ABTRACT Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Food Technology Student name Luong Nguyen Chinh Thesis Title Isolation of Lactic Acid Bacteria appl
Research rationale
Tofu, a vital food made from soy protein, is a traditional staple in Southeast Asia known for its high nutritional content and excellent digestibility Recognized for its health benefits by the FDA in 1999, tofu has gained popularity worldwide, leading to increased consumption in Western countries in recent years.
Research indicates that incorporating tofu and soy products into your diet can reduce the risk of chronic diseases such as cancer, heart disease, and osteoporosis Soy protein contains isoflavones and isoflavolesterol, which are effective in combating atherosclerosis, and it has a lower impact on serum total cholesterol, LDL, and triglycerides compared to animal proteins (Carrol, 1991; Potter, 1998) Additionally, the antioxidants in tofu, including isoflavones, aglycones, and proteins, help protect against lipid oxidation, promoting overall health (Jackson et al., 2002) Despite its health benefits and popularity among both urban and rural populations, tofu production in Vietnam is primarily small-scale, utilizing rudimentary technology and outdated equipment, resulting in products with limited shelf life—typically only 1-2 days—due to lack of quality registration, packaging, and proper storage.
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 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 utilize impure chemicals, which can result in metal residues contaminating the final product, raising concerns about food safety and quality.
Lactic bacteria and their benefits in food technology are of significant interest to scientists due to their ability to produce organic compounds with antibacterial properties Recent research focuses on utilizing lactic fermentation and its derived substances as natural biological preservatives to reduce mycotoxins in food, yielding promising and remarkable results.
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
This research was conducted at the Faculty of Food Biotechnology and Food Technology, Thai Nguyen University of Agriculture and Forestry However, the facility faces limitations due to the lack of specialized equipment and machinery essential for the research process.
REVIEW
OVERVIEW OF SOYBEAN
Soybeans are scientifically known as Glycine max Merril According to
Soybeans are legumes cultivated as short-term crops, typically harvested between 80 to 150 days, with plant heights ranging from 30 to 80 cm depending on the variety They are characterized by an upright growth habit, with fewer branches than other legumes, and produce fruits in clusters of 2 to 20 on each branch, totaling nearly 400 fruits per tree Each soybean fruit, slightly curved and measuring approximately 4 to 6 cm in length, contains 1 to 7 seeds and exhibits various shapes such as round, oval, long, or flat, with colors including yellow-green, gray, black, and predominantly yellow The soybean fruit comprises three parts: the shell, cotyledons, and embryo, with the cotyledons serving as the primary nutrient reserve.
Soybean seeds differ from cereal grains by lacking an aleurone layer; instead, the endosperm and embryo are separate structures The entire bean functions as a large embryo encased within a seed coat As a result, soybeans contain less starch compared to other legumes, but they have a significantly higher protein and lipid content, making them a unique and nutrient-dense legume.
2.1.2 Acreage, yield and demand for soybeans
2.1.2.1 Production situation in the world
Soybeans originated in East Asia, but nearly 60% of global soybean production takes place in the Americas, with the United States and Brazil leading as the top producers worldwide Recent data indicates that Brazil has surpassed the US, producing 124 million tons in the first half of 2020, with approximately two-thirds of its soybean output exported Other significant soybean-producing countries include Argentina, China, India, Paraguay, and Canada The soybean export volumes of major countries from 2017 to the first half of 2020 are detailed in Table 2.1, highlighting shifts in global soybean trade dynamics.
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 quick growth and wide adaptability, making them suitable for multiple cropping seasons throughout the year, including winter-spring, spring, summer-autumn, and spring-summer In Vietnam, soybeans are predominantly cultivated in the mountainous and midland regions of the northern provinces, such as 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 agricultural sector expands annually by approximately 100,000 hectares, primarily focusing on winter crops that yield around 160,000 tons as of 2017 Currently, these crops satisfy only about 8-10% of the country's total demand, which is increasing at an average rate of 53% annually Despite this growth, Vietnam still relies heavily on imports for soybeans, primarily sourcing them from countries such as China to meet domestic needs.
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, especially for tofu production, has outpaced domestic supply, making imported soybeans the primary source As local production cannot meet total consumption needs, reliance on imports has become essential to fulfill the growing market demand.
Soybeans are characterized by their higher protein and lipid content compared to other legumes, making them a valuable nutritional source Notably, soybeans have a lower starch content than many other legumes, which enhances their nutritional profile Among commercial legumes, soybeans exhibit the highest protein content, emphasizing their importance as a protein-rich crop (Yaklich 2001).
Soybean seeds exhibit a protein content ranging from 39.5% to 50.2% and an oil content between 16.3% and 21.6% on a dry matter basis, making them a valuable source of both nutrients In addition to these primary components, soybeans contain minor constituents such as phospholipids, vitamins, minerals, trypsin inhibitors, phytates, oligosaccharides, and isoflavones, which contribute to their nutritional and functional properties (Liu, 1997; Liu et al., 1995).
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 highlights that soybean seed proteins are predominantly concentrated in the endosperm and embryo, whereas the seed coat contains lower protein levels but has a higher carbohydrate content This distribution is important for understanding soybean seed composition and how it impacts nutritional value and processing.
In soybean seeds, the majority of protein is concentrated in the endosperm and embryo, while the seed coat contains less protein but higher levels of carbohydrates Soy protein is a significant component, primarily composed of non-substituting amino acids, with the exception of low methionine and tryptophan content The remaining amino acids in soy protein have a similar composition to those found in other key plant-based products.
Soy protein primarily consists of globulin, which makes up 85% to 95% of the protein content, though this varies among soybean varieties Additionally, soybeans contain smaller amounts of albumin, with negligible levels of prolamin and glutelin The overall protein content in soybeans ranges from 29.6% to 50.5%, averaging approximately 36% to 40%, making it a rich plant-based protein source.
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 contain a high proportion of unsaturated fatty acids, with 50-60% being linolenic acid (C18-2), which contributes to soybeans' classification as seeds of high biological value However, this high unsaturated fat content also makes soybeans prone to oxidation, increasing the risk of 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 constitute approximately 34% of the dry weight of the grain, with negligible starch content They are categorized into two types: soluble and insoluble carbohydrates, with water-soluble carbohydrates comprising only about 10% of the total The carbohydrate profile of soybean seeds is detailed in Table 2.7.
Table 2.7 Carbohydrate components in soybean
Soybeans are rich in essential vitamins necessary for overall body development, excluding vitamins C and D, which are present in minimal amounts and can be easily lost during processing The detailed vitamin composition of soybeans is provided in Table 2.8, highlighting their nutritional benefits.
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
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OVERVIEW OF TOFU
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Tofu is a nutritious plant-based protein rich in essential minerals like calcium, iron, and vitamin B, making it a popular healthy food choice It is naturally low in sodium and cholesterol-free, promoting heart health and dietary wellness Produced from soy protein gel, tofu's production methods are evolving from traditional manual techniques to advanced automatic processes Modern tofu manufacturing equipment, primarily developed in Japan, enhances efficiency and product consistency, supporting the global demand for high-quality tofu.
The first step in tofu production involves soaking soybeans, extracting the soy milk, and boiling the milk solution A coagulant such as CaCl2 or MgCl2 is then added to curdle the soy milk, forming a solid curd This curd is subsequently processed into tofu, typically shaped into blocks for convenient consumption.
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
In wet grinding, beans must undergo a soaking process to absorb water and swell, facilitating optimal grinding This involves two key stages: the solvate process, where soybean bonds remain intact, and the hydration process, where water molecules break molecular bonds, transforming the beans into a flexible colloidal state within the cells The effectiveness of soaking depends on three main factors: soaking time, the amount of water used, and the temperature of immersion Proper control of these factors ensures efficient water absorption and prepares the beans for the grinding process.
- Soaking time: outdoor temperature from 15 ÷ 25°C, soaking 5 ÷ 6 hours; outdoor temperature 25 ÷ 30°C, soaking 3 ÷ 4 hours
The optimal soaking water temperature for soybeans is between 20°C and 25°C, as soaking at higher temperatures causes rapid seed swelling but reduces their overall swelling capacity When soybeans are soaked at elevated temperatures, their ingredients tend to coagulate rather than remain in colloidal solution, making them harder to dissolve Therefore, maintaining the ideal temperature range ensures effective hydration and easier processing of soybeans.
For optimal soaking, use a water-to-bean ratio of 1:2.5, which promotes significant swelling and reduces acidity During the soaking process, the beans should absorb enough water to reach a moisture content of approximately 55% to 60%, ensuring they are properly hydrated for the next cooking stage.
Grinding is a mechanical process that breaks down cells to release proteins, lipids, and carbohydrates, which can then be turned into suspensions with dissolved water The key factor during this process is the amount of water added; adding too little water causes high friction and temperature rise, leading to protein denaturation and decreased solubility Conversely, adding excessive water increases dissolved substances but complicates subsequent processing stages For optimal results when grinding beans, a water-to-bean ratio of 1:6 is recommended.
After grinding, the suspension consists of a colloidal solution and water-insoluble solids During separation, solids may retain colloidal particles on their surface, making it essential to rinse the residue with water to remove residual colloids Care should be taken to use an appropriate amount of water during washing to prevent waste The filtration process involves two key steps: refining filtration and crude filtration, ensuring effective separation of solids from the colloidal solution.
Immediately heat the filtered milk solution to effectively break down enzymes like trypsin and toxins such as aflatoxin, while also killing microorganisms and deodorizing the fishy smell Rapid heating helps to disrupt the solvated layer, promoting easier coagulation of milk molecules for improved quality For optimal results, boil 100 liters of milk within 5 to 10 minutes, stirring continuously during the process to prevent burning and ensure uniform heating.
After boiling the soy milk must precipitate immediately The protein
The 19 isoelectric region plays a crucial role in protein precipitation, with salt significantly affecting the process During milk precipitation, heating to 95–100°C induces thermal denaturation of proteins, leading to effective coagulation Various agents can induce protein precipitation, including natural sour water, CaCl₂, CaSO₄, acetic acid, and lactic acid; among these, natural sour water is considered the most suitable However, using sour water requires considerable expertise to achieve optimal results Key conditions for soy milk precipitation include precise temperature control and appropriate choice of precipitation agents to ensure successful protein coagulation.
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 is crucial for efficient precipitation, as a high pH requires a larger volume of sour water, while a low pH reduces protein recovery efficiency To optimize the process, slowly add sour water to the milk solution once it reaches 95°C, following a three-phase approach In the first phase, incorporate half of the sour water to ensure controlled pH adjustment and maximize protein recovery.
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 precipitating, the tofu curd is ready to be shaped by placing it into a mold For optimal texture and shape formation, press the tofu curd at a temperature between 70°C and 80°C Temperatures below 60°C are insufficient for proper adhesion and shaping The ideal pressing duration is approximately 10 minutes to achieve firm, well-formed tofu.
After pressing the tofu to remove excess moisture, cut it into the desired sizes Then, soak the tofu in water to help achieve a firmer structure, allowing it to sit longer for the tofu to develop a slight sour taste This process ensures the tofu has a stable texture and enhanced flavor.
FUNDAMENTAL OF THE GEL PROTEIN FORMATION PROCESS OF
The formation of protein gel is essential for achieving the desired tofu texture, as soy proteins in their native state do not naturally form gels To create tofu, soy proteins must undergo heat denaturation and coagulation processes Thermal denaturation initiates protein unfolding, followed by proper protein ordering, which leads to gel formation These critical steps ensure that proteins are evenly dispersed within the gel network, resulting in high-quality tofu with the desired texture.
The process of making tofu involves the gelation of soybean protein through heat-induced denaturation of soy milk proteins This controlled thermogenesis triggers an orderly gel formation, transforming the soy milk into firm, cohesive tofu Proper control of this gel-forming process is essential for achieving the desired texture and quality of tofu.
Thermal denaturation aims to cause the protein structure to unfold, transforming a compact form into an open, diffused state that reveals the internal molecular architecture Heating exposes functional groups such as –SH groups, hydrophobic regions, carbonyl groups, peptide bond amines, and amide side chains, which directly influence the protein's network structure This process enhances understanding of protein behavior and interactions, crucial for applications in food science and biochemistry (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 mechanism of soy milk protein gel formation is determined mainly by temperature and consists of two main processes: dissociation and aggregation
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
Protein concentration plays a crucial role in determining the type and properties of soy milk protein gels Gel formation occurs at relatively low gelatin concentrations, but a higher globulin content enhances gel stability A minimum soy protein content of 8% is required for effective gelation, with higher glycinin levels promoting easier gel formation, while low glycinin content can hinder gel development due to protein separation Additionally, the mechanical strength of the gel increases with higher protein concentrations, as it depends on the degree of cross-linking within the protein network, exhibiting a linear relationship between gel strength and protein content.
Temperature significantly impacts soy protein by altering its quaternary structure, with glycinin and β-conglycinin denaturing at 85–95°C and 65–75°C, respectively Glycinin contains a large disulfide bridge, influencing its stability during heating If heating surpasses the minimum denaturation temperature necessary for gel formation, the gel's rheological properties are affected, leading to stronger gels; conversely, lower temperatures require longer times to form gels, resulting in weaker networks Insufficient heating fails to develop a robust three-dimensional protein network, while excessive heat above the soy milk protein denaturation point can cause structural metamorphosis, preventing proper gel formation.
Adding a 2% NaCl salt and soy milk solution enhances the ratio of gel glycin and conglycin, promoting gel formation However, increasing the salt concentration to 10% inhibits gel formation, indicating that high salt levels disrupt protein interactions Low concentrations of NaCl neutralize electrical charges within the protein structure, supporting gel development, whereas high salt content causes protein alterations and increases hydrophobic interactions, preventing gel formation.
Protein denaturation is significantly influenced by pH, which affects interactions between proteins and with solvents (Renkema, 2000) Proper pH adjustment is essential to balance denaturation and recombination, as well as to regulate the attraction and repulsion between adjacent protein chains When pH exceeds 12, the gel formation process is completely inhibited.
At high alkaline pH values, polypeptide chains become negatively charged, leading to electrostatic repulsion that can destabilize protein interactions during gel network formation, resulting in decreased gel strength Conversely, at neutral pH, the interaction between positively charged groups provides additional energy, promoting more effective gel formation.
Soy protein is classified into four segments: globulin 2S, 7S, 11S, and 15S Among these, the 7S and 11S fractions are the primary components of soy protein The 7S fraction is known as conglycinin in soy milk, while the 11S fraction corresponds to glycinin (Nielson, 1985) Together, glycinin and conglycinin make up approximately 65% to 85% of the total protein content in soybeans, highlighting their significance in soy protein composition.
Glycinin is an oligo protein with a complex molecular structure, comprising six monomers linked together to form a hexagonal shape This structure consists of two trimers, each made up of three monomers arranged in a specific sequence Glycinin's molecular architecture is detailed in diagram 2.3, illustrating its hexagonal conformation Additionally, Globulin 7S or 11S is a related protein composed of 12 lipophilic subunits, including six acidic (A) subunits and six basic subunits, contributing to its structural stability and functional properties.
"subunit" (B) The "sub the unit" are linked together by a disulfide bridge
2.3 The molecular structure of Glycinin
Beta-conglycinin is a major storage protein in seeds, representing approximately 35% of the seed's protein content and comprising a glycoprotein with around 5% carbohydrates Its structure consists of three main subunits—α, α', and β—which collectively form its fractional composition The organization of conglynin's sub-fractions is integral to understanding its functional properties and plays a significant role in seed physiology.
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+ ions as a coagulant to precipitate proteins in soy milk solutions can be effective, but chemical coagulation may introduce impurities into the tofu As a safer and more natural alternative, fermentation methods that lower pH—such as using lactic acid bacteria (LAB)—offer a promising substitute LAB is widely recognized in the food industry for its dual benefits: improving food quality and enhancing consumer health.
Lactic acid bacteria are Gram-positive, non-spore-forming, and non-motile bacteria known for their ability to ferment sugars into lactic acid They belong to the family Lactobacteriaceae and are classified into four main genera These beneficial bacteria play a crucial role in fermentation processes and supporting gut health, making them essential in probiotic and food industry applications Understanding their classification helps in optimizing their use in various health and dietary products.
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 typically exist in double forms, series, or as spherical and bacillus shapes, with a diameter ranging from 0.5 to 1.5 micrometers They can also display rod-like structures and form colonies that are small, round, glossy, and range in color from milky white to cream yellow, or larger, convex colonies with a milky appearance These bacteria are often characterized by their distinct acidic smell, which is a key indicator of their presence.
2.4.2 Common features of lactic acid bacteria
Lactic acid bacteria are generally homogeneous despite their morphological differences, sharing key characteristics such as being gram-positive, immobile, and non-spore-forming These bacteria have limited capacity to synthesize various substances but are notable for their ability to undergo both anaerobic and aerobic fermentation (Whittenbury, 1964) Additionally, they exhibit high tolerance to acidic environments, making them resilient in diverse conditions.
OVERVIEW OF HYDROPEROXIDE AND BACTERIOCIN DURING
Lactic fermentation reduces carbohydrate content and produces organic compounds with low molecular weight and antimicrobial properties, primarily lactic, acetic, and propionic acids Various lactic acid bacteria can synthesize additional antibacterial compounds that contribute to their effectiveness These compounds are characterized by their stability at different temperatures, efficacy at low pH, broad-spectrum antimicrobial activity, and solubility in acetone, making them valuable for food preservation and safety.
Bacteriocins, key antibacterial compounds, are extensively studied and used in food preservation due to their heat stability and ability to inhibit Gram-positive bacteria (Karpinski, 2016) In addition, organic acids like lactic acid produced during fermentation lower pH and contribute to antibacterial effects Furthermore, substances such as hydrogen peroxide also play a role in antimicrobial activity Consequently, lactic fermentation fluid used as a tofu coagulant possesses natural antibacterial properties, enabling proper storage without chemical preservatives and ensuring the safety and health of consumers.
Hydroperoxide (H2O2) is a byproduct formed during lactic fermentation, generated by lactic acid bacteria in the presence of oxygen Its strong oxidizing properties contribute to its bactericidal effect by damaging bacterial cell membranes and proteins through oxidation Additionally, reactions involving hydroperoxide can produce atomic oxygen, creating an anaerobic environment that inhibits the growth of certain microorganisms.
Lactic acid bacteria rely on a source of heme to produce catalase, which is essential for removing hydroperoxide Without heme, these bacteria cannot effectively break down hydroperoxide, leading to its accumulation Additionally, alternative hydroperoxide elimination systems in lactic acid bacteria are less active, contributing further to hydroperoxide build-up (Ouwehand, 2004).
Bacteriocins are antibacterial peptides produced by bacteria to inhibit the growth of competing bacteria (Karpinski, 2016) Bacteria capable of producing bacteriocins often develop resistance to them, contributing to their survival These peptides do not trigger allergic reactions in humans, pose no health risks, and are rapidly broken down by proteases and lipases Produced mainly by lactic acid bacteria such as Lactobacillus and Lactococcus, bacteriocins are small, positively charged protein molecules containing 30 to 60 amino acids with a high isoelectric point They are effective against bacteria closely related to the producing strain and can inhibit pathogens like Clostridium botulinum, Bacillus cereus, Bacillus alcalophilus, and Listeria monocytogenes Bacteriocins are present across all groups of lactic acid bacteria, with Lactobacillus and Lactococcus playing particularly significant roles in their production.
OVERVIEW OF TOFU PRESERVATION
The final stage of tofu production involves packaging and preservation, which are crucial in determining the product's taste, quality, and shelf life Choosing the right packaging materials and storage techniques ensures tofu remains fresh and maintains its flavor and texture during distribution and storage, ultimately enhancing consumer satisfaction Proper packaging and storage methods are essential for extending tofu's shelf life while preserving its nutritional value and overall quality.
Tofu products, with their high moisture and protein content, create an ideal environment for microbial growth As a result, even when stored under refrigeration, their shelf life is limited to just a few days (Rossi, Felis, Martinelli, Calcavecchia, & Torriani).
Extending the shelf life of tofu products remains a key concern for producers and consumers alike Today, various preservation methods are employed, ranging from the use of preservation chemicals to advanced packaging technologies, to effectively prolong tofu's freshness and ensure product quality.
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
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 atmosphere with a fixed initial composition that dynamically changes over time (Berk, 2018) This technique replaces the air in sealed packaging with a precisely controlled gas mixture—which may include carbon dioxide, oxygen, nitrogen, and other gases—to alter the gas contact with the food The primary goal of MAP is to inhibit physical, chemical, and biological deterioration, thereby extending the shelf life and maintaining food quality (Tiefenbacher, 2018; Ward, 2016).
MAP has been studied with tofu Stoops, Maes, Claes, and Van Campenhout
A 2012 study investigated Pseudomonas growth in MAP-packaged tofu, revealing that controlling carbon dioxide and oxygen levels during refrigeration struggles to eliminate Pseudomonas spoilage Another experiment utilizing a CO2 and N2 mixture (3:7 ratio) through flushing or vacuum packaging demonstrated that tofu stored under MAP showed significantly reduced microbial counts—up to four log cycles lower—compared to air-packed controls after 10 days The results indicated that MAP effectively inhibited microbial growth for up to 14 days, extending the shelf life of refrigerated tofu (Van Campenhout, Maes, & Claes).
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 potential of trimagnesium citrate (TMC) as an effective alternative to traditional tofu coagulants Their comprehensive study assessed various quality parameters, including yield, water-holding capacity (WHC), texture profile analysis (TPA), confocal microscopic analysis, and sensory evaluation, comparing raw and cooked tofu made with different coagulants The research revealed that TMC did not affect tofu yield but positively influenced textural properties and enhanced sensory quality, indicating its promise as a tofu coagulant.
Research by Yin and colleagues investigates the effects of fermenting tofu with Actinomucor elegans, highlighting significant increases in total and soluble phenolic contents and a reduction in insoluble phenolics post-fermentation The study demonstrates that fermented tofu exhibits higher antioxidant activities compared to unfermented varieties Metabolomic analysis reveals substantial enhancements in nutritional components such as carbohydrates, alcohols, fatty acids, organic acids, amino acids, and inorganic acids during fermentation These findings confirm that A elegans fermentation substantially improves the nutritional and functional properties of tofu, making it a promising method for functional food development.
In 2020, Elvira and colleagues expanded food preservation research by integrating machine learning and artificial intelligence technologies alongside traditional methods such as chemical preservatives and natural microbiological strains Their study evaluated the potential of plasma technology as an innovative preservation approach, highlighting its promise for enhancing food safety and extending shelf life through advanced technological solutions.
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 is a traditional and well-known food, but in Vietnam, research on this product has been limited In 2014, Nguyen Thi Minh Nguyet and Pham Thi Kim Ngoc studied the effects of coagulation agents on tofu quality, focusing on recovery efficiency and other properties Their findings demonstrated that sour water can be used to produce safe, high-quality tofu, revealing several unique characteristics of tofu made with plaster as a coagulation agent.
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 added to 100 mL of MRS broth medium to cultivate lactic acid bacteria present in yogurt and fermented soy milk The mixture was incubated at 37°C for 24 hours with shaking at 150 rpm to ensure optimal bacterial growth and fermentation.
The medium after incubation was diluted to a concentration of 10^-4, and then 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 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
Based on previous experimental results, the optimal pH for soy milk protein precipitation was established A high-performing fermentation strain was selected for its rapid fermentation ability and favorable sensory qualities in soy milk The fermentation process was then used to produce tofu, with samples collected to assess production efficiency The experiment was carefully structured to ensure accurate evaluation of both fermentation performance and tofu quality.
Figure 3.1 Tofu production process using lactic acid bacteria
1 Indicate the quantity of total aerobic bacteria
This study aims to evaluate the hygiene levels in tofu processing and storage, while also assessing the antibacterial properties of tofu produced with lactic acid bacteria Two samples were analyzed concurrently: one made with lactic acid bacteria and another produced using traditional plaster methods The findings provide insights into the hygienic quality and microbial safety of tofu, highlighting the potential benefits of lactic acid bacteria in enhancing product safety and antibacterial effectiveness.
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 group of Gram-negative, non-spore-forming bacteria that can be either aerobic or anaerobic and are capable of fermenting lactose These bacteria, including strains of E coli, Citrobacter, Klebsiella, and Enterobacter, produce gas within 48 hours at optimal culture temperatures The presence of coliforms is commonly used as an indicator of sanitary quality and potential contamination in water and food sources.
This study aims to evaluate the hygienic quality of water and sanitary conditions in food processing by assessing fecal pollution levels in water sources and monitoring fluctuations in coliform and fecal coliform bacteria from production through storage Using standardized enumeration methods as outlined in TCVN 4882: 2007 (Appendix 5), experiments were conducted on two tofu samples to determine the presence and levels of these microbiological indicators, ensuring food safety and compliance with health standards.
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 product evaluation based on the sensory method adheres to Vietnamese Standards (TCVN 3215 - 79) This standard employs a 20-point scale divided into six levels, ranging from 0 to 5, where 5 indicates the highest score and 0 the lowest for each indicator During assessment, each inspector records their evaluations accordingly, ensuring consistent and standardized measurement of product quality.
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
To assess the total number of aerobic bacteria in the sample, prepare 1 gram of tofu mixed with 9 milliliters of distilled water The bacterial count is expressed as CFU per gram, which allows for accurate comparison with established standards and criteria Use these results to evaluate the microbial quality of the sample and assign an appropriate score based on the observed bacterial levels Ensuring accurate measurement and adherence to testing protocols is essential for reliable and meaningful results.
When evaluating products with an odd number of testers, the average score is calculated by taking the mean of all individual scores, rounded to two decimal places The weighted average for each criterion is determined by multiplying the criterion's average score by its corresponding importance coefficient This approach ensures an accurate and standardized assessment of product quality based on multiple tester evaluations.
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 bean are an opaque yellow with a uniform color, free from any unusual spots on the surface When pressed, the water released is slightly cloudy, indicating freshness During frying, the beans turn an even yellow, ensuring a consistent and appealing appearance.
The crust is ivory white or opaque yellow, featuring no unusual color spots on the surface When cooked, the water released appears slightly opaque, and the frying process results in uneven yellowing with some areas exhibiting a lighter color, indicating the dish's doneness and quality.
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
Cooked soybeans should have a very sour and acrid flavor, which is characteristic of their typical taste A high-quality soybean product features a smooth crust with no cracks, smooth cuts, and a soft texture that is pleasant to eat When lightly pressed by hand, it exhibits elasticity without breaking, indicating good quality Additionally, the surface may be slightly rough, but the product remains intact after frying, ensuring optimal flavor and texture.
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
Sensory evaluation of the tofu samples was conducted three times, with each session involving two different samples produced at different times Panel members independently assessed each sample using a standardized sensory description scorecard, ensuring unbiased and accurate results The recorded evaluations provided valuable insights into the sensory attributes of the tofu, contributing to a comprehensive quality analysis.
- 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 added to 100 mL of MRS broth medium to cultivate lactic acid bacteria commonly found in yogurt and fermented soy milk The mixture was incubated at 37°C for 24 hours with shaking at 150 rpm to promote bacterial growth and fermentation.
After incubation, the medium was diluted to a concentration of 10^-4, and 100 µL was spread onto MRS agar plates, then incubated at 37°C Following 48 hours of incubation, characteristic colonies were observed and selected for repeated inoculation on MRS agar until uniform, homogeneous colonies were obtained, ensuring reliable and consistent results.
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
After establishing the optimal pH for soy milk protein precipitation, a fast-fermenting strain with excellent sensory qualities was selected for tofu production The fermentation process was carried out, followed by tofu manufacturing, with samples taken to assess production efficiency The experimental setup was organized to ensure accurate evaluation of both fermentation performance and tofu quality.
Figure 3.1 Tofu production process using lactic acid bacteria
1 Indicate the quantity of total aerobic bacteria
This study aims to evaluate the hygiene levels in tofu processing and storage, ensuring food safety standards are met Additionally, the antibacterial properties of lactic acid bacteria in tofu were assessed to determine their effectiveness in enhancing product safety Two samples were analyzed concurrently: one made with lactic acid bacteria and another produced using traditional plaster methods, allowing for a comparative analysis of microbial safety and hygiene quality.
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 group of Gram-negative, non-spore-forming bacteria that can be either aerobic or anaerobic, capable of fermenting lactose within 48 hours at suitable culture temperatures Key varieties include E coli, Citrobacter, Klebsiella, and Enterobacter, which are commonly used indicators of microbial contamination in food and water safety assessments.
This study aims to evaluate the hygienic quality of water and sanitary conditions in food processing environments by assessing fecal pollution in water sources and monitoring fluctuations of bacterial groups from production through storage The investigation involves experiments with two tofu samples, employing enumeration methods for coliforms and fecal coliforms as specified by TCVN 4882:2007 (Appendix 5) These microbiological indicators are crucial for ensuring food safety and maintaining proper hygiene standards in food processing.
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 complies with Vietnamese standards (TCVN 3215-79), which utilize a 20-point scale divided into six levels from 0 to 5 Each indicator can be scored between 0 (lowest) and 5 (highest), providing a standardized method for assessing product quality Evaluation is conducted by inspectors who record their scores accordingly, ensuring consistency and adherence to national quality standards.
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
To assess the microbial safety of the sample, we measured the total number of aerobic bacteria in the tofu sample, expressed as CFU/g The sample was prepared by mixing 1g of tofu with 9ml of distilled water to ensure accurate microbial analysis Results were compared against established standards and criteria to evaluate the quality and safety of the product Based on the comparison, a score from 1 to 10 was assigned, with higher scores indicating better compliance with safety standards This method provides a reliable assessment of the tofu's microbial content and overall quality.
When multiple testers evaluate a product, and the total number of testers (N) is an odd number, the average score is calculated as the mean of all test results, 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 This method ensures an accurate and meaningful assessment of product quality, adhering to best practices in scoring and data evaluation.
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 are an opaque yellow with a uniform color, free of any strange spots on the surface When pressed, they release slightly cloudy water, and during frying, they turn an even yellow, indicating quality and proper ripeness.
The crust is ivory white or opaque yellow, smooth without any unusual color spots on the surface When cooked, the water releases slightly opaque and yellowish, with uneven frying resulting in some areas appearing lighter in color.
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
A high-quality cooked soybean should have a very sour and acrid flavor, lacking the typical taste of standard cooked soybeans The ideal condition features a smooth crust without cracks, with smooth cuts that result in a soft, easily chewable texture When pressed lightly by hand, the soybean should demonstrate elasticity and a slightly rough surface, and it should not break easily during frying, indicating proper processing and quality.
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
Sensory evaluation was conducted three times, with each session involving the assessment of two different tofu samples produced at three separate time points Panelists independently evaluated the samples using a sensory description scorecard, ensuring unbiased and accurate results The recorded assessments provided valuable insights into the sensory qualities and consistency of the tofu samples across different production batches.
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
Bacterial isolates are characterized by their rod-shaped morphology, predominantly appearing as either short rods forming chains or longer rods These distinct cell forms are key identifiers in microbiological analysis A representative image illustrating some of these isolated bacterial cells can be seen in the accompanying figure Understanding the shape and arrangement of these bacteria is essential for accurate identification and classification.
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
Four gram-positive bacterial strains were tested for catalase activity; however, none of these strains produced bubbles upon exposure to 30% hydrogen peroxide, indicating a lack of catalase enzyme activity in all tested 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.
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 continues to grow effectively on soy milk medium, successfully lowering the pH A rapid decrease in pH within a short period is desirable during fermentation, highlighting the importance of selecting a strain that not only promotes efficient acidification but also ensures the quality of tofu is maintained.
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 successfully incubated in soy milk medium, rapidly lowering the pH during fermentation Despite its effective acidification, this strain did not improve the sensory qualities of soy milk, showing no positive impact on flavor or texture.
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 results confirmed the successful selection of the DP3 strain, which meets all the criteria established at the start of the research With this promising strain identified, the next step is to evaluate its effectiveness in improving tofu production efficiency This study aims to determine how the DP3 strain can optimize the fermentation process and enhance overall yield, contributing to improved tofu manufacturing methods.
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
DP3 is a Gram-positive, non-spore-forming bacterial strain that typically decreases pH through fermentation and exhibits catalase activity When tested for sugar fermentation, DP3 produced a yellow fluid without air bubbles, and the Durham tube remained green, indicating that this strain ferments sugars 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
Tofu production efficiency
The process of producing tofu with lactic fermented soy milk tested is
To produce tofu, 200g of soybeans are processed using a fermented milk solution, following the production diagram outlined in Figure 3.1 After pressing the tofu block, it is immersed in cold water to stabilize the product and prevent rapid souring The final recovered weight of the tofu product is approximately 400g, ensuring optimal quality and freshness.
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
The results presented in Figure 4.8 indicate a significant difference in total aerobic microorganism counts between the two tofu samples Both samples showed an increase in microbial counts over the storage period, but the experiment sample exhibited a slower rate of microbial growth compared to the control Specifically, the control sample reached a total aerobic microorganism count of 3.1×10³ CFU/g after nine days of storage, highlighting the improved microbial stability of the experiment sample.
Fermented soy milk produced with laboratory bacteria contains bacteriocins, which act as natural preservatives When used as a soy protein precipitation agent, these bacteriocins help inhibit the growth of aerobic microorganisms This natural antimicrobial property enhances the safety and shelf life of soy-based products, making them more appealing to consumers seeking health-conscious food options.
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 results indicated the presence of coliform bacteria, signaling contamination in the samples Over storage time, coliform levels increased, with the control sample exhibiting faster microbial growth compared to the lactic fermented soy milk The antibacterial compounds in the fermented soy milk effectively inhibited the growth of coliform bacteria, demonstrating its potential as a natural preservative.
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 clearly illustrates the significant difference in total mold growth between the two tofu samples The tofu produced with lactic fermented soy milk showed no visible mold development throughout the observation period, indicating excellent mold resistance In contrast, the control sample began showing mold growth by day 6, with a rapid increase in mold population thereafter These findings demonstrate that active compounds in fermented sour juices from DP3 strains possess strong mold-inhibitory properties, enhancing the shelf life and safety of tofu products.
4.4.4 Results comparing sensory quality between common tofu and tofu produced with lactic fermented soy milk
The overall sensory score results for control tofu and tofu made with lactic fermented soy milk are summarized in the table A statistical comparison of the mean sensory scores is provided in the appendix, highlighting significant differences between the two samples.
Table 4.5 Sensory score results between normal tofu and lactic fermented soy milk
Sample 1 st 2 nd 3 rd Average score
Lactic-fermented tofu achieved a higher sensory score of 17.44 compared to standard control tofu, which scored 17.14, indicating superior sensory quality Both types of tofu received good sensory ratings, demonstrating that lactic fermentation enhances the overall acceptability of tofu These findings suggest that using lactic fermentation in tofu production can improve sensory attributes, leading to a more satisfying product for consumers.