Isolation of lactic acid bacteria apply in tofu producing process

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 TABLE OF CONTENT ACKNOWLEDGEMENT iii TABLE OF CONTENT iv LIST OF S vii LIST OF TABLES viii PART I.INTRODUCTION 1.1 Research rationale 1.2 Research objective 1.3 Detail goals 1.4 Limitations PART II.LITERATURE REVIEW 2.1 OVERVIEW OF SOYBEAN 2.1.1 Characteristic of soybean 2.1.2 Acreage, yield and demand for soybeans 2.1.3 Chemical composition of soybeans 2.2 OVERVIEW OF TOFU 14 2.2.1 Process of producing tofu from natural sour water 16 2.2.2 Process explanation 17 2.3 FUNDAMENTAL OF THE GEL PROTEIN FORMATION PROCESS OF IN SOY MILK 20 2.3.1 Mechanism of formation 20 2.3.2 Factors affecting 21 2.3.3 Soy protein properties 23 2.4 LACTIC ACID BACTERIA OVERVIEW 24 2.4.1 General overview 24 2.4.2 Common features of lactic acid bacteria 25 2.4.3 Lactic fermentation 26 2.4.4 Nutritional requirements 29 2.5 OVERVIEW OF HYDROPEROXIDE AND BACTERIOCIN DURING LACTIC FERMENTATION 30 2.5.1 Hydroperoxide 31 2.5.2 Bacteriocin 31 2.6 OVERVIEW OF TOFU PRESERVATION 32 2.6.1 Vacuum packaging 33 2.6.2 Food freezing 33 2.7 RESEARCH SITUATION WORLDWIDE AND IN VIETNAM 34 2.7.1 Worldwide 34 2.7.2 In Vietnam 35 PART III METHOD 36 3.1 RESEARCH SUBJECT 36 3.1.1 Soybeans 36 3.1.2 Lactic acid bacteria 36 3.2 EQUIPMENT AND CHEMICAL REQUIRE FOR RESEARCH 36 3.3 LOCATION AND TIME PERIOD OF THE RESEARCH 38 3.4 RESEARCH CONTENT 38 3.5 RESEARCH METHODS 38 3.5.1 Content 1: Isolation of some strains of lactic acid bacteria that can be used in secondary production 38 3.5.2 Content 2: Selection of bacteria with good ferment ability for application in tofu production 3.5.3 Sensory evaluation (Sensory evaluation by the method of scoring TCVN 3215 - 79) 3.6 ANALYSIS METHOD 3.7 DATA PROCESSING METHODS PART IV.RESULTS AND DISCUSSION 4.1 Results of isolation and selection of lactic acid bacteria 4.1.1 Biological characteristics of lactic acid bacteria isolated 4.2 Experimental set up test for soy milk fermentation 4.2.1 Results of pH measurement 4.2.2 Sensory quality of fermented soy milk 4.2.3 Carbohydrate metabolism of selected strain 4.3 Tofu production efficiency 4.4 Determine the storage time 4.4.1 Indicate quantification of total aerobic bacteria 4.4.2 Indicate quantification for Coliforms 4.4.3 Quantify total molds 4.4.4 Results comparing sensory quality between common tofu and tofu produced with lactic fermented soy milk PART V CONCLUSION REFERENCES APPENDIXES 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 causes waste product, but also limits the scope of the distribution and the time it takes to commercialize the product On the other hand, the protein coagulation process in tofu is mainly using chemicals such as CaSO4, MgCl2, citric acid and CaCl2 Calcium sulphate (CS) (Murugkar, 2015), which are used in food Many tofu manufacturers use impure chemicals that lead to metal residues in the tofu product 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 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 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 to 20 fruits, and each tree has nearly 400 fruits One fruit has from to seeds The soybean fruit is slightly curved, the average length of it is about to 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 with the largest production in the world According to the latest data, Brazil has surpassed the US in soybean production, reaching 124 million tons in the first half of 2020 and nearly two-thirds of production is for export Some other major soybean producing countries such as, Argentina, China, India, Paraguay, Canada Soybean export volume of some major countries in the world from 2017 to the first half of 2020 is shown in Table 2.1 Table 2.1 Soybean export volume of some major countries in the world in the crops of 2017/2018, 2018/2019 and 2019/2020 (thousand tons) Country US Brazil Argentina China India Paraguay Canada Others Whole world (Source: FAS/USDA – statista) 2.1.2.2 Production situation in Vietnam Currently, soybean growing are forming in concentrated areas  Northern midland and mountainous provinces  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 810% 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 Season Cultivated area (thousand hectares) Yield (tons /hectare) Total quantity (thousand tons ) (Source: General Statistics Office of Vietnam, * FAS estimates) APPENDIXES Appendix 1: MRS broth medium pH of the complete medium at 250 ° C: 6.4 ± 0.2 - Soluble environment - Autoclave at 121 degrees for 15 minutes - Let it cool at room temperature Appendix 2: MRS agar STT 10 pH of the complete medium at 250 ° C: 6.4 ± 0.2 - Soluble environment - Autoclave at 121 degrees for 15 minutes - Let it cool at room temperature Appendix 3: Gram staining Required chemicals a) Fuchsin solution STT - Prepare solution (1) and (2) seperately Afterward, mix them together b) Gentian violet STT 1 - Prepare solution (1) and (2) seperately In the (1) solution, stir the slution until the gentian violet is completely disolve - Afterward, mix (1) and (2) c) Lugol STT - Dissolve KI in 5ml of distilled water to dissolve and add crystal iodine - Add 300ml of water after the iodine dissolves Gram staining method Label sample Aseptic technique, add a drop of water (about cm diameter) Sterilize the slide Use metal lube to transfer a colony of bacteria Sterilize the slide Let the liquid evaporate/ dry (about 10 minutes) Add crystal violet solution, let it stand for minute then wash Add iodine/lugol solution, let it stand for minute then wash Add few drop of decolorizer at a 45* angle, then rinse the slide in water Adding fuchin solution, let it stand for minute then wash Dry the slide Observe Gram staining method Appendix 4: Quantitative determination of total microorganisms Principle: Inoculate a certain quantity of sample or the diluted sample on nutrient agar at 30 ± 10 ° C under anaerobic conditions for a period of 48 - 72 h then count the number of colonies growing on it from there it is possible to count the number of viable cells present in the sample (pay attention to the appropriate dilution so that the number of colonies per Petri dish is between 30 and 300) Conducting method - Prepare culture medium: the medium for determining total microorganisms is TGA (Tripton - Glucose - Agar) Culture medium (TGA: Tripton - Glucose - Agar) STT Sterilization for 20 minutes at 1210C Sample dilution: take 5g in each recipe, crush with a ceramic mortar, mix well, weigh gram of sample, dilute to a concentration of 10 -1 -2 10 , 10 -3 (operation does not exceed 30 minutes) - Sample culture: take 100 μl of diluted sample and cultivate on Petri dishes with TGA medium, dilute each concentration to dishes, then incubate in incubator at 37° C after 48 to 72 hours, count all colonies appearing agar plates (number of colonies in each dish from 30 to 300 colonies) The average number of microorganisms in 1g sample is calculated by the formula N colony/g or ml = ΣC (n1 n2 ) f1V Where ∑C: total number of colonies counted on all plates n1: number of dishes counted at the first dilution concentration n2¬: the number of dishes counted at the 2nd dilution concentration f1 Dilution coefficient in first count plate V volume of inoculum in each Petri dish Appendix 5: Determination of coliform method MPN The MPN method is based on the principle of statistical probability distribution of microorganisms in different dilution concentrations The dilution is cultured repeatedly (3 - 10 times) Dilutions are chosen such that in the replicates there are a few positive, some negative The number of positive times are recorded and compared with statistics deduce the estimated value of the number of microorganisms in the sample Coliforms are a group of bacteria including a number of Gram-negative, nonspore-forming, aerobic or anaerobic bacteria that are capable of fermenting lactose, producing steam within 48 hours at the appropriate culture temperature varieties of E.coli, Citrobacter, Klebsiella and Enterobacter Principle: Endo medium containing sodium and Fucshin is capable of inhibiting Gram-positive bacteria during growth on this medium, Coliforms ferment lactose to form aldehydes and acids, aldehydes affect Fucshin-sulfite complex and the release of fucshin, which then stains colonies from pink to red lotus petals, round, evenly uniform, possibly iridescent or not Endo medium STT Pep Lac K2 H Nat Aga pH Sterilization for 20 minutes at 1210C Sample dilution: take 5g in each recipe, crush with a ceramic mortar, mix well, -1 -2 -3 weigh gram of sample, dilute to a concentration of 10 10 , 10 (operation does not exceed 30 minutes) - Sample culture: take 100 μl of diluted sample and cultivate on Petri dishes with TGA medium, dilute each concentration to dishes, then incubate in incubator at 37° C after 48 to 72 hours, count all pink color colonies appearing agar plates (number of colonies in each dish from 30 to 300 colonies) The average number of microorganisms in 1g sample is calculated by the formula N colony/g or ml = Where ∑C: total number of colonies counted on all plates n1: number of dishes counted at the first dilution concentration n2: the number of dishes counted at the 2nd dilution concentration f1 Dilution coefficient in first count plate V volume of inoculum in each Petri dish Appendix 6: Method of determining Yeast - Mold Principle: The culture medium contains inhibitors of bacteria (antibiotics such as Oxytetracylin or Chloramphenicol) cultured at 30 ± 10C under aerobic conditions after 48 - 72 hours Count the number of colonies on the Petri dish to determine the total number of yeasts - molds Prepare the culture medium: Culture medium (YGC: Yeast - Clucose - Chloramphenicol) STT G Y C A D - Sterilization for 20 minutes at 1210C - Sample dilution: take 5g in each recipe, crush with a ceramic mortar, mix well, -1 -2 -3 weigh gram of sample, dilute to a concentration of 10 10 , 10 (operation does not exceed 30 minutes) - Sample culture: take 100 μl of diluted sample and cultivate on Petri dishes with TGA medium, dilute each concentration to dishes, then incubate in incubator at 37° C after 48 to 72 hours, count all colonies appearing on agar plates (number of colonies in each dish from 30 to 300 colonies) The average number of microorganisms in 1g sample is calculated by the formula N colony/g or ml = ΣC (n1 n2 ) f1V Where ∑C: total number of colonies counted on all plates n1: number of dishes counted at the first dilution concentration n2: the number of dishes counted at the 2nd dilution concentration f1 Dilution coefficient in first count plate V volume of inoculum in each Petri dish Appendix 7: Carbohydrate ferment ability Composition Type of sugar Peptone Yeast extract Indicator Distilled water pH of solution: 7,0±0,2 Autoclaved the medium at 121 C in 15 minutes To prepared indicator solution, dilute 8g of bromthymol blue into the mixture of 250ml ethanol 90% and 250ml distilled water - Prepare test tubes containing various fermentation media, with Durham tubes placed and sterilized - Inoculate the microorganisms into the tube - Monitoring growth, acid-forming, and gas-generating capabilities in each test tube - Record and evaluate the results Appendix Numeration result of aerobic bacteria Test days 12 Appendix Numeration result of coliforms bacteria Test days 12 Appendix 10 Numeration result of molds bacteria 12 Appendix table 11: Sensory evaluation of tofu produced with lactic acid bacteria (1st time) Criteria Color Smell Taste Condition Quality score Appendix 12: Sensory evaluation of tofu produced with lactic acid bacteria (2nd time) Criteria Color Smell Taste Condition Quality score Appendix 13: Sensory evaluation of tofu produced with lactic acid bacteria (3rd time) Criteria Color Smell Taste Condition Quality score st Appendix 14: Sensory evaluation of common tofu (1 time) Criteria Color Smell Taste Condition Quality score Appendix 15: Sensory evaluation of common tofu (2 nd time) Criteria Color Smell Taste Condition Appendix 16: Sensory evaluation of common tofu (3 rd time) Criteria Color Smell Taste Condition Appendix 16 Picture form research process ... 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. .. 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 N 2-containing compounds... 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