Isolation of lactic acid bacteria apply in tofu producing process

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY LUONG NGUYEN CHINH ISOLATION OF LACTIC BACTERIA APPLY IN TOFU PRODUCING PROCESS BACHELOR THESIS Full name : Luong Nguyen Chinh Study mode: Full-time Major : Food Technology Falcuty : Advanced Education Program Batch : 2016 – 2020 Thai Nguyen, December 2020 ABTRACT Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Food Technology Student name Luong Nguyen Chinh Student ID DTN1654190002 Thesis Title Isolation of Lactic Acid Bacteria apply in tofu producing process Supervisor (s) Dr Luong Hung Tien Supervisor’s Signature Abstract: The aims of this study is isolating and screening the high acid producing ability LAB strains to apply in tofu’s protein coagulating and enhancing shelf life We conducted to isolate LAB from natural ferment yogurt and tofu sour water, and we were isolated 11 LAB strains with the colony and cell’s morphology: round, milky-white color, rods shape Totally, we are identified LAB by Bergey’s Classification of Bacteria, by characteristics of sugar using, Gram dying method, oxidase and catalase activity Isolated strains were testing on soy milk ferment capacity by incubating condition at 41°C for hours and comparing with control sample, protein coagulation capacity at 90 0C for 10 minutes The results showed that DP3 have highest ferment capacity and good coagulation activity Finally, DP3 strain was applied in tofu producing process to testing about tofu’s qualities, productivity, microbiological criteria and shelf-life characteristics Keywords: Lactic acid bacteria, fermentation, soy beans, tofu Number of pages: 81 Date of Submission: 04/12/2020 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 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 39 3.5.3 Sensory evaluation (Sensory evaluation by the method of scoring TCVN 3215 - 79) 43 3.6 ANALYSIS METHOD 46 3.7 DATA PROCESSING METHODS 46 PART IV.RESULTS AND DISCUSSION 47 4.1 Results of isolation and selection of lactic acid bacteria 47 4.1.1 Biological characteristics of lactic acid bacteria isolated 47 4.2 Experimental set up test for soy milk fermentation 50 4.2.1 Results of pH measurement 50 4.2.2 Sensory quality of fermented soy milk 51 4.2.3 Carbohydrate metabolism of selected strain 52 4.3 Tofu production efficiency 52 4.4 Determine the storage time 53 4.4.1 Indicate quantification of total aerobic bacteria 53 4.4.2 Indicate quantification for Coliforms 54 4.4.3 Quantify total molds 55 4.4.4 Results comparing sensory quality between common tofu and tofu produced with lactic fermented soy milk 56 PART V 57 CONCLUSION 57 REFERENCES 58 APPENDIXES 61 LIST OF FIGURES 2.1 Traditional tofu production process in Vietnam 16 2.2 Different gel network structures of protein 21 2.3 The molecular structure of Glycinin 23 2.4 The molecular structure of Conglycinin 24 3.1 Process of enumeration of total aerobic bacteria 41 3.2 Process of enumeration of Coliform 42 3.3 Process of enumeration of mold 43 4.1 Colony on MRS agar 47 4.2 and 4.3 Representative characteristic of bacteria cell 48 Figure 4.4 Gram possitive bacteria 49 Figure 4.5 Gram negative bacteria 49 4.6 and Figure 4.7 Negative catalase 50 Figure 4.8 Total Number of aerobic microorganism in types of tofu 54 Figure 4.9 Total number of coliforms in types of tofu 55 Figure 4.10 Total number of mold in types of tofu 55 LIST OF TABLES 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) Table 2.2 Soybean production in Vietnam from 2011 to 2017 Table 2.3 Production, supply and demand of soybeans in Vietnam Table 2.4 Chemical composition of some kind of beans Table 2.5 Chemical composition of soybean 10 Table 2.6 Non-substituting amino acids in soybeans compares to other important foods (g / 100g protein) 11 Table 2.7 Carbohydrate components in soybean 12 Table 2.8 Vitamin content in soybean 12 Table 2.9 Mineral contents in soybean 13 Table 3.1 Chemicals 36 Table 3.2 Experimental equipment 37 Table 3.3 Laboratory instruments 37 Table 3.4 Quality level specified standards 44 Table 3.5 Tofu description scorecard 45 Table 4.1 Gram staining result 49 Table 4.2 pH of fermented soy milk of bacteria strains after 8h 50 Table 4.3 Results of sensory evaluation of lactic fermented soy milk 51 Table 4.4 Sugar fermentation test result of DP3 52 Table 4.5 Sensory score results between normal tofu and lactic fermented soy milk 56 ABBREVIATIONS CS Calcium sulphate EMP Emble – Mayerhorf – Parnas FAS Foreign Agriculture Service FDA Food and Drug Administration LAB Lactic Acid Bacteria LDL Low Density Lipoprotein MAP Modified Atmosphere Packaging PAW Plasma Activated Water PP pathway Pento – phosphate pathway TI Trypsin Inhibitors TMC Trimagnesium Citrate TPA Texture profile Analysis USDA United State Department of Agriculture WHC Water Holding Capacity 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 APPENDIXES Appendix 1: MRS broth medium Composition Weight Peptone 20.0 g Yeast extract 5.0 g Glucose 20.0 g Tween 80 1.08 g Dipotassium phosphate 2.0 g Sodium acetate 5.0 g Ammonium citrate 2.0 g Magnesium sulfate 0.2 g Water liter 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 Composition Weight Meat extract 10.0 g Yeast extract 5.0 g Glucose 20.0 g Tween 80 1.08 g Dipotassium phosphate 2.0 g Sodium acetate 5.0 g Ammonium citrate 2.0 g Magnesium sulfate 0.2 g Bacteriological agar 15 g 10 Water liter 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 - Composition Weight Etylic 96° 10 ml Fuchsin base 0.5 g Phenol 5g Distilled water 95 ml Prepare solution (1) and (2) seperately Afterward, mix them together b) Gentian violet STT - Composition Weight Gentian violet 1g Etylic 10ml Phenol 5g Distilled water 1000 ml 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 Composition Weight Iodine 1g KI 2g Distilled water 300 ml - 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 Composition Weight Pepton 5g Glucose 4g Yeast extract 2,5g Agar 15g Distilled water litre 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 10-2, 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 = C (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 Composition Weight Pepton 10 g Lactose 10 g K2HPO4 2,5g Natrisulfit 3,3g Fucshin base 0,3g Agar 20g Distilled water liter pH 7,5 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 10-2, 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 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 = C (n1  0.1n2 ) 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 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 Composition Weight Glucose 20 g Yeast extract 5g Chloramphenicol 0,1g Agar 20g Distilled water liter - 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 10-2, 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 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 = C (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 gam/litre Type of sugar 5,00 Peptone 10,00 Yeast extract 5,00 Indicator mL Distilled water Liters pH of solution: 7,0±0,2 Autoclaved the medium at 1210C 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 Total aerobic Test days bacteria in Total aerobic Log CFU/g normal tofu bacteria in Log CFU/g LAB tofu 636 2,8 455 2,66 8532 3,93 2.773 3,44 50.065 4,7 11.500 4,06 ~ 31.818 4,5 12 ~ ~ Appendix Numeration result of coliforms bacteria Test days Total Total Coliform Coliforms in in Lactic tofu common tofu Log CFU/g 0 ~ ~ ~ ~ 45 1,65 ~ 91 1,96 45 1,65 12 227 2,36 182 2,26 Log CFU/g Appendix 10 Numeration result of molds bacteria Total mold Total mold in Log CFU/g normal tofu bacteria in LAB Log CFU/g tofu 0 ~ ~ ~ ~ ~ ~ 2,13 ~ 136 2,26 ~ 12 182 Appendix table 11: Sensory evaluation of tofu produced with lactic acid bacteria (1st time) Score from Criteria Average without Sum panelists important Impotant coefficient Average with important A B C D E Color 4 4 19 3,8 0,7 2,66 Smell 4 22 4,4 1,2 5,28 Taste 5 4 22 4,4 1,3 5,72 Condition 4 4 21 4,2 0,8 3,36 Quality score coefficient coefficient 17.02 Appendix 12: Sensory evaluation of tofu produced with lactic acid bacteria (2nd time) Score from Criteria Average without Sum panelists important Impotant coefficient Average with important A B C D E Color 5 5 24 4.8 0,7 3.36 Smell 5 5 22 4.4 1,2 5.28 Taste 5 4 22 4.4 1,3 5.72 Condition 5 21 4.2 0,8 3.36 coefficient coefficient Quality score 17.72 Appendix 13: Sensory evaluation of tofu produced with lactic acid bacteria (3rd time) Score from Criteria Average without Sum panelists important Impotant coefficient Average with important A B C D E Color 5 23 4.6 0,7 3.22 Smell 5 4 22 4.4 1,2 5.28 Taste 4 22 4.4 1,3 5.72 Condition 5 4 21 4.2 0,8 3.36 coefficient coefficient Quality score 17.58 Appendix 14: Sensory evaluation of common tofu (1st time) Score from Criteria Average without Sum panelists important Impotant coefficient Average with important A B C D E Color 4 3 18 3,6 0,7 2,52 Smell 4 22 4,4 1,2 5,28 Taste 5 4 22 4,4 1,3 5,72 Condition 4 4 21 4,2 0,8 3,36 Quality score coefficient coefficient 16.88 Appendix 15: Sensory evaluation of common tofu (2nd time) Score from Criteria Average without Sum panelists important Impotant coefficient Average with important A B C D E Color 5 4 21 4.2 0.7 2.94 Smell 3 4 22 4.4 1.2 5.28 Taste 3 22 4.4 1.3 5.72 Condition 4 21 4.2 0.8 3.36 coefficient coefficient Quality score 17.30 Appendix 16: Sensory evaluation of common tofu (3rd time) Score from Criteria Average without Sum panelists important Impotant coefficient Average with important A B C D E Color 4 4 21 4,2 0,7 2,94 Smell 4 5 22 4,4 1,2 5,28 Taste 5 23 4,6 1,3 5,98 Condition 4 4 19 3,8 0,8 3,04 coefficient Quality score Appendix 16 Picture form research process coefficient 17.24 ... 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 N2-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