Storage (Immediately Chilled Milk) 0.09±0.02d 0.39±0.06d 2.74±0.08e

After 3 Days of Storage (Delayed Chilled Milk) 0.16±0.04b 0.88±0.09b 4.14±0.11b

After 6 Days of Storage (Immediately Chilled Milk) 0.11±0.06c 0.42±0.05c 3.11±0.12d

After 6 Days of Storage (Delayed Chilled Milk) 0.19±0.07a 1.56±0.11a 7.89±0.50a

In a column, if a mean is expressed by a dissimilar letter, these are statistically significant (p<0.05)

27

EXPERMENT NO. 1Table 3.5: Effect of Immediate and Delayed Chilling of Raw Milk on Antioxidant Capacity of Pasteurized MilkStage of Sampling TFC

Rutin Equivalent

mg/ml

TAC%

DPPH%

Raw Milk (Immediately Chilled After Milking) 2.39±0.07a 49.8±0.10a 25.6±0.10a

Raw Milk Chilled After 2 Hrs of Storage (32±1oC) 1.95±0.02c 35.2±0.09d 14.2±0.09f

Raw Milk Chilled for 72 Hrs (Immediately Chilled Milk) 2.21±0.04b 45.6±0.13b 23.5±0.10b

Raw Milk Chilled for 72 Hrs (Delayed Chilled Milk) 1.72±0.01d 31.1±0.11d 11.9±0.08d

0 Day After Pasteurization (Immediately Chilled Milk) 2.18±0.04b 44.8±0.12b 23.1±0.11b

0 Day After Pasteurization Treatment (Delayed Chilled Milk) 1.69±0.03d 29.6±0.09e 11.7±0.07d

After 3 Days of Storage (Immediately Chilled Milk) 2.11±0.05b 41.4±0.15c 19.1±0.10c

After 3 Days of Storage (Delayed Chilled Milk) 1.55±0.01e 21.9±0.08e 6.95±0.08g

After 6 Days of Storage (Immediately Chilled Milk) 1.93±0.03c 37.2±0.10d 17.7±0.13e

After 6 Days of Storage (Delayed Chilled Milk) 1.27±0.06f 13.9±0.09f 2.67±0.07h

In a column, if a mean is expressed by dissimilar letter, these are statistically significant (p<0.05). TFC: Total flavonoid content, TAC: Total antioxidant capacity, DPPH: 1,1-diphenyl-2-picrylhydrazyl

28

CHAPTER 4EXPERIMENT No. 2

29

EXPERMENT NO. 2Background: In current investigation, the effect of UHT treatment and storage was determined by making a comparison in fatty acid profile, triglyceride composition, organic acids and lipid oxidation of the thermally treated and stored milk with raw milk, which was not reported in earlier investigations. Method: Raw milk samples were collected from the bulk storage facility of a dairy industry. The same milk was routed to UHT treatment and aseptically packaged samples were collected. The fatty acid profile, triglyceride composition, organic acids and lipid oxidation was determined in raw and UHT treated milk at 0, 30, 60 and 90 days. Fatty acid and triglyceride profile were determined on GC-MS while organic acids were determined by HPLC. For the measurement of induction period, professional Rancimat was used. Lipid oxidation was characterized through FFA, PV, AV and conjugated dienes. Results: Compositional attributes of milk remain unchanged during the entire length of storage. Concentrations of SCFA in raw and UHT milk were 10.49% and 9.62%. UHT treatment resulted in 8.3% loss of SCFA. Concentration of MCFA in raw and UHT treated milk was 54.98% and 51.87%. After 30, 60 and 90 days of storage, concentration of MCFA was found 51.23%, 47.23% and 42.82%, respectively. Concentration of C18:1 and C18:2 in raw and UHT milk was 26.86% and 25.43%, respectively. The loss of C18:1 and C18:2 in UHT treatment was 5.32%. Storage period of 30 days was found non-significant, while noticeable variations were found in triglyceride profile of 60- and 90-days old samples of UHT milk. Free fatty acids content of raw, UHT treated and 90 days old milk were 0.08%, 0.11% and 0.19%. UHT treated version of milk showed similar peroxide value. While, the storage remarkably affected the peroxide value. Induction period of raw, UHT and stored milk was strongly correlated with peroxide value and fatty acid profile. Mean value of lipase activity in raw milk was 0.73 ± 0.06 μmoles/ml. UHT treatments significantly decreased the lipase activity. The lipase activity of milk immediately after the UHT treatment was 0.18 ± 0.02 μmoles/ml. Color, flavor and smell score decreased through the storage of UHT milk for 90 days.Conclusion: The results of this investigation revealed that FA and triglyceride profile changed after 60 and 90 days of storage. Production of organic acids led to the drop of pH and sensory characteristics in UHT milk during the long-term storage. Induction period can be successfully used for the determination of anticipatory shelf life of UHT milk. Keywords: UHT Milk, Fatty acid profile, Triglyceride profile, Induction period, Lipid oxidation 4.1Background

Exposure of milk to UHT treatment and subsequent ambient storage leads to several biochemical changes such as proteolysis and lipolysis (Vazquez-Landaverde et al. 2006). Lipid oxidation is one of the major reasons for the spoilage of UHT milk. It has a strong negative impact on sensory characteristics of UHT milk (Deeth and Fitz-Gerald 2003). Volatile compounds generated from the breakdown of primary oxidation products induce oxidized flavor in UHT milk (Houghtby et al. 1992). During lipid oxidation, fatty acids are broken down to oxidation products (Nadeem et al. 2017). During lipid oxidation several biochemical changes take place and produced oxidation products. Scientific evidences have shown that oxidation products may promote cancers and cardiovascular diseases (Besbes et al. 2005). Rate of lipid oxidation mainly depends upon the fatty acid composition (Gulla and Waghray, 2011). Milk fat is highly complex, more than 400 fatty acids have been detected in it (Fox, 2011). Physico-chemical characteristics of milk fat is determined by the triglycerides (Tzompa-Sosa et al. 2016). Preventing lipid oxidation in UHT milk is a major challenge for the dairy industries. UHT treatment may lead to the degradation of lactose which can lead to the formation of OA (Celestino et al. 1997). Rancimat has been used for the estimation of shelf life of milk fat (Gulla and Waghray 2011; Chavan et al. 2011). 4.2. Methods 4.2.1. Materials and Experimental Plan

Raw milk samples were collected from the bulk storage facility of a dairy industry. The same milk was routed to UHT treatment and aseptically packaged samples were collected. Reagents used for this research work were procured from Sigma Aldrich, USA. UHT treated milk samples were stored at ambient conditions for a period of 90 days. 4.2.2. Milk Composition and FAP

30

EXPERMENT NO. 2Milk composition was determined on a lactoscan. For the measurement of FAP, fat was extracted

from milk. the dried fat (50 mg) was taken in a screw capped test tubes followed by the addition of 2 ml solution of 15%Hydrogen Chloride in Methanol (15%, Fluka). Tubes were tightly closed and put in the heating block for 60 min After 60 min 2 ml each deionized water and HPLC grade n-hexane were added, contents of the tubes Supernatant were transferred to GC-MS (7890-B, Agilent Technologies) (Kail et al. 2012)4.2.3. Triglycerides

Measurement of triglycerides was performed on GC-MS (7890-B, Agilent Technologies). Milk fat was dried over anhydrous sodium sulfate. The dried fat (50 mg) was then dissolved in n-hexane (1 ml) and 1 μL was injected into GC-MS through Auto Liquid Sampler (ALS) (Naviglio et al. 2017). 4.2.4. Lipase Activity

Lipase activity was determined by pH static titratable method and expressed in μ-moles i.e. the number of free fatty acids released from the triglyceride by lipases in 1 ml sample (McGregor and Fernandez-García 1994).4.2.5. Organic Acids

Milk samples was performed as per method described (McGregor and Fernandez-García, 1994). For the extraction of organic acids, 4 parts of UHT milk were treated with 1 part of 10 mM sulfuric acid. Supernatant was injected into HPLC. Standards of organic acids were purchased from Sigma-Aldrich. 4.2.6. Lipid Oxidation

FFA, PA and AV were measured at 0, 30, 60 and 90 days of storage period. 2m sample was taken in 50 mL test tube and mixed with 18 mL of 3.86% HCLO4. The sample was homogenized for 15 s and to prevent the oxidation of lipid, BHT was added. 2ml TBA mixed with 2 ml filtrate and measured the absorbance at 532 nm (AOCS, 1995)4.2.7. Induction Period

2.5 g fat sample was taken in reaction vessel, temperature and rate of oxygen was set at 120 °C and 20 l/hr and measured according to standard method (AOAC, 1987).4.2.8. Sensory Evaluation

Sensory evaluation of milk samples was performed in a sensory evaluation laboratory at 25 °C, a trained panel of judges for color, flavor and smell (Oupadissakoon et al. 2009).4.2.9. Statistical Analysis

This research work was planned in a CRD. Sampling stages were considered as treatments and replicated at least five times. Results were reported as Mean ± SD while for the estimation of significant difference among the treatments, DMR Test was used. Data were analyzed on SAS 9.1 software (Steel et al. 1997)4.3. Results and Discussion

Table 1 describes the results of chemical composition of raw milk, UHT treated and stored samples for 90 days. The difference in fat, protein and SNF content of raw and UHT treated milk was due to the standardization of milk at 3.5% fat content. Fat, protein and SNF content non-significantly decreased during the storage of 90 days. The pH of 90 days old samples of UHT milk was significantly higher than raw milk, UHT treated, 30- and 60-days old milk samples. Compositional attributes of UHT milk have been studied in earlier investigations. Hassan et al. (2009) reported that physico-chemical characteristics of UHT milk for a period of 180 days and non-significant changes were observed in the compositional attributes with little increase in acidity. Martins and van Boekel (2005) also reported similar results on the chemical composition of UHT milk. Anema and Li (2003) reported that pH of UHT milk decreased during the long-term storage. 4.3.1. FAP

FAP of raw, UHT treated and aseptically packaged milk samples stored for 90 days has been shown in Table 2. UHT treatment and storage period significantly affected the FAP of milk. However, the effect of storage period on FAP of UHT milk during ambient storage is not reported. According to the results, concentrations of SCFA in raw milk and UHT milk were 10.6% and 9.28%. SCFA are highly significant

31

EXPERMENT NO. 2from the flavor and sensory characteristics viewpoints, decline in their concentration may have a negative impact on flavor perspectives of milk (Gulla and Waghray 2011). Khan et al. (2017) compared the rate of oxidation in C18:1 and C18:2 and their study disclosed that rate of oxidation in C18:2 was ten times faster than C18:1. Storage induced oxidative deterioration in heat treated milk during the storage. UHT milk is stored at ambient temperature that can accelerate the auto-oxidation in UHT milk (Deeth and Fitz-Gerald 2003). Lipid oxidation is a serious problem of UHT milk (de-Wit and Nieuwenhuijse, 2008). Choe (2003) reported that oleic acid is the dominant unsaturated fatty acid in milk. Elevated storage temperature can accelerate the auto-oxidation in UHT milk. 4.3.2. Triglyceride Profile of UHT Milk

Fat content of bovine milk ranges from 2.5–6.5% that is comprised of triglycerides (Anonymous, 2000). Fat content and fatty acid composition considerably vary from breed to breed, season and stage of maturity (Moate et al. 2014). Milk fat is regarded as one of the most complex fat. More than 100 kinds of triglyceride species have been found in milk fat (Beccaria et al. 2014). The results of triglyceride profile of raw, fresh and stored UHT milk are given in Table 3. UHT treatment induced significant changes in triglyceride profile of milk. Storage duration up to 30 days was non-significant, while noticeable variations were found in triglyceride profile of 60- and 90-days old samples of UHT milk. Heat, moisture, metal ions and lipases have been recognized as catalysts for the hydrolysis of triglycerides. Milk contains about 87% water; lipases are also present in milk. Triglycerides are the key component of fat; all triglycerides have not same correlation with the fat content of milk (Zhou et al. 2014b). Milk fat is comprised of 97–98% triglycerides (Gutiérrez et al. 2009). UHT milk is usually toned and standardized at 3.5% fat content; therefore, TAG profile of UHT milk may be different from raw milk. Beccaria et al. (2014) studied the correlation between TAG and fatty acid profile in milk and stereospecific analyses were performed and strong connections were recorded in the content of fatty acid at all the three positions and content of similar fatty acids on the intact TAG. Lipid profile was significantly influenced by the storage period, amount of TAG decreased and FFA increased (Lu et al. 2018). 4.3.3. Organic Acids

During UHT treatment, lactose endures several chemical changes such as Maillard Reaction and formation of formic acid, pyruvic acid, acetic acid etc. Results of organic acids in raw, UHT milk at different stage of storage have been shown in Table 4. In current investigation organic acids were determined in raw milk, UHT milk, 30, 60 and 90 days old UHT milk. UHT treatment and storage period significantly affected the concentration of organic acids in milk. Claeys et al. (2013) reported that concentrations of citric acid in pasteurized milk was 1439 mg/L. Heat treatment may have an impact on citric acid content in milk, however, detailed investigation on this aspect should be performed (Islam et al. 2013). Islam et al. (2013) studied the concentration of organic acids in raw, pasteurized and UHT milk and they reported that concentrations of organic acids increased in the storage. Formic acid in heated milk is recognized as advanced product of Maillard reaction and amount of formic acid in sterilized milk was 150 times greater than normally pasteurized milk (Da-Silva et al. 2012). The decline in pH of UHT milk is mainly due to the production of formic acid and UHT milk having higher level of formic acid had lower pH and more acidity ( Claeys et al. 2013). Walstra et al. (2006) described that UHT treatment may lead to the degradation of lactose to organic acids. Succinic acid in UHT treated stored milk was less than raw milk (Nishimura et al. 2015)4.3.4. Lipid Oxidation

During the storage, UHT milk experienced oxidative and hydrolytic rancidity. For the measurement of hydrolytic rancidity, free fatty acids were used (Wang and Ho 2012). Lipases of bacterial and milk origin are mainly responsible for the hydrolysis of triglycerides and generation of free fatty acids. Poor quality raw milk has a greater number of lipases leading to the generation of more fatty acids. European Union has established a maximum limit of 0.2% free fatty acids in foods. In this research work, free fatty acids content of raw, UHT treated and 90 days old milk samples were 0.08%, 0.11% and 0.19%, respectively ( Rauh et al. 2014). Investigations have shown that milk fat is susceptible to auto-oxidation (Gulla and Waghray 2011). Milk fat contains about 23–25% oleic acid and 1.5–2% linoleic acids (Khan et al. 2017). For the assessment

32

EXPERMENT NO. 2of oxidative deterioration and oxidation status of foods, peroxide value is the method of choice for the researchers working in the field of analytical chemistry and food science (Dupont et al. 2007). UHT treatment had no effect on peroxide value of milk. While, the storage duration remarkably affected the peroxide value. For the estimation of primary oxidation products produced in UHT milk, conjugated dienes were determined. Values of conjugated dienes were not affected by the UHT treatment. However, the conjugated dienes considerably increased as the storage progressed. After 30, 60- and 90-days storage of UHT treated milk, conjugated dienes were 0.48, 0.61 and 1.18. For the determination of primary oxidation products in milk fat Chavan et al. (2001) used conjugated dienes. During auto-oxidation of fats and oils, ketones, alcohols and aldehydes are produced (O’Brien, 2008). Anisidine value is used to measure the number of aldehydes especially 2-alkenals that are produced during the course of auto-oxidation (Malmgren et al. 2017). The anti-oxidant properties of heated milk reduced and there was a considerable loss of the activity of gluthatione peroxidase, which is one of the major antioxidants. (Silvestre et al. 2008) 4.3.5. Induction Period (IP)

IP is used to estimate the oxidative stability index of fat and oils (Besbes et al. 2005). Research and development sections of dairy manufacturers have to wait for long time to know the oxidative stability of milk fat. The suitability of accelerated oxidation conditions to assess the oxidative stability of milk fat has already been established. For the shelf life assessment UHT milk, this technique is not previously for the measurement of oxidative stability of lipid phase of UHT milk. Induction period of raw, UHT and stored milk was strongly correlated with peroxide value and fatty acid profile. Measurement frequencies showing lower magnitude of oxidation products yielded high induction period. Induction period is influenced by the presence of antioxidants in food systems (Suetsuna et al. 2000). Milk also contains natural antioxidants which have been divided in two classes. First class is consisted of fat-soluble antioxidants such as, vitamin E, A, carotene. Second class is comprised of water-soluble substance which has antioxidant prospects. These include casein, whey proteins, vitamin C, amino acids, glutathione peroxidase (De-Wit and Nieuwenhuijse 2008). The lower values of induction period also indicated the weakening of antioxidant systems of milk during the storage. Strong correlations between the fatty acid profile and induction period was also recorded. Determination interval showing excessive breakdown of fatty acids had lower induction periods. These results offer promising opportunities for using induction period a useful method for the estimation of shelf life of UHT milk.4.3.6. Lipase Activity

This is the first study in which lipase activity and triglyceride profile of milk has been investigated for a period of 3 months. Quality of raw milk in developing countries is very poor, total plate count in the months of summer may be as high 50million/ml. Lipases are fat hydrolyzing enzymes, delayed chilling of raw milk, excessive agitation and rough handling of milk may lead to increased lipolytic activity. Lipases of milk origin are usually eliminated by the UHT treatment but the lipases of bacterial origin are heat resistant, they survive the traditional UHT process and cause serious problems in lipid fraction of UHT milk during the storage. Mean value of lipase activity in raw milk was 0.73 ± 0.06 μmoles/ml. UHT treatment significantly decreased the lipase activity. The lipase activity of UHT treated milk was 0.44 ± 0.03, 0.95±0.07 and 1.14±0.09 μmoles/ml. Lipase activity was strongly correlated with the triglyceride profile and free fatty acids. Testing intervals showing higher lipase activity also showed the higher concentration of FFA. During the storage, triglycerides of UHT milk decreased and all the testing intervals showed a decreasing trend. During ambient storage, the increased lipolytic activity may be connected to the increased lipase activity (Fitria et al. 2015). Lipases produced by the pseudomonas spp. and Bacillus spp. are thermostable and survive the orthodox UHT treatment (Gazi et al. 2014). 4.3.7. Sensory Evaluation

Color, flavor and smell score decreased through the storage of UHT milk for 90 days. After 90 days of storage duration, color, flavor and smell score of UHT treated milk was 6.5, 6.3 and 6.2. The lower score of UHT milk during the storage was due to the changes taking place in fatty acid, triglyceride profile, OA, FFA and oxidation products significantly affected the sensory characteristics of UHT milk. Lipase activity, FFA and PV of 90 days stored UHT milk was considerably higher, which was the reason for lower score of 90 days old samples of UHT milk. The flavor profile of UHT milk is different from raw and pasteurized

33

EXPERMENT NO. 2milk. UHT milk has been described as cooked and flat favor because during heat treatment various sulfur compound such as methyl ketones, and aliphatic aldehydes are produced (Zabbia et al. 2012)4.4. Conclusions

Fatty acid and triglyceride profile of raw milk and 60 days old UHT milk samples were significantly different from each other. During the storage, lipase activity went on increasing which led to the transition and triglyceride profile and formation of free fatty acids. Peroxide value was strongly correlated with induction period and samples having lower peroxide value showed higher induction period and vice versa. 4.5. REFERENCES Anema SG, Li Y. 2003. Effect of pH on the association of denatured whey proteins with casein micelles in

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EXPERMENT NO. 2Gulla S, Waghray K. 2011. Effect of storage on physicochemical characteristics and

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EXPERMENT NO. 2Steel R, Torrie J, Dickey DA. 1997. Principles and procedure of statistics. In: A

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polymorphs in milk fats with large differences in triacylglycerol profiles. J Agric Food Chem. 64:4152–4157 Celestino EL, Iyer M, Roginski H. 1997. Reconstituted UHT-Treated Milk: Effects of Raw Milk, Powder Quality and Storage Conditions of UHT Milk on Its Physicochemical Attributes and Flavour. Int Dairy J. 7:129–140.

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Table 4.1: Effect of UHT Treatment and Storage on Chemical Composition of MilkStage Fat% Protein% SNF% pH Acidity%Raw Milk 4.58 ± 0.08a 3.25 ± 0.13a 8.65±0.10a 6.82±0.17a 0.12±0.02c

UHT Milk 3.52 ± 0.05b 3.22 ± 0.09a 8.62±0.16a 6.79±0.09a 0.12±0.01c

30 Days Stored UHT Milk 3.51 ± 0.12b 3.22 ± 0.07a 8.61±0.19a 6.78±0.06a 0.12±0.05c

60 Days Stored UHT Milk 3.45 ± 0.03b 3.18 ± 0.04a 8.56±0.22a 6.65±0.18a 0.15±0.04b

90 Days Stored UHT Milk 3.41 ± 0.15b 3.11 ± 0.02b 8.42±0.02b 6.51±0.24c 0.18±0.01a

If the means in a column are expressed by a same letter these are non-significant (p>0.05)

Table 4.2: Impact of UHT Treatment and Storage on Fatty Acid Profile of MilkFatty Acid Profile mg/g

Raw Milk Immediately After UHT Treatment

30-Days 60-Days 90-Days

C4:0 3.79±0.06a 3.44±0.05b 3.39±0.03b 3.12±0.08c 2.88±0.13d

C6:0 2.52±0.26a 2.38±0.07b 2.35±0.05b 2.19±0.04c 2.04±0.17d

C8:0 1.41±0.15a 1.29±0.03b 1.22±0.11b 1.08±0.03c 1.02±0.08c

C10:0 2.77±0.05a 2.51±0.09b 2.43±0.10b 2.14±0.16c 1.95±0.02d

C12:0 3.11±0.09a 2.91±0.14b 2.84±0.17b 2.61±0.07c 2.44±0.01c

C14:0 10.22±0.23a 9.44±0.25b 9.18±0.16b 8.22±0.28c 7.56±0.25d

C16:0 25.38±0.77a 24.16±0.42b 24.10±0.35b 22.74±0.17c 21.53±0.64d

C18:0 16.27±0.19a 15.36±0.18b 15.11±0.33b 13.66±0.09c 11.29±0.15d

C18:1 24.69±0.55a 23.29±0.33b 22.87±0.66b 20.47±0.33c 18.52±0.13d

C18:2 2.17±0.05a 1.98±0.12b 1.73±0.02c 0.59±0.14d 0.14±0.01e

Means having same letter in a row are non-significant (p>0.05)

36

EXPERMENT NO. 2

37

EXPERMENT NO. 2Table 4.3: Effect of UHT Treatment and Storage on Triglyceride Profile of Milk Triglyceride Profile mg/g

Raw Milk Immediately After UHT Treatment

30-Days 60-Days 90-Days

TAG C24:0 0.20±0.01a 0.14±0.01b 0.12±0.02b 0.08±0.02c 0.03±0.01d

TAG C26:1 0.89±0.05a 0.81±0.03b 0.79±0.13b 0.64±0.08c 0.52±0.05d

TAG 28:0 1.29±0.08a 1.15±0.07b 1.12±0.19b 0.98±0.04c 0.85±0.11d

TAG C28:1 0.14±0.02a 0.12±0.03b 0.11±0.01b 0.06±0.02c 0.03±0.01d

TAG 30:0 1.91±0.06a 1.79±0.11b 1.75±0.05b 1.62±0.14c 1.54±0.02d

TAG C30:1 0.28±0.02a 0.24±0.05b 0.22±0.02b 0.17±0.04c 0.13±0.01d

TAG 32:0 3.35±0.12a 3.11±0.09b 3.07±0.08b 2.92±0.16c 2.61±0.19d

TAG C32:1 1.74±0.06a 1.55±0.06b 1.52±0.03b 1.31±0.09c 1.22±0.13d

TAG 34:0 7.56±0.10a 7.12±0.14b 7.08±0.28b 6.91±0.27c 6.58±0.04d

TAG C34:1 3.41±0.09a 3.18±0.05b 3.14±0.02b 2.89±0.012c 2.41±0.02d

TAG 36:0 11.39±0.35a 10.82±0.16b 10.77±0.31b 10.17±0.49c 9.42±0.37d

TAG C36:1 3.67±0.03a 3.49±0.07b 3.11±0.02b 2.88±0.06c 2.27±0.01d

TAG 38:0 14.66±0.45a 14.18±0.33b 14.11±0.56b 13.42±0.71c 12.54±0.43d

TAG C38:1 4.77±0.13a 4.22±0.16b 3.66±0.08b 2.99±0.03c 2.38±0.15d

TAG C40:2 10.55±0.18a 9.76±0.64b 9.71±0.73b 8.66±0.20c 7.44±0.26d

TAG C42:1 5.11±0.38a 4.62±0.28b 4.55±0.21b 4.19±0.07c 3.78±0.03d

TAG C44:1 4.62±0.19a 4.21±0.04b 4.17±0.08b 3.48±0.06c 3.17±0.02d

TAG C46:1 5.16±0.17a 4.58±0.02b 4.51±0.17b 4.13±0.22c 3.64±0.04d

TAG C48:3 7.66±0.21a 6.74±0.12b 7.63±0.09b 7.23±0.09c 6.74±0.05d

TAG C50:5 11.27±0.61a 11.25±0.16b 11.24±0.33b 10.51±0.31c 9.42±0.27d

TAG C52:1 10.24±0.34a 10.22±0.27b 10.21±0.67b 9.77±0.06c 8.27±0.15d

TAG C54:6 2.66±0.03a 2.65±0.04b 2.61±0.13b 2.27±0.01c 2.11±0.02d

In a row, if means are expressed by a different letter, that are statistically significant (p<0.05)

Table 4.4: Effect of UHT Treatment and Storage on Organic Acids in MilkOrganic Acids

Raw Milk Immediately After UHT Treatment

30-Days 60-Days 90-Days

Lactic Acid 60.43±0.57c 63.88±0.09b 64.17±0.04b 64.22±0.41b 67.73±0.82a

Acetic Acid 2.96±0.04c 3.62±0.13b 3.66±0.06b 3.71±0.50b 5.14±0.32a

Citric Acid 88.57±0.74c 92.34±0.69b 93.11±0.43b 93.44±0.98b 98.53±0.26a

Pyruvic Acid 3.61±0.08c 4.29±0.10b 4.32±0.24b 4.39±0.06b 7.19±0.18a

Formic Acid 84.27±0.43c 86.51±0.16b 86.97±0.33b 87.10±0.51b 95.64±0.13a

Succinic Acid 20.16±0.15c 22.67±0.35b 23.14±0.28b 23.19±0.40b 25.38±0.61a

Oxalic Acid 4.31±0.02c 5.94±0.12b 6.19±0.21b 6.24±0.31b 10.27±0.23a

In a row, if means are expressed by a different letter, that are statistically significant (p<0.05)

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EXPERMENT NO. 2Table 4.5: Effect of UHT Treatment and Storage on Lipid Oxidation of MilkStage Free Fatty

AcidsPeroxide

ValueAnisidine

ValueConjugated

DienesRaw Milk 0.08±0.01d 0.25±0.04c 3.59±0.13c 0.12±0.01c

UHT-Treated 0.11±0.02c 0.28±0.05c 3.88±0.0c 0.14±0.02c

30 Days Old 0.13±0.03b 0.42±0.07b 5.69±0.14b 0.48±0.06b

60 Days Old 0.16±0.02a 0.62±0.08b 7.56±0.03c 0.61±0.03c

90 Days Old 0.19±0.01a 1.18±0.11a 12.73±0.19a 1.18±0.16a

In a column, if means are expressed by a different letter, that are statistically significant (p<0.05)

Table 4.6: Effect of UHT Treatment and Storage on Sensory Characteristics of MilkStage Color Flavor SmellRaw 8.1±0.18a ND 8.0±0.15a

UHT-Treated 7.5±0.09b 7.4±0.12a 7.8±0.10a

30 Days old 7.3±0.13b 7.1±0.14b 7.2±0.16b

60 Days old 7.0±0.15b 6.6±0.22b 6.8±0.16b

90 Days old 6.9±0.21c 6.5±0.08c 6.7±0.19c

In a column, if means are expressed by a different letter, that are statistically significant (p < 0.05); ND = Not Determined

39

EXPERMENT NO. 2

Figure 4.1: Effect of UHT Treatment and Storage on Free Fatty Acids

Figure 4.2: Effect of UHT Treatment and Storage on Peroxide Value

40

EXPERMENT NO. 2

Figure 4.3: Effects of UHT Treatment and Storage on Anisidine Value

Figure 4.4: Effect of UHT Treatment and Storage on Conjugated Dienes

41

EXPERMENT NO. 2

Figure 4.5: Induction Period of Raw, UHT Treated Milk in Storage

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CHAPTER 5EXPERIMENT No. 3

Chemical Characteristics of Protein Fractions of Ultra High Temperature Treated Milk and Sedimentation

Muhammad Ajmal, Muhammad Nadeem1, Muhammad Tayyab and Nabila Gulzar1Department of Dairy Technology, University of Veterinary and Animal Sciences Lahore

Correspondence: muhammd.nadeem@uvas.edu.pkAbstract Impact of indirect Ultra-High Temperature Treatment (UHT; 142oC for 2sec) and 90 days ambient storage (30-35oC) on protein and AAP of UHT milk and sedimentation was investigated. After UHT treatment, β-lactoglobulin, α-lactalbumin, immunoglobulin and serum albumin decreased by 12.1%, 15.4%, 10.6% and 10.2% with no effect on casein fractions. Sedimentation was comprised of 14.9% αs1-Casein, 3.42% αs2-Casein, 9.88% β-casein, 2.27% κ-Casein, 4.36% β-Lactoglobulin, 1.88% α-lactalbumin, 1.84% immunoglobulin and 0.61% Serum Albumin. After 90 days of storage, out of 80% casein and 20% whey proteins, 38.5% and 8.7% became the part of sedimentation. After 3 months of storage of UHT milk at ambient temperature, about 40 and 14% threonine and lysine were transferred from milk to the sedimentation. Viscosity, NCN, NPN and plasmin activity increased during the long-term storage of UHT milk. Keywords: UHT Milk; Protein Profile; Amino Acid Profile; Sedimentation5.1. Introduction

UHT treatment leads to the destruction of enzymes and microbes in milk or bacterial origin survive the orthodox UHT treatment leads to several undesirable biochemical changes in UHT milk such as lipolysis and proteolysis (Button, 2011). Proteolytic events in milk consequences in the production of bitter flavor and age gelation. Alkaline proteinase, plasmin are the proteolytic enzymes of milk origin while, psychrotrophic bacteria also produce extracellular proteinases which causes breakdown of protein in ultra-high temperature (Chavan et al 2011). Plasmin causes proteolysis of casein which is the major reason for increased viscosity and flavor defect in UHT milk. Proteases cause the breakdown of casein and casein micelle to gamma casein, peptone and amino group (Aouadhi et al. 2012). Mastitis milk has higher concentration of plasmin as compared to the milk having lower concentration of somatic cells (Richmond, 2007). Psycrhroptrophic bacteria produce heat resistant proteolytic and lipolytic enzymes in milk. Psychrotrophic count of 6.9-7.2 log cfu/ml increased the thickness and oxidized flavor in UHT treated milk. Storage stability of UHT milk is a critical factor. In storage period flavor deterioration and age gelation, that is cause by the accumulation of micelles to make a three-dimensional network (Bhatt, 2014). However, gap between protein profiling and age gelation is not filled by any specific study. Thermal treatment of milk may lead to several changes in protein fraction such as, unfolding, rearrangement of disulfide linkages, denaturation, aggregation and glycation (Helstad et al. 2007; Smithers, 2008). Beta-lactoglobulin, alpha-lactalbumin, serum protein immunoglobulin is the major whey protein of milk that is about 20% of total milk protein. In addition to these major proteins, numerous minor proteins are also present in milk these include osteopontin, porteose peptone fraction and lactoferrin about 60 enzymes are also associated with whey proteins (Le et al. 2006). Due to the globular structure, whey proteins are strongly influenced by the temperature, significant denaturation of whey proteins may take place at 60oC. Thermal treatment of milk caused denature of whey protein and k-protein which leads to formation of aggregates with casein micelles and whey protein covered the casein micelles (Dupont et al. 2007). Solubility of heat-treated whey proteins in milk are in the order of porteose peptone > α-lactalbumin > bovine serum albumin > immunoglobulin. For the measurement of degree of denaturation in whey proteins, being highest in concentration (50%) denaturation of β-lactoglobulin is used as an indication of denaturation of whey proteins (Jovanovic et al. 2005). In UHT milk, sedimentation can happen right after thermal treatment or it takes place during the storage time. Sediment is composed of denatured protein, minerals, lactose and lipids with wide variation in composition. Degree of sedimentation depends upon the quality of raw milk, processing temperature and location of homogenizer in the UHT installations. For the characterization of casein and whey proteins, SDS-PAGE was used (Hulmi et

43

al. 2010). During heat treatment and storage, several changes take place in the protein profile of milk, therefore, a comprehensive investigation on protein profiling of UHT

44

EXPERMENT NO. 3treated milk and sedimentation during the storage should be studied. This study aimed to determine the effect of UHT treatment and storage period on protein and amino acid profiles of the sedimentation and UHT milk. 5.2. Materials and Methods5.2.1. Collection of Milk Samples

Samples of raw milk were obtained from a dairy industry maintaining bulk storage facility, the same milk was processed on a UHT plant (indirect heating; 142oC for 2 sec), aseptically packaged and filled in Tetra Brick Aseptic Packs of 250 ml capacity. The samples of UHT treated milk samples were collected and stored at room temperature for 90 days. Chemical and sensory analysis were performed at 0, 30, 60 and 90 days. 5.2.2. Milk Composition

Fat, protein, mineral and pH of UHT milk were measured by lactoscan (Julie Z-7 Salovakia) at 0, 30, 60 and 90 days. 5.2.3. Characterization of Proteins by SDS-PAGE

The whey and casein protein were precipitated at their isoelectric point with the help of 1 M HCl. The precipitate was solubilized at pH 7.0 by using 1M NaOH and dialyzed against deionized water. SDS-PAGE was performed. Sample 1g was dissolved in 1ml Tris-HCL buffer (pH 6.8) in the presence of β-mercaptoethanol (5%) and 0.1% SDS. After mixing the sample was heated 100°C for 3-5 min. Volumes of 20 µl of samples were loaded in the gels. Bio-Red electrophoresis was used for separation at 100 V for 2 hours. After completing the running time of gel separation placed the gel into staining solution with Commassie Brilliant Blue R-250 for 30 min and after staining time keep the gel overnight for de-staining (Laemmli and Favre 1973)5.2.4. Amino Acid Analysis

For the estimation of amino acid profile, freeze dried samples were used according to the method prescribed by Schuster (1988). Under vacuum, samples were hydrolyzed with 6N HCl at 110oC for 24hr, followed by drying on a rotary evaporator the acid (45oC) and dissolved in citrate buffer (pH 2.2) followed by filtration. Sample was injected in amino acid analyzer. 5.2.5. Non-Casein Nitrogen (NCN)

In raw, UHT treated milk and sedimentation samples, NCN was estimated, described method by Fox and McSweeney (1998). Fat was removed by centrifugation at 5000g for 20 min. Separation of casein was done by adjusting the pH of skim milk to 4.6 by 1M HCl. Casein was washed with distilled water for few times and filtrate containing NCN was analyzed by Kjeldahl’s method (AOAC, 2000). 5.2.6. Non-Protein Nitrogen (NPN)

Skim milk 10 ml was treated with 15% trichloro acetic acid was added to isolate the NPN from milk, followed by filtration on Whatman filter paper (No. 42) and determination of NPN by Kjeldahl’s method (AOAC, 2000).5.2.7. Plasmin Activity

For the estimation of plasmin activity in UHT milk, spectrophotometric method was used (Hassan et al. 2009). Plasmin breaks the bond between lysine and nitroanilide with the release of 4-nitroaniline that showed the absorbance of light at 405nm in visible region of spectrum. 5.2.8. Viscosity

Viscometer (Brookfield PRIME viscometer) in 16 ml adapter was used for the determination of viscosity of ultra-high temperature of milk at speed of 30, 60 and 90 RPM at 25oC (Datta and Deeth 2003).5.2.9. Sedimentation

Sedimentation in UHT treated milk samples was determined by following the method of (Chavan et al. 2011). Milk from up righted Tetra Brick packs was extracted with the help of 25 ml pipette without disturbing the layer of mass settled on the bottom, followed by drying at ambient temperature for 48 hrs. Packs were weighed, washed again, dried and weighed, the difference in the weight was used as sediment.5.2.10. Statistical Analysis

45

EXPERMENT NO. 3Two-way analysis of variance technique was used for data analysis; results were expressed as

Mean±SD. For denoting of significant difference, DMR Test was used on a SAS 9.1 software (Steel et al. 1997).5.3. Results and Discussion5.3.1. Chemical Composition

The results of fat, protein, mineral content and pH of milk before UHT, after UHT and subsequent storage of 90 days is mentioned in Table 1. Compositional parameters of milk were not affected by UHT treatment. However, fat and protein contents of 90 days of UHT treated milk sample was significantly less than after UHT and 45 days of storage. The decline in fat and protein contents may be attributed to the protein and lipid oxidation (Nadeem et al. 2017). pH of 90 days old milk samples was also less than other samples of UHT milk. pH of UHT milk usually decreases and OA increased as a function of heat treatment and storage. 5.3.2. Protein Profile of UHT Milk by SDS-PAGE

Casein is a major protein in milk, ratio of casein and whey proteins considerably vary from one species to the other. Casein to whey ratio in equine, human and bovine were 50:50, 40:60, and 80:20 respectively. Milk contained different types of enzymes more than 60 but they represent less 1 % of total protein in milk (Farrell et al. 2004). Table 3 describes the transition taking place in protein fractions of UHT milk in the storage phase. Storage also significantly affected the whey proteins, after 60 days of storage of UHT milk in ambient conditions, the loss of β-lactoglobulin, α-lactalbumin, Immunoglobulin and serum albumin was 36.12%, 46.7%, 38.31% and 64.76%. In this investigation, protein profiling of the sediment settling at the bottom of tetra brick aseptic package was performed to find out the proteins which settle down due to the heat treatment and storage. Protein profiling of UHT milk is not investigated in any of the earlier studies. It was found that sediment was comprised of 14.9% αs1-casein, 3.42% αs2-casein, 9.88% β-casein, 2.27% κ-casein, 4.36% β-lactoglobulin, 1.88% α-lactalbumin, 1.84% immunoglobulin and 0.61% Serum Albumin. Out of 80% casein, 30.5% was found to the part of sediment in 90 days old UHT milk and out of 20% whey proteins 8.7% became the part of sediment, when UHT milk was stored at ambient temperature for 90 days, 38.5% casein and 43.5% whey proteins became the part of the sediment. Existence of higher magnitude of casein and whey proteins in 90 days old UHT milk showed that it had lower nutritional value with compromised functional properties. Higher amount of sedimentation in 90 days old UHT milk may be due to the low quality of raw milk available in the market. Poor quality of raw milk is a serious problem of developing countries, adulteration, lack of cold chain facilities, rough and excessive handling of the milk are the major reasons for the lower quality of raw milk. The results of many studies showed that whey protein structure was unfolded due to heat treatment. Protein content of low-fat UHT milk decreased during the storage of 90 days (Hossain et al. 2011). When milk is heated acidic-basic group of milk constituent become increasingly more protonated and fraction of casein were produced. The strength of separation of protein fraction depend upon the extent of temperature. Heat treatment caused dissociation of milk protein into different fractions (Donato and Guyomarch 2009). Caseins are sensitive to concentration of calcium level in milk. Precipitation of casein directly depend upon the concertation of calcium level in milk (Considine and Flanagan, 2008). 5.3.3. Amino Acid Profile (AAP)

AAP of casein and whey protein has exceptional significance in human food regime. Due to their higher biological value (97-98%), these are regarded as quality proteins with fast metabolism with better ability to become the part of body. Amino acids in milk protein mostly exist in the form of branched chain which are extremely important for the maintenance and growth of tissues, some of the amino acids have antioxidant properties (Ha and Zemel 2000). The share of buffalo, cow, goat, sheep and camel milk in the total milk production of Pakistan is 62.04%, 34.56%, 1.65%, 0.08% and 1.81% (GOP, 2014-15). In South Asia, most of the milk is produced on small farms, dairy industry receives mixed milk of different species. Amino acid profile of raw milk, UHT treated, 30, 60- and 90-days old milk samples result expressed in Table 3. The difference was due to the fact, that milk transported to the dairy industries of Pakistan is a blend of

46

EXPERMENT NO. 3milk of various species. Tryptophane, threonine, proline and serine were not affected by the thermal heat treatment provided in the form of UHT. During the storage of 30 days, non-significant changes were recorded in amino acid profile of UHT milk. Tryptophane was not found in the sedimentation. Lysine and threonine are the limiting amino acids in many sources of protein, they are indispensable for human body and significant for the synthesis of protein (Fouillet et al. 2002). After 3 months of storage of UHT milk at ambient temperature, about 40 and 14% threonine and lysine were transferred from milk to the sedimentation. On the basis of migration of essential amino acids from milk to the sedimentation, it may be assessed that nutritional quality of UHT milk decreased after 3 months of storage. Amino acid profile of whey protein is superior to casein (Stancheva et al. 2011).5.3.4. NCN and NPN Content in UHT Milk and Sedimentation

Chemically, NCN is composed of soluble proteins. In current investigation, NCN and NPN contents of milk were compared with NCN and NPN content of sedimentation in UHT milk stored for 90 days (Table 4). UHT treatment had a non-significant effect on NCN content of milk, storage duration of 90 days considerably affected the NCN content of milk and sedimentation. Denaturation of whey protein during the heat treatment also leads to the formation of NCN (Walstra, 1999). Storage temperature also had a major effect on NCN and NPN content (Gaucher et al. 2008). NPN fraction of milk is composed of nitrogenous compounds soluble in trichloroacetic acid (12% solution). On an average basis, NPN accounts for 3-5% of the total nitrogen present in milk. The outcomes attained within the context of the investigation showed that NPN is variable and influenced by the several factors. NPN does not have any nutritional and economical significance. NCN and NPN contents of UHT milk is reported in literature, however, NCN and NPN contents of sedimentation not been previously reported. 5.3.5. Plasmin Activity

Psychrotrophic bacteria can grow below 7oC, since the induction of bulk raw milk collection and storage, these are the most predominant bacteria in milk (Walstra, 2003). However, extracellular enzymes produced by these bacteria are fairly thermostable, for example proteaseses and lipases. Increased plasmin activity in UHT milk promotes the off-flavor and increases viscosity, which increases with the progression of storage and results in the formation of a gel. Bitter flavor and age-gelation are the products of proteolysis of casein caused by the plasmin which (Walstra et al. 2006). Proteolytic activity is quite evident during the storage of milk (Manji and Kakuda 1988). Basically, these enzymes are produced at refrigerator temperature in raw milk. Age gelation is proteolysis, attributed to the milk enzymes namely milk plasmin, proteases, proteinase lipase were produced in raw milk due to psychrotrophic bacteria (Kelly and Fox 2006). Plasmin activity of milk before UHT, 0, 30, 60 and 90-days of storage was 4.32, 5.76, 7.45, 9.18 and 10.39 (µU/ml). 5.3.6. Sedimentation

Previous investigation on UHT milk have shown that native structure of casein micelle is modified along with the folding of globular proteins due to UHT treatment of milk (Hassan et al. 2009). The deposition of calcium phosphate onto the micelle lead to increase the weight of micelle and tendency to sediment in milk during storage. Proteases cause breakdown of protein that lead to age gelation and sedimentation in UHT milk. Lipase act on the triglyceride and produce rancid flavor (Lewis and Deeth 2008). Barraquio (2014) reported that some micronutrients of UHT milk decreased during processing age storage. In current investigation, the sedimentation in UHT milk was monitored for 90 days. It was recorded that amount of sedimentation increased as the duration of storage increased. Amount of sedimentation at different stages was as i.e. 30, 60 and 90 days of storage was 1.97g/250g, 4.13g/250g and 8.15g/250g, respectively. While, sedimentation was not found in Tetra Packs opened immediately after UHT treatment. Sediment was mainly comprised of protein, fat and lactose, chemical analysis of sediment formed in UHT milk after 90 days of storage revealed that protein, lactose and fat content were 42.3%, 14.7% and 38.5%, respectively. Analysis of UHT milk on 6week showed 0.22g sedimentation in 250ml pack, in the rest of storage period of 6 weeks, sedimentation continuously increased (Hassan et al. 2009). 5.3.7. Viscosity

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EXPERMENT NO. 3In UHT milk the major defect is increased viscosity during the passage of time at storage that

decreased the functional and sensory characteristics of UHT milk. Some major factors that were contributed to increase the viscosity of UHT milk such as proteolytic enzymes activity and storage temperature (Barbano et al. 2006). The formation of gel and increased thickness of UHT milk was caused by activity of psychotropic bacteria produced at storage temperature and survived the UHT temperature (Chavan et al.2011). The intensity of aggregation in proteins (casein-casein, casein-whey) was dependent in denaturation of protein during processing and plasmin activity at ambient temperature during storage. In present study, UHT milk was stored for period of 90 days, Viscosity of milk before UHT, after UHT and after 0, 30, 60 and 90 days of storage was 1.39, 1.42, 1.64, 1.81 and 1.92 centipoise. Kawady (2004) described that viscosity of UHT milk increased at room temperature during storage. Sato and others (2002) studied that heat treatment in milk partially denatures the whey proteins. Ryan and others (2013) suggested for the production of ideal protein drinks, it is necessary that drinks have low viscosity and turbidity. 5.4. Conclusions

Ultra-high temperature treatment had no effect on casein fractions of milk. Storage period up to 30 days had no pronounced effect on protein fractions of UHT treated milk. Sedimentation was not found in UHT treated milk till 30 days of storage in ambient conditions. AAP of milk of freshly UHT treated milk was different from 90 days old with a noticeable increase in viscosity. 5.5. Financial Assistance

By HEC (National Research Program for Universities, Grant No. 18/6978). 5.6. ReferencesAOAC. 2000. Official Methods of Analysis. 17th Rev. Ed. Association of Official Analytical Chemists,

Washington, DC.Aouadhi C, Simonin H, Prévost M, de Lamballerie A, Maaroufi S, Mejri .2012. Optimization of pressure-

induced germination of Bacillus sporothermodurans spores in water and milk. 30:1-7.Barbano D, Y Ma, Santos M .2006. Influence of Raw Milk Quality on Fluid Milk Shelf Life. J dairy sci.

89:15-19.Barraquio V. 2014. Which Milk is fresh? Int J Dairy Sci and Processing 1:201, 1-6 ISSN: 2379-1578. 105–

133Bhatt H. 2014. Prevention of plasmin-induced hydrolysis of caseins: a thesis presented in partial fulfilment

of the requirements of the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand. Massey University

Button PD, Roginski H, Deeth HC, Craven HM .2011. Improved shelf life estimation of UHT milk by prediction of proteolysis. J Food Quality. 34:229-235.

Chavan RS, Khedkar CD, Jana AH. 2011.UHT milk processing and effect of plasmin activity on shelf life: A review. Comprehensive Reviews in Food Sci. and Tech.10:251-268

Chavan SR, Khedkar CD, Jana AH. 2011. UHT milk processing and effect of plasmin activity on shelf life: a review. Compr Rev Food Sci Food Saf. 10(5):251–268.

Considine T, Flanagan J. 2008. Chapter 13 - Interaction between milk proteins and micronutrients.In Milk Proteins, A. Thompson, M. Boland, and H. Singh, eds. (San Diego: Academic Press), pp. 377–407.

Datta N, Deeth HC. 2003. Diagnosing the cause of proteolysis in UHT milk. Lebensm Wiss Tech. 36:173–182.

Donato L, Guyomarch F. 2009. Formation and properties of the whey protein/κ-casein complexes in heated skim milk - a review. Dairy Sci and Techn. 89(1):3-29.

Dupont D, Lugand D, Rolet-Repecaud O, Degelaen J. 2007. ELISA to detect proteolysis of Ultrahigh-Temperature milk upon storage. J Agr and Food Che. 55:6857-6862.

Farrell JrHM, Jimenez-Flores R, Bleck GT, Brown EM, Butler JE, Creamer LK, Hicks CL, HollarCM, Ng-Kwai-Hang KF, Swaisgood HE. 2004. Nomenclature of the Proteins of Cows’ Milk—Sixth

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EXPERMENT NO. 3Fouillet H, Mariotti F, Gaudichon C, Bos C, Tome D. 2002. Peripheral and splanchnic metabolism of dietary

nitrogen are differently affected by the protein source in humans as assessed by compartmental modeling. J Nutr. 132:125-133.

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Hassan A, Amjad I, Mahmood S. 2009. Microbiological and physicochemical analysis of different UHT milks available in market. Afri J Food Sci. 3(4):100-106.

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Kawady EA. 2004. Chemical and bacteriological studies on UHT Milk. Alexandria Science Exchange J. 35(2):107-114.

Kelly AL, Fox PF. 2006. Indigenous enzymes in milk: A synopsis of future research requirements. Int Dairy J. 16:707-715.

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Le TX, Datta N, Deeth HC .2006. A sensitive HPLC method for measuring bacterial proteolysis andproteinase activity in UHT milk. Food Res Int. 39:823-830.

Lewis M, Grandison A, Lin MJ, Tsioulpas A. 2011. Ionic calcium and pH as predictors of stability of milk to UHT processing. Milchwissenschaft. 66(2):197-200.

Manji B, Kakuda Y. 1988. The role of protein denaturation, extent ofproteolysis, and storage temperature on the mechanism of age gelation in amodel system. J Dairy Sci. 71:1455–1463.

Nadeem M, Imran M, Taj I, Ajmal M, Junaid M. 2017. Omega-3 fatty acids, phenolic compounds and antioxidant characteristics of chia oil supplemented margarine. Lip in Health and Dise. 16:102.

Richmond HD .2007. Dairy Chemistry a practical hand book for dairy chemists and other having control of dairies. Cole press USA

Ryan KN, Zhong QX, Foegeding EA. 2013. Use of whey protein soluble aggregates for thermal stability. A hypothesis papers. J Food Sci 78(8): 1105–15

Sato K, Imai H, Horikawa M, Kawanari M. 2002. Processed whey protein and process for manufacturing the same. Patent number US 6,495,194 B2. Assignee: Snow Brand Milk Products C Ltd

Schuster R. 1988. Determination of amino acids in biological, pharmaceutical, plant and food samples by automated precolumn derivatization and high-performance liquid chromatography. JChromatogr. 431:271–284.

Smithers GW .2008. Whey and whey proteins from ‘gutter-to-gold’. Int Dairy J. 18:695-704. Stancheva, Naydenova NN, Staikova G. 2011. Physicochemical composition, properties, and technological

characteristics of sheep milk from the Bulgarian dairy synthetic population. Macedonian J Anim Sci. 1:73-76.

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EXPERMENT NO. 3Steel R, Torrie J, Dickey DA. 1997. Principles and procedure of statistics. In: A biometrical approach. 3rd

ed. New York, USA: McGrraw Hill Book. ComWalstra P, Wouters JTM, Geurts TJ. 2006. Dairy Sci. And Technology. Boca Raton, USA: Taylor &

Francis. Walstra P. 2003. Physical chemistry of foods. Marcel Dekker, New York Walstra, P. 1999. Milk composition. In: Dairy technology: Principles of milk properties and processes.

Dekker, M. (ed.). CRC Press, New York, USA. p.27-107.

Table 5.1: Chemical Composition of UHT MilkStage of Sampling Fat% Protein% Minerals% pH

Before UHT 3.48±0.03a 3.17±0.02a 0.69±0.03a 6.82±0.11b

UHT Milk 3.48±0.04a 3.16±0.01a 0.69±0.01a 6.81±0.02b

30 Days Old 3.46±0.01a 3.16±0.03a 0.68±0.03a 6.67±0.05b

60 Days Old 3.42±0.05a 3.14±0.02a 0.65±0.04a 6.59±0.02a

90 Days Old 3.25±0.09b 3.04±0.05b 0.66±0.08a 6.51±0.10ba

Means presented in Table 1 are the outcome of triplicate treatment and triplicate analysis of every sample of Milk (3x3=9) If the means in a column are expressed by a same letter these are statistically non-significant (p>0.05)

Table 5.2: Protein Profile of UHT MilkProtein Fraction Before

UHTAfter UHT

30 Days Old

60 Days Old

90 Days Old

Sediment*

αs1-Casein% 32.5±0.08a 32.2±0.24a 31.8±0.22a 30.4±0.55b 28.1±0.42c 14.9±0.18d

αs2-Casein% 7.52±0.05a 7.47±0.12a 7.16±0.05a 6.44±0.17b 5.14±0.13c 3.42±0.06d

β-casein% 31.8±0.24a 31.5±0.61a 31.1±0.62a 29.4±0.73b 28.2±0.46c 9.88±0.34d

κ-Casein% 7.2±0.12a 6.94±0.08a 6.71±0.14a 6.44±0.21b 5.19±0.07c 2.27±0.03d

β-Lactoglobulin% 11.6±0.18a 10.2±0.27b 9.15±0.18b 8.26±0.04c 7.41±0.13d 4.36±0.16e

α-Lactalbumin% 3.49±0.06a 2.89±0.03b 2.21±0.02b 1.98±0.07c 1.54±0.09d 1.88±0.13c

Immunoglobulin% 2.92±0.14a 2.61±0.01b 2.25±0.05b 1.94±0.03c 1.61±0.02d 1.84±0.07c

Serum Albumin% 1.17±0.04a 1.05±0.02b 0.88±0.03b 0.55±0.02c 0.37±0.01d 0.61±0.02c

Total Protein Content%

3.17±0.02b 3.16±0.01b 3.16±0.03b 3.14±0.02b 3.04±0.05c 42.3±0.11a

*90 days old

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EXPERMENT NO. 3Means presented in Table 2 are the outcome of triplicate treatment and triplicate analysis of every sample of Milk (3x3=9) If the means in a row are expressed by a different letter these are statistically significant (p<0.05

51

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Table 5.3: Amino Acid Profile of UHT Milk at Different Stages of Processing and Storage (mg of N/g)Amino Acid Before UHT After UHT 30 Days 60 Days 90 Days SedimentationTryptophane 0.04±0.01a 0.04±0.01a 0.04±0.01a 0.03±0.01b 0.03±0.01b Not DetectedThreonine 0.15±0.02a 0.14±0.03a 0.13±0.01a 0.11±0.02b 0.09±0.01b 0.06±0.01c

Isoleucine 0.22±0.03a 0.15±0.02b 0.14±0.02b 0.12±0.03b 0.08±0.01c 0.05±0.02d

Leucine 0.34±0.04a 0.26±0.06b 0.25±0.01b 0.24±0.04b 0.17±0.03c 0.06±0.01d

Lysine 0.29±0.01a 0.21±0.02b 0.19±0.03b 0.14±0.01c 0.09±0.02c 0.04±0.02e

Methionine 0.14±0.02a 0.09±0.01b 0.08±0.02b 0.07±0.01b 0.05±0.02c 0.03±0.01d

Cystine 0.08±0.01a 0.05±0.02b 0.05±0.01b 0.04±0.01b 0.02±0.01c 0.04±0.02b

Phenylalanine 0.14±0.03a 0.11±0.02b 0.10±0.01b 0.09±0.03b 0.06±0.01c 0.05±0.01c

Tyrosine 0.17±0.01a 0.13±0.04b 0.12±0.02b 0.11±0.03b 0.08±0.02d 0.06±0.01d

Valine 0.25±0.06a 0.20±0.06b 0.19±0.02b 0.18±0.03b 0.12±0.01c 0.09±0.02d

Arginine 0.13±0.02a 0.10±0.01b 0.09±0.02b 0.08±0.01b 0.05±0.01c 0.03±0.01d

Histidine 0.08±0.01a 0.05±0.01b 0.05±0.01b 0.04±0.01b 0.02±0.01c 0.03±0.01b

Alanine 0.14±0.03a 0.09±0.02b 0.08±0.02b 0.07±0.01b 0.04±0.01c 0.06±0.01b

Aspartic Acid 0.28±0.05a 0.25±0.04a 0.24±0.03b 0.22±0.04b 0.16±0.03c 0.05±0.02d

Glutamic Acid 0.73±0.07a 0.71±0.07a 0.69±0.05b 0.67±0.06b 0.55±0.08c 0.14±0.03d

Glycine 0.09±0.01a 0.06±0.01b 0.05±0.01b 0.04±0.01b 0.02±0.01c 0.03±0.01b

Proline 0.34±0.03a 0.31±0.04a 0.29±0.04b 0.27±0.03b 0.18±0.02c 0.12±0.02d

Serine 0.21±0.02a 0.19±0.02a 0.18±0.01b 0.16±0.02b 0.11±0.02c 0.08±0.01d

Within one row, if the means carry a similar letter, that are non-significant (p>0.05)

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EXPERIMENT NO. 3

Table 5.4: Non-Casein Nitrogen and Non-Protein Nitrogen Content of UHT Milk and SedimentationStage of Sampling

Milk NCN% Sedimentation NCN%

Milk NPN% Sedimentation NPN%

Before UHT 0.47±0.02g Not Determined 0.17±0.01g Not Determined

After UHT 0.49±0.04g Not Determined* 0.18±.04g Not Determined**

30 Days Old 0.61±0.07f 1.16±0.08c 0.22±0.05f 0.62±0.05c

60 Days Old 0.77±0.03e 3.22±0.19b 0.29±0.09e 1.36±0.13b

90 Days Old 0.84±0.09d 5.59±0.24a 0.35±0.06d 2.98±0.16a

Within two columns of a same parameter, if the means carry a similar letter, that are non-significant (p>0.05)* NCN of sedimentation were not determined because sedimentation was not observed in UHT milk immediately after heat treatment** WPN of sedimentation were not determined because sedimentation was not observed in UHT milk immediately after heat treatment Total casein content in milk and sedimentation after 90 days of storage were 2.44% and 18.64%

Table 5.5: Plasmin Activity, Sedimentation and Viscosity of UHT MilkStorage period Plasmin activity

(µU/ml)Sedimentation

(g/250ml)Viscosity

(centipoise)

Before UHT 4.32±0.01c Not Found 1.44±0.02b

After UHT 5.76±0.02b Not Found 1.48±0.02b

30 days 7.45±0.05b 1.97±0.03c 1.54±0.05b

60 days 9.18±0.09b 4.13±0.02b 1.81±0.08a

90 days 10.39±0.01a 8.15±0.01a 1.92±0.03a

Means presented in Table 5 are the outcome of triplicate treatment and triplicate analysis of every sample of Milk (3x3=9) If the means in a column are expressed by a different letter these are statistically significant (p<0.05)

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CHAPTER 6EXPERIMENT No. 4

Changes in Fatty acids Composition, Antioxidant Potential and Induction Period of UHT-Treated Tea Whitener, Milk and Dairy Drink

Muhammad Ajmal1, Muhammad Nadeem1, Muhammad Imran2, Zarina Mushtaq2, Muhammad Haseeb Ahmad2, Muhammad Tayyab1, Muhammad Kamran Khan2 and Nabila Gulzar1

1Department of Dairy Technology, University of Veterinary and Animal Sciences LahoreCorrespondence: muhammd.nadeem@uvas.edu.pk

ABSTRACTBackground: In developing and developed countries, several versions of safe and shelf-stable UHT-treated products are manufactured. Terminologies and formulations of UHT-treated tea whitener, milk and dairy drink considerably vary. Comprehensive studies have been performed on UHT-treated milk; however, fatty acids compositional changes and oxidation status of UHT-treated tea whitener and dairy drink at different storage intervals have not been reported in literature. Methods: UHT-treated tea whitener, milk and dairy drink samples (450 each) of the same manufacturing date were purchased from the market and stored for 90 days. At the time of collection, all the samples were only week old. Samples of UHT-treated tea whitener, milk and dairy drink were regarded as treatments and every treatment was replicated five times. Chemical composition, FAP, (DPPH) radical scavenging activity, TAC, reducing power, antioxidant activity in linoleic acid system and induction period were determined at 0, 45 and 90 days of storage. Results: Fat content in freshly collected samples of UHT treated-tea whitener, milk and dairy drink were 6 and 3.5%. UHT treated milk had highest total antioxidant capacity, antioxidant activity in linoleic acid and 2, 2-Diphenyl-1-picrylhydrazyle (DPPH) free radical scavenging activity followed by UHT tea whitener and dairy drink. In freshly collected samples of UHT-treated milk, concentrations vitamin A and E were 0.46μg/100g and 0.63mg/100g, respectively. UHT-treated tea whitener had the lowest concentrations of vitamin A and E. With the progression of storage period, amount of vitamin A and E decreased. In freshly collected samples, amount of short, medium and unsaturated fatty acids in UHT-treated milk were 10.54%, 59.71% and 27.44%, respectively. After 45 days of storage of UHT-treated milk, the loss of short, medium and unsaturated fatty acid was 7%, 7.1 and 5.8%, respectively. After 45 days of storage of UHT-treated tea whitener, the loss of medium and unsaturated fatty acid was 1.6% and 0.99%, respectively. After 90 days of storage, the loss of medium and unsaturated fatty acids was 8.2% and 6.6%, respectively. The induction period of fresh UHT-treated tea whitener, milk and dairy drink was 15.67, 7.4 and 7.27 hrs. Strong correlations were recorded between induction period and peroxide value of UHT-treated products. Conclusion: This investigation disclosed that UHT-treated tea whitener had 6% fat content with no short-chain fatty acids. Antioxidant capacity of UHT-treated milk was higher than dairy drink and tea whitener. Due to the presence of partially hydrogenated fat, oxidative stability of UHT-treated tea whitener was better than UHT-treated milk and dairy drink. Vitamin A and E not found in UHT-treated tea whitener. For the anticipation of oxidative stability of UHT-treated milk, dairy drink and tea whitener, induction period/ Rancimat method can be used. Keywords: Tea Whitener, UHT-treated milk, Dairy Drink, Antioxidant Capacity, Fatty Acid Profile, Induction Period, Rancimat

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EXPERIMENT NO. 46.1 BACKGROUND

Thermal processes are applied for the long-term preservation of fluid milk and dairy products. Thermal processing is performed to reduce/ eliminate the bacteria, limit enzyme activity and enhance the shelf life (Walstra et al. 2006). The effectiveness of heat treatment depends upon the heating method employed and time-temperature combination. UHT treatment is commercially applied to manufacture long life milk (Sakkas et al. 2014). UHT treatment is used for the manufacturing of commercially sterile fluid milk, this involves heating the milk to 135-150ºC followed by aseptic packaging (Lewis and Deeth 2008). UHT treated products can be stored for 3-6 months without refrigeration (Bimbo et al. 2016). Heating of milk to UHT temperature consequences in declining the sensory attributes, nutritional value and oxidation of lipids (Nursten, 2005). Developing countries have not well-established cold chain facilities. Depriving food sources and ever-increasing population of developing countries have led to the commercial scale production of tea whiteners and dairy drinks. Tea-whiteners are usually formulated from skim milk powder, partially hydrogenated palm oil (6%), stabilizer and emulsifier followed by high pressure homogenization (<200 Bar), UHT treatment and aseptic packaging. The word UHT dairy drink is locally used to describe a product that is formulated from demineralized whey powder, anhydrous milk fat, stabilizer and emulsifier, upstream homogenization, UHT treatment and aseptic packaging (Lewis, 2011). Milk fat contains about 25-28% unsaturated fatty acids, hence it is liable to oxidation, storage temperature, light, metals and enzymes may lead to oxidative and hydrolytic rancidity (Shahidi and Zhong 2010). Sunds et al. (2016) reported that UHT-treated milk in accelerated storage conditions however, transition in antioxidant properties of milk was not reported. Former studies have shown that, the development of off-flavors is the consequence of an imbalance between the concentration of antioxidants and pro-oxidants (Gutierrez, 2015). When concentration of free radicals exceeds the defense system, state of oxidative stress occurs. Free radicals may cause carcinogenesis, cardiovascular diseases, necrosis and atherosclerosis (Hallwell, 2007). In body and food systems, activities of free radicals may be minimized by the antioxidants. Milk naturally contains two distinct antioxidant defense systems and these may be broadly classified in two categories as fat soluble and water-soluble antioxidant systems (Lindmark-Mansson and Akesson 2000). Fat soluble antioxidant system is comprehended of vitamin A, E, and carotenoids. While, water solvable antioxidant defense mechanism is made up of casein, whey proteins, vitamin C, cysteine, valine, lactase, glutathione peroxidase and superoxide dismutase, zinc and selenium (Richmond, 2007; Zaeroomali et al. 2014). Estimation of shelf life/oxidative stability of UHT-treated milk is a time taking phenomenon and sample of milk has to be stored for six months. Accelerated oxidation is widely used for the estimation of shelf life of fats, oils, cookies, potato chips and several foods, however, accelerated oxidations conditions are not formerly used. Khan et al. (2017) reported that heating method, storage period and spoilage agent of UHT products are different from pasteurized milk as UHT products are commercially sterile and aseptically packaged with no live organism. Detailed investigation on the oxidative variations in UHT-treated tea-whitener, milk and dairy drink is required. 6.2 METHODS6.2.1 Materials and experimental plan

UHT-treated tea whitener, milk and dairy drink samples (450 each) of the same manufacturing date were purchased from the market and stored at ambient temperature (25-30 oC) for 90 days. At the time of collection, all the samples were only week old. Samples of UHT-treated tea whitener, milk and dairy drink were regarded as treatments and every treatment was replicated five times. 6.2.2 Chemical composition of milk

Chemical composition of UHT-treated tea whitener, milk and dairy drinks was determined by wet chemistry. 6.2.3 Antioxidant assays6.2.3.1 (DPPH) radical scavenging activity

DPPH free radicals scavenging activity in UHT-treated tea whitener, milk and dairy drink samples were determined by technique prescribed by Ye et al. (2013). The sample was prepared in test tube by

55

EXPERIMENT NO. 4mixing the 200μL of milk sample in 1mL of 100 μM DPPH solution. Spectrophotometer was used for the measurement of absorption at 517nm.6.2.3.2 Total antioxidant activity

Sample (0.3 g) was measured in test tube and mixed with 3ml each H2SO4 (0.6M), ammonium molybdate (4mM) and sodium phosphate (28mM). Samples were incubated at 85oC for 30min, readings were taken on a spectrophotometer using ascorbic acid as standard (Nabasree and Bratati 2007).6.2.3.3 Reducing power

Sample (1ml) was mixed with 2.5ml potassium ferricyanide (1%) and incubated at 50 oC for 15min. Supernatant (2.5ml) was mixed with 0.5ml ferric chloride (0.1% in distilled water). Absorbance was measured on a double beam spectrophotometer at 700nm (Adesegun et al. 2008).6.2.3.4 Determination of antioxidant activity in linoleic acid system

Antioxidant activity was measured by the described method of Osawa and Namiki (1981)6.2.4 FAP

Fatty acid profile of UHT-treated tea whitener, milk and dairy drinks samples was determined on a GC-MS (7890-B, Agilent Technologies). Extracted fat 0.5mg was taken in a test tube, reacted with methanolic hydrogen chloride and put in the heating block for 1 hr. Extraction was performed by n-hexane, dried on sodium sulfate and 1µL was injected through front auto liquid sampler and injected into GC-MS. Temperature of the inlet and detector were set at 200 and 250oC, with a split ratio of 1:50. Quantification was done by FAME-37 standards (Qian, 2003)6.2.5 Use of accelerated oxidation in UHT-treated products

For the assessment of oxidative stability of UHT-treated products, Professional Rancimat (Model 892) was used. For the extraction of fat from samples, protocol of AOAC was used 1987). Briefly, 2.5g fat was taken in reaction vessels of Rancimat, a steady of dried oxygen was introduced at the flow rate of 20liter/hr using Synlab software. Induction period was calculated from the breakpoint in the curve (Choi et al. 2018).6.2.6 Statistical analysis

This experiment was conducted in a CRD. Data were analyzed by two-way variance technique and results were express as Mean ± SD. For the determination of significant difference DMR Test was performed on a SAS 9.1 software. 6.3 RESULTS AND DISCUSSION6.3.1 Effect of storage on chemical composition of UHT-treated products

Table 1 describes the chemical composition of UHT treated tea-whitener, milk and dairy drink. UHT-treated tea whitener showed significantly higher fat content, no significant difference was recorded in fat content of UHT treated milk and dairy drink. In UHT treated tea-whitener, fat content was toned at 6% level while, in UHT treated milk and dairy drink, fat content was set at 3.5%. Storage effect on fat and protein content of UHT treated tea-whitener, milk and dairy drink revealed a non-significant effect till 45 days of storage. After 90 days, fat and protein content in UHT treated tea-whitener, milk and dairy drink significantly decreased. After 90 days, lowest lactose content was found in UHT treated tea whitener followed by UHT milk and dairy drink. Tea-whitener had 5% sucrose; this could be the reason for lower lactose content. pH of UHT treated milk decreased during the long-term storage 6.3.2 Antioxidant capacity of UHT-treated products

Antioxidant systems of milk can be broadly classified into two categories, fat-soluble antioxidant system and the water-soluble antioxidant system. Lipid oxidation is a serious problem of UHT-treated milk in developing countries. Total antioxidant capacity (TAC) measures the capability of a substrate to counter with free radicals (Sies, 2007). In current investigation, TAC was used as a pointer of antioxidant capacity. It was found that TAC of UHT treated tea-whitener, milk and dairy drinks was influenced by the storage time. However, the decline in TAC was more in UHT treated milk and dairy drink samples. At zero-day, TAC of UHT treated tea-whitener, milk and dairy drink samples were 45.2%, 43.5% and 40.2%, respectively (Table 2). After 45 days of storage, TAC of UHT-treated tea whitener, milk and dairy drink were 41.8%, 35.9% and

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EXPERIMENT NO. 422.4%, respectively. After 90 days of storage, TAC of UHT-treated tea whitener, milk and dairy drink were 37.2%, 27.7% and 15.4%, respectively. Changes in TAC of UHT-treated tea whitener, milk and dairy drink during the storage are not previously studied. Peroxide value of UHT treated milk was intensely correlated with TAC. The determination intervals showing higher TAC showed lower peroxide value (R 2=0.9982). Antioxidant activity of milk is largely due to casein, whey, vitamin E, A, C, selenium, zinc and enzyme systems. During the storage period of 90 days concentration of fat, protein, α-tocopherol and vitamin A decreased that led to lower TAC in all types of UHT treated products investigated. In food system, free radicals cause impulsive oxidation and yield objectionable biochemical compounds which lead to the development of rancidity. At zero day, reducing power of UHT treated tea- whitener, milk and dairy drink were 6.82, 6.22 and 5.29%, respectively. However, it was strongly influenced by the temperature and storage length. After 45 days, reducing power of UHT-treated tea whitener, milk and dairy drink was 5.77, 4.59 and 1.17, respectively and it decreased to 0.24 when UHT-treated products were stored for 90 days. ALA acid of UHT-treated samples is shown in Table 2. At zero-day, ALA of UHT treated tea-whitener, milk and dairy drink was 10.6%, 9.88 and 8.43%, respectively and decreased with the rise of storage duration. After 45 days of storage, the loss of ALA in UHT treated-tea whitener, milk and dairy drink were 20.3%, 39.4% and 50.2%, respectively. After 90 days of storage, the loss of ALA in UHT-treated tea whitener, milk and dairy drink were 51.1%, 65.8% and 88.9%, respectively. DPPH free radical scavenging assay was used in several milk and dairy fat related studies (Nadeem et al., 2015; Nadeem and Ullah 2016). At zero days, DPPH free radical scavenging activity of UHT treated tea-whitener, milk and dairy drink was 25.6%, 23.6% and 21.8%, respectively. 6.3.3 Vitamin A and tocopherol content in UHT-treated products

In storage phase, several chemical changes take place. Rancidity or auto-oxidation is regarded as one of most significant cause of spoilage of UHT treated milk. Impact of storage length on vitamin A and vitamin E is not previously studied. In current investigation, the vitamin A and E were determined because of their antioxidant activities (Usta and Yilmaz-Ersan 2013; O’Connor and O’Brien 2006). At zero-day, the contents of vitamin A and E in UHT-treated milk were 0.46 μg/100g and 0.63mg/100g, respectively (Table 3). UHT treated tea-whitener had the lowest concentration of vitamin A and E. During partial hydrogenation, post hydrogenation refining and deodorization stages, palm oil is exposed to high temperature (>200 oC) for many hours and this sever heat treatment almost eliminates the naturally occurring vitamins and antioxidant activity (Onal and Ergin 2002). For the manufacturing of anhydrous milk fat, a heat treatment is applied which decreases the concentration of fat-soluble vitamins. Concentrations of vitamin A and E were significantly affected by the length of storage period. After 45 days of storage, UHT treated tea-whitener, milk and dairy drink, loss of vitamin A was 23.9%, 60.8% and 100%, respectively. After 90 days of storage of UHT-treated tea whitener, milk and dairy drink, the loss of vitamin A was 47.8%, 100% and 100%, respectively. After 45 days of storage, the loss of vitamin E in UHT-treated tea whitener, milk and dairy drink was 19.1%, 82.5% and 100%, accordingly. After 90 days of storage of UHT-treated tea whitener, milk and dairy drink, the loss of vitamin E was observed 69.8%, 100% and 100%, respectively. An investigation on UHT-treated milk showed that, after 14 days of storage, loss of vitamin A and E was 33% and 11% (Michlova et al. 2015). 6.3.4 Effect of storage duration on fatty acids profile of UHT-treated products

Milk fat contains two different types of FA and these can be broadly categorized into saturated and unsaturated fatty acids. From oxidative stability view point, unsaturated fatty acids are important (Datta and Deeth 2007). Lipid oxidation is a recognized as one of the most noticeable reason for the spoilage of UHT-treated milk (Datta and Deeth 2001). Changes in fatty acids profile of UHT-treated milk are mentioned in Table 4. At zero-day, amount of short, medium and unsaturated fatty acids in UHT-treated milk were 10.54%, 59.71% and 27.44%, respectively. After 90 days of storage, amount of medium and unsaturated fatty acids in UHT-treated tea whitener, milk and dairy drink were 54.8% and 25.62%, respectively. After 45 days of storage of UHT-treated tea whitener, the loss of medium and unsaturated fatty acid was 1.6% and 0.99%, respectively. After 90 days of storage of UHT-treated tea whitener, the loss of medium and unsaturated fatty acid was 8.2% and 6.6%, respectively. After 45 days of storage of UHT-treated milk, the

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EXPERIMENT NO. 4loss of short, medium and unsaturated fatty acid was 7%, 7.1 and 5.8%, respectively. After 90 days of storage of UHT-treated milk, the loss of short, medium and unsaturated fatty acid was 8.53%, 13.51% and 11.88%, respectively. Samples of UHT treated dairy drink underwent sever oxidation after 45 and 90 days of storage and the loss of C18:1 was 14.5% and 32.8%, respectively. Rate of lipid oxidation is mainly influenced by the fatty acids profile and therefore, partial hydrogenation of liquid oils is performed to increase their oxidative stability (Shahidi, 2005). The lower oxidation in UHT treated tea-whitener may be connected to the presence of partially hydrogenated palm oil (Timmons et al., 2001). Fatty acids profile of milk fat significantly changed after 180 days of storage (Nadeem et al. 2015; Pestana et al. 2015) In current investigation, the loss of unsaturated fatty acids was strongly connected with the peroxide value and assessment intervals showing higher losses of fatty acids had higher peroxide value. Lipid oxidation leads to the development of oxidized flavor that limit the shelf life and sensory characteristics of milk (Juhlin et al. 2010; O´Brien, 2009)6.3.5 Induction period and peroxide value of UHT-treated products

Induction period is used for the measurement of oxidative stability of oils and fats and this technique is recommended by the AOCA. It is used for the determination of oxidative stability of wide range of food products and depending upon the oil/content, the sample preparation is accordingly performed. However, this method was not previously used for the determination of oxidative stability of thermally treated milk. In current investigation, induction period of UHT treated milk was determined on a Rancimat at 120 oC with 20-liter O2/hr. For the determination of induction period of UHT treated milk, the fat was extracted from the milk and 2.5g sample was used for measurement of induction period on a Rancimat using lab StabNet 1.1 software. At zero-day, induction period of UHT treated tea-whitener, milk and dairy drink were 15.67, 9.74 and 7.27 hrs (Table 5). Induction period of UHT-treated milk was affected by the storage period. Strong correlations were observed between induction period and peroxide value. UHT treated product having higher induction period had lower peroxide value and vice versa. Use of induction period for the measurement of oxidative stability of milk is studied in a limited way. Peroxide value of UHT-treated samples increased with the advancement of storage duration and UHT-treated milk and dairy drink revealed higher peroxide value than UHT-treated tea whitener.6.4 CONCLUSION

Fat content of UHT-treated tea whitener were higher than UHT-treated milk and dairy drink. Fatty acid profile of UHT-treated tea whitener, milk and dairy drink were different from each other. UHT-treated tea whitener had no short-chain fatty acids. From the comparison of peroxide value of UHT-treated milk and dairy drink, it can be assumed that older fat was used in the formulation of dairy drink. Results of this detailed investigation on immitant UHT-treated dairy products indicated that these had lower vitamin A and tocopherol content and oxidized fat.6.5 REFERENCES Adesegun SA, Elechi NA, Coker HAB. 2008. Antioxidant activities of methanolic extract of Sapium

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Table 6.1 Effect of Storage Duration on Chemical Composition of UHT Treated Tea Whitener, Milk and Dairy Drink

UHT Dairy Product Storage Days Fat% Protein% Lactose% pH

Tea Whitener0 6.00±0.03a 3.28±0.07a 4.68±0.13a 6.65±0.08a

45 5.94±0.05a 3.27±0.04a 4.65±0.10a 6.64±0.02a

90 5.90±0.02a 3.15±0.09b 4.41±0.12c 6.52±0.01a

UHT Milk 0 3.50±0.03a 3.28±0.07a 4.68±0.13a 6.65±0.08a

45 3.42±0.01a 3.25±0.03a 4.64±0.06a 6.61±0.07a

90 3.35±0.04b 3.18±0.01b 4.55±0.14b 6.54±0.03b

Dairy Drink0 3.50±0.03a 3.28±0.07a 4.68±0.13a 6.65±0.08a

45 3.36±0.06b 3.24±0.05a 4.58±±0.09b 6.55±0.03b

90 3.24±0.08c 3.09±0.02b 4.57±0.04b 6.57±0.0b

If means are expressed by a non-uniform letter in a column, these are statistically significant (p<0.05)

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EXPERIMENT NO. 4Table 6.2 Antioxidant capacity of UHT Treated Products at different storage intervals

Storage Temperature Storage Days TAC% ALA% RP DPPH%

Tea Whitener0 45.2±0.41a 10.6±0.09a 6.82±0.16a 25.6±0.24a

45 41.8±0.29b 8.44±0.13b 5.77±0.14b 21.2±0.19b

90 37.2±0.55c 5.19±0.15d 3.69±0.18d 18.7±0.15c

UHT Milk 0 43.5±0.41a 9.88±0.09a 6.11±0.16a 23.3±0.24a

45 35.9±0.88d 6.42±0.11c 4.59±0.27c 16.1±0.13d

90 27.7±0.91e 3.62±0.21d 1.91±0.07e 11.1±0.49e

Dairy Drink0 40.2±0.41a 8.43±0.09a 5.29±0.16a 21.8±0.24a

45 22.4±0.39f 5.27±0.15e 1.17±0.08f 7.57±0.36f

90 15.4±0.69g 1.17±0.09f 0.24±0.03g 2.14±0.64g

If means are expressed by a non-uniform letter in a column, these are statistically significant (p<0.05)TAC: Total antioxidant capacity; ALA: Activity in linoleic acid; RP: Reducing power

Table 6.3 Vitamin A and E Contents of UHT Treated Tea Whitener, Milk and Dairy Drink at Different Stages of Storage

Storage Temperature Storage Days Vitamin A μg/100 g α-Tocopherol (mg/100)

Tea Whitener0 0.08±0.02a 0.13±0.06a

45 0.05±0.04b 0.15±0.09b

90 0.02±0.01c 0.01±0.03c

UHT Milk 0 0.46±0.02a 0.63±0.06a

45 0.23±0.06 d 0.29±0.02d

90 0.08±0.01e 0.15±0.01e

Dairy Drink0 0.11±0.02a 0.13±0.06a

45 Not Detected Not Detected90 Not Detected Not Detected

If means are expressed by a non-uniform letter in a column, these are statistically significant (p<0.05)

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63

Fatty Acid

Tea Whitener UHT Milk Dairy Drink0-Day 45-Days 90-Days 0-Day 45-Days 90-Days 0-Day 45-Days 90-Days

C4:0 ND ND ND 3.41±0.28a 3.32±0.05b 3.17±0.05c 2.65±0.12e 2.31±0.15d 2.05±0.12e

C6:0 ND ND ND 2.45±0.29a 2.19±0.11c 1.94±0.07d 1.14±0.03e 1.10±0.09e 1.03±0.03e

C8:0 ND ND ND 1.44±0.20a 1.22±0.05c 1.89±0.06d 0.62±0.04e 0.50±0.02e 0.42±0.04e

C10:0 ND ND ND 3.24±0.18a 3.07±0.03c 2.64±0.16d 1.32±0.13e 1.25±0.07e 1.12±0.13e

C12:0 0.29±0.11a 0.26±0.13a 0.19±0.16b 3.66±0.11a 3.35±0.19c 2.88±0.14d 1.59±0.02e 1.40±0.08e 1.29±0.02e

C14:0 1.01±0.55a 0.99±0.42a 0.76±0.26b 11.97±0.55a 10.42±0.36c 9.33±0.22d 7.64±0.16e 7.44±0.31e 6.74±0.16e

C16:0 42.5±1.17a 40.2±0.38a 37.87±0.33b 29.97±1.17a 28.33±0.41c 27.14±0.43d 25.73±0.49e 25.51±0.63e 24.73±0.49e

C18:0 3.8±0.99a 3.01±0.27a 2.89±0.21b 14.11±0.99a 13.39±0.53c 12.29±0.18d 10.24±0.37e 10.18±0.40e 9.24±0.37e

C18:1cC18:1t

35.8±1.24a

18.5±1.14a35±0.73a

18.4±1.13a31±0.47b

18.1±1.10a25.61±1.24a

ND*24.02±0.31c

ND23.37±0.45d

ND18.43±0.51e

ND18.27±0.22e

ND17.43±0.51e

NDC18:2 9.52±0.12a 8.89±0.03a 7.34±0.02b 1.37±0.12a 1.18±0.02c 0.76±0.04d ND ND NDC18:3 0.29±0.03a 0.21±0.01a 0.1±0.04b 0.46±0.03a 0.25±0.01c 0.05±0.01d ND ND ND

Table 6.4 Fatty Acids Profile of UHT Treated ProductsIn a row, if means are presented with a non-uniform letter, it indicates significant difference (p<0.05)ND: Not Detected

EX

PER

ME

NT

NO

. 4

EXPERMIMENT NO. 4Table 6.5 Induction period of UHT Treated Tea Whitener, Milk and Dairy Drink at Different Storage Intervals

Storage Temperature Storage Days Induction Period (Hrs) PV (MeqO2/kg)

Tea Whitener0 15.67±0.32a 0.22±0.02g

45 14.35±0.21b 0.48±0.04f

90 12.55±0.13d 1.71±0.08d

UHT Milk 0 9.74±0.11a 0.25±0.02g

45 8.61±0.05c 1.19±0.12e

90 6.55±0.09e 2.48±0.19c

Dairy Drink0 7.47±0.13a 0.45±0.02g

45 5.19±0.16f 3.27±0.16b

90 1.84±0.03g 5.44±0.13a

If means are expressed by a dissimilar letter, it shows non-significant effect (p>0.05)

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CHAPTER 7EXPERIMENT No. 5

Transition in Protein Profile, Maillard Reaction Products and Lipid Oxidation of Flavored Ultra High Temperature Treated Milk

Muhammad Ajmal, Muhammad Nadeem1, Muhammad Tayyab and Nabila Gulzar1Department of Dairy Technology, University of Veterinary and Animal Sciences Lahore

Correspondence: muhammd.nadeem@uvas.edu.pkAbstractChemical characteristics of mango flavored UHT milk were studied for 90 days and compared with values of normal UHT milk reported in literature. After 90 days, the decline in αs1-casein, αs2-casein, β-casein, κ-casein, β-lactoglobulin, α-lactalbumin, immunoglobulin and bovine serum albumin were 11.2%, 34.8%, 14.3%, 33.9%, 56.9%, 24.8%, 36.5% and 43.1%. Furosine content of flavored UHT milk at 0, 45 and 90 days were 278, 392 and 561 mg/100g protein. After 90 days of storage, magnitude of changes in protein fractions and Maillard reaction products in flavored UHT milk was higher than normal UHT milk. After 90 days of storage of flavored UHT milk, the loss of unsaturated fatty acids was 45.7%, from the initial values. PV of flavored UHT milk at 0, 45- and 90-days interval was 0.22, 0.65 and 2.88 (MeqO 2/kg). Appearance, flavor and mouth feel scores were not affected by the storage up to 45 days.Keywords: Flavored UHT Milk, Protein Profile, Maillard Reaction Products, Lipid OxidationPractical ApplicationsAll over the world, UHT treatment is performed to manufacture commercially sterile milk that can be stored in ambient conditions for the longer periods of time. UHT treatment and follow up storage induce several undesirable changes in milk. The magnitude of the changes taking place in protein profile, Maillard reaction products and lipid oxidation in normal UHT milk is present in literature, however literature is silent about flavored milk. Estimation of chemical changes in flavored UHT milk may be helpful for the industries to optimize the processing and storage conditions of favored UHT milk, short shelf version of flavored milk may be launched in the market. 7.1. Introduction

In advanced fluid milk processing, safety and shelf life of the products are assured by thermal treatment such as pasteurization and UHT Treatment (Pischetsrieder and Henle 2012). The latter is performed to destroy all forms of vegetative bacteria and to produce commercially sterile products for extended shelf life at ambient temperature (Toda et al. 2014). Flavored milk is produced from milk with the addition of sugar, stabilizer and flavors; it is popular all over the world due to its pleasant taste. Calcium, phosphorous, magnesium is potassium are required for the strengthening of bones (Murphy et al. 2008). Flavored UHT has another advantage of longer storage life as compared to pasteurized versions. Heat treatment induces several chemical changes in carbohydrates, proteins, vitamins and lipids. Heat treatment leads to structural changes, reorganization of disulfide bonds, glycation, denaturation and aggregation (De-Wet, 2009). Casein is present in four different fractions; αs1, αs2, β, and κ-casein (Huppertz et al. 2008). UHT treatment causes the creation of β-lg and κ-CN which consequences in the waning of linkages between κ-CN and αs-1-CN. Undesirable chemical changes in proteins lead to the separation of κ-casein along with linked β-lactoglobulin (Raynes et al. 2017). Due to the non-globular structure, casein is least affected by heat, while beta lactoglobulin is seriously affected by the heat treatment (Pinto et al. 2012). For pasteurized and UHT treated milk, minimum content of 2600mg/liter and 50mg/liter has been set by the International Dairy Federation. Thermal processing leads to lower the nutritional quality i.e. loss of vitamins, denaturation of globular proteins, precipitation of calcium phosphate and Maillard reaction products such as lactulose, hydroxyl methyl furfural and furosine (Claeys et al. 2013). UHT milk has slightly cooked flavor which is due to the production of excessive amounts of chemical compounds containing Sulphur (Vazques-Landaveerde et al. 2006). In the storage, cooked flavor is dominated by off/ stale flavor due to the generation of methyl ketones Vazques-Landaveerde et al. 2006). In milk, carbonyl group reacts with lysine residues of whey and casein proteins, the latter has more active lysine residues as compared to the former (Martins et al.

65

2001). Lactose also react with other amino acids such as, arginine, methionine, tryptophan and histidine. Alpha amino group of lysine residues react with lactose which

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EXPERMIMENT NO. 5lead to the formation of lactosyl lysine which is subsequently converted to α- N-deoxylactulosyl-l-lysine via Amadori rearrangements. Lactulosyl-lysine cannot be absorbed in the body due to it biological unavailability (Krause et al. 2003). Maillard reaction decreases the functional properties of protein by influencing the thermal stability, foaming, gelation, emulsification and textural properties of protein decreases (Jansson et al. 2014). Maillard reaction is influenced by time, pH and water activity (Mayer et al. 2010). Rate of Maillard reaction is mainly governed by the magnitude of carbohydrates and proteins in the processing and storage of milk. In the storage period, carbohydrate type significantly affected the formation of furosine and 2-methylbutanal (Basto et al. 2012). For the characterization of early and terminal stages of Maillard reaction, Furosine and CML are used, respectively (Rhfian-Henares et al. 2017). UHT milk may undergo hydrolytic and oxidative rancidity because of the production of FFAs, peroxides, hydro peroxides, aldehydes, ketones, alcohols (Elias et al. 2008). Application of heat treatment to milk showed a considerable influence in lipid fraction of milk (Ajmal et al. 2018b). This study aimed to compare the transition protein profile, Maillard reaction products and lipid oxidation in flavored UHT using advanced and conventional analytical techniques and compare them with values of normal UHT milk reported in literature. 7.2. Materials and Methods7.2.1. Collection of Milk Samples and Experimental Plan

Samples of mango flavored UHT milk 200 ml pack (475 Nos.) were purchased from the market and stored at ambient temperature for the duration of 90 days. Samples were analyzed at the frequency of 0, 45 and 90 days. 7.2.2. Milk Composition

Fat, protein, pH and total solids were determined by the standard methods given in (AOAC, 2000)7.2.3. Proteolysis

For the measurement of proteolysis, method suggested by Hull et al. (1947) was used, the degree of proteolysis in flavored UHT milk was expressed as µg tyrosine/ml.7.2.4. Characterization of Proteins

Protein fractions were determined using SDS- PAGE as described by (Laemmli, 1970). Milk samples were defatted by centrifugation (5000 rpm) for 15min. separated the whey protein from casein protein by adding the 1N of HCl until pH obtained 4.6. Supernatant was collected by centrifugation and then precipitated casein was washed with 4.6 pH distilled water and whey protein was precipitated at pH 5.2 adjust the pH with 1N NaOH. Casein and whey samples were freeze dried and stored at -30 0C. The protein sample (casein or whey) with sample buffer (0.6M Tris HCl, pH 6.7, 0.002% Bromophenol blue, 20% 2-β-mercaptoethano) was heated on boiling water for 10min. For the electrophoresis, resolving gel 15% and 4% stacking gel were prepared. Wells on the gel were loaded with 8μL Sample and 4μL standard. Power (90 volt) was applied to mini-protean to start the electrophoresis (2 hours) and gels were stained overnight. After staining, the gels were de-stained using an acetic acid solution 10%. 7.2.5. Hydroxy Methyl Furfural (HMF)

10 g sample was placed in volumetric flask and used distilled water for dilution of sample. After dilution 1 ml 30% and 1 ml 15% solution of potassium hexacyanoferrate were used. Then 1 ml of Carrez II (zinc acetate dehydrates) solution was added in flask to make clarity in the sample and made 100 ml with water. Filtrate mixed with the help of Whatman filter paper and 50μl filtered was injected to the HPLC for HMF analysis and a variable wavelength ultraviolet (UV) Detector (Varian 9050, Creek, California, USA), column (Agilent Bondesil, RP-C18, (4.6 mm, 5μm, 25cm). For measurement of HMF compared the peak areas of standard solution with samples (Sigma–Aldrich, USA). Methanol: water (10:90) where was (mobile phase) with flow rate 1 mL per min and measured the samples at wave length 285 nm 7.2.6. Estimation of Furosine (FUR)

The sample was hydrolyzed at 110 °C for 23 hrs. The hydrolyzed the sample with the help of 6N HCl about 5min at the ratio of 1ml HCl and 2.5μg of protein samples. Hydrolyzed sample was diluted with distilled water and centrifuge. After the drying 400 μL aliquots and its residues mixed with distilled water. Furosine content was quantified by HPLC with detection wavelength of 280 nm. The concentration of

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EXPERMIMENT NO. 5mobile phase sodium heptanesul-phonate with 20% acetonitrile and 0.2 % formic acid maintained the flow rate 1.2 ml per min. 7.2.7. Estimation of Nε-carboxymethyl lysine (CML)

Milk sample (10ml) was diluted with 100µL of NaBH4 (500mM NaBH4) in a borate buffer (0.2M). The sample was hydrolyzed for 24hr at 110°C in the presence of internal standard (100 µL of 1ppm solution). After filtration and drying, hydrolyzed sample dilute with water and analyzed using HPLC.7.2.8. Fatty Acid Profile

FAP was measured on a GC-MS (Agilent 7890-B). Extracted fat 50mg was taken in a test tube, reacted with methanolic hydrogen chloride and put in the heating block for 1 hr. Extraction was performed by n-hexane, dried over sodium sulfate and 1µl was injected through front auto liquid sampler. Temperature of the inlet and detector were set at 200 and 250oC, with a split ratio of 1:50 Identification and Quantification was performed by FAME-37 standards (Qian, 2003) 7.2.9. Oxidative Stability

Anisidine, PV and free fatty acids were measured by (AOCS, 1995)7.2.10. Sensory Evaluation

For sensory evaluation of UHT flavored milk, samples were tempered to 20oC for 2 hrs. Prior to sensory evaluation, briefing was conducted and terminology for sensory evaluation was standardized. Samples of milk were served in transparent glass cups in a completely randomized fashion for the estimation of appearance, flavor and color using 9-point scale. Every determination was performed at least three times (Larmond, 1987)7.2.11. Statistical Analysis

Data were analyzed according to ANOVA technique using PROG GLM by 9.2 software, means were compared using LSD test (Steel et al. 1997)7.3. Results and Discussion7.3.1. Composition

Fat and protein content of UHT flavored milk remains unchanged up to the duration of 45 days (Table 1). Analysis of fat and protein contents after 90 days of storage indicated that fat and protein contents significantly decreased during 90 days of storage. This significant decline in the fat and protein contents was due to the breakdown in storage. Hamad et al. (2017) described that fat content of UHT flavored milk during storage, decreased 3.49% to 3.35%. Storage interval and duration significantly affected the fat content of UHT flavored milk (Ammara et al. 2009). Kawady (2004) concluded that storage period significantly influenced the pH of milk samples. These results agree with (Aldubhany and Gouda 2014)7.3.2. Proteolysis

In this investigation, transition in tyrosine content was used as a marker of proteolysis in flavored UHT milk. Value of tyrosine found in this investigation are higher than those reported by (Al-saadi and Deeth 2008). Higher magnitude of proteolysis can be connected with existence of sugar in this study (sucrose 7%). Psychrotrophic bacteria are produced in milk during storage of milk such as protease, plasmin and negative impact on shelf life of dairy base products (Larsen et al. 2004). Proteases attack on protein, the resultant peptides cause bitterness in UHT milk. Proteolysis in UHT milk is caused by bacterial proteinases which are not activated by traditional UHT treatment (Rauh et al. 2014). 7.3.3. Protein Profile of UHT Flavored Milk

In modern dairy processing, thermal techniques are quite commonly used to manufacture long life milk, however, these techniques alter the physical, chemical and nutritional characteristics of treated products (Mohyuddin et al. 2016). In UHT treatment and follow-up ambient storage, many chemical changes take place that has a pronounced effect on shelf life and stability of end product. The chemical changes which take place in protein of UHT milk are denaturation of whey proteins, interaction between lactose and proteins, formation of sulphydryl compounds etc. (Datta and Deeth 2003). During UHT treatment, complex formation between β-lactglobulins / β-κ-casein takes place. Chemical changes taking place in UHT milk decreases the nutritional value. Nutritional value of recombined UHT milk decreased during long term storage under ambient conditions (Dalsgaard et al. 2007). Total protein content of flavored UHT milk at 0,

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EXPERMIMENT NO. 545 and 90 days of storage was 3.15%, 3.12% and 2.81%. Datta et al. (2002) result showed that UHT treatment significantly denatured the whey proteins especially β-lactoglobulin, α-lactalbumin. Czerwenka et al. (2005) studied that degradation of protein was more in the presence of milk sugar in thermal processing. Losito et al. (2010) result showed that the lysine residues reacted with lactose and unfolded the protein normal structure in the presence of UHT treatment. Rauh et al. (2015) studied that 92.4 % β-lactoglobulin and 71% α-lactalbumin was denatured during the UHT heat treatment milk. Elliott et al. (2005); Elliott et al. (2003) Described that amount of αs1-CN after three months of storage duration was 29.4%. According to the published research work, after three months, the value of αs1-cCN, αs2-CN, β-CN, κ-CN, β-lg, α-lb and BSA in three months old samples of UHT milk were 29.4%, 4.9%, 34.6%, 3.7%, 191mg/liter, 1011mg/liter, 34.2% and 57mg/liter. In case of flavored UHT milk, more decline in different fractions of protein were recorded, from their initial values, this may be connected to presence of higher amount of sugar in flavored (sugar content of flavored milk used in this investigation were 7%). 7.3.4. Maillard Reaction7.3.4.1. Hydroxy Methyl Furfural (HMF)

Maillard reaction is a multifaceted sequence of reaction between carbonyl group of milk sugar and amino group of lysine. Through various pathways, brown pigments are produced which are the major reasons for the brown color of UHT milk (Al-Saadi et al. 2013). Gokmen et al. (2006) reported that amount of HMF is related with severity of heat processing. In addition to temperature, rate of HMF production largely depends upon the type of sugar (Morales et al. 2000). Buhring et al. (2011) described a strong correlation between the development the development of browning and HMF content of foods. About 10% of the amadori product is converted into HMF and 30-40% is converted to furosine. Amount of HMF is directly connected to the intensity of heat applied to the food, it is used as a marker of thermal damage, it can also help to determine the thermal processes applied for the processing of foods (Rufian-Henares et al. 2002). Table 3 designates the outcomes of HMF content of UHT milk in long term storage. In this study, HMF content of UHT milk at 0, 45 and 90 days of storage were 1.56, 4.18 and 7.61 (µmol/L). HMF content of UHT flavored milk recorded in the current investigation are significantly higher than findings of (Saeed and Hashmi 2000). Higher HMF content of UHT flavored milk may be due to the presence of 7% sucrose in the formulation of flavored milk. Total carbohydrate content in the formulation of flavored milk was 11.67%. Maillard reaction is responsible for the production of HMF and its concentration mainly depends upon the intensity of heat. Concentration of HMF in UHT milk depends upon the amount of protein and sugar (Toda et al. 2014). Long term storage of UHT treated dairy products led to increase the amount of HMF. Concentration of lactose in UHT milk had a significant amount of HMF, milk samples having higher lactose led to the production of more HMF and vice versa (Troise et al. 2014). 7.3.4.2. Furosine

For the characterization of early stages of Maillard reaction, furosine is used as a chemical indicator. Lactulosyl-lysine, fructosyl-lysine, and tagatosyl-lysine are the amadori compounds produced due to the chemical reaction of lactose with lactose, glucose and galactose, acid hydrolysis of these compounds lead to the furosine (Mayer et al. 2010). UHT milk produced by direct UHT method had lower furosine than milk processed by the indirect method. After 14 weeks of storage, magnitude of furosine content increased from 29mg/100 g protein to 132 mg/100 g protein in directly UHT processing. After 14 weeks of storage, amount of furosine increased from 124mg/100 g protein to 183mg/ 100 g protein (Meltretter et al. 2014). Production of furosine largely depends upon the concentration of protein present in food matrix. Max limit of furosine in UHT milk is 250mg/ 100g protein (Claeys et al. 2013). HPLC characterization showed that furosine content of UHT milk were 130mg/ 100 g proteins were higher than raw milk samples. Furosine content of some lactose hydrolyzed dairy products were determined using reverse phase HPLC; values of furosine ranged from 235-82mg/ 100g protein, an increase of 74-90% was recorded after the storage of 4 months (Troise et al. 2014). Type of heat treatment and storage duration had a pronounced effect on furosine content of food . Table 3 describes the results of furosine content of flavored UHT milk stored for 90 days. Furosine content of flavored UHT milk at 0, 45 and 90 days of storage intervals were 278, 392 and 561 mg/100g protein. Furosine content of UHT milk and cream increased during the storage (Erbersdobler et al. 2002). The furosine content increased in infant baby powder during storage of 120 days from 720 to 1360 mg/100g

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EXPERMIMENT NO. 5protein. Furosine content of UHT milk increased with the advancement of storage period while, whey protein content decreased (Rauh et al. 2015)7.3.4.3. Nε-carboxymethyl-l-lysine (CML)

CML is low reacting, stable compounds which is produced during advanced Maillard product. CML is produced due to the degradation of Amadori compounds (Nguyen et al. 2014). In severely heat-treated food and dairy products, furosine and CML can be used to get detailed information regarding protein damage. CML is usually used as a criterion to adjudge the health risks of thermally processed foods (Meltretter et al. 2014). In this investigation, estimation of CML was performed to quantify the advanced stages of Maillard reaction. At 0, 45 and 90 days of storage, concentration of CML in flavored UHT milk was 67, 135 and 343 mg/kg protein. CML content of UHT milk at 0 and 120 days of storage were 40 and 40, 420 mg/kg protein (Fenaille et al. 2006). CML content in UHT sterilized infant formula increased during storage (Birlouez-Aragon et al. 2004).7.3.5. Fatty Acid Profile (FAP)

Heat treatment appreciably influenced the FAP (Nadeem et al. 2014). Fatty acid profile of cow and buffalo milk was influenced in the thermal processing and follows up storage (Nadeem et al. 2015). Ajmal et al. (2018a) results showed that alterations taking place in in FAP of UHT milk storage on aseptically packaged samples of milk, heat treatment and storage induced major changes in SCFA, MCFA and USFA, significant changes were also recorded in triglyceride profile. For the estimation of lipid oxidation in milk fat, FAP was considered a useful parameter (Nadeem et al. 2015). Transition in the fatty acid profile of milk fat was used as a marker of lipid oxidation. Literature regarding the alterations in FAP of UHT flavored milk is limited. At 0, 45 and 90 days, amount of SCFA were 9.57%, 9.46% and 8.29%. After 90 days of storage, the decline in short-chain fatty acids was 13.3% from the initial values. At 0, 45 and 90 days, concentrations of medium-chain fatty acids were 47.35%, 46.98% and 43.86%. After 90 days of storage, the decrease in the magnitude of medium-chain fatty acids was 7.37% from the starting values. Transition in the fatty acid profile indicated sever lipid oxidation of unsaturated fatty acids. Testing frequencies showing higher magnitude of transition in fatty acid profile also indicated higher peroxide value. Peroxide value of UHT flavored milk at the end of storage was 2.88 (MeqO2/kg). Ammara et al. (2009) studied that due oxidation of UHT milk concentration of peroxide also increased during storage at ambient temperature. 7.3.6. Oxidative Stability

Oxidative stability of foods is extremely important from viewpoints of consumer acceptability and healthfulness. Auto-oxidation of fats results in the production of several oxidation products that may lead to cardiovascular diseases and cancers (Ammara et al. 2009). Oxidation products such as hydro peroxides, peroxides, aldehydes, ketones and alcohols seriously affect the sensory characteristic of foods (Nadeem and Ullah, 2016) As compared to vegetable origin fats and oils, milk fat is highly complex as it is composed of more than one hundred fatty acids. Oxidation is a major problem of milk fat; this makes the food processor and researchers to continuously monitor the oxidation status of milk fat. In this investigation, lipid oxidation of UHT flavored milk was monitored for the duration of 90 days (Table 5). FFA, PV and AV were used as indicators of oxidative stability. At 0, 45 and 90 days, FFA content of flavored UHT milk were 0.08%, 0.11% and 0.16% (p<0.05). All the analysis intervals showed significant effect on the generation of FFA. From the shelf life and consumer acceptability viewpoints, concentration of free fatty acids is extremely important in dairy and food products. Excessive FFA may induce objectionable flavor in foods ( O’Connor et al. 2006). Psychrotrophic bacteria produce heat stable enzymes in milk that causes the degradation of lipids in the storage (Rauh et al. 2015). The maximum concentration of FFA in food matrix as allowed by the European Union is 0.2% max (oleic acid). FFA of 90 days old samples of UHT milk were less than the permissible limit of European Union. Rise in FFA of flavored UHT milk can be connected to the microbial lipases (which survive the UHT treatment), moisture, metal ions etc. (Nadeem et al. 2015). Tousova et al. (2013) studied that there were two types of lipolysis first one spontaneous lipolysis (caused by lipase) and second one induced lipolysis which is mainly caused by physical damage of fat membrane. Concentration of unsaturated fatty acids increased in summer so lipolysis of milk is also increased that time as compared to winter. When lipid oxidation occurred the value of TBARS significantly increased. In this investigation, lipid oxidation in flavored UHT milk was characterized through the estimation of PV and fatty acid composition.

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EXPERMIMENT NO. 5Strong correlations were recorded between the change in FAP and PV. The coefficient of correlation between the decline of unsaturated FA and PV was R2=0.9987. In our previous investigation on UHT milk, we correlated peroxide value with induction period, testing intervals showing higher peroxide value revealed lower induction period (Ajmal et al. 2018a). PV of flavored UHT milk at 0, 45- and 90-days interval was 0.22, 0.65 and 2.88 (MeqO2/kg). Lipid oxidation in UHT milk lead to higher peroxide value. Peroxide value of milk increased after one week of storage. Peroxide value of fluid milk significantly increased in storage (Nadeem and Ullah 2006). For the estimation of secondary and tertiary stages of auto-oxidation, anisidine value was used. Anisidine value and peroxide value may be combined to determine the total oxidation in fats. For the characterization of butter, ice cream and margarine, anisidine value was used (Nadeem et al. 2015). Anisidine value of flavored UHT milk at 0, 45 and 90 days of storage was 3.89, 5.72 and 9.26, respectively. Due to lipid oxidation of UHT milk, volatile fatty acids and off-odor compounds are produced (Datta et al. 2002). Enzymes and heat treatment may affect the stability of lipids. Lipid oxidation is catalyzed by the heat treatment in UHT milk (O’Connor et al. 2006). 7.3.7. Sensory Evaluation

Table 6 presents the sensory characteristics of UHT flavored milk. Sensory analysis of flavored UHT milk after 90 days indicated that appearance, flavor and mouth feel score significantly decreased from the initial values recorded at 0 day. After 90 days, the decline in appearance, flavor and mouth feel was due to fat separation, sedimentation of proteins, slight rancid flavor and poor mouth feel. Maillard reaction is responsible for the lower sensory characteristics of UHT milk (Costa et al. 2011). Singh et al. (2009) reported that aroma and color of UHT flavored milk is altered during processing and storage. Flavor of UHT treated dairy products is compromised due to thermal degradation of proteins, lipid oxidation and Maillard reaction (Zabbia et al. 2012). Oxidation of unsaturated fatty acids and Maillard reaction led to lower the sensory properties of UHT milk. 7.4. Conclusion

In this investigation, transition in protein profile, Maillard reaction products and lipid oxidation of flavored UHT milk was compared with normal UHT milk. It was recorded that magnitude of changes in various fractions of casein and whey proteins, Maillard reaction products was significantly higher in flavored UHT milk as compared to normal UHT milk. However, no connection was established between sugar of flavored milk and lipid oxidation. Findings of this investigation suggested that concentrations of protein fractions and Maillard reaction products increased during the storage. 7.5. Financial Assistance

HEC provided funding for this project through Grant No. NRPU/18/6978. 7.6. References Ajmal M,  Nadeem M, Imran M, Junaid M. 2018a. Lipid compositional changes and oxidation status of

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EXPERMIMENT NO. 5Table 7.1: Chemical Composition of Flavored UHT Milk Different Stages of Storage (n=475)Storage Days 0-day 45-days 90-days Normal UHT Milk, Values

Reported in Literature after 90 Days of Storage (Hamad et al.

2015)Fat% 3.5±0.05a 3.48±0.09a 3.25±0.12b 3.44a

Protein% 3.15±0.03a 3.12±0.06a 2.81±0.13b 2.85b

pH 6.68±0.09a 6.66±0.11a 6.55±0.15b 6.65a

Total Solids 15.7±0.18a 15.52±0.21a 14.29±0.25b 13.73c

If a row carries dissimilar letters on the means, these indicate significant difference (p<0.05)

Table 7.2: Transition in Protein Profile of UHT Flavored Milk Different Stages of Storage (n=475)Protein Fraction 0-day After 45

daysAfter 90

dayNormal UHT Milk, Values

Reported in Literature after 90 Days of Storage

αs1-Casein% 29.8±0.39a 28.78±0.79b 26.50±0.86c 29.4±0.03a (Jansson et al. 2014)

αs2-Casein% 5.43±0.44a 4.86±0.77b 3.54±0.45c 4.9±0.03b (Jansson et al. 2014)

β-casein% 35.6±0.46a 32.7±0.43b 30.5±0.76c 34.6±0.03a (Jansson et al. 2014)

κ-Casein% 4.45±0.03a 3.64±0.30b 2.94±0.05c 3.7±0.03b (Jansson et al. 2014)

β lacto globulin (mg/l)

188±0.02a 155±0.03b 113±0.06c 191±0.03a (Jansson et al. 2014)

α- Lactalbumin (mg/l)

1210±0.06a 960±0.07c 910±0.01d 1011±0.03b (Le et al. 2011)

Immunoglobulin% 40.12±0.01a 29.32±0.02c 25.45±0.04d 34.21±0.03b (Elliott et al. 2003)

Serum Albumin (mg/l)

86.28±0.09a 62.50±0.02b 49.12±0.06d 57±0.03c (Elliott et al. 2005)

If a row carries dissimilar letters on the means, these indicate significant difference (p<0.05)

Table 7.3: Transition in Maillard Reaction Products at Different Stages of Storage (n=475)75

EXPERMIMENT NO. 5Parameter 0-day After 45 days After 90 day Normal UHT

Milk, Values Reported in

Literature after UHT Treatment

Hydroxy Methyl Furfural (μg/100 mL)

1.56±0.04c 4.18±0.16b 7.61±0.22a 1.51 (Saeed and Hashmi, 2000)

Furosine (mg/100g protein) 278±1.26c 392±1.65b 561±2.36a 319.9 (Rauh et al. 2015)

Nε-carboxymethyl-l-lysine (mg/kg protein)

67±0.89c 135±0.75b 343±0.62a 9.81 (Hull et al. 2012)

If a row carries dissimilar letters on the means, these indicate significant difference (p<0.05)

Table 7.4: Fatty Acid Profile of Flavored UHT Milk at Different Stages of Storage (n=475)Fatty Acid 0-day After 45 days After 90 day Normal UHT Milk,

Values Reported in Literature after 90 Days of Storage (Ajmal et al.

2018a)C4:0 2.92±0.08a 2.89±0.03a 2.47±0.07b 2.88

C6:0 2.25±0.11a 2.23±0.02a 2.05±0.09b 2.04

C8:0 1.29±0.03a 1.25±0.05a 1.12±0.04b 1.02C10:0 3.11±0.06a 3.09±0.07a 2.65±0.13b 1.95

C12:0 3.69±0.04a 3.61±0.10a 3.31±0.21b 2.44

C14:0 15.39±0.19 a 15.24±0.26 a 14.11±0.30 b 7.56

C16:0 28.27±0.32 a 28.13±0.35 a 26.44±0.52 b 21.53C18:0 10.65±0.27 a 10.47±0.42a 9.36±0.24 b 11.29C18:1 24.72±0.16 a 23.36±0.49 b 14.51±0.38c 18.52C18:2 1.56±0.03 a 1.19±0.06b Not Detected 0.14

C18:3 0.48±0.01a 0.22±0.02 a Not Detected Not DetectedIf a row carries dissimilar letters on the means, these indicate significant difference (p<0.05)

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EXPERMIMENT NO. 5

Table 7.5: Oxidative Stability of Flavored UHT Milk (n=475)Fatty Acid 0-day After 45 days After 90 day Normal UHT Milk,

Values Reported in Literature after 90 Days of Storage (Ajmal et al. 2018)

Free Fatty Acids (oleic) 0.08±0.01c 0.11±0.02b 0.16±0.03a 0.19±0.03c

Peroxide Value (MeqO2/kg)

0.22±0.03c 0.65±0.16b 2.88±0.08a 1.18±0.03c

Anisidine Value 3.89±0.05c 5.72±0.13 b 9.26±0.14a 12.73±0.03c

If a row carries dissimilar letters on the means, these indicate significant difference (p<0.05)

Table 7.6: Sensory Characteristics of Flavored UHT Milk (n=475)Parameter 0-day After 45 days After 90 day Normal UHT Milk, Values

Reported in Literature after 90 Days of Storage (Drusch et al. 1999)

Appearance 8.2±0.19a 8.1±0.15a 7.4±0.05b 7.3±0.09b

Flavor 8.0±0.04a 7.8±0.06a 7.2±0.09b 7.6±0.09b

Mouth Feel 8.1±0.14a 7.9±0.08a 7.0±0.12c 7.5±0.09b

If a row carries dissimilar letters on the means, these indicate significant difference (p<0.05)

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CHAPTER 8SUMMARY

Several studies have been performed on various chemical aspects of UHT treated dairy products, however, detailed investigation on protein and lipid fractions of UHT treated dairy products requires. Keeping in view the above-mentioned facts, a comprehensive study was planned to study the biochemical changes in protein and lipid fractions of UHT treated milk at various stages of processing and storage and to study the protein and lipid profiling of flavored milk UHT treatment.

Impact of Ultra-High Temperature (UHT; 142oC for 2 sec) Treatment and 90 days ambient storage (30-35oC) on protein and amino acid profile of UHT milk and sedimentation was investigated. After UHT treatment, β-lactoglobulin, β-lactalbumin, immunoglobulin and serum albumin decreased by 12.1%, 15.4%, 10.6% and 10.2% with no effect on casein fractions. After 90 days, αs1-Casein, αs2-Casein, β-casein, κ-Casein, β- lactalbumin, Immunoglobulin and serum albumin decreased by 13.5%, 31.6%, 11.3% and 27.9%, 36.12%, 46.7% and 38.31%. Sedimentation was comprised of 14.9% αs1-casein, 3.42% αs2-casein, 9.88% β-casein, 2.27% κ-casein, 4.36% β-Lactoglobulin, 1.88% β-lactalbumin, 1.84% immunoglobulin and 0.61% Serum Albumin. Out of 80% casein and 20% whey, 38.5% and 8.7% became the part of sedimentation. After 90 days, except tryptophane, threonine, proline and serine, rest of the essential and non-essential amino acids were significantly affected by the UHT treatment and storage. In storage of UHT milk, viscosity and plasmin activity increased.

UHT-treated tea whitener, milk and dairy drink samples (450 each) of the same manufacturing date were purchased from the market and stored at ambient temperature (25-30 oC) for 90 days. At the time of collection, all the samples were only week old. Samples of UHT-treated tea whitener, milk and dairy drink were regarded as treatments and every treatment was replicated five times. Chemical composition, fatty acid profile, 2, 2-Diphenyl-1-picrylhydrazyle (DPPH) radical scavenging activity, total antioxidant activity, reducing power, determination of antioxidant activity in linoleic acid system and induction period was determined at 0, 45 and 90 days of storage. In freshly collected samples, the fat content in UHT-treated tea whitener was 6% as compared to 3.5% in UHT-treated milk and dairy drink samples while total antioxidant activity (40.2-45.2%), antioxidant activity in linoleic acid (8.43-10.6%) and 2, 2-Diphenyl-1-picrylhydrazyle (DPPH) free radical scavenging activity was found in the range of 21.8-25.6%. After 90 days of storage, the decreasing trend in these parameters was noted with the rise of storage duration. In freshly collected samples of UHT-treated milk, content of vitamin A and E were 0.46μg/100g and 0.63mg/100g, respectively. UHT-treated tea whitener had the lowest concentration of vitamin A and E which was significantly affected by the length of storage period. In freshly collected samples, amount of short, medium and unsaturated fatty acids in UHT-treated milk were 10.54%, 59.71% and 27.44%, accordingly. After 45 days of storage of UHT-treated milk, the loss of short, medium and unsaturated fatty acid was 7%, 7.1 and 5.8%, respectively. After 90 days of storage of UHT-treated milk, the loss of short, medium and unsaturated fatty acid was 8.53%, 13.51% and 11.88%, accordingly. After 45 days of storage of UHT-treated tea whitener, the loss of medium and unsaturated fatty acid was 1.6% and 0.99%, respectively. After 90 days of storage, UHT-treated tea whitener showed the loss of medium and unsaturated fatty acid as 8.2% and 6.6%, respectively. In freshly collected samples, induction period of UHT-treated tea whitener, milk and dairy drink was 15.67, 9.74 and 7.27 hrs, accordingly. Induction period of all types of UHT products showed decreasing trend in the storage period. Moreover, strong correlations were observed between induction period and peroxide value of UHT-treated product samples. This investigation disclosed that UHT-treated tea whitener had 6% fat content and no short-chain fatty acids. Antioxidant capacity of UHT-treated milk was higher than dairy drink and tea whitener. Due to the presence of partially hydrogenated fat, peroxide oxidative stability of UHT-treated tea whitener was better than UHT-treated milk and dairy drink. Vitamin A and E was not found in UHT-treated tea whitener and for the anticipation of oxidative stability of UHT-treated milk, dairy drink and tea whitener, induction period/ Rancimat method can be used.

Transition in protein profile, Maillard reaction products and lipid oxidation of flavored UHT treated milk was investigated. Samples of mango flavored UHT milk 200 ml pack (475 Nos.) were purchased from the market and stored at ambient temperature for the duration of 90 days. Proteolysis, hydroxyl methyl furfural furosine, Nε-carboxymethyl lysine, fatty acid profile, free fatty acids, peroxide value and sensory

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SUMMARYcharacteristics were studied at 0, 45 and 90 days of storage. In this investigation, transition in tyrosine content was used as a marker of proteolysis in flavored UHT milk. Tyrosine content of flavored UHT milk increased during the storage. Tyrosine content of flavored UHT milk at 0, 45 and 90 days of storage were 3.5, 6.9 and 15.2 µg tyrosine/ml. After 90 days of storage, the decline in αs1-casein, αs2-casein, β-casein, κ-casein, β-lactoglobulin, α-lactalbumin, immunoglobulin and bovine serum albumin were 11.2%, 34.8%, 14.3%, 33.9%, 56.9%, 24.8%, 36.5% and 43.1%. HMF content of UHT milk at 0, 45 and 90 days of storage were 1.56, 4.18 and 7.61 (µmol/L). Furosine content of flavored UHT milk at 0, 45 and 90 days of storage intervals were 278, 392 and 561 mg/100g protein. At 0, 45 and 90 days of storage, concentration of CML in flavored UHT milk was 67, 135 and 343 mg/kg protein. After 90 days of storage of flavored UHT milk, the loss of unsaturated fatty acids was 45.7%, from the initial values. PV of flavored UHT milk at 0, 45- and 90-days interval was 0.22, 0.65 and 2.88 (MeqO2/kg). Appearance, flavor and mouth feel score was not affected by the storage up to 45 days of storage.

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