(Luận văn thạc sĩ) isolation of lactic acid 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 1.3 Detail goals - Isolation

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

LUONG NGUYEN CHINH

Thai Nguyen, December 2020

ACKNOWLEDGEMENT

During my studies and internship at Thai Nguyen University of agriculture and forestry, I have now completed my graduation thesis In order to complete this thesis, I have been guided by the devoted guidance of my supervisor, along with the help of Thai Nguyen University of Agriculture and Forestry, Faculty of Food Technology and Biotechnology and the Advanced Program Office I also received the enthusiastic cooperation from colleagues, help and encouragement of my family members In response to that sentiment, through here I would like to express

my deep gratitude and respect to all collectives and individuals who created conditions to help me throughout the internship process

First of all, I would like to express my sincere thanks to the school management board, the Dean of the Faculty of Food Technology and the collective

of teachers in the Faculty of Food Technology, Thai Nguyen University of Agriculture and Forestry that taught and mentored me during these time, as well

as my graduation internship

In particular, I sincerely thank for the attention and guidance of the

instructing supervisor Dr Luong Hung Tien, who directly guided me to

implement this thesis successfully

Through this, I would also like to express my gratitude to family, relatives and friends for helping and encouraging me during my study and practice at school

Finally, I would like to respectfully send my sincere thanks and best wishes

to the teachers and teachers in the evaluation committee

Thank you sincerely!

Thai Nguyen, December 2, 2020 Student

LUONG NGUYEN CHINH

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PART I INTRODUCTION

1.1 Research rationale

Tofu is an important food made from soy protein (Kohyma et al., 1995) This

is an important traditional food for the people of Southeast Asia due to its high nutritional content and good digestibility (Tsai et al., 1981) The benefits of tofu

to human health were recognized by the FDA in 1999 Due to the recognized nutritional benefits of tofu, there has been an increase in tofu consumption among the western countries in the world in the recent years (Oboh, 2006)

Talking about the benefits of tofu, many researchers believe that using tofu

as well as soy products can reduce the number of chronic diseases such as cancer, heart disease, and osteoporosis Soy protein contains isoplavolesterol and isoflavones, which are effective against atherosclerosis (Carrol, 1991) It is also thought that the consumption of soybean protein has a lower effect on total cholesterol, cholesterol, LDL, and triglycerides in serum compared to animal protein (Potter, 1998) Isoflavones, aglycones, and proteins contained in tofu have antioxidant properties that protect against lipid oxidation (Jackson et al., 2002) Tofu brings many health benefits to consumers, tofu is a popular dish of both urban and rural residents, so it has a large consumer market and stability However,

in fact in Vietnam, tofu is mainly produced on a small scale, mainly on household scale with rudimentary technology, outdated equipment and machinery Tofu products are made without quality registration, no packaging, short storage time which only lasts 1-2 days after production The short shelf life of tofu not only

Moreover, the topic of lactic bacteria as well as a number of benefits related

to the use of lactic bacteria in food technology are of great interest to many scientists Several organic compounds produced during lactic fermentation are considered to be good antibacterial agents [7],[9],[16],[19] Therefore, there are more and more researches applying lactic fermentation or extracting substances from lactic fermentation to be used as biological preservatives, reducing mycotoxins in food more and more and with many remarkable results receive [5], [6], [7], [12]

Therefore, I conducted the topic: " Isolation of lactic bacteria apply in

tofu producing process"

1.2 Research objective

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

1.3 Detail goals

- Isolation some strains of lactic acid bacteria

- Selection of bacteria with good fermentation ability apply for tofu

production

1.4 Limitations

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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

- Equipment and chemicals provided: This study is conducted in the Faculty

of Food Biotechnology and Food Technology, Thai Nguyen university of Agriculture and Forestry Therefore, there inadequacy of specialized machines that are required for the research process

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PART II LITERATURE REVIEW

2.1 OVERVIEW OF SOYBEAN

2.1.1 Characteristic of soybean

Soybeans are scientifically known as Glycine max Merril According to

Phạm Văn Thiều (1993), Book of “Soybean plant, cultivation and product processing techniques” [4], soybeans are legumes, short-term crops (80 ÷ 150 days), and its stem is about 30 to 80 cm high, depending on the variety Soybean plants are relatively upright compared to other legumes Soybean trees have fewer branching than other legume trees; fruit grows in brunch, about 2 to 20 fruits, and each tree has nearly 400 fruits One fruit has from 1 to 7 seeds The soybean fruit

is slightly curved, the average length of it is about 4 to 6 cm Soybeans have many various shapes such as round, oval, long circle, and flat circle Similarly, the color also varies, including yellow-green, gray, black, but mostly yellow Soybeans have three parts: the shell, cotyledons, and the embryo Cotyledon is the part that reserve

of nutrients

Not as in cereal grains, in soybean seeds, there is no aleurone layer; endosperm and embryo stand separately The whole bean is a large embryo surrounded by a seed coat Therefore, compared with other legumes, soybeans contain less starch, while their protein and lipid content is much higher

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

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)

Currently, soybean growing are forming in 4 concentrated areas

 Northern midland and mountainous provinces

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 The Red River Delta region

 South East region

 The Mekong River Delta region

Soybean plants have short-time growth characteristics, wide adaptability, so

they are planted in many crops season a year such as: winter-spring, spring,

summer-autumn, spring-summer In Vietnam, soybeans are grown in the

mountainous and midland regions of the North provinces such as Cao Bang, 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, the country grows about 100 thousand hectares, mainly the winter

crop with an output of about 160 thousand tons (in 2017) meeting about 8-10% of

the country's demand, this demand increases by an average of 53% Soybeans are

now still imported Vietnam import soybean mainly from other 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)

Season 2010 2011 2012 2013 2014 2015 2016 2017

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

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Table 2.3 Production, supply and demand of soybeans in Vietnam

USDA Data

Adjusted Data

USDA Data

Adjusted Data

USDA Data

Adjusted Data

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(Source: Vietnam General Statistics Office, Global Trade Atlas, USDA adjusted statistics)

With the increasing demand for soybeans, domestically produced soybeans are not enough to meet the demand for soybeans in general and tofu products in particular Therefore, soybean material now depends mostly on imports

2.1.3 Chemical composition of soybeans

Soybeans have a lower starch content than other legumes, while their protein and lipid content is significantly higher Among other commercial legumes, the protein content in soybeans was the found to be the highest (Yaklich 2001) [27]

On a dry matter basis, the protein and oil content of soybean seed varies from 39.5% to 50.2% and 16.3% to 21.6% respectively The other minor components include phospholipids, vitamins, minerals, trypsin inhibitors, phytates,

oligosaccharides and isoflavones (Liu 1997, Liu et al 1995) [13]

Table 2.4 Chemical composition of some kind of beans

(Sources: USDA nutrient database)

Kind of beans

Content by percentage by weight of dry matter

Ash Cellulose Sugars Starch Protein Lipid

(Sources: USDA nutrient database)

As it can be seen from table 1.5 in soybean seed, protein amount

concentrate mostly on the endosperm and embryo while the seed coat contains

less protein and has higher amount of carbohydrates

2.1.3.1 Protein component

As it can be seen from table 2.5, in soybean seed, protein amount

concentrate mostly on the endosperm and embryo while the seed coat contains

less protein and has higher amount of carbohydrates Soy protein accounts for a

large amount Soy protein is composed of non-substituting amino acids (except

for low methionine and tryptophan content) while the remaining amino acids

have the same content as other important products

In soy protein, globulin accounts for 85 ÷ 95%, however there are

differences between soybeans There is also an amount of albumin, a negligible

amount of prolamin and glutelin In soybeans, the total protein content ranges

from 29.6% to 50.5%, with an average of 36% to 40%

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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

Soy lecithin makes up 3% of the grain weight A complex phosphatide, used

as an emulsifier, and antioxidant in food processing

2.1.3.3 Carbohydrate

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

Table 2.8 Vitamin content in soybean

(Sources: USDA nutrient database)

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2.1.3.5 Mineral

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

Soybean contain some enzyme, for example:

o Lipase: Hydrolysis glyceride into glycerin and fatty acids

o Phospholipase: Hydrolysis eter to acetic acid

o Lypoxygenas: Catalyst of H2 transfer in fatty acids

o Amylase: Include α-amylaza và β-amylaza

2.1.3.9 Trypsin Inhibitor TI

In soybeans, trypsin inhibitors account for about 6% of the total protein The inhibitory effect of a trypsin inhibitor is due to the fact that it binds to the trypsin to form an irreversible stable compound, whereas kimotrypsin produces

an unstable and reversible substance

2.1.3.10 Hemaglutinin

The hemaglutinin (also known as phytohemaglutinin lectin), which was first discovered in 1953 Hemaglutinin belong to the group of glucoprotein, containing 4.5% manose and 1% glucosaminn, has a molecular weight of 100.000 Dalton and two polypeptide chains Skim soybean contains about 3% hemaglutinin Hemalutinin is easily inactivated by the heat and fermented by temperature

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Tofu can be processed in many different ways such as frying, baking, boiling, steaming Three types of tofu are currently being produced: hard, soft, and silk tofu Depending on the water content, producers can form various structures of the tofu that tofu could become

Hard tofu maintains its shape when cooked that it can be stir-fried or baked Hard tofu contains the highest protein and lipid content Meanwhile, “Silk” tofu

is a slightly different product from the traditional product and was made of a creamy texture like silk The product is like custard When eating, there is no need for added flavor or added spices when cooking The tofu is sold as a cold item, packed in a tube that can be filled with water or a vacuum packaging

Tofu contains high levels of minerals such as calcium, iron, and vitamin B, but low in sodium and cholesterol-free, which has become a popular nutritional product Tofu is a nutritious product produced from soy protein gel Currently, the automatic production method of tofu is replacing the manual production method, and there is much modern equipment for tofu production made in Japan

The first step in tofu production is to soak the beans and extract the milk and boil the milk solution Then a coagulant is added to coagulate the milk Traditional soy milk protein coagulants commonly used are CaCl2, MgCl2 The resulting solid mass is processed into tofu in the desired forms, mainly in block form

2.2.2 Process explanation

2.2.2.1 Soaking seeds

In the wet grinding method, the beans must undergo a soaking phase Soaking the seeds is for the purpose of making the beans absorb water and swell Then the water molecules bipolar will act on molecules of proteins, lipids, carbohydrates, and cellulose This process consists of 2 phases: - The first stage is the solvate process, at this stage, the bonds in soybeans are not broken - Stage 2 is the hydration process: the water molecules continue to work and break the molecular bonds in soybean and transform them into a flexible colloidal state located in the cells There are 3 factors that affect the soaking process: soaking time, soaking water amount, 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 water temperature: if soybeans are soaked at a high temperature, the swelling speed of the seeds is fast, but the swell ability of the seeds is small Therefore, the ingredients in beans are only in the coagulated state, not the colloidal solution, so it is difficult to dissolve The best temperature for soaking beans is 20 ÷ 25°C

- The amount of soaking water commonly used is: beans / water = 1 / 2.5 This amount of water will help the beans have high swelling, low acidity At the end of the soaking phase, the best moisture is 55 ÷ 60%

2.2.2.2 Grind

Grinding is a mechanical process to break down cells, to release proteins, lipids, and carbohydrates By dissolved water, these substances could be turned

into suspensions The important influencing factor at this stage is the amount of water required to add When the amount of water added is low, it will create strong friction, causing the phenomenon of the temperature rise The increased heat makes the protein denatured, so the solubility of the protein becomes less If too much water is added, it will increase the number of dissolved substances but cause difficulties in the later stages The best water used to grind the bean should be according to the ratio: beans/water = 1/6

2.2.2.3 Filter

After grinding, we have a suspension solution, consisting of colloidal solution and water insoluble solids In the process of separating colloidal solution from solids, solids will retain on its surface colloidal particles, so it is necessary to rinse the residue with water The amount of water used for washing should not be too much In the filtration stage should go through 2 steps: refining and crude filtration

2.2.2.4 Heating and precipitation

Milk solution after filtration must be heated immediately Heating is required to break the enzyme trypsin and toxin aflatoxin, kill microorganisms, deodorize the fishy smell, break the solvated layer to create conditions for the milk molecules to come together and easier to coagulate The faster the heating time, the better soy milk quality The best time to boil 100 liters of milk is within 5 ÷ 10 minutes During the boiling process, stir stirring always to avoid burning the milk solution

After boiling the soy milk must precipitate immediately The protein precipitation has many causes such as the effect of heat, pH change in the

isoelectric region, the effect of salt In the process of precipitation, milk are boiled

to 95 ÷ 100°C to cause thermal denaturation and precipitate effects of proteins There are many types of protein precipitation agents such as natural sour water, CaCl2, CaSO4, acetic acid, lactic acid Of the above precipitates, natural sour water

is the most suitable When using sour water for precipitation requires users to have

a lot of experience The conditions for precipitation of soy milk are as follows:

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

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

 pH of sour water 4 ÷ 4.5

The pH of sour water is of great significance in precipitation If the pH is too high, the amount of sour water must be used much On the contrary, if the pH is too low, the protein recovery efficiency is low When the milk solution reaches 95°C, slowly add the sour water This process should be divided into 3 phases: In the first phase, use half of the sour water;

 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, we have tofu curd Put tofu curd into the mold The best temperature for pressed the tofu curd is 70 ÷ 80°C If below 60°C, the curd will not stick and cannot form shape The pressing time is usually 10 minutes

After pressing, remove the mold and cut the tofu the intended sizes, then soak the tofu in water for a stable structure and the longer time for the tofu to be sour

2.3 FUNDAMENTAL OF THE GEL PROTEIN FORMATION PROCESS

OF IN SOY MILK

2.3.1 Mechanism of formation

The formation of protein gel is the most important step to make tofu with the desired texture Soy proteins in their native state do not form a gel; they must be heat-denatured and then coagulated to form the tofu (Liu, 1997) [13] Thermal denaturation followed by ordering followed by gel formation are the important steps in ensuring all proteins are dispersed into the gel network

In the production of tofu, the gel-forming process of soybean protein involves thermogenesis of soy milk protein, followed by an orderly, gel-forming process to turn it into tofu

The purpose of thermal denaturation is to help the protein circuit to dilate Instead of the protein having a compact structure at first, after heating this structure

is opened, diffused and the internal structure of the protein molecules is revealed For example, the –SH functional groups, the hydrophobic part, the carbonyl group, the amine groups of the peptide bond and the amide group of the side chain become exposed It is these functional groups that directly affect the network structure (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 However, between glycinin and conglycinin, the mechanism of gel making is

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

2.3.2.1 Effect of protein concentration

Protein concentration is an important factor influencing the gel type and ultimate properties of soy milk protein gel Gelatin can form a gel at relatively low concentrations Meanwhile, the globulin concentration is higher than that of the newly formed gelatin To qualify for gel formation, the protein content of soy milk should be at least 8% In addition, if the glycinin content is high, the gel formation process is easy, otherwise, if the glycinin content is low, gel formation is difficult because they tend to separate The mechanical strength of the gel is also dependent

on the protein concentration, since strength is related to the number of linking formed on the protein chain There is a linear relationship between the mechanical strength of the gel and the protein concentration

cross-2.3.2.2 Effects of heating temperature

One of the effects of temperature on soy protein is to alter its quaternary

structure Glycinin and conglycinin are denatured at 85 ÷ 95°C and 65 ÷ 75°C respectively The difference between the two protein segments above is that in glycinin there is a large disulfite bridge If the heating temperature exceeds the minimum denaturation temperature required for gel formation, the rheological properties of the gel are affected Conversely, if the protein is heated to a lower temperature, a longer time for gel formation than when heated at high temperature

is required This also leads to weaker gel formation The low temperature is not enough to form the most robust three-dimensional network When heating protein dispersions at high temperatures exceeding the protein denaturation temperature

of soy milk, it can cause metamorphosis and no subsequent gel formation

2.3.2.3 Effect of strong ions

When adding 2% NaCl salt and soy milk solution, it increases the ratio of gel glycin and conglycin However, when the concentration was increased to 10%, gel formation did not take place The effect of adding low-content NaCl to the gel is

to neutralize the charge Conversely, the addition of salt with a high content will prevent gel formation due to some changes in the protein as well as increase the hydrophobic interaction

2.3.2.4 Effect of pH

The protein denaturation process, the interactions between proteins and proteins or between proteins and solvents are affected by pH (Renkema,2000) Adjustment of pH is necessary to achieve the right equilibrium between denaturation and recombination, as well as attraction and repulsion between adjacent protein chains When pH> 12 the gel forming process is completely inhibited This is explained that at acid pH values positively charged polypeptide

chains Also at high alkaline pH values polypeptide chains are negatively charged Under these conditions electrostatic repulsion can destabilize protein and protein interactions during gel network formation resulting in reduced gel strength At neutral pH the interaction between positively charged groups adds an additional energy that promotes gel formation

2.3.3 Soy protein properties

Soy protein is divided into 4 segments with the names as follows globulin 2S, 7S, 11S, 15S In these 4 segments, 7S and 11S segments are the main components of soy protein The 7S fraction is considered the conglycinin of soy milk, while 11S is the glycinin (Nielson, 1985) Glycinin and conglycinin account for about 65 ÷ 85%

of the total protein content of seeds

Glycinin - Globulin 11S

The molecular structure of glycinin is depicted in 2.3 Glycinin is an oligo protein and is structured from 6 monomers linked together A hexagonal structure of glycinin consists of 2 trimers, each of which consists of 3 monomers The monomers are arranged in a certain order as shown in 1.8 Globulin 7S or 11S is composed of 12 relatively lipophilic "subunits": Including 6 acidic "subunits" (A) and 6 bases

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

2.3 The molecular structure of Glycinin

Conglycinin - Globulin 7S

Is -conglicinin, usually accounting for nearly 35% of the protein weight of the seed, is a glucoprotein containing nearly 5% carbohydrates Conglynin segment is made up of 3 main "sub-parts" with the names a, a 'and b, respectively Conglycinin fractional structure is composed of 3 sub-fractions arranged as shown

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)

2.4 LACTIC ACID BACTERIA OVERVIEW

Using Ca2+ as a coagulant used to precipitated protein in soy milk solution may be productive However, using the chemicals to coagulate the tofu may lead

to impurity Therefore, using fermentation solutions to lower pH can be a substitution LAB is a familiar example It is wildly known in the food industry for its benefits to food itself as well as the health of consumers

2.4.1 General overview

Lactic bacteria are Gram-positive bacteria, no spore-forming, non-mobile,

capable of fermenting sugars to create lactic acid The lactic acid bacteria group is classified into the family Lactobacteriaccae and is classified 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

double form or in series; spherical or bacillus in the single, double, or clustered form, the diameter of the lactic cocci is usually from 0.5 ÷ 1.5 µm In addition, lactic acid bacteria have a rod shape Colonies of small round, glossy, medium color, milky white, cream yellow colonies or large round, milky convex colonies, especially the acidic smell colonies

2.4.2 Common features of lactic acid bacteria

Morphologically, there are many differences between lactic acid bacteria, but

in general, they are relatively homogeneous The gram-positive bacteria are immobile and do not produce spores The cell's ability to synthesize many substances is very weak As arbitrary anaerobic fermentation, it is able to ferment anaerobic as well as aerobic, with high tolerance to acidic environments (Whittenbury, 1964)

Lactic bacteria can live from anaerobic to micro aerobic Lactic bacteria have very complex growth requirements, not a single representative of this group can develop on pure glucose containing mineral media, most of them need a variety of vitamins (lactoflavin, tiamin, pantotenic acid, acid nicotinic, folic acid, biotin) and more complex amino acids or N2-containing compounds Therefore, the culture environment of bacteria is quite complex, containing a relatively large amount of yeast, tomato juice, It is the nutritional characteristics of vitamins, amino acids,

so the strains of lactic acid bacteria Used in the analysis of these two compounds

in different substrates

Lactic bacteria are fermented with mono and disaccharides The lactic acid bacteria cannot ferment starch and polysaccharides (only L delbrueckii can assimilate the starch) Others are able to use pentose and citric, which are mainly heterofermentative lactic acid bacteria Lactic bacteria also have protease property:

hydrolysis of milk proteins into peptides and amino acids, this activity is different

in different species, the permanent bacillus is highest

Lactic bacteria are resistant to drought, stable with CO2 and ethyl, many types can survive in the environment of 10 ÷ 15% alcohol or higher, some bacilli are stable with NaCl, can live in the environment from 7 ÷ 10% NaCl

The hygroscopic lactic acid bacteria have the optimal growth temperature in the range of 25 ÷ 35°C, the thermophilic group has the optimum temperature of 40

÷ 45°C, the cold group grows at a relatively low temperature (≤ 5°C) When heated

to 60 ÷ 80°C, it will die in 10 ÷ 30 minutes Some bacteria are capable of creating mucus membranes, others are resistant to saprophytic microorganisms, pathogenic microorganisms and food rot microorganisms Thus, in addition to the ability to create lactic acid of these types, it is also possible to produce compounds with antibacterial activity

Lactic bacteria are found in the air, soil, and water, but mainly in plants and food products such as pickled vegetables, yogurt Some lactic bacteria are found

in the human digestive tract and animal Currently, the classification of lactic acid bacteria is not complete, most of the classifiers are based on cell morphology, lactic bacteria are divided into 3 main groups:

 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

2.4.3 Lactic fermentation

It is the process of converting sugar into lactic acid by microorganisms, typically lactic acid bacteria Lactic fermentable bacteria belong to the

Lactobacterium family These are non-spore-forming bacilli and cocci and are

mostly non-movable, and oxygen-tolerant anaerobic bacteria They are capable of fermenting a wide variety of simple and double sugars but are not capable of fermenting complex carbohydrates and starches Growth of lactic acid bacteria requires the presence of peptones, amino acids or ammonium salts They have a special requirement for nutrients rich in vitamins, amino acids and minerals Fermentation occurs best in an environment of pH from 5.5 to 6 and is inhibited at around pH 5 and stops completely at pH <4.5 Suitable temperature for fermentation is in the range of 15 ÷ 50°C However, each species has a different suitable temperature range, if the temperature is greater than 80°C, the bacteria will be completely destroyed

2.4.3.1 Homogeneous lactic fermentation

Isomorphic lactic acid bacteria are bacteria that in the fermentation process produce only the main product, lactic acid In this case pyruvic acid is generated along the path Emble - Mayerhorf - Parnas (EMP) Then pyruvic acid will form lactic acid under the action of lactatdehydrogenase The amount of lactic acid formed accounts for more than 90% Only a small amount of pyruvate is carbonated to form acetic acid, ethanol, CO2, acetone The amount of byproducts formed depends on the presence of oxygen

Some strains of lactic isomeric fermentation

Streptococcus lactic (abbreviated as S lactic): Streptococcus or bacillus very

short, when young, the pair is paired or short chain This variety prefers moisture, grows best at 30 ÷ 35°C, coagulates milk after 10 ÷ 12 hours, in the environment

it accumulates 0.8 ÷ 1% acid, the optimal temperature for growth is 10°C, heat the

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 (abbreviated as L casein): small bacillus commonly

found in the long or short chain form, accumulating up to 1.5% acid, the optimum temperature is about 30 ÷ 35°C, thanks to its protease activity, hydrolysis is possible casein in milk into amino acids

L acidophilus: long heat resistant bacillus, the optimal temperature is about

30 ÷ 40°C, the minimum temperature is 20°C, in milk it accumulates up to 2.2% acid, this bacillus is isolated from the intestines of children and newborn babies are used to produce aucidophilus milk, capable of producing bacteriocin with inhibitory activity of intestinal pathogens, some strains capable of producing mucous membranes

2.4.3.2 Heterofermentative lactic fermentation

Occurs in the case of lactic bacteria without the basic enzyme in the Emblen

- Mayerhorf - Parnas (aldolase and triozophotphattizomerase) scheme, so phosphate xilulose will be formed in the pento-phosphate (pp) pathway In this case only 50% of the sugar is converted to lactic acid, in addition to other by-products such as acetic acid, ethanol, CO2 The amount of by-products completely depends on the cultivar, the vegetative environment and the external conditions, in general lactic acid accounts for 40% of the dissolved sugar, 20% sucxinic acid, ethyl alcohol 10%., acetic acid 10% and gaseous products account for about 20% The diversity of products formed during heterofermentative lactic fermentation proves that there are many enzyme systems in the body of lactic acid bacteria in this group, so the hydrolysis and glucose metabolism process is more complicated than isoforms of lactic acid bacteria

5-Some strains of heterofermentative lactic acid bacteria

Lactobacillus brevis: Found mainly in pickled foods like cabbage,

vegetables, and cucumber so it is also known as cabbage bacillus In the fermentation process, the product creates, in addition to lactic acid, acetic acid, ethyl alcohol, and CO2 give the product a pleasant aroma

2.4.4 Nutritional requirements

2.4.4.1 The need for carbon nutrition

Lactic bacteria can utilize a variety of carbohydrates from monosaccharides (glucose, fructose, manose), disaccharides (saccharose, lactose, maltose) to polysaccharides (starch, dextrin)

They use this carbon source to provide energy, build cell structures and serve

as substrates for the fermentation of organic acids

2.4.4.2 Needs for nitrogen nutrition

Most lactic acid bacteria cannot synthesize nitrogenous compounds So to ensure their growth and development, they must use nitrogen sources available in the environment

The sources of nitrogen of lactic acid bacteria can be used such as: meat extract, yeast extract, trypton, casein hydrolyzate from milk, pepton, Currently, yeast laf - nitrogen source is used the most and most effectively However, at the industrial scale it is not possible to use this nitrogen source because it is very expensive

2.4.4.4 Demand for other compounds

In addition to amino acids and vitamins, lactic acid bacteria also need other organic compounds for growth such as nitrogen bases or organic acids

Some organic acids have a favorable effect on the growth of lactic acid bacteria such as citric acid and oleic acid For now, people use citrate salts, derivatives of oleic acid as ingredients for the culture medium, isolate and preserve strains of lactic acid bacteria Similar to the two organic acids above, acetic acid also has important effects on cell growth So people often use acetic acid in the form of acetate salts as a buffer for the environment when culturing lactic acid bacteria

2.4.4.5 Requirements for other inorganic salts

In order to ensure adequate growth and development, inorganic salts are essential for lactic acid bacteria Mineral elements are needed such as copper, iron, sodium, potassium, phosphorus, sulfur, magnesium, especially manganese, because manganese helps prevent autolysis and stabilize cell structure

2.5 Overview of hydroperoxide and bacteriocin during lactic fermentation

Lactic fermentation reduces an appropriate amount of carbohydrates and produces a series of organic compounds with small molecular weight antimicrobial activity, most commonly lactic, acetic, and propionic acids There are also a number of other antibacterial compounds that can be synthesized by many different types of lactic acid bacteria In addition to their low molecular weight, they also have some other properties such as: antimicrobial activity at low pH, stable with temperature, broad spectrum of activity and soluble in acetone After lactic fermentation, in the fermentation medium there are many organic substances

with antibacterial effects When it comes to antibacterial compounds, bacteriocin cannot be ignored A compound that is being studied extensively and used in food

as a biological preservative Bacteriocin has heat stable properties that inhibit the Gram-positive bacteria group (Karpinski, 2016) In addition to this compound, organic acids, mostly lactic acid, produced during fermentation to lower pH also have antibacterial effects Besides, there is also an antibacterial role of hydroperoxide, and some other substances Thus, lactic fermentation fluid used as tofu precipitation agent has natural antibacterial properties Helps to prolong proper storage without the need to add any preservative chemicals Tofu products will be safe for the health of consumers

2.5.1 Hydroperoxide

Hydroperoxide is a byproduct formed during lactic fermentation Hydroperoxide has the molecular formula of H2O2 The bactericidal effect of hydroperoxide is due to its strong oxidizing ability on the bacterial cell Membrane lipids and protein groups may be oxidized Furthermore, some hydroperoxide reactions produce atomic oxygen thus creating an anaerobic environment, which

is unsuitable for certain species of microorganisms Hydroperoxide formation: in the presence of oxygen, lactic acid bacteria can synthesize hydroperoxide (H2O2)

In the absence of a source of heme, the lactic acid bacteria fail to produce catalase for the removal of hydroperoxide Other systems that remove hydroperoxide are less active than those that produce this organic compound leading to hydroperoxide build-up (Ouwehand,2004)

2.5.2 Bacteriocin

Bacteriocin is essentially an antibacterial peptide produced by bacteria to against other bacteria (Karpinski, 2016) Thus, the type of bacteria which capable

of producing bateriocin would have ability to resist to that bacteriocin In addition, bacteriocin does not cause allergic reactions in humans and health problems, is rapidly hydrolyzed by proteases and lipases Bacteriocin produced by lactic bacteria are protein molecules with positive charge, small size including 30 ÷ 60 amino acids, with high isoelectric point, capable of inhibiting bacteria that have close relationship with bacteria produced bacteriocin Bacteriocin is present in all

groups of lactic acid bacteria such as: Lactobacillus, Lactococcus, Enterococus,

Sterptococcus, Leuconostoc and Pediococcus In which 2 main groups are Lactobacillus, Lactococcus plays an important role Bacteriocin can inhibit some

bacteria such as: Clostridium botulinum, Bacillus cereus, Bacillus alcalophilus

and Listeria monocytogenes

2.6 Overview of tofu preservation [25]

The final tofu production process is packaging and preserving The method

of packaging material and storage technology will affect the final taste, quality, and shelf life of the tofu product

The high moisture and protein content (in tofu products) provide good surroundings for microbial growth, and therefore, even under refrigeration, the shelf life is only one few days (Rossi, Felis, Martinelli, Calcavecchia, & Torriani, 2016) However, how to extend the shelf life of tofu products has always been an important topic Nowadays, the preservation method for preserving tofu are diverse, from adding preservation chemicals to applying packaging technology

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

1 Choose packaging material

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

2.6.1 Vacuum packaging

Modified atmosphere packaging (MAP) is defined as the packaging of food

in an atmosphere whose composition is fixed initially but is constantly changing thereafter (Berk, 2018) It is based on replacing the air in sealed food packaging with a controlled gas mixture containing carbon dioxide, oxygen, nitrogen, and/or other gases to change the composition of the gas in contact with the food and prevent the deterioration of physical, chemical, and biological aspects of food quality (Tiefenbacher, 2018; Ward, 2016)

MAP has been studied with tofu Stoops, Maes, Claes, and Van Campenhout (2012) investigated the growth of Pseudomonas in tofu products in MAP They found that it was difficult to eliminate Pseudomonas spoilage by controlling the amount of carbon dioxide and oxygen with refrigerated product Another study was done using a mixture of CO2 and N2 (CO2: N2 = 3:7) using flushing or vacuum compensation, and the packaged tofu was refrigerated Tofu packed in air was used

as the control The results showed that the microbial count of air‐packed tofu was one to four log cycles higher than that of the MAP after 10 days of storage The MAP inhibited microbial growth up to 14 days (Van Campenhout, Maes, & Claes, 2013)

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

2.6.2 Food freezing

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)

2.7 RESEARCH SITUATION WORLDWIDE AND IN VIETNAM

2.7.1 Worldwide

In 2020, Kay Huyn Joo and partners investigates the potential of trimagnesium citrate (TMC) as an alternative tofu coagulant, comparing it with the traditional coagulants used in industry Yield, water-holding capacity (WHC), texture profile analysis (TPA), confocal microscopic analysis, and sensory evaluation were conducted to compare raw and cooked tofu made from different coagulants The research show that this new coagulant had no effect on yield but did influence textural properties and improve sensory quality

Meanwhile, Yin and partners implement on the topic of investigate the effects

of fermentation with Actinomucor elegans on the phenolic components, antioxidant

activities, and nutritional compounds of tofu The results show that a significant increase in the total and soluble phenolic content and a decrease in insoluble phenolic content in tofu were observed after fermentation The fermented tofu also showed higher antioxidant activities than the unfermented tofu Metabolomic analysis revealed that the nutritional composition including carbohydrates, alcohols, fatty acids, organic acids, inorganic acids, amino acids, and a miscellaneous of tofu was

significantly increased during fermentation These findings demonstrated that A

elegans fermentation could significantly enhance the nutritional and functional

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

2.7.2 In Vietnam

Tofu is a traditional and well- known However, in Vietnam, this product has not been studied much However, in 2014, the authors Nguyen Thi Minh Nguyet and Pham Thi Kim Ngoc studied the effects of coagulation agents on the recovery efficiency and the properties of tofu obtained, by examining the quality of tofu The results showed that sour water could be used to produce safe, quality tofu and at the same time showed many unusual properties of tofu obtained from plaster

PART III METHOD

3.1 Research subject

3.1.1 Soybeans

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

3.1.2 Lactic acid bacteria

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 3.1 Chemicals

Table 3.2 Experimental equipment

Table 3.3 Laboratory instruments