Influence of Ripening and Emulsifying Salts on Quality of Processed Cheddar from Cow and Buffalo Milk

By

ASIF MERAJ

M.Sc. (Hons.) Technology

A thesis submitted in partial fulfillment of the requirement for the degree of

DOCTOR OF PHILOSOPHY IN AND TECHNOLOGY

INSTITUTE OF FOOD SCIENCE & NUTRITION UNIVERSITY OF SARGODHA SARGODHA - PAKISTAN 2016

To

The Controller of Examinations, University of Sargodha, Sargodha-Pakistan

“We, the Supervisory Committee, certify that the contents and form of thesis submitted by ASIF MERAJ (Reg. # 09/US/PhD/FSc/3) have been found satisfactory and recommend that it be processed for evaluation, by the External Examiner(s) for the award of degree”.

Supervisory Committee

Supervisor: ______Dr. Mian Anjum Murtaza Institute of Food Science and Nutrition, University of Sargodha, Sargodha -Pakistan

Co-Supervisor: ______Prof. Dr. Nuzhat Huma National Institute of Food Sci. and Technology, University of Agriculture, Faisalabad -Pakistan

DECLERATION

It is stated that the research work contained in the thesis entitled “Influence of Ripening and Emulsifying Salts on Quality of Processed from Cow and Buffalo Milk ” is original and nothing has been stolen/copied/plagiarized from any source. The research work reported in the thesis has been completed according to the requirements of Higher Education Commission and University of Sargodha, Sargodha, Pakistan.

ASIF MERAJ Reg. # 09/US/PhD/FSc/3 Ph.D. Fellow

CERTIFICATE

It is certified that the research work contained in the thesis entitled “Influence of Ripening and Emulsifying Salts on Quality of Processed Cheddar Cheese from Cow and Buff alo Milk” by Mr. Asif Meraj (Reg. # 09/US/PhD/FSc/3) is original and nothing has been stolen/copied/plagiarized from any source. The research work reported in the thesis has been completed according to the requirements of Higher Education Commission and Un iversity of Sargodha, Sargodha, Pakistan.

______Dr. Mian Anjum Murtaza Prof. Dr. Nuzhat Huma (Supervisor) (Co-Supervisor)

CONTENTS

No. Title Page Acknowledgements 1 INTRODUCTION 01 2 REVIEW OF LITERATURE 06 2.1. Milk composition and properties for cheese 06 Making 2.2. Cow milk vs buffalo milk 07 2.3. Cheddar cheese 09 2.4. Cheddar cheese manufacturing 10 2.4.1. Pre-treatment of milk 10 2.4.2. Transformation of milk into cheese 13 2.5. Cheddar cheese ripening 16 2.5.1. Primary reactions 17 2.5.2. Secondary reactions 22 2.6. Processed Cheddar cheese 24 2.7. Manufacturing steps 25 2.7.1. Selection and formulation of ingredients 25 2.7.2. Processing, packing, cooling and storage of 26 process cheese 2.8. Factors affecting quality of processed Cheddar 27 Cheese 2.8.1. Natural cheese and its ripening 28 2.8.2. Formulation and processing conditions 28 2.8.3. Emulsifying salts 30 2.9. Sensory studies of cheese 32 3 MATERIALS AND METHODS 36 3.1. Procurement of raw materials 36 3.1.1. Raw milk 36 3.1.2. Starter cultures 37 3.1.3. Emulsifying salts 37 3.2. Manufacturing and ripening of natural Cheddar Cheese

No. Title Page 3.3. Quality evaluation of natural Cheddar cheese 40 3.3.1. Physico-chemical analysis 40 3.3.2. Organic acids 42 3.3.3. Assessment of proteolysis 43 3.4. Selection of emulsifying salts (combination) 44 3.5. Preparation of processed Cheddar cheese 45 3.6. Quality evaluation of processed Cheddar cheese 48 3.6.1. Physico-chemical analysis 48 3.6.2. Texture profile analysis (TPA) 48 3.6.3. Melt-ability 48 3.6.4. Assessment of proteolysis 49 3.6.5. Color analysis 49 3.6.6. Descriptive sensory evaluation 49 3.7 Statistical analysis 49 4 RESULTS AND DISCUSSION 50 4.1. Physico-chemical analysis of raw milk 50 4.2. Physico-chemical analysis of standardized milk 53 4.3. Cheddar cheese quality during ripening 56 4.4. Physico-chemical analysis 56 4.4.1. Moisture in Cheddar cheese 56 4.4.2. in Cheddar cheese 60 4.4.3. Crude protein in Cheddar cheese 60 4.4.4. Ash in Cheddar cheese 67 4.4.5. pH in Cheddar cheese 68 4.4.6. Acidity of Cheddar cheese 75 4.4.7. Salt in Cheddar cheese 76 4.5. Minerals in Cheddar cheese 83 4.5.1. Calcium in Cheddar cheese 83 4.5.2. Potassium in Cheddar cheese 84 4.5.3. Sodium in Cheddar cheese 91 4.5.4. Magnesium in Cheddar cheese 92 4.5.5. Phosphorus in Cheddar cheese 99 4.6. Organic acids in Cheddar cheese 103 4.6.1. Lactic acid in Cheddar cheese 103 4.6.2. Acetic acid in Cheddar cheese 104 4.6.3. Citric acid in Cheddar cheese 111

4.6.4. Butyric acid in Cheddar cheese 115 4.6.5. Pyruvic acid in Cheddar cheese 116 4.7. Proteolysis in Cheddar cheese 124 4.7.1.Water soluble nitrogen(primary proteolysis) 124 4.7.2. Total free amino acids (secondary 125 proteolysis) 4.8. Selection of emulsifying salts (combination) 133 4.9. Physico-chemical analysis of processed Cheddar 135 Cheese 4.9.1. Moisture content 137 4.9.2. Fat content 137 4.9.3. Protein content 145 4.9.4. Ash content 146 4.9.5. pH Value 153 4.9.6. Acidity content 154 4.9.7. Salt content 161 4.10. Minerals analysis 165 4.10.1. Calcium content 165 4.10.2. Potassium content 166 4.10.3. Sodium content 174 4.10.4. Magnesium content 178 4.10.5. Phosphorus content 179 4.11. Texture of processed Cheddar cheese 187 4.11.1. Hardness of processed Cheddar cheese 187 4.11.2. Cohesiveness of processed Cheddar cheese 188 4.11.3. Springiness of processed Cheddar cheese 192 4.11.4. Gumminess of processed Cheddar cheese 192 4.11.5. Chewiness of processed Cheddar cheese 193 4.12. Melt-ability of 197 4.13. Proteolysis in processed cheese 203 4.13.1. Water soluble nitrogen / total nitrogen (%) 203 (primary proteolysis) 4.13.2. Total free amino acids (secondary 204 proteolysis) 4.14. Color of processed cheese 212 4.14.1. Lightness (L*) value of color 212 4.14.2. Redness (a*) value for color 213 4.14.3. Yellowness (b*) value of color 214 4.15. Sensory evaluation of processed Cheddar cheese 219

4.15.1. Flavor of processed cheese 219 4.15.2. Odor of processed cheese 224 4.15.3. Texture of processed cheese 225 4.16. Multivariate analysis of sensory scores 232 5 SUMMARY 237 LITERATURE CITED 242 APPENDICES 292

ACKNOWLEDGEMENTS Over and above every thing, I offer my humble and sincerest gratitude to Almighty ALLAH, the most Merciful, Compassionate, Gracious and Beneficent, Who enabled me to complete the study. My special praises and respects are for Holy Prophet Hazrat Muhammad (Peace Be Upon Him) Who is the greatest scientist of this world and the most perfect and excellent among and of every born on the surface of earth forever. I would like to thank my Supervisor Dr. Mian Anjum Murtaza, Assistant Professor, Institute of Food Science and Nutrition, University of Sargodha, Sargodha, Pakistan, for his valuable and ever inspiring guidance, help and friendly attitude throughout my research and studies. His contributions for the successful completion of the manuscript are enormous. I am greatly thankful to my co-supervisor Prof. Dr. Nuzhat Huma, National Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan for her help and guidance during the course of my studies. I would like to thank all my teachers, friends and colleagues who inspired and helped me for achievement of the higher ideas of my life. Last but not the least; I pay cordial thanks to my loving and caring Parents, Brothers and Sisters, Wife and Daughter for inspiration to achieve my academic goals.

Asif Meraj

Chapter-1 INTRODUCTION

Pakistan is the 4th largest milk producing country in the world with as one of the fastest growing sectors. The annual milk production with about 3.2% increase is estimated as 50.99 million tonnes, out of which, 41.13 million tonnes are available for human consumption (GOP, 2014). Milk is mainly supplied to consumers in raw form. The milk products include heat treated milk, dried and concentrated milk, ice cream, butter, yoghurt and cheese. Cheese manufacturing is one of the classical examples of milk preservation and its history goes back to 6000-7000 BC. Cheese making is basically a dehydration process in which the fat and caseins of milk are concentrated 6-10-fold (Omotosho et al., 2011). Cheese is the coagulated and concentrated form of milk that is manufactured at appropriate temperature and humidity (Murtaza et al., 2014a). It is available in wide range of forms, flavors and textures (Raheem and Saris, 2009). Its popularity is because of its diversity in applications, nutritional values, convenience and good taste (Farkye, 2004; Murtaza et al., 2008; Singh et al., 2003). It is a complex food made from few simple ingredients since a wide range of cheese varieties exists in the world, each with unique characteristics (Falegan and Akere, 2014). About 2000 individual varieties are claimed (International Dairy Association, 2011) those are classified on basis of their form, manufacturing technology, ripening and chemical composition. On the basis of moisture level, cheese can be categorized as hard, semi-hard, semi-soft and soft (Manish and Srivastava, 2002; Walstra et al., 2006). Hard ripened cheese produced by acidification and concentration followed by gel formation with enzymes comes under the category of Cheddar cheese (Banks, 2002). Cheddar cheese is highly nutritious and a complex mixture and is rich in protein, fat, carbohydrates, vitamins and minerals (Murtaza et al., 2008; Murtaza et al., 2014a). Microbiological, biochemical and metabolic processes referred as glycolysis, lipolysis and proteolysis are accountable for the development of flavor and texture characteristics of Cheddar cheese during ripening (Singh et al., 2003; Smit et al., 2005; Murtaza et al., 2012).

Moisture, fat, peptides, salt, micro-flora, minerals and other insignificant ingredients, assorted within a casein matrix syndicate to make Cheddar a multifaceted food (Maarse et al., 1994; Murtaza et al., 2008). According to the component balance theory, a blend of the precise ingredients at the appropriate intensities produces the Cheddar aroma (result of various volatile components) and texture (House and Acree, 2002). Quality of cheese is highly affected by milk composition, starter cultures and manufacturing technology (Varnam and Sutherland, 1994). Structure of milk, specifically the substances of fat, protein, calcium and pH intensely influence the structure of cheese. The milk composition depends upon more than a few factors counting the species, breed, uniqueness, dietary status, health and phase of lactation of producing animal (Fox et al., 2000). Buffalo milk having high contents of fat and protein serves as very good raw material for preservation especially cheese making (Barłowska et al., 2011). It contains higher fat, lactose, caseins, calcium, magnesium and inorganic phosphate than cow milk (Ahmad et al., 2013; Murtaza et al., 2013) as well as the capacity of milk to be acidified is higher for buffalo than cow (Ahmad et al., 2008). On basis of the compositional profile and functional properties, buffalo milk compromises exceptional opportunities for the progress of fermented dairy products (Murtaza et al., 2008; Murtaza et al., 2015). Unlike the food products, for which constancy is the vital benchmark, cheese practices substantial changes throughout ripening (Murtaza et al., 2014a). Newly prepared curds of different cheese varieties have mild and mostly alike flavors and aroma, however; in the duration of ripening, the flavor and texture development occur, which are quality traits of each variety (McSweeney and Sousa, 2000; Smit et al., 2005). Unique characteristics of cheese are obtained through physical and chemical changes during ripening stage. The typical ripening temperatures for Cheddar cheese range from 6 to 8oC (Fox and Cogan, 2004) and the digestion of the curd components takes place through microbiological and chemical processes (Choisy et al., 2000). During cheese ripening, the biochemical changes taking place are divided into primary and secondary types. of residual lactose, lactate and citrate, lipolysis, and proteolysis come under primary actions. Secondary actions include breakdown of fatty

acids and amino acids, leading to the development of unstable flavor (McSweeney, 2004a, b). The conversion of lactose to lactate by starter bacteria is the key feature of ripening. Lactate produced is an essential substrate for a series of responses that occur during ripening (McSweeney, 2004). The amount of lipolysis in cheese is influenced by the variety of cheese and can vary from minor to extensive. Excessive lipolysis may yield unwanted off-flavors (McSweeney and Fox 1993). Most complex among primary events during cheese ripening is proteolysis particularly in inner bacterially ripened . Proteolysis consequences in the development of peptides, free amino acids and free ammonia. Peptides subsidize to both flavor and texture, while, the free amino acids and ammonia donate to flavor and aroma, and also switch pH (Murtaza et al., 2014b). Natural cheese is used to make processed cheese by blending in the existence of suitable emulsifying salts and other dairy and nondairy component shadowed by heat treatment and mechanical shear to ensure a smooth uniform product (Kapoor and Metzger, 2008). Processed cheese manufacturing involves the selection and preparation of ingredients and formulations. In addition to natural cheese and emulsifying salts, additional dairy (e.g. butter, cream, skim- milk powder, whey powder, caseinates, anhydrous milk fat) and non- dairy components (e.g. fish, , salt, colors, flavors, spices, food gums, mold inhibitors etc.) and additives (e.g., hydrocolloids, coloring, sensory active mixtures) are applied to modify the functional properties of the product (Nagyova et al., 2014). Various constituents influence the quality characteristics including texture, flavor and functional properties of processed cheese (Kapoor and Metzger, 2008). The uniformity of processed cheese is a vital quality attribute. Cheese composition, structural arrangements, physic- chemical status of components and macrostructure, which reflects the cracks and fissures, build the consistency (O’Callaghan and Guinee, 2004). The stability of processed cheese is exaggerated by processing conditions, pH of cheese melt, dry matter and fat content, occurrence and deliberation of calcium ions, usage of hydrocolloids, kind and application of emulsifying salts and cheese maturity etc. (Sadlikova et al., 2010). Apposite assortment of natural cheese is extreme important for the production of processed cheese. Mostly processed cheeses are formed from a mix of various natural cheese varieties however, processed cheeses manufacturing from only one variety of cheese at

various stages of ripening is also practiced (Caric and Kalab, 1993). In young, un-ripened cheese, casein molecules are in their unique state with their emulsifying properties unspoiled. During ripening, casein is hydrolyzed and drops its emulsifying properties affecting the ultimate structure (Chambre and Daurelles, 2000). Hence, the ripening of natural cheese amalgam is a significant aspect manipulating the texture of the processed cheese (Piska and Stetina, 2004). Key component for the production of processed cheese is the emulsifying salt (Nagyova et al., 2014). Presence of emulsifying salts results in uniform structure of processed cheese and enhances the emulsifying ability of cheese proteins by eliminating calcium from caseins and by peptizing, hydrating and dispersing the protein, buffering, stabilization of oil-in-water emulsion and structure formation (Guinee et al., 2004; Mulsow et al., 2007). The increase in the amount of emulsifying salts in processed Cheddar cheese improves the texture of the final product and vice versa but up to a limited extent since an overdose causes the bitterness (Everard et al., 2007). Emulsifying salts are ionic mixtures of monovalent cations and polyvalent anions (Kapoor and Metzger, 2008). These are significant in the processed cheese by playing a key role to ensure the desired consistency of the product. The vital task is to enhance the emulsifying potential of cheese proteins by exchanging the calcium cations of insoluble calcium paracaseinate with sodium ions, resulting in the formation of more soluble sodium paracaseinate (Cernikova et al., 2010; Mizuno and Lucey, 2007; Sadlikova et al., 2010). The complex series of chemical, bacterial and enzymatic reactions are responsible for the development of sensory characteristics that are typical of ripened cheese (Pollard et al., 2003). Determination of the degree of ripening and several sensory features is an imperative part of cheese quality assessment and at present implicates the use of trained sensory panelists or entities (Downey et al., 2005). Grading and judging are used extensively for sensory evaluation of dairy products (Bodyfelt et al., 1988) and a product is assessed based on the existence or nonexistence of particular defects and on total quality score. These quality scores are generally based on the estimations of one individual and the quality score is subjective rather than exactly defined (Drake et al., 2005).

Descriptive evaluation by a panel of trained assessors is a method of choice for determining the sensory profile of a cheese and can determine the influence of processing changes on individual sensory characteristics (Fox et al., 2000). It is a refined sensory test technique that generates a total sensory picture of a product (Stone and Sidel, 1985). This test approach can be used to govern the effect of individual modules on scores of descriptors of a complex product (Murtaza et al., 2013). Worldwide Cheddar cheese is produced from cow’s milk. However, in Pakistan, buffalo’s milk ranks at the top with annual production of 31.25 million tonnes followed by cow milk with annual production of 18.03 million tonnes (GOP, 2014). Keeping in view the above particulars, the project was designed to assess the influence of various ripening stages and emulsifying salts on the quality of processed Cheddar cheese prepared from cow and buffalo milk. Objectives: Hence the study was intended with the following main objectives:  Preparation of natural Cheddar cheese from cow and buffalo milk and its quality evaluation during ripening.  Preparation of processed cheese using raw, semi ripened and fully ripened cow and buffalo milk Cheddar cheese and different emulsifying salts.  Evaluation of texture and functional characteristics of processed cheese prepared from natural cheese of varying maturity and different emulsifying salts.  Evaluation of the proteolytic pattern of processed Cheddar cheeses as influenced by ripening.  Evaluation of the sensory quality of processed cheese produced from Cheddar Cheese of varying maturity.

Chapter-2 REVIEW OF LITERATURE

Cheese; the most assorted milk product is perhaps academically the most challenging and attention-grabbing. Multiple scientific disciplines like microbiology, biotechnology, biochemistry, enzymology, toxicology, flavor chemistry, nutrition, and chemical engineering involved in different aspects of cheese trapped the scientists to fell into cheese study. The present study was aimed to assess the influence of emulsifying salts and ripening periods on quality of processed Cheddar cheese manufactured from cow and buffalo milk. The relevant literature has been reviewed to support the studies under the following headings: 2.1 Milk composition and properties for cheese making 2.2 Cow milk vs buffalo milk 2.3 Cheddar cheese 2.4 Cheddar cheese manufacturing 2.4.1 Pre-treatment of milk 2.4.2 Transformation of milk into cheese 2.5 Cheddar cheese ripening 2.5.1 Primary reactions 2.5.2 Secondary reactions 2.6 Processed Cheddar cheese 2.7 Manufacturing steps 2.7.1 Selection and formulation of ingredients 2.7.2 Processing, packing, cooling and storage of process cheese 2.8 Factors affecting quality of processed Cheddar cheese 2.8.1 Natural cheese and its ripening 2.8.2 Formulation and processing conditions 2.8.3 Emulsifying salts 2.9 Sensory studies of cheese 2.1 Milk composition and properties for cheese making

Cheese composition, texture and flavor depend upon the composition of milk used for cheese manufacture (Lawrence et al., 1984; Lucey and Kelly, 1994). Several factors particularly the protein content of milk influence the processing quality of milk for cheese making (White, 2001). About 0.1% reduction in casein concentration reduces Cheddar cheese yield potential by 0.5 kg/100 kg milk (Amenu et al., 2003). During lactation stages milk have different concentrations of constituents (Lucey and Kelly, 1994). Protein content of milk increases (Guinee et al., 2007) and the lactose content decreases as lactation period progress. Varying composition of milk especially fat, caseins and their ratio affect cheese yielding capacity of milk (Ng- Kwai-Hang et al., 1987a). Dominant factor affecting curd firmness, synersis rate, moisture retention, and ultimately affecting cheese quality and yield is the casein fraction of milk (Lawrence, 1993). Milk coagulation properties like rennet coagulation time (RCT, min.), curd firmness time and curd firmness have significant impact on cheese production (Summer et al., 2002). Certain physical factors like milk quality, breed, season and herd, feeding, lactation also affect milk coagulation properties. Increase of milk protein and casein contents reduces the rennet coagulation time (Joudu et al., 2008). Season of the year has significant impact on milk coagulation properties (Chladek et al., 2011). Barlowska et al. (2012) reported that rennet coagulation time in cow milk is better in summer than in other seasons. Jersey milk has higher protein and fat contents than Friesian milk and therefore possesses better cheese making properties (Auldist et al., 2004). Milk with high somatic cell count (> 500 000 cells/ml) has high proteolytic activity, lower fat and casein concentration and high whey proteins especially serum albumin and immunoglobulin and results in low cheese yield (Auldist et al., 1996). Milk for cheese making should have low somatic cell count since proteases from somatic cells attacks α-S2 and β- caseins and reduce the cheese yield (Skeie, 2007). Fatty acid composition of milk fat affects the texture and flavor of cheese. Milk fat having higher amount of long chain fatty acids produces cheese with reduced firmness (Jaros et al., 1997). 2.2 Cow milk vs buffalo milk According to FAOSTAT (2010), Pakistan is the 2nd largest country of the world with respect to buffalo milk production. Pakistan and India are the main producers of water

buffalo (Bubalus bubalis) milk by producing 30% and 60% respectively in 2006 to 2009 (FAOSTAT, 2012). Buffalo shares the highest quantity of milk (62.8%) followed by cow (34.9%), goat (2.0%), camel (0.2%) and sheep (0.1%) in Pakistan (Ahmad et al., 2013). Conjugated linoleic acid, medium chain fatty acids, total protein and retinol and tocopherol contents are greater in buffalo milk than cow milk. Certain specific classes of gangliosides are also only present in buffalo milk (Berger et al., 2005). Sindhu (1998) reported that high percentages of more melting tri glycerides (9-12%) are present in buffalo milk as compared to cow milk (5-6%) resulting in buffalo milk as more thick in nature. Buffalo milk also contains high values of butyric acid containing triglycerides (50%) which are only 37% in cow milk (Ramamurthy, 1976). Triglycerides containing butyric acid present in higher amount increases the emulsifying capability of buffalo milk fat. Fat is the major component present in buffalo milk (Teixeira et al., 2005; Mattos, 2007) as the fat content in buffalo milk (7.5%) are twice as in cow (3.3%) making it more energy dense. High fat content make buffalo milk more suitable for processing. Buffalo milk contains bigger fat globules (4.15-4.6m) than cow milk (3.36-4.15m) while, cow milk have higher amount of free fatty acids (0.33%) than in buffalo milk (0.22%). Similarly Phospholipids are also present in rich amount (37.37 mg/100ml) in cow milk when compared with buffalo milk having 21 mg/100ml (Menard et al., 2010). Buffalo milk is superior in protein contents containing 11.42% higher proteins when matched with cow (Murtaza et al., 2008; Murtaza et al., 2015). Concentrations of whey proteins and caseins are higher in buffalo than cow milk. In buffalo milk, caseins are present in miceller form but in cow milk miceller casein are 90-95% and the rest exists in the serum part. Miceller caseins present in buffalo milk are of larger particle size (110- 160 nm) then the miceller caseins of cow milk (70-110 nm) (Ahmad, 2010). Moreover, the high amount of heat resistant whey proteins is present in buffalo milk. Proteose peptone content in buffalo milk is 330.5 mg/100ml while in cow milk are 240 mg/100ml (Aneja et al., 2002). Pandya and Haenlein (2009) reported that buffalo milk is wealthier in total proteins, predominantly the casein and whey proteins. Physico-chemical makeup of casein in cow and buffalo milks is different. Higher proportion of calcium present in buffalo milk casein makes it superior in whitening. Buffalo milk contains Biliverdin (a green-blue pigment) instead of carotene and also has bioactive penta saccharides and gangliosides, which are absent in cow

milk (Abd El-Salam and El-Shibiny, 2011; Araujo et al., 2012; Medhammar et al., 2012; Abd El-Salam and El-Shibiny, 2013). Sodium, potassium and chloride contents are comparable in both cow and buffalo milk while calcium and magnesium is higher in buffalo milk. Colloidal calcium and magnesium in buffalo milk is 160 mg/100g and 90 mg/100g paralleled to only 80mg/100g and 30mg/100g in cow milk. Buffalo milk contains high calcium to Phosphorus ratio (1.8) as compared to (1.2) cow milk (Sindhu, 1998). Buffalo milk possesses low lipase and alkaline phosphatase activity (Kumar and Bhatia, 1994). The free amino acids (FAAs) are existent in greater concentrations in buffalo milk (0.44%) associated to cow milk (0.15%). Buffalo milk has high specific gravity (1.0323), surface tension ( 45.50 dynes/cm at 20 ºC ) and viscosity (2.245 cP at 27 ºC) as compared to cow milk having (1.0317) , (42.50 dynes/cm at 20 ºC) and 1.450 cP at 27 ºC (Sindhu, 1998). 2.3 Cheddar cheese Sketches of historical description of cheese manufacture describe the production of cheese in goat skin bags hanging on the poles. Milk was contaminated by acid producing bacteria which lead to curdling and later on whey and curd produced by the subsequent motion in the bags were consumed preferably. In the Fertile Crescent present in today Iraq, cheese and bread were staples as early as in 6000-7000 BC (Scott, 1986). According to FAO and WHO (2010g) Cheddar cheese comes under the category of hard ripened cheese (Muehlhoff et al., 2013). Starter culture and rennet are used to produce acid and to coagulate the casein micelles in the milk to produce natural cheeses (Carolina et al., 2012). No other cheese is well known as Cheddar cheese which originated in the town of Cheddar, in Somerset, England. According to Jayadevan (2013) Cheddar can be anything just a month old to three years old cheese. Cheddar have sharp and mild flavor and can be smooth or dry depending on the age. It is used in cooking purposes and in soups and sauces. Cheddar cheese is a very acceptable and well-liked product on a worldwide basis. Banks (2002) described Cheddar cheese as hard ripened cheese formed by milk acidification, concentration and gel formation with rennet. Cheddar cheese is rich in casein concentration with all essential amino acids which makes it highly nutritional (Hughes and Willenberg, 1993). Fat is also a prominent part of Cheddar cheese along with minor quantity of other nutrients such as vitamins A, B2, B6 and B12. Because of its high protein and calcium

contents, cheese in moderation is an important component of balanced diet (Considine, 1982). Cheddar cheese comes under the classification of hard cheese without a surface flora and with long shelf life. It contains about 45-50% fat in dry matter and up to 62% of minimum dry matter (Spreer and Mixa, 1998). Cheddar cheese has firm body with close texture and a clean nutty flavor (Varnam and Sutherland, 2001). Moisture, fat, peptides, salt, minerals, microflora, and other negligible components, diversified within a casein medium pool to mark Cheddar a complex food (Maarse et al., 1994; Murtaza et al., 2008). Agreeing to the component balance theory, a combination of accurate constituents at the suitable intensities produces the Cheddar aroma and texture (House and Acree, 2002), since the cheese aroma is reflected to be the outcome of several unstable constituents, which separately do not imitate the overall odor (Adda, 1984). The incitement accountable for Cheddar cheese flavor is generally thought to be a type-specific and application of several odorless and non-volatile composites (O’Riordan and Delahunty, 2001). Structure and composition are related to the mechanical properties of cheese (Santillo and Albenzio, 2011). Color of Cheddar cheese varies from pale to deep yellow and flavor can be mild and creamy depending upon maturity (Kosikowski and Mistry 1997). Cheddar cheese also serves as an alternative to yoghurts and fermented milks as food vehicle for probiotic delivery. It creates favorable environment in the gastro intestinal tract for the survival of probiotic bacteria by creating buffer against the acids. High fat contents of cheese protect probiotic bacteria present in stomach (Bergamini et al., 2005 and Sharp et al., 2008). 2.4 Cheddar cheese manufacturing 2.4.1 Pre-treatment of milk Van Boekel (1994) presented reviews to know more about the transferal of milk constituents into cheese, these reviews were further analyzed by Van den Berg et al. (1994). He focused the treatments like bactofugation, enzyme addition, , addition of calcium chloride, denaturation of the whey proteins and showed that all these features have influence on the transmission of substances in the curd (Mona et al., 2011).

i) Standardization Three core procedures for standardizing cheese milk: like adding liquid skim milk and skimmed milk powder and the exclusion of cream have been outlined by Chapman (1981). In order to meet the regulatory standards and to achieve consistency in quality of a given variety of cheese, milk may be standardized on the basis of total protein to fat ratio or casein to fat ratio (Farkye, 2004; Guinee et al., 2006; McSweeney, 2007). Different methods used for the increase or decrease of fat and protein in cheese milk may include milk protein concentrate and skim milk powder addition or caseinate and cream. Casein to fat ratio of milk for production of Cheddar and Gouda variety cheese is 0.7 and in some cases may be 0.64 (Farkye, 2004). Protein to fat ratio of milk in the production of Cheddar cheese in Ireland industry varied from 0.84 to 1.02 and protein and fat contents were 2.99 to 3.59% and 3.26 to 4.2% (Guinee et al., 2006). When cheese is standardized by casein addition its yield is increased (Rehman et al., 2003). Standardization of milk reduces casein and fat losses into whey during cheese manufacture (Lucey and Kelly, 1994; Scott, 1998). Some disadvantages of Standardization have also been noticed; e.g., skimmed milk powder or condensed skim addition upturns quantity of lactose in cheese milk resulting an increase of remaining lactose in cheese which finally raises the probabilities of unwanted throughout ripening. Adding condensed skim indicates to a distinctive flavor in cheese and elevated intensities of lactose may cause in white crystal defect (Anderson et al., 1993). ii) Pasteurization High temperature pasteurization (85°C, 20 seconds), low temperature pasteurization (72°C, 15 seconds), thermization (65°C, few seconds) and ultra-high temperature treatment (145°C, 1 second) are techniques used for heat treatment of milk for cheese production. (Jong, 2010). Denaturation of whey proteins takes place during pasteurization resulting in β- lactoglobulin–κ Casein which might lead to certain insignificant variances in amino acid shapes in lactic and soft cheese (Raynal- Ljutovac et al., 2008). Milk undergoes numerous changes during pasteurization which affect the characteristics of cheese. Pasteurization reduces mesophillic aerobic organisms, it

incapacitates or declines the action of milk lipase and other enzymes and activates plasmin (Andrews et al., 1987; Chavarri et al., 1998; Fox et al., 1993). Proteolytic and lipolytic processes are also affected by pasteurization without depending on the origin of milk used (Gaya et al., 1990; Krause et al., 1997; McSweeney et al., 1993; Sousa and Malcata, 1997). It also affects the flavor; cheese prepared after milk pasteurization is less strong in flavor than their indigenous raw milk flavor (Grappin and Beuvier 1997). Gonzalez et al. (2009) reported that the pasteurization increases the protein content, moisture retention and increased yield of resultant cheese. Milk is The purpose of milk pasteurization for cheese production is to shrink microbial load, increase yield, quality and ripening at higher temperature (Ordonez et al., 1999; Blumenthal, 2002). Pasteurization, through the use of plate heat exchanger and holding tubes with typical time temperature relations of 72°C/15 sec. is standard practice to kill pathogens (Maubois, 2002). Pasteurization >72°C have generally not been used in milk for cheese making due to their contrary effects on curd formation (Guinee et al., 1997) and synersis (Pearse et al., 1985, Pears and MacKinlay, 1985). Mona et al. (2011) described that nitrogen recovery is high for cheese made from pasteurized milk whereas pasteurization has no effect on fat recovery. Quality of Cheddar cheese made from fresh milk, heated milk and micro filtered milk has been studied by McSweeney et al. (1993). Superior quality cheese was achieved from micro-filtered and pasteurized milk whereas cheese made from fresh milk was of low-grade due to its unusual and intense flavor which advanced more rapidly than that of other cheeses. iii) Homogenization homogenized at 60ºC to disrupt milk fat drops into acceptable lipid droplets, inhibiting cream split-up, thus projecting stability and lifetime of milk emulsion. Two types of homogenization (single stage and two stages) are usually used in milk processing. In two stage homogenization, primary stage decreases the mass of fat balls and the secondary stage unsettles fat masses that may be generated (Zamora et al., 2007). In raw milk, size of fat globules normally stuck between 0.2–15 μm, and homogenization usually targets to condense it to < 2 μm. In most cases two step valve homogenizers are used for this purpose operated at pressure of 20MPa (Huppertz and Kelly, 2006).

Milk homogenization permits the fat globules to act together with casein matrix to produce more flexible cheese with a reduced amount of free oil formation. Homogenized milk when used for cheese-making will produce higher yields since extra moisture will finally be recollected in the protein system (Everett and Auty, 2008). Ultra high pressure homogenization (UHPH) of whole and skimmed milk yields fine emulsion units (Hayes, et al., 2005; Thiebaud et al., 2003 and Hayes and Kelly, 2003a) alters protein assembly and physical characteristics and inactivates enzymes (Hayes and Kelly, 2003b). All of these indirectly affect milk clotting properties and attributes of cheese microstructure. 2.4.2 Transformation of milk into cheese Milk is considered colloidal suspension of casein micelles and the curd formed as a result of three step process; (i) acidification (ii) coagulation (iii) synersis convert this colloidal suspension into cheese (Fox et al., 2000a). i) Acidification Basic step in the production of numerous varieties of cheese is the acidification. It is an essential step in processing of good quality cheese and has significant impact on several aspects of cheese manufacturing:  Spoilage and pathogenic macro organisms are controlled  Coagulant activity during manufacturing and ripening gets affected  Helps in determination of cheese composition by promoting synersis  Enzyme activity is influenced by acidification which affect the flavor and quality (Fox et al., 2000a; McSweeney, 2007) Acidification is achieved through production lactic acid by starter cultures (Fox et al, 2004). Different types of starter cultures are used in cheese making. They play specific role in acidification of cheese to get the desired pH during cheese making. They also play vital role in the ripening and flavor improvement. Lactic acid bacteria are mostly associated with fermented foods like yoghurt, sour cream, cheese and butter milk and contribute to taste, flavor and shelf life of fermented foods (Shah and Prajapati, 2014). Starter cultures are now commercially available in liquid and freeze dried form. Freeze dried cultures are available as concentrated or unconcentrated and injected straight into milk, also entitled as direct vat cultures (Farkye, 2004). The best suited and extensively

used starter cultures for Cheddar cheese production are Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris (Michel and Martley, 2001). ii) Coagulation Prime step in the improvement of cheese texture and flavor is milk clotting or coagulation. It is influenced by particular enzymatic proteolytic degradation of milk composites specifically proteins (Ozcan and Eren-Vapur, 2013). The principal milk clotting enzyme in cheese making is chymocin (Farkye, 2004). Chymocin is the most highly active enzyme in animal rennet. Rennet coagulation involves two stages; a proteolytic stage and calcium mediated stage. Hydrolysis of K-casein occurs which destabilizes the casein micelles and yields Para- K-casein micelles in proteolytic stage. Para casein micelles become aggregated during calcium mediated stage. Hydrolysis of K-casein causes the clotting of milk by slashing Phe105-Met106 bond of k- casein (Fox et al., 2000a; Horne and Banks, 2004). In cheese making, k-casein polymorphism is a precarious aspect in creating total casein and k-casein applications, casein micelle size and clotting features (Caroli et al., 2009). Cheeses are mostly made by enzymatic (rennet) coagulation. Enzyme (rennet) traditionally used for cheese making has been obtained from the stomachs of young calves, buffalo and lamb (Fox and McSweeney, 2004). Enzymes developed by genetic engineering provide broad spectrum. As an alternative of traditional (old-fashioned) animal source, calf rennet-chymocin can be articulated by genetically reformed microorganisms, similar to yeasts or fungi (Beerman and Harting, 2012). The practice of calf rennet and microbial rennet (e.g., R. miehei) and their inference on proteolysis throughout cheese production have been confirmed by Yesilyurt (1992), Eren-Vapur and Ozcan (2012). Reduction in the quantity of milk clotting enzyme sources and the growth and development of the industry has created the demand for new alternative sources of chymocin. This situation has led to use recombinant enzymes. Coagulation takes approximately 35 minutes at 30°C after the addition of the enzymatic cultures and rennet, a small temperature fluctuation of 2°C from the temperature at which the milk treatment ended (Murtaza et al., 2014a). In order to formulate the solid curd and attain the pH up to 6.4 the mixture is left un- disturbed.

iii) Synersis Curd is exposed to synersis (Drying out). Main steps leading to curd synersis include coagulum cutting, cooking the curd, and stirring, pressing, salting and gel synersis promoting actions (Fox and McSweeney, 2004). Two properties of milky gel result in synersis are contraction capacity of the protein framework formed by casein micelles and ability of the gel to evacuate interstitial whey which is a function of permeability and porosity (Felfoul et al., 2013). The curd is kept alone for some time after cutting; this is known as curing period. A crust is formed on outer side of the curd during curing period to prevent exudation of fat and moisture from the curd. The stirring and cooking is followed to withdraw moisture and promote protein network shrinkage (Brown, 2002). Curd is cut into smaller pieces which increases the surface area of the curd and allows it to expel water and whey more effectively. Four series of cuts are applied on the curd; (i) along the length of the vat (ii) Perpendicular to the 1st cut (iii) along another angle (iv) along the edge to remove the curd from walls. Whey expulsion takes place upon cutting the curd and causes shrinkage of mesh like structure around the fat globules (McMahon et al., 1993). Curd particle size and/or fat loss are affected by cutting and stirring speeds. Early cutting of gel at constant cutting and stirring speed enhances the curd fines and fat losses into whey. Overdue cutting yields stable gel which is unable to collapse and rises curd moisture content (Castillo, 2001). After draining, the curd is washed depending up on the cheese type and manufacturing technology. Curds of different Cheddar and Monterey cheese are pounded by addition of cold water, ≤ 18ºC, @ ~25% of the milk volume on the upper side of the curd bed. The curd and wash water mixture undergoes agitation and water is drained after 5-15 minutes. The principal role of curd washing is cooling and dejection of moisture loss. Reduction of lactic acid or lactose may take place during this process. Generally curd washing is not applied in conventional Cheddar cheese making (Hou et al., 2014). The curd is cooked at 39-40ºC for 15 minutes and then stirred for 45-50 minutes. Cooking promotes the synersis while stirring is necessary for uniform heat distribution throughout the curd particles (Muhammad et al., 2010). The curd is cooked after cutting to

facilitate the starter growth and development. Sometimes salt is added to lower down the starter activity to enhance the flavor of cheese (Farkye, 2004). Cheddar cheese undergoes a process called “Cheddaring” in which curd is allowed to “matt” together. In this process small particles are gathered, binded to become more compact. The acidity is between 0.45% to 0.60 %. After attaining the desirable texture, the curd is cut into slabs which are later on piled on top of one another, promoting the removal of whey from the bottom slabs. Repetition of this process continues till whole slabs attain similar texture and consistency (Stine, 1950). After Cheddaring the Curd is broken up into small pieces to make it able to be evenly salted. Salt is used in solid form and is evenly sprinkled on the curd pieces. It helps in preservation and enhancing taste (Kongo, 2013). Salt controls lactose metabolism, fresh cheese pH, the maturity rate and cheese quality (Fox, 1987; Murtaza et al., 2014b). Proteins which show a dynamic role in texture and protein solubility is modified by salt (Fox et al., 2000a). Moulding and pressing is the last step in cheese manufacturing. Metal, plastic or wooden moulds are used to give proper shape to the salted curd (Scott, 1986). Continuous mass of the curd is formed during moulding (Fox et al., 2000a).Cheese mould is accompanied with cloth and curd is filled into moulds. After filling cheese mould is put below cheese press and pressure is slowly increased (NIIR Board, 2009). Sudden high pressure should be avoided because it compresses the exterior layer and captures moisture in the body of cheese. Sometimes soaked muslin cloth in warm and somewhat salted water is aligned with stainless steel moulds to let the residual whey to discharge. After 18 hours cheese blocks are taken out from the moulds and then enveloped in plastic film and sealed (Kosikowski and Mistry, 1997b). 2.5 Cheddar cheese ripening Post coagulation operations are the determinants of the cheese classification. Ripening is the main distinguishing factor among the rennet coagulated cheeses. According to the CODEX general standard for cheese (FAO and WHO, 2010g): “Ripened cheese is cheese which is not set for intake soon after manufacturing but which must be seized for certain time, at certain temperature, and under such other circumstances as

will consequence in the compulsory biochemical and physical modifications exemplifying the cheese in question.” Ripening comprises bacteriological and biochemical modifications which change organoleptic and texture possessions of the cheese (Murtaza et al., 2014a: Pachlova et al., 2012). Cheese ripening is a result of combined effect of several biological, bacteriological, fundamental, physical and sensory variations taking place during post manufacturing storage and transforms the fresh curd to a cheese with wanted norms (Guinee and Callaghan, 2010). It is a critical process for most of the rennet curd cheeses and varies from 2 weeks to 2 years for different cheese varieties (Murtaza et al., 2014a). Ripening agents such as endogenous milk enzymes, coagulants, auxiliary lipases and proteases, enzyme arrangements of individual starter and non-starter bacteria and other micro flora are the determinants of the diversity of chemical components (Pappa et al., 2006). Ripening can be divided into two steps of reactions: 2.5.1 Primary reactions During ripening period cheese undergoes numerous primary changes to facilitate the transformation which include: (1) glycolysis ( metabolism), (2) proteolysis (hydrolysis of protein and peptides) and (3) lipolysis (hydrolysis of triacylglycerols) (Fox et al., 1996; McSweeney, 2004). These primary events are followed by secondary biochemical events which develop various unstable flavor combinations and are metabolic rate of fatty acids and amino acids (Murtaza et al., 2014a). Changes in texture and flavor production of cheese are associated with the primary reactions whereas better-quality traits of cheese flavor and textural amendments are affected by secondary transformations (Singh et al., 2003). Hundreds of combinations take part in cheese flavor development and cheese flavor is very complex. Chemical reactions like proteolysis, lipolysis and glycolysis produce flavor compounds. Flavor compounds can be distributed into volatiles and non-volatiles. Nonvolatile compounds are involved in taste and include Peptides, free amino acids, long and medium-chain fatty acids, and a lot of organic acids whereas volatile compounds are involved in aroma development and include alcohols, aldehydes, volatile organic acids, ketones, sulfides, short-chain fatty acids, and other byproducts from fatty acids, amino acids and lactose (Karimi et al., 2012). i) Glycolysis

Sugars or sugar products are broken down by enzymes of starter cultures or subordinate cultures during glycolysis (Fox et al., 2000b). During glycolysis one or other of subsequent changes may occur depending upon cheese varieties:  Left over lactose is converted into L-Lactate by starter cultures  L (+)-lactate may be racemized to unsolvable d (−)-lactate by non-starter lactic acid bacteria (NSLAB), Increase in hydrostatic pressure decreases D (-)- and L(+)-lactate (Rodriguez et al., 2012)

 Lactate may be converted to propionic acid, acetic acid, CO2 and H2O by Propionibacterium spp. During maturation of cheese, presence of lactose is not suitable because NSLAB can utilize it as growth substrate and can contribute to undesirable flavor and quality especially when present in large amounts e.g. > 108 cfu g−1 (Beresford and Williams, 2004). Further existence of lactose in cheese throughout ripening makes it not much suitable to lactose intolerant customers (Lomer et al., 2008). During cheese making lactose is converted into lactic acid by starter culture (Azarnia et al., 2006) and lactic acid is ample organic acid in all type of cheese (Izco et al., 2002). Conversion of residual lactose is into lactate depends up on temperature and salt in moisture level (McSweeney, 2004a; Murtaza et al., 2014c). At low salt- in-moisture applications and low inhabitants of non-starter lactic acid bacteria (NSLAB), left over lactose is changed primarily to L (+) lactate by the starter bacteria (Choisy et al., 2000). Nonstarter lactic acid bacteria (NSLAB) grow in all cheeses depending upon the temperature. They influence the concentration of lactate or lactose when their number exceeds 106 to 107 cfu/g (Fox et al., 1990). Lactate undergoes several reactions by several pathways and can be oxidized by lactic acid bacteria in cheese to ethanol, acetate, formic acid and carbon dioxide at rate dependent on oxygen availability (Fox et al., 2000b). Other pathways include conversion to propionate, acetate, water and carbon dioxide by Propioni bacterium spp., carbon dioxide and water by Penicillium spp. yeasts and butyric acid and hydrogen by Clostridium spp. Numerous flavoring compounds made after lactose degradation include acetic acid, propionic acid and diacetyl (Forde and Fitz-Gerald, 2000). Source of citrate in cheese is milk and 0.2 to 0.5% citrate is present in Cheddar cheese curd. In cheeses manufactured by using mesophillic starter cultures, citrate acts as precursor

for flavor development (McSweeney et al., 2004; McSweeney, 2004a). NSLAB metabolize citrates to butanediol, acetate, acetoin, and diacetyl (Cogan and Hill, 1993; Palles et al., 1998). Succinate accumulation and citrate catabolism have close resemblance to each other. Citrate metabolism depends upon succinate producing bacteria because citrate utilization and succinate formation proceeds at an almost 1:1 molar ratio. Succinate is formed in the citrate pool in cheese. Succinate production was significantly increased during 42 days of ripening when both Lb. pentosus CHCC 13992 and Lb. helveticus CHCC 4481 were used as adjuncts. Rate of succinate production was increased due to a stimulation of Lb. pentosus CHCC 13992 by Lb. helveticus CHCC 4481, through a larger pool of free amino acids (FAA) in the cheese (Moller et al., 2013). Acetate is reflected to donate cheese flavor but high concentrations may lay down foundation for off-flavors.(Karimi et al., 2012.) Diacetyl produced by citrate metabolism may be compacted to acetoin and 2, 3-butanediol in cheese medium (Thage et al., 2005). ii) Lipolysis Esterases and lipases carry out a bio-chemical incident which results in hydrolysis of ester bond in milk triglycerides and yields glycerol, free fatty acids and mono and diglycerides is called Lipolysis (Avila et al., 2007). Lipolysis is main biochemical changes occurring for the period of cheese ripening. During lipolysis free fatty acids (FFA) are liberated. Acetic acid is also extracted with fatty acids and is a chief volatile acid. During early stages of ripening, acetic acid is produced through biochemical pathways. FFA along with proteolysis products and volatile compounds impart flavor to cheese (McSweeney and Sousa, 2000; Urbach, 1993). Main lipolytic managers in cheese are lipases, lipolytic enzymes set up in milk, rennet pregastric esterases (PGE) and microflora (Collins et al., 2003b). Enzymes responsible for the production of free fatty acids are present in milk, rennet and microflora (Aminifar et al., 2014). Ozcan and Kurdal (2012) reported that addition of lipase increased the rate of lipolysis in Cheddar cheese. Ripening stages play a significant role on lipolysis and concentration of free fatty acids (C4:0–C18:2) and acetic acid, both are gradually increased during ripening (El Galiou

et al., 2013). Ozer et al. (2008) described that microencapsulation encouraged the creation of diacetyl and enlarged the concentration of long-chain free fatty acids (FFAs). Voigt et al. (2010) showed the reduction of free fatty acids in cheese treated by high hydrostatic pressure reflecting the reduced lipolytic activity. This may be due to decreased water accessibility for the enzyme or lipid protein connections which would have sheltered lipid hydrolysis by enzyme action. Lipolytic enzymes of LAB hydrolyze esters of mono, di and triglycerides and free fatty acids in Cheddar cheese. Pseudomonas, Acetobacter and Flavobacterium species of LAB have high lipolytic activity than Lactobacillus and Lactococcus spp. They are answerable for the deliverance of substantial level of free fatty acids due to high number and extended ripening period (Collins et al., 2003a; Alewijn et al., 2005). An increase in the level of free fatty acids till end of maturing has been stated by Yilmaz et al. ( 2005), Atasoy and Turkoglu (2008) , Turkoglu (2011) and Guizani et al. (2006). Increase in number of short chain free fatty acids (C4:0 to C8:0) is in high proportion than medium chain free fatty acids (C10:0 to C14:0) and long chain free fatty acids (C16:0 to C18:2). Among short chain, butyric acid is the main contributor of the characteristic aroma during ripening. Milk lipoprotein has the specificity towards FFA located at the positions sn- 1 and sn-3 of the triglyceride to impart aroma to cheese (Lavasani and Ehsani, 2012). iii) Proteolysis Proteolysis the most important process since it directly affects the flavor and texture improvement and characteristic sensory features in most cheese varieties (Sousa et al., 2001). Different enzymes catalyze the proteolysis during cheese ripening. These enzymes may be from (i) milk (ii) starter cultures (iii) nonstarter culture (iv) secondary cultures (v) coagulant like chymocin, pepsin, microbial or plant acid proteinases and (vi) exogenous proteinases or peptidases (Sousa et al., 2001). Most complex process during cheese ripening is proteolysis. Steps involved in proteolysis reaction are (i) hydrolysis of caseins to large peptides by remaining coagulant and plasmin (ii) breakdown of outsized peptides to middle and small peptides by proteinases and peptidases (iii) hydrolysis of intermediate and small peptides into free amino acids, dipeptides and tripeptides by starter peptidases (Farkye, 2004).

Extent of proteolysis is reflected by “ripening index” which is proportion of water soluble nitrogen (WSN). Milk proteases and coagulant enzymes cause the casein hydrolysis which is determined by ripening index (El-Galiou, 2013). Proteolytic activity of lactic acid microorganisms of different taxonomic groups (Lactococcus, Lactobacillus and Leuconostoc) has been studied by Prosekov et al. (2013). Showing that strains of Lactococcus lactis decompose soluble proteins existing in the environment. Proteinases of commercial rennet and the action of the enzymes of the starter culture produce cheeses with intense proteolysis (Solak et al., 2013). During ripening there is progressive breakdown of casein which increases the casein digestibility (Henning et al., 2006). Ripening and fermentation induces the proteolysis and upsurges volumes of bioactive peptides and FAAs existing in the cheese (Raynal-Ljutovac et al., 2008). Caseins (αs1-, αs2-, β-, and k-casein) are hydrolyzed by enzymes leading to the development of large and intermediate-size peptides that may be hydrolyzed by proteolytic enzymes, originating from the microflora (SLAB, NSLAB, and probiotic adjuncts) of the cheeses, into smaller peptides and FAAs. Small peptides and FAAs are involved in flavor development (Lynch et al., 1999; McSweeney 2004a). Flavor and texture of cheeses can also be changed by the adding probiotics that affect the proteolysis pattern in cheese (Karimi et al., 2012). Certain microorganisms release small peptides and free amino acids (FAA) due to their proteolytic capability. These include L. acidophilus, L. casei, and L. paracasei (Gomes et al., 1998). Small oligopeptides develop beneficial and characteristics flavor of cheese whereas large casein-derived peptides exhibit a bitter flavor (Law and Tamime, 2011). Proteolysis remains low at low ripening temperatures (4ºC) as related to usual maturing temperature (8 to 10 ⁰C) of cheese (Ong et al., 2006). Nonstarter bacteria have capability of replacing the starter’s activity by their proteolytic activity and produce peptides and amino acids of same molecular weight to those of starter’s activity. Progress of proteolysis during ripening can be determined by peptide/protein hydrolysis providing amino acids and small peptides (Mikulec et al., 2010). Casein fractions αs1-I-CN and αs1-II-CN increases during 30 days of ripening by chymocin activity which is then hydrolyzed into smaller peptides (Mikulec et al., 2008). Smaller γ-CN becomes visible on the gel after 30 days of ripening due to decomposition of β-CN by

plasmin activity (Radeljevic et al., 2013). Proteinases and peptidases activity of lactic acid bacteria and non-starter bacteria cause secondary proteolysis (Sousa et al., 2001). Volatile sulphur compound (VSC) has profound effects on organoleptic quality of ripened cheese. Caseins contain methionine and cysteine which are catabolized to produce VSCs (Landaud et al., 2008). Main sulphur amino acid found in cheese curd is methionine (Bonnarme et al., 2001; Bonnarme et al., 2000). Degradation of methionine to methanethiol (MTL) give rise to the VSCs. MTL is later on oxidized to produce dimethyl trisulfide (DMTS), dimethyl disulfide (DMDS) and other VSCs, such as thioethers and thioesters (Landaud et al., 2008). 2.5.2 Secondary reactions Secondary actions leading to the creation of volatile flavor compounds include: i) Metabolism of free fatty acids Free fatty acids (FFAs) act as predecessors for making of impulsive flavor compounds from side to side reactions jointly known as metabolism of fatty acids. Fat also influences the flavor and flavor development in Cheddar cheese as studied by Drake et al. (2010). In most of cheese varieties, fat is the main component which directly and indirectly contributes to texture, rheology and cooking properties and flavor. Free Fatty acids act as ancestors for other volatile flavor complexes, such as hydroxyacids, n-methyl ketones (alkan- 2-ones), lactones, secondary alcohols, thioesters and esters (Guinee and McSweeney, 2006). Reaction of free fatty acids with alcohol produces esters. propyl, butyl and methyl esters have been found in cheese. During cheese ripening esters are made by transesterification of FFA (Holland et al., (2002). FFAs react with sulfhydryl compounds to form thioesters generally methanethiol (CH3SH) thus creating methylthioesters. (McSweeney and Sousa, 2000; Collins et al., 2003a). Hydroxy acids act as precursors to form lactones. Lactones with five and six-sided rings (γ- and δ-lactones), respectively have been found in cheese (Collins et al., 2003a). P-oxidation of free fatty acids liberate methyl ketones such as 2-Pentanone, 2- Nonanone that impart typical flavor to cheese. Eleven methyl ketones have been recognized in cheese (Collins et al., 2004). Numerous factors affect the degree of production of methyl ketones. These include physical state of the mould, temperature and concentration of forerunner free fatty acid. Maximum methyl ketones are produced at the pH of 5 and 7

(Gripon, 1991). Enzymes of P. roqueforti reduce methyl ketone to secondary alcohols (e.g. pentan-2-ol, heptan- 2-ol and nonan-2-ol) (Martelli, 1989; Engels et al., 1997). Cullere et al. (2009) reported that l-Hepten-3-one was identified as odorant responsible for the mushroom note. ii) Metabolism of amino acids Amino acids catabolism is another secondary event during ripening as a result of which flavoring compounds are produced. Fernandez and Zuniga (2006) reported that each amino acid undergoes a separate catabolic pathway to form the specific compounds. Compounds formed during catabolism include aldehydes, indole, ammonia, phenols, amines and alcohols, which subsidize as a whole to cheese flavor (Urbach, 1995). Three steps have been recognized in this complex process by Tavaria et al. (2002) as follow:  Hydrolysis of side chain, desulfuration, transamination, deamination and decarboxylation  Transformation of the resultant compounds (amines and α-ketoacidosis)  Decline of aldehydes to alcohols or oxidation of aldehydes to carboxylic acids Formation of flavor compounds by amino acid catabolism pathways has been studied by several authors (Smit et al., 2005; Yvon and Rejien, 2001; McSweeney and Sousa 2000). Aroma of different cheeses is enhanced by the flavor produced through above metabolism and has been studied through many research works (Randazzo et al., 2010; Di-Cagno et al., 2003; Qian and Reineccius, 2002; Moio et al., 2000; Moio and Addeo, 1998; Bosset and Gauch, 1993). Curtin and McSweeney (2004) studied the pathways for the catabolism of the aromatic amino acids Phe, Trp, Tyrosine. Amino acids are converted into flavor compounds by two major pathways:  Elimination reactions catalyzed by amino acid lyases  Pathways initiated by amino acid aminotransferases Production of methanethiol from methionine contributes to cheese flavor. Almost all LAB contain two distinct C–S lyases, cystathionine b-lyase and cystathionine g-lyase which is involved in flavor formation in cheeses (Liu et al., 2008). Methionine is converted to ∞-keto acids by aromatic and branched-chain aminotransferases (Smit et al., 2005). ∞-keto acids play important role in aroma development and are converted into acetyl-CoA derivatives, a- hydroxyacids and aldehydes. Aldehydes are then transformed into alcohols and carboxylic

acids. Aroma compounds formed through above pathways are important for development of beneficial and detrimental flavor in cheese (Ardo, 2006). 2.6 Processed Cheddar cheese The term processed cheese is often used to refer to different types of cheese as explained by the code of federal regulations (CFR) of USA. These categories of cheese differ from each other on the basis of minimum fat contents on dry matter basis, extreme moisture content, and lowest final pH, as well as amount and the number of elective constituents that can be used in the manufacture of cheese (21CFR133.169 to 133.180) (FDA, 2006). According to CFR, three major categories of processed cheese are pasteurized processed cheese (PC), pasteurized processed cheese food (PCF), and pasteurized processed (PCS). Process cheese was first manufactured in Europe in 1890. Commercial manufacturing of processed cheese took place in Europe and USA in 1910 and 1920. Process cheese was developed to be shipped to warmer climate and during wartime due to its long shelf life (Ustunol, 2011). Availability of emulsifying salts increased the popularity of processed cheese with the production of a variety of cheeses using the Cheddar cheese as main constituent. Processed cheese production contributed to 20% of total cheese produced in 2010 in USA (USDA, 2011). Technological and developmental aspects of processed cheese have been reviewed by many authors (Marchesseau et al., 1997; Schar and Bosset, 2002; Dimitreli and Thomareis, 2007; Kapoor and Metzger, 2008). High quality natural cheese is used to make process cheese and it provides important nutrients such as calcium, phosphorus and protein. Shelf life of processed cheese is longer than unprocessed cheeses. It is very tasty, smooth and medium firm and is used in different ways to form wide variety of culinary dishes (Goyal and Goyal, 2013). Mixing dairy and non-dairy ingredients including sodium chloride, emulsifying salts and one or more than one varieties of cheese followed by heating process to make a homogeneous product for longer shelf life leads to the formation of processed cheese. Physicochemical characteristics of which can be attributed to its microstructure (Kapoor and Metzger, 2008). Awad et al. (2004) described the manufacturing of processed cheese by mixing one or more varieties of natural cheese of same or different ageing periods, color, emulsifying salts

and water followed by heating to produce uniform mixture. Process cheese is an oil-in water type emulsion and can be packed in different forms including blocks, cakes, slices and sticks depending upon end use (Tamime et al., 2011). Sodium chloride has been used for many years as preservative and flavor enhancer to make process cheese (Ayyash and Shah, 2010). Pasteurized process cheese is a dairy product having 1% moisture contents in excess from the natural cheese used for its production and not exceeding 43% moisture in final product. Processed cheese essentially comprise at least 47% fat in dry matter and emulsifying salts (Phosphate or citrate based) up to 3% w/w. Cream, anhydrous milk fat, acidifying agents to modify the pH to 5.3 or greater, salt, water, mold inhibitors, artificial colors, as an antisticking agent and enzyme-modified cheese are the noncompulsory ingredients (Glass and Doyle, 2013). 2.7 Manufacturing steps Process cheese manufacturing comprises on numerous steps among which most critical is the selection of natural cheese as it is the major. As mature cheeses are expensive and take much more time to reach the maturity so the manufacturers seek alternatives to use young cheeses and blend them with mature cheeses to produce processed cheese (Tamime et al., 2011). 2.7.1 Selection and formulation of ingredients Different parameters like, texture, flavor and quality of the end product are prominently affected by the blend of ingredients selected for process cheese making. According to Chambre and Duurelles (2000) processed cheese is a product formed by combining cheeses of dissimilar type and ripeness with emulsifying salts. A processed cheese made by using mozzarella and Cheddar cheeses up to 60-75% provided not only suitable flavor, but also indorsed good melting properties (Callahan and Metzger, 1992). The main characteristics of natural cheeses used for processed cheese manufacture are variety, flavor, ripeness, uniformity, pH and texture (Fox and McSweeney, 1998; Guinee, 2002). Moreover the natural cheese must have optimum fat and moisture contents to impart texture and flavor to the resultant processed cheese (Fox and Fox, 2000; FAO and WHO 2007). Degree of maturation, milk composition and processing conditions may cause variation in natural cheese composition e.g., calcium-to-casein ratio, pH and level of integral casein (Guinee, 2000). High hydrostatic treatment increases shreddability of natural cheese

by reducing manufacturing costs and time to acquire the desired cooking properties (Rodriguez et al., 2012). Natural cheese is grinded to ensure good and maximum contact between the emulsifying salts and cheese blends (Caric and Kalab, 1993). Formulations of process cheese are reliant on the type and end usage application of the final product. Final functional properties of processed cheese are affected by compositional and chemical properties in numerous ways. Selection of appropriate ingredients is key step in process cheese formulation and includes natural cheese (type and age), price and convenience of supplementary ingredients. Computer based formulation programs are available which use cost and composition of a component as a benchmark for mounting a formulation (Kapoor and Metzger, 2008). Before the heat processing in the making of the cheese blend, adding emulsifying salts is last step. Emulsifying salts are necessary for process cheese manufacture, because in the absence of emulsifying salts when natural cheese is heated a gummy like substance is formed that causes separation of fat. Emulsifying salts remove the problem of fat separation and calcium binding and caseins solubility is increased. Better solubility of caseins increases its emulsification properties. With the help of emulsifying salts protein environs the fat drops; stabilizes fat and results in a plane homogenized cheese (Fox et al., 2000a). Calcium binding is very much essential because calcium decreases water solubility of the casein and lowers down emulsifying influence of protein. Emulsifying salts replace calcium with sodium or bind calcium in order to cover its effects (Shimp, 1985). Suitable combination of emulsifying salts enhances pH of the cheese blend which in result increases the calcium-masking aptitude of the emulsifying salts and provides a stable product (Mulsow et al., 2007). In the absence of emulsifying salts diverse, gluey, pudding- like mass is formed that go through extensive oiling-off and moisture exudation during manufacture and on cooling (Guinee, 2007). 2.7.2 Processing, packing, cooling and storage of process cheese Process parameters that control the emulsion formulation and different functional properties of process cheese include cooking temperature, cooking time, mixing and rate of cooling (Glenn et al., 2003; Shirashoji et al., 2006; Garimella et al., 2006; Zhong et al., 2004). According to Lee et al. (2004) mixture of natural cheeses, chelating salts, and

water was heated and stirred at a temperature between 70ºC and 95ºC for a short period of time (about 5–10 min) to form process cheese. Fagan et al. (2007) made processed cheese by mixing ingredients for 1 min. and cooking to 80ºC by indirect steam heating for 2 min. During cooking process, blend was continuously agitated using a knife at 300 rpm and baffle mixer at 80 rpm. After cooking, blend was decanted into food grade plastic containers which were later on lidded, cooled and stored at 4ºC. Different designs and operating conditions of the cookers used for the preparation of process cheese have altered the cooking temperature range between 70 ºC to > 100ºC dependent on the cooker design and the variety of process cheese (Kapoor and Metzger, 2008). The hot process cheese is usually surrounded in lacquered foil which is then retained in cartons, cans or tubes (Caric, 1993). Processed cheese is cooled immediately after manufacture as the slow cooling results in the firm body of the processed cheese. Nature of cooler considerably marks the functional properties of processed cheese. Forced convection cooler provides less firm and more meltable process cheese as compared to the processed cheese cooled in the free convection cooler (Zhong et al., 2004). Omar et al. (2012) manually filled process cheese into glass cups protected with aluminum foil, cooled and then kept at about 5°C -7 °C. Process cheese should be put in storage beneath 10°C (Caric, 1993). Normally it is stored between 8-10°C. Labeling of process cheese depends upon the natural cheese and amount of salt used for making process cheese (FDA, 2008). 2.8 Factors affecting quality of processed Cheddar cheese Amount of raw ingredients and the type of processing method used for the formulation, determines the quality of processed cheese. Compositional changes and processing conditions mainly contribute to the characteristics and functional properties of processed cheese (Kapoor and Metzger, 2008). Quality standards of the cheese are based on the fat and moisture contents of the final product which comes from the basic ingredient, cheese from which they are made (Fox and Fox, 2000; FAO and WHO; 2007). Three main factors which affect the quality and functional properties of process cheese are (i) natural cheese (ii) processing conditions and (iii) emulsifying salts (type and amount).

2.8.1 Natural cheese Process cheese might have different forms i.e. slice, block, spread but based upon natural cheese (Mihaela et al., 2013). Amount of natural cheese varies from 51 to 80% in processed cheese making depending on type of processed cheese (FDA, 2006). Oldness of natural cheese affects the properties of processed cheese (Brickley et al., 2008). Intact casein level existing in the natural cheese mainly affects the process cheese functionality. During ripening the casein which is not hydrolyzed by enzymes is the intact casein and it forms the structural network of processed cheese (Berger et al., 1998). There is an inverse relation between age and intact casein, hence as the age of natural cheese increases the intact casein decreases (Garimella et al., 2006). Natural cheese influences the melt-ability and melted texture of the process cheese (Kapoor and Metzger, 2008). Type, maturity and physical properties of cheese are the basic elements that affect the properties of processed cheese (Tamime et al., 2011). Textural, viscoelastic, functional, microstructural, and sensorial properties of processed cheese are measured by the brand and age of the natural cheese used for making it (Bowland and Foegeding, 2001; Acharya and Mistry 2005; French et al., 2002; Glenn et al., 2003). Exo-polysaccharides (EPS) modify the protein network of the Cheddar cheese and affect the hardness of processed cheese. Hardness of processed cheese prepared by using one month old Cheddar cheese having EPS was lower than processed cheese manufactured with one day old Cheddar cheese. Moreover hardness of the one month old Cheddar having EPS was lower than Cheddar of the same age prepared without EPS. Reduction in hardness of cheese by EPS producing cultures may be due to increase in moisture contents (Awad et al., 2013). 2.8.2 Processing conditions Final quality of processed cheese is significantly exaggerated by the formulation and the processing method used in the manufacturing of processed cheese. The desired final characteristics of processed cheese that determine its end use applications are characterized by the ingredients and processing method (Garimella et al., 2006). Formulation is characterized by the type and amount of ingredients used. Process conditions include the temperature during mixing and cooking and the speed of mixing and cooling rate. All these

parameters have emotional impact on the quality of the processed cheese (Kapoor and Metzger, 2008). The uniform melting quality of the processed Cheddar cheese is a desirable property. Meltability of processed cheese can be explained by two-component system which includes melting casein network and un-melting whey protein network. Aggregation of whey proteins or possible interaction between whey proteins and casein causes increase in elasticity of the cheeses. Cooking time and temperature play important role in melting properties of cheese. Increased cooking time produces softer and more meltable cheese whereas increased cooking temperature decreases firmness and increased meltability and spreadability (Swenson et al., 2000). Whey protein isolate and whey protein polymers when added to process cheese caused a decrease in its meltability (Mleko and Foegeding, 2000). When concentration of whey protein concentrate (WPC) was increased, the meltability of processed cheese was decreased (Gupta and Reuter, 1993; Shirashoji et al., 2006). During processing, viscosity, firmness, flow behavior and meltability of processed cheese food (PCF) are affected by the mixing speed. Viscosity and the firmness of the PCF, increased, whereas melting and flow properties of PCF declined with the increase in the mixing speed. A uniform distribution in fat globule was observed when PCF manufactured using high mixing speed (Garimella et al., 2006). There are numerous factors including pH that affect the textural and melting properties of processed Cheddar cheese (Mulsow et al., 2007). Process cheese must ensure pH below or equal to 5.6 as described by Food and Drug administration (2006) pH of the processed cheese also play role in the body and texture development. If pH is too low i.e. <5.4 it will cause brittle, crumbly and grainy texture (Berger et al., 1998; Shirashoji et al., 2006). Proteins become closer to their isoelectric point at low pH causing decrease in net negative charge. Repulsive forces between protein molecules are diminished resulting in increase of protein-protein interactions and causing proteins to shrink. As proteins are aggregated, give fragile process cheese emulsions with a brittle and coarse texture (Shirashoji et al., 2006). Low concluding pH of the process cheese causes oil separation (Meyer, 1973), however toxin production and microbial spoilage is also prevented due to enhancement of the

inhibitory activity of sorbic acid (Glass and Doyle, 2013). El-bakry et al. (2010) described that decrease in level of salt causes decrease in the pH. 2.8.3 Emulsifying salts Most important among the ingredients of process cheese are the emulsifying salts and the natural cheese. Most commonly used salts in cheese are the sodium salts of citrate and phosphate (Gupta et al., 1984). Thirteen (13) emulsifying salts have been identified by the CFR (U.S. department of health and human services, 2003) which include: sodium metaphosphate (sodium hexametaphosphate), sodium aluminum phosphate, sodium citrate, tetra sodium pyrophosphate, monosodium phosphate, disodium phosphate, tri sodium phosphate, di potassium phosphate, sodium acid pyrophosphate, potassium citrate, calcium citrate, sodium potassium tartrate and sodium tartrate. Emulsifying salts have been divided into three main groups by Kapoor and Metzger (2008) as citrates, polyphosphates and monophosphates. These emulsifying salts are used singly or in combination up to 3% w/w. These emulsifying salts perform the functions of ion exchangers, buffers, and Ca sequestrants and peptization. According to Dalgleish (2006) emulsifying salts perform following functions during process cheese manufacture: 1) Break casein micelle to release Ca from micelle 2) Make the proteins to become soluble 3) Emulsification of fat to make stable emulsion 4) Adjustment and control of pH 5) Produce homogeneous plastic mass upon cooling Chandan and Kapoor (2011) declared the emulsifying salts to perform two main functions i.e. chelation of calcium (to disrupt the cross-linked protein network of calcium- phosphate existing in natural cheese) and adjustment of pH. Meltability of processed cheese is not affected by the kind and extent of emulsifying salts as declared by Shirashoji et al. (2006). Trisodium citrate and disodium phosphate are most commonly used emulsifying salts in USA. In process cheese varieties which are manufactured for slice, trisodium citrate is preferred whereas di sodium phosphate or a combination of disodium phosphate and trisodium phosphate is used in varieties which acquire loaf type properties and storage at

room temperature (Kapoor and Metzger, 2008). Pal et al. (2002) reported that disodium phosphate and trisodium phosphate produced most pronounced melting effects. When processed cheese made with sodium hexametaphosphate (SHMP) the increase in pH increases the meltability (Lu et al., 2008). When concentration of SHMP is increased it will increase the hardness of process cheese (Shirashoji et al., 2010). The main function of SHMP is to disperse the insoluble CN matrix in natural cheese. Orthophosphates or TSC have low CN dispersing ability as compared to polyphosphates (Dimitreli et al., 2005 and Mizuno and Lucey, 2005). Hardness of processed cheese decreased in the succeeding manner polyphosphate > triphosphate > diphosphate > monophosphate when individual phosphate salts were applied (Dimitreli and Thomareis, 2009; Sadlikova et al., 2010). When mono, di and tri phosphates mixed with poly phosphate as binary mixtures, the increase in polyphosphate in the mixture increased the hardness of the processed cheese (Weiserova et al., 2011). Phosphate salts containing two or more phosphorus atoms in a molecule when hydrolyzed cause an increase in the hardness of processed cheese (Awad et al., 2002; Guinee et al., 2004; Shirashoji et al., 2010 and Weiserova et al., 2011). Adhesiveness, Cohesiveness and hardness of the processed cheese depend up on arrangement of ternary emulsifying salts of sodium phosphates. Specific ratios of monophosphate to diphosphate have emotional impact on the texture properties of block processed cheese. Cohesiveness and adhesiveness of processed cheese decreased when monophosphate and diphosphate used at ratio of 3:4 while 1:1 ratio of polyphosphates causes increase in the hardness (Bunka et al., 2013). Commonly used emulsifying salts are the sodium based phosphate salts. Although phosphorus is an essential nutrient but its large quantity causes damage to bone structure resulting in osteoporosis particularly if calcium consumption is very low (Cashman, 2006). Large quantity of phosphorus in processed cheese lowers the calcium to phosphorus ratio (Schaffer et al., 1999). Moreover, common emulsifying salts have sodium in large concentrations which is considered as a risk factor in human nutrition nowadays (Palar and Sturm, 2009). In order to increase the calcium to phosphorus ratio and decrease sodium utilization, efforts have been made to prepare processed cheese without the adding emulsifying salts. Many studies describe the partial and complete substitution of emulsifying

salts with monoglycerides, hydrocolloids, lecithin and gums (Cernikova et al., 2010). Emulsifying salts serve as additional source of sodium in processed cheese. (Cruz et al., 2011b). Appropriate labeling of sodium contents on the processed cheese packaging is necessary for the consumers to purchase these products according to their needs (Chand et al., 2012; Kim et al., 2012; Drake et al., 2011; McLean et al., 2012; Sanz-Valero et al., 2012; Dunbar, 2010). El-Bakry et al. (2010) stated that potassium chloride when used as emulsifying salt replacer had a substantial effect on the salt content of process cheese. Emulsifying salts should be used according to the dosage as provided by the supplier on the package to achieve typical chemical and sensorial characteristics (Mihaela et al., 2013). 2.9 Sensory studies of cheese Scientific discipline used to suggest amount, examine and take to mean those replies to products as professed through the senses of sight, touch, smell, taste and hearing is defined as sensory evaluation (Sidel and Stone, 1993). covers vide areas like; raw material inspection, selection of packaging material, Product development and improvement, quality control, cost reduction and shelf life/ storage studies. Humans are the measuring instruments for all sensory methods (Ackbarali and Maharaj, 2014). Sensory attributes are helpful for the identification of cheeses and are widely appreciated by consumers (Barcenas et al., 2004; Murtaza et al., 2013). Parameters used to define the sensory include texture, color, taste, aroma and visual appearance (Di- Monaco et al., 2008). Cheeses are characterized by using sensory methodologies. Descriptive sensory analysis (Barcenas et al., 2001; Barcenas et al., 2003a) and free profiles (Barcenas et al., 2003b) have been used to characterize cheeses. Sensory judgments involve four stages: Coded sensation, physical concentration, overt response and perceived concentration (Van Trijp and Schifferstein, 1995). As products or materials are evaluated by people during sensory evaluation so these must have qualities: Shouldn’t have personal prejudice, preferences to ensure an objective evaluation and differentiation between and description of product characteristics. Should be trained to perform sensory evaluation repeatedly and should be audited and evaluated to determine their competencies. Selected to be representative (target market) consumers / respondents (Ackbarali and Maharaj, 2014).

Sensory properties of processed cheese are measured by the variety, characteristic, and ripeness of the base cheese (Bowland and Foegeding, 2001; Acharya and Mistry, 2005; French et al., 2002; Glenn et al., 2003). Among sensory properties of process cheese central aspect is taste which is delivered by salt. Salt (NaCl) not only contribute towards good sensory score but also increases shelf life of processed cheese (Liem et al., 2011). Kamleh et al. (2012) used KCl as a replacement for NaCl during process cheese manufacture and found that KCl bounces crumbliness, bitterness, and moistness to the process cheese. Emulsifying salts have pronounced effects on the sensory and textural properties of the processed cheese. Reduction in emulsifying salt quantity causes the increased processing time and hardening of the resultant product. High rate of reduction of emulsifying salts up to 40% caused the very hard texture of cheese (El- Bakry et al., 2010). Potassium containing emulsifying salts has a substantial impact on the sensory feature of process cheese (Hoffmann et al., 2012). Color is a part of quality control point in cheese. Milk composition and cheese making techniques affect texture and flavor and to some extent color of cheese (Drake et al., 2009). Cheese color is influenced by intrinsic factors (carotenoids, β-Carotene, biliverdin, bilirubin, animal species, animal feed and light scattering) of milk used for cheese making and extrinsic factors (colorants, packaging and storage of cheese). Casein micelles and the milk fat globules are the primary contributors to the milk opacity and its color by contributing to the higher refractive index of milk than water. Cows accumulate high concentration of carotenoids, especially β-carotene due to lower vitamin A synthesis in enterocytes (Hincu et al., 2010). This gives cow milk a slight yellow tint as matched to buffalo milk. Fresh buffalo milk holds a blue green pigment (biliverdin) which gives buffalo milk entirely different appearance than the cow milk (Sahai, 1996). Appearance of the product is the key factor which attracts the consumers at the time of buying. Sensory attributes like color, size, shape and surface texture collectively contributes to the overall appearance of the product (Schroder, 2003). Almost 300-400 receptors are present in the nose which responds to volatile flavor components (Brand and Bryant, 1994; Laing and Jinks, 1996). When volatiles pass through nasal passages the resultant odor is called aroma of the product (Meilgaard et al., 1999).

Series of chemical, enzymatic and bacterial reactions taking place during ripening of Cheddar cheese are responsible for developing sensory characteristics (Pollard et al., 2003; Smit et al., 2005; Azarnia et al., 2006). Coagulant used for cheese manufacture, color and texture interact with each other to give appearance to cheese (Delahunty and Drake, 2004). During storage period, the texture integrity of the cheese is lost due to proteolytic activities and low water availability. However, gumminess increases during storage period whereas chewiness increases after 3 month of storage period (El-Baz et al., 2011; Awad et al., 2014). Acceptability of processed cheese decreases with the extension in storage period (Awad and Salama, 2010a and 2010b). Descriptive sensory analysis is the most influential device in cheese flavor study. In this method sensory aspects of foods including texture, flavor, appearance, aroma or other aspect are identified by a panel of 6 to 12 trained individuals (Meilgaard et al., 1999). Panel of these trained individuals act as an instrument. It may take 75-100 hrs. to train the panel for detection of good cheese flavor (Drake and Civille, 2003). To ensure the reliable results there is need of validation and monitoring of the panel personnel and sometime retraining (Castillo et al., 2008). Castaneda et al. (2007) used descriptive quantitative analysis for sensory evaluation using a trained panel of 13 people. In their studies sensory profile of texture included the following properties: The: springiness (ability to recover its initial thickness rapidly after compression and deformation), firmness (resistance that the sample presents with small displacements of the jaws), friability (ability to generate several pieces from the beginning of chewing), deformability (ability of the sample to deform successively or stretch easily before breaking the sample in the buccal cavity), adhesiveness (moving the tongue to detach the sample stuck in the palate or teeth), and cohesiveness (firmness of the internal joints in the cheese sample). Salari and Mortazavi (2008) performed sensory evaluation of processed cheese after 60 days of storage by cutting cheese into small cubes of (20x20x20) mm and keeping cheese samples at room temperature 25 ± 1 ºC. They used deionized water to take away palate during sensory assessment of samples. They assigned marks for texture, flavor, odor, body and appearance as very good (5), good (4), acceptable (3), bad (2) and very bad (1).

Consumer behavior helps the scientists and product developers to produce the desired products and the sensory tests are helpful to assess the potential buyers of a specific product (Piggot, 1988). Quantitative tests are helpful to get consumers liking and disliking data within short time through set of questions about consumer preferences, liking and sensory attributes etc. (Meilgaard et al., 1999). Consumer acceptance is quantified using tools like 9 point hedonic scale has been used in industry for several years in sensory evaluation of foods. It is used to compare the rating provided by judges. Its use has been validated in scientific literature (Stone et al., 1993). Different scales like 5-, 7- and 9-point have been used and for adults due to the adults’ behavior to avoid extreme rating of scale while judging food products (Resurreccion, 1998).

Chapter 3 MATERIALS AND METHODS

Research work was carried out in processing hall, Institute of Food Science and Nutrition and Hi-Tech. laboratory, University of Sargodha, Sargodha, Hi-Tech. laboratory, University of Agriculture, Faisalabad and Noon Pakistan Limited Bhalwal, District Sargodha. The details of materials used and methodology applied are given below; 3.1 Procurement of raw materials 3.1.1 Raw milk Raw milk from cows and buffaloes was procured from local dairy farms of Bhalwal region of Sargodha for the preparation of Cheddar cheese. Milk was analyzed in its original form and fat content were standardized at 3% level. The standardized milk was also analyzed to assess its suitability for cheese making. Following tests were performed. a) pH The pH of Milk was measured through electronic digital pH meter (Inolab WTW Series 720). Buffer solution of pH 4 and 7 were used to calibrate the pH meter. Milk sample was taken in a beaker; electrode of pH meter was immersed in the sample to determine pH (AOAC, 2000). b) Acidity Acidity in milk samples was determined by the method (No. 947.05) given in AOAC (2000). Nine ml of milk sample was taken in a titration flask and 2-3 drops of phenolphthalein were added to it. The sample containing indicator was titrated against 0.1N NaOH until light pink end point appeared and for few seconds. Volume of 0.1 N NaOH used was recorded to determine acidity of milk in terms of lactic acid by using following expression: % Acidity (as lactic acid) = Volume of NaOH used X 0.1 c) Fat The Gerber method was used to determine fat content in milk (Marshall, 1993). Sulphuric acid (10ml) was poured into butyrometer and then 10.94ml of milk sample was

slowly added into butyrometer followed by 1ml of amyl alcohol. The contents of butyrometer were thoroughly mixed and centrifuged at 1100 rpm for 5 minutes. Butyrometer was transferred to a water bath at 65°C for at least 3 minutes and the percent fat was recorded directly from the butyrometer scale. d) Crude protein The nitrogen content in milk sample was estimated by using Kjeltech (Model: D- 40599, Behr Labor Technik GmbH, Germany) based on Kjeldahl’s method (AOAC, 2000; No. 991.20). One ml of milk sample was digested in digestion tubes using two digestion tablets and 20 ml of sulphuric acid. Digested sample was distilled with 40% NaOH. The distillate was collected in 50 ml 4% boric acid solution and titrated against 0.1 N HCl. The protein content in milk was estimated by multiplying the percent nitrogen with 6.38. Vol. (sample-blank) HCl x normality of HCl x 0.014 Nitrogen (%) = ------x 100 Weight of sample Protein (%) = N (%) x 6.38 e) Total solids The total solids content in milk was determined by drying the milk to constant weight at 105oC overnight using heating oven (AOAC, 2000). f) Solids-not-fat (SNF) The SNF content in milk was calculated by the difference in total solids and fat content of milk. 3.1.2 Starter cultures A direct Vat Set (DVS) mesophillic homofermentative starter culture R-704 of CHR- Hansen, Denmark was purchased from the distributor of CHR-Hansen in Pakistan. 3.1.3 Emulsifying salts Different emulsifying salts (trisodium citrate, disodium phosphate and trisodium phosphate) were purchased from local market. 3.2 Manufacturing and ripening of natural Cheddar cheese Natural Cheddar cheese was prepared following the standard protocol, with some modifications, as described by Murtaza et al. (2014a) at Noon Pakistan Limited Bhalwal near Sargodha. Three batches of Cheddar cheese were prepared from cow milk and three from buffalo milk at different (one month) intervals to get Cheddar cheese with different ripening

periods. Raw milk of cow and buffalo was purchased from local dairy farms having chilling facilities. It was transported to Noon Pakistan Limited Bhalwal through vehicle containing insulated double jacket SS-316 tank. Initial quality tests of raw milk were performed in milk reception laboratory, Noon Pakistan Limited. Milk was then pasteurized and clarified through plate type pasteurizer and cream separator at the temperature of 82ºC for 8 Sec. After pasteurization 500 liter milk was standardized to maintain fat level at 3% and shifted to cheese vat. Milk was heated to 31°C through indirect steam in the vat and inoculated with freeze dried mesophillic homofermentative microbial culture R-704 (CHR-Hansen, Denmark). After addition of culture, agitators were turned on for uniform distribution of culture into milk. Milk was held at this temperature for 30-40 min. for the optimum growth of culture. After inoculation and incubation of milk the chymocin powder (Extra NB 5000 from CHR- Hansen, Denmark) was added @ 1g / 50 liter of milk. After addition of chymocin powder the milk was agitated for 10 min. for its uniform distribution and milk was held at 31°C for 40- 50 min. to be coagulated. Coagulation was checked by inserting the SS knife into curd. When sufficient coagulation was done and the curd attained the pH of 6.4, it was cut into cubes using screens made of SS wires. Cutting facilitates to expel whey and water more effectively. After cutting the curd was heated to 37-38 ºC for without stirring. The curd was then stirred for ½ hr. to promote synersis and to release maximum water and whey and for starter growth and development. Whey was removed when curd attained the pH of 6.0-6.1. The curd pieces were then gathered at one corner of the vat to get compact shape. On attaining the desired shape and the acidity up to 0.60% the curd was cut into slabs to perform the Cheddaring process during which the curd slabs were piled on one other by changing sides of the slabs. This process continued till all slabs got similar texture. When it reached the 1.0% acidity, the curd was broken into small pieces called milling process by using milling machine and at the same time salt NaCl was sprinkled at 2% rate.to control the pH. Salted curd was then put into moulds made of SS plates to give it proper shape. After molding the curd was pressed using hydraulic press at a pressure of 4-5 bars. Muslin cloth was used as lining of the mould to facilitate the removal of remaining whey. Pressing was done for 18 hours and then removed from moulds and vacuum packed in polythene bags and stored at 4-6 ºC for 3 months. The manufacturing procedure is also outlined in Fig. 3.1.

Addition of salt Raw milk Milling 2%

Standardization Cheddaring Moulding

Pasteurization Whey removing Pressing (82ᵒC for 8Sec.) at pH 6.0-6.1 (5Bar 12-18 Hrs.)

Analysis of Cooking the curd standardized milk 37-38 ºC for Vacuum packing 40-45 min.

Addition of starter culture Cutting the curd Ripening 4-6 ºC 31ᵒC

Fig. 3.1 Incubation at Addition of Natural Cheddar 31ᵒC for rennet 1g/50Ltr. cheese 30-40 min. 31ᵒC 40-50 min.

Fig.3.1 Flow chart of natural Cheddar cheese manufacturing

3.3 Quality evaluation of natural Cheddar cheese 3.3.1 Physico-chemical analysis During ripening, natural Cheddar cheese was evaluated for moisture, fat, protein, ash, pH, acidity, salt and minerals content with the interval of one month during ripening. The details of the procedures followed are as under. a) Moisture Moisture content was determined by oven drying (Method No. 926.08; AOAC, 1990). 3 g of grated and shredded cheese sample were weighed in a round, flat bottom metal dish and placed in oven at 100°C and dried to constant weight, cooled and weighed. Loss in weight was expressed as moisture. Weight of sample before drying - Weight of sample after drying Moisture (%) = 100 X ------Weight of sample before drying b) Fat The fat content in cheese was determined by Gerber method as described by Marshall (1993). 10 ml sulphuric acid was added into butyrometer followed by 3 ml distilled water at 60°C. Then cheese sample wrapped in grease proof cellophane paper was inserted to butyrometer and again 5 ml distilled water at 60°C and 1 ml of amyl alcohol was added. The butyrometer was shaken vigorously to dissolve cheese particles completely and then centrifuged it at 1300 rpm for 5 minutes. The butyrometer was placed in water bath at 65°C with the stopper downwards for 2 to 3 minutes and recorded the percentage of fat by measuring fat column. c) Crude protein Crude protein was determined by Kjeldahl method (Method No. 920.123; AOAC, 1990). One g of grated and shredded cheese sample was digested in Kjeldhal digestion flask with concentrated sulfuric acid and digestion tablets until clear solution was obtained. The ammonium trapped in sulfuric acid was released by adding 40% sodium hydroxide through distillation and collected in boric acid solution. Then it was titrated against sulfuric acid to determine the amount of Nitrogen. Nitrogen was calculated by the following formula. 0.0014X ml of 0.1N H2SO4 used X Volume of diluted sample N% = ------Weight of sample X Volume distilled

Protein content was determined by multiplying N% to 6.38. Protein % = N % X 6.38 d) Ash Ash content was determined by igniting the cheese sample (Method No. 935.42; AOAC, 1990). 5 g of grated and shredded cheese sample were weighed in a china dish and first ignited on flame and then in a furnace at 550°C until white ash was obtained. Sample was cooled and weighed. Weight of ash Ash % = 100 X ------Weight of cheese sample e) pH

The pH of cheese slurry prepared by blending 20 g of grated and shredded cheese sample with 12 ml of water, was measured at 25°C with a pH meter (inoLab WTW Series 720) after calibrating with fresh pH 4.0 and 7.0 standard buffers (Ong et al., 2007b). f) Acidity Acidity in cheese was determined by titration (Method No. 920.124; AOAC, 1990). To 1 g of grated and shredded cheese sample, warm water was added to a volume of 10 ml, vigorously shaked and filtered. Filtrate was titrated with 0.1N NaOH using phenolphthalein as indicator. Acidity was expressed as Lactic acid %. ml of 0.1N NaOH X 0.009 X 10 Acidity (Lactic acid) % = 100 X ------10 X ml of Filtrate g) Salt The salt contents in Cheddar cheese samples were determined by Volhard method (Method No. 975.20; AOAC, 1990). Two g of grated and shredded cheese sample was weighed in 50 ml beaker; 20 ml warm water was added and stirred to break the particles to make slurry. The slurry was transferred to 250 ml Erlenmeyer flask. The beaker was rinsed with 10 ml warm 91 water and added to the Erlenmeyer flask. 25 ml of 0.1N silver nitrate, 10 ml of nitric acid and 50 ml of water was added to sample in Erlenmeyer flask. Then it was placed on a hot plate for boiling in a hood. When solution boiled, potassium permanganate was added in 5 ml portions until the solution turned brown and remained brown for at least 5 minutes on gentle boiling. Heating was continued until the brown color disappeared and resulted in a straw colored clear solution. There was white curd like particles in the solution

indicating the silver chloride aggregates. Then the hot solution was filtered into a clean 250 ml Erlenmeyer flask. Filter paper was washed thoroughly with hot water. Solution was cooled to room temperature and 2 ml ferric ammonium sulfate was added as an indicator. The excess silver nitrate was titrated with 0.1N potassium thiocyanate (KSCN) to the first pale reddish brown color that lasted for 30 seconds. The volume of 0.1N potassium thiocyanate used for titration against sample and blank was recorded. The salt (sodium chloride) content was calculated by using the following formula: [(ml of 0.1 N AgNO3) – (ml of 0.1N KSCN)] X 0.0585 Sodium chloride % = ------Weight of Sample (g) h) Minerals content in Cheddar cheese Minerals (calcium, potassium, sodium, magnesium and phosphorus) content were determined using flame photometer and atomic absorption spectrophotometer as described by Kirk and Sawyer (1991). 1 g of grated and shredded cheese sample was taken in conical flask. 20 ml concentrated nitric acid was added and heated slowly till it discolored. Then 5 ml perchloric acid was added and heated strongly till 1-2 ml solution remained. Deionized water was used to make the volume 100 ml. Standard solutions of known concentration were prepared separately and run on photometers followed by sample dilutions. 3.3.2 Organic acids Organic acids (lactic, acetic, citric, Butyric and Pyruvic) content in cheese were determined after one month intervals during ripening using high performance liquid chromatography (HPLC) following the method described by Akalin et al. (2002) with some modifications. 7 g of grated sample was added to 40 ml of buffer-acetonitrile mobile phase (0.5% (W/V) (NH4)2HPO4 (0.038 M) − 0.2% (V/V) acetonitrile (0.049 M), at pH 2.24 with H3PO4), extracted for 1 h, agitated on a shaker (JK Ultra Turrax IKA T18 basic) and then centrifuged at 6000 × g for 5 min. The supernatant was filtered once through Whatman #1 filter paper and twice through a 0.45-µm membrane filter (Sartorious SM 11606, Goettingen, Germany) and then used directly for HPLC analysis. A SHIMADZU liquid chromatograph (LC-10 AT VP Series, SHIMADZU Corporation, Japan) was equipped with a SCL-10AVP

system controller injector-fitted with a 20 µl sample loop and a SPD-10AVP UV-VIS detector. The detector was set at 214 nm. Operating conditions were: mobile phase, aqueous 0.5% (wt. / vol.) (NH4)2HPO4 (0.038 M) − 0.2% (vol. / vol.) acetonitrile (0.049 M) adjusted to pH 2.24 with H3PO4; flow rate 0.5 ml/min and ambient column temperature. A reverse-phase Skim-Pack C18 (LC) column (SHIMADZU Corporation, Japan) was used. The mobile phase was prepared by dissolving analytical-grade (NH4)2HPO4 in distilled water, HPLC-grade acetonitrile and H3PO4. HPLC-grade reagents were used as standards (Sigma Chemical Co., St. Louis, MO). Solvents were degassed under vacuum. Both solvents and standard solutions were filtered through a 0.45-µm membrane filter (Sartorious SM 11606). 3.3.3 Assessment of proteolysis Proteolysis in natural Cheddar cheese was evaluated during ripening after 30 days intervals using High Performance Liquid Chromatography as detailed by Hickey et al. (2006) and Rulikowska et al. (2013). 5 g sample was homogenized in 20 ml of 0.1 M citrate buffer at pH 3.6 at 40°C for 1 hour to separate the fat. The whole suspension was centrifuged at 3000 g for 30 min at 4°C. The resulting pellet (100 mg) was dissociated in 5 mL of disassociating solution and held in a water bath at 40°C for 1 hour with periodic mixing. This mixture was then centrifuged at 20,000 g for 10 min at room temperature, filtered through a nylon 0.45 mm syringe filter. An aliquot of 20 ml was injected into Shimadzu liquid chromatograph (LC-10 AT VP Series, Shimadzu Corporation, Kyoto, Japan) with a UV/Vis detector at 214 nm (Shimadzu Corporation, Kyoto, Japan). Separation was achieved using a gradient of two mobile phases: (A) 90% water and 10% acetonitrile with 0.1% trifluoroacetic acid (B) 90% acetonitrile and 10% water with 0.1% trifluoroacetic acid at a flow rate of 0.8 mL min-1. The gradient was 75% A for 12 min, reduced to 51% A over 31 min, followed by a further reduction to 20% A over the next 13 min, held at 20% A for another 3 min followed by an increase to 75% A in 3 min and held at this concentration for a further 2 min: total run time of 54 min. The column used was a C4 Jupiter 5 mm 300 A (Shimadzu Corporation, Kyoto, Japan). Water soluble nitrogen and total free amino acids were calculated.

3.4 Selection of emulsifying salts (combination) Based on the literature available, three emulsifying salts were selected and used as single and in different combinations to get the most suitable combination of emulsifying salts for manufacturing of processed Cheddar cheese in further studies. The emulsifying salts selected were: 1. Sodium Citrate (commonly used for slice applications) 2. Di-Sodium Phosphate (commonly used in loaf applications) 3. Tri-Sodium Phosphate

The blend of 90 days ripened Cheddar cheese of cow and buffalo milk (1:1) was used to prepare the processed cheese. Total amount of emulsifying salts used in each sample (treatment) was 3% (of total cheese weight) and different combinations formed were as given below (table 3.1).

Table 3.1 Detail of treatments (combinations) of emulsifying salts used

Sodium citrate Di-sodium Phosphate Tri-sodium Treatments (%) (%) Phosphate (%)

T1 100 - -

T2 - 100 -

T3 - - 100

T4 50 50 -

T5 50 - 50

T6 - 50 50

T7 33.33 33.33 33.33

T8 25 25 50

T9 25 50 25

T10 50 25 25

Processed Cheddar cheese samples prepared by using different ratio/combinations of emulsifying salts were analyzed to discriminate the effect of salt combinations on functional (hardness, gumminess and melt-ability) and sensory (color and flavor) properties following the standard protocols as described in further studies.

3.5 Preparation of processed Cheddar cheese Processed Cheddar cheese was manufactured from blends of raw, semi-ripened and fully ripened natural Cheddar cheese (of cow and buffalo milk) using selected emulsifying salts combination following the procedure described by Kapoor and Metzger (2008). Natural Cheddar cheese of desired ripening period was taken out from cold store. Cheese was unwrapped and cleaned with knife. Cheese blocks were cut into small pieces, milled and put into mincing machine to be minced. Minced cheese was transferred into kettle for heating and melting. Calculated amount of water and emulsifying salts were added to the kettle to get desired fat and moisture percentage in the final product. Mixture of natural Cheddar cheese, emulsifying salts and water was heated to 80 ºC for 10 to 15 min. with continuous stirring to get the homogeneous mixture. During heating process the mixture was examined by opening the base valve of kettle to know either homogeneous mixture has attained or not and the natural cheese has been properly mixed. On getting the homogeneous mixture the cheese was poured into SS trays and transferred to cold storage for cooling at 4-6 ºC for 12 hours. When cheese was properly cooled it was cut into cakes and vacuum sealed in polythene bags. Processed cheese was stored at 4-6 ºC for further analysis. The manufacturing procedure for processed Cheddar cheese is outlined in Fig. 3.2 and the detail of different samples (treatments of processed cheese) prepared is given in table 3.2.

Selection, cleaning, trimming and Storage at 4-6 ºC cutting of natural Cheddar cheese

Mincing, milling and grinding of Packing into polythene bags cheese

Transfer into kettle for heating Cutting into cakes and melting

Blending of cheese with added Cooling for 10-12 Hrs. at 6 ºC water and emulsifying salts

Processing of cheese at 80ºC for 10-15 Min. Filling into trays

Fig. 3.2 Flow chart of processed Cheddar cheese manufacturing

Table 3.2 Detail of samples (treatments of processed Cheddar cheese) prepared

Cow milk Cheddar cheese Treatments Ripening periods 30 Days 60 Days 90 Days

TC1 0 0 100%

TC2 0 25% 75%

TC3 25% 0 75%

TC4 25% 25% 50%

TC5 0 50% 50%

TC6 50% 0 50% Buffalo milk Cheddar cheese Treatments Ripening periods 30 Days 60 Days 90 Days

TB1 0 0 100%

TB2 0 25% 75%

TB3 25% 0 75%

TB4 25% 25% 50%

TB5 0 50% 50%

TB6 50% 0 50% Cow + Buffalo milk Cheddar cheese Treatments Ripening periods 30 Days 60 Days 90 Days

TC1B1 0 0 100%

TC2B2 0 25% 75%

TC3B3 25% 0 75%

TC4B4 25% 25% 50%

TC5B5 0 50% 50%

TC6B6 50% 0 50%

3.6 Quality evaluation of processed Cheddar cheese 3.6.1 Physico-chemical analysis Processed Cheddar cheese was analyzed for moisture, fat, protein, ash, pH, acidity, salt and minerals content with the interval of one month during storage following the standard procedures as detailed above for natural Cheddar cheese. 3.6.2 Texture profile analysis (TPA) The effect of ripening and emulsifying salts on texture of processed cheese was evaluated by performing the texture profile analysis (TPA) of the cheese samples using TA- XT Plus Texture Analyzer (Stable Micro Systems, Godalming, Surrey, UK) using Compression plate Probe P-75 as described by O’Mahony et al. (2005). Cheese samples were placed in airtight plastic bags and equilibrated at 8°C for 18 h. Cubes of 25mm length, width and height were cut from each sample using a stainless steel wire cutter and equilibrated at 8°C for a further 30 min before analysis. Samples were removed from the incubator and immediately compressed to 30% of the original height in 2 consecutive cycles (i.e., double compression) at a rate of 1 mm/s. Definitions and calculated TPA parameters of the TA-XT Texture Analyzer used to measure the texture of processed cheese are as below: Hardness: The force required to compress the cheese sample to 30% of its original height during the first compression cycle. Cohesiveness: The ratio of the area under the positive region (during application of force) of the second compression curve to that of the first compression curve. Springiness: The ratio of the time taken to compress the sample to 30% of its original height during the second compression cycle to that of the first compression cycle. Gumminess: The product of hardness and cohesiveness. Chewiness: The product of gumminess and springiness 3.6.3 Melt-ability Melt-ability of the processed cheese was determined by placing grated cheese plugs weighing approximately 3g into test tubes, the test tubes were covered with aluminum foil, and holes were made to let the hot gas escape during heating (Koca and Metin, 2004). The

test tubes were placed vertically in a refrigerator at 5°C for 30 min and then horizontally in an oven and heated at 100°C for 90 min. Melt-ability was measured in millimeters from the bottom of the test tube to the point at which the cheese has stopped flowing (Poduval and Mistry, 1999). 3.6.4 Assessment of proteolysis Proteolysis in processed Cheddar cheese was assessed using High Performance Liquid Chromatography as done for natural Cheddar cheese (Hickey et al., 2006; Rulikowska et al., 2013). 3.6.5 Color analysis Cheese color was analyzed using a Miniscan portable colorimeter (Hunter Associates Laboratory, Inc., Reston, VA). Color standardization was performed using white and black standard plates (Hunter Associates Laboratory, Inc.) inserted into a plastic bag (QME355 3.5 mil, Vilutis & Co., Inc., Frankfurt, IL) used for cheese packaging. Color measurements were made using CIE (1978) L*, a* and b* values using illuminant D65. The a* value is an indicator of green (-) and red (+) whereas b* is an indicator of blue (–) and yellow (+) (Hunterlab, 2011). 3.6.6 Descriptive sensory evaluation Processed Cheddar cheese was subjected to descriptive sensory evaluation at 0 day (fresh) and 90 days of storage by a panel of 10 evaluators drawn from faculty members and post-graduate students of the institute, following the method given by Muir and Hunter (1992). The evaluators were first trained on commercially available cheese samples and a descriptive sensory language was established for different parameters (Odor, Flavor and Texture) of cheese. Then the processed cheese samples were evaluated for the developed parameters at room temperature in well lighted and properly ventilated sensory evaluation laboratory. Scores were awarded for different descriptive features for each parameter from total of 100 on a designed performa (attached as appendix). 3.7 Statistical analysis Data obtained from the study was subjected to statistical analysis using Completely Randomized Design (CRD), Analysis of Variance (ANOVA) and other suitable statistical techniques to assess the significance of research (Statistica 8.1; Steel et al., 1997).

Chapter-4 RESULTS AND DISCUSSION

The research work was performed in two phases; the preparation and quality evaluation of natural Cheddar cheese during ripening and the manufacturing and evaluation of processed cheese. The results have been discussed accordingly under the following headings: 4.1 Physico-chemical analysis of raw milk Raw milk quality depends on composition of milk and is imperative because it affects the quality of milk products (Galvao et al., 2010). Milk is a dissimilar masterpiece of nutrients. The milk composition is exposed to change in reaction of genetics, breeding, feeding, number and stage of lactation and health status of the animal (Islam et al., 2014). Bovine milk contributes upto 85% of the world milk production whereas buffalo is the 2nd largest (11% of total) milk producing animal (Gerosa and Skoet, 2012). Raw milk of cows and buffaloes procured from local dairy farms. Milk was analyzed for physico-chemical composition to check the suitability for cheese production. The physico-chemical composition of cow and buffalo milk is given in table 4.1 and 4.2 respectively. Cow milk contained lower total solids (12.68±0.032%, 12.98±0.035, 12.74±0.049) as compared to buffalo milk. Buffalo milk was rich in fat and protein matter and these varied from 5.48±0.029% to 5.90±0.050% and 4.04±0.067% to 4.16±0.015% when matched with cow milk samples. Very little variation in mean values of acidity was observed in all samples of both types of milk and it ranged between 0.12±0.006 % to 0.13±0.006 % in cow milk and was almost same 0.13±0.006 % in buffalo milk. Considerably higher mean pH was observed in all samples of buffalo milk starting from 6.78±0.006 to 6.87±0.020 and was less in cow milk samples and was between 6.62±0.026 to 6.71±0.020. On the whole, the significant dissimilarities in compositions were perceived in both the species and buffalo milk was found superior in nutrient profile. Buffalo milk is categorized by higher fat, total solids, proteins, caseins, lactose and ash contents than cow, goat, camel and human milk. Buffalo milk hold almost all valuable combinations found in other milks, e.g., proteins, free amino acids, naturally occurring peptides,

50

Table 4.1 Physico-chemical composition of cow milk samples

Raw milk used for batch # 1 Raw milk used for batch # 2 Raw milk used for batch # 3 Analysis S1 S2 S3 Means±SD S1 S2 S3 Means±SD S1 S2 S3 Means±SD pH 6.71 6.73 6.7 6.71±0.015 6.73 6.69 6.71 6.71±0.020 6.61 6.65 6.6 6.62±0.026 Acidity% 0.12 0.11 0.12 0.12±0.006 0.12 0.13 0.12 0.12±0.006 0.15 0.14 0.14 0.14±0.006 Fat% 4.1 4.1 4.05 4.08±0.029 4.5 4.55 4.5 4.52±0.029 4.20 4.25 4.15 4.20±0.050 Crude protein % 3.62 3.65 3.6 3.62±0.025 3.54 3.52 3.55 3.54±0.015 3.58 3.61 3.55 3.58±0.030 Total solids% 12.72 12.67 12.66 12.68±0.032 12.96 13.02 12.96 12.98±0.035 12.72 12.8 12.71 12.74±0.049 SNF% 8.62 8.57 8.61 8.60±0.026 8.46 8.47 8.46 8.46±0.006 8.52 8.55 8.56 8.54±0.021

51

Table 4.2 Physico-chemical composition of buffalo milk samples

Raw milk used for batch # 1 Raw milk used for batch # 2 Raw milk used for batch # 3 Analysis S1 S2 S3 Means±SD S1 S2 S3 Means±SD S1 S2 S3 Means±SD pH 6.78 6.79 6.78 6.78±0.006 6.81 6.79 6.8 6.80±0.010 6.87 6.89 6.85 6.87±0.020

Acidity% 0.13 0.13 0.13 0.13±0.000 0.13 0.14 0.13 0.13±0.006 0.13 0.12 0.13 0.13±0.006

Fat% 5.5 5.5 5.45 5.48±0.029 5.9 5.85 5.95 5.90±0.050 5.4 5.45 5.4 5.42±0.029

Crude protein % 3.97 4.06 4.1 4.04±0.067 4.16 4.18 4.15 4.16±0.015 3.98 4.08 4.12 4.06±0.072

Total solids% 14.18 14.13 14.13 14.15±0.029 14.72 14.6 14.72 14.68±0.069 13.81 13.86 13.84 13.84±0.025

SNF% 8.68 8.63 8.68 8.66±0.029 8.82 8.75 8.77 8.78±0.036 8.41 8.41 8.44 8.42±0.017

52 fatty acids, vitamins, conjugated linoleic acid, Minerals (Ca, Mg, P, Mn and Zn) and other bioactive compounds (Ahmad et al., 2013; Islam et al., 2014). Due to rich nutrients buffalo milk is preferred by the consumers and is readily drunk or converted into products like cheese and yoghurt in India (Han et al., 2014; Bilal et al., 2006). 4.2 Physico-chemical analysis of standardized milk Milk standardization offers manufacturer the ability to deploy the composition of the final cheese by adjusting the composition of the starting milk in order to meet the legal definition of the specific variety and to recover yields. In addition, the use of standardized milk sidesteps the manufacture of cheese containing excess fat and reduces fat and casein losses into the whey (Lucey and Kelly 1994, Scott 1998). In current research studies the raw milk of cow and buffalo was pasteurized at 85ºC for 16 Seconds and standardized at 3.0% fat through centrifugal cream separator. Standardized milk was analyzed physico-chemically to know the variation in other milk components. Results of the statistical analysis of standardized cow and buffalo milk are given in table 4.3 and table 4.4 respectively. After standardization, subsequent changes observed in both types of milk though fat brought to 3%. Total solids decreased in both cases and were between 11.58±0.044% to 11.65±0.046% in cow milk samples and 11.53±0.046% to 11.80±0.015% in buffalo milk samples. Considerable increase in proteins was noted ranging between 3.66±0.021% to 3.72±0.021% in standardized cow milk and 4.11±0.021% to 4.23±0.021% in standardized buffalo milk. Acidity and pH decreased in both milk types after standardization, acidity values were 0.11±0.006% and 0.12±0.006% in standardized cow milk whereas 0.12±0.006% in standardized buffalo milk. pH values were 6.63±0.025 to 6.73±0.015 in standardized cow milk and 6.72±0.015 to 6.80±0.025 in standardized buffalo milk. Milk standardization minimizes the influence of the dissimilarities in milk on the composition and quality of cheese (Ong et al., 2013). As fat is the chief constituent in buffalo milk (Teixeira et al., 2005; Mattos, 2007) any modification in its concentration may influence total solid levels. Total solids are essential parameters for industry, since their greater amount in the milk specifies better yield in the manufacture of dairy (Campanile et al., 2007) hence buffalo

53

Table 4.3 Physico-chemical analysis of standardized cow milk

Raw milk used for batch # 1 Raw milk used for batch # 2 Raw milk used for batch # 3 Analysis S1 S2 S3 Means±SD S1 S2 S3 Means±SD S1 S2 S3 Means±SD pH 6.72 6.75 6.73 6.73±0.015 6.7 6.68 6.66 6.68±0.020 6.6 6.65 6.63 6.63±0.025 Acidity% 0.11 0.10 0.11 0.11±0.006 0.12 0.13 0.13 0.13±0.006 0.15 0.14 0.14 0.14±0.006 Fat% 3.00 3.00 2.95 2.98±0.029 3.05 3.00 3.05 3.03±0.029 3.05 2.95 3.00 3.00±0.050 Crude protein % 3.71 3.70 3.74 3.72±0.021 3.64 3.65 3.68 3.66±0.021 3.65 3.68 3.70 3.68±0.025 Total solids% 11.68 11.68 11.60 11.65±0.046 11.55 11.56 11.63 11.58±0.044 11.65 11.53 11.59 11.59±0.060 SNF% 8.68 8.68 8.65 8.67±0.017 8.5 8.56 8.58 8.55±0.042 8.60 8.58 8.59 8.59±0.010

54

Table 4.4 Physico-chemical analysis of standardized buffalo milk

Raw milk used for batch # 1 Raw milk used for batch # 2 Raw milk used for batch # 3 Analysis S1 S2 S3 Means±SD S1 S2 S3 Means±SD S1 S2 S3 Means±SD pH 6.72 6.73 6.70 6.72±0.015 6.83 6.78 6.80 6.80±0.025 6.76 6.81 6.80 6.79±0.026

Acidity% 0.10 0.10 0.11 0.10±0.006 0.11 0.12 0.12 0.12±0.006 0.12 0.11 0.12 0.12±0.006

Fat% 3.00 3.00 3.05 3.02±0.029 2.95 3.00 3.10 3.02±0.076 3.00 3.05 2.95 3.00±0.050

Crude protein % 4.12 4.09 4.13 4.11±0.021 4.21 4.24 4.25 4.23±0.021 4.13 4.16 4.15 4.15±0.015

Total solids% 11.78 11.80 11.81 11.80±0.015 11.87 11.90 12.00 11.92±0.068 11.50 11.58 11.50 11.53±0.046

SNF% 8.78 8.80 8.76 8.78±0.020 8.92 8.90 8.90 8.91±0.012 8.50 8.53 8.55 8.53±0.025

55 milk receives increasing research interest and investment in various countries, owing mainly to its attractive nutrient content (Murtaza et al., 2008). 4.3 CHEDDAR CHEESE QUALITY DURING RIPENING 4.4 PHYSICO-CHEMICAL ANALYSIS In order to determine the quality, legal standards and the customer requirements the analysis of cheese is necessary as discussed by Mehta (2014). Cheese assists as a vehicle for preserving the valuable nutrients in milk and making them available throughout the year (Krupa et al., 2011). Moisture, fat, peptides, salt, micro flora, minerals and other minor constituents, mixed within a casein matrix combine to make Cheddar a complex food (Murtaza et al., 2008). Proximate composition (Moisture, Fat, Protein, Ash) pH, acidity and salt contents were determined at 0 day, 30 day, 60 day and 90 day interval to assess the physicochemical changes in Cheddar cheese prepared from cow and buffalo milk in three batches. 4.4.1 Moisture in Cheddar cheese Analysis of moisture is not only necessary to know the end product specifications but variation in moisture also affects shelf life and textural properties of cheese (Lee et al., 2004). Analysis of variance table (4.5) shows the significant (P<0.05) difference of moisture in cheese prepared from cow and buffalo milk and non-significant during ripening period. Results showed higher moisture contents (39.51±0.333%) in cow cheese and lower (38.850±0.312%) in cheese prepared from Buffalo milk in batch one and similar trend was observed in other batches. The results of the moisture in present study are very close to the results of Lawrence et al. (2004) and Moller et al. (2013a), who kept moisture at level of 37.7%. USFDA (2002) described the maximum moisture content in Cheddar cheese can be 39 % by weight. Low moisture in buffalo cheese may be due to the high fat contents (Souza et al., 2012; Simoes et al., 2013). Karahan et al. (2011) described that cheese prepared from 100% buffalo milk had low moisture as compared to the cheese prepared by mixing cow and buffalo milk. The main cause of significant difference may be the variation in milk composition of cow and buffalo as reported by Ahmad et al. (2008). Storage days have non-significant (P>0.05) effect on the moisture contents of both cheeses prepared from cow and buffalo milk. Moisture contents decreased from 39.51±0.333 % to 38.640±0.312 % from 0 day to 90 days in cheese from cow milk and similarly from 38.850±0.270 % to 37.910±0.195 % from 0 day to 90 days in cheese prepared from buffalo milk

56 Table 4.5 Analysis of variance for moisture in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square NS NS NS Days (D) 3 1.0511 2 0.4299 1 0.1776 Milk (M) 1 2.7744* 1 3.7083** 1 3.2865** NS NS NS D x M 3 0.0249 2 0.0376 1 0.0003 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.6a Effect of milk types and ripening period on moisture (%) of Cheddar

cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 39.51±0.333 38.85±0.270 39.18±0.241 30 39.50±0.318 38.69±0.346 39.10±0.278 60 38.91±0.507 38.40±0.313 38.65±0.290 90 38.64±0.312 37.91±0.195 38.28±0.232 Means±SE 39.14±0.197A 38.46±0.163B

57 Table 4.6b Effect of milk types and ripening period on moisture (%) of Cheddar

cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 40.14±0.373 39.41±0.252 39.78±0.259 30 40.09±0.207 39.13±0.111 39.61±0.239 60 39.77±0.232 38.74±0.194 39.25±0.268 Means±SE 40.00±0.152A 39.09±0.138B

Table 4.6c Effect of milk types and ripening period on moisture (%) of Cheddar cheese (Batch # 3)

Storage Milk Means±SE Days Cow Buffalo 0 40.98±0.184 39.92±0.294 40.45±0.283 30 40.73±0.101 39.69±0.105 40.21±0.241 Mean±SE 40.85±0.110A 39.81±0.149B Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

58

Cow milk Buffalo milk

42

41

40

39

e

r

u

t

s

i

o M 38

37

36

35

Fig. 4.1 Effect of milk types and ripening period on moisture (%) of Cheddar cheese

59 (Table 4.6a; Fig. 4.1). Such decrease in moisture during ripening has also been reported by Tarakci and Kucukoner (2006) that moisture ratio was not affected due to wrapping in polythene bag. 4.4.2 Fat in Cheddar cheese Source of milk, age and fat contents have profound effects on the Cheddar cheese flavor among which the fat is responsible for the development of correct flavor (Hassan et al., 2013). Fat also influences the body and texture of cheese by filling interstitial spaces in the protein and mineral structural mesh (McGregor and White, 1990; Kucukoner and Haque, 2006). Analysis of variance table 4.7 shows a significant (P<0.05) effect of milk types and non-significant (P>0.05) effect of ripening on fat contents of cheese. Fat contents were significantly higher in cheese prepared from buffalo milk in batch 1 and 2 as 30.50±0.289 % and 30.50±0.351 % than cow milk 30.07±0.233 % and 29.83±0.328. Non-significant impact of storage days was observed on fat contents of Cheddar cheese prepared from cow and buffalo milk showing a slight decrease in fat content in all three batches (Fig. 4.2). Results of present study are close to the findings of Murtaza et al. (2008) ; Simoes, et al. (2013); Hamid, (2014) and Hickey et al. (2006), who reported fat contents in Cheddar cheese 30.58% (Cow) and 32.62 % (Buffalo) , 29% ( Buffalo 70% + Cow 30%), 29.04% and 30.99% respectively. The milk for all batches was standardized at 3.0% fat level but slightly higher fat values were observed in cheese prepared from buffalo milk which might be due to the richness of the buffalo milk in all ingredients as compared to the cow milk (Islam et al., 2014., Ahmad et al., 2013.) causing retention of fat in cheese matrix along with proteins. In the present study non-significant (P>0.05) decrease in fat during ripening was noted which may be correlated to the findings of the Hamid and Abdelrahman (2012), who noted decrease in fat content of the cheese during ripening owing to the lipolytic effect of the cheese microflora. Studies are also in correlation with the results of Miocinovic et al. (2014), who reported a non-significant decrease in the fat composition of cheese during 8 weeks of ripening. 4.4.3 Crude protein in Cheddar cheese Dominant protein in cheese is casein as the most of whey proteins are lost in whey. During coagulation of milk almost all caseins go into the curd in concentrated form and their ratio to the total proteins can fluctuate depending on the genetic and physiological factors (Metz et al.,

60 Table 4.7 Analysis of variance for fat in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square NS NS NS Days (D) 3 0.5204 2 0.8750 1 0.33333 NS Milk (M) 1 1.6538* 1 2.0000* 1 0.00000 NS NS NS D x M 3 0.0149 2 0.0417 1 0.00000 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.8a Effect of milk types and ripening period on fat (%) of Cheddar cheese

(Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 30.07±0.233 30.50±0.289 30.28±0.192 30 29.83±0.167 30.33±0.376 30.08±0.215 60 29.67±0.285 30.33±0.333 30.00±0.246 90 29.33±0.219 29.83±0.176 29.58±0.168 Means±SE 29.73±0.127B 30.25±0.149A

61 Table 4.8b Effect of milk types and ripening period on fat (%) of Cheddar cheese

(Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 29.83±0.328 30.50±0.351 30.17±0.262 30 29.50±0.252 30.33±0.176 29.92±0.232 60 29.17±0.333 29.67±0.418 29.42±0.264 Means±SE 29.50±0.181B 30.17±0.209A

Table 4.8c Effect of milk types and ripening period on fat (%) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 30.00±0.115 30.00±0.200 30.00±0.103 30 29.67±0.145 29.67±0.145 29.67±0.092 Means±SE 29.83±0.112A 29.83±0.133A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

62

Cow milk Buffalo milk

31

30.5

30

29.5

t

a 29 F

28.5

28

27.5

27

Fig.4.2 Effect of milk types and ripening period on fat (%) of Cheddar cheese

63 Table 4.9 Analysis of variance for crude protein in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square NS NS NS Days (D) 3 0.0056 2 0.01576 1 0.00187 Milk (M) 1 12.6150** 1 5.71220** 1 4.77541** NS NS NS D x M 3 0.0004 2 0.00540 1 0.00041 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.10a Effect of milk types and ripening period on crude protein (%) of

Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 25.18±0.050 26.607±0.050 25.89±0.321 30 25.14±0.035 26.597±0.087 25.87±0.328 60 25.12±0.038 26.577±0.072 25.85±0.328 90 25.09±0.058 26.550±0.081 25.82±0.329 Means±SE 25.13±0.022B 26.583±0.032A

64 Table 4.10b Effect of milk types and ripening period on crude protein (%) of

Cheddar cheese (Batch # 2)

Storage Days Milk Mean±SE Cow Buffalo 0 25.22±0.059 26.407±0.015 25.81±0.267 30 25.24±0.035 26.367±0.033 25.80±0.253 60 25.19±0.052 26.253±0.129 25.72±0.246 Mean±SE 25.22±0.026B 26.342±0.045A

Table 4.10c Effect of milk types and ripening period on crude protein (%) of

Cheddar cheese (Batch # 3)

Storage Days Milk Mean±SE Cow Buffalo 0 25.05±0.085 26.320±0.074 25.68±0.289 30 25.03±0.072 26.283±0.055 25.66±0.282 Mean±SE 25.04±0.050B 26.302±0.042A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

65

Cow milk Buffalo milk

27

26.5

26

n

i

e t 25.5

o r P

25

24.5

24

Fig.4.3 Effect of milk types and ripening period on crude protein (%) of

Cheddar cheese

66 2001). Milk having high protein concentration shows high buffering capacity and less whey expulsion during cheese making (Lucey et al., 2004; Ong et al., 2013). Analysis of variance table (4.9) shows that milk types exerted a highly significant (P<0.01) difference on crude protein while ripening showed a non-significant effect. There is highly significant difference of the protein values in cheese from both types of milk showing high protein content in buffalo cheese in all three batches with 26.607±0.050%, 26.407±0.015% and 26.320±0.074% at 0 day while 25.18±0.050%, 25.22±0.059% and 25.05±0.085% in cow cheese (Table 4.10a, b, c; Fig. 4.3). Protein contents of the cheese in present study are justified by the results of Fenelon et al. (2000) and Ong et al. (2007b), who reported 25.5% and 27.46% protein in full fat Cheddar cheese. Murtaza et al. (2008) described the proteins in cow and buffalo milk Cheddar as 25.51% and 26.6% respectively Highly significant difference of proteins in cheese prepared from cow and buffalo milk is due to the difference in the milk composition of both animals. Buffalo milk is rich in proteins than cow milk and the high concentration and colloidal form of casein in buffalo milk causes the increased number of casein micelles resulting in higher protein in the cheese (Ahmad et al., 2013). Ripening periods showed a non-significant (P>0.05) difference in the protein values of both types of cheeses during the entire storage period of 90 days. A slight decrease was noted in cow milk cheese ranging from 25.18±0.050 % to 25.09±0.058 % during storage in batch # 1 and 26.607±0.050 % to 26.550±0.081 % in buffalo milk cheese. Similar trend was observed in other batches in both types of cheeses. Protein contents decreased as the storage period increased (Murtaza et al., 2008 and Altahir et al., 2014). 4.4.4 Ash in Cheddar cheese In any food system ash is an important property because it the beginning point to determine the mineral profile in any food stuff (El-Nasri et al., 2012). Ash is obtained as in organic matter resulting from burning of organic matter in any food stuff. It is not the true representative of the minerals in the food stuff (Kirk and Sawyer, 1991). Analysis of variance (table 4.11) reveals highly significant difference (P<0.01) in the results of Ash in both types of cheese prepared from cow and buffalo milk. High ash contents observed in cheese prepared from buffalo milk as compared to the cow milk as given in table 4.12a. Buffalo milk cheese showed the ash contents between

67 4.080±0.015% to 4.140±0.026 % in three batches whereas cow milk cheese had the ash contents between 3.73±0.020 % to 3.78±0.009 %. The results of ash contents in the current study are in line with the results of Kucukoner and Haque (2006) and Nateghi et al. (2012), who reported 3.0% and 3.4% ash in full fat and half fat Cheddar cheese and 4.16% and 4.67% ash in dry matter in full fat and half fat Cheddar cheese, respectively (Fig. 4.4). Variation in ash contents in both types of cheese may be due to variation in mineral composition of both types of milk used for the cheese preparation. Buffalo milk has high levels of minerals as compared to cow milk (Medhammar et al., 2012; Ahmad et al., 2013). A non-significant variation in the ash was observed during entire ripening period having 3.73±0.020 % to 3.74±0.031 % from 0 to 90 day in cow milk cheese (batch # 1) and 4.140±0.026 % to 4.120±0.015 % in buffalo milk cheese (batch # 1). Similar trend was observed in other cheese batches. Results of the present study dis-agree with the studies of Hassanein et al. (2014) and resemble with results of Singh, et al. (2003) and Farkye (2004). Former showed the significant decrease in ash contents whereas later described that ash contents remain un-changed during ripening period. 4.4.5 pH of Cheddar cheese pH is one of the important parameters used for determining the quality of cheese. Lactic acid produced from starter cultures determines the ultimate pH of the cheese (Pandeya et al., 2003). Rennet clotting of milk is controlled by numerous factors among which pH plays a vital role (Jacob et al., 2011; Odile et al., 2012). Both enzymatic and aggregation phases of milk coagulation are affected by pH (Castillo et al., 2000). Rennet concentration also influences the intensity of primary proteolysis and is pH dependent. During cheese manufacture the curd firming rate is increased by lowering the pH (Bittante, 2011). Analysis of variance table 4.13 shows highly significant (P<0.01) effect of milk types and ripening on pH of the cheese prepared from cow and buffalo milk while non- significant effect (P>0.05) on interaction between milk types and ripening. A non- significant (P>0.05) variation in pH results was observed in both types of cheese having pH value 5.26±0.028 in cow milk cheese and 5.147±0.023 in buffalo milk cheese in batch no. 1. Similarly 5.20±0.015 and 5.213±0.013 in second batch and 5.27±0.009 and 5.213±0.009 third batch of cow and buffalo cheese,

68 Table 4.11 Analysis of variance for ash in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square NS NS Days (D) 3 0.00008 2 0.01452 1 0.00067V Milk (M) 1 0.93220** 1 0.58320** 1 0.42941** NS NS NS D x M 3 0.00025 2 0.01622 1 0.00021 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.12a Effect of milk types and ripening period on ash (%) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 3.73±0.020 4.140±0.026 3.94±0.092 30 3.73±0.018 4.130±0.021 3.93±0.091 60 3.74±0.023 4.123±0.019 3.93±0.087 90 3.74±0.031 4.120±0.015 3.93±0.086 Means±SE 3.73±0.010B 4.128±0.009A

69 Table 4.12b Effect of milk types and ripening period on ash (%) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 3.78±0.009 4.080±0.015 3.93±0.068 30 3.78±0.030 4.257±0.172 4.02±0.133 60 3.79±0.026 4.083±0.012 3.94±0.068 Means±SE 3.78±0.012B 4.140±0.058A

Table 4.12c Effect of milk types and ripening period on ash (%) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 3.73±0.021 4.100±0.010 3.92±0.083 30 3.74±0.009 4.123±0.012 3.93±0.087 Means±SE 3.73±0.010B 4.112±0.009A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

70

Cow milk Buffalo milk

4.4

4.2

4

3.8

h

s

A 3.6

3.4

3.2

3 0 day 30 day 60 day 90 day 0 day 30 day 60 day 0 day

Fig.4.4 Effect of milk types and ripening period on ash (%) of Cheddar

cheese

71 Table 4.13 Analysis of variance for pH of Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square Days (D) 3 0.02473** 2 0.01574** 1 0.01841** NS Milk (M) 1 0.13500** 1 0.00036 1 0.01141** NS NS NS D x M 3 0.00160 2 0.00001 1 0.00021 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.14a Effect of milk types and ripening period for pH of Cheddar cheese (Batch # 1)

Storage Milk Means±SE Days Cow Buffalo 0 5.26±0.028 5.147±0.023 5.20±0.030A 30 5.23±0.015 5.090±0.010 5.16±0.032A 60 5.19±0.032 5.013±0.038 5.10±0.045B 90 5.14±0.026 4.967±0.015 5.06±0.042B Means±SE 5.20±0.017A 5.054±0.023B

72 Table 4.14b Effect of milk types and ripening period for pH of Cheddar cheese

(Batch # 2)

Storage Milk Means±SE Days Cow Buffalo 0 5.20±0.015 5.213±0.013 5.21±0.009A 30 5.16±0.017 5.173±0.009 5.17±0.009B 60 5.10±0.019 5.110±0.015 5.11±0.011C Means±SE 5.16±0.017 5.166±0.016

Table 4.14c Effect of milk types and ripening period for pH of Cheddar cheese (Batch # 3)

Storage Milk Means±SE Days Cow Buffalo 0 5.27±0.009 5.213±0.009 5.24±0.013A 30 5.20±0.017 5.127±0.020 5.16±0.020B Means±SE 5.23±0.018A 5.170±0.022B Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

73

Cow milk Buffalo milk

5.3

5.2

5.1

5

pH

4.9

4.8

4.7

Fig 4.5 Effect of milk types and ripening period for pH of Cheddar cheese

74 respectively (Table 4.14a, b, c; Fig. 4.5). pH values of the present study resembles with the results of Papetti and Carelli, (2013) reported pH from 5.12 to 5.58, Ong et al. (2007b) 5.1- 5.4 and Kucukoner and Haque (2006) 5.0-5.1 in various Cheddar cheese samples. pH decreased during ripening in both cheese samples showing a highly significant (P<0.01) trend. pH values decreased from 5.26±0.028 to 5.14±0.026 in cow milk cheese in 90 day of ripening and 5.147±0.023 to 4.967±0.015 in buffalo milk cheese in batch #1 and the similar trend was observed in other two batches in both cheese types. Decline in cheese pH is caused by the fermentation of lactose and degradation of fats during ripening period (Azarnia et al., 2006). Decrease in pH has been justified by the production of lactic acid by lactic acid bacteria (Souza et al., 2003). 4.4.6 Acidity of Cheddar cheese Numerous researches are in process to improve the quality of cheese, producing specific flavor of cheese by using potential starters. During cheese manufacture the starter bacteria produce acid, flavor and contribute in ripening (Hols et al., 2005; Kongo, 2013 and Steel et al., 2013). Lactic acid is produced from milk (lactose) by homofermentative bacteria whereas lactose is converted into lactic acid, acetic acid, ethanol and CO2 by heterofermentative species (Widyastuti et al., 2014). Lactic acid improves cheese structure, contributes to the taste and protects against microbiological spoilage (Ceylan et al., 2003). Analysis of variance table 4.15 shows that milk types have highly significant (P<0.01) effect on acidity whereas ripening have significant effect on acidity of Cheddar cheese. High level of acidity observed in buffalo cheese 0.900±0.012% and low acidity level in cow cheese 0.84±0.010% in batch # 1 (Table 4.16a; Fig. 4.6) and similarly in other two batches 0.867±0.012% and 0.850±0.012% in buffalo cheese and 0.83±0.015% and 0.81±0.007% in cow cheese (Table 4.16b and 4.16c). The results of the present study are justified by the studies of Souza et al. (2003); El-Nasri et al. (2012) and Delamare et al. (2012), who reported the acidity as 0.90%, 0.81%, and 0.83% respectively in different cheese varieties. Variations in the concentration of the acidity may be due to the varied composition and properties of milk used for the preparation of cheese (Ceylan et al., 2003). A highly significant (P<0.01) increase in acidity was observed in both cheese types during ripening upto 90 days. Acidity increased from 0.900±0.012% to 0.967±0.007% in batch # 1 of buffalo milk cheese and 0.84±0.010% to 0.91±0.007% in batch # 1 of cow milk cheese (Table 4.16a) and a similar highly significant increase in acidity was noted in other two batches of both types of cheese.

75 Increase in acidity during ripening period might be due to the growth of lactic acid bacteria which resulted in the production of lactic acid and thus increase in acidity (Bilal, 2000; Warsama et al., 2006; El-Owni and Hamid 2008; Hamid and Abdelrahman 2012). Altahir et al. (2014) also reported the significant increase in titratable acidity during storage period up to 90 days. 4.4.7 Salt in Cheddar cheese The most reliable and common method for the preservation of cheese is salting (Salam and Benkerroum, 2006). Salt is a source of dietary sodium, imparts flavor and also acts as preservative (Guinee, 2004). Salt plays important role in controlling synersis and water activity in cheese (Guinee and Fox, 2004). Manipulation with the salt concentration can be helpful in controlling the bitterness, wide sapid flavor and sensory properties (Moller et al., 2013b; Moller et al., 2012). In the present study cheese was prepared from cow and buffalo milk using 1.5% salt (NaCl) during moulding process. Results of the salt concentration are given in the analysis of variance table 4.17. Milk types showed significant (P<0.05) effect whereas ripening showed non-significant (P>0.05) effect on salt contents of cheese. Results revealed that salt percentage was significantly (P<0.01) different among cow and buffalo milk cheese showing a higher salt concentration in buffalo milk cheese in all batches as given in tables 4.18a, b and c, whereas low concentration of salt was observed in Cheddar cheese prepared from cow milk ranging from 1.72±0.031 to 1.79±0.019 at 0 day analysis (Table 4.18 a-c; Fig. 4.7). The difference of the salt in both cheeses may be due to the variability of the salting method as powdered NaCl was sprinkled on the milled cheese during moulding process which might have not been equally distributed (Aquilanti et al., 2013). A very little (non-significant) increase in salt was observed in both types of cheeses in all batches ranging between 1.72±0.031 to 1.77±0.012 in cow milk cheese batch # 1 and 1.82±0.032 to 1.87±0.019 in buffalo milk cheese batch # 1. A similar trend was observed in other two batches of both types of cheese. The present study is supported by the results of Altahir et al. (2014), who showed an increase in the salt concentration with the increase in ripening period. The salt values of Cheddar cheese in this study are justified by the studies of Ong et al. (2007b), who declared salt in Cheddar cheese as 1.78% while Papetti and Carelli (2013) showed salt 2.12% at 90 days storage. Further difference may be due to the composition of cow and buffalo milk used for the preparation of Cheddar cheese owing to the higher levels of sodium and chlorides in buffalo milk.

76 Table 4.15 Analysis of variance for acidity in cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square Days (D) 3 0.00489** 2 0.00276* 1 0.00141* Milk (M) 1 0.02535** 1 0.00500** 1 0.00521** NS NS NS D x M 3 0.00005 2 0.00007 1 0.00008 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.16a Effect of milk types and ripening period on acidity (%) of Cheddar cheese (Batch # 1)

Storage Milk Means±SE Days Cow Buffalo 0 0.84±0.010 0.900±0.012 0.87±0.015D 30 0.85±0.007 0.923±0.003 0.89±0.016C 60 0.87±0.003 0.943±0.003 0.91±0.016B 90 0.91±0.007 0.967±0.007 0.94±0.014A Means±SE 0.87±0.008B 0.933±0.008A

77 Table 4.16b Effect of milk types and ripening period on acidity (%) of Cheddar cheese (Batch # 2)

Storage Milk Means±SE Days Cow Buffalo 0 0.83±0.015 0.867±0.012 0.85±0.012B 30 0.84±0.012 0.870±0.012 0.85±0.011B 60 0.87±0.007 0.900±0.012 0.89±0.008A Means±SE 0.85±0.009B 0.879±0.008A

Table 4.16c Effect of milk types and ripening period on acidity (%) of Cheddar cheese (Batch # 3)

Storage Milk Means±SE Days Cow Buffalo 0 0.81±0.007 0.850±0.012 0.83±0.010B 30 0.83±0.010 0.877±0.003 0.85±0.011A Means±SE 0.82±0.007B 0.863±0.008A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

78

Cow milk Buffalo milk

1

0.95

0.9

0.85

y

t

i

d

i 0.8 c

A

0.75

0.7

0.65

0.6

0 day 30 day 60 day 90 day 0 day 30 day 60 day 0 day

Fig 4.6 Effect of milk types and ripening period on acidity (%) of Cheddar cheese

79 Table 4.17 Analysis of variance for salt in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square NS NS NS Days (D) 3 0.003449 2 0.00185 1 0.00120 Milk (M) 1 0.055104** 1 0.02961** 1 0.00853* NS NS NS D x M 3 0.000093 2 0.00004 1 0.00003 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.18a Effect of milk types and ripening period on salt (%) of Cheddar cheese (Batch # 1)

Storage Milk Mean±SE Days Cow Buffalo 0 1.72±0.031 1.82±0.032 1.77±0.029 30 1.73±0.006 1.82±0.010 1.78±0.021 60 1.76±0.012 1.85±0.019 1.80±0.022 90 1.77±0.012 1.87±0.019 1.82±0.026 Mean±SE 1.74±0.009B 1.84±0.011A

80 Table 4.18b Effect of milk types and ripening period on salt (%) of Cheddar cheese (Batch # 2)

Storage Milk Mean±SE Days Cow Buffalo 0 1.66±0.027 1.743±0.018 1.70±0.023 30 1.68±0.012 1.767±0.009 1.72±0.020 60 1.70±0.012 1.777±0.015 1.74±0.019 Mean±SE 1.68±0.010B 1.762±0.009A

Table 4.18c Effect of milk types and ripening period on salt (%) of Cheddar cheese (Batch # 3)

Storage Milk Mean±SE Days Cow Buffalo 0 1.79±0.019 1.837±0.015 1.81±0.015 30 1.80±0.020 1.860±0.012 1.83±0.016 Mean±SE 1.80±0.013B 1.848±0.010A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

81

Cow milk Buffalo milk

1.9

1.85

1.8

1.75

t

l

a 1.7 S

1.65

1.6

1.55

1.5

Fig.4.7 Effect of milk types and ripening period on salt (%) of Cheddar cheese

82 4.5 MINERALS IN CHEDDAR CHEESE Dairy products are rich source of minerals (Soyeurt et al., 2008). Many of the constructive nutrients are taken from milk, cheese and yoghurt in variable quantities. Numerous nutritional benefits are obtained by consuming the cheese due to its micro- nutrients and minerals (Lucas et al., 2008). Certain factors such as milk origin (Cow, buffalo), geography of the area and the processing equipment have considerable effects on the variation of mineral composition of cheese (Tarakci and Temiz, 2009).The minerals influence the heat solidity and clotting capacity of the cheese curd and take part in the coagulation by influencing the whey draining and texture of the curd (Patino et al., 2005). In the present study mineral analysis was performed after at 0, 30, 60 and 90 days to check the changes in the mineral composition during ripening. Five minerals (Ca, Mg, K, P and Na) were analyzed. 4.5.1 Calcium in Cheddar cheese Calcium is considered as major mineral in milk and dairy products and intricate in various metabolic functions in human body like blood coagulation and muscular contraction (Soyeurt et al., 2008). Low intake levels of calcium in diet can exert numerous effects on human health such as osteoporosis (Lanou et al., 2005; Devriese et al., 2006; Huth et al., 2006) arterial hypertension, colon cancer (Gueguen and Pointillart, 2000; Huth et al., 2006) and regulation of body fat and weight (Huth et al., 2006). Cheese is cherished source of calcium, especially bioavailable calcium. Calcium plays role in the texture of the cheese, being hard having low calcium level and vice versa (McSweeney, 2007). Analysis of variance (table 4.19) showed that milk types highly significantly influenced the calcium contents whereas ripening had non-significant effect on calcium. Data shows the significantly higher (P<0.01) calcium contents in buffalo milk cheese (743.223±1.530 mg/100g) then cow milk cheese (710.77±1.038 mg/100g) in batch #1 (table 4.20a; Fig. 4.8). Batch #2 and batch # 3 also showed highly significant difference in Ca contents in both types of cheese as given in table 4.20 b-c. Variation in calcium is due to the difference in composition of milk used for the cheese preparation (Upreti et al., 2006). Caro et al. (2014) presented calcium level as 717 mg/100g, 769 mg/100g, 770 mg/100g and 771 mg/100g in different Mexican cheeses and Altahir et al. (2014) declared Ca as 880mg/100g in Sudanese cheese and O’Connor and

83 O’Brien (2000), 720mg/100g in Cheddar cheese. Hard cheeses contain approximately 800mg/ 100g of cheese (Fox and McSweeney, 2004). Storage days showed non-significant (P>0.05) effect on calcium concentration which increased from 743.223±1.530 mg/100g to 747.247±1.109 mg/100g in buffalo milk cheese and 710.77±1.038 mg/100g to 714.57±1.972 mg/100g in cow milk cheese (batch#1) as mentioned in table 4.20a and other two batches as given in table 4.20b and c. Singh et al. (2003) and Farkye (2004) declared that minerals don’t undergo primary and secondary catabolic changes during ripening. Present studies are close to the results of Altahir et al. (2014), who affirmed non-significant (P>0.05) change in calcium during 60 days of ripening. 4.5.2 Potassium in Cheddar cheese As cheese consumption is increasing in the world so there is need of reducing sodium carrier salts (Johnson et al., 2009; Agarwal et al., 2011; Cruz et al., 2011; Drake et al., 2011). Potassium serves as best sodium replacer in diet to reduce the sodium- induced hypertension, lessens urinary calcium excretion and protects skeletal mass (Karagozlu et al., 2008). Potassium chloride has been extensively and successfully used as replacement of NaCl in cheese (Ayyash et al., 2011; Gomes et al., 2011b). Results concerning to the potassium analysis in Cheddar cheese prepared from cow milk and buffalo milk are given in the table 4.21 and 4.22a-c. A highly significant difference was observed in both kinds of cheese with potassium concentration being higher in buffalo milk Cheddar. Potassium values recorded in buffalo cheese were 104.85±2.361mg/100g, 96.86±0.758 mg/100g and 100.21±1.010 mg/100g in three batches whereas 94.62±1.677 mg/100g, 86.23±0.412 mg/100g and 88.13±0.566 mg/100g in cow milk cheese at 0 day of analysis (Fig. 4.9). Mean values of potassium in the current study are analogous to the studies of Dhuol and Hamid (2014), who analyzed potassium concentration in soft white cheese as 98.3 mg/100g and Caro et al. (2014) showed Potassium concentration as 95mg/100g ,100mg/100g and 102mg/100g in Botanero, Manchego and Morral (Mexican cheeses). Such difference is due to the inter species metamorphosis in composition of milk which is replicated in the products manufactured therefrom (Fox and McSweeney, 2004).

84 Table 4.19 Analysis of variance for calcium in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square NS NS NS Days (D) 3 16.69 2 4.1087 1 2.3144 Milk (M) 1 6356.04** 1 4117.1800** 1 2951.2900** NS NS NS D x M 3 0.06 2 0.0014 1 0.0004 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

4.20a Effect of milk types and ripening period on calcium (mg/100g) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 710.77±1.038 743.223±1.530 727.00±7.303 30 711.75±0.853 744.487±1.863 728.12±7.378 60 713.08±1.235 745.403±2.125 729.24±7.310 90 714.57±1.972 747.247±1.109 730.91±7.377 Means±SE 712.54±0.716B 745.090±0.849A

85 4.20b Effect of milk types and ripening period on calcium (mg/100g) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 718.13±0.565 748.410±1.264 733.27±6.800 30 718.97±0.691 749.203±0.598 734.09±6.773 60 719.81±0.517 750.037±1.273 734.92±6.787 Means±SE 718.97±0.384B 749.217±0.594A

4.20c Effect of milk types and ripening period on calcium (mg/100g) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 710.14±0.660 741.517±1.054 725.83±7.038 30 711.03±1.342 742.383±0.714 726.71±7.044 Means±SE 710.59±0.698B 741.950±0.601A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

86

Cow milk Buffalo milk

760

740

720

700

680

Ca

660

640

620

600

Fig.4.8 Effect of milk types and ripening period on calcium (mg/100g) of

Cheddar cheese

87 Table 4.21 Analysis of variance for potassium in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square NS NS NS Days (D) 3 12.96 2 3.429 1 1.896 Milk (M) 1 685.12** 1 483.398** 1 435.969** NS NS NS D x M 3 0.19 2 0.090 1 0.001 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

4.22a Effect of milk types and ripening period on potassium (mg/100g) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 94.62±1.677 104.85±2.361 99.73±2.628 30 94.84±1.015 105.62±1.617 100.23±2.557 60 96.26±0.621 106.92±1.041 101.59±2.444 90 97.46±0.571 108.54±1.792 103.00±2.616 Means±SE 95.80±0.572B 106.48±0.864A

88 4.22b Effect of milk types and ripening period on potassium (mg/100g) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 86.23±0.412 96.86±0.758 91.55±2.408 30 87.12±0.765 97.44±0.635 92.28±2.349 60 87.99±0.557 98.13±0.110 93.06±2.283 Means±SE 87.11±0.391B 97.48±0.341A

4.22c Effect of milk types and ripening period on potassium (mg/100g) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 88.13±0.566 100.21±1.010 94.17±2.750 30 88.95±0.061 100.98±1.345 94.96±2.757 Means±SE 88.54±0.313B 100.59±0.772A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

89

Cow milk Buffalo milk

120

110

100

90

K 80

70

60

50

40

Fig.4.9 Effect of milk types and ripening period on potassium (mg/100g) of Cheddar cheese

90 Ripening presented a non-significant effect on the potassium concentration of both forms of cheese. Potassium contents in cow milk cheese (batch # 1) at 0 day was 94.62±1.677 mg/100g which non-significantly increased to 97.46±0.571 mg/100g at 90th day. In buffalo milk cheese potassium content was 104.85±2.361 mg/100g at 0 day and 108.54±1.792 mg/100g at 90th day. Similar no-significant increase was observed in other two batches in both types of cheese showing mean value of K concentration 87.11±0.391 mg/100g in batch # 2 (table 4.22b) and 88.54±0.313 mg/100g in batch # 3 (table 4.22c) of cow milk cheese and 97.48±0.341 mg/100g in batch # 2 and 100.59±0.772 mg/100g in batch # 3 of buffalo milk cheese. As it is obvious that during ripening minerals doesn’t undergo major chemical reactions while minor changes in composition of mineral occur may be due to bio-chemical and microbiological changes during storage (Farkye, 2004; McSweeney, 2004; Ong et al., 2007a). 4.5.3 Sodium in Cheddar cheese Sodium induced hypertension is common in persons who consume foods rich in sodium. As consumption of cheese is increasing day by day through different sources, there is need of sodium reduction by replacing it with suitable salts without affecting the acceptability of cheese (Johnson et al., 2009; Agarwal et al., 2011; Cruz et al., 2011; Drake et al., 2011). Analysis of variance Table 4.23 shows results regarding sodium (Na) concentration in the Cheddar cheese prepared from cow and buffalo milk. Data showed highly significant (p<0.01) difference in the Na concentration in both types of cheese with higher concentration (731.673±1.124 mg/100g) in buffalo milk Cheddar (batch # 1) and lower (690.16±1.207 mg/100g) in cow milk cheese and contrarily in other two batches in both types of cheese (Fig. 4.10). Results are close to the findings of Murtaza et al. (2008), Altahir et al. (2014) and Caro et al. (2014), who reported Na conc. 662mg/100g (cow Cheddar), 680mg/100g (buffalo Cheddar), 830mg/100g and 686mg/100g Na, respectively. Highly significant difference in the concentration of Na in both cheese types is due to the difference in composition of milk as declared by Ahmad et al. (2013) that buffalo milk is rich in Na than cow milk. Ripening showed non-significant (p>0.05) effect on the concentration of Na in both types of cheese from 0 to 90 days. Sodium contents varied from 731.673±1.124 mg/100g to 736.617±0.944 mg/100g in buffalo milk cheese in ripening while 690.16±1.207 mg/100g to 694.76±0.621 mg/100g in case of cow milk cheese in batch #1.

91 A similar variation in sodium (Na) contents was seen in other two batches in both cheese types. Slight change in the concentration of sodium during storage may be due to the decomposition of different components by lipolysis and proteolysis (Altahir et al., 2014). Ripening has non-significant impact on sodium as minerals don’t undergo any major chemical reaction during ripening (McSweeney and Sousa, 2000; Singh et al., 2003). 4.5.4 Magnesium in Cheddar cheese Magnesium is involved to some extent in bone health and human development. Minerals play vital role in controlling blood pressure when calcium, magnesium and sodium taken in combination with other milk components (Huth et al., 2006). Magnesium depletion from diet causes reduced bone growth. Reformed magnesium stability can be found in diabetes mellitus, chronic renal failure, nephrolithiasis, osteoporosis, aplastic osteopathy, and heart and vascular disease (Musso, 2009). Analysis of variance table 4.25 shows that ripening had non-significant (P>0.05) whereas milk types had highly significant (P<0.01) effect on Mg contents in Cheddar cheese. Significantly high level of magnesium contents (32.61±0.382 mg/100g) in cheese prepared from buffalo milk while low level of Mg (25.13±0.363 mg/100g) was noted in cow milk cheese in batch # 1 (table 4.26a; Fig. 4.11). A similar concentration of magnesium found in other two batches which is given in table 4.26b-c. Values of magnesium found in the present study are parallel to the findings of Caro et al. (2014), who stated Mg concentration as 22mg/100g, 30mg/100g and 31mg/100g in different Mexican cheeses. Altahir et al. (2014) found Mg concentration as 34 mg/100g and Gonzalez et al. (2009) found 25.8mg/100g in cheese. Variation in magnesium concentration in both types of cheese is due to the variation in the milk composition from which they have been prepared. Murtaza et al. (2008) revealed that buffalo milk is richer in minerals like magnesium and in-organic phosphate. Ripening periods exerted a non-significant (P>0.05) impact on the concentration of cow and buffalo milk cheese during 90 days of storage. The magnesium contents increased from 32.61±0.382 mg/100g to 34.18±0.510 mg/100g in buffalo cheese whereas 25.13±0.363 mg/100g to 26.23±0.370 mg/100g in cow milk cheese (batch # 1). Current studies resemble with the findings of Gonzalez et al. (2009), who depicted the fact that ripening periods don’t influence the average concentration of magnesium in cheese.

92 Table 4.23 Analysis of variance for Sodium in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square NS NS Days (D) 3 24.8* 2 5.31 1 1.86 Milk (M) 1 10345.5** 1 6876.52** 1 4170.51** NS NS NS D x M 3 0.1 2 0.17 1 0.01 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

4.24a Effect of milk types and ripening period on sodium (mg/100g) of Cheddar cheese (Batch # 1)

Day Milk Means±SE Cow Buffalo 0 690.16±1.207 731.673±1.124 710.92±9.312B 30 691.72±2.481 733.067±1.758 712.40±9.344B 60 693.09±1.198 734.470±2.121 713.78±9.317AB 90 694.76±0.621 736.617±0.944 715.69±9.374A Means±SE 692.43±0.831B 733.957±0.864A

93 4.24b Effect of milk types and ripening period on sodium (mg/100g) of Cheddar cheese (Batch # 2)

Day Milk Means±SE Cow Buffalo 0 685.01±0.759 724.230±1.017 704.62±8.789 30 686.18±1.513 725.523±1.037 705.85±8.836 60 687.11±0.577 725.817±0.722 706.47±8.664 Means±SE 686.10±0.600B 725.190±0.528A

4.24c Effect of milk types and ripening period on sodium (mg/100g) of Cheddar cheese (Batch # 3)

Day Milk Means±SE Cow Buffalo 0 688.95±1.199 726.183±0.953 707.57±8.353 30 689.69±1.686 727.027±0.488 708.36±8.386 Means±SE 689.32±0.940B 726.605±0.515A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

94

Cow milk Buffalo milk

740

720

700

680

Na 660

640

620

600

Fig.4.10 Effect of milk types and ripening period on sodium (mg/100g) of

Cheddar cheese

95 Table 4.25 Analysis of variance for magnesium in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square NS NS NS Days (D) 3 2.994 2 2.50 1 1.613 Milk (M) 1 331.155** 1 527.69** 1 260.587** NS NS NS D x M 3 0.498 2 0.00 1 0.003 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

4.26a Effect of milk types and ripening period on magnesium (mg/100g) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 25.13±0.363 32.61±0.382 28.87±1.691 30 25.44±0.252 32.05±1.071 28.74±1.558 60 25.94±0.555 33.60±0.569 29.77±1.751 90 26.23±0.370 34.18±0.510 30.21±1.800 Means±SE 25.68±0.214B 33.11±0.385A

96 4.26b Effect of milk types and ripening period on magnesium (mg/100g) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 22.33±0.394 33.14±0.577 27.74±2.438 30 22.91±0.110 33.78±0.600 28.35±2.447 60 23.63±0.360 34.43±0.663 29.03±2.438 Means±SE 22.96±0.245B 33.78±0.359A

4.26c Effect of milk types and ripening period on magnesium (mg/100g) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 26.06±0.474 35.41±0.477 30.74±2.112 30 26.83±0.226 36.12±0.335 31.47±2.085 Means±SE 26.45±0.290B 35.77±0.304A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

97

Cow milk Buffalo milk

38

34

30

26

Mg 22

18

14

10

Fig.4.11 Effect of milk types and ripening period on magnesium (mg/100g) of

Cheddar cheese

98 4.5.5 Phosphorus in Cheddar cheese

Foods may contain both natural phosphorus and phosphate additives. Dairy foods are rich in phosphorus especially both types of cheese, natural and processed. On an average basis the cheese provides 522 mg P/100g. In milk, phosphorus is present in different fractions which affect its bioavailability e.g. Casein contains phosphopeptide, which is impervious to enzymatic hydrolyses (Uribarri and Calvo 2003). Phosphorus present in cheese is divided into two arrangements: Free (accompanying with the serum phase of cheese) and bound (linked with milk proteins). Bound phosphorus is further divided into two forms: organic P and bound in-organic P. Bound in-organic phosphate is lost during whey drainage whereas organic phosphate is not solubilized as it is associated with the phosphoserine (Rahimi et al., 2013).

Analysis of variance table 4.27 for phosphorus indicated a highly significant difference in both categories of cheese prepared from cow and buffalo milk presenting higher phosphorus content in buffalo milk cheese. Phosphorus was recorded as 480.773±0.660 mg/100g (table 4.28a), 478.033±1.059 mg/100g (table 4.28b) and 482.997±1.182 mg/100g (table 4.28c) in buffalo milk cheese batches at 0th day of analysis whereas 453.16±1.709 mg/100g, 445.25±1.262 mg/100g and 449.51±1.314 mg/100g in cow milk cheese at 0th day analysis (Fig. 4.12).

Significantly higher variation in phosphorus concentration in buffalo milk cheese is due to the less P drainage in whey as buffalo milk might have high amount of organic phosphate. Furthermore buffalo milk contains 18.5 mM of colloidal phosphate out of 27.7 mM which causes high P concentration in cheese prepared from buffalo milk (Ahmad et al., 2013).

Mean values of phosphorus in the present study reveal similar phosphorus concentration as declared by Caro et al. (2014) as 459mg/100g and 481mg/100g and Rahimi et al. (2013) as 488mg/100g in different cheese varieties.

A non-significant (P>0.05) trend in variation of phosphorus concentration was observed in both types of cheese during ripening upto 90 days. A non-significant increase in buffalo milk cheese from 480.773±0.660 mg/100g to 484.250±1.900 mg/100 while 453.16±1.709 mg/100g to 455.63±2.407 mg/100g in cow milk cheese was observed.

Sloublization of Ca and P takes place during acidification but P is less solubilized than Ca and goes into whey in lesser amount which reveals the non-significant changes. (Rahimi et al., 2013).

99 Table 4.27 Analysis of variance for phosphorus in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square NS NS NS Days (D) 3 9.47 2 3.1215 1 1.1163 Milk (M) 1 4705.40** 1 4842.5900** 1 3363.4000** NS NS NS D x M 3 0.28 2 0.0012 1 0.0001 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

4.28a Effect of milk types and ripening period on phosphorus (mg/100g) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 453.16±1.709 480.773±0.660 466.97±6.229 30 453.98±2.215 481.930±0.675 467.96±6.335 60 454.81±2.345 482.643±0.958 468.73±6.325 90 455.63±2.407 484.250±1.900 469.94±6.545 Means±SE 454.40±0.973B 482.399±0.625A

100 4.28b Effect of milk types and ripening period on phosphorus (mg/100g) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 445.25±1.262 478.033±1.059 461.64±7.368 30 446.01±0.652 478.800±0.687 462.41±7.344 60 446.66±0.976 479.500±0.647 463.08±7.361 Means±SE 445.97±0.538B 478.778±0.461A

4.28c Effect of milk types and ripening period on phosphorus (mg/100g) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 449.51±1.314 482.997±1.182 466.25±7.530 30 450.12±0.583 483.600±1.390 466.86±7.516 Means±SE 449.82±0.657B 483.298±0.827A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

101

Cow milk Buffalo milk

490

480

470

460

450

P 440

430

420

410

400

Fig.4.12 Effect of milk types and ripening period on phosphorus (mg/100g) of

Cheddar cheese

102 4.6 ORGANIC ACIDS IN CHEDDAR CHEESE The organic acids denote the third main group among the fermentation products (Soccol et al., 2008). Organic acids can be obtained from biomass-derivative sugars through fermentation (Du et al., 2014). Acetic acid bacteria, lactic acid bacteria and certain fungi are the producers of organic acids. Organic acids play an important role in fermented milks and cheese and the total aroma intensity is interrelated with organic acid intensities in aged cheeses (Akalin et al., 2002). Organic acid were determined after 30 day interval upto 90 days during ripening of Cheddar cheese prepared from cow and buffalo milk. 4.6.1 Lactic acid in Cheddar cheese Lactose (sugar) present in milk is converted into lactic acid by homo fermentative lactic acid bacteria. Lactic acid bacteria acidify milk and generate flavor and texture (Griffiths and Tellez 2013; Kongo, 2013). During ripening process of cheese, the lactic acid contribute to cheese classification (Widyastuti et al., 2014) and the most abundant organic acid in all cheeses is lactic acid (Izco et al., 2002). Analysis of variance table 4.29 shows the highly significant (P<0.01) difference among the lactic acid contents in all batches of cheeses prepared from cow and buffalo milk. The highest values of lactic acid were recorded in all cheese batches prepared from buffalo milk as 16549.33±29.45 ppm, 15450.00±52.92 ppm and 15916.67±66.92 ppm whereas lactic acid contents in cheese prepared from cow milk were 15135.00±30.14 ppm, 14313.33±89.50 ppm and 14943.33±34.80 ppm, respectively (Fig. 4.13). Lactic acid concentration in current study are close to the findings of Izco et al. (2002), who reported 21852 ppm lactic acid contents in Cheddar cheese and Caro et al. (2014) reported lactic acid as 17300 ppm and 18900 ppm in Mexican cheeses. Sharma et al. (2013) studied the production of lactic acid and reported much higher lactic acid percentage in buffalo milk as compared to cow milk. During ripening, a highly significant (P<0.01) increase in lactic acid concentration was observed in cheese prepared from buffalo milk ranging from 16549.33±29.45 ppm to 18118.33±50.85 ppm in batch # 1 (0-90 day) , 15450.00±52.92 ppm to 16610.00±90.18 ppm in batch # 2 (0-60 day) and 15916.67±66.92 ppm to 16730.00±87.37 ppm in batch # 3 (0-30 day). Same trend of increase in lactic acid concentration during ripening was observed in cheese prepared from cow milk showing an increase of 15135.00±30.14 ppm to 16871.67±60.85 ppm in batch #1 (0-90 d ay), 14313.33±89.50 ppm to 15570.00±40.41

103 ppm in batch # 2 (0-60 day) and 14943.33±34.80 ppm to 15653.33±81.10 ppm in batch # 3 during 0-30 day of ripening. Upreti et al. (2006) described a significant increase in lactic acid in Cheddar cheese during ripening period upto 48 weeks. Lipolysis, proteolysis and lactose, lactate and citrate metabolism causes an increase in lactic acid concentration during ripening. Lactose is changed into lactate which later on produces organic acids through racemization, oxidation or microbial metabolism (McSweeney, 2004; Ong et al., 2007a). 4.6.2 Acetic acid in Cheddar cheese During glycolysis numerous flavoring compounds like diacetyl and acetic acid are produced. Acetic acid and certain other compounds are affected by the degradation of lactose (Forde and Fitz-Gerald, 2000). Results regarding acetic acid concentration of cow and buffalo milk cheese have been presented in the table 4.31 and 4.32a-c. A highly significant difference in cow and buffalo milk cheese was recorded with reference acetic acid, being high in buffalo milk cheese as compared to cow milk. Acetic acid in cow milk cheese was 181.33±3.180 ppm in batch #1, 215.33±6.064 ppm in batch # 2and 194.33±3.180 ppm in batch # 3 (Fig. 4.14).

Citrate is quickly metabolized to produce acetic acid, diacetyl and CO2 in the presence of fermentable carbohydrates (Cogan and Hill 1993). Highly significant acetic acid content in buffalo milk Cheddar is due to the increased citrate concentration in buffalo milk (144-224 mg/100ml) as compared to the cow milk (160-176.8 mg/100ml) as reported by Claeys et al. (2014). Values of acetic acid determined in the present study are comparable to the findings of Izco et al. (2002) as 434.00 ppm (in Cheddar Cheese); Desfosses-Foucault et al. (2012) as 400 ppm (in Cheddar cheese) and Caro et al. (2014) as 300ppm - 600 ppm (in Mexican cheeses). During ripening up to 90 days, a highly significant increase in acetic acid concentration was observed in both types of cheese. Acetic acid concentration in cow milk cheese increased from 181.33±3.180 ppm to 322.00±3.512 ppm (batch # 1) from 0 to 90 days, 215.33±6.064 ppm to 311.00±2.082 ppm (batch # 2) from 0-60 days and 194.33±3.180 ppm to 219.33±3.528 ppm (batch # 3) from 0-30 day. A similar increase in acetic acid concentration was observed in buffalo

104 Table 4.29 Analysis of variance for lactic acid in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square Days (D) 3 3063740** 2 2208154** 1 1740408** Milk (M) 1 9898357** 1 5308368** 1 3151875** NS NS NS D x M 3 13182 2 3526 1 8008 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.30a Effect of milk types and ripening period on lactic acid (ppm) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 15135.00±30.14 16549.33±29.45 15842.17±316.82D 30 15801.67±55.25 17083.67±42.23 16442.67±288.35C 60 16415.33±98.87 17610.00±75.06 17012.67±272.84B 90 16871.67±60.85 18118.33±50.85 17495.00±281.01A Means±SE 16055.92±198.9B 17340.33±177.8A

105 Table 4.30b Effect of milk types and ripening period on lactic acid (ppm) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 14313.33±89.50 15450.00±52.92 14881.67±258.38C 30 14850.00±80.83 15931.67±25.22 15390.83±244.81B 60 15570.00±40.41 16610.00±90.18 16090.00±236.71A Means±SE 14911.11±185.6B 15997.22±171.0A

Table 4.30c Effect of milk types and ripening period on lactic acid (ppm) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 14943.33±34.80 15916.67±66.92 15430.00±220.24B 30 15653.33±81.10 16730.00±87.37 16191.67±246.58A Means±SE 15298.33±163.5B 16323.33±188.4A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

106

Cow milk Buffalo milk

18500

17000

15500

d

i 14000

c

A

c

i t

ac

L 12500

11000

9500

8000

Fig.4.13 Effect of milk types and ripening period on lactic acid (ppm) of

Cheddar cheese

107 Table 4.31 Analysis of variance for acetic acid in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square Days (D) 3 24481** 2 13774.9** 1 64.55** Milk (M) 1 147580** 1 67957.6** 1 1466.25** NS NS NS D x M 3 118 2 64.2 1 4.65 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.32a Effect of milk types and ripening period on acetic acid (ppm) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 181.33±3.180 327.667±4.631 254.50±32.817D 30 225.33±4.842 379.333±4.631 302.33±34.566C 60 271.33±4.096 438.667±4.667 355.00±37.520B 90 322.00±3.512 481.667±5.840 401.83±35.832A Means±SE 250.00±15.87B 406.833±17.73A

108 Table 4.32b Effect of milk types and ripening period on acetic acid (ppm) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 215.33±6.064 342.000±2.646 278.67±28.478C 30 264.00±3.786 379.333±5.812 321.67±25.975B 60 311.00±2.082 437.667±4.333 374.33±28.405A Means±SE 263.44±13.97B 386.333±14.09A

Table 4.32c Effect of milk types and ripening period on acetic acid (ppm) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 194.33±3.180 348.000±4.041 271.17±34.438B 30 219.33±3.528 391.333±5.783 305.33±38.579A Means±SE 206.83±5.980B 369.667±10.19A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

109

Cow milk Buffalo milk

500

450

400

350

300

d

i

c

A

c

i 250 t ce

A 200

150

100

50

0

Fig.4.14 Effect of milk types and ripening period on acetic acid (ppm) of

Cheddar cheese

110 milk cheese having 327.667±4.631 ppm to 481.667±5.840 ppm (batch # 1) from 0-90 day, 342.000±2.646 ppm to 437.667±4.333 ppm (batch # 2) from 0-60 day and 348.000±4.041 ppm to 391.333±5.783 (batch # 3) from 0-30 days respectively. Upturn of acetic conforms to the studies of Ong et al. (2007a), who showed an increase in the acetic acid as ripening progressed being highest at the end of ripening period. A significant increase in the acetic acid during ripening of Cheddar cheese has also been observed by Upreti et al. (2006) due to the lactose fermentation. Biochemical changes occurring during cheese ripening influence the production of organic acids (Murtaza et al., 2012). 4.6.3 Citric acid in Cheddar cheese Citric acid is the major product of carbohydrate catabolism of lactic acid bacteria. Transition of the culture from logarithmic phase to the retardation phase causes synthesis of citric acid and iso-citric acid (Otto et al., 2013). Citric acid is also produced by some fungi and the best substrate for citric acid production is glucose. Acidity is increased with the production of citric acid which retards the growth of the spoilage and pathogenic microorganisms hence improve the hygienic quality of cheese (Izco et al., 2002). Analysis of variance table 4.33 shows that milk types and ripening, both have highly significant effect on citric acid contents in Cheddar cheese. A highly significant difference of citric acid concentration was observed in both types of cheese having highest values in buffalo milk cheese. The range of citric acid in buffalo milk cheese was 728.000±5.132 ppm to 758.333±4.410 ppm while 542.00±3.512 ppm to 572.67±5.333 ppm was observed in cow milk cheese (Fig. 4.15). Results of citric acid concentration in the present study are within range of 600- 800 ppm (in Cheddar cheese) as established by Murtaza et al. (2012) 577-3020 ppm as by Lues (2000) and 1954 ppm as depicted by Izco et al. (2002). Much better nutrient profile of buffalo milk provides an opportunity for microorganisms to quickly multiply (Han, et al., 2007). Raj and Chaudhary (2014) illustrated the production of citric acid from dairy products and showed that dairy products having high concentration of Lactose (Disaccharide having Glucose and Galactose), Calcium to phosphate ratio and riboflavin are rich in citric acid synthesis. Increased amount of these substances in buffalo milk has been reported by Ahmad et al. (2013) which is the reason of high citric acid concentration in buffalo milk cheese.

111 Table 4.33 Analysis of variance for citric acid in cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square Days (D) 3 63604** 2 37061** 1 14840** Milk (M) 1 213759** 1 194064** 1 100101** NS D x M 3 152 2 928** 1 5NS Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.34a Effect of milk types and ripening period on citric acid (ppm) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 542.00±3.512 728.000±5.132 635.00±41.684D 30 615.33±6.064 812.000±3.512 713.67±44.088C 60 701.00±7.371 897.667±7.219 799.33±44.217B 90 784.33±2.963 960.000±5.508 872.17±39.380A Means±SE 660.67±27.50B 849.417±26.52A

112 Table 4.34b Effect of milk types and ripening period on citric acid (ppm) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 572.67±5.333f 758.333±4.410c 665.50±41.632C 30 640.67±4.631e 843.333±5.696b 742.00±45.436B 60 705.33±7.860d 940.000±5.292a 822.67±52.644A Means±SE 639.56±19.39B 847.222±26.36A

Table 4.34c Effect of milk types and ripening period on citric acid (ppm) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 557.67±4.333 739.000±4.509 648.33±40.644B 30 626.67±3.844 810.667±6.360 718.67±41.278A Means±SE 592.17±15.64B 774.833±16.40A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

113

Cow milk Buffalo milk

1000

900

800

700

600

d

i

c

A

c 500

i

tr i

C 400

300

200

100

0

Fig.4.15 Effect of milk types and ripening period on citric acid (ppm) of

Cheddar cheese

114 Highly significant increase in the citric acid concentration was also noted in both types of cheese during ripening period from 0-90 days. Increase recorded in buffalo milk cheese was 728.000±5.132 ppm to 960.000±5.508 ppm in batch # 1(0-90 days), 758.333±4.410 ppm to 940.000 ±5.292 ppm in batch # 2, (0-60 days) and 739.000±4.509 ppm to 810.667±6.360 ppm in batch # 3, (0-30 days). Likewise increase was noted in cow milk cheese during entire ripening period showing 542.00±3.512 ppm to 784.33±2.963 ppm in batch #1 (0-90 days), 572.67±5.333 to 705.33±7.860 ppm in batch # 2 (0-60 days) and 557.67±4.333 ppm to 626.67±3.844 ppm in batch # 3 (0-30 days). A substantial increase in citric acid concentration in Cheddar cheese during week 4 to week 48 was also recorded by Upreti et al. (2006). Citrate fermenting microorganisms convert citrate to pyruvate, CO2 and acetic acid influencing the citric acid contents. Lues and Bekker (2002) noted a decrease in citric acid upto 40 day ripening and then increased dramatically at the end of ripening period. Several metabolites such as lactate and citrate serve as originators of series of reactions that causes production of various organic acids lactic, acetic, citric, formic and pyruvic acids. Generally organic acid production increases with the progress of ripening period (Akalin et al., 2002; Lues and Bekker 2002; Singh et al., 2003; Farkye, 2004 and Ong et al., 2007). 4.6.4 Butyric acid in Cheddar cheese Butyric acid is not only formed by fats but proteolysis and deamination of protein (Casein) degradation products lead to butyric acid production (Califano and Bevilacqua, 2000; Akalin et al., 2002). There are certain Clostridial strains which can transform sugars to butyric acid (Zigova and Sturdik 2000). Short chain free fatty acids are directly involved in aroma production in most of the aged cheeses as described by Jung et al. (2013). Butyric acid is mostly involved in flavor production in industrial applications (Du et al., 2014). Fruity flavor has been attributed to cheese because of butyric acid (Hayaloglu, 2009). The analysis of variance table 4.35 shows the results of the butyric acid produced in cheese prepared from cow and buffalo milk during ripening period upto 90 days. A highly significant difference in both cheeses (cow and buffalo). The highest amounts of butyric acid were detected in cheese (1355.00±9.29 ppm, 1358.00±16.80 ppm and 1358.33±9.28 ppm) prepared from buffalo milk than the concentration (1022.67±04.33 ppm, 1047.67±10.27 ppm and 1044.33±6.84 ppm) in cow milk cheese (Fig. 4.16).

115 The outcomes of the present study regarding butyric acid are close to the findings of Murtaza et al. (2012), who reported butyric acid as 1500-2000 ppm in Cheddar cheese and Izco et al. (2002), who declared butyric acid as 892 ppm in Cheddar cheese. Andic et al. (2011) studied the butyric acid concentration ranging from 1041-1825 ppm in Kashar (Turkish) cheese packed as vacuum sealed in 120 days ripening. As mentioned earlier that fat, casein and lactose are converted into butyric acid during cheese fermentation and are higher in buffalo milk when compared with cow milk. High amounts of these substances in buffalo milk are the reason of significantly higher amounts of butyric acid in buffalo milk cheese (Murtaza et al., 2012). During ripening periods, significantly high rise in butyric acid values was noted in both cheeses. Butyric acid increased from 1355.00±9.29 ppm to 1862.33±7.22 ppm in batch # 1 (0-90 days), 1358.00±16.80 ppm to 1701.67±18.33 ppm in batch # 2 (0-60 days) and 1358.33±9.28 ppm to 1566.67±7.69 ppm in batch # 3 (0-30 days) in buffalo milk cheese and likewise 1022.67±04.33 ppm to 1572.33±10.11 ppm in batch # 1 (0-90 days), 1047.67±10.27 ppm to 1420.00±10.07 ppm in batch # 2 (0-60 days) and 1044.33±6.84 ppm to 1234.67±8.97 ppm in batch # 3 (0-30 days) in cow milk cheese, respectively. Significantly higher (P<0.01) increase in the both types of cheese during ripening is justified by studies of Kara et al. (2014), who reported highly significant increase in butyric acid in Tulum cheese during ripening upto 90 days. A highly significant increase in butyric acid during ripening upto 90 days has also been observed by Andic et al. (2011). Lipase is the main lipolytic agent in raw milk cheeses (Turkoglu, 2011) and significant increase of short chain fatty acids (butyric acid C4:0, Caproic acid C6:0 and Caprillic acid C8:0) during ripening depended up on the lipase concentration. Lipolysis rate of Cheddar cheese is increased by increased concentration of lipase as detected by Ozcan and Kurdal (2012). 4.6.5 Pyruvic acid in Cheddar cheese A key position in cell metabolism is occupied by the pyruvic acid. Pyruvic acid is involved in many biochemical pathways including glycolysis, gluconeogenesis, amino acid, and protein metabolism. Homo fermentative lactic acid bacteria ferment galactose and fructose which undergo many biochemical pathways (glycolysis) to produce pyruvic acid. Pyruvate is produced in cheese through various metabolic paths using carbon sources i.e. through metabolism of citrate, lactate, amino acids, and nucleotides (Lazzi et al., 2014). Pyruvate is also produced in cheese matrix by starter lysis (Liu et al., 2003).

116 Milk types showed significant, while ripening showed highly significant impact on Cheddar cheese as given in analysis of variance table 4.37. Significant (P<0.05) difference was observed in both types of cheese with pyruvic acid being high in cheese batch # 1 and 2 produced from buffalo milk and batch # 3 produced from cow milk. Values of pyruvic acid were 86.833±1.740 ppm (batch # 1), 87.000±0.866 ppm (batch # 2) and 88.500±1.443 ppm (batch # 3) in buffalo milk while 82.43±0.348 ppm (batch # 1), 86.33±0.882 ppm (batch # 2) and 94.50±0.866 ppm (batch # 3) in cow milk cheese (Fig. 4.17). Results of pyruvic acid in the current study are in connection with the findings of Akalin et al. (2002), who declared the mean pyruvic acid concentration of pickled white cheese within 100-180 ppm. Dolci et al. (2008) described the concentration of pyruvic acid ranges from 38-303 ppm during ripening upto 90 days of a traditional Italian cheese. Variation in the pyruvic acid concentration is justified by Islam et al. (2013) they studied the effect of feed on the metabolites (pyruvic, acetic, citric, orotic, succinic, uric acid) present in buffalo, holstein cross, indigenous and red Chittagong cattle and found pyruvic acid only in milk of buffalo. Absence of pyruvic acid in the milk of other animals is related to feed and in the current study an increased amount of pyruvic acid in batch # 3 of cow milk cheese might be the feeding effect. Ripening periods highly significantly (P<0.01) influenced the pyruvic acid contents in both types of cheese. Pyruvic acid increased highly significantly during entire ripening period upto 90 days. Increase was started from 86.833±1.740 ppm to 155.667±2.906 ppm in batch # 1 (0-90 days), 87.000±0.866 ppm to 134.000±2.598 ppm in batch # 2 (0-60 days) and 88.500±1.443 ppm to 109.167±1.590 ppm in batch # 3, (0- 30 days) in buffalo milk cheese whereas 82.43±0.348 ppm to 150.07±3.652 ppm in batch # 1 (0-90 days), 86.33±0.882 ppm to 127.00±2.082 ppm in batch # 2 (0-60 days) and 94.50±0.866 ppm to 111.67±2.804 ppm pyruvic acid in cow milk cheese. As pyruvate is the core metabolite in sugar metabolism and is formed through glycolytic pathway. It can serve as substrate for various metabolic reactions hence, its decrease or increase is not at the same rate during ripening (Hutkins, 2001). At initial stages of ripening pyruvic acid production is not at regular pattern however cheeses with high levels of Ca and P exhibit a higher increase in pyruvic acid (as in the present study in case of buffalo milk cheese) concentration during later stages of ripening (Upreti et al., 2006).

117 Table 4.35 Analysis of variance for butyric acid in cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square Days (D) 3 307877** 2 192254** 1 119201** Milk (M) 1 480534** 1 387493** 1 312987** NS NS D x M 3 2329** 2 338 1 243 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.36a Effect of milk types and ripening period on butyric acid (ppm) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 1022.67±04.33h 1355.00±9.29f 1188.83±74.45D 30 1273.00±11.50g 1510.67±9.68d 1391.83±53.57C 60 1422.00±12.77e 1694.00±13.6b 1558.00±61.39B 90 1572.33±10.11c 1862.33±7.22a 1717.33±65.08A Means±SE 1322.50±61.33B 1605.50±57.6A

118 Table 4.36b Effect of milk types and ripening period on butyric acid (ppm) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 1047.67±10.27 1358.00±16.80 1202.83±69.95C 30 1235.67±16.19 1524.00±9.07 1379.83±65.01B 60 1420.00±10.07 1701.67±18.33 1560.83±63.67A Means±SE 1234.44±54.10B 1527.89±50.20A

Table 4.36c Effect of milk types and ripening period on butyric acid (ppm) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 1044.33±6.84 1358.33±9.28 1201.33±70.40B 30 1234.67±8.97 1566.67±7.69 1400.67±74.43A Means±SE 1139.50±42.8B 1462.50±46.9A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

119

Cow milk Buffalo milk

2000

1800

1600

1400

1200

d

i c

A

c

i 1000

r

y

t

u B 800

600

400

200

0

Fig.4.16 Effect of milk types and ripening period on butyric acid (ppm) of Cheddar cheese

120 Table 4.37 Analysis of variance for pyruvic acid in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square Days (D) 3 5209.27** 2 2898.10** 1 1073.52** Milk (M) 1 98.01* 1 136.13** 1 54.19** NS NS NS D x M 3 2.20 2 27.54 1 9.19 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.38a Effect of milk types and ripening period on pyruvic acid (ppm) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 82.43±0.348 86.833±1.740 84.63±1.264D 30 103.00±1.607 106.167±2.205 104.58±1.411C 60 126.33±2.186 129.333±3.180 127.83±1.851B 90 150.07±3.652 155.667±2.906 152.87±2.434A Means±SE 115.46±7.692B 119.500±7.837A

121

Table 4.38b Effect of milk types and ripening period on pyruvic acid (ppm) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 86.33±0.882 87.000±0.866 86.67±0.573C 30 107.00±1.732 115.833±1.740 111.42±2.260B 60 127.00±2.082 134.000±2.598 130.50±2.160A Means±SE 106.75.927B 112.278±6.906A

Table 4.38c Effect of milk types and ripening period on pyruvic acid (ppm) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 94.50±0.866 88.500±1.443 91.50±1.538B 30 111.67±2.804 109.167±1.590 110.42±1.546A Means±SE 103.08±4.057A 98.833±4.720B Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

122

Cow milk Buffalo milk

160

140

120

100

d i

c

A

c i 80

v

u

r

y P 60

40

20

0

Fig.4.17 Effect of milk types and ripening period on pyruvic acid (ppm) of

Cheddar cheese

123 4.7 PROTEOLYSIS IN CHEDDAR CHEESE Proteolysis is acute process in the manufacture of all types of cheeses. It is accountable for structural changes and significantly affects the cheese flavor and taste formation (Orlyuk and Stepanishchev, 2014). Numerous factors playing role in proteolysis are raw milk microflora, instigation of plasminogen into plasmin, industrialized process and technology involved, nature of microbial culture used, ripening interval and the conditions during ripening. High temperature, increased amount of rennet, water activity in cheese, lower salt and encouraging pH cause increased proteolysis (Kalit et al., 2005). Proteolysis determines the organoleptic properties of cheese i.e. formation of cheese taste and flavor (Pitt and Hocking 2009; Heather 2012). During ripening of Cheddar cheese, proteolysis develops texture and flavor of cheese. Proteolysis is also involved in softening of Cheddar cheese texture by breaking intact casein into polypeptides and small water soluble peptides that are not added to the protein matrix (Upreti and Metzger, 2006). Proteolysis intensity is greater in mold ripened cheeses as compared to other cheeses (Lison, 2013). During proteolysis, proteins are tarnished to primary products (polypeptides) and afterwards to secondary products like small and medium sized peptides and ultimately to amino acids. Residual enzymes from milk and rennet, salt in moisture (S/M), ripening temperature and pH changes during ripening are the main parameters which cause protein hydrolysis (Lawrence and Gilles, 1987). Evolution of proteolysis in Cheddar cheese made from cow and buffalo milk was followed at 0, 30, 60 and 90 day interval by determining water soluble nitrogen (WSN) as percentage of total nitrogen (WSN/TN%) and total free amino acids (TFAA%). Data presented in tables showed highly significant variations (P<0.01) related to ripening periods (days) in both parameters (WSN/TN% and TFAA%) in all batches of Cheddar cheese prepared from cow and buffalo milk. A significant effect was also observed in all batches of cheese with respect to milk sources for these parameters. 4.7.1 Water soluble nitrogen (primary proteolysis) Water soluble fraction is very diverse in footings of composition and contains whey proteins, amino acids and high, medium and low molecular weight peptides (Tavaria et al., 2004). Extent of secondary proteolysis i.e. formation of small sized residues and free amino acids is often measured by the water soluble nitrogen (WSN) (Furtado and Partridge, 1988). Peptide / protein hydrolysis products generate water soluble nitrogen and amino acids (Muehlenkamp-Ulate and Warthesen, 1999; Mikulec, 2010).

124 Analysis of variance (ANOVA) table 4.39 for water soluble nitrogen as percentage of total nitrogen (WSN/TN%) showed a highly significant (P<0.01) difference in batch #1 (table 4.40a) of both cheeses being high in cheese prepared from cow milk as 5.55±0.18 and low in cheese prepared from buffalo milk as 4.98±0.29 at 0 day. Similarly significant difference (P<0.05) in WSN/TN% was also observed in batch # 2 (table 4.40b) and batch # 3 (Table 4.40c) in both types of cheeses. Results are close to the findings of Miocinovic et al. (2014), they found WSN/TN% as 8.55±0.07 and 8.65±0.09 in low fat ultra-filtered cheese at 0 day analysis. Greater amount of WSN/TN% in cow milk cheese might be due to the greater amount of water soluble proteins in cow milk as compared to buffalo milk discussed by (Kumar et al., 2012). As ripening progressed, there was highly significant (P<0.01) increase in WSN/TN % of both types of cheeses in all batches as given in (Table 4.40a, 4.40b and 4.40c). Value of WSN/TN% increased in buffalo milk cheese from 5.55±0.18 to 15.40±0.31, 5.13±0.11 to 11.92±0.30 and 5.18±0.06 to 8.72±0.16 in batch # 1, 2 and 3, respectively (Fig. 4.18). Similarly in cow milk cheese these values were as 4.98±0.29 to 14.82±0.11 (0-90 days), 4.82±0.16 to 11.42±0.26 (0-60 days) and 4.92±0.13 to 8.30±0.19 (0-30 days). Water soluble nitrogen expressed as total nitrogen (as primary proteolysis product) increased highly significantly because degradation of casein to low molecular weight water soluble peptides and amino acids take place by milk enzymes, lactic acid and non-starter lactic acid bacteria (Azarnia et al., 2010). Rehman et al. (1998) declared that remaining coagulant and plasminogen are liable for production of WSN. 4.7.2 Total free amino acids (secondary proteolysis) Small peptides and free amino acids are produced by the action of microbial peptidases and proteases. Free amino acids are the final products of the proteolysis and their concentration during ripening depends upon the liberation of amino acids from casein and transformation into catabolic products (Hassan et al., 2013). Amino acids, peptides, organic acids and amines contain the flavor components (Mikulec et al., 2010). Texture and organoleptic properties of cheese are also influenced by amino acids and soluble peptides (Law, 1999). Lactic acid bacteria (LAB) and non-starter lactic acid bacteria (NSLAB) produce enzymes like proteinases and peptidases and their activity induces secondary proteolysis (Sousa et al., 2001). Analysis of variance (ANOVA) table 4.41 shows the results of total free amino acids in Cheddar cheese prepared from cow and buffalo milk. Milk sources showed a highly significant (P<0.01) variability of total free amino acids in batch # 1 and 2 in both

125 types of cheese and a significant (P<0.05) difference in batch # 3. Concentration of total free amino acids (TFAA) in cow milk cheese at 0 day was 1248.33±23.15µg/g (batch # 1) 1325.00±37.53 µg/g (batch # 2) and 1271.67±20.88 µg/g (batch # 3) while lesser amount of TFAA was found in buffalo milk Cheddar cheese having 1098.33±50.69µg/g (batch # 1), 1138.33±26.82µg/g (batch # 2) and 1130.00±30.41 µg/g (batch # 3) in fresh cheese (Fig. 4.19). Hou et al. (2014) studied the proteolysis in full fat (31%) Cheddar cheese and found the total amino acids within the range of 1000 µg/g to 5000 µg/g during 90 days of ripening which are comparable to the current results. Comparable results were also shown by Rulikowska et al. (2013) studied the impact of reduced sodium chloride on Cheddar cheese and found TFAA concentration between 1000 µg/g to 3000 µg/g during ripening of 112 days. Difference in the concentration of TFAA may be due to the dissimilarity in peptidase activity affected by pH and non-starter lactic acid bacteria (Gobbetti et al., 1999 a,b), penetrability of starter and non-starter bacteria and the point of autolysis (Doolan and Wilkinson, 2009). Higher amount of TFAA in cow milk cheese might be due to the rapid proteolysis in cow milk and having more amounts of soluble proteins as compared to buffalo milk. TFAA increased highly significantly (P>0.01) in all batches of cheese prepared from cow and buffalo milk during ripening as given in ANOVA Table 4.41. In cow milk cheese, TFAA increased from 1248.33±23.15 µg/g to 2501.67±53.57 µg/g from 0-90 days in batch # 1 (table 4.42a), 1325.00±37 µg/g to 2200.00±53.93 µg/g from 0-60 days in batch # 2 (table 4.42b) and 1271.67±20.88 µg/g to 1668.33±63.40 µg/g 0-30 days in batch # 3 (table 4.42c). Buffalo milk cheese also presented a similar increasing trend as 1098.33±50.69 µg/g to 2433.33±29.63 µg/g in batch # 1 at (0-90 days), 1138.33±26.82 µg/g to 2023.33±72.19 µg/g in batch # 2 at (0-60 days) and 1130.00±30.41 µg/g to 1578.33±30.32 µg/g in batch # 3 during (0-30 days). Free amino acids are mostly released by the action of rennet and starter cultures. Peptidase from milk also releases FAA to some extent as a result the amino acid concentration increases during ripening (Kalit et al., 2005). Rehman et al. (2003) also stated that amino acids are released by the starter and non-starter peptidases.

126 Table 4.39 Analysis of variance for water soluble nitrogen / total nitrogen in

Cheddar cheese.

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square Days (D) 3 108.731** 2 67.1735** 1 35.8802** Milk (M) 1 2.802** 1 0.7401* 1 0.3502* NS NS NS D x M 3 0.029 2 0.0126 1 0.0169 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.40a Effect of milk types and ripening period on water soluble nitrogen /

total nitrogen (%) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 5.55±0.18 4.98±0.29 5.27±0.20D 30 9.10±0.25 8.38±0.22 8.74±0.22C 60 12.60±0.31 11.73±0.19 12.17±0.25B 90 15.40±0.31 14.82±0.11 15.11±0.20A Means±SE 10.66±1.12A 9.98±1.11B

127 Table 4.40b Effect of milk types and ripening period on water soluble nitrogen /

total nitrogen (%) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 5.13±0.11 4.82±0.16 4.98±0.11C 30 8.47±0.22 8.07±0.16 8.27±0.15B 60 11.92±0.30 11.42±0.26 11.67±0.21A Means±SE 8.51±0.99A 8.10±0.96B

Table 4.40c Effect of milk types and ripening period on water soluble nitrogen /

total nitrogen (%) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 5.18±0.06 4.92±0.13 5.05±0.09B 30 8.72±0.16 8.30±0.19 8.51±0.14A Means±SE 6.95±0.79A 6.61±0.76B Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

128

Cow milk Buffalo milk

18

16

%

14 n

e

og

r

t i 12

N

l

a t

To

/ 10

n e

og

r

t

i 8

N

e l

ub l 6

So

r

e t a 4 W

2

0

Fig.4.18 Effect of milk types and ripening period on water soluble

nitrogen / total nitrogen (%) of Cheddar cheese

129 Table 4.41 Analysis of variance for total free amino acids in Cheddar cheese

Source of Batch # 1 Batch # 2 Batch # 3 variation d.f Mean square d.f Mean square d.f Mean square Days (D) 3 1816569** 2 1170050** 1 535519** Milk (M) 1 97538** 1 168200** 1 40252* NS NS NS D x M 3 4146 2 650 1 2002 Error 16 12 8 Total 23 17 11 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

Table 4.42a Effect of milk types and ripening period on total free amino acids

(µg/g) of Cheddar cheese (Batch # 1)

Storage Days Milk Means±SE Cow Buffalo 0 1248.33±23.15 1098.33±50.69 1173.33±41.79D 30 1698.33±50.53 1510.00±18.93 1604.17±48.54C 60 2030.00±27.54 1926.67±24.89 1978.33±28.45B 90 2501.67±53.57 2433.33±29.63 2467.50±31.35A Means±SE 1869.58±139.31A 1742.08±149.93B

130

Table 4.42b Effect of milk types and ripening period on total free amino acids

(µg/g) of Cheddar cheese (Batch # 2)

Storage Days Milk Means±SE Cow Buffalo 0 1325.00±37.53 1138.33±26.82 1231.67±46.56C 30 1715.00±51.07 1498.33±33.21 1606.67±55.58B 60 2200.00±53.93 2023.33±72.19 2111.67±56.43A Means±SE 1746.67±128.8A 1553.33±130.7B

Table 4.42c Effect of milk types and ripening period on total free amino acids

(µg/g) of Cheddar cheese (Batch # 3)

Storage Days Milk Means±SE Cow Buffalo 0 1271.67±20.88 1130.00±30.41 1200.83±35.72B 30 1668.33±63.40 1578.33±30.32 1623.33±37.32A Means±SE 1470.00±93.59A 1354.17±102.07B Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

131

Cow milk Buffalo milk

2700

2400

2100

) 1800

g /

g

µ

(

d i 1500

c

A

o

n

i

m 1200

A

l

a

t

To 900

600

300

0

Fig.4.19 Effect of milk types and ripening period on total free amino acids

(µg/g) of Cheddar cheese

132 4.8 SELECTION OF EMULSIFYING SALTS (COMBINATION) The results of functional (hardness, gumminess and melt-ability) and sensory (color and flavor) characteristics of processed Cheddar cheese prepared by using different combinations of emulsifying salts are presented in table (4.43). It is obvious from the results that when emulsifying salts used singly, they showed maximum or minimum values for some properties of processed cheese which is undesirable. Sodium citrate when used alone (3.0%), it gained maximum values for hardness, gumminess and melt- ability, whereas di- sodium phosphate attained minimum values for these parameters. Brickley et al. (2008) also found that when di-sodium phosphate was used more than 2.0%, showed crystallization and reduced melt-ability while the cheese manufactured by using more than 3.0% sodium citrate was more meltable. As the sensory perception is concerned, the processed cheese samples having single emulsifying salts were unable to get good scores while those made by using combinations of emulsifying salts conquered better scoring. The highest scores were attained by treatment (T9) having sodium citrate (50%), di-sodium phosphate (25%) and tri-sodium phosphate (25%). The cheese sample (treatment) also showed optimum values for functional properties. The results obtained are supported by the previous studies since larger fat pockets; grainy texture and weaker body were obtained in cheese when di-sodium phosphate was used in greater amounts (Rayan et al., 1980). Similarly crystals were observed and melt- ability was low in cheese prepared using di-sodium phosphate more than 2% while cheese processed with more than 3% sodium citrate was more meltable (Brickley et al., 2008). Tri-sodium phosphate produced more firm cheese when used singly as compared to di-sodium phosphate and sodium citrate (Mizuno and Lucey, 2007; Bunka et al., 2013). Increased amount of sodium citrate led to the much better body, texture, melt- ability and sensory properties (Shirashoji et al., 2006). Hence, based on the present results and literature, the combination of sodium citrate (50%), di-sodium phosphate (25%) and tri-sodium phosphate (25%) was selected as emulsifying salt for the manufacturing of processed Cheddar cheese in further studies.

133

Table 4.43 Effect of emulsifying salts on functional and sensory properties (Means±SE) of processed Cheddar cheese

Treatments Hardness (g) Gumminess Meltability (mm) Color (Hedonic) Flavor (Hedonic)

T1 3810±78 3890±50 65±0.78 6.5±0.48 7.0±0.52

T2 3550±67 3610±85 45±0.56 7.0±0.52 6.5±0.45

T3 3700±54 3750±102 54±0.83 6.5±0.61 7.0±0.38

T4 3685±86 3775±60 58±0.66 7.0±0.45 6.0±0.40

T5 3770±93 3830±105 51±0.92 7.0±0.58 7.5±0.62

T6 3640±90 3735±70 61±0.58 6.5±0.51 5.5±0.35

T7 3660±45 3750±92 56±0.70 7.5±0.42 7.5±0.55

T8 3680±73 3690±68 49±0.46 7.0±0.27 7.0±0.26

T9 3665±104 3710±55 60±0.62 8.0±0.45 8.0±0.40

T10 3740±66 3770±64 50±0.78 7.0±0.38 7.5±0.46

134

4.9 PHYSICO-CHEMICAL ANALYSIS OF PROCESSED CHEDDAR CHEESE

Viscoelastic matrix containing cheese of different maturity periods as basic ingredient is characterized as processed cheese (Nagyova et al., 2014). It is made from good quality natural cheeses using blends of fresh, young and matured natural cheese (Tamime et al., 2011). Key constituents for the manufacture of processed cheeses are emulsifying salts (ES), typically sodium salts of phosphates, polyphosphates, or citrates (Guinee et al., 2004; Mizuno and Lucey, 2007). Numerous factors such as chemical and compositional properties affect the functional properties of processed cheese (Kapoor and Metzger, 2008). Uniform structure of processed cheese is obtained by using emulsifying salts and most commonly used are sodium phosphates, polyphosphates and citrate (Sadlikova et al., 2010). Emulsifying salts are used in processed cheese at the rate of 2-3 % in the final product (Guinee et al., 2004; Mizuno and Lucey, 2005). In the current study blends of Cheddar cheese made from cow and buffalo milk (Ripened at 0-90 days) were used in the manufacture of processed Cheddar cheese using phosphate containing emulsifying salts @ 3% (di-sodium phosphate 50% , tri-sodium phosphate 25% and sodium citrate 25%). Physico-chemical analysis including moisture, fat, protein, ash, pH, acidity and salt content were determined in fresh processed cheese and with one month interval during storage of 90 days. Statistical analysis of physico-chemical composition of processed Cheddar cheese given in table 4.44 showed that the storage period had non-significant (p>0.05) effect on moisture, fat, protein and ash content and highly-significant (p<0.01) effect on all other parameters. Milk types (cow, buffalo and blend) showed highly significant (p<0.01) effect on all parameters except salt content. Various treatments (based on ripening periods) of processed cheese highly significantly (p<0.01) affected the moisture, pH, acidity and salt content, while significantly (p<0.05) influenced the fat content. However, protein and ash content were affected non-significantly (p>0.05). The interaction between milk types and storage periods highly significantly influenced the protein content while the interaction between milk type and cheese treatments highly significantly affected the moisture, fat, pH, acidity and salt content. However, all other interactions had non- significant influence on physico-chemical composition of processed cheese.

135

Table 4.44 Mean sum of squares for physico-chemical composition of processed Cheddar cheese

Source of variation d.f Moisture Fat Protein Ash pH Acidity Salt

NS NS NS NS Storage Days (D) 3 0.24463 0.21759 0.3357 0.01760 0.06072** 0.00954** 0.02470**

NS Milk type (M) 2 4.11051** 1.14699** 93.7978** 1.03488** 0.04482** 0.01658** 0.00480

NS NS Treatments (T) 5 0.70365** 0.40185* 0.2402 0.01118 0.10507** 0.02700** 0.04266**

NS NS NS NS NS NS D × M 6 0.00424 0.02199 0.7275** 0.00023 0.00071 0.00006 0.00316

NS NS NS NS NS NS NS D × T 15 0.00340 0.06667 0.2841 0.00044 0.00141 0.00022 0.00110

NS NS M × T 10 2.86174** 1.00949** 0.3329 0.00689 0.00745** 0.00535** 0.02698**

NS NS NS NS NS NS NS D × M × T 30 0.00348 0.05301 0.2957 0.00061 0.00157 0.00012 0.00096

Error 144

Total 215

136

4.9.1 Moisture content Mean moisture content table 4.45a indicated a significant difference between buffalo (44.83±0.068%), cow (44.74±0.047%) and combined milk cheese (44.38±0.058%). However, moisture content increased non-significantly from 44.60±0.073% (at 0 day) to 44.73±0.065% after 90 days of storage (Fig. 4.20). The moisture level was highly- significantly influenced by cheese treatments (table 4.45b) with the lowest content

(44.44±0.090%) in T1 and the highest (44.84±0.101%) in T6. With reference to the interactions between milk types and treatments, the highest moisture content

(45.45±0.092%) were in T4 of buffalo milk cheese while the lowest content

(44.10±0.115%) and (44.10±0.123%) in T1 of buffalo milk cheese and T6 of combined milk cheese, respectively (Table 4.25b). The non-significant variation of the moisture content in the interaction between all the parameters is obvious in table 4.45c. The results are very close to the declaration of (FDA 2006), in which clearly mentioned that processed cheese should have the maximum moisture contents as 43.0%. Moreover, the results of the present study are according to the findings of Kapoor et al. (2007); Pinto et al. (2007) and Suleiman et al. (2011), who reported the mean moisture contents of the processed cheese as 38.2-64.21% depending upon the quality of the ingredients used in process cheese manufacture. Dimitreli and Thomareis (2008) also reported the moisture content of spreadable type processed cheese as 42.0% to 61.8%. Guinee and O’Kennedy (2009) reported the moisture contents of processed cheese made from Cheddar cheese as 46.9-47.3 %. Significant difference in the moisture values in cheese treatments is due to the variation in moisture contents of the Cheddar cheese of different ripening periods and milk types used for the manufacturing of processed cheese. Emulsifying salts phosphates and citrate) showed no effect on the moisture content of the processed cheese (Al-Otaibi et al., 2006). Cunha and Viotto’s (2010) also declared a non-significant effect of emulsifying salts on processed Cheddar cheese. 4.9.2 Fat content In traditional processed cheeses, plentiful small particles of fat (spherical in nature) are uniformly distributed in the protein complex. Reduced diameter makes the fat globules evenly distributed throughout protein-matrix causing an increase in protein-

137 protein and protein-fat interactions hence increasing the elasticity of the processed cheese (Pereira et al., 2001; Cunha et al., 2010). Results concerning the fat content of processed cheese prepared from cow, buffalo and mix Cheddar cheese of variable maturity is given in the table 4.46 a-c and Fig. 4.21. Results described a significant difference in fat composition of processed cheese (being prepared from natural cheese of cow, buffalo and combination of cow and buffalo) showing a mean value as buffalo milk (29.92±0.056%), cow milk (29.74±0.049%) and blended milk cheese (29.99±0.057%). Fat contents decreased non-significantly during storage of 0-90 days from 30.14±0.120% to 29.69±0.082% with a mean value 29.92±0.056% (buffalo milk cheese), 30.03±0.095% to 29.56±0.098% with a mean value 29.74±0.049% (cow milk cheese) and 30.22±0.116% to 29.78±0.092% with a mean value 29.99±0.057% (blended milk cheese). Overall reduction in fat during 0-90 days storage mean value was 30.13±0.064% to 29.68±0.053% as per table 4.46 a. A highly significant difference in fat contents was observed in cheese treatments (table 4.26b). With reference to the interaction between milk type and treatments the highest fat level (30.50±0.107a %) was observed in T1 (blended milk) and the lowest value

(29.58±0.104%) was observed in T1 (cow) and (29.58±0.120%) in T3 (blended milk). Table 4.46c shows a non-significant variation in fat contents between interactions of all parameters. The results of the current study are in agreement with the conclusions of Kwak et al. (2002), who reported the average fat contents of the processed cheese between 29.0% to 33.0% prepared from Cheddar and different ratios of emulsifying salts. The results of present study are also very close to the findings of Suleiman et al. (2011), who concluded fat contents between 29.0% - 29.83% in processed cheese. Guinee and O’Callaghan (2013) reported the fat contents 22.4% - 30.9% in process cheese product made by using Cheddar cheese. Non-significant decrease in fat contents during storage period in the present study is in connection with the results of Hamed et al. (1997) and El-Diam et al. (2010). They reported a decrease in fat contents during storage. The decrease in fat content might be due to slight increase in moisture level.

138 Table 4.45a Effect of milk types and storage periods on moisture content (%) of processed Cheddar cheese (Means ± SE)

Milk types Storage days Means ± SE Buffalo Cow Cow + Buffalo 0 44.78±0.164 44.70±0.089 44.32±0.087 44.60±0.073 30 44.75±0.119 44.69±0.085 44.31±0.159 44.58±0.076 60 44.88±0.134 44.77±0.115 44.41±0.112 44.68±0.074 90 44.91±0.132 44.80±0.086 44.47±0.096 44.73±0.065 Means ± SE 44.83±0.068A 44.74±0.047A 44.38±0.058B

Table 4.45b Effect of milk types and treatments on moisture content (%) of processed Cheddar cheese (Means ± SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 44.10±0.115h 45.04±0.107bc 44.16±0.052h 44.44±0.090C

T2 44.30±0.077gh 44.48±0.081efg 45.14±0.048b 44.64±0.072B

T3 45.09±0.125bc 44.37±0.094fgh 44.36±0.079fgh 44.61±0.081B

T4 45.45±0.092a 44.64±0.072def 44.18±0.125h 44.76±0.104AB

T5 44.80±0.060cd 44.73±0.048de 44.32±0.138gh 44.61±0.062B

T6 45.23±0.055ab 45.19±0.061ab 44.10±0.123h 44.84±0.101A

Means ± SE 44.83±0.068A 44.74±0.047A 44.38±0.058B

139 Table 4.45c Effect of milk types, treatments and storage periods on moisture content (%) of processed Cheddar cheese (Means ± SE)

Storage Milk types Treatments Means ± SE days Buffalo Cow Cow + Buffalo

T1 44.04 ± 0.447 45.03 ± 0.147 44.13 ± 0.114 44.40 ± 0.211

T2 44.24 ± 0.137 44.44 ± 0.127 45.00 ± 0.105 44.56 ± 0.129

T3 45.05 ± 0.554 44.33 ± 0.257 44.31 ± 0.136 44.56 ± 0.218 0 T4 45.37 ± 0.130 44.58 ± 0.121 44.17 ± 0.156 44.71 ± 0.189

T5 44.79 ± 0.245 44.68 ± 0.067 44.30 ± 0.044 44.59 ± 0.105

T6 45.22 ± 0.187 45.17 ± 0.070 44.03 ± 0.121 44.81 ± 0.206

T1 44.03 ± 0.118 44.93 ± 0.162 44.12 ± 0.171 44.36 ± 0.161

T2 44.23 ± 0.027 44.43 ± 0.224 45.14 ± 0.086 44.60 ± 0.155

T3 45.01 ± 0.062 44.35 ± 0.109 44.27 ± 0.196 44.55 ± 0.135 30 T4 45.35 ± 0.103 44.60 ± 0.209 44.08 ± 0.511 44.68 ± 0.246

T5 44.70 ± 0.039 44.74 ± 0.099 44.24 ± 0.484 44.56 ± 0.164

T6 45.15 ± 0.059 45.10 ± 0.153 44.01 ± 0.528 44.75 ± 0.245

T1 44.15 ± 0.240 45.10 ± 0.440 44.17 ± 0.104 44.47 ± 0.215

T2 44.35 ± 0.211 44.49 ± 0.270 45.18 ± 0.121 44.67 ± 0.166

T3 45.11 ± 0.116 44.39 ± 0.276 44.41 ± 0.263 44.63 ± 0.165 60 T4 45.52 ± 0.269 44.69 ± 0.159 44.22 ± 0.218 44.81 ± 0.219

T5 44.87 ± 0.085 44.72 ± 0.163 44.34 ± 0.325 44.64 ± 0.133

T6 45.26 ± 0.136 45.21 ± 0.135 44.12 ± 0.038 44.86 ± 0.193

T1 44.18 ± 0.115 45.12 ± 0.022 44.23 ± 0.047 44.51 ± 0.156

T2 44.38 ± 0.236 44.55 ± 0.042 45.23 ± 0.037 44.72 ± 0.148

T3 45.18 ± 0.118 44.41 ± 0.193 44.45 ± 0.035 44.68 ± 0.141 90 T4 45.55 ± 0.270 44.69 ± 0.156 44.26 ± 0.043 44.83 ± 0.210

T5 44.83 ± 0.043 44.77 ± 0.091 44.39 ± 0.268 44.66 ± 0.107

T6 45.31 ± 0.061 45.28 ± 0.161 44.25 ± 0.131 44.95 ± 0.186

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

140

B C C+B

46

45.5

45

44.5

e

r 44

oistu M 43.5

43

42.5

42

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90

Fig.4.20 Effect of milk types, treatments and storage periods on moisture content (%) of processed Cheddar cheese

141 Table 4.46a Effect of milk types and storage period on fat content (%) of processed Cheddar cheese (Means ± SE)

Storage Milk type Means ± SE days Buffalo Cow Cow + Buffalo

0 30.14±0.120 30.03±0.095 30.22±0.116 30.13±0.064

30 30.03±0.103 29.81±0.082 30.06±0.121 29.96±0.060

60 29.83±0.114 29.58±0.083 29.89±0.103 29.77±0.060

90 29.69±0.082 29.56±0.098 29.78±0.092 29.68±0.053

Means ± SE 29.92±0.056A 29.74±0.049B 29.99±0.057A

Table 4.46b Effect of milk types and treatments on fat content (%) of processed Cheddar cheese (Means ± SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 29.92±0.120cde 29.58±0.104fg 30.50±0.107a 30.00±0.089A

T2 29.79±0.130d-g 29.67±0.128d-g 29.96±0.096cd 29.81±0.070B

T3 30.13±0.109bc 29.96±0.130cd 29.58±0.120fg 29.89±0.077AB

T4 29.54±0.096g 29.88±0.090c-f 30.13±0.109bc 29.85±0.068AB

T5 29.79±0.114d-g 29.63±0.139efg 29.83±0.155c-g 29.75±0.078B

T6 30.38±0.125ab 29.75±0.115d-g 29.92±0.104cde 30.01±0.078A

Means ± SE 29.92±0.056A 29.74±0.049B 29.99±0.057A

142

Table 4.46c Effect of milk types, treatments and storage period on fat content (%) of processed Cheddar cheese (Means ± SE)

Milk types Storage Treatments Cow + Means ± SE days Buffalo Cow Buffalo

T1 30.17 ± 0.167 29.83 ± 0.167 30.83 ± 0.167 30.28 ± 0.169

T2 30.00 ± 0.289 30.00 ± 0.289 29.83 ± 0.167 29.94 ± 0.130

T3 30.33 ± 0.167 30.17 ± 0.167 29.83 ± 0.167 30.11 ± 0.111 0 T4 29.50 ± 0.289 30.00 ± 0.000 30.33 ± 0.167 29.94 ± 0.155

T5 30.17 ± 0.333 30.00 ± 0.500 30.33 ± 0.441 30.17 ± 0.220

T6 30.67 ± 0.167 30.17 ± 0.167 30.17 ± 0.167 30.33 ± 0.118

T1 30.00 ± 0.289 29.50 ± 0.289 30.67 ± 0.167 30.06 ± 0.212

T2 29.83 ± 0.167 29.83 ± 0.167 30.00 ± 0.289 29.89 ± 0.111

T3 30.33 ± 0.167 30.00 ± 0.000 29.50 ± 0.289 29.94 ± 0.155 30 T4 29.67 ± 0.167 30.00 ± 0.289 30.33 ± 0.167 30.00 ± 0.144

T5 29.83 ± 0.167 29.67 ± 0.167 29.83 ± 0.167 29.78 ± 0.088

T6 30.50 ± 0.289 29.83 ± 0.167 30.00 ± 0.289 30.11 ± 0.162

T1 29.83 ± 0.333 29.33 ± 0.167 30.33 ± 0.167 29.83 ± 0.186

T2 29.67 ± 0.441 29.50 ± 0.289 30.00 ± 0.289 29.72 ± 0.188

T3 30.00 ± 0.289 29.83 ± 0.333 29.50 ± 0.289 29.78 ± 0.169 60 T4 29.50 ± 0.000 29.83 ± 0.167 30.00 ± 0.289 29.78 ± 0.121

T5 29.67 ± 0.167 29.50 ± 0.000 29.67 ± 0.167 29.61 ± 0.073

T6 30.33 ± 0.167 29.50 ± 0.000 29.83 ± 0.167 29.89 ± 0.139

T1 29.67 ± 0.167 29.67 ± 0.167 30.17 ± 0.167 29.83 ± 0.118

T2 29.67 ± 0.167 29.33 ± 0.167 30.00 ± 0.000 29.67 ± 0.118

T3 29.83 ± 0.167 29.83 ± 0.441 29.50 ± 0.289 29.72 ± 0.169 90 T4 29.50 ± 0.289 29.67 ± 0.167 29.83 ± 0.167 29.67 ± 0.118

T5 29.50 ± 0.000 29.33 ± 0.167 29.50 ± 0.289 29.44 ± 0.100

T6 30.00 ± 0.289 29.50 ± 0.289 29.67 ± 0.167 29.72 ± 0.147

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

143

B C C+B

31.5

31

30.5

30

Fat 29.5

29

28.5

28

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90

Fig.4.21 Effect of milk types, treatments and storage period on fat content (%) of processed Cheddar cheese

144 4.9.3 Protein content Cheese is considered as paracasein network comprising fat and aqueous phases. Solublized casein is capable to intermingle with water and fat under agitation and heating resulting a gel structure on cooling (Siew et al., 2004). Results regarding protein content of the processed Cheddar cheese are given in the table 4.47 a, b, c and Fig. 4.22. A highly significant difference among the processed cheese of different milk types was observed showing highest mean values of protein content (22.87±0.048%) in buffalo milk cheese followed by (21.67±0.037%) in cheese from milk blend and 20.59±0.080% in cow milk cheese (table 4.47a). Protein contents decreased non-significantly in all cheese types during storage period up to 90 days. Decrease was observed as 22.98±0.149% to 22.79±0.045% (0-90 days) in buffalo milk cheese, 21.74±0.061% to 21.59±0.092% (0-90 days) in cow + buffalo milk cheese and 20.51±0.123% to 21.03±0.246% (0-90 days) in cow milk cheese as given in table 4.27a. Cheese treatments showed a non-significant difference regarding protein content table 4.24b. The highest protein value (22.95±0.156%) was recorded in buffalo milk cheese treatment (T4) whereas the lowest protein value (20.25±0.114%) was recorded in cow milk cheese (T6). A non-significant difference of protein contents among interaction of all parameters is given in table 4.47c. Protein values recorded in present study are covenant to consequences of Guinee and O’Kennedy (2009), who reported the protein content as 20.7-21.6% in processed cheese made from Cheddar. Awad et al. (2013) prepared processed cheese from Cheddar cheese containing EPS (Exo-polysaccharide) culture and the protein values (21.42- 22.41%) of their study are very similar to the results of the current study. Age of Cheddar cheese has profound effects on the properties of processed cheese.

During this period ∞S1-casein is degraded causing softness of cheese (Osthoff et al., 2011). Decrease in protein during storage period is due to assimilation or degradation of protein in cheese. Maillard reaction and amino acid degradation causes the reduction in amino acid contents resulting in decrease in protein contents of processed cheese during storage (Schar and Bosset, 2002). Storage duration and temperature also affect the amino acid degradation resulting in decrease of protein (Frantisek et al., 2004). Decrease in

145 protein amount may also be due to the addition of water during production of processed cheese (El-Diam et al., 2010). Emulsifying salt has capability to modify the protein assembly and stabilize the melted mass of cheese, however Ayyash and Shah (2011) demonstrated that changing the emulsifying salts doesn’t affect the cheese composition. 4.9.4 Ash content Results for the ash content of the processed cheese prepared from cow, buffalo and mixture of cow and buffalo milk cheese are given in the table 4.48a, b, c and Fig. 4.23. A highly significant difference of ash values was observed in cheese from different milk types showing the highest values (3.46±0.009%) in buffalo milk process cheese whereas the lowest (3.22±0.006%) were recorded in cow milk cheese (table 4.48a). Storage periods showed a non-significant decrease in ash contents as 3.35±0.017% (at 0-day) to 3.31±0.016% (at 90th day) as given in table 4.48a. A non-significant effect of ash contents was seen in cheese treatments (table 4.48b) with the highest ash content (3.358±0.024%) in T4 while the lowest ash contents were recorded in T6 as 3.304±0.019%. Inter-relation of cheese treatments and milk types also exhibited a non-significant difference among the ash contents. Non-significant difference of ash contents between interactions of all parameters is given in the table 4.48c. Results of the ash content in the present study are in line with the investigations of the Acharya and Mistry (2005); Shirashoji et al. (2006) and Pinto et al. (2007), they concluded the ash content in processed cheese as 1.33 - 4.82% depending upon the ingredients used. Ash content of the processed cheese in the study is also comparable with the results of El- Diam et al. (2010), who reported ash values between 1.75 - 5.85% in processed Cheddar cheese. Non-significant difference in ash contents of the processed cheese during storage for 90 days has been reported by Suleiman et al. (2011). The highest ash content was found in treatment having maximum amount of 90 days ripened cheese which shows that ripening affects the ash content of the cheese and at the same time the lowest amount of ash content was found in cheese having maximum 0 day ripened natural cheese. Non- significant change in ash contents is due to the fact that least biochemical changes occur in process cheese during storage.

146 Table 4.47a Effect of milk types and storage period on protein content (%) of processed Cheddar cheese (Means ± SE)

Milk type Storage days Means ± SE Buffalo Cow Cow + Buffalo 0 22.98±0.149a 20.51±0.123d 21.74±0.061b 21.74±0.153 30 22.88±0.105a 20.45±0.085d 21.67±0.093b 21.67±0.147 60 22.84±0.046a 20.37±0.093d 21.66±0.038b 21.62±0.143 90 22.79±0.045a 21.03±0.246c 21.59±0.092b 21.80±0.133 Means ± SE 22.87±0.048A 20.59±0.080C 21.67±0.037B

Table 4.47b Effect of milk types and treatments on protein content (%) of processed Cheddar cheese (Means ± SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 22.83±0.046 20.96±0.279 21.70±0.100 21.83±0.162

T2 22.78±0.070 20.84±0.281 21.72±0.077 21.78±0.165

T3 22.89±0.120 20.51±0.115 21.64±0.094 21.68±0.175

T4 22.9±0.156 20.50±0.113 21.64±0.103 21.70±0.184

T5 22.8±0.125 20.49±0.150 21.71±0.087 21.68±0.176

T6 22.94±0.161 20.25±0.114 21.60±0.098 21.60±0.199

Means ± SE 22.87±0.048A 20.59±0.080C 21.67±0.037B

147

Table 4.47c Effect of milk types, treatments and storage period on protein content (%) of processed Cheddar cheese (Means ± SE)

Storage Treatments Milk types Means ± SE days Buffalo Cow Cow + Buffalo

T1 22.93 ±0.030 20.55 ±0.279 21.75 ±0.166 21.74 ± 0.356

T2 22.88 ±0.090 20.49 ±0.226 21.80 ±0.135 21.72 ± 0.355

T 23.04 ±0.505 20.53 ±0.268 21.71 ±0.147 21.76 ± 0.401 0 3 T4 23.05 ±0.517 20.56 ±0.275 21.74 ±0.187 21.78 ± 0.401

T5 22.91 ±0.527 20.61 ±0.494 21.77 ±0.173 21.77 ± 0.395

T6 23.06 ±0.560 20.32 ±0.471 21.68 ±0.228 21.68 ± 0.453

T1 22.84 ±0.130 20.49 ±0.266 21.69 ±0.247 21.67 ± 0.357

T2 22.79 ±0.168 20.42 ±0.238 21.72 ±0.188 21.64 ± 0.357

T 22.89 ±0.075 20.44 ±0.254 21.66 ±0.314 21.66 ± 0.373 30 3 T4 22.98 ±0.504 20.53 ±0.225 21.65 ±0.327 21.72 ± 0.400

T5 22.86 ±0.080 20.54 ±0.251 21.72 ±0.235 21.71 ± 0.350

T6 22.94 ±0.486 20.29 ±0.194 21.60 ±0.286 21.61 ± 0.420

T1 22.80 ±0.139 20.39 ±0.288 21.72 ±0.206 21.64 ± 0.365

T2 22.74 ±0.172 20.13 ±0.096 21.74 ±0.129 21.54 ± 0.387

T 22.83 ±0.145 20.73 ±0.218 21.61 ±0.021 21.72 ± 0.313 60 3 T4 22.92 ±0.054 20.46 ±0.285 21.61 ±0.043 21.66 ± 0.365

T5 22.82 ±0.137 20.37 ±0.310 21.70 ±0.032 21.63 ± 0.368

T6 22.94 ±0.034 20.16 ±0.053 21.58 ±0.040 21.56 ± 0.402

T1 22.75 ±0.030 22.40 ±0.293 21.62 ±0.294 22.26 ± 0.205

T2 22.69 ±0.176 22.34 ±0.307 21.62 ±0.226 22.22 ± 0.199

T 22.79 ±0.127 20.35 ±0.256 21.57 ±0.258 21.57 ± 0.370 90 3 T4 22.87 ±0.035 20.44 ±0.261 21.54 ±0.278 21.61 ± 0.369

T5 22.78 ±0.192 20.45 ±0.269 21.65 ±0.274 21.63 ± 0.359

T6 22.84 ±0.090 20.22 ±0.127 21.54 ±0.269 21.53 ± 0.389

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

148

B C C+B

24

23

22

21

otein Pr 20

19

18

17 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 Day 0 Day 30 Day 60 Day 90 Protein_P

Fig.4.22 Effect of milk types, treatments and storage period on protein content (%) of processed Cheddar cheese

149 Table 4.48a Effect of milk types and storage period on ash content (%) of processed Cheddar cheese (Means ± SE)

Storage Milk type Means ± SE days Buffalo Cow Cow + Buffalo 0 3.48 ± 0.027 3.24 ± 0.015 3.33 ± 0.010 3.35 ± 0.017

30 3.46 ± 0.011 3.23 ± 0.008 3.31 ± 0.005 3.33 ± 0.014

60 3.45 ± 0.011 3.21 ± 0.009 3.31 ± 0.005 3.32 ± 0.014

90 3.44 ± 0.014 3.21 ± 0.013 3.28 ± 0.019 3.31 ± 0.016

Means ± SE 3.46 ± 0.009A 3.22 ± 0.006C 3.31 ± 0.006B

Table 4.48b Effect of milk types and treatments on ash content (%) of processed Cheddar cheese (Means±SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 3.48 ± 0.011 3.20 ± 0.009 3.30 ± 0.014 3.329 ± 0.020

T2 3.46 ± 0.014 3.21 ± 0.013 3.32 ± 0.013 3.332 ± 0.019

T3 3.46 ± 0.013 3.23 ± 0.014 3.31 ± 0.012 3.332 ± 0.018

T4 3.52 ± 0.036 3.24 ± 0.015 3.31 ± 0.017 3.358 ± 0.024

T5 3.40 ± 0.014 3.24 ± 0.016 3.31 ± 0.013 3.318 ± 0.014

T6 3.43 ± 0.014 3.19 ± 0.014 3.29 ± 0.018 3.304 ± 0.019 Means ± SE 3.46 ± 0.009A 3.22 ± 0.006C 3.31 ± 0.006B

150 Table 4.48c Effect of milk types, treatments and storage period on ash content (%) of processed Cheddar cheese (Means ± SE)

Storage Milk types Treatments Means ± SE days Buffalo Cow Cow + Buffalo

T1 3.50 ± 0.042 3.21 ± 0.032 3.33 ± 0.017 3.34 ± 0.045

T2 3.46 ± 0.052 3.24 ± 0.049 3.34 ± 0.032 3.35 ± 0.038

T3 3.51 ± 0.026 3.25 ± 0.052 3.32 ± 0.030 3.36 ± 0.044 0 T4 3.53 ± 0.161 3.27 ± 0.035 3.34 ± 0.042 3.38 ± 0.063

T5 3.43 ± 0.018 3.26 ± 0.052 3.31 ± 0.007 3.34 ± 0.030

T6 3.45 ± 0.046 3.23 ± 0.012 3.33 ± 0.027 3.34 ± 0.036

T1 3.48 ± 0.021 3.20 ± 0.009 3.31 ± 0.021 3.33 ± 0.041

T2 3.47 ± 0.023 3.22 ± 0.023 3.33 ± 0.017 3.34 ± 0.038

T3 3.47 ± 0.019 3.23 ± 0.018 3.31 ± 0.010 3.34 ± 0.035 30 T4 3.52 ± 0.015 3.25 ± 0.015 3.32 ± 0.003 3.36 ± 0.041

T5 3.42 ± 0.017 3.24 ± 0.028 3.31 ± 0.007 3.32 ± 0.028

T6 3.42 ± 0.019 3.20 ± 0.019 3.31 ± 0.012 3.31 ± 0.033

T1 3.47 ± 0.017 3.20 ± 0.012 3.29 ± 0.022 3.32 ± 0.041

T2 3.46 ± 0.019 3.20 ± 0.009 3.31 ± 0.009 3.32 ± 0.039

T3 3.44 ± 0.009 3.22 ± 0.022 3.30 ± 0.003 3.32 ± 0.033 60 T4 3.51 ± 0.024 3.23 ± 0.036 3.31 ± 0.007 3.35 ± 0.044

T5 3.38 ± 0.018 3.23 ± 0.025 3.32 ± 0.015 3.31 ± 0.024

T6 3.43 ± 0.012 3.18 ± 0.009 3.29 ± 0.006 3.30 ± 0.037

T1 3.47 ± 0.012 3.20 ± 0.023 3.28 ± 0.051 3.32 ± 0.044

T2 3.45 ± 0.020 3.20 ± 0.009 3.31 ± 0.046 3.32 ± 0.040

T3 3.42 ± 0.015 3.23 ± 0.028 3.30 ± 0.047 3.32 ± 0.033 90 T4 3.49 ± 0.040 3.22 ± 0.040 3.28 ± 0.057 3.33 ± 0.047

T5 3.37 ± 0.044 3.22 ± 0.034 3.30 ± 0.055 3.30 ± 0.031

T6 3.41 ± 0.039 3.16 ± 0.049 3.24 ± 0.063 3.27 ± 0.045

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

151

B C C+B

3.7

3.6

3.5

3.4

3.3

sh

A 3.2

3.1

3

2.9

2.8

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90

Fig.4.23 Effect of milk types, treatments and storage period on ash content (%) of processed Cheddar cheese

152 4.9.5 pH Value

Besides compositional and processing conditions, pH of the cheese melt also affects the consistency of the processed cheese (Cernikova et al., 2008; Gustaw and Mleko, 2007). Moreover the pH values play role in the viscoelastic properties of cheese (Lee and Klostermeyer, 2001). Ingredients used for the production of processed cheese and the emulsifying salts are key controllers of the pH in processed cheese (Nagyova et al., 2014). Mean values of pH in the processed cheese prepared from cow, buffalo and mixture of cow and buffalo milk Cheddar cheese is given in table 4.49a, b, c. Milk types showed highly-significant difference (Fig. 4.24) among the pH values with the highest mean pH value (5.481±0.007) in cow milk process cheese and the lowest value (5.435±0.010) in buffalo milk cheese (table 4.49a). Non-significant decrease in pH values was observed during storage of processed cheese up to 90 days. Table 4.49a shows a decrease in mean values of pH from 5.494±0.009 (at 0-Day) to 5.416±0.011 (at 90-Day). Treatment means showed a highly significant difference of pH values of processed cheese (table 4.49b). The highest pH recorded in T6 was (5.514±0.008) while the lowest pH value (5.371±0.013) was in T1. Interaction between milk types and treatments of processed cheese also showed a highly significant difference with respect to pH values as given in table 4.49b. Interaction of storage, milk types and treatments presented a non- significant effect on the pH of processed Cheddar cheese (table 4.49c). Results regarding pH values of the current study are comparable to the findings of Mihaela et al. (2013). They stated the pH of processed cheese between 5.3-5.9 for the traditional cheesed prepared by using 50% fresh and 50% ripened (110 Days) cheese and emulsifying salts. Awad et al. (2013) prepared processed cheese by using 1-month old Cheddar cheese and recorded pH as 5.61- 5.68. Results of pH are also justified by the Food and Drug Administration (FDA), according to which the processed Cheddar should have pH equal to or less than 5.6. Overall increase in the pH of the processed cheese is due to the addition of the emulsifying salts. Phosphate salts cause pH stabilization due to their buffering capacity (Guinee et al., 2004; Mulsow et al., 2007). Sadlikova et al. (2010), who declared that use of phosphate, increased the pH of the processed cheese. The highest pH was observed in treatments having high amount of fresh cheese (0-day ripened) and the lowest was in processed Cheddar having maximum amount of (60-90 days) ripened cheese. Thus it can be concluded that Cheddar cheese ripening also influences the pH of the process cheese.

153 4.9.6 Acidity content The formulation for processed cheese includes the different ratios of Cheddar cheese of varying maturity (i.e. mild, medium or mature) and the criteria for natural cheese selection include flavor, texture, consistency and level of acidity. Natural cheese of very high acidity (degraded, off-flavored) should not be used for the manufacture of processed cheese to obtain the finest quality end product (Kapoor et al., 2007; Tamime et al., 2011). Results regarding the acidity of the cheese in current study are given in (tables 4.50 a, b and c). Milk types exhibited a highly significant difference of the acidity in all types of natural cheese (Fig. 4.25). The highest mean values of acidity (1.139±0.002%) were recorded in cow + buffalo milk cheese whereas lowest (1.110±0.005%) mean values of acidity were recorded in cow milk cheese. Acidity increased significantly from 1.110±0.005% (at 0 day) to 1.141±0.004% (at 90-day) during storage (table 4.50a). Cheese treatments (table 4.50b) showed a highly-significant effect on the acidity content showing the highest values (1.161±0.003%) in T1 and the lowest acidity values (1.087±0.007%) in

T6. With reference to the interaction between milk types and cheese treatments, the highest acidity values (1.173±0.003%) were recorded in T1 (buffalo) and the lowest acidity values

(1.033±0.007%) in T6 (cow). Interaction of all parameters including milk types, storage days and treatments showed a non-significant effect as declared in Table 4.50c. The results relating to acidity are in line with the consequences of El-Diam et al. (2010). They reported the acidity of the processed cheese made from natural cheese (having 4.4% fat) ranging from 0.62% to 1.42% during 0-90 days of storage and 0.62- 1.38% acidity in case of processed cheese prepared from natural cheese (having 2.2% fat) during storage from 0-90 days. Krumov et al. (2010) noted the acidity of processed cheese as lactic acid (%) ranging between 0.88% to 1.23%, which also resembles with the outcomes of the current study. Processed Cheddar cheese during storage is influenced by product composition, processing, packing and storage conditions (Schar and Bosset, 2002). Increase in acidity is correlated to the acidity of the natural cheese used in the processed cheese manufacture. Maximum acidity was noted in the processed cheese prepared from natural cheese of 90 day maturity in case of cow milk cheese. Highly significant difference of acidity of the processed cheese is due to variable

154 Table 4.49a Effect of milk types and storage period on pH value of processed Cheddar cheese (Means ± SE)

Milk type Storage days Means ± SE Buffalo Cow Cow + Buffalo 0 5.473 ± 0.018 5.518 ± 0.012 5.489 ± 0.013 5.494 ± 0.009A 30 5.449 ± 0.019 5.488 ± 0.015 5.453 ± 0.015 5.464 ± 0.010B 60 5.417 ± 0.017 5.469 ± 0.012 5.426 ± 0.020 5.437 ± 0.010C 90 5.403 ± 0.021 5.449 ± 0.013 5.396 ± 0.018 5.416 ± 0.011D Means ± SE 5.435 ± 0.010B 5.481 ± 0.007A 5.441 ± 0.009B

Table 4.49b Effect of milk types and treatments on pH value of processed Cheddar cheese (Means ± SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 5.328 ± 0.019g 5.432 ± 0.015ef 5.353 ± 0.019g 5.371 ± 0.013D

T2 5.366 ± 0.015g 5.456 ± 0.018cde 5.411 ± 0.019f 5.411 ± 0.012C

T3 5.453 ± 0.018de 5.458 ± 0.014cde 5.456 ± 0.015cde 5.456 ± 0.009B

T4 5.518 ± 0.008ab 5.498 ± 0.012b 5.487 ± 0.018bcd 5.501 ± 0.008A

T5 5.449 ± 0.012de 5.493 ± 0.010bc 5.448 ± 0.019ef 5.463 ± 0.009B

T6 5.498 ± 0.011b 5.550 ± 0.012a 5.493 ± 0.015bc 5.514 ± 0.008A Means ± SE 5.435 ± 0.010B 5.481 ± 0.007A 5.441 ± 0.009B

155 Table 4.49c Effect of milk types, treatments and storage period on pH value of processed Cheddar cheese (Means ± SE)

Storage Treatments Milk types Means ± SE days Buffalo Cow Cow + Buffalo

T1 5.387 ± 0.049 5.473 ± 0.038 5.427 ± 0.012 5.429 ± 0.022

T2 5.430 ± 0.021 5.510 ± 0.006 5.470 ± 0.035 5.470 ± 0.016

T3 5.487 ± 0.052 5.510 ± 0.017 5.510 ± 0.035 5.502 ± 0.019 0 T4 5.540 ± 0.026 5.523 ± 0.015 5.533 ± 0.028 5.532 ± 0.012

T5 5.470 ± 0.040 5.493 ± 0.009 5.510 ± 0.035 5.491 ± 0.017

T6 5.523 ± 0.015 5.600 ± 0.012 5.487 ± 0.012 5.537 ± 0.018

T1 5.353 ± 0.032 5.413 ± 0.033 5.377 ± 0.030 5.381 ± 0.018

T2 5.363 ± 0.033 5.480 ± 0.051 5.423 ± 0.012 5.422 ± 0.025

T3 5.477 ± 0.043 5.467 ± 0.009 5.473 ± 0.029 5.472 ± 0.015 30 T4 5.517 ± 0.009 5.503 ± 0.029 5.507 ± 0.018 5.509 ± 0.010

T5 5.477 ± 0.012 5.517 ± 0.012 5.447 ± 0.026 5.480 ± 0.014

T6 5.510 ± 0.040 5.550 ± 0.029 5.493 ± 0.049 5.518 ± 0.022

T1 5.317 ± 0.012 5.410 ± 0.035 5.337 ± 0.023 5.354 ± 0.019

T2 5.337 ± 0.009 5.433 ± 0.020 5.383 ± 0.041 5.384 ± 0.019

T3 5.430 ± 0.015 5.453 ± 0.029 5.417 ± 0.009 5.433 ± 0.011 60 T4 5.507 ± 0.009 5.503 ± 0.012 5.477 ± 0.048 5.496 ± 0.015

T5 5.433 ± 0.012 5.493 ± 0.015 5.410 ± 0.053 5.446 ± 0.020

T6 5.477 ± 0.003 5.523 ± 0.015 5.533 ± 0.018 5.511 ± 0.011

T1 5.257 ± 0.012 5.430 ± 0.012 5.273 ± 0.018 5.320 ± 0.028

T2 5.333 ± 0.024 5.400 ± 0.015 5.367 ± 0.044 5.367 ± 0.018

T3 5.420 ± 0.023 5.403 ± 0.007 5.423 ± 0.015 5.416 ± 0.009 90 T4 5.507 ± 0.009 5.463 ± 0.035 5.430 ± 0.030 5.467 ± 0.017

T5 5.417 ± 0.012 5.470 ± 0.038 5.423 ± 0.015 5.437 ± 0.015

T6 5.483 ± 0.009 5.527 ± 0.015 5.460 ± 0.031 5.490 ± 0.014

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

156

B C C+B

5.7

5.6

5.5

5.4

pH 5.3

5.2

5.1

5

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90 pH_P

Fig.4.24 Effect of milk types, treatments and storage period on pH value of processed Cheddar cheese

157

Table 4.50a Effect of milk types and storage period on acidity (%) of processed Cheddar cheese (Means ± SE)

Storage Milk type Means ± SE days Buffalo Cow Cow + Buffalo

0 1.113 ± 0.011 1.092 ± 0.011 1.125 ± 0.004 1.110 ± 0.005D

30 1.128 ± 0.009 1.106 ± 0.009 1.135 ± 0.004 1.123 ± 0.005C

60 1.139 ± 0.007 1.115 ± 0.009 1.144 ± 0.004 1.133 ± 0.004B

90 1.146 ± 0.006 1.125 ± 0.008 1.151 ± 0.004 1.141 ± 0.004A

Means ± SE 1.132±0.004B 1.110 ± 0.005C 1.139±0.002A

Table 4.50b Effect of milk types and treatments on acidity (%) of processed Cheddar cheese (Means ± SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 1.173 ± 0.003a 1.143 ± 0.004bc 1.167 ± 0.004a 1.161 ± 0.003A

T2 1.166 ± 0.004a 1.131 ± 0.004de 1.150 ± 0.004b 1.149 ± 0.003B

T3 1.136 ± 0.002cd 1.125 ± 0.004efg 1.133 ± 0.003de 1.131 ± 0.002C

T4 1.076 ± 0.009j 1.109 ± 0.005i 1.126 ± 0.004ef 1.104 ± 0.005D

T5 1.131 ± 0.003de 1.118 ± 0.003gh 1.136 ± 0.004cd 1.128 ± 0.002C

T6 1.110 ± 0.007hi 1.033 ± 0.007k 1.120 ± 0.003fg 1.087 ± 0.007E Means ± SE 1.132 ± 0.004B 1.110 ± 0.005C 1.139 ± 0.002A

158 Table 4.50c Effect of milk types, treatments and storage period on acidity (%) of processed Cheddar cheese (Means ± SE)

Storage Treatme Milk types Means ± SE days nts Buffalo Cow Cow + Buffalo

T1 1.161 ± 0.002 1.130 ± 0.005 1.153 ± 0.007 1.148 ± 0.005

T2 1.156 ± 0.012 1.116 ± 0.005 1.135 ± 0.008 1.136 ± 0.007

T3 1.128 ± 0.001 1.110 ± 0.006 1.120 ± 0.006 1.119 ± 0.004 0 T4 1.038 ± 0.007 1.091 ± 0.005 1.113 ± 0.005 1.081 ± 0.012

T5 1.120 ± 0.006 1.106 ± 0.003 1.120 ± 0.005 1.115 ± 0.003

T6 1.077 ± 0.015 1.001 ± 0.010 1.106 ± 0.004 1.061 ± 0.017

T1 1.170 ± 0.005 1.140 ± 0.007 1.162 ± 0.005 1.157 ± 0.005

T2 1.163 ± 0.006 1.130 ± 0.005 1.147 ± 0.007 1.147 ± 0.006

T3 1.133 ± 0.006 1.122 ± 0.007 1.129 ± 0.006 1.128 ± 0.003 30 T4 1.060 ± 0.006 1.101 ± 0.006 1.123 ± 0.006 1.095 ± 0.010

T5 1.130 ± 0.007 1.114 ± 0.007 1.133 ± 0.006 1.126 ± 0.004

T6 1.114 ± 0.007 1.028 ± 0.007 1.117 ± 0.006 1.086 ± 0.015

T1 1.178 ± 0.005 1.146 ± 0.006 1.174 ± 0.005 1.166 ± 0.006

T2 1.169 ± 0.006 1.137 ± 0.010 1.156 ± 0.000 1.154 ± 0.006

T3 1.139 ± 0.005 1.130 ± 0.005 1.137 ± 0.000 1.135 ± 0.003 60 T4 1.096 ± 0.007 1.118 ± 0.008 1.130 ± 0.000 1.115 ± 0.006

T5 1.134 ± 0.007 1.120 ± 0.006 1.143 ± 0.005 1.132 ± 0.004

T6 1.120 ± 0.005 1.042 ± 0.011 1.126 ± 0.003 1.096 ± 0.014

T1 1.183 ± 0.004 1.155 ± 0.003 1.179 ± 0.005 1.172 ± 0.005

T2 1.175 ± 0.003 1.141 ± 0.005 1.162 ± 0.001 1.159 ± 0.005

T3 1.144 ± 0.003 1.139 ± 0.005 1.144 ± 0.003 1.143 ± 0.002 90 T4 1.109 ± 0.005 1.126 ± 0.004 1.139 ± 0.006 1.125 ± 0.005

T5 1.140 ± 0.006 1.131 ± 0.003 1.150 ± 0.005 1.140 ± 0.004

T6 1.129 ± 0.005 1.060 ± 0.005 1.131 ± 0.003 1.106 ± 0.012

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

159

B C C+B

1.25

1.2

1.15

1.1

1.05

cidity

A

1

0.95

0.9

0.85

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90

Fig.4.25 Effect of milk types, treatments and storage period on acidity (%) of processed Cheddar cheese

160 acidity of the natural cheese, being higher in processed cheese having increased amount of mature cheeses. Singh and Kanawjia (1989) also reported the increase in the acidity of the processed cheese during storage. 4.9.7 Salt content Salt represents the sodium amount in the process cheese, provides salty taste and maintains water activity by controlling microbial and enzymatic stability (Grummer and Schoenfuss, 2011). Salt is one of the primogenital and most operational preservative for process cheese (Taormina, 2010). It is the low-cast and superlative flavoring substances for food, including process cheese since it deliver additional paybacks to process cheese, including flavor enrichment (Liem et al., 2011). Salt reduction in process cheese affects the insoluble calcium and phosphate (Shirashoji et al., 2006). Results of the salt content in processed cheese have been elaborated in table 4.51 a, b, c. A non-significant impact of the salt concentration was observed with respect to milk (Fig. 4.26). The highest mean value of salt (1.090±0.010%) was observed in cow + buffalo milk cheese followed by 1.076±0.009% and 1.075±0.007% buffalo and cow milk processed cheese respectively (table 4.51a). Salt increased highly significantly during storage period from 0-90 with mean value as 1.058±0.012% (at 0-Day) to 1.103±0.008% (at 90th Day) as given in the Table 4.51a. Cheese treatments showed a highly significant impression on the salt concentration

(table 4.28b) with the highest mean salt content (1.109±0.011%) in T2 and the lowest

1.023±0.010% in T6. Interaction between cheese treatments and milk types (table 4.28b) showed highly significant difference with highest salt contents (1.160±0.014%) in T2 (cow

+ buffalo) and the lowest salt contents (0.988±0.014%) in T6 of cow + buffalo milk cheese. Interaction of milk types, cheese treatments and storage periods exerted a non-significant trend as shown in the table 4.51c. The values for salt content in the current study are very close to the findings of Awad et al. (2013), who reported the salt concentration between 1.10 % to 1.21% in the processed cheese prepared by using Cheddar cheese. El-Sayed (1996) stated the salt within the range of 2.7-3.5 % in the processed cheese prepared from Cheddar and young Ras cheese available in Egypt. Increase in the salt concentration during storage may be due to the reaction of insoluble calcium and phosphate to chlorides. In addition to the sodium chloride, processed cheeses have another source of sodium which comes from emulsifying salts added to the processed cheese as raw material (Cruz et al., 2011b). El-Bakry (2010) found that potassium chloride when used as an emulsifying salt replacer had a significant effect on the salt content of process cheese.

161 Table 4.51a Effect of milk types and storage period on salt (%) of processed Cheddar cheese (Means ± SE)

Storage Milk type Means ± SE days Buffalo Cow Cow + Buffalo 0 1.054 ± 0.019 1.066 ± 0.018 1.054 ± 0.025 1.058 ± 0.012B 30 1.050 ± 0.017 1.068 ± 0.014 1.082 ± 0.019 1.067 ± 0.010B 60 1.097 ± 0.016 1.083 ± 0.014 1.099 ± 0.015 1.093 ± 0.009A 90 1.104 ± 0.016 1.083 ± 0.009 1.123 ± 0.016 1.103 ± 0.008A Means ± SE 1.076 ± 0.009 1.075 ± 0.007 1.090 ± 0.010

Table 4.51b Effect of milk types and treatments on salt (%) of processed Cheddar cheese (Means ± SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 1.125 ± 0.013abc 1.050 ± 0.015ef 1.132 ± 0.019abc 1.102 ± 0.011A

T2 1.057 ± 0.022ef 1.109 ± 0.008bcd 1.160 ± 0.014a 1.109 ± 0.011A

T3 1.002 ± 0.015g 1.112 ± 0.017bcd 1.053 ± 0.023ef 1.056 ± 0.013B

T4 1.144 ± 0.008ab 1.052 ± 0.020ef 1.121 ± 0.013abc 1.106 ± 0.011A

T5 1.108 ± 0.016bcd 1.070 ± 0.015de 1.084 ± 0.022cde 1.088 ± 0.010A

T6 1.022 ± 0.015fg 1.058 ± 0.017ef 0.988 ± 0.014g 1.023 ± 0.010C Means ± SE 1.076 ± 0.009 1.075 ± 0.007 1.090 ± 0.010

162

Table 4.51c Effect of milk types, treatments and storage period on salt (%) of processed Cheddar cheese (Means ± SE)

Storage Treatments Milk types Means ± SE days Buffalo Cow Cow + Buffalo

T1 1.107 ± 0.047 1.057 ± 0.030 1.090 ± 0.050 1.084 ± 0.023

T2 1.020 ± 0.021 1.113 ± 0.003 1.140 ± 0.040 1.091 ± 0.022

T3 0.983 ± 0.038 1.080 ± 0.067 1.000 ± 0.050 1.021 ± 0.030 0 T4 1.143 ± 0.028 1.053 ± 0.067 1.123 ± 0.052 1.107 ± 0.029

T5 1.093 ± 0.041 1.060 ± 0.055 1.003 ± 0.080 1.052 ± 0.033

T6 0.977 ± 0.012 1.033 ± 0.034 0.970 ± 0.049 0.993 ± 0.020

T1 1.103 ± 0.017 1.033 ± 0.039 1.117 ± 0.003 1.084 ± 0.018

T2 1.003 ± 0.038 1.097 ± 0.024 1.157 ± 0.037 1.086 ± 0.028

T3 0.977 ± 0.012 1.117 ± 0.027 1.030 ± 0.069 1.041 ± 0.030 30 T4 1.133 ± 0.009 1.037 ± 0.044 1.113 ± 0.032 1.094 ± 0.022

T5 1.077 ± 0.044 1.060 ± 0.032 1.103 ± 0.007 1.080 ± 0.017

T6 1.007 ± 0.012 1.063 ± 0.032 0.973 ± 0.009 1.014 ± 0.017

T1 1.140 ± 0.012 1.050 ± 0.045 1.127 ± 0.007 1.106 ± 0.020

T2 1.097 ± 0.030 1.110 ± 0.012 1.173 ± 0.012 1.127 ± 0.015

T3 1.017 ± 0.047 1.127 ± 0.007 1.077 ± 0.028 1.073 ± 0.023 60 T4 1.147 ± 0.013 1.060 ± 0.046 1.120 ± 0.006 1.109 ± 0.019

T5 1.127 ± 0.027 1.083 ± 0.027 1.107 ± 0.003 1.106 ± 0.013

T6 1.057 ± 0.044 1.070 ± 0.056 0.990 ± 0.021 1.039 ± 0.025

T1 1.150 ± 0.015 1.060 ± 0.017 1.193 ± 0.054 1.134 ± 0.026

T2 1.107 ± 0.064 1.117 ± 0.022 1.170 ± 0.032 1.131 ± 0.024

T3 1.030 ± 0.012 1.123 ± 0.015 1.107 ± 0.003 1.087 ± 0.015 90 T4 1.153 ± 0.012 1.057 ± 0.018 1.127 ± 0.003 1.112 ± 0.016

T5 1.137 ± 0.007 1.077 ± 0.009 1.123 ± 0.009 1.112 ± 0.010

T6 1.047 ± 0.024 1.063 ± 0.022 1.020 ± 0.020 1.043 ± 0.013

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

163

B C C+B

1.3

1.2

1.1

1

alt S 0.9

0.8

0.7

0.6

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90

Fig.4.26 Effect of milk types, treatments and storage period on salt (%) of processed Cheddar cheese

164 4.10 MINERALS ANALYSIS

Milk and cheese deliver many fruitful nutrients to diet in variable amounts. The consumption of cheese is of great nutritional concern due to its composition of micronutrients (Gonzalez et al., 2011). As cheese is manufactured by heating and adding salts in the mixture of ingredients, so these conditions also affect the mineral composition of cheese (Altahir et al., 2014). In present study, Ca, K, Na, Mg and P content in processed Cheddar cheese were determined. Analysis of variance table 4.52 shows that milk types highly significantly (P<0.01) influenced all minerals under study while storage showed significant (P<0.05) effect on Ca, Mg and K and non-significant effect on Na and P. Treatment combinations also significantly affected all minerals except Phosphorus. 4.10.1 Calcium content Physical properties of natural cheese are prominently affected by protein and calcium- casein ratio. Generally calcium content is not taken into consideration during selection of natural cheese for processing (Joshi et al., 2004; Kindstedt et al., 2004 and Guinee and O’ Kennedy, 2009). Cow milk provides calcium which is well utilized in both Cheddar and processed Cheddar cheese. Cheese serves as good medium to provide absorbable calcium due to having CPP (Casein Phosphor peptides) which is formed by proteolysis digestion of casein in the gut (Kato et al., 2002). Results for calcium content in processed Cheddar cheese prepared from cow, buffalo and mixture of cow and buffalo Cheddar cheese are given in tables 4.53a, b, c and Fig. 4.27. Highly significant difference of calcium content was observed with respect to milk types showing highest mean value (564.40±0.825 mg/100g) in processed cheese of buffalo milk while the lowest content (483.64±0.788 mg/100g) was recorded in cheese from cow milk Cheddar. Calcium content decreased significantly during storage period from 526.69±4.589 mg/100g to 523.61±4.724 mg/100g (table 4.53a). Highly significant difference of calcium regarding treatments (table 4.53b) showed the highest mean value of calcium (529.86±5.549 mg/100g) in treatment T1 and the lowest values of calcium (519.47±5.451 mg/100g) in treatment T6. Interaction of milk types and treatments displayed highest values of calcium 570.58±1.190 mg/100g in T5 (buffalo milk cheese) and the lowest calcium values 477.83±1.319 mg/100g in T6 (cow milk cheese). Table 4.53c shows a non-significant difference of calcium contents for all parameters (storage, milk types and treatments).

165 Results regarding calcium content match with the results of Guinee and O’Callaghan (2013), who formulated the processed cheese product by using different percentage of the Cheddar cheese and skim milk cheeses and declared the Ca content ranging between 363- 766 mg/100g. Guinee and O’Kennedy (2009) prepared processed cheese by using Cheddar and found Ca content between 375-615 mg/100g which is in accordance with the results of the current study. Significant decrease in calcium contents during storage period (0-90 days) has also been noted by Dhoul and Hamed (2014) in the white soft cheese prepared from cow milk. Similar decrease in calcium content during storage up to 0-180 days has also been declared by Abdalla and Hassan (2013) in Sudanese white soft cheese. Decrease in calcium is attributed to its solubility in acidic medium due to increase in acidity or its dissolution to pickling solution (El-Abd et al., 1982 and Dariani et al., 1980). However increase in calcium by using calcium containing salts in processed cheese food increase the firmness by decreasing the melt-ability (Kapoor et al., 2007). 4.10.2 Potassium content Potassium is present in aqueous solution and is involved in ionic equilibrium of the body fluids. De La Fuente et al. (1997) pointed out that more than 90% of sodium and potassium are present in free sate in dairy products and are immediately released from organic matter. Potassium can be added to the cheese as potassium chloride or part of dietetic salt and has many advantages (Naismith and Braschi, 2003; Reps et al., 1998 and 2009). Results concerning potassium content of processed Cheddar cheese are given in table 4.54a, b, c and Fig. 4.28. The highest values of the potassium (70.57±0.301 mg/100g) were found in buffalo milk processed cheese and the lowest (60.15±0.247 mg/100g) in cow milk processed cheese. During storage, significant decrease was observed in potassium content which decreased from 65.78±0.639 mg/100g (at 0-day) to 64.43±0.665 mg/100g (at 90- day) as mentioned in table 4.54a. The significant difference of potassium contents between treatments was noted with the highest potassium content (66.22±0.841 mg/100g) in treatment T1 and the lowest

(64.28±0.834 mg/100g) in treatment T4. The highest values of potassium with respect to interaction of milk types and treatments were recorded as 72.00±0.739 mg/100g in

Treatment T1 (buffalo milk cheese) while the lowest values (60.25±0.708 mg/100g) were exhibited by treatment T3 (cow milk cheese) as mentioned in table 4.54b. Interaction of

166

Table 4.52 Mean sum of squares for minerals content of processed Cheddar cheese

Source of variation d.f Ca K Na Mg P

NS NS Storage Days (D) 3 86.0* 19.42* 0.2557 16.13* 0.03096

Milk type (M) 2 117644.0** 1957.29** 54194.00** 3931.17** 60640.20**

NS Treatments (T) 5 514.0** 18.54* 11.0361** 49.72** 0.75299

NS NS NS NS NS D × M 6 9.3 0.45 0.0856 1.18 0.00035

NS NS NS NS NS D × T 15 8.0 0.29 0.0420 0.60 0.08783

NS NS M × T 10 156** 2.87 6.1593* 17.91** 0.61500

NS NS NS NS NS D × M × T 30 6 0.27 0.0436 0.69 0.08677

Error 144 30 6.86 3.0336 5.24 3.15680

Total 215

167

Table 4.53a Effect of milk types and storage period on calcium (mg/100g) of processed Cheddar cheese (Means ± SE)

Storage Milk type Means ± SE days Buffalo Cow Cow + Buffalo 0 566.06±1.470 485.72±1.643 528.28±1.425 526.69±4.589A

30 563.44±1.429 484.28±1.120 527.33±1.066 525.02±4.498AB

60 564.44±1.710 483.06±1.479 527.28±1.578 524.93±4.658AB

90 563.67±1.998 481.50±1.915 525.67±1.469 523.61±4.724B

Means ± SE 564.40±0.825A 483.64±0.788C 527.14±0.693B

Table 4.53b Effect of milk types and treatments on calcium (mg/100g) of processed Cheddar cheese (Means ± SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 569.33 ± 1.163a 489.58 ± 1.090g 530.67 ± 1.494d 529.86 ± 5.549A

T2 564.08 ± 1.328b 488.08 ± 1.076gh 529.25 ± 1.533de 527.14 ± 5.303B

T3 559.25 ± 1.219c 484.92 ± 1.438hi 522.00 ± 1.537f 522.06 ± 5.190C

T4 567.83 ± 1.542ab 480.50 ± 1.395ij 526.17 ± 1.419ef 524.83 ± 6.083B

T5 570.58 ± 1.190a 480.92 ± 2.482ij 529.50 ± 1.340de 527.00 ± 6.274B

T6 555.33 ± 1.137c 477.83 ± 1.319j 525.25 ± 1.728ef 519.47 ± 5.451D Means ± SE 564.40 ± 0.825A 483.64 ± 0.788C 527.14 ± 0.693B

168 Table 4.53c Effect of milk types, treatments and storage period on calcium (mg/100g) of processed Cheddar cheese (Means ± SE)

Storage Milk types Treatments Means ± SE days Buffalo Cow Cow + Buffalo

T1 570.67 ± 1.764 492.33 ± 1.453 533.33 ± 4.667 532.11 ± 11.410

T2 566.00 ± 1.155 489.33 ± 1.453 529.33 ± 3.528 528.22 ± 11.129

T3 561.00 ± 2.082 487.67 ± 3.180 525.00 ± 2.082 524.56 ± 10.659 0 T4 569.00 ± 2.887 482.00 ± 2.082 525.00 ± 2.646 525.33 ± 12.623

T5 571.67 ± 3.756 485.00 ± 7.095 531.67 ± 0.882 529.44 ± 12.737

T6 558.00 ± 1.732 478.00 ± 1.155 525.33 ± 4.807 520.44 ± 11.710

T1 567.00 ± 2.082 488.67 ± 1.202 529.00 ± 2.082 528.22 ± 11.345

T2 561.33 ± 1.856 489.33 ± 1.202 530.00 ± 2.082 526.89 ± 10.458

T3 561.67 ± 1.453 486.00 ± 1.732 520.33 ± 0.882 522.67 ± 10.960 30 T4 567.33 ± 2.404 480.00 ± 0.577 527.67 ± 1.764 525.00 ± 12.654

T5 569.00 ± 1.528 482.00 ± 3.055 529.67 ± 1.764 526.89 ± 12.625

T6 554.33 ± 3.383 479.67 ± 1.453 527.33 ± 3.180 520.44 ± 11.004

T1 570.33 ± 3.756 489.33 ± 0.882 529.33 ± 2.028 529.67 ± 11.759

T2 566.00 ± 2.646 487.00 ± 3.215 529.33 ± 5.239 527.44 ± 11.575

T3 558.33 ± 1.764 484.00 ± 3.215 523.33 ± 5.783 521.89 ± 10.916 60 T4 566.67 ± 4.055 481.33 ± 2.028 526.33 ± 3.180 524.78 ± 12.426

T5 570.67 ± 0.882 479.00 ± 3.786 530.67 ± 4.485 526.78 ± 13.377

T6 554.67 ± 0.882 477.67 ± 4.410 524.67 ± 3.180 519.00 ± 11.316

T1 569.33 ± 2.028 488.00 ± 4.041 531.00 ± 3.606 529.44 ± 11.864

T2 563.00 ± 4.359 486.67 ± 2.906 528.33 ± 2.603 526.00 ± 11.162

T3 556.00 ± 3.606 482.00 ± 3.606 519.33 ± 1.764 519.11 ± 10.794 90 T4 568.33 ± 4.485 478.67 ± 5.548 525.67 ± 4.702 524.22 ± 13.180

T5 571.00 ± 3.464 477.67 ± 6.741 526.00 ± 2.517 524.89 ± 13.670

T6 554.33 ± 2.906 476.00 ± 3.464 523.67 ± 4.333 518.00 ± 11.537

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

169

B C C+B

600

580

560

540

520

ium

c 500

Cal 480

460

440

420

400

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90 Ca_P

Fig.4.27 Effect of milk types, treatments and storage period on calcium (mg/100g) of processed Cheddar cheese

170

Table 4.54a Effect of milk types and storage period on potassium (mg/100g) of processed Cheddar cheese (Means ± SE)

Storage Milk type Means ± SE days Buffalo Cow Cow + Buffalo 0 71.17 ± 0.466 60.72 ± 0.403 65.44 ± 0.473 65.78 ± 0.639A 30 70.94 ± 0.707 60.39 ± 0.405 65.39 ± 0.486 65.57 ± 0.669A 60 70.61 ± 0.531 59.94 ± 0.557 64.78 ± 0.619 65.11 ± 0.680AB 90 69.56 ± 0.648 59.56 ± 0.579 64.17 ± 0.658 64.43 ± 0.665B Means ± SE 70.57 ± 0.301A 60.15 ± 0.247C 64.94 ± 0.283B

Table 4.54b Effect of milk types and treatments on potassium (mg/100g) of processed Cheddar cheese (Means ± SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 72.00 ± 0.739 60.75 ± 0.552 65.92 ± 0.358 66.22 ± 0.841A

T2 69.83 ± 0.796 60.00 ± 0.537 64.50 ± 0.584 64.78 ± 0.771BC

T3 70.42 ± 0.679 60.25 ± 0.708 64.17 ± 0.869 64.94 ± 0.825BC

T4 70.08 ± 0.583 59.08 ± 0.543 63.67 ± 0.678 64.28 ± 0.834C

T5 71.25 ± 0.641 60.50 ± 0.680 65.83 ± 0.796 65.86 ± 0.842AB

T6 69.83 ± 0.869 60.33 ± 0.595 65.58 ± 0.609 65.25 ± 0.766ABC

Means ± SE 70.57 ± 0.301A 60.15 ± 0.247C 64.94 ±0.283B

171 Table 4.54c Effect of milk types, treatments and storage period on potassium (mg/100g) of processed Cheddar cheese (Means ± SE)

Milk types Storage Treatments Cow + Means ± SE days Buffalo Cow Buffalo

T1 72.67 ± 1.764 61.33 ± 0.882 66.33 ± 0.333 66.78 ± 1.738

T2 70.33 ± 1.453 60.67 ± 1.202 64.67 ± 1.202 65.22 ± 1.544

T3 70.67 ± 0.333 60.67 ± 1.453 65.00 ± 2.646 65.44 ± 1.692 0 T4 70.33 ± 0.882 59.67 ± 0.333 64.33 ± 0.333 64.78 ± 1.570

T5 72.00 ± 1.528 61.33 ± 1.202 66.33 ± 0.667 66.56 ± 1.651

T6 71.00 ± 0.577 60.67 ± 1.202 66.00 ± 0.577 65.89 ± 1.550

T1 72.00 ± 2.082 60.67 ± 1.202 66.00 ± 1.155 66.22 ± 1.809

T2 70.33 ± 2.028 60.00 ± 0.577 65.00 ± 1.155 65.11 ± 1.645

T3 71.00 ± 1.732 61.00 ± 1.000 64.33 ± 2.333 65.44 ± 1.717 30 T4 70.67 ± 0.882 59.33 ± 0.667 64.33 ± 0.667 64.78 ± 1.681

T5 71.33 ± 1.856 60.33 ± 1.764 66.67 ± 0.333 66.11 ± 1.759

T6 70.33 ± 2.963 61.00 ± 1.000 66.00 ± 1.000 65.78 ± 1.648

T1 72.33 ± 1.202 60.67 ± 1.453 66.00 ± 1.000 66.33 ± 1.795

T2 70.00 ± 1.155 59.67 ± 2.028 64.33 ± 1.453 64.67 ± 1.691

T3 70.33 ± 0.882 60.00 ± 1.732 64.00 ± 1.155 64.78 ± 1.640 60 T4 70.33 ± 1.453 59.00 ± 1.528 63.33 ± 2.603 64.22 ± 1.913

T5 71.00 ± 1.528 60.33 ± 1.453 65.33 ± 2.028 65.56 ± 1.757

T6 69.67 ± 2.028 60.00 ± 1.155 65.67 ± 1.202 65.11 ± 1.594

T1 71.00 ± 1.528 60.33 ± 1.453 65.33 ± 0.333 65.56 ± 1.659

T2 68.67 ± 2.333 59.67 ± 0.333 64.00 ± 1.528 64.11 ± 1.532

T3 69.67 ± 2.404 59.33 ± 2.028 63.33 ± 1.453 64.11 ± 1.806 90 T4 69.00 ± 1.732 58.33 ± 1.764 62.67 ± 1.333 63.33 ± 1.748

T5 70.67 ± 0.667 60.00 ± 1.732 65.00 ± 2.887 65.22 ± 1.832

T6 68.33 ± 1.202 59.67 ± 1.856 64.67 ± 2.186 64.22 ± 1.544

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

172

B C C+B

80

75

70

65

sium 60

s

ota

P 55

50

45

40 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90 K_P

Fig.4.28 Effect of milk types, treatments and storage period on potassium (mg/100g) of processed Cheddar cheese

173 all parameters (treatments, milk types and storage days) showed a non-significant trend as shown in table 4.54c. Potassium content of cheese in the existing study are very close to the results of Dhoul and Hamid et al. (2013), who studied the mineral content of white cheese and found potassium content were between 79.5±1.37 mg/100g to 81.6±1.53 mg/100g. Guo et al. (1997), found the potassium as 67mg/100g in Mozzarella cheese which is also in accordance to the current studies. Almenara et al. (2007) reported that potassium concentration depends upon the animal origin of milk. The highest potassium values found in buffalo milk cheese prove the fact. Decrease in potassium during storage might be due to the lipolytic activity of micro- organisms on potassium as a result of which some potassium might be leaked into pickling whey (Khalid, 1991; Abdalla, 1992 and Nuser et al., 2001). 4.10.3 Sodium content Sodium and fat perform essential part in the manufacture of cheese and processed cheese (Johnson et al., 2009). Main sodium contributors in processed cheese are natural cheese (Approximately 28%-37%), added emulsifying salts (44% to 48%) and sodium chloride (ranging from 15% to 24%). Sodium intake occupies a major part with respect to the health discussions and it should not exceed 2000 mg per day (equal to 5g of salt) as stated by World Health Organization (2011). Sodium when added in the form of sodium chloride also takes part in safety of processed cheese and 50% reduction of sodium chloride significantly affect the microbiological constancy by facilitating survival of pathogens (Ilhak et al., 2011 and Shrestha et al., 2011). Buffalo milk cheese contained the highest amount (311.52±0.236 mg/100g) of sodium and the lowest amount (257.26±0.154 mg/100g) was observed in cow milk cheese (table 4.55a). Mean values of sodium content remained almost un-changed as 281.95±3.080 mg/100g at 0-day to 282.12±3.096 mg/100g after 90-days of storage showing a non-significant effect of storage as shown in table 4.55a. Highly significant effect on sodium content was seen by treatments with the highest value (283.10±3.898 mg/100g) in T1 and the lowest (281.50±3.672 mg/100g) in T3 (Fig. 4.29). Interaction of milk types and treatments also presented highly significant difference in sodium contents of processed cheese with the lowest content (256.97±0.272 mg/100g) in T5 (cow milk cheese) and the highest (313.56±0.470 mg/100g) in T1 (buffalo milk cheese) as documented in

174 Table 4.55a Effect of milk types and storage period on sodium (mg/100g) of processed Cheddar cheese (Means ± SE)

Storage Milk type Means ± SE days Buffalo Cow Cow + Buffalo 0 311.40±0.538 257.20±0.331 277.26±0.140 281.95±3.080A

30 311.47±0.481 257.27±0.308 277.32±0.383 282.02±3.081A

60 311.49±0.404 257.28±0.195 277.34±0.456 282.03±3.081A

90 311.73±0.497 257.30±0.391 277.34±0.341 282.12±3.096A

Means ± SE 311.52±0.236A 257.26±0.154C 277.31±0.171B

Table 4.55b Effect of milk types and treatments on sodium (mg/100g) of processed Cheddar cheese (Means ± SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 313.56 ± 0.470a 257.95 ± 0.383e 277.79 ± 0.528d 283.10 ± 3.898A

T2 310.79 ± 0.429bc 257.42 ± 0.544e 277.08 ± 0.457d 281.76 ± 3.735B

T3 309.70 ± 0.559c 256.99 ± 0.381e 277.81 ± 0.361d 281.50 ± 3.672B

T4 311.30 ± 0.368b 257.20 ± 0.249e 277.05 ± 0.440d 281.85 ± 3.782B

T5 311.85 ± 0.611b 256.97 ± 0.272e 277.05 ± 0.388d 281.96 ± 3.840B

T6 311.94 ± 0.428b 257.04 ± 0.359e 277.11 ± 0.334d 282.03 ± 3.839B Means ± SE 311.52 ± 0.236A 257.26 ± 0.154C 277.31 ± 0.171B

175 Table 4.55c Effect of milk types, treatments and storage period on sodium (mg/100g) of processed Cheddar cheese (Means ± SE)

Storage Milk types Treatments Means ± SE days Buffalo Cow Cow + Buffalo

T1 313.50 ± 1.781 257.87 ± 0.543 277.71 ± 0.248 283.03 ± 8.157

T2 310.45 ± 0.462 257.35 ± 2.152 277.04 ± 0.264 281.61 ± 7.776

T3 309.66 ± 0.777 256.94 ± 0.086 277.76 ± 0.559 281.45 ± 7.671 0 T4 311.23 ± 0.760 257.14 ± 0.275 276.98 ± 0.157 281.79 ± 7.903

T5 311.68 ± 2.407 256.92 ± 0.142 277.00 ± 0.353 281.87 ± 8.029

T6 311.89 ± 0.179 256.98 ± 0.459 277.06 ± 0.250 281.98 ± 8.021

T1 313.55 ± 0.386 257.95 ± 1.314 277.79 ± 1.342 283.10 ± 8.153

T2 310.54 ± 1.400 257.42 ± 0.954 277.09 ± 1.314 281.68 ± 7.776

T3 309.70 ± 1.412 257.01 ± 1.316 277.81 ± 0.656 281.51 ± 7.684 30 T4 311.30 ± 0.868 257.21 ± 0.267 277.06 ± 1.223 281.86 ± 7.911

T5 311.77 ± 1.123 256.97 ± 0.158 277.07 ± 1.191 281.94 ± 8.017

T6 311.93 ± 1.167 257.06 ± 0.131 277.11 ± 0.524 282.03 ± 8.023

T1 313.58 ± 0.185 257.98 ± 0.125 277.81 ± 1.662 283.12 ± 8.149

T2 310.56 ± 0.208 257.44 ± 0.117 277.10 ± 1.243 281.70 ± 7.761

T3 309.72 ± 1.560 257.01 ± 0.136 277.83 ± 1.323 281.52 ± 7.687 60 T4 311.32 ± 1.199 257.22 ± 0.167 277.07 ± 1.140 281.87 ± 7.915

T5 311.78 ± 0.133 256.99 ± 0.063 277.08 ± 1.071 281.95 ± 8.008

T6 311.95 ± 0.105 257.06 ± 1.222 277.12 ± 1.312 282.04 ± 8.035

T1 313.60 ± 1.227 258.01 ± 1.086 277.83 ± 1.228 283.15 ± 8.154

T2 311.59 ± 1.185 257.46 ± 0.975 277.10 ± 1.115 282.05 ± 7.930

T3 309.73 ± 1.355 257.02 ± 1.196 277.82 ± 0.611 281.53 ± 7.684 90 T4 311.34 ± 0.450 257.23 ± 1.088 277.07 ± 1.195 281.88 ± 7.917

T5 312.14 ± 1.039 257.00 ± 1.256 277.07 ± 0.783 282.07 ± 8.073

T6 311.98 ± 1.618 257.07 ± 1.054 277.14 ± 0.622 282.06 ± 8.042

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

176

B C C+B

340

320

300

280

odium

S 260

240

220

200

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90 Na_P

Fig.4.29 Effect of milk types, treatments and storage period on sodium (mg/100g) of processed Cheddar cheese

177 Table 4.55b. Non-significant effect of interactions between all parameters relating to sodium content of processed cheese is mentioned in Table 4.55c.

Sodium contents of the recent study are within the range of the results by Gonzalez et al. (2011) reporting sodium between 276-1392 mg/100g in cheese prepared from cow, ewe and goat milk. Code of federal regulation (FDA, 2008-b) explained that processed cheese should have sodium contents within range of 325-798 mg/50g in processed cheese.

Non-significant increase in sodium content was noted in the results of present study which is in accordance with Abdel Razig (2000), who declared a significant increase of sodium content during storage up to 90 days. Such increase in sodium may be due to the use of emulsifying salts which prevent the separation of fat from moisture thus preventing the loss of moisture during storage and also acting as extra source of sodium.

4.10.4 Magnesium content

Magnesium and phosphate are distributed between soluble and colloidal phase and interact with the milk proteins by exerting profound effects on quality and stability of milk and milk products (Varnam and Sutherland, 2001). Milk contains about 10g/kg of magnesium while cheese 23g/100g (Kato et al., 2002). Chloride salts of potassium, magnesium and calcium can be used to impart salty taste and as sodium replacers in reduced sodium cheese since these (K, Mg and Ca) are cation substitute (Johnson et al., 2009) but can’t provide same saltiness level as sodium. However sometimes acceptability of these salt replacers is not preferred in cheese due to their inherent bitter and metallic off- flavors (Fitzgerald and Buckley, 1985; Grummer et al., 2013).

Tables 4.56a, b and c, declare the results of magnesium content of processed Cheddar cheese from cow, buffalo and mixture of cow and buffalo milk Cheddar. Highly significant difference between milk types was observed with respect to magnesium values with the highest (37.24±0.309 mg/100g) in buffalo milk cheese and the lowest (22.49±0.245 mg/100g) value was in cow milk cheese (Fig. 4.30). Storage days showed a significant variation in magnesium content showing a decreasing trend with mean values as 30.15±0.857 mg/100g (at 0-day) to 28.96±0.905 mg/100g (at 90th day).

178 The highly significant difference of magnesium between treatments was noted (table 4.56b). The highest mean values of (31.36±1.001 mg/100g) was found in treatment

T1 and the lowest (28.39±1.036 mg/100g) in T6. Interaction between milk types and treatments also disclosed highly significant difference regarding magnesium content with the highest (38.17±0.613 mg/100g) in T1 (buffalo milk cheese) and the lowest (20.92±0.452 mg/100g) in T6 (cow milk cheese) as mentioned in table 4.56b. Non- significant difference of interactions between milk types, treatments and storage periods was noted in table 4.56c.

Magnesium content as in the present study are quite close to the results of the

McMahon et al. (2014), who studied the effect of different salt cations containing MgCl2 on certain parameters of full fat Cheddar cheese during ripening and reported Mg content between 30 mg/100g in control having no cation salt to 113 mg/100g in cheese with salts. Gonzalez et al. (2011) also studied the mineral composition of cheese and noted the magnesium content between 25.785 mg/100g to 68.669 mg/100g which also corresponds to the results of the current study.

Significant decrease in Mg content during storage is in disagreement to the findings of Gonzalez et al. (2009), who stated that magnesium content remained un- changed throughout the storage period but are in accordance with the results of Merdivan et al. (2004), they declared a significant decrease in Mg during storage period.

As stated earlier the magnesium and calcium are bound to casein micelles and are between soluble and colloidal phase so these get solubilized and lost in curd. Magnesium is partially associated with phosphates and citrates. Most cheeses have reduced magnesium contents containing 10-50 mg/100g (IDF, 2008). Variation in the amount of magnesium in processed cheese is due to its variation in Cheddar cheese from cow, buffalo and their mixture (blend).

4.10.5 Phosphorus content

Phosphate is present in foods in two forms as natural and phosphate additives. Foods rich in protein have high amounts of phosphate (Suurseppa et al., 2001). Absorption of phosphorus is reduced by high intake of calcium (Spencer et al., 1984).

179 National nutritional council (2005) suggested the Ca: P weight ratio 1.3 and are highest in dairy products however addition of phosphate additives decrease Ca: P ratio.

Milk types showed a highly significant difference of phosphorus content being the highest (372.37±0.198 mg/100g) in buffalo milk processed cheese while the lowest (314.38±0.163 mg/100g) on cow milk processed cheese (Fig. 4.31). Phosphorus contents remained un-changed showing a non-significant impact of storage period (342.63±3.264 mg/100g to 342.68±3.258 mg/100g) from 0-90 days as given in table 4.57a.

Treatments table 4.57b also presented a non-significant difference regarding phosphorus content of the processed cheese showing the highest mean value (342.85±4.037 mg/100g) in T2 and the lowest (342.45±3.990 mg/100g) in T4. Interaction of milk types and treatments also revealed a non-significant change in phosphorus with highest content

(372.83±0.659 mg/100g) in T2 (buffalo milk cheese) and lowest (341.17±0.409 mg/100g) content in T2 (cow + buffalo milk cheese).

According to the National Institute for Health and Welfare (2009), processed cheese having 20-24g fat contains 610mg/100g of phosphorus and the processed cheese having 27-35g fat contains the P content up to 360mg/100g which are in line with the results of the current study. Guinee and O’Kennedy (2009) prepared the processed cheeses by using natural Cheddar cheeses with different levels of calcium phosphate and showed much high phosphate contents (605-736 mg/100g). They further analyzed water soluble (WSP) and water insoluble phases (WISP) of the processed cheese and recorded (367-426 mg/100g) in WSP and 394-554 mg/100g in WISP which are quite close to the outcomes of present study.

Phosphate content didn’t exhibit any change throughout the storage period which may be due to the fact that in-organic phosphate is completely solubilized at pH 5.2 during acidification. As the calcium level in cheese increases most of the P in emulsifying salts becomes insoluble which leads to the formation of insoluble complex between orthophosphate content of emulsifying salt and the Ca of cheese (Le Graet and Brule 1993).

180

Table 4.56a Effect of milk types and storage period on magnesium (mg/100g) of processed Cheddar cheese (Means ± SE)

Milk type Storage days Means ± SE Buffalo Cow Cow + Buffalo 0 37.61 ± 0.444 23.17 ± 0.414 29.67 ± 0.583 30.15 ± 0.857A 30 37.50 ± 0.785 22.67 ± 0.478 29.67 ± 0.610 29.94 ± 0.907A 60 36.94 ± 0.639 22.17 ± 0.550 28.89 ± 0.554 29.33 ± 0.893AB 90 36.89 ± 0.593 21.94 ± 0.501 28.06 ± 0.644 28.96 ± 0.905B Means ± SE 37.24 ± 0.309A 22.49 ± 0.245C 29.07 ± 0.303B

Table 4.56b Effect of milk types and treatments on magnesium (mg/100g) of processed Cheddar cheese (Means ± SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 38.17 ± 0.613a 24.25 ± 0.392f 31.67 ± 0.466c 31.36 ± 1.001A

T2 36.67 ± 0.582ab 22.08 ± 0.417g 29.00 ± 0.492d 29.25 ± 1.045B

T3 38.50 ± 0.702a 22.67 ± 0.678fg 27.00 ± 0.577e 29.39 ± 1.187B

T4 36.83 ± 0.878ab 22.33 ± 0.700g 26.50 ± 0.452e 28.56 ± 1.102B

T5 37.92 ± 0.900a 22.67 ± 0.569fg 31.33 ± 0.466c 30.64 ± 1.121A

T6 35.33 ± 0.527b 20.92 ± 0.452g 28.92 ± 0.529d 28.39 ± 1.036B Means ± SE 37.24 ± 0.309A 22.49 ± 0.245C 29.07 ± 0.303B

181 Table 4.56c Effect of milk types, treatments and storage period on magnesium (mg/100g) of processed Cheddar cheese (Means ± SE)

Milk types Storage Treatments Cow + Means ± SE days Buffalo Cow Buffalo

T1 39.00 ± 0.577 24.00 ± 0.577 32.00 ± 1.528 31.67 ± 2.224

T2 37.00 ± 1.000 22.33 ± 0.882 29.00 ± 0.577 29.44 ± 2.161

T3 39.00 ± 0.577 23.33 ± 0.882 28.33 ± 1.453 30.22 ± 2.367 0 T4 37.00 ± 1.155 23.00 ± 1.528 27.33 ± 1.202 29.11 ± 2.170

T5 38.67 ± 0.667 24.00 ± 1.528 32.00 ± 1.000 31.56 ± 2.193

T6 35.00 ± 0.577 22.33 ± 0.882 29.33 ± 0.882 28.89 ± 1.874

T1 38.33 ± 0.882 24.33 ± 1.202 32.67 ± 0.333 31.78 ± 2.080

T2 36.67 ± 2.028 22.33 ± 0.882 29.33 ± 0.882 29.44 ± 2.180

T3 38.67 ± 2.028 22.33 ± 1.856 27.33 ± 0.667 29.44 ± 2.550 30 T4 37.00 ± 2.082 23.00 ± 1.000 27.00 ± 1.000 29.00 ± 2.205

T5 38.67 ± 3.480 22.33 ± 1.453 32.33 ± 1.202 31.11 ± 2.638

T6 35.67 ± 1.453 21.67 ± 0.882 29.33 ± 0.882 28.89 ± 2.098

T1 37.33 ± 1.856 24.33 ± 1.202 31.33 ± 0.882 31.00 ± 2.000

T2 36.67 ± 1.202 21.67 ± 0.882 29.00 ± 1.000 29.11 ± 2.226

T3 38.33 ± 1.764 22.67 ± 1.856 26.67 ± 1.202 29.22 ± 2.488 60 T4 36.67 ± 2.333 22.00 ± 2.082 26.33 ± 0.333 28.33 ± 2.357

T5 37.33 ± 1.333 22.33 ± 0.333 31.00 ± 0.577 30.22 ± 2.216

T6 35.33 ± 1.764 20.00 ± 0.577 29.00 ± 1.000 28.11 ± 2.306

T1 38.00 ± 1.732 24.33 ± 0.333 30.67 ± 0.667 31.00 ± 2.048

T2 36.33 ± 0.882 22.00 ± 1.155 28.67 ± 1.764 29.00 ± 2.173

T3 38.00 ± 1.732 22.33 ± 1.453 25.67 ± 1.202 28.67 ± 2.494 90 T4 36.67 ± 2.404 21.33 ± 1.453 25.33 ± 0.882 27.78 ± 2.448

T5 37.00 ± 1.528 22.00 ± 1.155 30.00 ± 0.577 29.67 ± 2.242

T6 35.33 ± 0.667 19.67 ± 0.333 28.00 ± 1.732 27.67 ± 2.327

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

182

B C C+B

45

40

35

30

25

Mg 20

15

10

5

0 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90 Mg_P

Fig.4.30 Effect of milk types, treatments and storage period on magnesium (mg/100g) of processed Cheddar cheese

183

Table 4.57a Effect of milk types and storage period on phosphorus (mg/100g) of processed Cheddar cheese (Means ± SE)

Storage Milk type Means ± SE days Buffalo Cow Cow + Buffalo 0 372.34 ± 0.380 314.35 ± 0.420 341.19 ± 0.464 342.63 ± 3.264A 30 372.37 ± 0.427 314.38 ± 0.336 341.22 ± 0.331 342.66 ± 3.261A 60 372.38 ± 0.440 314.40 ± 0.319 341.24 ± 0.280 342.67 ± 3.261A 90 372.39 ± 0.366 314.40 ± 0.231 341.25 ± 0.170 342.68 ± 3.258A Means ± SE 372.37 ± 0.198A 314.38 ± 0.163C 341.22 ± 0.161B

Table 4.57b Effect of milk types and treatments on phosphorus (mg/100g) of processed Cheddar cheese (Means ± SE)

Milk types Treatments Means ± SE Buffalo Cow Cow + Buffalo

T1 372.25 ± 0.507 314.76 ± 0.363 341.18 ± 0.489 342.73 ± 3.980A

T2 372.83 ± 0.659 314.55 ± 0.458 341.17 ± 0.409 342.85 ± 4.037A

T3 372.29 ± 0.490 314.27 ± 0.470 341.24 ± 0.318 342.60 ± 4.014A

T4 371.91 ± 0.347 314.21 ± 0.412 341.23 ± 0.332 342.45 ± 3.990A

T5 372.68 ± 0.446 314.34 ± 0.435 341.23 ± 0.431 342.75 ± 4.038A

T6 372.26 ± 0.465 314.16 ± 0.291 341.29 ± 0.445 342.57 ± 4.019A Means ± SE 372.37 ± 0.198A 314.38 ± 0.163C 341.22 ± 0.161B

184 Table 4.57c Effect of milk types, treatments and storage period on phosphorus (mg/100g) of processed Cheddar cheese (Means ± SE)

Storage Milk types Treatments Means ± SE days Buffalo Cow Cow + Buffalo

T1 372.22 ± 1.267 314.73 ± 1.286 341.21 ± 1.264 342.72 ± 8.331

T2 372.79 ± 1.600 314.51 ± 1.357 341.10 ± 1.337 342.80 ± 8.454

T3 372.77 ± 0.870 314.22 ± 1.066 341.17 ± 1.309 342.72 ± 8.478 0 T4 371.14 ± 0.478 314.17 ± 1.325 341.19 ± 1.295 342.16 ± 8.245

T5 372.90 ± 0.583 314.32 ± 1.358 341.20 ± 1.290 342.81 ± 8.484

T6 372.24 ± 0.894 314.12 ± 0.799 341.26 ± 1.589 342.54 ± 8.415

T1 372.24 ± 1.242 314.76 ± 0.528 341.26 ± 1.324 342.75 ± 8.323

T2 372.82 ± 1.624 314.55 ± 1.118 341.11 ± 1.208 342.83 ± 8.448

T3 372.11 ± 1.204 314.28 ± 1.238 341.20 ± 0.133 342.53 ± 8.369 30 T4 372.15 ± 0.983 314.22 ± 1.116 341.23 ± 0.612 342.53 ± 8.382

T5 372.61 ± 1.118 314.33 ± 1.035 341.24 ± 1.029 342.72 ± 8.436

T6 372.26 ± 1.114 314.15 ± 0.508 341.28 ± 0.964 342.56 ± 8.407

T1 372.27 ± 1.292 314.79 ± 0.705 341.28 ± 1.190 342.78 ± 8.324

T2 372.84 ± 1.700 314.57 ± 1.031 341.14 ± 0.517 342.85 ± 8.442

T3 372.13 ± 1.278 314.28 ± 1.206 341.23 ± 0.635 342.54 ± 8.374 60 T4 372.17 ± 0.815 314.23 ± 0.695 341.25 ± 0.606 342.55 ± 8.378

T5 372.61 ± 1.115 314.34 ± 1.019 341.25 ± 1.009 342.73 ± 8.436

T6 372.27 ± 1.259 314.17 ± 0.706 341.30 ± 0.720 342.58 ± 8.405

T1 372.27 ± 0.913 314.77 ± 0.680 340.96 ± 0.673 342.67 ± 8.319

T2 372.85 ± 1.207 314.57 ± 0.683 341.35 ± 0.389 342.92 ± 8.431

T3 372.13 ± 1.135 314.29 ± 0.852 341.35 ± 0.284 342.59 ± 8.365 90 T4 372.18 ± 0.623 314.24 ± 0.509 341.26 ± 0.038 342.56 ± 8.373

T5 372.61 ± 1.229 314.36 ± 0.451 341.24 ± 0.592 342.73 ± 8.426

T6 372.28 ± 1.068 314.19 ± 0.686 341.32 ± 0.614 342.59 ± 8.401

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

185

B C C+B

380

370

360

350

340

us

r 330

hospho P 320

310

300

290

280

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90

Fig.4.31 Effect of milk types, treatments and storage period on phosphorus (mg/100g) of processed Cheddar cheese

186 4.11 TEXTURE OF PROCESSED CHEDDAR CHEESE Combination of sensory elements resulting from physical properties professed by sense of light and touch is defined as the texture (Fox et al., 2000a). Texture is the most essential characteristic defining the product identity as well as attributes of food material resulting from combination of physical and chemical properties, especially for cheese (Buffa et al., 2001; Quigleya et al., 2011). Texture of cheese is greatly influenced by both compositional and processing parameters (Wium et al., 2003).

Results of statistical analysis of processed Cheddar cheese have been illustrated in analysis of variance table 4.58. Processed cheese manufactured from Cheddar cheese of cow, buffalo and their mixture showed highly significant (P<0.01) variation with respect to hardness, gumminess, chewiness while non-significant difference for cohesiveness and springiness. Treatment means showed a non-significant change (P>0.05) in hardness, cohesiveness, gumminess, chewiness and springiness. Interaction means between cheese types and treatments also exhibited a non-significant variation of results for all texture attributes. The results for each attribute of cheese texture are discussed below.

4.11.1 Hardness of processed Cheddar cheese

Hardness is the force necessary to bite entirely through sample when placed in the middle of molar teeth (Meullenet and Gross 1999). Results of cheese hardness (Table 4.59 and Fig. 4.32) showed that the highest mean value (3780.0±22.24g) in buffalo milk followed by 3666.7±32.93g in mixture of cow and buffalo milk and 3596.7±26.79g cow milk cheese. Regarding cheese treatments the highest mean hardness was noted in T1 and

T4 which non-significantly decreased in other treatments. The interactions also showed non-significant variations in hardness values (Table 4.59).

Hardness decreases with the use of polyphosphates in greater amounts since they give multiple negative charges to caseins, reducing hydrophobic interactions of dispersed caseins which in return reduce the hardness of the final matrix (Shirashoji et al., 2010 and Bunka et al., 2013). Increased hardness in processed cheese of buffalo milk Cheddar is due to the buffalo milk fat, which is harder than cow milk fat. El-Alfy et al. (2008) concluded that cheese made from cow milk is less firm and more meltable due to

187 characteristics of cow milk fat. Cheese hardness is also influenced with the ease of proteolysis. Due to proteolysis “New” ionic peptides are created which undergo a competition to occupy available water resulting lesser amount required to solvate the protein chains. Thus the resultant cheese becomes harder and less deformable (Creamer and Olson, 1982). Free water accessibility is reduced with increase in ripening time eventually increasing the cheese resistance to deformation (McMahon et al., 1999; Beal and Mittal, 2000). Increased hardness was observed in treatments having high amount of 60 and 90 days ripened cheese may be due to the above mentioned phenomenon.

4.11.2 Cohesiveness of processed Cheddar cheese

Cohesiveness of the cheese is the ease with which a sample crumbles (Zoon, 1991). Strength of internal bonds that makes up the body of the product is considered cohesiveness (Szczesniak et al. 1963 and Bourne, 1978).

The results for cohesiveness of processed Cheddar cheese (Table 4.60; Fig. 4.33) showed non-significant variation due to milk types. The highest cohesiveness value (1.046±0.008) in buffalo milk cheese and the lowest (1.029±0.006) in cow milk Cheddar.

With respect to treatments the highest cohesiveness (1.043±0.009) was noted in T6 and the lowest in T1 (1.029±0.004). Overall the non-significant differences were recorded among cheese treatments as well as the interaction between milk types and treatments.

Reduced cohesiveness observed in processed cheese having high amounts of 30, 60 and 90 day ripened cheese was due to the disruption of casein matrix and nature of fat dispersion which causes the cheese to adhere to itself. Cheddar cheese becomes less cohesiveness with increasing the ripening time (Awad et al., 2005) as confirmed in the current study. It is also obvious in processed cheese having high amount of 90-day ripened Cheddar that high proteolysis (free amino acids) interrupts the structural integrity of protein matrix and lessens cohesiveness (Irudayaraj et al., 1999).

Processed cheese prepared from Cheddar cheese of buffalo milk showed high cohesiveness as Simoes et al. (2013) concluded that cohesiveness decreased with partial addition of cow milk in buffalo milk for cheese preparation.

188

Table 4.58 Analysis of variance for texture of processed Cheddar cheese

Source of d.f Mean squares variation Hardness Cohesiveness Gumminess Chewiness Springiness

NS NS Milk (M) 2 154067** 0.00157 295043** 197206** 0.00057

NS NS NS NS NS Treat (T) 5 4797 0.00018 922 4051 0.00013

NS NS NS NS NS M × T 10 1012 0.00005 1771 2382 0.00010

Error 36 18577 0.00112 13831 16192 0.00134

NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01)

189 Table 4.59 Effect of milk types and treatments on hardness (g) of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB

T1 3810.0±31.18 3620.0±103.92 3670.0±101.61 3700.0±51.48A

T2 3795.0±73.90 3605.0±72.17 3670.0±38.68 3690.0±42.32A

T3 3765.0±48.50 3590.0±91.22 3670.0±99.30 3675.0±48.49A

T4 3790.0±49.07 3590.0±27.14 3720.0±128.75 3700.0±50.02A

T5 3785.0±69.86 3600.0±43.30 3665.0±88.91 3683.3±44.22A

T6 3735.0±86.03 3575.0±95.26 3605.0±76.21 3638.3±49.60A Means ± SE 3780.0±22.24A 3596.7±26.79B 3666.7±32.93B Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

4000

3900

3800

3700

s 3600 C

es 3500

dn B r a C+B H 3400

3300

3200

3100

3000 1 2 3 4 5 6 Treatment

Fig.4.32 Effect of milk types and treatments on hardness (g) of processed Cheddar cheese

190

Table 4.60 Effect of milk types and treatments on cohesiveness of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB

T1 1.039±0.006 1.024±0.002 1.025±0.010 1.029±0.004A

T2 1.041±0.027 1.029±0.020 1.028±0.015 1.033±0.011A

T3 1.044±0.011 1.030±0.026 1.034±0.031 1.036±0.012A

T4 1.045±0.033 1.027±0.014 1.030±0.009 1.034±0.011A

T5 1.043±0.020 1.031±0.015 1.030±0.025 1.034±0.010A

T6 1.062±0.019 1.032±0.020 1.034±0.004 1.043±0.009A Means ± SE 1.046±0.008A 1.029±0.006A 1.030±0.006A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

1.1

1.05

1 s

es

n C

ve i 0.95 B es C+B

oh

C 0.9

0.85

0.8 1 2 3 4 5 6 Treatment

Fig.4.33 Effect of milk types and treatments on cohesiveness of processed Cheddar cheese

191

4.11.3 Springiness of processed Cheddar cheese Springiness is the bouncing property of sample through several consecutive bites (Civille and Szczesniak, 1973). The springiness values for processed Cheddar cheese (table 4.61; Fig. 4.34) showed the non-significant variation among the cheese samples regarding treatments as well as milk types. The mean springiness values were 0.991±0.005 for cow milk cheese, 0.981±0.009 for buffalo milk cheese and 0.989±0.007 for cheese prepared from cow and buffalo mixture. Results of current study are in contrast with the outcomes of Jung et al. (2013), and Awad et al. (2005) showed a decreased springiness with the advancement of ripening time.

Treatment T1 of processed cheese made with 100% (90-day) ripened buffalo milk Cheddar cheese and treatment T1 of processed cheese made with mixture of 100% (90- day) ripened cow and buffalo milk Cheddar showed the highest springiness values than other treatments. Different springiness behavior expressed by processed cheese made from cow, buffalo and mixture of cow and buffalo milk Cheddar might be due to variation in pH and moisture of Cheddar cheese used for processed cheese making. Elasticity of cheese decreases with the progress of ripening because proteolysis breaks down the casein network (Tunick et al., 1990; Hort and Le Grys, 2001). Due to protein degradation availability of free water is reduced resulting in firmness of fat molecules and ultimately springiness is reduced (Tarakci and Temiz, 2009). Texture of cheese is also affected by pH. Solubility of caseins is high at high pH resulting a soft texture (Fathollahi et al., 2010). Increase in moisture contents decreases hardness, springiness and chewiness as explained by Korish and Abd-ElHamid, (2012). 4.11.4 Gumminess of processed Cheddar cheese Gumminess and chewiness are related to cheese hardness (Awad et al., 2013). Gumminess has been defined as the confrontation offered by a cube of cheese during cuddling between thumb and fore finger or resistance offered by cube of cheese during normal mastication (Wium et al., 1997). The mean values (Table 4.62; Fig. 4.35) showed the highest gumminess (3952.1±26.23) in buffalo milk processed cheese while the lowest in cow milk cheese as 3702.5±21.18. Among the treatments, mean values showed non-significant variation in cheese gumminess with the lowest value (3794.7±51.08) in treatment T6. The interaction between milk types and cheese treatments also non-significantly affected the gumminess in processed Cheddar cheese.

192 All types of cheeses presented different behavior with respect to gumminess. Processed cheese prepared from 50% (30-day) and 50% (90-day) ripened buffalo milk Cheddar showed the highest gumminess values while processed cheese manufactured from 50% (30-day) and 50 % (90-day) ripened cow milk Cheddar showed quite low values. The overall results of processed cheese manufactured from buffalo milk Cheddar were high as compared to processed cheese prepared from cow milk Cheddar and the mixture of cow and buffalo milk. Results of current study has been supported by Madadlou et al. (2005), who revealed the fact that fat and moisture act as plaster in casein matrix of cheese texture and provide it softness and lubricity. More over di- phosphates and tri phosphates as used in current research support gel formation which affects gumminess (Mizuno and Lucey, 2007 and Bunka et al., 2013). 4.11.5 Chewiness of processed Cheddar cheese Time required to grind the sample in such a way that it becomes ready for swallowing is called chewiness (Zoon, 1991). Beal and Mittal (2000) explained a correlation between hardness and chewiness by stating “harder cheese is more difficult to chew”. Table 4.63 depicted the significantly higher mean chewiness value (3875.3±27.37) in processed cheese from buffalo milk followed by 3735.0±25.13 and 3670.7±25.86 in mixture of cow + buffalo milk cheese and cow milk processed cheese, respectively. The treatments based on ripening periods showed non-significant differences in mean chewiness values for cheese samples. The interaction between milk type and treatments also showed the similar trend (Fig. 4.36). Chewiness values were high in all treatments having rich amount of 60-day and 90- day ripened cheese which conforms to the studies of Jung et al. (2013), that chewiness increases with passage of ripening time in cheese. Awad et al. (2005) also showed an increase in chewiness of reduced fat Cheddar during ripening. High chewiness values in processed cheese made from buffalo milk Cheddar coincide with findings of Simoes et al. (2013), who declared a decrease in chewiness with decline in cow milk quantity during manufacture of artisanal marajo butter cheese.

193 Table 4.61 Effect of milk types and treatments on springiness of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB

T1 0.990±0.015 0.991±0.009 0.997±0.014 0.993±0.006A

T2 0.977±0.026 0.992±0.016 0.990±0.023 0.986±0.011A

T3 0.984±0.031 1.001±0.008 0.984±0.012 0.990±0.010A

T4 0.983±0.025 0.992±0.018 0.985±0.037 0.987±0.014A

T5 0.970±0.033 0.989±0.012 0.994±0.019 0.985±0.012A

T6 0.979±0.022 0.984±0.020 0.983±0.010 0.982±0.009A Means ± SE 0.981±0.009A 0.991±0.005A 0.989±0.007A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

1.05

1

0.95

s

es 0.9 C

n i B

ng

i r 0.85 C+B Sp 0.8

0.75

0.7 1 2 3 4 5 6 Treatment

Fig.4.34 Effect of milk types and treatments on springiness of processed Cheddar cheese

194 Table 4.62 Effect of milk types and treatments on gumminess of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB

T1 3960.2±45.27 3718.5±24.26 3762.4±03.26 3813.7±40.03A

T2 3950.4±82.11 3711.3±93.42 3772.2±33.98 3811.3±51.68A

T3 3931.1±127.7 3696.9±27.96 3795.9±87.49 3808.0±56.69A

T4 3959.0±38.40 3688.1±75.72 3831.6±109.1 3826.2±55.88A

T5 3946.5±65.62 3709.9±79.25 3775.0±36.62 3810.5±47.32A

T6 3965.2±61.81 3690.4±19.52 3728.7±61.17 3794.7±50.08A Means ± SE 3952.1±26.23A 3702.5±21.18B 3777.6±23.74B Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

4200

4000

3800

s

es C

n

i 3600 B

mm

u C+B

G 3400

3200

3000 1 2 3 4 5 6 Treatment

Fig.4.35 Effect of milk types and treatments on gumminess of processed Cheddar cheese

195 Table 4.63 Effect of milk types and treatments on chewiness of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB

T1 3920.0±62.69 3683.9±79.59 3750.0±115.8 3784.6±56.65A

T2 3860.0±23.94 3680.0±39.73 3735.0±53.25 3758.3±33.54A

T3 3870.0±111.3 3700.0±68.36 3733.4±14.23 3767.8±46.00A

T4 3892.1±86.96 3660.0±91.28 3775.3±42.43 3775.8±50.96A

T5 3830.0±92.87 3670.0±73.41 3752.4±62.09 3750.8±44.97A

T6 3880.0±52.87 3630.0±79.64 3664.1±80.65 3724.7±53.25A Means ± SE 3875.3±27.37A 3670.7±25.86B 3735.0±25.13B Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

4200

4000

3800

s

es C

n i 3600 B w

e

h C+B

C 3400

3200

3000 1 2 3 4 5 6 Treatment

Fig.4.36 Effect of milk types and treatments on chewiness of processed Cheddar cheese

196 4.12 MELT-ABILITY OF PROCESSED CHEESE

Quality of Cheddar and processed cheese is determined for Specific product claims and one of the determining factors for such claims is the melt-ability (Kuo et al., 2000). Melt-ability is term used to specify the range to which melted cheese flows and spreads on heating (Kuo et al., 2001). It reflects the functional quality of cheese and is often defined by the consumers according to their requirement (Dorota and Pikul, 2009). It becomes utmost important when cheese used as ingredient in ready to eat foods like (Farahmandfar et al., 2011). Analysis of variance table 4.64 for melt-ability of processed cheese showed a non- significant (P>0.05) effect of storage period while highly significant (P<0.01) effect of milk types and treatments based on ripening. According to the results of present study the highest mean value (57.96±0.67 mm) of melt-ability was seen in processed cheese prepared from cow milk Cheddar while the lowest mean value (47.33±0.51 mm) was observed in processed cheese from buffalo milk Cheddar. Melt-ability increased non-significantly from 46.78±1.04 mm to 47.61±1.07 mm in 90 days with mean value 47.33±0.51 mm in cheese manufactured from cow milk, 57.50±1.35 mm to 58.17±1.40 mm (0- 90 days) with mean value of 57.96±0.68 mm in cheese from buffalo milk, 52.39±1.19 mm to 52.83±1.28 mm (0-90 days) with mean value of 52.78±0.61 mm in processed cheese from mixture of cow and buffalo Cheddar (Fig. 4.37). Values of melt-ability correspond to the results of Cais-Sokolinska and Pikul (2008), who used test tube method to assess the melt-ability of curd ripened fried cheese and recorded 55.5±0.8 mm to 42.3±2.3 mm but a decrease was observed during storage up to 42 days in contrast to the current study. Olson et al. (2007) declared that during storage, protein is degraded to free amino acids and small peptides consequently the protein matrix is changed. Such change in protein-peptide level can affect the melt-ability and sliceability of Cheddar cheese during storage. Initially Cheddar cheese has firm texture which becomes soft with the development in proteolysis resulting the increase in melt-ability with the passage of time. McSweeney (2007) advocated that Protein-protein interactions bind para-casein fibers

197 together to govern the melt-ability. Cheese becomes tough when these interactions are strong but a soupy appearance is observed when these interactions are weak. Increase in melt-ability of mozzarella cheese prepared from cow and buffalo milk during storage up to 30 days has been reported by El-Alfy et al. (2008). Highly significant (P<0.01) variation between interaction means of cheese types and treatments has been elaborated in table 4.65b. The highest melt-ability values

(67.08±0.38 mm) were observed in treatment T1 of processed cheese prepared from cow milk and the lowest (42.08±0.45 mm) were found in treatment T6 of processed cheese prepared from buffalo milk. Van Hekken et al. (2007) showed an increase in melt-ability in cheese by increasing fat content. Koca and Metin (2004) declared that decrease in fat causes decreased melt- ability. In current study, fat content was almost same in all treatments thus increased melt- ability shown by processed cheese prepared from cow milk is due to the characteristics of cow milk fat that makes cheese less firm and more meltable as discussed by El-Alfy et al. (2008). Age of the cheese plays specific role in determining the amount of emulsifying salts to be added, melting time and cheese quality and safety (Mihaela et al., 2013). High melt- ability values were observed in all treatments having increased amount of 60-90 days ripened cheese in current study. High melt-ability values may also be due to the right amount of emulsifying salts used in the formation of processed cheese. Mihaela et al. (2013) made processed cheese by using different emulsifying salts and declared that the most pronounced effects of melt-ability were produced by di-sodium phosphate (DSP) and tri-sodium phosphate (TSP) as used in the current study. Emulsifying salts play important role in the formation of processed cheese gel network (Chandan and Kapoor, 2011). Phosphate salts when added at the rate of 1% causes casein dispersion appropriate for sufficient melting whereas increase in phosphate salts increases the casein dispersion resulting decreased melting of processed cheese. Some phosphate salts have the crosslinking ability with casein molecules which can also affect melt-ability.

198

Table 4.64 Analysis of variance for melt-ability of processed Cheddar cheese

Source of d.f Sum of squares Mean squares F-value variation

Storage Days (D) 3 22.05 7.35 NS Milk Types (M) 2 4064.90 2.16 2032.45 Treatments (T) 5 4899.63 597.29** 979.93 D × M 6 1.99 287.98** 0.33 NS D × T 15 9.87 0.10 0.66 NS M × T 10 133.60 0.19 13.36 D × M × T 30 30.18 3.93** 1.01 NS Error 144 490.00 0.30 3.40 Total 215 9652.22

NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant

(P<0.01)

199 Table 4.65a Effect of milk types and storage period on melt-ability (mm) of processed Cheddar cheese (Means ± SE)

Milk Storage Days Means ± SE B C CB 0 46.78±1.04 57.50±1.35 52.39±1.19 52.22±0.91A 30 47.33±1.00 57.89±1.39 52.56±1.26 52.59±0.91A 60 47.61±1.05 58.28±1.40 53.33±1.23 53.07±0.92A 90 47.61±1.07 58.17±1.40 52.83±1.28 52.87±0.93A Means ± SE 47.33±0.51C 57.96±0.68A 52.78±0.61B

Table 4.65b Effect of milk types and treatments on melt-ability (mm) of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB

T1 54.00±0.46e 67.08±0.38a 60.00±0.55c 60.36±0.94A

T2 50.00±0.49fg 62.25±0.43b 56.92±0.34d 56.39±0.88B

T3 48.17±0.44h 57.58±0.42d 53.92±0.56e 53.22±0.71C

T4 43.25±0.54j 53.67±0.50e 49.42±0.74gh 48.78±0.80E

T5 46.50±0.31i 57.08±0.51d 51.00±0.48f 51.53±0.77D

T6 42.08±0.45j 50.08±0.47fg 45.42±0.47i 45.86±0.61F Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

200 Table 4.65c Effect of milk types, treatments and storage period on melt-ability (mm) of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB D1T1 53.67±0.88 66.33±0.88 59.33±0.88 59.78±1.88 D1T2 49.33±0.88 61.67±0.88 57.00±0.58 56.00±1.84 D1T3 47.33±1.20 57.33±0.88 53.33±1.20 52.67±1.55 D1T4 43.00±1.15 53.67±0.88 48.33±0.88 48.33±1.62 D1T5 46.00±0.58 56.67±0.88 50.67±0.88 51.11±1.59 D1T6 41.33±0.88 49.33±0.88 45.67±0.88 45.44±1.24 D2T1 54.00±0.58 67.00±0.58 60.00±2.00 60.33±1.98 D2T2 50.00±0.58 62.0±1.15 56.67±0.88 56.22±1.79 D2T3 48.00±0.58 57.67±0.88 54.00±0.58 53.22±1.45 D2T4 43.67±0.88 53.33±0.88 48.33±0.88 48.44±1.46 D2T5 46.33±0.67 57.33±1.45 50.67±1.20 51.44±1.70 D2T6 42.00±1.00 50.00±1.15 45.67±1.45 45.89±1.31 D3T1 54.33±0.67 67.33±0.88 60.33±0.88 60.67±1.92 D3T2 50.33±0.88 62.67±0.88 57.00±0.58 56.67±1.83 D3T3 48.67±0.33 57.67±1.20 54.00±2.00 53.44±1.47 D3T4 43.33±1.67 54.00±1.53 52.33±2.03 49.89±1.87 D3T5 46.67±0.88 57.67±1.45 51.00±1.00 51.78±1.70 D3T6 42.33±0.88 50.33±1.20 45.33±0.67 46.00±1.26 D4T1 54.00±1.73 67.67±0.88 60.33±0.88 60.67±2.07 D4T2 50.33±1.76 62.67±0.88 57.00±1.00 56.67±1.89 D4T3 48.67±1.33 57.67±0.88 54.33±0.88 53.56±1.42 D4T4 43.00±1.15 53.67±1.20 48.67±0.88 48.44±1.63 D4T5 47.00±0.58 56.67±0.67 51.67±1.20 51.78±1.46 D4T6 42.67±1.20 50.67±0.88 45.00±1.15 46.11±1.31 Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

201 B C C+B

80

70

60

50

(mm)

40

ability

-

elt

M 30

20

10

0

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Day 0 Day 30 Day 60 Day 90

Fig.4.37 Effect of milk types, treatments and storage period on melt-ability (mm) of processed Cheddar cheese

202 4.13 PROTEOLYSIS IN PROCESSED CHEESE

Proteolysis is catalyzed by enzymes that are originated from different sources. Those coming from milk are acid phosphatases, lipoprotein lipases, and plasmin and xanthine oxidase. Enzymes are also produced by propionic acid bacteria, bacterium linens strains and moulds, natural milk bacteria and non-starter microflora. Enzymes remained or added in cheese making process like chymocin, pepsin or microbial proteinases also affect proteolysis (Radeljevic et al., 2013). Enzymatic activity depends upon active acidity, redox potential, water activity of cheese mass and ripening environments (Tamime, 2011, Law and Tamime, 2011, Carroll and Hobson, 2012).

Proteolysis in processed cheese prepared from Cheddar cheese of cow, buffalo and mixture of cow and buffalo was determined by assessing the concentration of water soluble nitrogen as percentage of total nitrogen (WSN/TN %) and total free amino acids (TFAA) .

4.13.1 Water soluble nitrogen / total nitrogen (%) (primary proteolysis)

Results of statistical analysis for water soluble nitrogen as percentage of total nitrogen (WSN/TN%) in processed has been elaborated in analysis of variance (ANOVA) table 4.66. Storage days showed a non-significant (P>0.05) effect while cheese types and treatments showed highly significant (P<0.01) effect on WSN/TN (%). Interaction of means between storage days and cheese types, storage days and treatments, cheese types and treatments and the storage days, cheese types and treatments exhibited a non- significant effect on WSN/TN (%) of processed cheese.

The results of WSN/TN (%) table 4.67a showed non-significant variation of WSN/TN (%) during storage up to 90 days at 6ºC. Among milk types the highest mean values (69.07±0.10%) of WSN/TN (%) were seen in cow milk processed Cheddar cheese while lowest (66.58±0.10%) WSN/TN (%) was present in buffalo milk processed cheese. The results of current study are close to the findings of Guinee and O’Kennedy (2009), who noted the WSN/TN (%) in low calcium, medium calcium and high calcium processed Cheddar cheese as 70.9%, 76.3% and 75.1%, respectively.

203 As the concentration of medium ripened or the fresh Cheddar increased in processed cheese treatments, the WSN/TN% decreased. Since, among the treatments the mean highest (68.81±0.19 %) WSN/TN (%) was found in T1 and the lowest (66.97±0.21%) in T6.

In processed cheese, emulsifying salts interrupt the casein matrix of cheese by exhausting casein calcium and colloidal calcium phosphate resulting in solubilization of large portion of casein (Gaucher et al., 2007; Panouille et al., 2004). Addition of water during processed cheese manufacture facilitates the reaction of emulsifying salts with casein matrix and boosts the release of WSN hence increasing the WSN levels in processed cheese as compared to the Cheddar cheese used. High WSN/TN (%) values in processed cheese prepared from cow milk Cheddar cheese are due to high concentration of WSN/TN (%) in cow milk Cheddar cheese used for its manufacture.

Upadhyay et al. (2004) stated that Cheddar cheese possesses the typical pattern of proteolysis, Fenelon and Guinee (2000) declared that ∞S1-CN is hydrolyzed more than β- CN when milk is coagulated by chymocin which is active against∞S1-CN. Hinz et al. (2012) determined that β-CN is hydrolyzed by the native milk proteinase such as plasmin. During proteolysis ∞S1 casein is degraded which is the cause of softness of processed cheese. Increased intensities of intact proteins provide necessary strength to processed cheese by making it little bit harder but more springy and easy to crack (Tamime et al., 2011).

4.13.2 Total free amino acids (secondary proteolysis)

End products achieved during secondary proteolysis of cheese are free amino acids which serve as originators for development of aroma in cheese and ultimately affect the cheese flavor (McSweeney and Sousa, 2000). Free amino acids are converted to volatile flavor components by amino-acid converting enzymes (Smit et al., 2005).

Analysis of variance (ANOVA) table 4.68 reveals the statistical analysis of total free amino acids (TFAA) content of processed Cheddar cheese. According to the analysis, the storage days showed a non-significant (P>0.05) while cheese types (prepared from cow, buffalo and mixture of cow and buffalo milk Cheddar) and

204 treatments exhibited a highly significant (P< 0.01) impact on the total free amino acid content. Interaction between storage days and cheese types, storage days and treatments, cheese types and treatments presented non-significant (P>0.05) differences.

Mean values for TFAA are presented in the tables 4.69 a, b and c. Non-significant variation (P>0.05) in all types of processed cheeses prepared from Cheddar cheese of various milk sources is evident during storage period of 90 days. The concentration of total free amino acids remained almost same from 0-60 days while a non-significant decrease was noted during 60 to 90 days of storage. Total free amino acids decreased from 4015.3±15.68 (µg/g) to 4012.7±16.14 (µg/g) in processed cheese prepared from cow milk Cheddar while, 3926.5±11.16 (µg/g) to 3923.3±12.67 (µg/g) in cheese manufactured from buffalo milk. Overall mean showed a decrease from 3970.8±09.40 (µg/g) at 0 day to 3968.9±09.37 (µg/g) at 90th day of storage.

In comparison of the milk types, the highest mean value for TFAA (4014.90±08.01 µg/g) was observed in cow milk cheese followed by mixture of cow and buffalo milk (3970.7±07.25 µg/g) and buffalo milk processed cheese (3924.6±06.39 µg/g). As the treatments of processed cheese based on Cheddar of varying ripening periods are concerned, the highest mean TFAA value (4022.20±12.12 µg/g) was noted in T1 which gradually decreased as the concentration of medium ripened and fresh Cheddar increased.

The lowest mean TFAA content (3922.80±08.65 µg/g) was found in T6 of processed cheese.

The final products of proteolysis are free amino acids and their concentration depends up on the cheese variety and ripening indices (McSweeney and Fox, 1997). Similar trend as for WSN/TN was found in case of total free amino acids because concentration of the TFAA was higher in all processed cheeses when compared with the TFAA concentration in Cheddar cheeses used for their manufacture. It may be due to the rapid solubilization of larger portions of caseins in the presence of free water and reactions of emulsifying salts resulting in liberation of free amino acids. Such liberated amino acids directly contribute to the flavor development or serve as flavor precursor (Lane et al., 1997).

205 Table 4.66 Analysis of variance for water soluble nitrogen / total nitrogen

(WSN/TN) of processed Cheddar cheese

Source of d.f Sum of squares Mean squares F-value variation

NS Storage Days (D) 3 0.531 0.177 0.54

Milk Types (M) 2 225.790 112.895 344.62**

Treatments (T) 5 89.835 17.967 54.85**

NS D × M 6 0.930 0.155 0.47

NS D × T 15 2.514 0.168 0.51

NS M × T 10 2.947 0.295 0.90

NS D × M × T 30 4.972 0.166 0.51

Error 144 47.173 0.328

Total 215 374.693

NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant

(P<0.01)

206 Table 4.67a Effect of milk types and storage period on water soluble nitrogen / total nitrogen (%) of processed Cheddar cheese (Means ± SE)

Milk Storage Days Means ± SE B C CB 0 66.59±0.18 69.08±0.19 67.93±0.20 67.86±0.18A 30 66.59±0.20 69.07±0.19 67.93±0.21 67.86±0.18A 60 66.56±0.21 69.07±0.21 67.93±0.23 67.85±0.19A 90 66.59±0.20 69.08±0.21 68.26±0.17 67.97±0.18A Means ± SE 66.58±0.10C 69.07±0.10A 68.01±0.10B

Table 4.67b Effect of milk types and treatments on water soluble nitrogen / total nitrogen (%) of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB

T1 67.51±0.09 70.02±0.19 68.91±0.13 68.81±0.19A

T2 67.06±0.23 69.69±0.10 68.66±0.18 68.47±0.21B

T3 66.73±0.07 69.45±0.13 68.10±0.20 68.09±0.20C

T4 66.55±0.09 68.88±0.11 67.74±0.13 67.72±0.17D

T5 66.05±0.20 68.46±0.05 67.28±0.11 67.26±0.18E

T6 65.57±0.15 67.95±0.12 67.39±0.29 66.97±0.21F Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean

207 Table 4.67c Effect of milk types, treatments and storage period on water soluble nitrogen / total nitrogen (%) of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB D1T1 67.51±0.10 70.02±0.48 68.91±0.02 68.81±0.39 D1T2 67.09±0.51 69.69±0.16 68.65±0.55 68.48±0.44 D1T3 66.72±0.17 69.46±0.02 68.11±0.55 68.09±0.43 D1T4 66.56±0.25 68.89±0.02 67.72±0.03 67.72±0.34 D1T5 66.07±0.09 68.46±0.03 67.29±0.02 67.27±0.35 D1T6 65.57±0.25 67.96±0.07 66.91±0.02 66.81±0.35 D2T1 67.52±0.28 70.01±0.05 68.91±0.04 68.81±0.37 D2T2 67.05±0.55 69.69±0.05 68.67±0.19 68.47±0.42 D2T3 66.74±0.23 69.44±0.26 68.09±0.56 68.09±0.43 D2T4 66.56±0.24 68.87±0.53 67.76±0.54 67.73±0.40 D2T5 66.05±0.55 68.45±0.03 67.28±0.01 67.26±0.38 D2T6 65.58±0.24 67.94±0.03 66.88±0.07 66.80±0.35 D3T1 67.50±0.27 70.02±0.55 68.92±0.59 68.81±0.44 D3T2 67.01±0.50 69.68±0.40 68.67±0.56 68.45±0.46 D3T3 66.73±0.00 69.45±0.47 68.10±0.02 68.09±0.41 D3T4 66.52±0.26 68.88±0.07 67.75±0.01 67.72±0.35 D3T5 66.01±0.50 68.46±0.03 67.28±0.44 67.25±0.40 D3T6 65.56±0.56 67.95±0.02 66.88±0.59 66.80±0.42 D4T1 67.52±0.02 70.01±0.48 68.91±0.02 68.82±0.39 D4T2 67.09±0.56 69.69±0.16 68.65±0.19 68.48±0.42 D4T3 66.73±0.14 69.44±0.25 68.11±0.51 68.09±0.43 D4T4 66.54±0.03 68.88±0.06 67.73±0.23 67.72±0.35 D4T5 66.07±0.60 68.47±0.25 67.27±0.25 67.27±0.40 D4T6 65.58±0.28 67.96±0.55 68.88±0.07 67.47±0.52 Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

208 Table 4.68 Analysis of variance for total free amino acids of processed Cheddar

cheese

Source of d.f Sum of Mean squares F-value

variation squares

NS Storage Days (D) 3 102 34 0.01

Milk Types (M) 2 293536 146768 37.78**

Treatments (T) 5 223592 44718 11.51**

NS D × M 6 145 24 0.01

NS D × T 15 584 39 0.01

NS M × T 10 20428 2043 0.53

NS D × M × T 30 976 33 0.01

Error 144 559378 3885

Total 215 1098741

NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant

(P<0.01)

209 Table 4.69a Effect of milk types and storage period on total free amino acids (µg/g) of processed Cheddar cheese (Means ± SE)

Milk Storage Days Means ± SE B C CB 0 3926.5±11.16 4015.3±15.68 3970.5±14.98 3970.8±09.40A 30 3924.7±13.46 4016.0±17.05 3970.0±15.51 3970.2±10.12A 60 3924.0±14.66 4015.7±16.52 3971.5±15.76 3970.4±10.25A 90 3923.3±12.67 4012.7±16.14 3970.8±12.87 3968.9±09.37A Means ± SE 3924.6±06.39C 4014.9±08.01A 3970.7±07.25B

Table 4.69b Effect of milk types and treatments on total free amino acids (µg/g) of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB

T1 3962.3±14.99 4083.0±18.08 4021.3±13.67 4022.2±12.12A

T2 3940.3±16.55 4057.0±16.31 3991.5±18.27 3996.3±12.52AB

T3 3923.8±15.59 4011.0±12.54 3979.8±15.90 3971.5±10.28BC

T4 3908.8±14.07 3988.8±15.87 3949.5±15.64 3949.0±10.16CD

T5 3920.5±13.68 4001.5±15.79 3954.5±16.96 3958.8±10.36C

T6 3892.3±13.52 3948.3±12.83 3927.8±14.85 3922.8±08.65D Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

210 Table 4.69c Effect of milk types, treatments and storage period on total free amino acids (µg/g) of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB D1T1 3962.0±19.67 4086.0±52.13 4020.0±29.47 4022.7±25.53 D1T2 3945.0±30.29 4052.0±35.09 3995.0±44.74 3997.3±24.18 D1T3 3926.0±32.87 4015.0±25.50 3978.0±40.42 3973.0±21.14 D1T4 3910.0±22.35 3990.0±25.34 3952.0±38.56 3950.7±18.77 D1T5 3926.0±36.94 3997.0±27.69 3955.0±47.49 3959.3±21.72 D1T6 3890.0±22.46 3952.0±29.43 3923.0±14.95 3921.7±14.60 D2T1 3968.0±27.80 4088.0±45.35 4026.0±36.03 4027.3±25.38 D2T2 3939.0±40.71 4055.0±29.50 3987.0±4.15 3993.7±22.25 D2T3 3922.0±17.44 4009.0±28.70 3982.0±39.54 3971.0±19.74 D2T4 3908.0±37.68 3990.0±46.07 3949.0±39.90 3949.0±23.83 D2T5 3918.0±42.48 4006.0±45.56 3951.0±56.11 3958.3±27.39 D2T6 3893.0±39.69 3948.0±28.42 3925.0±38.07 3922.0±19.57 D3T1 3959.0±58.06 4080.0±23.32 4019.0±43.39 4019.3±28.07 D3T2 3937.0±32.28 4061.0±52.75 3994.0±48.42 3997.3±28.90 D3T3 3926.0±50.77 4012.0±32.43 3982.0±38.97 3973.3±24.24 D3T4 3909.0±43.56 3991.0±47.24 3946.0±32.81 3948.7±23.96 D3T5 3919.0±5.88 4004.0±27.74 3956.0±11.88 3959.7±15.17 D3T6 3894.0±27.66 3946.0±25.29 3932.0±55.17 3924.0±20.76 D4T1 3960.0±19.66 4078.0±42.85 4020.0±6.73 4019.3±21.89 D4T2 3940.0±48.88 4060.0±30.71 3990.0±54.40 3996.7±28.76 D4T3 3921.0±37.04 4008.0±30.31 3977.0±28.93 3968.7±20.56 D4T4 3908.0±23.24 3984.0±23.00 3951.0±34.90 3947.7±17.65 D4T5 3919.0±29.87 3999.0±42.94 3956.0±27.86 3958.0±20.65 D4T6 3892.0±34.21 3947.0±36.00 3931.0±10.21 3923.3±16.76 Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

211 4.14 COLOR OF PROCESSED CHEESE

Color is an imperative sensual element of dairy products that provides vital evidences of the essential reactions taking place in food items particularly processed foods. Color grows due to the innate chemical reactions in many foods (Vyawahare et al., 2013). The first sensation that consumer identifies and uses as tool to accept or reject the product is the color (Gokmen and Sugut, 2007). Milk fat and its color are the determinants of the cheese color (Carpino et al., 2004). Variations in the amount and the assembly of the skim phase of cheese affect the intensity of light absorption of cheese (Rudan et al., 1998). Difference in cheese color may be due to the addition of color and vegetables. Natural colorants added into milk and milk products consequently increase the a* and b* values in the resultant products (Petersn et al., 1999). The types of emulsifying salts significantly influence the cheese color. The final color of the cheese depends upon the size of the fat particles and the fat content distributed in the protein matrix (Chen and Liu, 2012). Results for the color analysis of processed Cheddar cheese manufactured from fully ripened, semi ripened and un ripened Cheddar cheese prepared from cow and buffalo milk are given in analysis of variance (ANOVA) table 4.70. Mean squares for milk sources showed significant (P<0.05) difference for lightness (L*) and highly significant (P<0.01) difference between redness (a*) and yellowness (b*). Treatments also showed a highly significant (P<0.01) effect for L*, a* and b* however interaction between milk sources and treatments showed non-significant difference for L* and b. 4.14.1 Lightness (L*) value of color In cheese, riboflavin acts as photosensitizer and may cause increase in L* values along with the carotenoids but in case of Cheddar cheese, it is photo-oxidized (Deger and Ashoor, 1987; Trobetas et al., 2008). Results (table 4.71; Fig. 4.38) showed the significant (P<0.05) difference was recorded in mean lightness (L*) values of cow milk cheese (84.63±0.36), buffalo milk cheese (83.68±0.32) and cow+ buffalo milk cheese (83.45±0.32). The highest L* value

(85.81±0.59) was noted for treatment T6 of cow milk cheese and the lowest value

(81.73±0.71) was found in treatment T1 of buffalo milk cheese.

212 Results of L* values in the current study are very close to the findings of Rinaldoni et al. (2014), who prepared soft cheese like product and found L* values 84.99 in the control treatment. Simoes et al. (2013) prepared cheese with 100% buffalo milk and a combination of cow+ buffalo milk and found L* values as 88.67 ± 4.90 and 84.85 ± 4.96, respectively.

The highest values of color (L*) observed in cow milk treatment T6 are due to the high amount of 0 day ripened cheese (50%) as compared to the T1 of buffalo milk cheese having 100% fully ripened (90 day) cheese. This phenomenon has been confirmed by Jung et al. (2013), who concluded that L* value significantly decreases resulting color changes with the progress of ripening in cheese. The L* (Lightness) value of the processed cheese decreases with passage of time even at low temperature up to 5ºC and is independent to light exposure (Kristensen et al., 2001). The composition of riboflavin and carotenoids is better in cow than buffalo milk is also the cause of high L* values for cow milk cheese (Rohm and Jaros, 1997; Trobetas et al., 2008). High L* values may also be due to sodium citrate used in all treatments. Chen and Liu (2012) demonstrated the processed cheese prepared with tri-sodium citrate were whiter and less yellow as compared to the processed cheeses prepared with other E- Salts. McClements (2005) stressed that more whiter product is obtained when there is large number of small fat particles and the smallest fat particle size was observed in cheese having di-Sodium phosphate (Chen and Liu, 2012). 4.14.2 Redness (a*) value for color a* value (Red-Green) is temperature dependent and increases with the increase in storage temperature and also influenced by light exposure being increases with exposure to light (Kristensen et al., 2001). Mean redness (a*) values for processed Cheddar cheese prepared from cow and buffalo milk are given in table 4.72. Highly significant difference (P<0.01) was noted for Color (a*) redness with mean value of 1.359±0.171 in buffalo milk, 0.578±0.055 in cow milk and 0.737±0.046 in cow + buffalo milk cheese. Treatment T1 of buffalo milk processed Cheddar cheese exhibited the highest a* value (2.803±0.090) while the lowest value (0.253±0.027) was recorded in treatment T6 of cow milk processed Cheddar cheese (Fig. 4.39). Results of current study are very close to the findings of Jung et al. (2013),

213 who found a* redness values in cheese between 2.21 to 3.50 during six months of ripening. Buffalo milk processed Cheddar cheese showed higher values of redness (a*) in all treatments as compared to cow milk and mixture of buffalo and cow milk processed Cheddar cheese. Increase in redness (a*) value during ripening was noted by Kesenkas et al. (2012) and Jung et al. (2013) which is evidence of increased redness (a*) values in treatments having high amount of ripened cheese used in the manufacture of processed Cheddar cheese. High redness (a*) observed in buffalo milk is due to the presence of blue-green pigment (biliverdin) which is absent in cow milk (Abd El-Salam and El- Shibiny, 2011). 4.14.3 Yellowness (b*) value of color The mean yellowness (b*) values of processed cheese prepared from cow, buffalo and a mixture of cow and buffalo milk Cheddar cheese are presented in table 4.73. The highest value for yellowness (13.11±0.18) was recorded in cow milk cheese followed by 12.33±0.21 in mixture of cow and buffalo milk and 11.95±0.22 in buffalo milk Cheddar cheese. Among the treatments, the highest yellowness (b*) was present in T6 as 13.19±0.13 and the lowest in T1 as 10.90±0.25 (Fig. 4.40). Results of current study for yellowness (b*) values are very close to the conclusions of Cardenas et al. (2014), who found yellowness (b*) values within range of 10.75± 0.17 to 10.92±0.34 in probiotic cheese. Buffa et al. (2001) declared that yellowness (b*) increases during ripening which supports the increased yellowness (b*) values in processed cheese treatments having more amount of 60 and 90 day ripened Cheddar cheese in the study. Overall increased yellowness (b*) as observed in cow milk processed cheese is due to the presence of increased amount of β-carotene in cow milk which causes increased yellow chromaticity in the products as mentioned by Simoes et al. (2013).

214 Table 4.70 Analysis of variance for color of processed Cheddar cheese

Source of d.f Mean squares variation L* a* b*

Milk Types (M) 2 7.058* 3.06449** 6.3333**

Treatments (T) 5 8.798** 1.36979** 6.5277**

NS NS M × T 10 0.625 0.33539** 0.1263

Error 36 1.407 0.00814 0.1140

NS = Non-significant(P>0.05); *= Significant (P<0.05); ** = Highly significant (P<0.01)

215 Table 4.71 Effect of milk types and treatments on color (lightness L*) of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB

T1 81.73±0.71 82.37±0.56 82.35±0.97 82.15±0.40B

T2 83.93±0.56 83.99±0.63 83.34±0.99 83.75±0.39AB

T3 83.73±0.75 84.69±0.60 83.21±0.56 83.88±0.39A

T4 83.60±0.35 85.46±0.55 83.35±0.83 84.14±0.45A

T5 84.50±0.75 85.46±0.84 83.66±0.60 84.54±0.45A

T6 84.59±0.57 85.81±0.59 84.78±0.57 85.06±0.35A Means ± SE 83.68±0.32AB 84.63±0.36A 83.45±0.32B Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

87

86

85

84

83 C

L* B 82 C+B

81

80

79

78 1 2 3 4 5 6 Treatment

Fig.4.38 Effect of milk types and treatments on color (lightness L*) of processed Cheddar cheese

216 Table 4.72 Effect of milk types and treatments on color (redness a*) of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB

T1 2.803±0.090a 0.917±0.059c-f 1.087±0.064c 1.60±0.303A

T2 1.633±0.090b 0.773±0.045d-h 0.757±0.062d-h 1.054±0.149B

T3 1.027±0.033cd 0.587±0.052ghi 0.717±0.054e-h 0.777±0.069C

T4 0.977±0.030cde 0.527±0.049g-j 0.703±0.032e-h 0.736±0.068C

T5 0.917±0.045c-f 0.413±0.026ij 0.643±0.035f-i 0.658±0.075C

T6 0.797±0.049d-g 0.253±0.027j 0.513±0.038hij 0.521±0.081D Means ± SE 1.359±0.171A 0.578±0.055C 0.737±0.046B Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

3.5

3

2.5

2 C

a* B 1.5 C+B

1

0.5

0 1 2 3 4 5 6 Treatment

Fig.4.39 Effect of milk types and treatments on color (redness a*) of processed Cheddar cheese

217 Table 4.73 Effect of milk types and treatments on color (yellowness b*) of processed Cheddar cheese (Means ± SE)

Milk Treatment Means ± SE B C CB

T1 10.24±0.15 11.81±0.13 10.65±0.19 10.90±0.25D

T2 11.42±0.25 13.04±0.48 11.92±0.02 12.13±0.29C

T3 12.12±0.08 13.37±0.24 12.61±0.02 12.70±0.19B

T4 12.76±0.13 13.53±0.28 12.94±0.03 13.08±0.15AB

T5 12.28±0.15 13.36±0.23 12.72±0.05 12.79±0.17AB

T6 12.88±0.08 13.58±0.26 13.12±0.07 13.19±0.13A Means ± SE 11.95±0.22C 13.11±0.18A 12.33±0.21B Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

16

14

12

10 C

8 B b* C+B 6

4

2

0 1 2 3 4 5 6

Treatment

Fig.4.40 Effect of milk types and treatments on color (yellowness b*) of processed Cheddar cheese

218 4.15 SENSORY EVALUATION OF PROCESSED CHEDDAR CHEESE

Quality judgment by consumers has undergone changes from the concept of quality of produce to the quality of production process (Rijswijk et al., 2008). Espejel et al. (2009) declared that product knowledge has become fundamental in decision making process by consumers. Sensory properties play vital role in product differentiation and different groups of consumers (older, educated, wealthy etc.) show different behavior towards product acceptability (Braghieri et al., 2014). Sensory perception is often used to assess the consumer liking and dis-liking (Drake et al., 2006). Finished products such as traditional cheeses are mostly categorized by their sensory possessions (SISS, 2012). Barcenas et al. (2004) acknowledged that cheeses are mostly identified by their definite sensory aspects. Sensory examination is grounded on the use of trained panels to measure the sensory stuffs of food (Stone et al., 2012). Descriptive sensory analysis and multivariate method has been found suitable to explore sensory variances mounting from the numerous treatments (Hannon et al., 2005). This part of the study was conducted to evaluate the effect of ripening periods and emulsifying salts on sensory quality of processed Cheddar cheese prepared from Cheddar cheese of cow, buffalo and mixture of cow and buffalo milk through descriptive analysis. Processed cheese was assessed for descriptive sensory characteristics at 0 and 90 days of storage by a panel of 10 trained evaluators. The evaluators established a descriptive sensory language and scores were given from total of 100 for each parameter (odor, flavor and texture). The descriptive sensory analysis was supported with multivariate statistical analysis to provide a useful contrivance in judging the sensory changes ascending from different treatments 4.15.1 Flavor of processed cheese Flavor is the combined effect of taste, smell and chemical irritation (Keast et al., 2004). Taste is stimulated by non-volatile sugars and acids; smell is motivated by volatile aromas and chemical irritation by produced gases. Such amalgamation between taste, smell and chemical irritation guarantees the prospect of interactions between senses (Keast and Breslin 2003). Quality and taste of processed cheese hangs on upon the age of cheese that has been used for making it (Tamime et al., 2011). Flavor is one of three leading sensory stuffs that decide selection, acceptance and ingestion of food (Hassan et al., 2013).

219 Analysis of variance (ANOVA) table 4.74 showed the statistical analysis of scores achieved by the different traits of processed Cheddar cheese. Highly significant (P<0.01) difference in all flavor attributes observed with respect to milk sources while storage periods showed non-significant (P>0.05) variation on “creamy”, “bitter”, “salty”, “sweet”, “sour” and “smokey” attribute however “rancid” and “soapy” attributes were highly significantly (P<0.01) affected during storage up to 90 days. Treatment means exhibited highly significant (P<0.01) disparity for “creamy” and “rancid” flavor attribute, significant (P<0.05) inequality for “sweet”, “sour”, “soapy” and “smokey” flavor element and non- significant deviation for “bitter” and “salty” flavor aspect. The interaction between storage period and milk sources as well as storage period and treatments unveiled non-significant (P>0.05) difference for all flavor characteristics. Sensory scores attained by various treatments of processed Cheddar cheese are given in the table 4.75. Processed cheese prepared from Cheddar cheese of buffalo milk gained the highest mean sensory flavor scores (9.44) followed by scores of processed cheese made from mixture of cow and buffalo milk Cheddar cheese (9.40) and the lowest scores (9.27) were achieved by processed cheese from cow milk Cheddar. On the whole “creamy”, “salty”, “sweet” and “sour” attributes of flavor gained the higher scores than other parameters. All flavor attributes showed a slight increase in sensory scores from 0 to

90 day of storage. Treatment T1 (having 100% 90-day ripened cheese) gained highest score in all types of processed cheeses which is indication of development of cheese flavor with the passage of time. Some researchers consider creaminess as flavor attribute while others classify it as texture attribute. Anyhow it involves several senses and in liquid and semi-solid dairy products, it is much influential on textural properties. Tournier et al. (2007) concluded creaminess is involved in texture and pleasantness. Though Cheddar cheese used for processed cheese making (in current study) was made after standardization of milk fat at 3%, the high “creaminess” scores in buffalo milk might be due to buffalo milk fat characteristics since Fenelon et al. (2000) correlated the fat content of Cheddar cheese to creamy, buttery and caramel flavor. Saint-Eve et al. (2009) also concluded that fat content affects both sensory and textural properties of cheese. Drake et al. (2001) defined nutty flavor as “(nonspecific) nut-like aromatic associated with nuts”. Several compounds like ketones, lactones, esters, alcohols, aldehydes, free fatty acids, free amino acids and salts have been considered to be associated with the production of nutty flavor but still it is not clear whether one or combination of these compounds is involved for nutty flavor production. Rychlik and Bosset (2001a) and Avsar et al. (2004) declared that for Cheddar type cheese, acetic and

220 propionic acid are involved in nutty flavor development. High scores of nutty flavor in processed cheese made from buffalo milk Cheddar cheese are due to the higher amount of acetic acid and propionic acid present in buffalo milk Cheddar as compared to cow milk. Bitterness is customarily treated as defect in Cheddar cheese usually perceived as after taste (Suriyaphan et al., 2001). Sweetness is inversely related to bitterness as demarcated by Drake et al. (2001). Non-starter lactic acid bacteria are mainly involved in producing bitterness. Less bitterness observed in processed cheese manufactured from Cheddar cheese of buffalo milk is verified by the outcomes of Murtaza et al. (2014c), who clarified that reduction of bitterness prevails with the addition of salt in Cheddar cheese made from buffalo milk. Reduced salt concentration reduces the aggregation of caseins and increases casein hydrolysis which amplifies bitterness (Guinee and O’Kennedy, 2007). Coagulant (rennet, chymocin etc.) is often involved in the production of bitter peptides in cheese. Casein structures are hydrophobic but when expunged by proteinases can produce bitterness (Hassan et al., 2013). Unsaturated fatty acids produce rancid flavors when oxidized (Allred et al., 2006). Animal diet play important role in the development of oxidized and rancid flavor. Cheddar cheese manufactured from milk of cows fed on hay and supplemented with concentrate having calcium salts of palm and fish oil developed oxidized and rancid flavor during 30 and 90 day ripening. Rancid flavor attribute attained the second lowest score and was more in processed cheese made from Cheddar cheese of buffalo milk which might be due to the diet consumed by animals. Free fatty acids resulting from the lipolytic changes cause rancid flavor. Sodium serves as stimulus for salty taste due to its exclusive sodium-specific transduction mechanism concerning epithelial sodium channels (ENaCs) on the taste receptor cells (Chandrashekar et al., 2010). The highest sensory scores were awarded to the salty flavor trait of all types of processed cheeses in the present study, might be due to salt and extra sodium source included in the form of emulsifying salts. Sourness was observed more in processed cheese of buffalo milk Cheddar as compared to cow milk might be due to more lactic acid produced in buffalo milk. Lactic acid promotes acidic taste in cheese and when present in greater amounts can render cheese with sour taste (Hassan et al., 2013).

221 Table 4.74 Mean sum of squares for flavor (sensory scores) of Cheddar cheese

SOV d.f Creamy Rancid Bitter Salty Sweet Sour Soapy Smokey

NS NS NS NS NS NS Storage Days (D) 1 1.1000 1.77803** 0.04947 1.6013 0.02147 0.01213 2.517** 0.0514

Milk (M) 2 88.8200** 1.40103** 1.67463** 10.9611** 1.35025** 1.53346** 102.905** 84.5613**

NS NS Treatments (T) 5 3.7828** 0.28076** 0.06988 0.1828 0.22062* 0.18567* 0.183* 0.1418*

NS NS NS NS NS NS NS NS D × M 2 0.3969 0.06603 0.00487 0.0252 0.00758 0.00063 0.010 0.0058

NS NS NS NS NS NS NS NS D × T 5 0.2714 0.00223 0.00324 0.0234 0.00292 0.00973 0.030 0.0032

NS NS NS NS NS NS NS M × T 10 1.3250** 0.00853 0.00511 0.0125 0.01197 0.01054 0.013 0.0044

NS NS NS NS NS NS NS NS D × M × T 10 0.2840 0.00393 0.00202 0.0091 0.00381 0.00205 0.005 0.0023

Error 324 0.3720 0.02495 0.04686 0.4547 0.09698 0.07104 0.070 0.0561

Total 359

** = Highly significant (p<0.01) * = Significant (p<0.05) NS = Non-significant (p>0.05)

222 Table 4.75 Mean sensory scores for flavor of Cheddar cheese

Storage Cow Milk Buffalo Milk Cow + Buffalo Milk Flavor Means Days T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 0 17.70 17.40 16.90 16.70 17.30 16.65 19.20 18.90 18.82 18.79 18.92 18.72 18.52 18.40 18.32 18.30 18.45 18.25 18.12±0.071 Creamy 90 17.82 18.47 16.94 16.80 17.35 16.72 19.25 18.92 18.86 18.85 18.93 18.70 18.52 18.46 18.37 18.39 18.56 18.32 18.24±0.073 0 4.10 4.02 3.98 3.92 4.00 3.85 4.30 4.28 4.24 4.18 4.26 4.15 4.22 4.18 4.15 4.10 4.20 4.08 4.12±0.013 Rancid 90 4.28 4.22 4.18 4.11 4.19 4.03 4.46 4.32 4.30 4.28 4.38 4.25 4.35 4.32 4.28 4.25 4.34 4.20 4.26±0.014 0 7.52 7.48 7.45 7.45 7.50 7.40 7.30 7.25 7.22 7.20 7.28 7.20 7.36 7.32 7.28 7.31 7.35 7.28 7.34±0.017 Bitter 90 7.55 7.50 7.50 7.48 7.53 7.45 7.33 7.30 7.28 7.22 7.26 7.21 7.35 7.30 7.32 7.33 7.36 7.30 7.36±0.018 0 20.21 20.12 20.03 19.95 20.10 19.90 19.52 19.45 19.40 19.38 19.44 19.35 19.85 19.82 19.80 19.75 19.81 19.71 19.76±0.049 Salty 90 19.95 19.93 19.90 19.86 19.91 19.85 19.36 19.33 19.28 19.35 19.32 19.30 19.70 19.68 19.65 19.60 19.67 19.55 19.62±0.054 0 9.82 9.74 9.71 9.68 9.75 9.64 9.99 9.95 9.92 9.88 9.95 9.80 9.91 9.88 9.85 9.84 9.89 9.76 9.83±0.024 Sweet 90 9.86 9.70 9.70 9.71 9.74 9.64 10.03 10.00 9.98 9.92 9.94 9.83 9.89 9.90 9.90 9.86 9.91 9.74 9.85±0.023 0 8.40 8.34 8.26 8.28 8.34 8.22 8.58 8.55 8.51 8.50 8.54 8.46 8.52 8.49 8.47 8.45 8.48 8.40 8.43±0.019 Sour 90 8.42 8.38 8.30 8.30 8.35 8.16 8.60 8.61 8.55 8.50 8.55 8.43 8.50 8.50 8.51 8.46 8.48 8.40 8.44±0.022 0 5.21 5.18 5.13 5.09 5.17 5.05 3.40 3.36 3.28 3.25 3.37 3.21 4.10 4.08 4.12 4.10 4.10 4.02 4.18±0.059 Soapy 90 5.40 5.40 5.35 5.30 5.30 5.20 3.55 3.50 3.52 3.45 3.40 3.35 4.28 4.30 4.30 4.25 4.20 4.18 4.35±0.060 0 2.20 2.18 2.12 2.10 2.16 2.05 3.90 3.84 3.80 3.79 3.85 3.75 3.10 3.08 3.05 3.00 3.07 2.97 3.00±0.053 Smokey 90 2.24 2.20 2.16 2.10 2.18 2.10 3.92 3.85 3.82 3.80 3.80 3.79 3.12 3.10 3.10 3.06 3.10 3.00 3.02±0.055 9.42 9.39 9.23 9.18 9.30 9.12 9.54 9.46 9.42 9.40 9.45 9.34 9.46 9.43 9.40 9.38 9.44 9.32 Means 9.27 9.44 9.40

223 The lowest flavor score was awarded to smokiness in all types of processed cheeses manufactured in the current study as it develops very slowly. Young et al. (2004) detected cooked / milky flavor in one, two and four month ripened Cheddar cheese. Young un- developed cooked flavor was sensed by a panel through descriptive sensory analysis of seven and nine month old cheese during studies by Caspia et al. (2006). 4.15.2 Odor of processed cheese Most of the odors we distinguish daily are combination of multiple odorous constituents, all donating to the overall smell. Odors originate from different chemical reactions and products can be designed to cover up smell but can’t be eliminated. Breakdown and transformation of chemical compounds directly affect the sensory evaluation (Fox et al., 2000a). Generally pleasant and un-pleasant odors prevail in foods and are processed differently (Bensafi et al., 2002). Increase in understanding with odors, increases the ability to name different odors (Homewood and Stevenson, 2001). Statistical analysis of odor attributes of processed Cheddar cheese is given in the table 4.76. Results revealed that storage time (0-90 days) has non-significant (P>0.05) effect on “rancid” and “sweet” odor characteristic whereas significant (P<0.05) effect on “creamy” and “pungent” odor trait. “Nutty” and “sour” attributes were highly significantly (P<0.01) affected by storage time. Milk types presented a highly significant (P<0.01) effect on all odor attributes. Interaction between milk types and storage days, treatments and storage days and milk types and treatments exhibited a non-significant (P>0.05) effect on the all odor attributes of processed cheese. The mean sensory scores obtained by odor features for all treatments are given in the table 4.77. The highest sensory scores were awarded to the “creaminess” in treatment

T1 of processed cheese prepared from 100% (90-day ripened) buffalo milk Cheddar cheese as 26.00, followed by 25.00 in T5 of processed cheese prepared from buffalo milk Cheddar and T1 of processed cheese prepared from mixture of cow and buffalo milk Cheddar (90- day ripened) cheese. “Creaminess” increased significantly during storage up to 45 days. After “creaminess”, the highest scores were awarded to “sweet” trait being highest (19.10) in treatment T1 of processed cheese made from Cheddar cheese (90-day ripened) of buffalo milk. “Sweetness” decreased non-significantly in all treatments during storage period. Cheese ripening seemed to have significant effect on all treatments

224 of processed cheese as highest scores for all odor attributes were attained by Treatment T1( having 90-day ripened cheese) in all types of processed cheeses prepared in current study. Overall least scores were awarded to “rancid” and “nutty” odor traits in all treatments and processed cheese prepared from Cheddar cheese of buffalo milk was more “rancid” and less “nutty” than processed cheese made from Cheddar of cow milk and mixture of cow and buffalo milk. Processed cheese prepared from mixture of cow and buffalo milk Cheddar gained overall high scores as compared to process cheese prepared from Cheddar cheese of cow or buffalo milk separately. This difference is due to variation in composition of cow and buffalo milk as buffalo milk is wealthier in fat, lactose, protein and minerals than cow and the capability of milk to be acidified is higher for buffalo than cow milk (Ahmad et al., 2008). In Cheddar cheese, biochemical reactions like lipolysis and proteolysis produce odors. Free fatty acids along with volatile compounds and products from proteolysis cycle directly affect the cheese smell and taste (Sousa and McSweeney, 2001). A variety of cheese aroma compounds is formed from lipid degradation (Sympoura et al., 2009). Fresno et al. (2014) made cheese by using rennet paste of the young lamb and noticed high concentration of short chain free fatty acids resulted strong sensory attributes, “pungent” odor and intense flavor. “Pungent” odor may be due to the occurrence of pregastric lipase in the rennet paste as discussed by Pirisi et al. (2007); Addis et al. (2008) and Ferrandini et al. (2012). Caspia et al. (2006) also observed “pungent” odor in twelve month ripened Cheddar cheese. Esposito et al. (2014) studied the effect of grazing animals on pastures and confinement and concluded that a low intensity of butter and smoked odor was observed in cheese made from milk of animals kept on pasture as compared with cheese from confined animals. 4.15.3 Texture of processed cheese Texture discernment of food product is very critical for its acceptability (Engelen et al., 2003, Vliet and Primo-Martin, 2011 Ishihara et al., 2011). Hence, proper knowledge of texture is necessary to develop products to attract consumers (Ares et al., 2010; Galmarini et al., 2013) .When texture is assumed as sensory property, it gives proper direction of quality aspects. Szczesniak (2002) defined texture as sensory and

225 functional expression of physical, mechanical and surface properties of food sensed through the senses of vision, hearing, touch and kinesthetic. Tongue activities, Mouth temperature and saliva composition are the main factors which effecting texture perception in mouth by flouting down the food (Salles et al., 2011). Statistical analysis of scores awarded to the sensory texture attributes of the processed cheese is presented in table 4.78. Storage period of processed cheese showed non-significant (P>0.05) change in all texture parameters (“crumbly”, “firmness”, “grainy”, “mouth-coating”, “maturity” and “pasty”) from 0-90 days of storage. Milk types revealed highly significant (P<0.01) difference in all texture attributes of all types of processed cheeses prepared in current study. Treatments showed significant (P<0.05) variation for “crumbly” and “grainy” character whereas non-significant (P>0.05) change for other texture elements. Interaction of means for storage days and milk types, storage days and treatments and milk types and treatments exhibited non-significant (P>0.05) variability of all texture aspects. Sensory scores awarded to processed cheese texture characteristics given in the table 4.79 revealed that processed cheese manufactured from Cheddar cheese of buffalo milk attained the highest sensory texture scores 18.76 followed by processed cheese made from mixture of cow and buffalo milk (17.55) and cheese made from cow milk Cheddar (16.67). “Firmness” gained maximum sensory scores (26.80) in processed cheese treatment

T1 prepared from buffalo milk Cheddar cheese of 90-day maturity followed by “pasty” (high in processed cheese made from Cheddar cheese of cow milk with 90-day maturity) and “mouth-coating” (high in processed cheese prepared from Cheddar cheese of buffalo milk with 90-day maturity). Non-significant increase was observed in “firmness”,” crumbly”, “mouth-coating” and “maturity” traits whereas non- significant decrease seen in “pasty” and “grainy” texture elements during storage up to 90 days. Proteolysis is the most significant one and plays a dynamic part in the development of texture (Sousa et al., 2001). A well-balanced breakdown of the curd protein, mainly casein, into small peptides and amino acids is essential in progress of suitable cheese texture (Visser, 1993). Primary degradation of major caseins; for

226 example, αs1-caseins (Singh et al., 2003) and demineralization of the casein micelles has major consequences for cheese texture (Pastorino et al., 2003). Ripening of the cheese has positive effects on firmness being increased with increased ripening time which is evident from current study, since processed cheese having 90-day mature Cheddar cheese was more firm than other treatments (Murtaza et al., 2013). Aminifar et al. (2010) studied sensory properties of feta cheese and disclosed that in 2nd month of storage, due to proteolysis, big molecules of caseins and oligopeptides are converted into small peptides and amino acids causing softer texture of cheese during this time span. Metabolic processes are responsible for flavor and texture changes during ripening (Smit et al., 2005). Papetti and Carelli, (2013) noticed gradual decrease in the grainy and rubbery texture attributes with the progress of ripening time and increase in the firmness which corresponds to the results of current study. Rehman et al. (2000) determined that ripening period considerably inclined "firmness”, “rubbery” and “grainy” textures and “mouth-coating” properties of Cheddar cheese. Maturity parameter of sensory texture was similar in both cases (cow and buffalo milk Cheddar cheese) during ripening in studies by Murtaza et al. (2013). Pasty parameter relates to mouth feel and is explained by resistance to tongue movements. Pasty behavior of cheese decreases with the ease of ripening especially at high temperature as deliberated by Murtaza et al. (2013). Similar decrease in “pasty” trait was observed in all types of processed cheeses prepared in the current study. Caspia et al. (2006) also found that firmness and crumbliness of cheese changed significantly during aging from one to twelve months.

227

Table 4.76 Mean sum of squares for odor (sensory scores) of Cheddar cheese

SOV d.f Creamy Rancid Pungent Nutty Sweet Sour

NS NS Storage Days (D) 1 5.620* 0.09637 0.6682* 18.0052** 1.6714 1.7222**

Milk (M) 2 116.730** 3.68255** 13.4749** 1.6602** 25.7962** 25.0917**

NS Treatments (T) 5 59.234** 1.39301** 0.2957* 0.1892* 0.4447 0.3483**

NS NS NS NS NS NS D × M 2 0.209 0.00283 0.0136 0.0135 0.0227 0.0406

NS NS NS NS NS NS D × T 5 1.792 0.00083 0.0015 0.0105 0.0155 0.0064

NS NS NS NS NS NS M × T 10 1.403 0.14735 0.0117 0.0025 0.0356 0.0078

NS NS NS NS NS NS D × M × T 10 0.375 0.00130 0.0063 0.0031 0.0118 0.0011

Error 324 1.383 0.08552 0.1025 0.0682 0.4788 0.0439

Total 359

** = Highly significant (p<0.01) * = Significant (p<0.05) NS = Non-significant (p>0.05)

228

Table 4.77 Mean sensory scores for odor of Cheddar cheese

Storage Cow Milk Buffalo Milk Cow + Buffalo Milk Odor Means Days T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 0 23.50 22.50 21.50 22.00 23.00 21.00 26.00 24.00 23.50 23.00 25.00 22.50 25.00 24.00 23.50 24.00 24.50 22.50 23.39±0.111 Creamy 90 24.00 23.00 22.00 21.50 23.00 21.50 26.00 24.50 24.00 23.00 24.50 23.00 25.50 24.50 24.00 23.50 25.00 23.00 23.64±0.137 0 3.00 2.75 2.50 2.65 2.85 2.35 3.15 2.90 2.80 2.85 3.00 2.75 3.10 3.05 3.00 3.05 3.12 2.90 2.88±0.020 Rancid 90 3.03 2.80 2.53 2.70 2.89 2.38 3.19 2.96 2.84 2.88 3.03 2.78 3.16 3.06 3.03 3.05 3.13 2.92 2.91±0.031 0 8.13 8.08 8.05 8.05 8.10 8.00 7.50 7.40 7.37 7.38 7.41 7.25 7.84 7.77 7.73 7.72 7.80 7.62 7.73±0.029 Pungent 90 8.27 8.18 8.12 8.10 8.16 8.08 7.57 7.51 7.50 7.50 7.58 7.31 7.90 7.85 7.80 7.78 7.85 7.71 7.82±0.033 0 3.40 3.34 3.31 3.30 3.38 3.26 3.15 3.09 3.05 3.05 3.12 3.01 3.28 3.25 3.21 3.20 3.27 3.18 3.21±0.012 Nutty 90 3.82 3.80 3.78 3.65 3.84 3.70 3.62 3.56 3.52 3.50 3.62 3.48 3.75 3.70 3.67 3.60 3.72 3.57 3.66±0.026 0 18.20 18.09 18.00 17.95 18.15 17.80 19.10 18.92 18.98 18.95 19.04 18.85 18.62 18.50 18.45 18.51 18.58 18.43 18.51±0.054 Sweet 90 18.05 17.98 17.85 17.88 18.00 17.78 18.88 18.90 18.88 18.78 18.88 18.70 18.45 18.34 18.30 18.34 18.40 18.28 18.37±0.060 0 5.20 5.08 5.00 5.04 5.10 5.00 4.30 4.21 4.16 4.15 4.19 4.06 4.80 4.73 4.67 4.70 4.72 4.60 4.65±0.032 Sour 90 5.38 5.29 5.21 5.18 5.27 5.15 4.45 4.36 4.31 4.27 4.31 4.18 4.92 4.86 4.78 4.80 4.78 4.70 4.79±0.033 10.33 10.07 9.82 9.83 10.15 9.67 10.58 10.19 10.08 9.94 10.31 9.82 10.53 10.30 10.18 10.19 10.41 9.95 Means 9.98 10.15 10.26

229

Table 4.78 Mean sum of squares for texture (sensory scores) of Cheddar cheese

SOV d.f Crumbly Firmness Grainy Mouth-coating Maturity Pasty

NS NS NS NS NS NS Storage Days (D) 1 0.251 0.051 0.021 0.521 0.9589 1.9375

Milk (M) 2 399.955** 147.494** 150.047** 721.627** 20.6772** 41.6298**

NS NS NS NS Treatments (T) 5 0.647* 0.505 0.413* 0.539 0.3255 0.3801

NS NS NS NS NS NS D × M 2 0.010 0.004 0.025 0.025 0.0113 0.0182

NS NS NS NS NS NS D × T 5 0.008 0.003 0.022 0.006 0.0059 0.0065

NS NS NS NS NS NS M × T 10 0.031 0.014 0.022 0.010 0.0159 0.0084

NS NS NS NS NS NS D × M × T 10 0.006 0.002 0.018 0.008 0.0064 0.0033

Error 324 0.230 0.816 0.154 0.697 0.2796 0.8686

Total 359

** = Highly significant (p<0.01) * = Significant (p<0.05) NS = Non-significant (p>0.05)

230

Table 4.79 Mean sensory scores for texture of Cheddar cheese

Cow Milk Buffalo Milk Cow + Buffalo Milk Storage Texture Means Days T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

0 12.05 11.85 11.78 11.70 11.83 11.65 15.62 15.52 15.45 15.40 15.50 15.36 13.73 13.64 13.60 13.57 13.65 13.53 13.64±0.117 Crumbly 90 12.12 11.99 11.82 11.84 11.85 11.68 15.69 15.55 15.50 15.48 15.52 15.38 13.78 13.68 13.62 13.60 13.70 13.58 13.69±0.116

0 24.62 24.54 24.46 24.40 24.58 24.35 26.80 26.76 26.68 26.61 26.75 26.55 25.53 25.47 25.40 25.38 25.45 25.35 25.54±0.094 Firmness 90 24.60 24.55 24.50 24.42 24.60 24.34 26.86 26.80 26.72 26.60 26.78 26.57 25.55 25.50 25.45 25.41 25.50 25.36 25.56±0.093

0 6.42 6.37 6.30 6.26 6.39 6.25 8.71 8.62 8.55 8.50 8.68 8.50 7.65 7.60 7.52 7.48 7.60 7.43 7.49±0.075 Grainy 90 6.38 6.60 6.25 6.30 6.40 6.28 8.65 8.60 8.55 8.48 8.65 8.52 7.69 7.64 7.50 7.50 7.66 7.45 7.51±0.073

0 20.66 20.54 20.45 20.38 20.62 20.35 25.40 25.32 25.28 25.24 25.38 25.20 22.10 22.01 21.96 21.90 22.07 21.85 22.60±0.160 Mouth- Coating 90 20.72 20.65 20.60 20.50 20.70 20.48 25.50 25.35 25.30 25.30 25.50 25.28 22.15 22.00 22.00 22.01 22.10 21.94 22.67±0.162

0 13.52 13.45 13.37 13.30 13.49 13.28 14.32 14.27 14.22 14.18 14.30 14.15 13.91 13.84 13.80 13.78 13.84 13.75 13.82±0.044 Maturity 90 13.65 13.52 13.50 13.45 13.60 13.42 14.40 14.40 14.34 14.30 14.39 14.24 14.01 13.98 13.87 13.91 13.89 13.76 13.92±0.047

0 23.50 23.41 23.34 23.30 23.44 23.25 22.32 22.25 22.20 22.16 22.28 22.10 22.98 22.90 22.84 22.80 22.86 22.75 22.82±0.075 Pasty 90 23.35 23.26 23.24 23.18 23.30 23.10 22.17 22.09 22.03 22.00 22.04 21.93 22.82 22.78 22.70 22.73 22.70 22.62 22.67±0.076

16.80 16.73 16.63 16.59 16.73 16.54 18.87 18.79 18.74 18.69 18.81 18.65 17.66 17.59 17.52 17.51 17.59 17.45 Means 16.67 18.76 17.55

231

4.16 MULTIVARIATE ANALYSIS OF SENSORY SCORES

Principal component analysis (PCA) displaying the first two principal components (PCs) of descriptive sensory analysis of processed Cheddar cheese are presented in table 4.80. PC1 and PC2 significantly (P<0.05) distinguished among the cheeses and accounted for a variation of 78.55% and 13.12%, respectively. Fig. 4.41 shows a bi-plot of scores of processed cheeses and loadings of the sensory attributes. Hierarchical cluster analysis (HCA) on the sensory scores was used to group the narrowly correlated (Fig. 4.42) samples in terms of sensory attributes and engendered three main and two sub-clusters. PC1 illuminates the major difference (78.55%) among the sensory characteristics of the processed Cheddar cheese samples incoherent on the basis of milk types (Cheddar made from cow milk, buffalo milk and their mixture) used for making processed cheese and storage days cheese. Concerning the sensory scores, process cheese samples prepared from cow milk Cheddar were grouped on one side while the samples of cheese processed from buffalo milk Cheddar on other side. The processed cheese from mixture of cow and buffalo milk Cheddar occupied the central position on the plot. It shows that Cheddar cheese based on milk types significantly influenced the sensory discernment of all the samples irrespective of storage days. PC2 enlightens 13.12% of the dissimilarity between the treatments of processed cheese samples based on the Cheddar cheese of varying maturity. The Processed cheese samples prepared from fully and medium ripened Cheddar fell on upper side while those having medium ripened and fresh Cheddar in more quantities positioned on lower side showing substantial discrepancy. The processed cheese samples positioned asymmetrically and nearby on the bi-plot with regard to storage days (0 and 90 days) showing the non-significant differences in sensory scores awarded. The comparative positions of the cheese samples on the bi-plot is a valuable index of the influence of the milk sources, cheese treatments and storage periods on the sensory profile of processed Cheddar cheese. Hierarchical cluster analysis deals with foundations for understanding bi-plot and credentials of clusters of narrowly related samples. Three main clusters were generated. The first cluster recognized by the HCA congregated the processed cheese samples manufactured from the Cheddar cheese of cow milk and stored at 6ºC for 90 days. It comprised the sub-groups of processed cheese differentiated by the Cheddar cheese of varying maturity and storage days.

232 Cluster two contained the processed cheese samples prepared from the mixture of Cheddar cheese of cow and buffalo milk and stored for 90 days. Cluster three enclosed the cheese samples manufactured from buffalo milk Cheddar cheese at different maturity levels. These clusters encircled the subgroups; some containing processed cheese made from 100% 90-days ripened Cheddar and the others having Cheddar of 60-days and 30 days maturity in different concentrations. Cluster1 and 2 were grouped in to one main conglomerate which is linked with cluster 3 showing the sensory association of cluster 1 and 2 with cluster 3. Grouping and sub grouping on the dendrogram showed that milk types and processed cheese treatments based on Cheddar of varying maturity considerably influenced the sensory characteristics and played their role in laying down the foundations of clusters. Likewise the spots of cheese samples on PCA plot also suggest the significant effect of these factors on sensory discernment of processed Cheddar cheese.

233 Table 4.80 Principal Component Analysis (PCA) of the descriptive sensory scores showing the loadings of each variable on first two principal components (PCs)

Variable PC1 PC2 Creamy -0.9329 0.2351

Rancid -0.7254 0.5924

Bitter 0.8707 0.3530

Salty 0.9032 0.1894 Flavor Sweet -0.8736 0.3913

Sour -0.8971 0.3729

Soapy 0.9718 0.2041

Smokey -0.9972 -0.0577

Creamy -0.6644 0.6699

Rancid -0.5550 0.7106

Pungent 0.9190 0.3743 Odor Nutty 0.3368 0.6255

Sweet -0.9717 -0.0457

Sour 0.9235 0.3761

Crumbliness -0.9949 -0.0789

Firmness -0.9883 -0.0824

Grainy -0.9969 -0.0544 Texture Mouth Coating -0.9558 -0.1643

Maturity -0.9821 0.0823

Pasty 0.9438 0.2055

Variance 78.55% 13.12%

234

Projection of the cases on the factor-plane ( 1 x 2) Cases with sum of cosine square >= 0.00 4

D2C1

3 D2CB1

D2B1 D2CB5

2 D2CB2 D2DC2C5 D1DC2BC1B3 D1C1

D1DC2BC5B4 1 D1B1

D2BB25

2% D1CB2

1 . D1C5

13 D2C3

: 0 D1CB4 2 D2B3 D1CB3 D1CD22C4 r D1B5 D2CB6

o

ct

a

F

D1BD22B4 -1

D1CB6 D1C4 D2C6 D1 C3 D1B3D2B6 -2 D1B4

-3 D1B6 D1C6

-4 -6 -4 -2 0 2 4 6 8 Factor 1: 78.55%

Fig.4.41 PCA showing the first two PCs of descriptive sensory scores

235

Ward`s method Euclidean distance

D1C1 D2C1 D2C2 D1C2 D1C5 D2C5 D1C3 D1C4 D2C3 D2C4 D2C6 D1C6 D1CB1 D1CB5 D2CB2 D2CB5 D2CB1 D1CB2 D1CB4 D1CB3 D2CB3 D2CB4 D1CB6 D2CB6 D1B1 D2B1 D1B2 D2B3 D2B2 D2B5 D1B5 D1B3 D1B4 D1B6 D2B4 D2B6

0 10 20 30 40 50 60 70 Linkage Distance

Fig.4.42 Dendrogram obtained from Heirarchical Cluster Analysis (HCA) of sensory scores

236

Chapter-5 SUMMARY

Cheddar cheese; a highly nutritious food is a complex mixture of protein, fat, carbohydrates, vitamins and minerals. Quality of cheese is highly affected by milk composition, starter cultures, manufacturing technology and ripening period. Cheddar cheese undergoes significant changes during ripening which is the result of numerous microbiological, biochemical and metabolic processes referred as glycolysis, lipolysis and proteolysis, accountable for unique texture, aroma, appearance and taste. Manufacturing of processed Cheddar cheese involves natural Cheddar cheese and emulsifying salts in major proportions and other dairy (like butter, cream, skim- milk powder, whey powder, caseinates and anhydrous milk fat) and non-dairy (like fish, vegetables, salt, colors, flavors, spices, food gums, mold inhibitors, hydrocolloids and coloring etc.) components as optional ingredients, followed by heat treatment and mechanical shear to have a smooth homogeneous product. Various constituents influence the quality and functionality of processed cheese. The present study was envisioned to assess the effect of various ripening stages of natural Cheddar cheese prepared from cow and buffalo milk and selected emulsifying salts on the quality and functionality of processed Cheddar cheese. Raw milk from cows and buffaloes was standardized at 3% fat level and analyzed for pH, acidity, fat, protein, total solids and solids-not-fat content to assess the suitability for cheese making. Three batches of natural Cheddar cheese were prepared from cow milk and buffalo milk individually at different (one month) intervals and ripened at 4-6oC for 3 months. During ripening, cheese was evaluated for moisture, fat, protein, ash, pH, acidity, salt, minerals and organic acids (lactic, acetic, citric, butyric and pyruvic) content and proteolysis (water soluble nitrogen and total free amino acids) after one month intervals during ripening. Buffalo milk Cheddar cheese contained significantly higher values of fat, protein, ash, acidity, salt and minerals content as compared to cow. Cheese from buffalo milk was also considerably rich in organic acid contents; particularly lactic (16549.33±29.45 ppm), acetic (348.000±4.041 ppm), citric (728.000±5.132 ppm) and butyric (1355.00±9.29 ppm) acids than in cow milk cheese having 15135.00±30.14 ppm, 181.33±3.180 ppm, 542.00±3.512 ppm and 1022.67±04.33 ppm, respectively.

237 Water soluble nitrogen in total nitrogen (WSN/TN %) and total free amino acids (TFAA) content were significantly higher in cow milk Cheddar than buffalo. During ripening, acidity increased highly significantly from 0.900±0.012% to 0.967±0.007% in buffalo milk cheese and 0.84±0.010% to 0.91±0.007% in cow milk cheese while pH decreased from 5.26±0.028 to 5.14±0.026 in cow milk and 5.147±0.023 to 4.967±0.015 in buffalo milk cheese. However, fat protein, ash, salt and minerals content changed non-significantly in both types of cheese during 90 days ripening. The concentration of organic acids and onset of proteolysis (WSN/TN % and TFAA content) increased highly significantly in both cow and buffalo milk Cheddar cheese during the course of ripening owing to microbial and biochemical changes. Three emulsifying salts (Sodium Citrate, Di-Sodium Phosphate and Tri- Sodium Phosphate) were used as single and in different combinations to get the most suitable combination of emulsifying salts for manufacturing of processed Cheddar cheese. Processed cheese samples were analyzed to discriminate the effect of salt combinations on functional (hardness, gumminess and melt-ability) and sensory (color and flavor) properties. The combination of sodium citrate (50%), di-sodium phosphate (25%) and tri-sodium phosphate (25%) was selected for the manufacturing of processed Cheddar cheese in further studies. Processed cheese was manufactured from blends of raw, semi-ripened and fully ripened natural Cheddar cheese (of cow and buffalo milk) using selected emulsifying salts combination and stored for 3 months at 4-6 ºC for further analysis. The processed cheese was analyzed for moisture, fat, protein, ash, pH, acidity, salt and minerals content, texture profile, melt-ability, color and proteolysis during storage. Cheese was subjected to descriptive sensory evaluation at 0 and 90 days of storage for odor, flavor and texture. The results obtained were subjected to statistical analysis to assess the significance of the study. Buffalo milk processed cheese retained the highest mean moisture (44.83±0.068%), protein (22.87±0.048), ash (3.46±0.009), calcium (564.40±0.825 mg/100g), potassium (70.57±0.301 mg/100g), sodium (311.52±0.236 mg/100g), magnesium (37.24±0.309 mg/100g) and phosphorus (372.37±0.198 mg/100g) content, the blend of cow and buffalo milk cheese showed the highest mean fat (29.99±0.057%), salt (1.090±0.010%) and acidity (1.139±0.002%) while the highest pH value (5.481±0.007) was recorded in case of cow milk processed cheese. The

238 acidity increased significantly from 1.110±0.005% to 1.141±0.004% and salt content increased highly-significantly from 1.058±0.012% to 1.103±0.008% in 90 days. Significant decrease was noted in calcium, potassium and magnesium content during storage of processed cheese. However, fat, protein, ash and pH decreased non- significantly during 90 days storage. Processed cheese treatments (based on maturity of Cheddar) showed highly significant differences in moisture, fat, salt, acidity, pH, calcium, potassium, sodium and magnesium content whereas non-significant variation in protein, ash and phosphorus content. Various textural parameters were highly significantly different in cow, buffalo and blend of cow and buffalo milk processed cheese. Cheese hardness (3810.0±15.28g), cohesiveness (1.062±0.019), gumminess (3965.2±61.81) and chewiness (3920.0±62.69) were higher in processed cheese manufactured from buffalo milk Cheddar, while, the highest springiness value (1.001±0.008) was calculated in cow milk cheese. Treatment means showed a non-significant variation in the results for all the textural parameters. Melt-ability was found considerably diverse with respect to milk types and cheese treatments. The highest mean value (57.96±0.67 mm) was noted in cow milk and the lowest (47.33±0.51 mm) in buffalo milk processed cheese. Storage revealed a non-significant increase in melt-ability in all the samples. Assessment of proteolysis based on water soluble nitrogen (WSN/TN %) and total free amino acids (TFAA) showed non-significant effect of cheese storage. Regarding milk types, both WSN/TN (%) and TFAA (µg/g) content, were significantly higher in cow milk processed cheese as compared to buffalo and their blends. Milk sources and cheese treatments exhibited significant difference for lightness (L*), redness (a*) and yellowness (b*) as color parameters. Lightness (L*) and yellowness (b*) was recorded the highest (84.63±0.36 and 13.11±0.18, respectively) in cow while the redness (a*) in buffalo milk processed cheese (1.359±0.171). Processed cheese was judged for sensory perception at 0 and 90 days of storage. The evaluators established a descriptive sensory language and awarded scores from total of 100 for each parameter (odor, flavor and texture). Highly significant differences in all flavor, odor and texture attributes were observed with respect to milk sources and cheese treatments. However, storage periods showed significant and

239 non-significant variations in the mean scores awarded to different traits of sensory parameters. Cheese treatments exhibited considerable disparity for “creamy”, “rancid”, “sweet”, “sour”, “soapy” and “smokey” flavor attribute. Processed cheese from buffalo Cheddar gained the highest mean sensory flavor scores (9.44) followed by blend of cow and buffalo milk (9.40) and cow milk Cheddar (9.27). All flavor attributes showed a slight increase in sensory scores from 0 to 90 day of storage. Cheese sample (having 100% fully ripened cheese) gained the highest score in all types of processed cheeses indicating the development of flavor with the course of ripening. The highest scores for odor were awarded to the “creaminess” in cheese treatment prepared from 100% fully ripened buffalo milk Cheddar as 26.00. “Sweetness” decreased non-significantly in all treatments during storage period. Overall least scores were awarded to “rancid” and “nutty” odor traits in all treatments and processed cheese prepared from buffalo Cheddar was more “rancid” and less “nutty” than of cow milk cheese and their blend. Processed cheese from buffalo Cheddar attained the highest scores (18.76) for sensory texture followed by processed cheese from cow and buffalo milk blend (17.55) and cow milk Cheddar (16.67). Cheese treatments showed noteworthy disparity for “crumbly” and “grainy” characters but non-significant variation for other texture elements. “Firmness” gained maximum sensory scores (26.80) in processed cheese treatment having fully ripened buffalo milk Cheddar followed by “pasty” prepared from cow milk Cheddar cheese of 90-day maturity. The descriptive sensory analysis was supported with principal component analysis (PCA) and Hierarchical cluster analysis (HCA). First two principal components of descriptive analysis ominously distinguished the sensory variations. PC1 illuminates the major difference (78.55%) among the sensory characteristics of the processed cheese samples incoherent on the basis of milk types and storage days. PC2 enlightens 13.12% of the dissimilarity between the treatments of processed cheese based on Cheddar of varying ripening periods. The development of clusters, (grouping and sub grouping) on dendrogram also showed the significant effect of milk types and cheese treatments on sensory characteristics.

240 Conclusions and findings: It was concluded that;  Natural Cheddar cheese produced from buffalo milk was nutritionally (with regard to composition) superior than cow milk cheese.  Proteolysis was significantly higher in cow milk cheese while the production of organic acids was more in buffalo milk Cheddar. Both increased significantly during the course of ripening.  In processed Cheddar, meltability was significantly higher in case of cow milk cheese and treatments prepared from fully ripened natural cheese.  Processed cheese prepared from buffalo milk Cheddar exhibited more hardness, cohesiveness and gumminess, while springiness was higher in cow milk cheese. However, the combinations of fresh and ripened cheese showed non-significant variation in texture profile.  More proteolysis was determined in processed cheese treatments having fully ripened Cheddar and gradually decreased as the proportion of semi ripened and fresh cheese increased.  Sensory perception of the processed cheese was non-significantly influenced by the proportions of ripened/semi ripened natural Cheddar, however, buffalo milk cheese was scored significantly higher for most of the traits.  The cluster analysis showed that using cow and buffalo milk cheese blends improved the sensory characteristics since they were awarded optimum descriptive scores.  The selected combination of emulsifying salts produced the processed Cheddar with improved quality and functionality.

Hence, it is recommended that combinations of emulsifying salts, blends of cow and buffalo milk cheese with proportional ripening can be used to improve the functionality of processed Cheddar and particularly to reduce the cost of production.

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291 APPENDIX-I

INSTITUTE OF FOOD SCIENCE AND NUTRITION UNIVERSITY OF SARGODHA, SARGODHA- PAKISTAN DESCRIPTIVE SENSORY EVALUATION OF PROCESSED CHEDDAR CHEESE NAME OF THE JUDGE SIGNATURE & DATE

* Take water and crackers after each sample during evaluation Milk type Buffalo cheese / Cow cheese / Cow & Buffalo Mixture CHEESE SAMPLES 1 2 3 4 5 6 ODOR (Score 1-100) Creamy Sour Sweet Nutty Pungent Rancid FLAVOR (Score 1-100) Creamy Sweet Salty Sour Bitter Soapy Smokey Rancid TEXTURE (Score 1-100) Firmness Crumbly Grainy Mouth/Teeth-coating Pasty Maturity

292