Isolation of lactic acid bacteria apply in tofu producing process

Research rationale

Tofu, a key food derived from soy protein, is highly valued in Southeast Asia for its exceptional nutritional content and digestibility Recognized by the FDA in 1999 for its health benefits, tofu has seen a surge in popularity in Western countries in recent years, reflecting a growing awareness of its nutritional advantages.

Tofu and soy products are believed to offer significant health benefits, potentially reducing the risk of chronic diseases such as cancer, heart disease, and osteoporosis Research indicates that soy protein, rich in isoflavones and isoplavolesterol, can effectively combat atherosclerosis Additionally, soybean protein has a lesser impact on total cholesterol, LDL, and triglycerides compared to animal protein The antioxidant properties of isoflavones, aglycones, and proteins found in tofu also play a crucial role in protecting against lipid oxidation.

Tofu offers numerous health benefits and is widely consumed by both urban and rural populations in Vietnam, creating a stable market for this popular dish However, tofu production in the country is predominantly small-scale and relies on outdated technology and equipment Most tofu products lack quality registration and proper packaging, resulting in a limited shelf life of just 1-2 days post-production This short shelf life not only leads to product waste but also restricts distribution and commercialization opportunities for tofu in the market.

The protein coagulation process in tofu primarily involves the use of food-grade chemicals such as calcium sulfate (CaSO4), magnesium chloride (MgCl2), citric acid, and calcium chloride (CaCl2) (Murugkar, 2015) However, some tofu manufacturers may utilize impure chemicals, resulting in metal residues in the final tofu product.

Lactic bacteria are gaining significant attention in food technology due to their numerous benefits, particularly as effective antibacterial agents produced during lactic fermentation Ongoing research is increasingly focused on utilizing lactic fermentation and its byproducts as biological preservatives, which have shown promising results in reducing mycotoxins in food.

Therefore, I conducted the topic: " Isolation of lactic bacteria apply in tofu producing process".

Research objective

Isolation and selection of lactic acid bacteria with good fermentation ability and application in tofu production process as a coagulant

Detail goals

- Isolation some strains of lactic acid bacteria

- Selection of bacteria with good fermentation ability apply for tofu production

Limitation that are expected to be encountered throughout the study

- Language barrier: Since this report is conducted in English, so the study would have some obstacles due to the difference of language.

The research conducted at the Faculty of Food Biotechnology and Food Technology at Thai Nguyen University of Agriculture and Forestry faces challenges due to a lack of specialized equipment and chemicals essential for the study.

Limitations

Soybeans, scientifically known as Glycine max Merril, are short-term legumes that typically grow between 80 to 150 days, reaching heights of 30 to 80 cm depending on the variety Characterized by their relatively upright growth and fewer branches compared to other legumes, soybean plants produce fruits in clusters, with each plant yielding nearly 400 fruits Each fruit, slightly curved and averaging 4 to 6 cm in length, contains 1 to 7 seeds Soybeans come in various shapes, including round, oval, long circle, and flat circle, with colors ranging from yellow-green and gray to black, though yellow is the most common The soybean structure consists of three parts: the shell, cotyledons, which serve as nutrient reserves, and the embryo.

Unlike cereal grains, soybean seeds lack an aleurone layer, with the endosperm and embryo existing separately The entire soybean is essentially a large embryo encased in a seed coat As a result, soybeans have lower starch content compared to other legumes, but significantly higher levels of protein and lipids.

2.1.2 Acreage, yield and demand for soybeans

2.1.2.1 Production situation in the world

The homeland of soybeans is East Asia, but nearly 60% of the world soybean

REVIEW

OVERVIEW OF SOYBEAN

Soybeans, scientifically known as Glycine max Merril, are short-term legumes that typically mature in 80 to 150 days They grow upright, reaching heights of 30 to 80 cm, and have fewer branches compared to other legumes Each soybean plant produces approximately 400 fruits, with each fruit containing 1 to 7 seeds The fruits, which are slightly curved and measure about 4 to 6 cm in length, come in various shapes including round, oval, and flat, and can be yellow-green, gray, or black, although yellow is the most common color Structurally, soybeans consist of three parts: the shell, cotyledons, and embryo, with the cotyledons serving as the nutrient reserve.

Unlike cereal grains, soybean seeds lack an aleurone layer, with the endosperm and embryo existing separately The entire soybean is essentially a large embryo encased in a seed coat As a result, soybeans have lower starch content compared to other legumes, but they are significantly richer in protein and lipids.

2.1.2 Acreage, yield and demand for soybeans

2.1.2.1 Production situation in the world

Soybeans originated in East Asia, but today, Brazil leads global production, surpassing the United States with 124 million tons produced in the first half of 2020, of which nearly two-thirds is exported Other significant soybean-producing countries include Argentina, China, India, Paraguay, and Canada The export volumes of these major soybean-producing nations from 2017 to mid-2020 are detailed in Table 2.1.

Table 2.1 Soybean export volume of some major countries in the world in the crops of 2017/2018, 2018/2019 and 2019/2020 (thousand tons)

(Source: FAS/USDA – statista) 2.1.2.2 Production situation in Vietnam

Currently, soybean growing are forming in 4 concentrated areas

 Northern midland and mountainous provinces

The Red River Delta region

The Mekong River Delta region

Soybean plants exhibit rapid growth and adaptability, allowing them to be cultivated in multiple seasons throughout the year, including winter-spring, spring, summer-autumn, and spring-summer In Vietnam, these versatile crops thrive particularly well in the mountainous and midland regions of northern provinces like Cao Bang and Son La.

La, Bac Giang accounts 40% of the total area of the country In addition, soybeans are also grown in some regions such as Dong Nai, Daklak, Dong Thap.

Vietnam's soybean production has been steadily increasing, with an annual growth of approximately 100,000 hectares and a yield of around 160,000 tons in 2017, which only meets 8-10% of the country's demand that is rising by an average of 53% The country continues to rely on imports for soybeans, primarily sourcing from markets such as China, Cambodia, Thailand, Canada, and the United States Notably, Brazil stands out as the largest supplier, with imports reaching 584.6 thousand tons in 2012 For a detailed overview of soybean production trends in Vietnam from 2010 to 2017, refer to table 2.2.

Table 2.2 Soybean production in Vietnam from 2011 to 2017

(Source: General Statistics Office of Vietnam, * FAS estimates)

Table 2.3 Production, supply and demand of soybeans in Vietnam

Vietn am Gene ral Statis tics Offic e, Glob al Trad e Atlas, USD

With the incre dem and for soyb eans

, dom estic ally prod uced soyb eans are not eno ugh to mee t the dem and for soyb eans g e n e r a l a n d t o f u p r o d u c t s i n p a r t i c u l a r T h e r e f o r e , s y b e a n m a t e r i a l n o w d e p e n d s

S oybea ns have a lower starch conten t than other legum es, while their protei n and lipid conten t is signifi cantly higher.

, the prot ein cont ent in soyb eans was the foun d to be the high est

The other minor compo nents includ e phosp holipi ds, vitami ns, miner als, trypsi n inhibit ors,

Chemi cal compos ition of some kind of beans

Kind of beans Đậu Hà Lan (Pisum sativum ) Đậu đỗ

Table 2.5 Chemical composition of soybean

Table 1.5 illustrates that in soybean seeds, the protein is predominantly concentrated in the endosperm and embryo, whereas the seed coat has a lower protein content and a higher carbohydrate composition.

Table 2.5 illustrates that in soybean seeds, protein is predominantly concentrated in the endosperm and embryo, whereas the seed coat contains lower protein levels and higher carbohydrates Soy protein is significant, consisting primarily of essential amino acids, although it has lower levels of methionine and tryptophan compared to other key products.

Soy protein primarily consists of globulin, which constitutes 85% to 95% of its content, although variations exist among different soybean types Additionally, soybeans contain a small amount of albumin and trace amounts of prolamin and glutelin The overall protein content in soybeans varies between 29.6% and 50.5%, with an average protein level of 36% to 40%.

Soy protein is almost identical to the protein of an egg The amino acid content of soybeans when compared to other foods is shown in Table 2.6

Table 2.6 Non-substituting amino acids in soybeans compares to other important foods (g / 100g protein)

Soybeans contain a lipid content ranging from 13.5% to 24%, with an average of 18% of their dry weight, primarily composed of glycerides and lecithin These glycerides are rich in unsaturated fatty acids, particularly 50 to 60% linolenic acid (C18-2), which contributes to the high biological value of soybeans However, this high lipid content also makes soybeans prone to oxidation, resulting in spoilage during storage.

Soy lecithin makes up 3% of the grain weight A complex phosphatide, used as an emulsifier, and antioxidant in food processing

Carbohydrates account for about 34% of the dry weight of the grain, and the

11 insoluble Water-soluble types make up only about 10% of total carbohydrates The carbohydrate content of soybean seeds is shown in Table 2.7

Table 2.7 Carbohydrate components in soybean

Soybeans are rich in essential vitamins that support bodily development, although they lack vitamins C and D The vitamin content in soybeans is relatively low and can be diminished during processing For a detailed breakdown of the vitamin composition, please refer to Table 2.8.

Table 2.8 Vitamin content in soybean Vitamin

Soybean seeds consist of 5% mineral content in their dry weight, primarily featuring essential minerals such as calcium, phosphorus, manganese, zinc, and iron Notably, soybeans are particularly rich in iron and zinc, as detailed in Table 2.9.

Table 2.9 Mineral contents in soybean

Soybeans are rich in various enzymes that play crucial roles in biochemical processes Key enzymes include lipase, which hydrolyzes glycerides into glycerin and fatty acids; phospholipase, responsible for converting ether to acetic acid; and lipoxygenase, a catalyst for hydrogen transfer in fatty acids Additionally, soybeans contain amylase, which includes both α-amylase and β-amylase, as well as urease, contributing to their nutritional and functional properties.

Saponins, found in soybean seeds, are glycoside compounds known for their unpleasant bitter taste, with total content ranging from 0.62% to 6.12%, influenced by the soybean variety The distribution of saponins varies within the cotyledons and subcotyls, depending on seed maturity Notably, some soybean varieties are entirely free of saponins Additionally, saponins act as a foaming agent during milling, complicating subsequent processing stages.

Raffinose and stachyose, two types of oligosaccharides found in soybeans, are known to cause gas in humans The absence of the enzyme α-galactosidase in the human large intestine means that non-bacterial galactooligosaccharides ferment, resulting in the production of gases such as CO2, H2, and occasionally CH4.

Trypsin inhibitors in soybeans constitute approximately 6% of the total protein content These inhibitors exert their effects by binding irreversibly to trypsin, forming a stable compound, while their interaction with chymotrypsin results in an unstable and reversible complex.

OVERVIEW OF TOFU

Tofu, a soft cheese-like product made by coagulating soy milk protein, is a dietary staple in many Asian countries Its popularity is rapidly increasing in America and Europe, highlighting its growing market presence.

Tofu is a versatile ingredient that can be prepared through various cooking methods, including frying, baking, boiling, and steaming There are three main types of tofu available: hard, soft, and silken, each differing in texture and water content This variety allows producers to create tofu with distinct structures, catering to different culinary needs and preferences.

Hard tofu retains its shape during cooking, making it ideal for stir-frying or baking, and is rich in protein and lipids In contrast, silk tofu features a creamy, custard-like texture and does not require additional flavors or spices when prepared Typically sold chilled, silk tofu is packaged in a tube filled with water or vacuum-sealed for freshness.

Tofu is a highly nutritious food rich in essential minerals like calcium and iron, as well as vitamin B, while being low in sodium and free from cholesterol This soy protein gel product has gained popularity as a health-conscious option The shift from traditional manual production to automated methods has been facilitated by advanced equipment, particularly from Japan, enhancing the efficiency of tofu manufacturing.

Tofu production begins with soaking soybeans, extracting soy milk, and boiling the milk solution A coagulant, typically calcium chloride (CaCl2) or magnesium chloride (MgCl2), is then added to coagulate the milk The resulting solid mass is subsequently processed into various forms of tofu, predominantly in block form.

2.2.1 Process of producing tofu from natural sour water [1]

(Công nghệ sản xuất mì chính và các sản phẩm lên men cổ truyền (1))

2.1 Traditional tofu production process in Vietnam

The wet grinding method begins with a crucial soaking phase, where beans absorb water and swell, allowing water molecules to interact with proteins, lipids, carbohydrates, and cellulose This process occurs in two stages: the first stage, known as the solvate process, involves the initial absorption without breaking molecular bonds, while the second stage, the hydration process, sees water molecules breaking these bonds, transforming the soybeans into a flexible colloidal state within the cells Three key factors influence the soaking process: soaking time, the amount of soaking water, and immersion temperature.

- Soaking time: outdoor temperature from 15 ÷ 25°C, soaking 5 ÷ 6 hours; outdoor temperature 25 ÷ 30°C, soaking 3 ÷ 4 hours.

Soaking soybeans at high temperatures accelerates the swelling process, but results in a lower swelling capacity This causes the ingredients in the beans to remain in a coagulated state rather than dissolving into a colloidal solution, making them harder to digest The optimal temperature for soaking soybeans is between 20°C and 25°C.

For optimal soaking, use a water-to-bean ratio of 2.5:1, which enhances bean swelling and reduces acidity After soaking, aim for a moisture content of 55% to 60% for the best results.

Grinding is a mechanical process that breaks down cells to release proteins, lipids, and carbohydrates, which can then be transformed into suspensions with the addition of water The key factor in this process is the amount of water used; insufficient water leads to increased friction and temperature, resulting in protein denaturation and reduced solubility Conversely, excessive water enhances the number of dissolved substances but complicates subsequent stages of processing The optimal water-to-bean ratio for grinding is 1:6.

After grinding, a suspension is formed that includes a colloidal solution and water-insoluble solids To separate the colloidal solution from the solids, it's essential to rinse the solids with water to remove the colloidal particles adhering to their surface However, the amount of water used for rinsing should be limited The filtration process should consist of two stages: refining and crude filtration, ensuring effective separation of components.

Immediately heating the milk solution after filtration is crucial for several reasons This process effectively inactivates the enzyme trypsin and eliminates the toxin aflatoxin, ensuring the safety of the product Additionally, heating kills harmful microorganisms, deodorizes any fishy odors, and disrupts the solvated layer, facilitating the aggregation of milk molecules for easier coagulation To achieve optimal soy milk quality, it is essential to boil 100 liters of milk as quickly as possible.

5 ÷ 10 minutes During the boiling process, stir stirring always to avoid burning the milk solution.

After boiling soy milk, it is essential to precipitate the proteins immediately This protein precipitation can occur due to several factors, including heat application, changes in pH within the isoelectric region, and the influence of salt During the precipitation process, boiling the milk is crucial for achieving the desired results.

Thermal denaturation and protein precipitation occur at temperatures of 95 ÷ 100°C, and various agents such as natural sour water, CaCl2, CaSO4, acetic acid, and lactic acid can be used for this process Among these agents, natural sour water is the most effective for protein precipitation, although it requires users to possess considerable experience to achieve optimal results Specific conditions must be met for successful precipitation of soy milk.

 The temperature of the soy milk solution when precipitating is> 95°C;

 pH of the aqueous solution when precipitation is greater than 6;

The pH level of sour water plays a crucial role in the precipitation process, as a high pH requires larger quantities of sour water, while a low pH results in reduced protein recovery efficiency To effectively integrate sour water into the milk solution, which should be heated to 95°C, it is essential to add the sour water gradually in three phases, starting with the addition of half of the total sour water in the first phase.

 After 3 minutes add half of the remaining sour water;

 After 3 minutes, add the remaining sour water.

Usually, the amount of sour water accounts for 20 to 22% of the bean milk to be precipitated.

2.2.2.5 Squeeze the tofu and soak into water

To create tofu, start with tofu curd after precipitation and place it into a mold The optimal pressing temperature for the curd is between 70°C and 80°C; temperatures below 60°C will prevent the curd from adhering and shaping properly Typically, the pressing process lasts for about 10 minutes.

FUNDAMENTAL OF THE GEL PROTEIN FORMATION PROCESS OF

The key to achieving the desired texture in tofu lies in the formation of protein gel, which requires soy proteins to undergo heat denaturation and coagulation, as they do not gel in their native state (Liu, 1997) This process involves thermal denaturation, followed by ordering, and ultimately gel formation, ensuring that all proteins are effectively dispersed within the gel network.

The production of tofu involves a crucial gel-forming process where soybean protein undergoes thermogenesis, transforming soy milk into a cohesive gel that ultimately results in the creation of tofu.

Thermal denaturation serves to expand the protein structure, transitioning it from a compact form to a more open and diffuse configuration upon heating This process reveals the internal structure of protein molecules, exposing key functional groups such as –SH, hydrophobic regions, carbonyl groups, amine groups from peptide bonds, and amide groups from side chains These exposed functional groups play a crucial role in influencing the overall network structure of the protein (Wang and Damodaran, 1991).

After dissociation and then regroup during the heat denaturation the protein molecules are transformed into fibers Interaction of protein molecules, fibers in a certain order form the three-dimensional network.

The formation of soy milk protein gel is primarily influenced by temperature and involves two key processes: dissociation and aggregation Notably, the gel-making mechanism varies between the two main protein fractions, glycinin and conglycinin In tofu production, the balance between these fractions plays a crucial role; a higher ratio of glycinin to conglycinin results in a firmer tofu gel.

2.2 Different gel network structures of protein 2.3.2 Factors affecting

Protein concentration plays a crucial role in determining the type and properties of soy milk protein gel While gelatin can create a gel at lower concentrations, soy milk requires a minimum protein content of 8% for effective gel formation The presence of glycinin significantly influences this process; higher glycinin levels facilitate gel formation, whereas lower levels hinder it due to separation tendencies Additionally, the mechanical strength of the gel is directly linked to protein concentration, with a linear relationship existing between the two, as increased protein leads to more cross-linking within the protein chains.

Temperature significantly impacts the quaternary structure of soy protein, particularly glycinin and β-conglycinin, which denature at temperatures of 85-95°C and 65-75°C, respectively Glycinin features a prominent disulfide bridge, which differentiates it from β-conglycinin When the heating temperature surpasses the minimum required for gel formation, it adversely affects the gel's rheological properties Conversely, heating at lower temperatures necessitates a longer time for gel formation, resulting in weaker gels due to insufficient development of a robust three-dimensional network Additionally, heating soy milk protein dispersions beyond the denaturation temperature can lead to structural changes that inhibit gel formation.

Adding a 2% NaCl salt solution to soy milk enhances the gelation of glycin and conglycin, but increasing the salt concentration to 10% inhibits gel formation Low concentrations of NaCl help neutralize protein charges, facilitating gel formation, while higher salt levels disrupt protein structure and increase hydrophobic interactions, ultimately preventing gelation.

The protein denaturation process is significantly influenced by pH, affecting interactions between proteins and solvents (Renkema, 2000) To achieve a balance between denaturation and recombination, as well as attraction and repulsion among protein chains, pH adjustment is essential At pH levels above 12, gel formation is entirely inhibited due to the negative charge on polypeptide chains in high alkaline conditions and the positive charge at acidic pH values, leading to electrostatic repulsion that destabilizes protein interactions and weakens gel strength Conversely, at neutral pH, the interaction between positively charged groups enhances energy and promotes effective gel formation.

Soy protein is categorized into four segments: 2S, 7S, 11S, and 15S, with the 7S and 11S segments being the primary components The 7S fraction, known as conglycinin, is found in soy milk, while the 11S fraction is referred to as glycinin Together, glycinin and conglycinin constitute approximately 65% to 85% of the total protein content in soy seeds.

Glycinin is an oligo protein characterized by a hexagonal structure formed from six monomers linked together This structure consists of two trimers, each made up of three monomers arranged in a specific sequence Glycinin is classified as a globulin, specifically 7S or 11S, highlighting its significance in protein composition.

12 relatively lipophilic "subunits": Including 6 acidic "subunits" (A) and 6 bases

"subunit" (B) The "sub the unit" are linked together by a disulfide bridge.

2.3 The molecular structure of Glycinin

β-conglicinin, a glycoprotein that constitutes approximately 35% of the protein content in seeds, contains around 5% carbohydrates Its structure is divided into three primary subunits, referred to as a, a', and b, which collectively form the fractional arrangement of conglycinin.

2.4 The molecular structure of Conglycinin

The subunits are linked together through hydrophobic, hydrogen-linked interactions without any disufite bonds (Thanh and Shibasaki,1978).

Using Ca2+ as a coagulant to precipitate proteins in soy milk can be effective, but it may introduce impurities in tofu As an alternative, fermentation solutions that lower pH can be utilized, with lactic acid bacteria (LAB) being a well-known example LAB is widely recognized in the food industry for its beneficial effects on both food quality and consumer health.

Lactic bacteria are non-spore-forming, Gram-positive, and non-mobile microorganisms that ferment sugars to produce lactic acid This group falls under the Lactobacillaceae family and is divided into four distinct genera.

Lactic acid bacteria, including Streptococcus, Pediococcus, Lactobacillus, and Leuconostoc, exhibit a variety of shapes such as short or long bacilli in single or double forms, as well as spherical or bacillus shapes in clusters Typically, the diameter of lactic cocci ranges from 0.5 to 1.5 µm These bacteria often form colonies that are small, round, glossy, and vary in color from milky white to cream yellow, with some colonies being large and convex, often accompanied by a distinct acidic odor.

2.4.2 Common features of lactic acid bacteria

Lactic acid bacteria exhibit morphological differences but are generally homogeneous, characterized as gram-positive, non-motile, and non-spore-forming Their capacity to synthesize various substances is limited, and they can ferment both anaerobically and aerobically, demonstrating a high tolerance for acidic environments.

OVERVIEW OF HYDROPEROXIDE AND BACTERIOCIN DURING

Lactic fermentation effectively reduces carbohydrates while generating various organic compounds with antimicrobial properties, including lactic, acetic, and propionic acids These compounds, along with other antibacterial substances synthesized by lactic acid bacteria, exhibit low molecular weight and maintain antimicrobial activity at low pH levels, demonstrating stability at elevated temperatures and broad-spectrum efficacy Notably, bacteriocin, a heat-stable compound extensively studied for its use as a biological preservative, inhibits Gram-positive bacteria Additionally, organic acids produced during fermentation, particularly lactic acid, contribute to antibacterial effects by lowering pH The presence of hydroperoxide and other substances further enhances these antibacterial properties Consequently, lactic fermentation fluid serves as a natural tofu precipitation agent, extending shelf life without the need for chemical preservatives, ensuring consumer safety.

Hydroperoxide (H₂O₂) is a byproduct of lactic fermentation, known for its potent bactericidal properties due to its strong oxidizing ability, which can oxidize membrane lipids and proteins This compound can also produce atomic oxygen, creating an anaerobic environment that is detrimental to certain microorganisms Lactic acid bacteria synthesize hydroperoxide in the presence of oxygen, but without a heme source, they cannot produce catalase to eliminate it Consequently, the less effective hydroperoxide removal systems lead to its accumulation (Ouwehand, 2004).

Bacteriocins are antibacterial peptides produced by bacteria to combat other bacterial strains, showcasing the ability of bacteriocin-producing bacteria to resist their own toxins (Karpinski, 2016) These peptides are non-allergenic and do not pose health risks to humans, as they are quickly hydrolyzed by proteases and lipases Specifically, bacteriocins from lactic acid bacteria are positively charged protein molecules that are small in size, making them effective in various applications.

Bacteriocins, which are produced by various groups of lactic acid bacteria including Lactobacillus and Lactococcus, consist of 30 to 60 amino acids and have a high isoelectric point These compounds are effective in inhibiting bacteria that share a close relationship with the bacteria that produce them.

Enterococcus, Streptococcus, Leuconostoc, and Pediococcus are key bacterial genera, with Lactobacillus and Lactococcus comprising two major groups that play crucial roles in fermentation These bacteria produce bacteriocins, which can effectively inhibit harmful bacteria, including Clostridium botulinum, Bacillus cereus, Bacillus alcalophilus, and Listeria monocytogenes.

OVERVIEW OF TOFU PRESERVATION

The final stage of tofu production involves packaging and preservation, where the choice of packaging materials and storage techniques significantly influences the taste, quality, and shelf life of the tofu product.

Tofu products, due to their high moisture and protein content, create an ideal environment for microbial growth As a result, even when refrigerated, their shelf life is limited to just a few days.

Extending the shelf life of tofu products remains a significant concern in the food industry Various preservation methods have emerged, including the use of preservation chemicals and advanced packaging technologies, to enhance the longevity of tofu.

In general, prolonging shelf life can be divided into two parts.

2 Apply storage preservative during or after packaging

These methods of preservation include physics, chemistry, and a combination of physical and chemical methods.

Together with using lactic bacteria, 2 preservation method will be used to prolong the shelf life of tofu

Modified atmosphere packaging (MAP) involves sealing food in a controlled gas mixture that initially alters the atmospheric composition to preserve its quality This method replaces the air inside the packaging with gases such as carbon dioxide, oxygen, and nitrogen, which helps to prevent the deterioration of the food's physical, chemical, and biological properties By maintaining a stable environment, MAP effectively extends the shelf life of various food products.

MAP has been studied with tofu Stoops, Maes, Claes, and Van Campenhout

A study conducted in 2012 examined the growth of Pseudomonas in tofu products stored in modified atmosphere packaging (MAP) and found that controlling carbon dioxide and oxygen levels in refrigerated products was ineffective in eliminating Pseudomonas spoilage Subsequent research utilized a CO2 and N2 mixture (3:7 ratio) with flushing or vacuum compensation for packaging and refrigeration, comparing it to air-packed tofu as a control The findings revealed that the microbial count in air-packed tofu was one to four log cycles higher than that in MAP after 10 days of storage, with MAP successfully inhibiting microbial growth for up to 14 days (Van Campenhout, Maes, & Claes, 2013).

In summary, MAP has been successfully used for shelf life extension and freshness preservation of tofu products.

Food freezing technology is used as a food preservation method It can increase the storage time and extend the shelf life of food (Kobayashi, Ishiguro,

Ozeki, Kawai, & Suzuki, 2020) Frozen tofu is a delicious and famous Asian food made by freezing soft or firm tofu (Ji et al., 2017).

RESEARCH SITUATION WORLDWIDE AND IN VIETNAM

In 2020, Kay Huyn Joo and colleagues explored trimagnesium citrate (TMC) as a potential alternative to traditional tofu coagulants Their study involved a comprehensive comparison of yield, water-holding capacity (WHC), texture profile analysis (TPA), confocal microscopic analysis, and sensory evaluation of raw and cooked tofu produced with various coagulants The findings revealed that while TMC did not affect the yield, it significantly influenced the textural properties and enhanced the sensory quality of the tofu.

Yin and colleagues investigated the effects of fermentation with Actinomucor elegans on the phenolic components, antioxidant activities, and nutritional compounds of tofu Their findings revealed a significant increase in both total and soluble phenolic content, alongside a decrease in insoluble phenolic content post-fermentation Additionally, the antioxidant activities of the fermented tofu surpassed those of the unfermented variety Metabolomic analysis indicated that fermentation significantly enhanced the nutritional composition of tofu, including carbohydrates, alcohols, fatty acids, organic acids, inorganic acids, and amino acids These results highlight that A elegans fermentation can notably improve the nutritional and functional properties of tofu.

In 2020, Elvira and colleagues explored the use of Plasma Activated Water (PAW) as an immersion solution for preserving tofu, alongside traditional chemical preservatives and natural microbiological strains Their research demonstrated that PAW is a promising non-thermal technology that effectively controls pathogenic microorganisms while maintaining the physical and functional properties of tofu.

Tofu is a traditional food that is well-known globally, yet it has not been extensively studied in Vietnam In 2014, researchers Nguyen Thi Minh Nguyet and Pham Thi Kim Ngoc explored the impact of coagulation agents on tofu's recovery efficiency and quality Their findings revealed that sour water can be effectively used to produce safe, high-quality tofu, while also highlighting the unique properties of tofu made from plaster.

METHOD

RESEARCH SUBJECT

Soybeans are scientifically known as Glycine max Merril.

Soybeans choose good quality (round beans, uniform, light yellow color, poor quality seeds such as beetles, little damage, low ratio of flat seeds, low cracked seeds).

Soybeans were purchased in Thai Nguyen

Using lactic bacteria strains isolated from fermented sour products such as pickles, yogurt, fermented soy milk

3.2 Equipments and chemicals required for research

3.3 Location and time period of the research

- Location: Faculty of Biotechnology - Food Technology, Thai Nguyen University of Agriculture and Forestry

Content 1: Isolation of some strains of lactic acid bacteria that can be used in tofu production

Content 2: Selection of bacteria with good ferment ability for application in tofu production

3.5.1 Content 1: Isolation of some strains of lactic acid bacteria that can be used in secondary production

3.5.1.1 Method of isolation and selection of lactic bacteria

To cultivate lactic acid bacteria from yogurt and fermented soy milk, a sample was prepared by mixing 20 mL of solution with 100 mL of MRS broth medium The mixture was then incubated at 37°C for 24 hours while being shaken at 150 rpm.

The medium after incubation was diluted at a concentration of 10 -4 , hence spread 100 àL on a plate containing MRS agar (appendix) and incubated at 37°

C After 48 hours, observe and select characteristic colonies for repeated inoculation on MRS agar until homogeneous colonies are obtained.

3.5.1.2 Determination of morphological, physiological and biochemical features

- Characteristics of colony morphology and bacterial cells: The isolated colonies were examined for cell morphology under the microscope on the oil objective lens X100.

- Morphological, physiological and biochemical characteristics: Check some characteristics of lactic acid bacteria

3.5.2 Content 2: Selection of bacteria with good ferment ability for application in tofu production

3.5.2.1 Experimental set up test for soy milk fermentation

Prequalified strains of lactic acid bacteria were incubated to increase the biomass in 10 ml of MRS broth for 24 hours at 37 ° C.

Prepare the soy milk solution (soybeans: distilled water = 1: 2.5) Pour into each glass jar 50 mL milk solution Afterward, add 5 ml of lactic bacteria strains and incubate at 41°C for 6-7 hours.

The treatments were performed 3 times

3.3.5.2 Tofu production efficiency using lactic bacteria

The optimal pH for precipitating soy milk protein was established through prior experiments Subsequently, a strain with excellent fermentation capabilities and favorable sensory evaluation for soy milk was selected to produce tofu Samples were then taken to assess production efficiency, with the experimental setup detailed below.

Figure 3.1 Tofu production process using lactic acid bacteria

1 Indicate the quantity of total aerobic bacteria.

The study aims to evaluate hygiene levels in tofu processing and storage while examining the antibacterial properties of tofu produced using lactic acid bacteria Two samples were analyzed concurrently: one made with lactic acid bacteria and another produced using plaster.

The method of enumeration of total aerobic bacteria is specified according to TCVN 5165 - 90 (Appendix 4).

Prepare sample: 1g of tofu + 9ml of distilled water

From the above dilutions, transfer 100 μl of sample to a Petri dish containing TGA medium (2 dishes each).

Count the colonies growing on the plate

Result: Total number of aerobic bacteria in the sample (CFU / g)

Figure 3.2 Process of enumeration of total aerobic bacteria

Coliforms are a diverse group of Gram-negative bacteria, including various strains of E coli, Citrobacter, Klebsiella, and Enterobacter These non-spore-forming bacteria can thrive in both aerobic and anaerobic environments and are characterized by their ability to ferment lactose, producing gas within 48 hours under suitable culture conditions.

The purpose of this study is to evaluate the hygienic quality of water and the sanitary conditions in food processing, specifically assessing fecal contamination in water sources It involves monitoring fluctuations in two groups of bacteria from production through storage, similar to other microbiological indicators Experiments were conducted using two tofu samples, and the enumeration methods for Coliforms and Fecal Coliforms were carried out in accordance with TCVN 4882: 2007 (Appendix 5).

Figure 3.3 Process of enumeration of Coliform

3 Indicate the quantity of total mold

Principle: Culture media containing inhibitors of bacteria (antibiotics such as Oxytetracylin or Chloramphenicol) are cultured at 30 ± 10C under aerobic conditions after 48 - 72 hours (Appendix 6)

From the above dilutions, transfer 100 μl of sample to a Petri dish containing YGC medium (2 dishes each).

Figure 3.4 Process of enumeration of mold 3.5.3 Sensory evaluation (Sensory evaluation by the method of scoring TCVN

For tofu, to assess the sensory quality, the assessment was conducted through the following four criteria: smell, taste, state, and color.

The product evaluation according to the sensory method complies with Vietnamese standards (TCVN 3215 - 79) The Vietnamese standard uses a scale of

The evaluation process involves scoring each inspector on a scale from 0 to 5, where 5 represents the highest level of performance and 0 signifies the lowest This assessment is based on recorded results, which are compared against established criteria and descriptions to assign an appropriate integer score within the specified range.

When evaluating a product with an odd number of testers (N), the average score is calculated by taking the mean of the scores from all testers, rounded to two decimal places The weighted average point for each indicator is determined by multiplying the average score of that criterion by its corresponding importance coefficient.

Common point is the total weighted scores of all sensory parameters The six rating ranks are equivalent to the description content Vietnamese standard

3215 - 79 specifies the quality grades for products that have common points and weightless points for some corresponding criteria.

Table 3.4 Quality level specified standards

To meet the quality requirements (medium grade), the average score without important coefficient for each sensory criteria is 2.8 and the average score is at least 11.2 for each product.

A sensory evaluation was performed three times, during which two distinct tofu samples produced at three different times were prepared The sensory panel assessed these samples using a sensory description scorecard, with each member independently evaluating and recording their results in the sensory assessment.

-Determine the pH index with a pH meter according to TCVN 6492: 1999

-Method of sensory assessment: according to the criteria of TCVN 7030: 2002

PART IV RESULTS AND DISCUSSION 4.1 Results of isolation and selection of lactic acid bacteria

The results were isolated 11 strains of bacteria from 1 sample of yogurt and

1 sample of sour water (acidic water) Specifically, the bacteria strains are denoted as follows:

- Yogurt samples isolated 6 bacteria strains (symbols: SC1, SC2, SC3, SC4, SC5, SC6)

- Acidic water samples isolated 5 bacteria strains (symbols: DP1, DP2, DP3, DP4, DP5)

4.1.1 Biological characteristics of lactic acid bacteria isolated

4.1.1.1 Morphological characteristics of colonies and bacterial cells

Through the isolation process, I conduct to select the colonies with round shape, opaque white color, ivory surface and smooth edges.

Figure 4.1 Colony on MRS agar

The bacterial isolates are characterized by their rod-shaped morphology, predominantly existing in two forms: short rod chains and long rods A representative image of some of these isolated cells is provided in the accompanying figure.

Figure 4.2 and 4.3 Representative characteristic of bacteria cell

Some of the characteristics biochemical tests to determine the physiological and biochemical properties of lactic acid bacteria include:

- Gram staining: All strains isolated were stained Gram, in which 5/12 strains caught the purple color of the dye, indicating that these strains belonged to gram-positive bacteria.

Bacteria strains SC1 SC2 SC3 SC4 SC5 SC6

Figure 4.4 Gram possitive bacteria Figure 4.5 Gram negative bacteria

The investigation into catalase activity revealed that four strains of gram-positive bacteria exhibited no bubble formation when exposed to 30% hydrogen peroxide (H2O2), indicating a lack of catalase activity in these strains.

Figure 4.6 and Figure 4.7 Negative catalase

Base on physiological and biochemical characteristics of these 4 strains of bacteria, it can be concluding that they belong to the Lactobacillus Sp Family.

4.2 Experimental set up test for soy milk fermentation

After 8 hours of incubation, the pH is shown in Table.

Table 4.2 pH of fermented soy milk of 4 bacteria strains after 8h

4.2.2 Sensory quality of fermented soy milk

The pH value table indicates that SC4 thrives in soy milk, resulting in a significant decrease in pH levels This rapid reduction in pH is crucial, but it is equally important to select a strain that maintains a desirable flavor during fermentation to ensure the quality of the final tofu product.

The sensory quality of lactic fermented soy milk is shown in Table 3.3.

Table 4.3 Results of sensory evaluation of lactic fermented soy milk

It was shown that lactic acid bacteria SC4 was able to grow on soy milk medium, lowering pH in a short time However, the result of this strain on the

DP3 is the one which got the finest sensory evaluation, and fermentation is only inferior to strains SC4 In overall, this strain has better ability among all.

The initial findings confirmed the selection of the DP3 strain, which meets the research's initial criteria The next phase involves exploring the tofu production efficiency utilizing this specific strain.

4.2.3 Carbohydrate metabolism of selected strain

The carbohydrate metabolism is shown in the table below

Table 4.4 Sugar fermentation test result of DP3

The DP3 strain is a Gram-positive, non-spore forming bacterium that typically reduces pH through fermentation and exhibits catalase activity When tested with three types of sugar, the DP3 strain produced a yellow fluid without any gas bubbles, as indicated by the green Durham tube, confirming that it ferments sugar solely to produce acid without generating gas.

Using the Bergey’s Manual of Determinative Bacteriology, it can be concluded DP3 strain belongs to the Lactobacillus genus It can either be

Lactobacillus casei or Lactobacillus delbroeckii

The process of producing tofu with lactic fermented soy milk tested is summarized as shown in the diagram in Figure 3.1.

Tofu production utilizes 200g of soybeans combined with a fermented milk solution, following the process outlined in Figure 3.1 After the tofu block is pressed, it is immersed in cold water to stabilize the product and prevent rapid souring, resulting in a total recovery weight of 400g Freshly made LAB tofu exhibits distinct characteristics that highlight its quality and freshness.

- Condition: The shell is smooth, without cracks, smooth cuts, soft

- Color: Ivory white crust, milky white cut, uniform color, absent no strange color spots on the tofu piece.

- Flavor: typical aroma of cooked soybeans, with no sour odor, or any other strange odor

To determine shelf life products, we are using tofu sample and control sample to conduct storage The storage process are combined cooling condition and packaging

4.4.1 Indicate quantification of total aerobic bacteria

The results of determining the total number of aerobic microorganisms are shown in the chart Figure 4.8 and Appendix Table 8.

Figure 4.8 Total Number of aerobic microorganism in 2 types of tofu

The results depicted in Figure 4.8 indicate a significant difference in total aerobic microorganism counts between the two tofu samples Over the storage period, the total aerobic microorganisms in both samples increased; however, the experimental sample exhibited a slower growth rate compared to the control sample, reaching 3.1 x 10³ CFU/g after 9 days of storage.

LOCATION AND TIME PERIOD OF THE RESEARCH

- Location: Faculty of Biotechnology - Food Technology, Thai Nguyen University of Agriculture and Forestry

RESEARCH CONTENT

Content 1: Isolation of some strains of lactic acid bacteria that can be used in tofu production

Content 2: Selection of bacteria with good ferment ability for application in tofu production

RESEARCH METHODS

3.5.1 Content 1: Isolation of some strains of lactic acid bacteria that can be used in secondary production

3.5.1.1 Method of isolation and selection of lactic bacteria

To cultivate lactic acid bacteria from yogurt and fermented soy milk, a sample was combined with 20 mL of solution and added to 100 mL of MRS broth medium The mixture was incubated at 37°C for 24 hours while being shaken at 150 rpm.

The medium after incubation was diluted at a concentration of 10 -4 , hence spread 100 àL on a plate containing MRS agar (appendix) and incubated at 37°

C After 48 hours, observe and select characteristic colonies for repeated inoculation on MRS agar until homogeneous colonies are obtained.

3.5.1.2 Determination of morphological, physiological and biochemical features

- Characteristics of colony morphology and bacterial cells: The isolated colonies were examined for cell morphology under the microscope on the oil objective lens X100.

- Morphological, physiological and biochemical characteristics: Check some characteristics of lactic acid bacteria

3.5.2 Content 2: Selection of bacteria with good ferment ability for application in tofu production

3.5.2.1 Experimental set up test for soy milk fermentation

Prequalified strains of lactic acid bacteria were incubated to increase the biomass in 10 ml of MRS broth for 24 hours at 37 ° C.

Prepare the soy milk solution (soybeans: distilled water = 1: 2.5) Pour into each glass jar 50 mL milk solution Afterward, add 5 ml of lactic bacteria strains and incubate at 41°C for 6-7 hours.

The treatments were performed 3 times

3.3.5.2 Tofu production efficiency using lactic bacteria

The optimal pH for precipitating soy milk protein was established through prior experiments Subsequently, a strain with rapid fermentation capabilities and favorable sensory evaluation for soy milk was selected Tofu production was then carried out, followed by sampling to assess production efficiency, as outlined in the experimental setup.

Figure 3.1 Tofu production process using lactic acid bacteria

1 Indicate the quantity of total aerobic bacteria.

The purpose of this study was to evaluate the hygiene levels in tofu processing and storage, while also examining the antibacterial properties of tofu produced using lactic acid bacteria Two samples were analyzed concurrently: one sample of tofu made with lactic acid bacteria and another sample produced using plaster.

The method of enumeration of total aerobic bacteria is specified according to TCVN 5165 - 90 (Appendix 4).

From the above dilutions, transfer 100 μl of sample to a Petri dish containing TGA medium (2 dishes each).

Figure 3.2 Process of enumeration of total aerobic bacteria

Coliforms are a diverse group of Gram-negative bacteria that can ferment lactose, including varieties such as E coli, Citrobacter, Klebsiella, and Enterobacter These non-spore-forming bacteria can thrive in both aerobic and anaerobic environments and typically produce gas within 48 hours when cultured at the appropriate temperature.

The purpose of this study is to evaluate the hygienic quality of water and the sanitary conditions in food processing, specifically by assessing fecal pollution in water sources The research aims to monitor fluctuations in two groups of bacteria from production through storage, similar to other microbiological indicators Experiments were conducted using two tofu samples, with enumeration methods for Coliforms and Fecal coliforms following the standards outlined in TCVN 4882: 2007 (Appendix 5).

Figure 3.3 Process of enumeration of Coliform

3 Indicate the quantity of total mold

Principle: Culture media containing inhibitors of bacteria (antibiotics such as Oxytetracylin or Chloramphenicol) are cultured at 30 ± 10C under aerobic conditions after 48 - 72 hours (Appendix 6)

From the above dilutions, transfer 100 μl of sample to a Petri dish containing YGC medium (2 dishes each).

Figure 3.4 Process of enumeration of mold 3.5.3 Sensory evaluation (Sensory evaluation by the method of scoring TCVN

For tofu, to assess the sensory quality, the assessment was conducted through the following four criteria: smell, taste, state, and color.

The product evaluation according to the sensory method complies with Vietnamese standards (TCVN 3215 - 79) The Vietnamese standard uses a scale of

The evaluation process involves scoring inspectors on a scale of 0 to 5, where 5 represents the highest performance and 0 indicates the lowest Each inspector's performance is assessed based on recorded results, which are then compared against established descriptions and criteria This systematic approach ensures that each inspector receives an integer score reflecting their level of achievement.

When multiple testers evaluate a product, with an odd number of participants (N), the average score is calculated as the mean of the scores from all testers, rounded to two decimal places The weighted average score for each criterion is determined by multiplying the average score of that criterion by its corresponding importance coefficient.

Common point is the total weighted scores of all sensory parameters The six rating ranks are equivalent to the description content Vietnamese standard

3215 - 79 specifies the quality grades for products that have common points and weightless points for some corresponding criteria.

Table 3.4 Quality level specified standards

To meet the quality requirements (medium grade), the average score without important coefficient for each sensory criteria is 2.8 and the average score is at least 11.2 for each product.

A sensory evaluation was performed three times, with each session involving the preparation of two distinct tofu samples produced at three different times The sensory panel was instructed to assess the samples using a sensory description scorecard, allowing each member to independently evaluate and document their findings in the sensory assessment.