MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING CAPSTONE PROJECT FOOD TECHNOLOGY SOFT CHEESE PRODUCTION U
INTRODUCTION
The study scientific imperative
The Vietnam government has also launched a project that meets 40% of domestic fresh milk demand (1.4 million litres of milk) in 2025 (Bộ Công thương, 2010) The Vietnam dairy industry has decided to modernize dairy cow farms and improve raw milk productivity to meet local dairy demand Raw milk materials for dairy products are mostly purchased from small-scale farmers; however, 20 – 50% of the milk from small – scale suppliers did not meet the quality requirement (according to the Institute of Policy and Strategy for Agriculture and Rural Development) Thus, Vinamilk and other local dairy companies began investing in dairy farms and importing cows to Vietnam to ramp up production in 2007 For example, Vinamilk built 12 International standard farms across the country, with 130000 total dairy cows and 950 – 1000 tons of raw fresh milk per day (Vinamilk, 2012) Despite those positive signs, milk domestically production has still been insufficient to meet the growing demand Locally produced milk meets approximately 80% of the drinking milk production and 35% of milk domestic consumption demand Generally, Vietnam does not have favorable conditions for the dairy industry due to the tropical climate, limited land and undeveloped technologies Therefore, exported milk serves as an input to ensure a stable product supply in dairy processing
Compared to milk and other dairy products, cheese is a higher content of protein which is a good source of nutrients for the human daily diet Currently, dairy consumption in Vietnam has significantly increased because of the increasing concern for health and nutrition However, domestic milk does not provide enough the amount of milk for cheese production Processed cheese and imported cheese are the cheese that is familiar to Vietnamese consumers Processed cheese is natural cheese mixed with non – cheese ingredients such as salt, preservatives, emulsifiers to enhance the texture and flavour of the cheese; therefore, processed cheese is less nutritious than natural cheese In terms of imported cheese, the large amount of imported cheese is hard cheese varieties Hard cheese is low moisture content, as it is easy to transport and store in tropical regions like Vietnam Compared to hard cheese, soft cheese is rich in protein, and it has lower fat content appropriate for everyone, especially for the elderly, children and those on a diet Besides, soft cheese is well – suited to being processed into cheese preparations or various dishes such as cheesecakes, sauces, desserts Despite its short shelf – life, there are not many choices of imported soft cheese in the Vietnam market
Soft cheese is mainly divided based on the coagulation methods: acid – coagulated, acid/ heat coagulated, rennet – coagulated Rennet – coagulated soft cheese is firmer than other soft cheese varieties because acid and rennet interaction results in better draining than acid-rennet coagulated soft cheese The cheese is better draining resulting in a lower moisture content along with firmer curds that leads to improving cheese texture The high pH of rennet – coagulated cheese also results in a less acidic flavour of the final cheese product (Fox et al., 2017)
To sum up, cheese is a nutritious food that is an excellent source of protein, calcium, minerals and vitamins Currently, the demand for the rapid cheese consumption growth; however, domestic cheese production is still limited Besides, the advantage of rennet – coagulated cheese far outweighs acid – coagulated soft cheese regarding texture and sensory Thus, we had an idea of developing a kind of rennet coagulated – soft cheese made of skim milk powders that have been suitable for milk production in Vietnam Additionally, the product is locally produced, which is accessible to domestic consumers and is an appropriate option for all aged consumers.
Objectives and tasks of the study
The study was done to build soft cheese technological processing from recombined milk coagulated by rennet and citric acid
The thesis was done with the following study tasks:
- Determining the chemical composition of ingredients
- Investigating the effect of citric acid concentration on casein coagulation
- Investigating the influence of rennet (incubation temperature and enzyme concentration) on soft cheese criteria
- Evaluating the quality of soft cheese
- Evaluating the quality changes of soft cheese during storage time.
Research methodology
- Microstructure observation (Scanning Electron Micrograph)
Study contribution
The research results provide technical parameters for the production process of soft cheese from recombined milk coagulated by acid citric and rennet These data could be used as a reference for relative research purposes at universities
This soft cheese production process could be applied to pilot production or industrial- scale manufacturing
Study structure
The study has 5 chapters Chapter 1 – Introduction; Chapter 2 – Literature review; Chapter 3 – Materials and methods; Chapter 4 – Experience results and analysis; Chapter 5 – Conclusion and recommendations.
LITERATURE REVIEW
Cheese and soft cheese overview
Dairy products are among the most widely consumed food throughout the world since they are rich in calcium and protein, which increases human metabolism and physical performance (Gharibzahedi and Jafari, 2017) Cheese is one of the popular dairy products, with a complex matrix and a rich supply of nutritious compounds (Nájera et al., 2003) Cheese has a variety of flavors and textures; therefore, it can use as a main course, a dessert, or an ingredient in other food products (Fox et al., 2017) In comparison to whole milk, with
3 – 4% protein and fat, it has 30 – 40% protein and fat, as well as other minerals and fat- soluble vitamins
Cheese is defined as a product coagulated wholly or parly the protein of milk, skimmed milk, partly skimmed milk, cream, whey cream or buttermilk, or any combination of these materials, via the action of rennet or other suitable coagulants, and by partially draining the whey resulting from the coagulation, while respecting the principle that cheese- making results in a concentration of milk protein (Codex Alimentarius, 1973) Consequenly, the protein content of the cheese will be distinctly higher than the protein level of the above milk materials from which the cheese was made Cheese is divided into two major groups, namely unripended and ripended cheese Ripened cheese is cheese which is not ready for consumption shortly after manufacture The ripened cheese has to be held for such a period time, at such temperature, and under such other conditions as will result in the necessary biochemical and physical changes characterizing the cheese Unlike ripened cheese, unripened cheese is cheese that can be consumed shortly after manufacture
Our study focuses on unripened cheese type, especially soft cheese Soft cheese is a kind of cheese without ripening, which is produced by coagulating milk, cream, or whey through acid coagulation; coagulation by heating and acidification, a combination of acid and rennet coagulation Soft cheese is ready- to eat after production Some traditional and unique kinds of cheese present the culture and characteristics of their origins These days, these unique soft cheese varieties are widespread due to globalization and tourism increase
In terms of nutritious, soft cheese is rich in proteins, calcium, phosphate and low in calories and it also is easy to digeset Therefore, it is highly recommended for everyone to use, especially for the elderly, children and those on diet Besides, soft cheese can use on their own or in sweet or savoury dishes Soft cheese is versatile and well – suited to being processed into cheese preparations or various dishes (e.g., cheesecakes, sauces, desserts)
Soft cheese is classified into different groups based on factors such as the method of coagulation: rennet – coagulated cheese, acid – coagulated cheese, acid/heat coagulated; structure of final product: gel, paste, or grain; manufacturing ingredient: whey or milk (Fox et al., 2004)
Acid – coagulation, no presence, or a small amount of rennet is added Acid is created from lactic acid bacteria (Labelle), however, some soft cheese varieties are direct – acidified by gluconodelta – lactone
Quark, Cream cheese and Northern- America soft cheese
Rennet – coagulation, no presence, or a small amount of LAB is added
Quesco, Balnco, Quesco Fresco, Halloumi, Cottage
Acid/heat coagulation These products are firm but have a high moisture content
Ricotta (Italy), Channe and Panner (India)
Paste Tvorog, cream cheese, double cream cheese
Adapted from Fox et al (2004)
Cottage cheese, Quark, and Tvorog are the more well-known soft cheese varieties than others in the marketplace (Fox et al., 2004) Quark (in German-speaking countries) or Tvorog (in Eastern European countries) is essentially a milk protein paste It has milky white to faintly yellowish colour; a smooth, homogenously soft, mildly supple and elastic texture,
Figure 2.2 Quark cheese mildly acidic and clean flavour Because of the high moisture content (82% w/w), the shelf life of Quark or Tvorog is about 2 – 4 weeks (Guinee et al., 1993) Quark cheese has a high nutritional value due to its high concentration of proteins
Cottage cheese is a soft, unripened, mildly – acid tasting cheese with discrete curd particles of relatively uniform size Cream Cottage cheese is dry- curd Cottage cheese covered with cream dressing Cottage cheese is made by coagulating acidically pasteurized skim - milk or reconstituted extra low – heat skim milk powder Therefore, cottage cheese is a nutritious and low-calorie food that contains less than 110 kcal/100 g (Farkye, 2004)
2.1.3 Chemical compositions of some soft cheese varieties
Cheese is a nutritious food that has a good deal of calcium, lipid and protein The nutritional value of cheese can be affected by milk such as milk source (cow, goat, or buffalo), milk type (whole milk, part – skim milk, or skim milk), manufacturing and ripening level
Insoluble nutrients of milk (coagulated casein, fat, insoluble minerals, fat-soluble vitamins) exist in final cheese products; whereas, soluble components (whey protein, lactose, water-soluble vitamin sand minerals) dissolve in water (O’Brien and O’Connor, 2004) The total solids content of soft cheese from 14 – 55% (Fox et al., 2004) is lower than that of ripened cheese varieties such as cheddar (66%) (Pastorino et al., 2003), blue cheese (57 – 60%) (Robin et al., 1993)
Table 2.2 Chemical composition of some cheese varieties
Total solid Fat Protein Sodium Calcium pH w/w (Carlson et al.) mg/ 100g
Coagulation by acidification and heating
Adapted from Fox et al (2017)
The chemical compositions of soft cheese in most of the countries has been regularized by Codex Standard For example, in Germany, Quark is defined as cheese with total solid at least 18% (w/w), protein content at least 12% (w/w) and a maximum of 18.5% of whey protein content in total nitrogen content (Schulz-Collins and Senge, 2004) In some countries, the regulation of cheese definition is less restricted, which moisture and protein content are required For example, Quark or Verse Kaas (Enwa et al., 2013) in Holland requires 87% (w/w) maximum moisture content and 60% (w/w) minimum protein content) in total non – fat solid (Schulz-Collins and Senge, 2004) American cream cheese (33% lipid, 45% total solids); Neufchatel (20 – 33% lipid, 35% total solid) (Kosikowski and Mistry,
Generally, the procedure of cheese production follows the below steps, described in Figure 2.3
Figure 2.3 The general procedure of soft cheese
Cottage cheese is a typical rennet coagulated soft cheese since commercial cottage cheese has been used as a reference product in this study
The official Codex Alimentarius standard for Cottage cheese lists pasteurized bovine skim milk as the raw material for manufacture, as well as the following authorized ingredients: lactic acid bacteria or direct-acidification, rennet or other suitable coagulating agents, CaC12 (maximum of 200 mg/kg milk), NaCl and water The coagulum is sliced into various – sized curd particles with specific knives, heated, then held for a sufficient amount of time to allow the curd to firm and the whey to drain The whey is drained until the curds reach the desired consistency (firmness) The curd is then washed, creamed (typically), salted and packed (Bodyfelt and Potter, 2008) Cottage cheese is consumed as soon as it is made (within 3 – 4 weeks)
2.1.5 Cheese production and consumption in Vietnam a Cheese consumption in Vietnam
Traditionally, cheese is not a staple in the Vietnamese diet since Vietnamese consumers only consume cheese occasionally rather than daily In recent years cheese consumption demand has gone an upward trend in Vietnam In 2019, the cheese sector in Vietnam grew relatively well, with consumption reaching more than 2,000 billion VND, an increase of 11% over 2018 (Virac, 2020) In comparison to other dairy products, consumption value in Vietnam still accounts for a small proportion Despite modest growth, the industry predicts to remain at a consistent growth rate of roughly 13% per year between
2020 and 2024 The steady growth of the segment reflected a gradual change in local tastes as more locals became exposed to new international foods
Demand for dairy products in Vietnam, cheese varieties especially, involves the general economic situation Vietnam has been growing young population (an estimated 56% of the Vietnam population is under 30 years old) and heightening income levels are good conditions for Vietnam to become a market for prospective consumption in the area According to YCP Solidiance Research and Analysis, the biggest motivators for people to purchase dairy were overwhelmingly health advantages and nutrition Furthermore, long shelf – life and ease of corporate with other foods may have contributed to dairy products being more than a staple in an average Vietnamese family
Ingredients of cheese manufacturing
Most cheeses in the world are made from bovine milk, and the remains are made from goat’s milk, buffalo’s milk and others The percentage of bovine milk made cheeses in the world accounts for 81%; while, percentages of cheese made from buffalo, sheep, goat’s milk is 11%, 2% and 1.5%, respectively (Fox et al., 2017)
Milk is the secretion of mammary glands in mammals, with the function of nourishing the young Milk from some animals such as cows, buffaloes, goats and sheep has been consumed by humans as milk liquid and other dairy products The main composition of milk is water, and the remains include lipid, lactose and protein (whey protein and casein) (Robinson et al., 2002) Lipid plays a significant role in human nutrition Unlike lipid from flora and fauna sources, milk lipid contains several fatty acids, vitamins (Thanh, 2003) Milk lipid absorbs easier than lipid of animals and plants because of its lower melting point Triglycerides have the highest amount in milk lipid, about 98% and 2% of milk lipid is phospholipids, monoglycerides, ganglioside and free fatty acid (Robinson et al., 2002)
Good-quality milk is a must for good – quality cheese production The milk for cheese production has to be free of contaminants, off – flavors, abnormal colour and odour The main factors include genetic factors, the stage of lactation, illness of the cow and feed is responsible for natural variation in milk composition These mentioned factors can affect the quality properties and functionality of cheese (Farkye, 2004)
Table 2.4 presents chemical compositions of fresh bovine milk
Table 2.4 Chemical compositions of fresh bovine milk
Moisture 84.93 – 85.07 Minerals 2.87 – 2.89 Proteins 3.27 – 3.31 Lipids 2.75 – 2.79 Lactose 4.96 – 4.98 Total solid 12.43 – 12.57
Adapted from Siddig et al (2016)
As fresh milk is considered as a perishable product, cheese made of fresh milk is produced mostly in countries or regions there are self – sufficient in raw milk production
Many developing countries such as East and Southeast Asia, North Africa and sub – Saharan Africa are unlikely to achieve self – sufficiency in dairy products because they could not be able to increase production as quickly as population and economic growth requirements In non – self – sufficiency dairy countries, the local milk businesses produce with imported milk powders material Processors prefer imported milk powder because they are often price-competitive compared to local suppliers, convenient, consistent in quality, constantly available and can be used to make almost every dairy product Skim milk powder is more likely used in dairy products production because it is easy to substitute and adjust fat
Skim milk is milk separated fat from the milk Skim milk powder is manufactured by evaporating skim milk into concentrated skim milk and subsequently drying it into powders The purpose of milk powder production is to turn a perishable liquid raw material into a commodity that can store for a couple of years without any quality loss Besides, milk powder is easy to handle, which decreases transportation costs
One of the crucial characteristics of powder products is insolubility It is the powder dispersion, meaning that the powder particles will become thoroughly wetted As a result, the soluble components dissolve and the colloidal particles (fat globules and casein micelles) disperse throughout the solution The insolubility index can be estimated by the fraction that cannot dissolve after centrifugation when milk powder dissolve in standardized conditions (Walstra, 1999) Insolubility varies from different kinds of milk, depending on drying method For example, the insolubility index of drum drying is 80 – 85 %, compared with 98 – 99.5 % of spray drying (Thanh, 2003)
Table 2.5 Chemical components of milk powder vary (calculated on %)
Components Whole milk powder Skim milk powder
In Vietnam, the current Vietnamese domestic raw milk has only been able to fulfil 30-40% of consumer demand has met the only production of drinking milk; therefore, the import of milk powder for dairy production is inevitable Milk powder is used widely in the Vietnamese food industry; for example, a) reconstituted milk and formulated milk production; b) chocolate production; c) a substitute of egg in the bakery industry; d) dairy products manufacturing (Thanh, 2003)
Milk powder has a low moisture content, about 2.5 – 3.0%, which is easy to handle and long self-life, making research more convenient Besides, it is easier to adjust the fat of reconstituted skim milk powder than reconstituted whole milk powder Based on practical and current trends in domestic production, in this research, we built a process of producing soft cheese using whole milk powder
Recombined and reconstituted milk provide a nutritious and high – quality source of dairy products in areas where a fresh raw milk supply is not readily available or is in short supply Reconstitued milk are prepared by dissolving single dried ingredient (e.g., whole milk powder or skim milk powder) into water; while recombined products can be prepared by combining more than one liquid or dried ingredients Recombined milk refers to the product obtained from recombining dairy fat source, skim milk powder and water in correct proportion to obtain fluid milk (Walstra, 1999) Within this research, we use recombined milk to produce soft cheese.
Casein coagulated mechanism
Milk protein has a high biological value, making it a great source of essential amino acids Milk protein, making up 3.3 – 3.9% of milk, is divided into two primary categories consisting of casein and whey proteins (also called serum proteins) Protein accounts for 3.3 – 3.9%, including 80% casein and 20% whey protein in bovine milk Casein and whey protein in milk can change during the feeding period Table 2.6 shows the concentration of milk protein compositions
Table 2.6 Protein compositions of bovine milk
Major milk protein Grams/ liter % of Total protein
Total whey proteins 𝛂-Lactalbumin 𝛃-Lactaglobumin BSA Immunoglobulins Protease peptone
Casein is a protein that precipitates from milk near pH 4.6 Casein is extensively associated and found in milk in the large aggregates, known as casein micelles containing colloidal calcium phosphate The biological functions of casein are to transport calcium and phosphate and form a clot in the stomach for efficient digestion
Casein micelles are hydrophobic and slightly charged These micelles exhibit considerable linkages, including both self-linkage and linkage with one another via hydrophobic links The phosphate groups contribute to casein's high charge The groups bind divalent ions like Ca 2+ efficiently, especially at higher pH Because of the highly charged, casein remains in the solution (Walstra, 1999)
Casein is formed from numerous components, mainly αs1-, αs2-, β-,k-casein with the molar ratio, is about 11 : 3 : 10 : 4 respectively The αs- and β- phosphoproteins with phosphate groups esterified to serine residues They precipitate with Ca 2+ , but k – casein prevents them from precipitation However, k-casein is easily attacked by rennet enzyme chymosin which splits off a portion of the k – casein molecules; it thereby loses its protective ability Since the caseins precipitate in the presence of Ca ions These reactions are the basis of the clotting of milk by rennet and thus cheese making At pH = 4.6 and above 20℃, casein is coagulated, which leads to curd formation during yoghurt or cheese making (Walstra, 1999)
Whey proteins have the following properties including heat-sensitive, globular, water-soluble proteins and enzymes (Hui, 1993) The main components of whey proteins are β-lactoglobulin, α-lactoglobulin, albumin and immunoglobulin (Walstra, 1993) Whey proteins are capable interact with themselves and k – casein to form heat – induced protein aggregates β-lactoglobulin is the predominant serum protein, therefore, its properties tend to dominate those of whey protein preparations, particularly the reactions with k- casein occurring during heat treatment (Walstra, 1999) Small β – lactoglobulin aggregates form, leading to larger β -lactoglobulin aggregates are formed (Jang and Swaisgood, 1990) during heat treatment Once the heating temperature and/ or heating duration are raised further, forming complexes with large denatured β – lactoglobulin aggregates and both proteins bind to the surface of casein micelles (Fox, 2003) Following denaturation of whey proteins, a reaction between the whey proteins and the k-caseins initially happen on the surfaces of the casein micelles, resulting in complexes of whey protein and k – casein (Oldfield et al., 2000) Additionally, whey protein aggregates form alone (Mahmoudi et al., 2007; Vasbinder and
De Kruif, 2003) Furthermore, hydrophobic interactions or disulfide between the casein fraction and the heat-denatured and whey proteins form when k – casein is present
2.3.2 Casein coagulation types in soft cheese manufacturing
Coagulation of casein of the milk protein system is a crucial step in the production of all cheese varieties, forming a gel that entraps the fat if present Coagulation can be accomplished by: a) limited proteolysis by rennet; b) acidification to pH 4.6; c) acidification to a pH value greater than 4.6 (perhaps 5.2) with heating to 90 ℃ Likely other cheese varieties, soft cheese is also produced by coagulation of milk, cream, or whey through acidification, acidification with a small amount of rennet, or acid and heat combination Soft cheese is ready for consumption once its production finishes
Adapted from Fox et al (2017)
Figure 2.5 Fresh/ soft cheese varieties classified through methods of coagulation
Quark and Labneh are typical and conventional soft cheese products in that they are fermented/acidified milk from which some whey is removed to recover the gelled solids in a more concentrated form These products differ in production specifics (e.g., temperature, shear), resulting in sensory changes that lead to distinctive products suited to different regions, the most notable being Chakka (India)
Acid/heat coagulated cheeses, such as Mascarpone, Ricotta, Paneer are typically made by acidifying the cheese milk to a pH of 5.4 – 6.0, heating to 80 – 90 ℃ and recovering the curds
The most well-known rennet – coagulated soft cheese is Cottage cheese It is a soft granular, unripened cheese with curd granules thinly covered in a salted cream dressing; the flavor ranges from cream-like blandness to moderate acidity Cottage cheese is produced of coagulated skim milk with acid and a small amount of rennet Rennet serves two major purposes: provides a firmer gel and minimizes casein losses during subsequent whey separation (Fox et al., 2017) Table 2.7 distinguishes rennet - coagulated soft cheese from rennet – coagulated hard cheese (or ripened cheese)
Table 2.7 Rennet – coagulated soft cheese and ripened cheese comparison
Characteristic Rennet – coagulated cheese Ripened cheese
Texture Softer and less chewy Fimer and more chewy
Shelf – life Shorter shelf – life (4 weeks) Long shelf – life (up to 2 years) Others Do not require ripening
Are consumed immediately after producing
Must be ripened at time period Are not ready to consume after producing
Adapted from Fox et al (2017)
The focus of this study is on acid and rennet – coagulated cheese; therefore, the mechanism of gel formation using acid and rennet is briefed in the following parts Acid usage aims to create an optimal pH for rennet action at the first stage (Fox et al., 2017) Various kinds of acid can be used in cheesemaking, such as citric acid, malic acid, lactic acid, etc Citric acid is used within this study since it is accessible, affordable, effective and commonly used in food production.
Citric acid
Citric acid, a tricarboxylic acid (C6H8O7.H2O), is a metabolite of plants and animals and is found in citrus and pineapple juice Citric acid is colorless, soluble in water and has a molecular weight of 210.14 g/mol
Citric acid manufacturing via fermentation is the most cost – effective and widely used method to produce this product More than 90% of the citric acid produced in the world is derived through fermentation, which has several advantages: operations are simple and steady, the plant is generally less complicated and requires less sophisticated control systems, the technical skills required are fewer and frequent blackout do not harm the plant's operation (Soccol et al., 2006) A wide range of microorganisms, including fungus and bacteria have been applied for citric acid production Aspergillus and Candida are some of the most popular microorganisms for the biosynthesis of citric acid (Angumeenal and Venkappayya, 2013)
Citric acid is a functional and safe food additive It is accepted worldwide as GRAS (generally recognized as safe) and has been authorized by the Joint FAO/WHO Expert Committee on Food Additives (Soccol and Vandenberghe, 2003; Soccol et al., 2008; Vandenberghe et al., 1999) Citric acid is widely used in the food and pharmaceutical industries due to its general safety, pleasant acid taste, high water solubility and chelating and buffering properties Other uses for citric acid include detergents and cleaning products, cosmetics and toiletries and others Table 2.8 presents applications of citric acid in food and beverage industry
Table 2.8 Acid citric applications in food and beverage industry
Beverages Wine and ciders Prevents browning in some white wines
Prevents the turbidity of wines and ciders Used as pH adjustment
Provide tartness Impart natural fruit flavor
As acidulant in carbonated and sucrose- based beverages
Food Jellies, jam and preservative
Used as pH adjustment Act as an acidulant Impart the desired level of tang, tartness and flavor Enhance the effectiveness of antimicrobial preservatives
Candies Act as an acidulant Impart tartness
Minimize sucrose inversion Create yellowish color in hard candies Prevent crystallization of sucrose
Frozen fruits Protects ascorbic acid by inactivating trace metals Reduce pH value to inactivate oxidative enzymes
Fats and oils Synergist for other antioxidants, as a sequestrant Stabilizing action
Dairy products As emulsifiers in ice creams and processed cheese Acidifying agent and antioxidant in many cheese products
Adapted from Soccol et al (2006)
There are some kinds of cheese produced by acid and heat coagulation such as Quark, Paneer (India), or Ricotta (Italy) Acid/ heat coagulated cheese generally is fresh, softand unripened (Farkye, 2004) Besides, unripened rennet coagulated cheese such as “Italian soft cheese” and Queso Blanco (Latin America) also is considered as soft cheese
Direct acidification can be used as an alternative to biological acidification and is commercially used in Cottage, Quark, Feta – type cheese production The obvious benefit of direct acidification is increaseing cheesemaking efficiency and eliminating potential difficulties connected with starter performance in cheese vats (such as agglutination, bacteriophage and antibiotics) (Sharma et al., 1980) The direct acidification method can improve soft cheese quality (increase the fat content and total solid cheese) and bring economic benefits through reducing production costs and time.
Rennet
Rennet and coagulants are proteolytic enzyme preparations that have been employed in cheesemaking for thousands of years and they appear to be the oldest known application of enzymes Historically, the name "rennet" was referred to as the crude extract from a bovine calf stomach However, an FAO Committee has proposed the use of the term "rennet" preceded by the name or source of the particular enzyme for all milk – clotting enzyme preparations, e.g., calf rennet, animal rennet, plant rennet, microbial rennet (Garg and Johri,
1994) After that, a study suggested that the name ‘rennet’ should be reserved for enzyme preparations from ruminant stomachs, whereas other milk-clotting enzymes should be named coagulants (Andrén, 1998)
Chymosin (EC 3.4.23.4) is a gastric proteinase that is released from the mucosa of the abomasum of newborn ruminants and other mammals during the first days of life (Foltmann, 1992) Chymosin is the primary clotting enzyme component of calf rennet Chymosin has a low overall proteolytic activity but is especially active in the hydrolysis of the Phe105-Met106 in the milk protein k – casein
2.5.2 Types of rennet or coagulants
Cheese production has increased, as has demand for rennet However, the scarcity of calf rennet (derived from calves' stomachs) and the growing vegetarian preferences, besides some cultural and religious use restrictions, have incentivized for new natural sources of milk-clotting enzymes New natural coagulants come from different sources such as animal, microbial, fermented – produced and vegetable The difference between milk-clotting enzymes is the rate that continuously degrades caseins after the hydrolysis to initiate gel degree during cheese manufacture depending on the amount of the curd is in contact with the whey and the curd pH at drainage (McSweeney, 2007) Table 2.9 gives information about rennets that are substitutes for calf rennet
Table 2.9 Enzyme coagulants varieties of milk
Animal rennet Microbial rennet Fermented –produced chymosin (FPC)
Origins From dried stomachs or frozen stomachs of adult cow, goat and sheep
Rhizomucor miehei is common microbial coagulant use in cheesemaking
Fermented by Genetically Modified Organisms (GMO), namely
Aspergillus niger and Kluyveromyces marxianus var lactis
Advantage Slight differences compared to calf rennet (Harboe et al., 2010)
Are used to a larger extent, primarily due to a relatively low price (Andrén,
High milk-clotting activity ratio, with a slight difference in cheese yield compared to calf rennet (Banks, 1992;
Emmons and Binns, 1990; Green, 1985; Hicks et al., 1988; Vandenberg,
High specificity, purity, robust curd formation that fulfil the requirements from various consumer groups/segments, e.g religious or vegetarian
Disadvantage Fluctuated price due to varying availability of stomachs, or due to veterinarian crises, such as mad cow disease
High proteolytic activity, leading to lower cheese yield compared to animal rennet (Emmons and Binns, 1990;
Depend on the acceptance of products GMOs products by consumer
These mentioned enzymes can clot milk; however, excessive proteolysis can result in undesirable tastes in cheese (Jacob et al., 2011) As a result, an appropriate selection of milk - clotting enzyme for cheesemaking is crucial, as it can affect yield and cheese qualities Animal rennet is similar to calf rennet in term of yield, cheese quality and sensory characteristics Besides, rennet extracted from animal has been used for a long time, so it is popular Cheese coagulated by animal rennet is more acceptable and suitable for the majority consumers compared with other coagulants Thus, we decided to use animal rennet as a coagulant in soft cheese production.
Mechanism of gel formation during combined acidification and renneting
The coagulation of milk by proteolysis is a key operation of cheesemaking Figure 2.6 discusses the principile of the rennet coagulation process into various steps
Figure 2.6 Description of gel formation during combined acidification and renneting
Adapted from Schulz-Collins and Senge (2004)
The enzymatic (rennet – induced) coagulation of milk is generally separated into two phases: (1) hydrolysis of the micelle-stabilizing protein, k – casein, and (2) aggregation and gelation of the rennet-altered micelles, with the formation of a particulate gel The primary enzymatic phase and the secondary phase (non – enzymatic phase) is discussed in the following parts
2.6.1 Primary enzymatic phase κ-casein is the only protein hydrolyzed during the rennet-induced coagulation of milk and that it is hydrolyzed specifically at the Phe105-Met106 bond The k-casein molecules provide a steric stabilizing layer with their hydrophilic C – terminal peptides protruding into the aqueous phase The proteolysis of the k – casein molecules initiates gel formation, followed by the release of a hydrophilic peptide known as the casein micelle properties into the serum phase (or the whey phase) The remaining N – terminal segment of k – casein, known as para-k-casein, is still connected to the casein network Gradual loss of the casein micelle properties is accompanied by a drop in the micellar zeta potential, resulting in micelle instability and aggregation, eventually forming a gel (Horne and Lucey, 2017)
During the primary phase of rennet action, hydrolysis of k-casein by chymosin or related enzymes releases the highly charged, hydrophilic C – terminal portion of k-casein (glycomacropeptide), lowering the zeta potential of the casein micelles and removing projecting peptides hairs from their surfaces, leading to destroy the principal micelle- stabilizing factors (electrostatic and steric) and their colloidal stability When about 85% of the total k-casein is hydrolyzed, the stability of the micelles is lowered to the point at which they collide, they stay in contact and eventually form a three-dimensional network known as a coagulum or gel (Fox et al., 2017)
Figure 2.7 Summary of the rennet coagulation of milk The primary phase involves enzymatic hydrolysis of κ – casein, while the secondary phase involves aggregation of the rennet- altered (para – casein) micelles into a three-dimensional gel network (coagulum)
Research situation
Rennet-coagulated cheese has been studied continually for decades and continue to be an interest
Lablee (1980) described the use of recombined milk made from skim milk powder and anhydrous milk fat in the ultrafiltration production of soft surface – mold cheeses, semi – cooked pressed cheeses, processed cheese and white cheese with 35% solids The procedures produced satisfactory results and they are suggested for usage in areas where local milk production is limited
Guinee et al (1993) showed that numerous cheese varieties can be produced satisfactorily from recombined milk using low heat skim milk powder and anhydrous milkfat However, unripened cheese usually has a faint "anhydrous milk fat" or "powder" flavor, hence, that is not acceptable for the manufacturing of fresh, white cheese On the other hand, they also pointed that the use of starter culture is advantageous to reduce off – flavor which comes from the oxidation of anhydrous fat used Starter culture lows down pH value due to acid development, leading to reduce active oxygen
Jelen and Renz-Schauen (1989) presented that a small amount of used rennet can improve the draining characteristics of the curd, to reduce casein fines and increase curd firmness in the production of Quark (German – type cheese) and Tvorog (Russia)
Guinee et al (1993) pointed that during acidification, the rennet activity promotes the destabilization and aggregation of casein micelles For example, the ratio of aggregation to disaggregation forces is increased in the early phases of acidification Other studies also showed that when a small amount of rennet is added at the beginning of acidification, the enzymatic reaction is hastened by decreasing the pH, resulting in much firmer gels (Herbert et al., 1999; Lehmann et al., 1991; Lucey et al., 2000; Schulz et al., 1999; Tranchant et al., 2001)
Krekker (2020) conducted the possibility of obtaining soft cheese prepared by acid- rennet coagulation and using calcium and magnesium salts, as well as a combination of starter cultures with the addition of streptobacteria The results showed that the optimal conditions for producing acid-rennet coagulated soft cheese under conditions of combined usage of calcium and magnesium salts have been identified, which reduces the protein loss in whey and increases magnesium and calcium content in the final soft cheese
Pakulski and Bagnicka (2017) determined the effect of the production technology of soft white unripened from sheep milk on amino acids The milk – sheep cheeses were produced with four following methods: (i) “traditional” rennet method control (Lehtinen et with heat treatment of the curd As a result, acid-rennet coagulated cheese had the highest content of essential and non-essential amino acids
Cheese coagulated by citric acid and rennet has been found for a couple of years Additionally, due to the abundance of raw milk sources in these countries, fresh raw milk is mainly used as the cheesemaking ingredient in the studies There are not many types of research about cheese produced by milk powder Besides, studies also revealed that there are some advantages regarding texture and amino acids content of rennet-coagulated cheese compared with acid-coagulated cheese
There is no soft cheese produced from local dairy companies in the domestic market Soft cheese varieties in the local market are mainly imported from Italy, Poland, Germany, France The reason is that the amount of domestic – produced raw milk is only enough to meet the need for drinking consumption In addition, the demand for soft cheese products of Vietnamese people is still not high
There has been study about cheese products of our research team which is the study of Hoan et al (2020) and the master thesis of Chi (2019) The authors studied MTGases applied to produce fresh cheese made from whole milk powder The purpose of theses studies was optimization of dairy treatment process with transglutaminase in the manufacture of fresh cheese coagulated by acid from yogurt starter culture However, acid – coagulated cheese has the disadvantage that it has acidic flavor and its texture is grainy and is not smooth There has been no published papers in our country about soft cheese production from skim milk powder and coagulated by rennet and acid
Thus, this is also another approach of our research team into the soft cheese variety Our study aims to produce a kind of soft cheese that is coagulated by acid and rennet combination The purpose of this new soft cheese kind is to diversify cheese products in the Vietnam market and increase cheese demand in daily life Skim milk powder is the main ingredient in our soft cheese production That seems to be proper for the current Vietnam dairy industry since raw milk material is not enough to meet the production and consumption.
MATERIALS AND METHODS
Materials, chemicals and equipment
Ingredients for soft cheese manufacturing include skim milk powder, whipping cream, animal rennet and citric acid
Skim milk powder was supplied by Fonterra Ltd company, 109 Fanshawe Street, Auckland, New Zealand and distributed by Khoi Minh Food company at 108/799 Nguyen Kiem Street, Ward 3, Go Vap District, Ho Chi Minh City The compositions of skim milk powder are presented in Table 3.1
Table 3.1 Compositions of skim milk powder
Adapted from the COA of skimmed milk powder (Appendix 2)
Whipping cream powder is manufactured by NBC Food Industries Sdn Bhd and bought at Bep Nha Sau store at 53, 106 street, Tang Nhon Phu A ward, 9 District, Ho Chi Minh city The compositions of whipping cream powder are shown in Table 3.2
Table 3.2 Compositions of whipping cream powder
Adapted from COA of whipping cream powder (Appendix 3)
Naturen Extra 220 Rennet extract was manufactured by Chr Hansen company, Denmark and we bought this product from Russia (website: https://moskva.pro- syr.ru/pepsin-i-sychuzhnyj-ferment/zhidkiy-ferment-30-mL/) The rennet product is extracted from the animal with the ratio chymosin: pepsin = 95:5
Powdered citric acid was purchased at Hoa Nam Laboratory Chemical Equipment Trading Company, located at 22/76 Cu Xa Lu Gia, Ward 15, District 11, Ho Chi Minh city The quality criteria of citric acid are shown in appendix 4
Chemicals were sodium hydroxide (NaOH); hydrochloric acid (HCl) 0.1N; Zinc oxide (ZnO); 0.1N sulfuric acid (H2SO4); ammonia solution (NH4OH); diethyl ether (C4H10O); petroleum ether 60 – 90; cooper sulfate pentahydrate (CuSO4.5H2O); phenolphthalein (C20H14O4); lactose; acetone (C3H6O); which were purchased at Anh San Service Trading Co., Ltd, located at 270A Ly Thuong Kiet Street, Ward 14, District 10, Ho Chi Minh city
Equipment included Memmert water baths (Germany); Brookfield CT3 Texture Analyzer; CR – 400 Chroma Meter; Hanna HI9124 Waterproof Portable pH Meter; Sartorius BL210S Analytical Balance; IKA disperser; Memmert drying oven (Germany); incubator, water distiller, freeze dryer, vortex shaker, autoclave machine, refrigerator, electric stove
Instruments were micro pipets 10 – 1000 àl; micropipettes 20 – 200 àl, tips, test tubes, Eppendorf tubes, pipettes, burets, beakers, measuring cylinders, dispensing bottles, Petri plates, erlens, pipet bulbs, glass stirring rods.
Soft cheese manufacturing
The soft cheese manufacturing is illustrated in Figure 3.1
Enzyme concentration and temperature in survey range
Citric concentration in survey range equivalent to a decrease pH from 6.5 to 5.5
Figure 3.1 The flowchart of soft cheese production coagulated by rennet and citric acid
Soft cheese production involves these following steps i Recombination and Standardization
Skimmed milk powder was mixed with whipping cream powder with a ratio of 12.5 to 2.5 Then the mixture was dissolved with 85 portion of water to obtain recombined milk with the total solid content approximately 15%
The recombined milk (300g) was made by mixing 39 g of skimmed milk powder and
9 g of whipping powder in 252 g of water at 45-50 ℃ The milk solution was stirred and stood for 30 minutes until completely dissolved The obtained recombined milk had the total solid content approximately 15% ii Homogenization
The purpose of homogenization is to reduce the size of fat globules, helping them evenly to distribute in the milk
The recombined milk solution (300 g) was homogenized by an IKA disperser whose setting value was 4000 rpm for 10 minutes iii Pasteurization
Pasteurization is a heat treatment process that destroys pathogens in food, increasing food safety for consumers Pasteurization also increases food quality by destroying microorganisms and enzymes that might reduce food quality
The homogenized milk (300 g) was pasteurized at 85℃ for 15 minutes (Şanlı et al., 2011; Yüksel and Erdem, 2010) iv Cooling
The milk (300 g) after pasteurization was lowered to the room temperature v Acidification
Citric acid is directly added to the milk to achieve the desired pH
The pasteurized milk (300 g) was lowered to the room temperature The amount of citric acid (10 mL) with the survey concentration ranges (0.05, 0.1, 0.15, 0.2, 0.25%) was added to the milk vi Coagulation
Coagulation of casein forms a gel, which is usually achieved by rennet addition Rennet cleaves the glycomacropeptide from k – casein during coagulation, causing the destabilization of casein micelles and subsequent aggregation
Rennet was added to the recombined milk (300 g) with the survey concentration ranges (0.01; 0.025; 0.04; 0.055%, v/v) followed acidification The milk was stood for 45 minutes until the milk is completely curdled and proceed to extract the whey vii Whey separation
Dehydration or syneresis of the coagulum, which is the loss of whey as the result of pressing the curd This step purpose is to separate whey protein and shape soft cheese
The whey separation method was carried out in the same way as described by Everard et al (2011) with some modifications as described in Figure 3.2 The formed curd after acid and rennet coagulation was pressed into a cylindrical mold with a height of 2.0 cm and a diameter of 5.0 The curd was placed in the mentioned cylindrical mold with perforated holes with a diameter of 2.0 mm on the mold wall (Figure 3.2), the mold inner surface was lined with a thin cloth and the bottom of the mold is sealed
Figure 3.2 Schematic of cheese curd pressing apparatus
Adapted from Everard et al (2011)
The curd was gently inserted into the mold At the initial period of the process, whey came out on its as a result of being compressed by the weight of overlapped curds and whey escaped through holes in the mold wall When the whey was no longer separated easily, a cylindrical block weighs 1 kilogram was placed on the top of the curd and the cloth The cheese was press continuously with the block for about 15 minutes After that, the final cheese was collected The final cheese was stored in the fridge at 4 ± 2℃
Figure 3.3 The cylindrical draining mold (left) and the 1-kg cylindrical block
Research plan
Note: H is cheese yield (Carlson et al.), %TSwhey and % TScheese are total solid whey and cheese respectively, CSY (Carlson et al.) is the percentage of total cheese solid compared to total milk cheese solid
Experiment 1 : Investigate the effect of citric acid concentration (0.05; 0.1; 0.15; 0.2 and 0.25%) on protein coagulation
Experiment 2 : Investigate the effect of temperature (30;
Experiment 3 : Investigate the effect of rennet enzyme concentration (0.01; 0.025; 0.04 and 0.055%) on protein coagulation
Determine %H, %TSwhey, %TScheese, CSY (soild cheese yield), texture analysis
Investigation of the effect of citric acid and rennet on protein coagulation
Determine chemical compositions (moisture, ash, proteins, lipids, carbohydrates) of ingredients
Chemical compositions: dry matter , protein, lipid, acidity, pH
Microbiological criteria: Listeria monocycogenes and coagulase-positive Staphylococci
Microstructure observation of soft cheese (SEM)
Sensory properties of soft cheese
Evaluation of soft cheese quality during storage time
Evaluation of soft cheese quality
Evaluate soft cheese quality (acidity, whey separation, texture, colorness) change after 1, 7, 14, 21, and 28 days of storage
Experimental designs
3.4.1 Experiment 1: The effect of citric acid concentration on protein coagulation
The experiment is adapted from the study of Dussault-Chouinard et al (2019) and optimized for our study
The purpose of this experiment is to determine appropriate citric acid concentration when combined with rennet for casein coagulation in soft cheese production Fixed factors were recombined milk weight (300 g), total solid content of recombined milk (15%) and rennet concentration (77 μL) Investigated factors were citric acid concentration 0.05 (CA05); 0.1(CA10); 0.15 (CA15); 0.2 (CA20) and 0.25 % (CA25) (respectively decreasing pH from 6.5 to 5.5)
Procedure: Experiment 1 was implemented as follows Citric acid was first prepared with the concentrations shown above (0.05; 0.1; 0.15; 0.2 and 0.25%) The milk was recorded pH and acidity through titration with 0.1N NaOH before and after citric acid
Figure 3.4 Experiment 1 layout addition Following that, rennet was added and stood for 45 minutes until pale green whey appeared; coagulation was completed
The following criteria of soft cheese were determined including cheese yield,
%TSwhey, %TScheese, %CSY and cheese texture analysis by CT3 Texture Analyzer After that, the citric acid concentration used for casein coagulation in soft cheese production was conducted
3.4.2 Experiment 2: The effect of incubation temperature on protein coagulation
The experiment was designed based on studies of Nájera et al (2003) and Ong et al
The experiment's purpose is to determine the appropriate incubation temperature for the soft cheese coagulation step Experiment 2 was performed with the same layout as experiment 1 Fixed factors were recombined milk weight(300 g), the citric acid concentration conducted from experiment 1 (section 3.4.1), rennet concentration (0.025%) The investigated factor was incubation temperature of the milk during coagulation including
10 mL citric acid with determined concentration in experiment 1 (0.01%) and 0.025% (77 μL) of rennet were added into the pasteurized milk The milk was subsequently incubated at different mentioned temperatures for 45 minutes
Figure 3.5 Experiment 2 layout (A) and milk coagulation image (B)
The following criteria of soft cheese were determined including cheese yield,
%TSwhey, %TScheese, %CSY and cheese texture analysis by CT3 Texture Analyzer After that, the appropriate incubation temperature used for casein coagulation in soft cheese production was conducted
3.4.3 Experiment 3: The effect of rennet concentration on protein coagulation
The experiment was adapted from the study of Santoso et al (2020) and optimized for our study with some modifications
The experiment’s purpose is to determine the appropriate rennet concentration for casein coagulation of soft cheese production Fixed factors were recombined milk weight(300 g), citric acid concentration and incubation temperature are chose from the results of experiments 1 (section 3.4.1) and experiment 2 (section 3.4.2), respectively The investigated factor was rennet concentration including 0.010% (E01), 0.025% (E025);
Procedure: 10 mL citric acid concentration conducted from experiment 1 (0.01%) and rennet concentration of 0.010%; 0.025%; 0.04% and 0.055% (v/v) were added into pasteurized milk The milk was then incubated at 37 ℃ determined temperature in experiment 2 (section 3.4.2) until pale green whey appeared; the coagulation was completed
The following criteria of soft cheese were determined including cheese yield,
%TSwhey, %TScheese, %CSY and cheese texture analysis by CT3 Texture Analyzer After that, the appropriate incubation temperature used for casein coagulation in soft cheese production was conducted.
Analysis methods
Cheese yield is defined as the amount of cheese in kilograms, obtained from 100 kg of milk It is an important parameter in cheese production The higher cheese yield means the larger amount of cheese obtained, gaining economic benefits Cheese yield can evaluate the economic efficiency of cheese making In addition, several factors affect cheese yield including milk composition, amount and genetic variants of casein, milk quality, milk pasteurization, coagulant type, vat design, curd firmness at cutting and manufacturing parameters (Abd El-Gawad and Ahmed, 2011)
Soft cheese weight after whey separation was determined by a 4 – digit analytical balance The yield of soft cheese was calculated according to the following equation:
With, m is the weight of soft cheese (g)
M is the weight of recombined milk (g)
Cheese solid yield (CSY) is defined as the ratio of total soils of soft cheese to the total solids in milk (Salinas‐Valdés et al., 2015) CSY is calculated according to the following formula
H is the cheese yield (Carlson et al.) m TS cheese is the total solid in cheese (g) m TS milk is the total solid of recombined milk (g)
Whey separation is determined following the method of the study of El-Kholy
(2005) Soft cheese samples were weighed (with a precision of 0.01 g) and placed in zip – lock bags The whey leached out of the sample was recorded after 20 hours at 25 ℃ Based on the weight differences, the percentage of separated whey was determined (Dmytrów et al., 2010)
Whey separation is equal to the net change in weight divided by the original weight of cheese after cutting
Where, m 1 is the weight of whey separated from the soft cheese sample (g) m 0 is the weight of the initial cheese sample (g)
Soft cheese acidity was determined according to TCVN 6509:2013 (ISO/TS 11869:2012)
Acidity is the volume, in milliliters, of 0.1 mol/l sodium hydroxide solution required to titrate 10 grams of product to pH 8.3 ± 0.1 (titrated acidity is expressed in millimoles per
Cheese samples were cooled to about 22 ± 2℃ Approximately 10g (with a precision of 0.01 g) of the prepared sample was then put in a zip-lock bag 10mL of distilled water was added to the zip bag containing the sample and mixed well The sampled solution was transferred to an erlen and some drops of phenolphthalein were added and mixed well The amount of acid contained in the cheese sample was titrated with 0.1N NaOH solution until the solution turns pink and the color was not disappeared in 30 seconds The used NaOH volume was recorded, in milliliters, with an accuracy of 0.05 mL The experiment was triplicated
The titratable acidity is calculated as follows:
V is the volume of NaOH used for titration m is the sample weight (g)
Texture profile analysis was performed on cheese samples using the Brookfield CT3-
4500 Texture Analyzer to evaluate hardness, adhesiveness and springiness with a probe TA- AACC36 (36 mm in diameter) and fixture TA-BT-KIT Parameter for measurement of cheese texture was presented in Table 3.3
Table 3.3 Parameters and setting values of Brookfield CT3 Texture Analyzer
A cylinder probe (TA-AACC36) 36 mm in diameter
The cheese sample was cut into a shape of a cylinder (20 mm in diameter, 20 mm in height) from the cheese center by using a sharp knife and kept refrigerated (4 ± 2℃) in a closed container before analysis to prevent moisture loss After being cut, the cheese sample was left at room temperature (25 ℃) for 30 minutes before analyzing texture (Buriti et al.,
1 : Cylindrical probe; 2: Soft cheese sample; 3: Fixture Figure 3.6 Texture analysis layout of soft cheese samples 3.5.6 Sensory evaluation
Sensory properties of cheese was evaluated based on TCVN 10565-3:2015 (ISO 22935-3:2009) with a panel of 5 people who had highly trained Each panelist was independent of the others and used a separate 5 – point scale to estimate the standard deviation of products sensory properties compared with the established product sensory specification The result of the sensory evaluation was the mean values of the panel members
Evaluated sensory attributes were appearance, texture and flavor The appearance was evaluated through the color and smoothness of the cheeses The texture was judged by hardness (defined as the force required to bite entirely through a sample placed between molar teeth) and firmness (defined as the amount of resistance to compression offered by a
1 cm thick slice of cheese when pushed between the thumb and the index finger until fingers touch each other) (Clark et al., 2009) The term ‘‘flavor’’ has many definitions but within this study, this term will be defined as the ‘‘impressions perceived via the chemical senses from a product in the mouth’’(Clark et al., 2009) Sensory evaluation sessions were conducted in the separated booth under fluorescent light The samples were coded with three random digit numbers and presented nomadically The testing room was cleaned without a strange odor Technical definitions of sensory attributes of soft cheese are attached in Appendix 1
According to Vietnamese standard (QCVN 5/3:2010/BYT), the microbiological criteria of soft cheese product made from milk or whey (heat – treated milk or whey) are specified in Appendix 6
Listeria monocytogenes was determined according to TCVN 7700-2:2007 (ISO
Principle: The quantitative detection of L monocytogenes consists of five consecutive stages The first stage is preparation of an initial suspension in each specified dilutions; After that, a defined amount of the initial suspension is inoculated on the petri dish which contained the culture of listeria agar Next, other plates are prepared, while using decimal dilutions from the initial suspension under the same conditions These plates are incubated at 37 ℃ and checked after 24 h and 48 h In addition, some specified tests are implemented to confirm colonies of presumptive L Monocytogenes From the number of
Figure 3.7 Preparation of soft cheese sample for sensory evaluation confirmed colonies, we calculate the number of L monocytogenes present in one gram of the test sample
Enumeration of coagulase-positive Staphylococci
Coagulase-positive Staphylococci was determined according to TCVN 4830-1:2005 (ISO 6888-1 : 1999, AMD 1 : 2003)
Principle: The amount of initial suspension was inoculated on the surface of a selective solid culture Decimal dilutions of the initial suspension were inoculated Then the plates were incubated aerobically at 35 ℃ (or 37 ℃) and checked after 24 hours and 48 hours Eventually, we calculate the number of coagulase – positive Staphylococci in one gram of sample from the number of typical and/or typical colonies on the plates in the selected dilutions The result is significant and confirmed with a positive coagulase test
The color space for the fresh cheese samples was determined by a CR – 400 chroma meter (Minolta, Japan) according to the study of Mokrzycki and Tatol (2011) The CR – 400 tristimulus colorimeter employs the D65 illuminant method, which is extremely similar to conventional visual evaluation and may be utilized with a wide range of samples The CR –
400 can take an average color reading from an 11 mm diameter measuring area that roughly matches CIE 1913 observers (Zhu et al., 2009)
Currently, the color of food is represented using two – and three dimensional spatial value and color systems Although there are many color spaces, the most widely used color space in the food industry is L*a*b* color space (Tarlak et al., 2016)
L* is the value representing the color from black (0) to white (100) a* is a chromatic component ranging between -120 and +120, from green to red b* is a chromatic component ranging between -120 and +120, from blue to yellow
The color meter was calibrated before measuring the color of the samples L*, a*and b* value of samples was recorded The difference in color is defined as ∆E* that is calculated according to the following formula (Rhim et al., 1999)
Based on the △E value, the difference in color between the samples was expressed as (Adekunte et al., 2010)
• 0 < △E