The effect of passion fruit juice on quality of mozzarella cheese

MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITYUNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING GRADUATION THESIS FOOD TECHNOLOGY THE EFFECT OF PASSION FRUIT

MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY

UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR

HIGH QUALITY TRAINING

GRADUATION THESIS FOOD TECHNOLOGY

THE EFFECT OF PASSION FRUIT JUICE ON

QUALITY OF MOZZARELLA CHEESE

ADVISOR: PHAM THI HOAN STUDENT: TRAN THI THUY DUONG

NGUYEN THI MINH THU

SKL008938

HO CHI MINH UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING FOOD TECHNOLOGY

DEPARTMENT OF FOOD TECHNOLOGY

GRADUATION THESIS Thesis ID: 2022-18116008

THE EFFECT OF PASSION FRUIT JUICE ON QUALITY

OF MOZZARELLA CHEESE

TRẦN THỊ THÙY DƯƠNG NGUYỄN THỊ MINH THƯ Major: FOOD TECHNOLOGY

HO CHI MINH CITY – AUGUST 2022

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HO CHI MINH UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING FOOD TECHNOLOGY

DEPARTMENT OF FOOD TECHNOLOGY

GRADUATION THESIS Thesis ID: 2022-18116008

THE EFFECT OF PASSION FRUIT JUICE ON QUALITY

OF MOZZARELLA CHEESE

TRẦN THỊ THÙY DƯƠNG NGUYỄN THỊ MINH THƯ Major: FOOD TECHNOLOGY

HO CHI MINH CITY – AUGUST 2022

THE SOCIALIST REPUBLIC OF VIETNAM

Independence – Freedom– Happiness

-GRADUATION THESIS ASSIGNMENT

Major: Food Technology Class: 18116CLA

Supervisor: Phạm Thị Hoàn, Ph.D Email: hoanpt@hcmute.edu.vn

Date of assignment: 14/02/2022 Date of submission: 08/08/2022

1 Thesis title:

Effect of Passion fruit juice on quality of Mozzarella cheese

2 Thesis assignment:

Effect of passion fruit juice on qualities of mozzarella includes the following contents:

+Analysis of the chemical composition of materials (ash, moisture, protein, lipid, pH, titrableacidity and microbiological indicators, fruit weight and diameter)

+ Investigate the influence of passion fruit juice volume on properties of passion fruit mozzarellacheese (%H, %TSwhey, %TScheese, CSY (soild cheese yield), colorspace measurement andtexture analysis)

+ Investigate the influence of coagulation temperature on passion fruit mozzarella

+ Evaluate the qualities of passion fruit mozzarella during 21 days of storage.

The content and requirements of the graduation thesis have been approved by the Chair of the Food

Technology program

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Thanks to the devotion of the teachers and the knowledge accumulated after more thanthree years sitting on the lecture chair of Ho Chi Minh City University of Technical Education.Besides my efforts to learn and research, I also receive a lot of support and encouragementfromthe school, teachers, friends and family First of all, I would like to express my sincerethanks to Ph.D, Phạm Thị Hoàn for teaching, guiding and helping us throughout the projectimplementation process

We would like to thank the teachers and students of the Department of Chemical andFood Technology, Ho Chi Minh City University of Technical Education for their dedication,teaching and building of the basic knowledge needed in during the four years of study andpractice at the school, create the premises and conditions for the successful implementation ofthis graduation thesis Lastly, we would like to sincerely thank all those who have helped andassisted us in completing this project in this food technology

Although we have tried a lot to complete the project, with limited time and ability, this isthe first time we have entered into researching, calculating and perfecting a technology product,

so it is unavoidable errors We look forward to receiving the contributions of teachers

Sincerely,Trần Thị Thùy Dương & Nguyễn Thị Minh Thư

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STATEMENT OF ORGINALITY

I hereby declare that the Graduation Thesis “Effect of Passion fruit juice on quality ofMozzarella cheese is the results of my own research and has never been published in any work ofothers During the implementation process of this project, I have seriously taken research ethics;all findings of this projects are results of my own research and surveys; all references in thisproject are clearly cited according to regulations

I bear full responsibility for the fidelity of the number and data and other contents of mygraduation project

Date:

Student

Trần Thị Thùy Dương Nguyễn Thị Minh Thư

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

STATEMENT OF ORGINALITY ii

ASSESSEMENT FORM GRADUATION THESIS OF FOOD TECHNOLOGY (FOR COMMITTEE MEMBER) iii

ASSESSEMENT FORM GRADUATION THESIS OF FOOD TECHNOLOGY (FOR REVIEWER) ix

ASSESSEMENT FORM GRADUATION THESIS OF FOOD TECHNOLOGY (SUPERVISOR) ix

TABLE OF CONTENTS xv

LIST OF FIGURES xviii

LIST OF TABLES xix

LIST OF ABBREVIATIONS xx

ABSTRACT xxi

Chapter 1: INTRODUCTION 1

1.1 Pose the problem 1

1.2 Topic goal 2

1.3 Subject and scope of research 2

1.4 Research content 2

1.5 The scientific and practical values of the topic 3

Chapter 2: LITERATURE REVIEW 4

2.1 Cheese 4

2.1.1 Overview of cheese 4

2.1.2 Classification 5

2.2 Mozzarella Cheese 6

2.2.1 Classification 7

2.2.2 Casein coagulation 8

2.3 Raw material 11

2.3.1 Raw milk 12

2.3.2 Passion fruit 14

2.5 Research situations of the topic 17

2.5.1 Domestic situation 17

2.5.2 Overseas situation 18

2.6 Reasons for choosing the topic 18

Chapter 3: MATERIALS AND RESEARCH METHODS 20

3.1 Locations, chemicals, laboratory equipment 20

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3.1.1 Locations

3.1.2 Chemicals

3.1.3 Laboratory tools and equipments

3.2 Materials

3.2.1 Fresh cow's milk

3.2.2 Passion fruit

3.2.3 Citric acid

3.2.4 Rennet

3.3 Research schema

3.4 Manufacturing of passion fruit mozzarella

3.5 Experimental arrangements

3.5.1 Experiment 1: The effect of the passion fruit juice volume on the properties mozzarella

3.5.2 Experiment 2: The effect of temperature in coagulation process on the properties of mozzarella

3.5.3 Experiment 3: The effect of rennet concentration on the propertiesof mozzarella 29 3.5.4 Evaluation of the qualities of passion fruit mozzarella during storage period

3.6 Research methods

3.6.1 Method for determining chemical composition

3.6.2 Determining the yield of cheese efficiency

3.6.3 Determination of cheese solid yield (CSY)

3.6.4 Determining titratable acidity

3.6.5 Colorspace measurement

3.6.6 Microstructure analysis using scanning electron microscope (SEM)

3.6.7 Texture profile analysis (TPA)

3.6.8 Sensory evaluation

3.6.9 Microbiological analysis

3.6.10 Data analysis

CHAPTER 4: RESULTS AND DISCUSSION

4.1 The chemical composition and physicochemical properties of raw materials

4.1.1 Chemical composition of raw milk

4.1.2 Physicochemical properties of passion fruit juice

4.2 The effect of the passion fruit juice volume on the quality properties of mozzarella

4.2.1 Effect of passion fruit juice volume

4.2.2 Effect of passion fruit juice proportion on mozzarella texture properties

4.2.3 Effect of passion fruit proportion on color change of mozzarella 394.3 The effect of temperature in coagulation process on the properties of mozzarela 414.3.1 Effect of temperature in coagulation process 414.3.2 Effect of temperature in coagulation process on mozzarella texture properties 424.3.3 Effect of temperature in coagulation process on color change of mozzarella 434.4 The effect of rennet concentration on the quality of mozzarella 454.4.1 Effect of rennet concentration 454.4.2 Effect of rennet concentration on mozzarella texture properties 464.4.3 Effect of rennet concentration on color change of mozzarella 474.5 Conclusion on the production process of passion fruit mozzarella 484.6 Evaluation of the quality properties of passion fruit mozzarella 494.6.1 Chemical composition and microbiological criteria 494.6.2 Evaluation of the microstructure of mozzarella 514.6.3 Sensory evaluation of passsion fruit mozzarella 514.7.1 Color change of passion fruit mozzarella during storage 53Chapter 5: CONCLUSION AND RECOMMENDATIONS 555.1 Conclusion 555.2 Recommendations 55

REFERENCES Error! Bookmark not defined.

APPENDIX 59

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LIST OF FIGURES

Figure 1 1 U.S Consumption of Cheese (1975-2018) 1Figure 2 1 Illustrations of cheese products 4Figure 2 2 Mozzarella cheese 7Figure 2 3 Purple passion fruit 15Figure 2 4 Yellow Passion Fruit 16Figure 2 5 Illustrate the mechanism of coagulation by rennet 10Figure 3 1 Nong Lam Milk 20Figure 3 2 Passion fruit (Passiflora edulis) 21Figure 3 3 Naturen Extra 220 rennet 21Figure 3 4 Research schema 23Figure 3.5 Flowchart of technological process of PM 24Figure 3.6 Protein coagulation 25Figure 3.7 Curds 26Figure 3 8 Curd after aging 26Figure 3 9 Draining & kneading 27Figure 3 10 Moulding 27Figure 3 11 Shema of experimental arrangement 1 28Figure 3 12 Shema of experimental arrangement 2 29Figure 3 13 Colorspace measurement 32Figure 3 14 Texture profile analysis 33Figure 4 1 The surface of milk curd at different PJ volume in the coagulation process 38Figure 4 2 Mozzarella samples at different volume of Passion fruit juice 39Figure 4 3 Milk curds at different coagulation temperatures 42Figure 4 4 Milk curds surface at different coagulation temperatures 42Figure 4 5 Milk curdling surface at different rennet concentration 46Figure 4 6 Mozzarella samples at different rennet concentration 48Figure 4 7 Flowchart of technological process of passion fruit mozzarella (PM) 49

Figure 4 8 Microstructure of the first reference sample (RS1), the second reference sample

(RS2) passion fruit mozzarella (PM) 51Figure 4.9 The result of sensory evaluation of mozzarella samples 52Figure 4 11 Changes in Lightness value (L*) of RS1, RS2, PM samples during storage 53Figure 4 12 Changes in a* value of RS1, RS2, PM samples during storage 53Figure 4 13 Changes in b* value of RS1, RS2, PM samples during storage 54

LIST OF TABLES

Table 2 1 Classification of cheese by water content 5Table 2 2 Classification of cheese by fat content 5Table 2 3 Classification of cheese by processing 6Table 2 4 Classification of cheese by coagulation method 6Table 2 5 Mozzarella identity standards from CFR (Code of Federal Regulations 8

Table 2 6 Chemical composition ( Mean   SD) of raw camel and cow milk 12Table 2 7 The nutritional value of passion fruit juice 16Table 3 1 The technical parameters of rennet 22Table 3 2 Parameter and setting value of structural measuring device 33Table 4 1 Chemical composition of fresh cow's milk 35Table 4 2 Physicochemical properties of PJ 35Table 4 3 The effect of passion fruit juice (PJ) volume on mozzarella quality properties 37Table 4 4 Effect of PJ on texture properties of mozzarella 38Table 4 5 Colorspace results of mozzarella changed with casein coagulation temperature 39Table 4 6 The effect of coagulation temperature on mozzarella quality properties 41Table 4.7 Effect of coagulation temperature on texture properties of Mozzarella 43Table 4 8 Color change results of PM and the reference samples 43Table 4 9 The effect of rennet concentration on the mozzarella quality properties 45Table 4 10 Effect of rennet concentration on texture properties of Mozzarella 46Table 4.11 Color change results of PMs and the reference samples 47Table 4 12 Quality criteria of PM and RS2 50Table 4 14 The result of sensory evaluations of mozzarella samples 52

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Texture profile analysisThe first reference sampleThe second reference sample

The research topic "Effect of Passion fruit juice on quality of Mozzarella cheese" was

carried out: a) to evaluate the effect of passion fruit juice concentration on the mozzarellaproperties; b) to determine the technological parameters (temperature, rennet concentration) ofcasein coagulation by rennet; c) to evaluate some of cheese qualities during 21 days storage

The analytical results showed that the passion fruit mozzarella (PM) made with passionfruit juice (PJ) volume of 9 (mL) had appropriate yield efficiency at 8.23% and reached the pH at5.9 Next to cheese made with rennet concentration of 75L/300 mL of milk and coagulationtemperature at 37  1 ; as a result, the cheese yield efficiency (%H), total solids cheese(%TScheese), cheese solids yields (%CSY) was 9.47%; 55.86%; 45.94%, respectively.Furthermore, the finished cheese had proteins, lipids, dry matter content: 24.10%, 21.30%,52.20%, respectively Besides that, microbiological analysis also was conducted and gave the

result that no detected of harmful pathogens such as: Escherichia coli, Salmonella spp, Listeria

monocytogenes, Coagulase-positive Staphylococci The microstructure of PM had many sharp

edges, the shape of the structure resembles a cluster of particles Moreover, in descriptivesensory test, the PM samples had the difference in term of texture and smell compared with 2other reference samples (RS1 and RS2) In addition, the average acceptability of PM sampleswere the highest, at 6.77/9, and RS1, RS2 samples were 5.87; 5.95 in grade 9 respectively

Through all the above results, we found that passion fruit juice had the influence onmozzarella making-process such as: chemical composition, texture, microstructurecharacteristics, sensory and nutritional value of the product Therefore, passion fruit juice isbelieved that it will have many potential applications in the food industry as whole and dairyproduct industry in particular

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Chapter 1: INTRODUCTION 1.1 Pose the problem

Cheese is a high nutritional value food with a wide variety of flavors and textures Amongthem, Mozzarella is one of the most consumed cheeses worldwid They play a large role inseveral world markets, especially the North American marke Cheese factories typically producemore than 100,000 kg of pizza cheese per day (Fox et al., 2017c)

Source: USDA ERS.

Figure 1.1 U.S Consumption of cheese (1975-2018)

With the development of the economy and the cultural exchange between the West and theEast, cheese has become popular all over the world, including in Vietnam Cheese in general andmozzarella, in particular, are products rich in essential nutrients: especially proteins, amino acids,fats, fatty acids, vitamins, and minerals (Walther et al., 2008) Currently, the demand forMozzarella cheese is increasing not only because of its health benefits but also can be used tocombine with other dishes such as pizza, hotdogs, and hamburgers Therefore, the demand forcheese from customers is constantly increasing, requiring cheese manufacturers to makeappropriate changes to meet the demand for that increase With hundreds of cheeses in differentflavors, consumers will find the product that meets their needs During the information collectionprocess, we found that the collection of documents on the production process of mozzarella andthe influence of passion fruit juice on mozzarella was minimal There are few domestic studies

on the production process of mozzarella from raw milk in Vietnam with the conditions inVietnam In addition, there have not been any direct studies in Vietnam on adding passion fruitjuice to improve the quality and structure of mozzarella Therefore, it is necessary to perfect amozzarella production process in the actual conditions of our country In mozzarella makingprocess, the micelle casein will coagulate when the pH is reduced to a critical point by adding anacidic agent, typically citric acid - a flavor enhancer to food products, also a natural preservative,inhibiting the growth of bacteria and preventing oxidation in food products In Vietnam, citricacid is abundant in passion fruit, a popular fruit with a characteristic flavor and aroma Passionfruit juice contains citric acid, malate acid, lactic acid, ascorbic acid, and other organic acids(Casimir et al., 1981) In addition, passion fruit juice is a source of vitamin C ascorbic acid andvitamin A carotenoids (Knight & Sauls, 1994) This is also the second purpose of this research,

with the desire to create mozzarella cheese products that not only achieve improvements inperformance and texture but also bring high nutritional values and new flavors In this study,

we used a combination of both rennet and passion fruit juice as coagulants for fresh cheese

+ Evaluate the qualities of passion fruit mozzarella (Chemical composition: total dry mattercontent, proteins, lipids, acidity, pH, microstructure of products, sensory test, microbiologicalcriteria); the qualities of passion fruit mozzarella during 21 days of storage

1.3 Subject and scope of research

 Subjects:

 Fresh cow's milk imported from the breeding unit of Nong Lam University, Ho Chi Minh

City

 Passion fruit juice which added to the Mozzarella production process

 Passion fruit mozzarella production process

Effect of passion fruit juice on qualities of mozzarella includes the following contents:

+Analysis of the chemical composition of materials (ash, moisture, protein, lipid, pH, titrableacidity and microbiological indicators, fruit weight and diameter)

+Investigate the influence of passion fruit juice volume on properties of passion fruit mozzarellacheese (%H, %TSwhey, %TScheese, CSY (soild cheese yield), colorspace measurement andtexture analysis)

+ Investigate the influence of coagulation temperature on passion fruit mozzarella qualities

+ Investigate the influence of rennet concentration on the qualities of mozzarella cheese

qualities

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+ Evaluate the qualities of passion fruit mozzarella (Chemical composition: total dry mattercontent, proteins, lipids, acidity, pH, microstructure of products, sensory test, microbiologicalcriteria).

+ Evaluate the qualities of passion fruit mozzarella during 21 days of storage

1.5 The scientific and practical values of the topic

 Scientific significance:

- The process of producing fresh cheese with the addition of passion fruit juice can be used

as a reference for research and study at universities

Chapter 2: LITERATURE REVIEW 2.1 Cheese

2.1.1 Overview of cheese

Cheese is called name for a group of highly nutritious dairy products (Le, 2010) Thecollection name of chese is derived a group of food products made from fermented milk,produced in a variety of flavors and forms around the world (Fox, 1993) (Khanal et al., 2019).Cheese is produced by coagulating milk with the help of yeast or suitable probiotics or vegetablecoagulants, vinegar and lemon and partially depleting the whey (Hickey, 2017) Initially, this wassimply a method of preserving milk (Patrick F Fox, 1993), until now cheese has developed into apopular food due to its variety of applications, value nutritious, convenient and palatable toconsumers This diversity is due to developments in cheese production technology, biochemistryand microbiology of cheese ripening (Farkye, 2004) Since ancient times, cheese has been anneccesary ingredient in the diet in a lot of country on the world Cheese is made from foodproduct rich in proteins, fats, minerals and vitamins, besides containing bioactive amino acids,peptides and lactic acid bacteria (Malcata et al., 2016)

Figure 2 1 Illustrations of cheese products Fresh cheese is a cheese without biochemical ripening (Thanh, 2003), produced by

coagulating milk, cream or whey through acidification, acidification with small amounts ofrennet, or a combination of acid and temperature, can be used immediately after production iscomplete (P F Fox et al., 2017 )

There are many high nutritional value of fress cheese in dairy product In which, theprotein content is about 15-20% including all kinds of important amino acids such as metionin,lizin, In addition, fresh cheese also has a significant amount of minerals Ca, P, Fe, Mg, needed for cell growth, bone formation and metabolism in the body Whey is separated fromfresh cheese by gravity rather than mechanical pressure to help maintain a smoother texture and

higher moisture content Therefore, fresh cheese has the highest moisture content of all cheeses.

In our country, fresh cheese has begun to be produced but not much Currently, on the market,there are many fresh cheese products from different companies with unique quality and taste

2.1.2 Classification

Currently, there are many types of cheese in the world Cheese products differ in structure,odor, taste, color, physicochemical and microbiological parameters (Le, 2010) There aredifferent methods for classifying cheese: grading based on texture (depending mainly onmoisture content), grading based on coagulation method and grading based on ripeness of thecheese indicators (Khanal et al., 2019)

Table 2.1 Classification of cheese by water content

Source: (Le, 2011)Table 2.2 Classification of cheese by fat content

Product Type

Cheese has a very high fat content

Cheese with high fat content

Cheese with medium fat content

Cheese with low fat content

Skinny cheese

Source: (Le, 2011)

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Source: (P F Fox et al., 2017)

2.2 Mozzarella Cheese

Mozzarella cheese is type of a soft, unripe of the Pasta-filata family that originates fromthe Battipalia region of Italy Cheese in general and mozzarella in particular is made by freezingand fermenting (incubating) the milk of some animals, such as cows, buffaloes, sheep, goats Traditionally, mozzarella is made from buffalo milk Buffalo milk is a high Vitamin A, proteinand low cholesterol, thus this traditional cheese made from buffalo milk has more preferredcharacteristics

Mozzarella is taken into account a fress cheese as well as it is famous for a mild flavor andrubbery texture Mozzarella cheeses dated back in the southern areas of Italy, from the Battipaliaregion manufacturing from milk of cow however traditional cheese with main ingredient is thatfrom buffalo milk, i.e., Mozzarella di Bufala Until now, mozzarella is created throughout Italy,another European countries and the United States from a different of dairy materials , but theproduct line produced from buffalo milk is still very valuable around the world (Sameen and cs,

2008) Mozzarella cheese is sort of a soft, unripened and white cheese whose melting and

stretching propertie, especially mozzarella cheese is highly suitable for pizza making (Kindstedt,

1995) These functional properties of mozzarella cheese are effected by a variety of elements thatcontains cheese composition particularly moisture, fat content, pH, coagulating enzyme, starterculture, cooking and stretching, salt content and the changes occurring during aging and storage(McMahon et al., 1996)

The finished cheese is lightly salted, white, soft with a very lively surface sheen and has uniquepropery of stretchability Due to this, it can be used to make pizza toppings, lasagna, and veal cutletalla Parmigiana Since mozzarella cheese naturally stretches, it is often used as a topping on pizza.The popularity of pizza joints, especially among young people, has increased mozzarella cheesemanufacturing In Italy, 78% of the country's entire cheese production is made up of mozzarella andpizza cheese (Merrill et al, 1994) In the production of this cheese, after fermentation the curd(pH∼5.1 – 5.4) undergoes a plasticization step at the end of production by kneading and spinning inhot water ( ∼80 – 85℃) or dilute saline (Guinee et al., 2002) It was the low pH andhigh temperature that facilitated the formation of para-casein fibers with relatively high tensilestrength (Kimura et al., 1992; Taneya et al., 1992) After the cheese is cooked on the pizza, thesethreads give the melted cheese its spinning, stringiness, and chewiness (Pf Fox et al., 2000)

Figure 2 2 Mozzarella cheese

2.2.1 Classification

According to Codex standards (WHO/FAO) mozzarella is divided into two main typesbased on usage:

1 Mozzarella has a high relative humidity, produced for direct consumption

Mozzarella has high moisture content (52 – 60%), high pH (5.6 – 5.8), high moisturecontent, low salt content (0.1 – 0.5%) and short shelf life (21 days) (Clark et al., 2009) Freshmozzarella is usually originated by adding acid directly to acidify milk rather than acidifyingbacteria Products are usually formed into small spheres with the size of different sizes and areconsumed immediately after production Fresh mozzarella is frequently consumed raw because

of its delicate milky flavor and tender texture, which make it a good choice for salads,sandwiches, and appetizers This cheese is not commonly used as an accompaniment to other

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dishes because the protein matrix contracts when heated, releasing the whey, making the dish overly watery (Clark et al., 2009).

2 Mozzarella has low moisture content and is frequently used as a component in foods (Clark et al., 2009).

Low moisture mozzarella (45 - 52%) is a product with lower pH (5.2 -5.4), lowermoisture content, produced by using bacterial culture method (Clark et al., 2009) Because of itslower pH, lower moisture content, and higher salt content, it has a longer shelf life Lowmoisture mozzarella can be produced to have a variety of spinning and melting properties, issolid enough to be chopped or sliced, and can be cooked without the need of excessive water.Due to its special processing and cooking qualities, mozzarella is frequently used Low-moisturemozzarella that has not been cooked can be used in sandwiches, as a topping for pizza, as a fillerfor lasagna, and even breaded for use as an appetizer (Clark et al., 2009)

Beside that, due to code of Federal Regulations, mozzarella can devide into 4 groups:

Table 2 5 Mozzarella identity standards from CFR (Code of Federal Regulations (CFR) (b) Title

21, Part 133.155 thru 133.158)

Type of cheese

Mozzarella (Regular)

Mozzarella ( Part skim)

Mozzarella ( Low moisture)

Mozzarella ( Low moisture, part skim)

2.2.2 Casein coagulation

The method of changing casein from a colloidal state to a curd with a gel structure occursduring casein coagulation in milk (Le, 2010) This is one of the important stages of cheeseproduction in general and mozzarella in particular The structure of casein micelles is verysensitive to changes in factors such as pH, temperature, and ionic strength Therefore, the study

of the modification of casein micelles has been applied in the food industry to improve productquality There are many different methods applied and in the cheese industry, milk coagulationmethods are often used: isoelectric (acid) coagulation, heat and acid coagulation, rennet(enzyme) coagulation

2.2.2.1 Coagulation by acid

By lowering their charge, dissolving part of the insoluble calcium phosphate crosslinks,and altering internal protein bonds, acidification has a direct impact on the stability of caseinmicelles At some crucial point, when electrostatic repulsion is diminished and is unable to repelattractive forces like hydrophobic contacts, aggregates and eventually gels begin to develop.Acid-induced milk gels become more rigid over time as a result of continuing casein particle-to-casein link formation inside the network

Normal milk contains between 80 and 95 percent casein in the type of micelles, which arecolloidal dispersions that precipitate at a pH of 4.6 (isoelectric pH) at 30ºC (Brunner, 1977;Dalgleish, 1982; Patrick F Fox et al., 1998) Milk acidification can be performed using bacterialcultures, fermenting lactose to lactic acid, direct addition of acids, such as HCl, or usingglucono-δ-lactone (GDL) (J Lucey et al., 1997) is hydrolyzed to gluconic acid, leading to adecrease in pH We bring the milk pH to the isoelectric value of casein 4.6, it will reduce theelectrostatic repulsion between the molecules, then the interaction between the hydrophobicradicals of the protein molecules will prevail, the casein will switch to insoluble state and theappearance of milk coagulation (Le, 2010) The process of coagulating casein in milk byadjusting the milk pH to the casein isoelectric point, acid coagulation (lactic or citric) method isused in production technology of some fresh cheeses such as Cottage, Quarg, Cream cheese, etc.Acid coagulation represents 25% of total cheese production and is commonly used fresh(Mcsweeney et al., 2004) Manufacturers will use lactic acid bacteria to create lactic acid,decrease the pH value of milk, causing coagulation of casein (Le, 2010) In addition, the use oflactic acid bacteria also serves the purpose of biosynthesis of some metabolic products tocontribute to the characteristic flavor of cheese.

2.2.2.2 Coagulation by acid and heat

Review of component and processing parameters' influence on the textural characteristics

of fresh acid cheeses and acid milk gels (Lucey & Singh, 2003) Fresh acid cheeses use a mix ofheat and acid to coagulate milk, cream, or whey Pretreatments, such as heating the milk orhomogenizing it, delayed acidification often using mesophilic starter cultures and gelation, wheyseparation, and/or curd treatment are frequently required for the creation of fresh acid cheeses(Sheehan et al., 2004) Coagulation by a combination of heat and acid is used for a limitednumber of cheeses, including ricotta and manouri (Fox et al., 2017a) Queso blanco is a cheesefrom Latin America that is created by boiling milk to a temperature of 80 to 85°C, adding acid,and then gently churning the milk to create a curd Acetic, citric, phosphoric, among other acids,

or simply lemon juice, might be

2.2.2.3 Coagulation by rennet

Currently, the rennet enzyme used to coagulate casein in cheese production can be obtainedfrom three sources: animal sources, plant sources, and microbial sources The majority of maturecheeses are made with rennet-coagulated cheese, which accounts for 75% of all cheeseproduction (McSweeney, 2004) The two stages of the enzymatic coagulation process, whichinvolves the modification of casein micelles, are the cessation of milk serum separation and thetransformation of milk into curd

The κ-casein molecules are crucial for maintaining the milk's micelle structure (Le, 2010)

It has been discovered that during the rennet coagulation of milk, only κ-casein is hydrolyzedand that it is hydrolyzed to para-κ-casein and macropeptides also known as glycomacropeptidesbecause they include all or most sugar groups associated with κ-casein (Fox et al., 2015) At thisstage, only κ-casein molecules are hydrolyzed at the above specific site, hydrophilicmacropeptides diffuse into the surrounding medium while para-κ-casein remains attached to themicelle moieties, makes micelles “easy to assemble” together in preparation for agglomeration

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(Le, 2010) The micelles become less stable and start to coagulate when 85% of thetotal κ-casein in milk has been hydrolyzed (the coagulation temperature is usually 30℃) During the

coagulation process, the micelles will drag fat particles and some other compounds in milk including lactose, vitamins, salt, etc

Casein

Rennet

Para-casein + macropeptides

Ca2+ , 20oCGel

Figure 2 3 The mechanism of coagulation by rennetWith the right temperature and time, the milk curd has been formed, the milk serumseparation stage is performed For milk casein coagulation agglomeration of milk with a particlegel structure, casein gels are induced by enzymes formed by filaments, each consisting of manyinteracting casein particles, and each granule considered as aggregates of micelles in milk Thisgranular gel structure contains straight fibers, through which part of the milk serum from withinthe coagulants will escape Therefore, cutting the curd will make more milk serum escape, thefinished cheese has low moisture content, high hardness (Le, 2010) Besides the curd massformation process, the enzymatic coagulation of casein is also influenced by external factors.They depend on temperature, at <15℃, the milk solution will not be able tocoagulate (Le, 2010), at a temperature of >55-60℃ depending on the pH andconcentration, the enzyme will be denatured When the pH of milk is decreased, calciumphosphate is dissociated from the micelle structure, reducing the charge of the micelle, theenzyme moves closer and closer to its optimal pH, increasing the reaction rate (Fox et al., 2015).Increasing the concentration of Ca2+ also changes the equilibrium of the system, they reduce thecharge of the casein, reduce the electrostatic repulsion between the micelles and promotecoagulation The higher the amount of enzyme used, the faster the hydrolysisreaction of κ-casein, the more micelles that can release macropeptides on the surface, thefaster the coagulation rate (Le, 2010)

The complex group of enzymes generated by the rennet in the stomachs of young ruminantmammals, mainly calves (Uniacke-Lowe & Fox, 2017) The most important component of rennet

is Chymosin - a protease enzyme used to coagulate caseins in milk (A Kumar et al., 2010).Pepsin and lipase are two more enzymes found in rennet in addition to chymosin

Rennet's main functions include the protease chymosin Its cleavage cleaves thecaseinomacropeptide from the κ-casein chain, causing destabilization of the casein micelles(Corredig & Salvatore, 2016) Milk coagulates as a result of the casein's adhesion to othercleaved casein molecules and formation of a lattice (Corredig and Salvatore, 2016) They canclot better in the presence of calcium and phosphate, which is why these minerals areoccasionally added when making cheese (Lucey, 2002) The network of solid casein proteins iscut that holds together the other components of milk, such as fats and minerals, to make cheese

Animal rennet : is a complex mixture of enzymes, the main one being chymosin, which is

normally obtained from the stomach of calves (O’Connor, 1993) Young animals' gastricglands secrete animal rennet; when adults consume milk, rennet also occurs in their stomachs

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rennet from each species has somewhat varying chemical properties, utilizing sheep's rennet tocoagulate milk is the best option, but the difference is not significant Milk coagulates quicklywith animal rennet at a pH that is natural and with minimum degradation of milk proteins (Garg

& Johri, 1994) Acids, neutral calcium and barium salts, and the occurrence of coagulation allspeed up this process Slower when mixed with distilled water, potassium, sodium, or base salts

In addition, animal rennet added during cheese making improves whey separation and increasesdensity hardening of curd mass (Fox et al., 2017b) Based on the above advantages, we selectedanimal rennet sources to use for this study

Plant rennet: There are numerous recognized milk-derived proteolytic enzymes, including

bromelain (EC 3.4.4.24) from pineapple, ficin (EC 3.4.4.12) from Ficus species, and papain (EC3.4.4.10) from papaya These enzymes appear to be present everywhere in plant structures Buds,flowers, fruits, latex, leaves, roots, sap, and seeds all contain them Ash gourd, fig, milkweed,papaya, pineapple, prickly artichoke, pumpkin, and soybean are a few of the most well-knownplants Sadly, the majority of plant coagulants have shown to have non-specific proteolyticaction Most plant rennets are unsuitable for manufacturing cheese because the ratio of milk-clotting function to proteolytic activity is so low; excessive proteolysis results in bitter products,but bitterness lessens over the course of 3-6 months of storage (Garg & Johri, 1994)

Microbial rennet: used for milk fermentation has also been developed through genetic

engineering by genetically modifying the fermenting microorganisms, providing the market withmany innovative products (Islam et al., 2021) However, in certain nations, microbial coagulantsare not permitted for the manufacturing of cheese because it is thought that they will result inpoorer yields and poorly performing (Garg et al., 1994)

Although rennet's primary role in cheese making is to coagulate milk, it also contributes toimproved flavor and texture by participating in proteolysis during cheese ripening (Fox, 1988).Coagulation is directly affected by the type of enzyme used, the ratio and composition ofsubstances present in milk fat, protein, whey and casein, the presence of calcium ions and pH(Fox et al., 2017a)

In the Mozzarella production process, coagulation of milk casein to create curd is animportant process Nowadays, many coagulation agents have been studied and found Amongthem, the most common is the enzyme coagulant (rennet) with 75% of the total cheese yield (Fox

et al., 2017d), along with previous studies by D'Alessandro (2019), Mme (2018) aboutMozzarella, the coagulation agent is rennet In addition, we use passion fruit juice as the source

of acid for this process to lower the pH of the milk, it is effective in the coagulation

2.3 Raw material

Traditional mozzarella is made from buffalo milk (McSweeney et al., 2017) andMozzarella produced from buffalo milk is the best quality cheese until now (Zicarelli, 2004).Nevertheless, the source of raw milk depends on the region that to use the vary of mammals likebuffalo, cow, sheep, goat, or camel

2.3.1 Raw milk

Milk is a liquid secreted from the mammary glands of animals, containing high levels ofnutrients The variations in breed, environmental factors, and breeding conditions all affect theessential ingredients in milk The milk from various animals varies greatly Although dairy cattleand goats have been raised in our country for a long time, for many reasons, the production ofthese types of milk is still very low So far, the raw material for the dairy industry in Vietnam isstill cow's milk There are several factors that significantly influence milk composition includingbreed, feed composition and lactation stage

Milk is a very complicated liquid that is made up of four primary parts namely water, lipid,protein, lactose and about 100 minor components Among them, the most important ingredientfor cheese production is calcium phosphateThe characteristics of one of the milk's casein andprotein groups, in particular, affect the creation and quality of cheese Whey, that containssoluble components, removes the majority of the water present in milk 90% of it, i.e wheyprotein, lactose and some inorganic salts (P F Fox et al., 2017)

Good quality milk serves as the foundation for making cheese of top quality, which has aclean taste, is low in somatic cells, contains no antibiotics, and has a relatively low bacterialcount The yield, quality, and functional attributes of cheese are influenced by the milk'scomposition, which is influenced by the breed of the lactating animal, season, lactation stage,disease, and genetics To guarantee constant cheese quality and depending on the raw milk'smicrobiological quality and its source, hydrogen peroxide (H2O2) can be added to the raw milk(Moatsou & Moschopoulou, 2014) In addition, the production of mozzarella is also impact bythe lipid and protein/casein content of the milk The higher the casein content in milk, the greaterthe cheese recovery in the cheese making process (Le, 2010)

Table 2 6 Chemical composition (Means SD) of raw camel’s and cow’s milk

Soure: Salma M Siddig et al, 2016

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