Research rationale
Tofu, a vital food derived from soy protein, is a traditional staple in Southeast Asia celebrated for its high nutritional value and excellent digestibility Recognized by the FDA for its health benefits in 1999, 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 associated with numerous health benefits, including a potential reduction in chronic diseases like cancer, heart disease, and osteoporosis Research indicates that soy protein, rich in isoflavones and isoplavolesterol, may lower cholesterol levels and protect against atherosclerosis Additionally, the antioxidant properties of isoflavones and proteins in tofu help guard against lipid oxidation Despite its popularity and significant consumer market in Vietnam, tofu production remains largely small-scale and outdated, lacking quality registration and proper packaging, resulting in a short shelf life of just 1-2 days.
3 causes waste product, but also limits the scope of the distribution and the time it takes to commercialize the product
The protein coagulation process in tofu primarily involves food-safe chemicals like calcium sulfate (CaSO4), magnesium chloride (MgCl2), citric acid, and calcium chloride (CaCl2) (Murugkar, 2015) However, some tofu manufacturers utilize impure chemicals, resulting in metal residues in the final product.
Lactic bacteria are gaining significant attention among scientists due to their numerous benefits in food technology The organic compounds generated during lactic fermentation are recognized as effective antibacterial agents Consequently, research is increasingly focused on utilizing lactic fermentation and its extracts 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
Limitations
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 was carried out at the Faculty of Food Biotechnology and Food Technology at Thai Nguyen University of Agriculture and Forestry However, the study faced challenges due to a lack of specialized equipment necessary for the research process.
REVIEW
OVERVIEW OF SOYBEAN
Soybeans are scientifically known as Glycine max Merril According to
Phạm Văn Thiều (1993) in his book "Soybean Plant, Cultivation and Product Processing Techniques" describes soybeans as short-term legumes that grow within 80 to 150 days, reaching heights of 30 to 80 cm depending on the variety Characterized by their upright growth and fewer branches compared to other legumes, soybean plants produce clusters of fruit containing 2 to 20 pods, with each plant yielding nearly 400 fruits Each pod can hold 1 to 7 seeds, which are slightly curved and measure approximately 4 to 6 cm in length Soybean seeds come in various shapes, including round, oval, long, and flat, with colors ranging from yellow-green and gray to black, though yellow is the most common The soybean consists of three main 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
The homeland of soybeans is East Asia, but nearly 60% of the world soybean production is located in the Americas, in which the US and Brazil are the countries
Brazil has emerged as the world's leading soybean producer, surpassing the United States with a remarkable output of 124 million tons in the first half of 2020, of which nearly two-thirds is destined for export Other significant soybean-producing nations include Argentina, China, India, Paraguay, and Canada A detailed analysis of soybean export volumes from these major countries from 2017 to mid-2020 is presented 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 are known for their rapid growth and adaptability, allowing them to be cultivated in multiple cropping seasons throughout the year, including winter-spring, spring, summer-autumn, and spring-summer In Vietnam, these versatile crops thrive in the mountainous and midland areas of northern provinces, such as Cao Bang and Son.
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
Each year, Vietnam expands its agricultural land by approximately 100,000 hectares, primarily for winter crops, yielding around 160,000 tons in 2017 This production satisfies only 8-10% of the country's growing demand, which has risen by an average of 53% Currently, Vietnam still relies on soybean imports, mainly sourcing from markets like China.
Cambodia, Thailand, Canada and the United States In particular, Brazil is the largest soybean supplier to Vietnam with soybeans imported from this market in
2012 reaching 584.6 thousand tons Situation of soybean production in Vietnam from 2010 to 2017 can be observed in table 2.2
Table 2.2 Soybean production in Vietnam from 2011 to 2017
(Source: General Statistics Office of Vietnam, * FAS estimates)
Cultivated area (thousand hectares) 197,8 181,1 120,8 180 200 100,8 94 100 Yield (tons /hectare) 1,51 1,47 1,45 1,5 1,5 1,45 1,57 1,57 Total quantity (thousand tons ) 298,6 266,9 175,3 270 300 146,4 147,5 157
Table 2.3 Production, supply and demand of soybeans in Vietnam
(Source: Vietnam General Statistics Office, Global Trade Atlas, USDA adjusted statistics)
The rising demand for soybeans, particularly for tofu products, has outpaced domestic production, leading to a significant reliance on imports to fulfill the market needs.
Soybeans are unique among legumes due to their lower starch content and significantly higher levels of protein and lipids In fact, research indicates that soybeans possess the highest protein content compared to other commercial legumes (Yaklich 2001) [27].
Soybean seeds contain protein levels ranging from 39.5% to 50.2% and oil content between 16.3% and 21.6% on a dry matter basis Additionally, they include minor components such as phospholipids, vitamins, minerals, trypsin inhibitors, phytates, oligosaccharides, and isoflavones.
Table 2.4 Chemical composition of some kind of beans
Content by percentage by weight of dry matter Ash Cellulose Sugars Starch Protein Lipid Đậu Hà Lan
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 concentration.
Table 2.5 illustrates that soybean seeds primarily concentrate protein in the endosperm and embryo, while the seed coat has lower protein levels and higher carbohydrate content Soy protein constitutes a significant portion of the seed and is made up of essential amino acids, although it has lower levels of methionine and tryptophan compared to other key products.
Soy protein consists primarily of globulin, which makes up 85% to 95% of its content, although variations exist among different soybean types Additionally, soybeans contain a small amount of albumin, with minimal prolamin and glutelin present The total protein content in soybeans ranges from 29.6% to 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)
Amino Acid Soybean Egg Beef Milk Rice Necessary value
Soybeans have 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, including 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 starch content is negligible Carbohydrate can be divided into 2 types: soluble and
12 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 for bodily development, although they lack vitamins C and D, and their vitamin content is low and can diminish during processing For a detailed breakdown of the vitamin composition, refer to Table 2.8.
Table 2.8 Vitamin content in soybean
Folic acid 1,9 mg/g Vitamin PP 2,3 mg%
The mineral content accounts for 5% of the dry weight of the soybean seed
In which, notably calcium, phosphorus, manganese, zinc, iron Soybeans are rich in iron and zinc The content of these minerals is shown 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 hydrolyzing ethers to acetic acid; and lipoxygenase, a catalyst for hydrogen transfer in fatty acids Additionally, soybeans contain amylase, which encompasses both α-amylase and β-amylase, as well as urease, contributing to their overall nutritional profile and functionality.
Saponins, which are glucose components found in soybean seeds, contribute to an unpleasant bitter taste The total saponin content in soybean seeds varies between 0.62% and 6.12%, largely influenced by the soybean variety Additionally, the distribution of saponins is uneven within the cotyledons and subcotyls, depending on the seeds' maturity Certain soybean varieties exhibit unique saponin characteristics.
14 completely saponin-free Saponin is a foaming factor in the milling process, so it makes difficulty for the further processing stages
OVERVIEW OF TOFU
Tofu, a soft cheese-like product made by coagulating soy milk protein with a coagulant, is a staple food in Asian countries and is rapidly gaining popularity in America and Europe.
Tofu is a versatile ingredient that can be prepared through various methods including frying, baking, boiling, and steaming There are three main types of tofu available: hard, soft, and silken, each distinguished by its water content, which influences the texture and structure of the final product.
Hard tofu is ideal for stir-frying or baking due to its ability to maintain shape during cooking, and it boasts the highest protein and lipid content among tofu varieties In contrast, silken tofu features a creamy, custard-like texture and does not require additional flavors or spices when prepared Typically sold cold, silken tofu is packaged in tubes filled with water or vacuum-sealed for freshness.
Tofu is a highly nutritious food, rich in essential minerals like calcium and iron, and packed with vitamin B, while being low in sodium and cholesterol-free This soy protein gel product has gained popularity as a healthful dietary choice The production of tofu is increasingly shifting from traditional manual methods to automated processes, utilizing advanced equipment primarily developed in Japan.
Tofu production begins with soaking soybeans to extract soy milk, which is then boiled A coagulant, typically calcium chloride (CaCl2) or magnesium chloride (MgCl2), is added to the heated milk to facilitate coagulation The resulting solid mass is then processed into various forms of tofu, predominantly in block shape.
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 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 solvate process, where the bonds in the soybeans remain intact, followed by the hydration process, where water molecules break these molecular bonds, transforming the beans into a flexible colloidal state Key factors influencing the soaking process include 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 swelling but reduces their overall swelling capacity At elevated temperatures, the components of the beans remain in a coagulated state rather than forming a colloidal solution, making them harder to dissolve The optimal soaking temperature for beans is between 20°C and 25°C.
For optimal bean preparation, use a soaking water ratio of 1 part beans to 2.5 parts water This proportion enhances bean swelling and reduces acidity, ensuring that at the end of the soaking phase, the ideal moisture content reaches between 55% and 60%.
Grinding is a mechanical process to break down cells, to release proteins, lipids, and carbohydrates By dissolved water, these substances could be turned
The process of creating suspensions is significantly influenced by the amount of water added Insufficient water results in high friction, leading to increased temperatures that can denature proteins and reduce their solubility Conversely, excessive water may enhance the number of dissolved substances but complicate subsequent stages The optimal water-to-bean ratio for grinding is 1:6, ensuring the best results in suspension formation.
After grinding, a suspension is created 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 adhering colloidal particles, while ensuring that the water used for washing is minimal The filtration process involves two key steps: refining and crude filtration.
After filtration, milk must be heated immediately to effectively deactivate the enzyme trypsin and eliminate the toxin aflatoxin, while also killing microorganisms and deodorizing any fishy odors This heating process helps break the solvated layer, facilitating the coagulation of milk molecules For optimal soy milk quality, it's crucial to boil 100 liters of milk within 5 to 10 minutes, and continuous stirring during boiling is essential to prevent burning.
After boiling the soy milk must precipitate immediately The protein precipitation has many causes such as the effect of heat, pH change in the
The isoelectric region at pH 19 plays a significant role in the precipitation of proteins in milk, which is typically heated to temperatures between 95 and 100°C to induce thermal denaturation Various agents can facilitate protein precipitation, including natural sour water, CaCl2, CaSO4, acetic acid, and lactic acid; among these, natural sour water is the most effective However, successful use of sour water for precipitation requires considerable expertise Specific conditions must be met for the effective 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; a high pH requires a larger volume of sour water, while a low pH results in reduced protein recovery efficiency To effectively incorporate sour water, gradually add it to the milk solution at 95°C in three phases, starting with the addition of half of the 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
After the precipitation process, tofu curd is obtained and placed into a mold For optimal pressing, the temperature should be maintained between 70°C and 80°C; temperatures below 60°C will prevent the curd from adhering properly and shaping The typical pressing duration is around 10 minutes.
After pressing the tofu, carefully remove it from the mold and cut it into your desired sizes Soak the tofu in water to enhance its stability, allowing it to sit longer for a slightly sour flavor.
FUNDAMENTAL OF THE GEL PROTEIN FORMATION PROCESS OF
The key to achieving the desired texture in tofu lies in the formation of protein gel Native soy proteins do not gel on their own; they require heat denaturation followed by coagulation to create tofu (Liu, 1997) The process involves thermal denaturation, ordering, and gel formation, which are crucial for dispersing all proteins into the gel network effectively.
The production of tofu involves a crucial gel-forming process where soybean protein undergoes thermogenesis, transforming soy milk protein into a structured gel that ultimately becomes tofu.
Thermal denaturation serves to expand the protein structure, transitioning it from a compact form to a more open and diffused state upon heating This process reveals the internal architecture of protein molecules, exposing functional groups such as –SH groups, 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 formation mechanism differs between the proteins glycinin and conglycinin.
21 different Gel formation in tofu production is affected by both glycinin and conglycinin fractions If the glycinin ratio is higher than conglycinin, the tofu gel will form harder
2.2 Different gel network structures of protein 2.3.2 Factors affecting
The concentration of protein plays a crucial role in determining the type and properties of soy milk protein gel Gelatin can create a gel at lower concentrations compared to globulin, which requires a minimum soy milk protein content of 8% for effective gel formation High levels of glycinin facilitate this process, while low glycinin content can hinder gel formation due to separation tendencies Additionally, the mechanical strength of the gel is directly influenced by protein concentration, with a linear correlation existing between the two, as increased protein leads to more cross-linking in the protein chains.
One of the effects of temperature on soy protein is to alter its quaternary
Glycinin and β-conglycinin, two key proteins in soy, denature at temperatures of 85-95°C and 65-75°C, respectively, with glycinin containing a significant disulfide bridge Exceeding the minimum denaturation temperature necessary for gel formation alters the gel's rheological properties, while heating at lower temperatures requires a longer time for gel formation, resulting in weaker gels due to insufficient development of a robust three-dimensional network Additionally, heating protein dispersions in soy milk beyond the denaturation temperature can lead to structural changes that prevent gel formation altogether.
Adding a 2% NaCl salt solution to soy milk enhances the gel formation of glycin and conglycin However, increasing the salt concentration to 10% inhibits gel formation Low levels of NaCl help neutralize protein charges, promoting gel formation, while high salt concentrations disrupt this process by altering protein structure and increasing hydrophobic interactions.
The protein denaturation process is significantly influenced by pH levels, impacting interactions between proteins and solvents (Renkema, 2000) Proper pH adjustment is essential for balancing denaturation and recombination, as well as the attraction and repulsion of adjacent protein chains Notably, when pH exceeds 12, the gel formation process is entirely inhibited, as positively charged polypeptides dominate at acidic pH values.
At high alkaline pH values, polypeptide chains carry a negative charge, leading to electrostatic repulsion that can destabilize protein interactions and weaken gel network formation In contrast, at neutral pH, the presence of positively charged groups enhances energy levels, thereby promoting stronger gel formation.
Soy protein is classified into four segments: globulin 2S, 7S, 11S, and 15S, with the 7S and 11S fractions being the primary components The 7S fraction, known as conglycinin, and the 11S fraction, referred to as glycinin, together contribute approximately 65% to 85% of the total protein content in soy seeds (Nielson, 1985).
Glycinin, an oligoprotein, has a molecular structure that consists of six monomers linked together, forming a hexagonal configuration This structure is composed of two trimers, each containing three monomers arranged in a specific sequence Additionally, globulin 7S or 11S is made up of 12 relatively lipophilic subunits, which include six acidic subunits (A) and six basic subunits.
"subunit" (B) The "sub the unit" are linked together by a disulfide bridge
2.3 The molecular structure of Glycinin
β-conglycinin, which constitutes approximately 35% of the protein content in seeds, is a glycoprotein that contains nearly 5% carbohydrates This protein is organized into three main subunits, referred to as a, a', and b, forming its fractional structure.
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).
LACTIC ACID BACTERIA OVERVIEW
Using Ca2+ as a coagulant in soy milk can effectively precipitate protein, but it may introduce impurities in tofu An alternative method involves using fermentation solutions to lower pH, with lactic acid bacteria (LAB) being a popular choice LAB is well-regarded in the food industry for its benefits not only to food quality but also to consumer health.
Lactic acid bacteria are non-spore-forming, Gram-positive, and non-motile bacteria that ferment sugars to produce lactic acid This group belongs to the Lactobacillaceae family and is divided into four genera.
Streptococus, Pediococcus, Lactobacillus and Leuconostoc This group of bacteria has many different shapes, including short or long bacillus shaped in single or
Lactic acid bacteria are characterized by their diverse forms, including double or clustered cocci, as well as rod-shaped structures Typically, the diameter of lactic cocci ranges from 0.5 to 1.5 micrometers These bacteria can form colonies that appear small, round, and glossy, with colors varying from milky white to cream yellow Additionally, larger, convex colonies with a distinct acidic smell are also common among lactic acid bacteria.
2.4.2 Common features of lactic acid bacteria
Lactic acid bacteria exhibit various morphological differences, yet they share a general homogeneity These gram-positive, non-motile bacteria do not produce spores and have limited capacity for synthesizing substances They can ferment both anaerobically and aerobically, demonstrating a high tolerance for acidic environments (Whittenbury, 1964).
Lactic bacteria thrive in both anaerobic and microaerobic environments, but they have complex growth requirements that cannot be met by pure glucose mineral media alone Most lactic bacteria require a variety of vitamins, such as riboflavin, thiamine, pantothenic acid, niacin, folic acid, and biotin, along with more complex amino acids or nitrogen-containing compounds Consequently, their culture environment is intricate, often enriched with ingredients like yeast and tomato juice The nutritional characteristics of these vitamins and amino acids play a crucial role in the growth of lactic acid bacteria, which are essential for analyzing these compounds in various substrates.
Lactic acid bacteria primarily ferment mono and disaccharides, with the exception of L delbrueckii, which can assimilate starch Most lactic bacteria are unable to ferment starch and polysaccharides but can utilize pentose and citric acid, particularly the heterofermentative types Additionally, lactic acid bacteria possess protease activity, contributing to their functional properties.
26 hydrolysis of milk proteins into peptides and amino acids, this activity is different in different species, the permanent bacillus is highest
Lactic bacteria exhibit remarkable resilience, thriving in drought conditions and demonstrating stability in the presence of carbon dioxide and ethyl Many species can endure environments with alcohol concentrations ranging from 10% to 15% or higher, while certain bacilli can survive in saline conditions with sodium chloride levels between 7% and 10%.
Hygroscopic lactic acid bacteria thrive optimally at temperatures between 25°C and 35°C, while thermophilic bacteria prefer 40°C to 45°C, and cold-adapted strains can grow at temperatures as low as 5°C These bacteria are sensitive to heat, dying within 10 to 30 minutes when exposed to temperatures between 60°C and 80°C Additionally, some strains can produce mucus membranes and exhibit resistance to saprophytic and pathogenic microorganisms, as well as those that cause food spoilage Beyond their lactic acid production, these bacteria also have the potential to generate compounds with antibacterial properties.
Lactic acid bacteria are naturally present in various environments, including air, soil, and water, with significant concentrations in plants and fermented food products like pickled vegetables and yogurt These bacteria also inhabit the human and animal digestive systems Although the classification of lactic acid bacteria is still evolving, they are primarily categorized into three main groups based on their cell morphology.
Coccus (Coccus): double, quadruple, arranged in clusters or chains, rarely stand separately
Bacilli (Lactobacillus): Gram-positive, non-spore forming, arranged in chains
Leuconostoc: oocyte, in addition to lactic acid production also produces polysaccharide mucus
It is the process of converting sugar into lactic acid by microorganisms, typically lactic acid bacteria Lactic fermentable bacteria belong to the
The Lactobacterium family consists of non-spore-forming bacilli and cocci that are primarily non-motile and oxygen-tolerant anaerobic bacteria These organisms can ferment a variety of simple and double sugars but are unable to break down complex carbohydrates and starches Their growth necessitates the presence of peptones, amino acids, or ammonium salts, along with a nutrient-rich environment containing vitamins and minerals Optimal fermentation occurs at a pH range of 5.5 to 6, with inhibition at pH 5 and complete cessation below pH 4.5 The ideal fermentation temperature ranges from 15 to 50°C, although each species has its specific temperature preferences, and exposure to temperatures above 80°C can destroy the bacteria.
Isomorphic lactic acid bacteria are specialized microorganisms that primarily produce lactic acid during fermentation, following the Emble-Mayerhof-Parnas (EMP) pathway to generate pyruvic acid The enzyme lactate dehydrogenase then converts most of this pyruvic acid into lactic acid, which constitutes over 90% of the end products A minor fraction of pyruvate is converted into byproducts such as acetic acid, ethanol, CO2, and acetone, with the quantity of these byproducts influenced by the availability of oxygen.
Some strains of lactic isomeric fermentation
Streptococcus lactic, commonly referred to as S lactic, is a short bacillus that typically appears in pairs or short chains when young This bacterium thrives in moist environments, with optimal growth occurring at temperatures between 30°C and 35°C It is known for coagulating milk within 10 to 12 hours and can produce an acid concentration of 0.8% to 1% in its surroundings, with an ideal growth temperature of 10°C.
28 maximum degree is 40 ÷ 45°C Some strains form bacteriocin in the form of nisin
S lactic: Streptococcus is widely used in the processing of products such as yogurt, sour cream, and cheese
Lactobacillus casein (L casein) is a small bacillus that exists in both long and short chain forms, capable of accumulating up to 1.5% acid Its optimal growth temperature ranges from 30 to 35°C, and due to its protease activity, L casein effectively hydrolyzes casein in milk into amino acids.
L acidophilus: long heat resistant bacillus, the optimal temperature is about
Milk should be maintained at temperatures between 30°C and 40°C, with a minimum of 20°C, to prevent the accumulation of up to 2.2% acid The bacillus responsible for this process is isolated from the intestines of children and newborns and is used to produce acidophilus milk This type of milk is known for its ability to produce bacteriocin, which exhibits inhibitory activity against intestinal pathogens, while certain strains can also promote the health of mucous membranes.
Occurs in the case of lactic bacteria without the basic enzyme in the Emblen
The Mayerhof-Parnas pathway involves the conversion of 5-phosphate xylulose in the pentose phosphate pathway, resulting in the production of lactic acid and various by-products In this process, approximately 50% of the sugar is transformed into lactic acid, while the remaining products include acetic acid, ethanol, and carbon dioxide, with lactic acid representing about 40% of the dissolved sugars The distribution of by-products, which also includes 20% succinic acid and 10% ethyl alcohol, is influenced by factors such as cultivar, vegetative environment, and external conditions The variety of products generated during heterofermentative lactic fermentation highlights the complexity of enzyme systems present in lactic acid bacteria, indicating a more intricate hydrolysis and glucose metabolism process compared to other lactic acid bacteria isoforms.
Some strains of heterofermentative lactic acid bacteria
OVERVIEW OF HYDROPEROXIDE AND BACTERIOCIN DURING
Lactic fermentation effectively reduces carbohydrates while generating various organic compounds with antimicrobial properties, primarily lactic, acetic, and propionic acids Numerous lactic acid bacteria can synthesize additional antibacterial compounds, which exhibit low molecular weight and possess unique characteristics such as antimicrobial activity at low pH, thermal stability, a broad spectrum of action, and solubility in acetone The fermentation medium is enriched with these beneficial organic substances following the process.
Bacteriocin is a significant antibacterial compound extensively studied and utilized in food as a biological preservative, particularly due to its heat-stable properties that effectively inhibit Gram-positive bacteria (Karpinski, 2016) Additionally, organic acids, especially lactic acid produced during fermentation, contribute to antibacterial effects by lowering pH levels Hydroperoxide and other substances also play a role in this antibacterial activity Consequently, lactic fermentation fluid, used as a tofu precipitation agent, possesses natural antibacterial properties that help extend the storage life of tofu without the need for chemical preservatives, ensuring the safety and health of consumers.
Hydroperoxide (H2O2) is a byproduct of lactic fermentation, known for its strong oxidizing properties that exhibit a bactericidal effect by oxidizing membrane lipids and protein groups Its reactions can generate atomic oxygen, leading to an anaerobic environment that is unfavorable for various microorganisms Lactic acid bacteria synthesize hydroperoxide in the presence of oxygen, contributing to its formation during fermentation processes.
Lactic acid bacteria require a source of heme to produce catalase, which is essential for the removal of hydroperoxide Without heme, these bacteria struggle to eliminate hydroperoxide effectively, resulting in its accumulation, as other removal systems are less efficient (Ouwehand, 2004).
Bacteriocin is essentially an antibacterial peptide produced by bacteria to against other bacteria (Karpinski, 2016) Thus, the type of bacteria which capable
Bacteriocin, produced by lactic acid bacteria such as Lactobacillus and Lactococcus, is a positively charged protein molecule composed of 30 to 60 amino acids, known for its ability to inhibit harmful bacteria like Clostridium botulinum and Listeria monocytogenes This antimicrobial peptide is safe for human consumption, as it does not trigger allergic reactions or health issues and is rapidly broken down by proteases and lipases All groups of lactic acid bacteria, including Enterococcus, Streptococcus, Leuconostoc, and Pediococcus, produce bacteriocin, with Lactobacillus and Lactococcus being the most significant contributors.
OVERVIEW OF TOFU PRESERVATION
The final stage of tofu production involves packaging and preservation, which significantly influences the taste, quality, and shelf life of the product The choice of packaging materials and storage techniques plays a crucial role in maintaining the integrity of the tofu.
Tofu products have a high moisture and protein content, creating 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 crucial topic in food preservation Currently, there are various methods employed to preserve tofu, including the use of preservation chemicals and advanced packaging technologies.
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 an environment where the gas composition is initially controlled but changes over time This technique replaces the air in food packaging with a specific mixture of gases, including carbon dioxide, oxygen, and nitrogen, to enhance food quality and prevent deterioration By altering the gas composition surrounding the food, MAP helps maintain its physical, chemical, and biological integrity.
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), revealing challenges in eliminating spoilage despite controlling carbon dioxide and oxygen levels in refrigerated conditions Another experiment utilized a CO2 and N2 mixture (3:7 ratio) with flushing or vacuum compensation for packaging, while air-packed tofu served as the control Findings indicated that the microbial count in air-packed tofu was one to four log cycles higher than in MAP after 10 days of storage, with MAP effectively inhibiting microbial growth for up to 14 days.
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 the viability of trimagnesium citrate (TMC) as a substitute coagulant for tofu, benchmarking it against conventional industry coagulants Their study assessed yield, water-holding capacity (WHC), texture profile analysis (TPA), confocal microscopy, and sensory evaluation of both raw and cooked tofu produced with various coagulants The findings revealed that while TMC did not affect the yield, it positively impacted the textural characteristics and enhanced the sensory quality of the tofu.
Yin and partners investigated the effects of fermentation with Actinomucor elegans on the phenolic components, antioxidant activities, and nutritional compounds of tofu Their results indicated a significant increase in total and soluble phenolic content, along with a decrease in insoluble phenolic content post-fermentation Additionally, the fermented tofu exhibited higher antioxidant activities compared to unfermented tofu Metabolomic analysis revealed a notable enhancement in the nutritional composition of tofu, including carbohydrates, alcohols, fatty acids, organic acids, inorganic acids, and amino acids during fermentation These findings suggest that fermentation with A elegans can significantly improve the nutritional and functional properties of tofu.
In 2020, Elvira and colleagues explored the integration of machine learning and artificial intelligence in their research, examining the effectiveness of Plasma Activated Water (PAW) as an immersion solution, alongside the use of chemical preservatives and natural microbiological strains.
35 these results evidence that PAW is a promising non-thermal technology which can facilitate the control of pathogenic microorganisms on tofu while retaining its physical and functional properties
Tofu, a traditional and popular food, has not been extensively studied in Vietnam However, a significant study conducted in 2014 by Nguyen Thi Minh Nguyet and Pham Thi Kim Ngoc explored the effects of coagulation agents on tofu's recovery efficiency and properties Their findings revealed that sour water can be effectively used to produce safe and high-quality tofu, while also highlighting the unique characteristics 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
Table 3.3 Laboratory instruments Number Laboratory instruments Origin
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
A 20 mL sample was combined with 100 mL of MRS broth medium to cultivate lactic acid bacteria from yogurt and fermented soy milk The mixture was incubated at 37°C for 24 hours while being shaken at 150 rpm.
After diluting the incubation medium to a concentration of 10^-4, 100 µL was spread onto an MRS agar plate and incubated at 37°C After 48 hours, characteristic colonies were observed and selected for repeated inoculation on MRS agar until homogeneous colonies were achieved.
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 to produce tofu Samples were then collected to assess production efficiency, following the experimental arrangement outlined below.
Figure 3.1 Tofu production process using lactic acid bacteria
1 Indicate the quantity of total aerobic bacteria
The study aimed to evaluate hygiene levels in tofu processing and storage while also assessing the antibacterial properties of tofu produced using lactic acid bacteria Two parallel samples were analyzed: one made with lactic acid bacteria and another produced with plaster.
The method of enumeration of total aerobic bacteria is specified according to TCVN 5165 - 90 (Appendix 4)
Percentage of fermented soy milk used = 20%
Figure 3.2 Process of enumeration of total aerobic bacteria
Coliforms are a diverse group of Gram-negative bacteria, including varieties such as E coli, Citrobacter, Klebsiella, and Enterobacter These non-spore-forming bacteria can ferment lactose and produce gas within 48 hours when cultured at the appropriate temperature, demonstrating their ability to thrive in various environments.
The purpose of this study is to evaluate the hygienic quality of water and the sanitary conditions in food processing, specifically focusing on the fecal pollution of water sources The research involves monitoring the fluctuations of two groups of bacteria from the production stage through storage, similar to other microbiological indicators Experiments were conducted on two tofu samples, utilizing enumeration methods for Coliforms and Fecal coliforms as outlined in TCVN 4882: 2007 (Appendix 5).
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)
Prepare sample: 1g of tofu + 9ml of distilled water
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)
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 sensory evaluation of the product adheres to Vietnamese standards (TCVN 3215 - 79), utilizing a 20-point scale with six levels ranging from 0 to 5 In this system, the highest score an indicator can receive is 5, while the lowest is 0 Each inspector's evaluation is based on recorded observations.
From the above dilutions, transfer 100 μl of sample to a Petri dish containing YGC medium (2 dishes each)
Count the colonies growing on the plate
Result: Total number of aerobic bacteria in the sample (CFU / g) Prepare sample: 1g of tofu + 9ml of distilled water
44 results, compare with the description and criteria and use an integer to score from
When evaluating a product with an odd number of testers (N), the average score is calculated as the mean of the scores provided by the testers, rounded to two decimal places The weighted average point 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
Requirement about average score (without important coefficient)
Excellent 18.6 ÷ 20 Important criteria have higher than 4.7 score Good 15.2 ÷ 18.5 Important criteria have higher than 3.8 score Fairly good 11.2 ÷ 15.1 Each criteria have higher than 2.8 score
Bad 7.2 ÷ 11.1 Each criteria have higher than 1.8 score Very bad 4.0 ÷ 7.1 Each criteria have higher than 1.0 score
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
Color 5 Ivory white crust, milky white cut, uniform color, without any strange color spots on the bean, water escapes clear, when frying is even yellow
4 The crust and inside coat are ivory-white, uniform color, without any strange spots on the surface of the bean, the water escapes clear, when frying is even yellow
The crust and inner coat of the beans exhibit a uniform opaque yellow color, free from any unusual spots When cooked, the water appears slightly cloudy, and during frying, the beans maintain an even yellow hue.
The crust appears ivory white or opaque yellow, free from any unusual color spots When frying, the water may become slightly opaque and yellow, indicating uneven cooking, with some areas exhibiting lighter colors.
1 The crust color is not uniform, the surface is a bit glossy, due to the viscous layer coming out, slightly opaque water escapes
0 The crust is black, white, speckled with pink color due to the development of mold, the water coming out have the color of rice water
Odor 5 The characteristic aroma of cooked soybeans, without any burning, sour, or any strange smell
4 The smell of beans fades, appears a burning odor, no smell of sour, strange smell
3 Clear burning smell, no sour odor
2 Clear burning smell, slightly sour
1 Clear burning smell, much sour
Taste 5 Typical taste of cooked soybeans , with no sour, acrid taste and have aftertaste
4 Typical taste of cooked soybeans , with no sour, acrid taste and have no aftertaste
3 Typical taste of cooked soybeans , a bit sour and acrid taste
2 Typical taste of cooked soybeans , have sour and acrid taste
1 Sour and acrid taste, no more typical taste of cooked soybeans
The dish has a distinctly sour and acrid flavor, lacking the traditional taste of cooked soybeans Its texture features a smooth crust with no cracks, and it cuts easily while remaining soft to the bite When pressed lightly, it demonstrates elasticity and a slightly rough surface, ensuring it does not break apart during frying.
4 Smooth crust, no cracks, smooth cuts, a little tough when eating, when pressing lightly by hands show elasticity, slightly rough, not broken when frying
3 Smooth crust, no cracks, smooth cuts, a little tough when eating, when pressing lightly by hands show no elasticity, slightly rough, not broken when frying
2 Smooth crust, no cracks, cuts are no longer smooth, hard to eat, broken when frying
1 The surface is not smooth, the cuts are not smooth, the structure is broken when frying
0 Too hard or too soft, the structure is broken when frying
A sensory evaluation was performed on three occasions, with each session featuring two distinct tofu samples produced at different times Panel members independently assessed the samples using a sensory description scorecard and documented their evaluations in the sensory assessment records.
- 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
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
A 20 mL sample was combined with 100 mL of MRS broth medium to cultivate lactic acid bacteria from yogurt and fermented soy milk The mixture was incubated at 37° C for 24 hours while being shaken at 150 rpm.
After diluting the incubation medium to a concentration of 10^-4, 100 µL was spread onto MRS agar plates and incubated at 37°C After 48 hours, characteristic colonies were observed and selected for repeated inoculation on MRS agar until homogeneous colonies were achieved.
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 fermentation was selected to produce tofu Samples were then taken to assess production efficiency, with the experimental setup outlined as follows.
Figure 3.1 Tofu production process using lactic acid bacteria
1 Indicate the quantity of total aerobic bacteria
The study aimed to evaluate hygiene levels in the processing and storage of tofu while also assessing the antibacterial properties of tofu made with lactic acid bacteria Two parallel samples were analyzed: one tofu sample produced using lactic acid bacteria and another produced with plaster.
The method of enumeration of total aerobic bacteria is specified according to TCVN 5165 - 90 (Appendix 4)
Percentage of fermented soy milk used = 20%
Figure 3.2 Process of enumeration of total aerobic bacteria
Coliforms are a diverse group of Gram-negative, non-spore-forming bacteria that can ferment lactose, producing gas within 48 hours under suitable culture conditions This group includes various species such as E coli, Citrobacter, Klebsiella, and Enterobacter.
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 and monitoring the fluctuations of two bacterial groups from production through storage Experiments were conducted using two tofu samples, with enumeration methods for Coliforms and Fecal coliforms following TCVN 4882: 2007 (Appendix 5).
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)
Prepare sample: 1g of tofu + 9ml of distilled water
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)
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 sensory evaluation of the product adheres to Vietnamese standards (TCVN 3215 - 79), utilizing a 20-point scale with six levels ranging from 0 to 5 In this system, the highest score for any indicator is 5, while the lowest is 0 Each inspector's evaluation is based on the recorded results.
From the above dilutions, transfer 100 μl of sample to a Petri dish containing YGC medium (2 dishes each)
Count the colonies growing on the plate
Result: Total number of aerobic bacteria in the sample (CFU / g) Prepare sample: 1g of tofu + 9ml of distilled water
44 results, compare with the description and criteria and use an integer to score from
When evaluating a product with an odd number of testers (N), the average score is calculated by taking the mean of the scores given by the testers, rounded to two decimal places The weighted average point for a specific indicator is derived 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
Requirement about average score (without important coefficient)
Excellent 18.6 ÷ 20 Important criteria have higher than 4.7 score Good 15.2 ÷ 18.5 Important criteria have higher than 3.8 score Fairly good 11.2 ÷ 15.1 Each criteria have higher than 2.8 score
Bad 7.2 ÷ 11.1 Each criteria have higher than 1.8 score Very bad 4.0 ÷ 7.1 Each criteria have higher than 1.0 score
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
Color 5 Ivory white crust, milky white cut, uniform color, without any strange color spots on the bean, water escapes clear, when frying is even yellow
4 The crust and inside coat are ivory-white, uniform color, without any strange spots on the surface of the bean, the water escapes clear, when frying is even yellow
The crust and inner coat of the beans exhibit a consistent, opaque yellow color without any unusual spots When cooked, the water released is slightly cloudy, and the frying process results in an even yellow hue.
The crust appears ivory white or opaque yellow, free from any unusual color spots When frying, the water may become slightly opaque and yellow, indicating uneven cooking, with some areas exhibiting lighter hues.
1 The crust color is not uniform, the surface is a bit glossy, due to the viscous layer coming out, slightly opaque water escapes
0 The crust is black, white, speckled with pink color due to the development of mold, the water coming out have the color of rice water
Odor 5 The characteristic aroma of cooked soybeans, without any burning, sour, or any strange smell
4 The smell of beans fades, appears a burning odor, no smell of sour, strange smell
3 Clear burning smell, no sour odor
2 Clear burning smell, slightly sour
1 Clear burning smell, much sour
Taste 5 Typical taste of cooked soybeans , with no sour, acrid taste and have aftertaste
4 Typical taste of cooked soybeans , with no sour, acrid taste and have no aftertaste
3 Typical taste of cooked soybeans , a bit sour and acrid taste
2 Typical taste of cooked soybeans , have sour and acrid taste
1 Sour and acrid taste, no more typical taste of cooked soybeans
The dish exhibits a very sour and acrid flavor, lacking the typical taste of cooked soybeans It features a smooth crust with no cracks and soft texture, demonstrating elasticity when lightly pressed by hand Additionally, the surface remains intact and does not break apart during frying.
4 Smooth crust, no cracks, smooth cuts, a little tough when eating, when pressing lightly by hands show elasticity, slightly rough, not broken when frying
3 Smooth crust, no cracks, smooth cuts, a little tough when eating, when pressing lightly by hands show no elasticity, slightly rough, not broken when frying
2 Smooth crust, no cracks, cuts are no longer smooth, hard to eat, broken when frying
1 The surface is not smooth, the cuts are not smooth, the structure is broken when frying
0 Too hard or too soft, the structure is broken when frying
A sensory evaluation was performed three times, with each session featuring two distinct tofu samples produced at three different times The sensory panel utilized a scorecard to assess the samples based on specific sensory descriptions Each panel member independently evaluated the tofu and documented their findings in the sensory assessment.
ANALYSIS METHOD
- 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
DATA PROCESSING METHODS
AND DISCUSSION
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-like shapes, predominantly appearing in two forms: short rod chains and long rods A representative image 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
SC1 Pink color DP1 Purple color
SC2 Pink color DP2 Pink color
SC3 Pink color DP3 Purple color
SC4 Purple color DP4 Pink color
SC5 Pink color DP5 Pink color
Figure 4.4 Gram possitive bacteria Figure 4.5 Gram negative bacteria
In a study assessing catalase activity, four strains of gram-positive bacteria were tested with 30% hydrogen peroxide (H2O2) The results indicated that none of these strains exhibited bubble formation, confirming the absence of catalase activity among them.
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.
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 Moreover, the selected strain must not only ferment quickly but also possess a desirable flavor profile to ensure the quality of the final tofu product is not compromised.
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
SC4 Unpleasant smell, acrid sour
Bright yellow solution floating on top
The soymilk strongly precipitates on the bottom, smooth and without effervescence
DP1 Softened fermented flavor Milky white color The soymilk slightly precipitated on the bottom
Bright yellow solution floating on top, layer classification
The soymilk strongly precipitates on the bottom, smooth and without effervescence
Precipitated soymilk, with a suspension layer
Lactic acid bacteria SC4 demonstrated the ability to thrive in soy milk medium, rapidly decreasing the pH levels However, sensory evaluations of the soy milk fermentation revealed that this strain did not yield favorable results.
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 results confirmed the selection of the DP3 strain, which meets the initial research requirements The next step involves investigating the tofu production efficiency using this selected 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 exhibits catalase activity and typically lowers pH through fermentation Testing with three types of sugar revealed that DP3 ferments sugar to produce acid, as indicated by the yellow fluid and the absence of gas bubbles, while the Durham tube remained green.
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
Tofu production efficiency
The process of producing tofu with lactic fermented soy milk tested is summarized as shown in the diagram in Figure 3.1
Tofu production begins with 200g of soybeans and 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.
Characteristics of freshly made LAB tofu
- 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
Determine the storage time
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
Figure 4.8 illustrates a notable difference in total aerobic microorganism counts between the two tofu samples, with both samples showing an increase in microbial counts over time However, the experimental sample exhibited a slower rate of increase compared to the control sample, reaching 3.1 x 10³ CFU/g after 9 days of storage.
Fermented soy milk, produced by laboratory bacteria that generate bacteriocin, serves as an effective soy protein precipitation agent The presence of bacteriocin in the soy milk helps to inhibit the growth of aerobic microorganisms, contributing to the product's preservation and safety.
The results of determining the total number of Coliforms are shown in the chart Figure 4.9 and Appendix Table 9
Figure 4.9 Total number of coliforms in 2 types of tofu
The study revealed the presence of Coliform bacteria, indicating contamination in the samples Over the storage period, Coliform levels increased, with the control sample exhibiting a faster growth rate than the others However, the lactic fermented soy milk samples demonstrated antibacterial properties that effectively inhibited the growth of these microorganisms.
The results of determining the total number of mold are shown in the chart Figure 4.10 and Appendix Table 10
Figure 4.10 Total number of mold in 2 types of tofu
DAY 0 DAY 3 DAY 6 DAY 9 DAY 12
DAY 0 DAY 3 DAY 6 DAY 9 DAY 12
Figure 4.10 illustrates a significant disparity in total mold presence between two tofu samples The tofu made with lactic fermented soy milk exhibited no mold growth, while the control sample showed no mold until day 6, when a sharp increase in mold levels was observed These findings indicate that the active compounds in fermented sour juices from DP3 strains possess strong antifungal properties.
4.4.4 Results comparing sensory quality between common tofu and tofu produced with lactic fermented soy milk
The overall sensory scores for control tofu and tofu made from lactic fermented soy milk are presented in the accompanying table, while a statistical comparison of the mean values for the common sensory score can be found in the appendix.
Table 4.5 Sensory score results between normal tofu and lactic fermented soy milk
Sample 1 st 2 nd 3 rd Average score
The study revealed that both control tofu and lactic-fermented tofu achieved favorable sensory ratings, with lactic-fermented tofu scoring 17.44, surpassing the 17.14 score of conventional tofu This indicates that lactic fermentation enhances the sensory quality of tofu, resulting in a more satisfactory product.