(Luận văn thạc sĩ) 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 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 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) Season Country 2017/18 2018/19 2019/20 US 120,070 120,520 96,790 Brazil 122,000 119,000 124,000 Argentina 37,800 55,300 51,000 China 15,580 15,970 18,100 India 8,350 10,930 9,300 Paraguay 10,480 8,850 9,900 Canada 7,720 7,270 6,000 Others 20,050 22,430 21,020 Whole world 342,050 360,270 336,110 (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 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 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 (Source: General Statistics Office of Vietnam, * FAS estimates) Table 2.3 Production, supply and demand of soybeans in Vietnam 2009/2010 USDA Data 2010/2011 Adjusted Data USDA Data 2011/2012 Adjusted Data USDA Data Adjusted Data Cultivated Area 198 215 235 200 198 200 215 235 13 13 28 28 28 Production 300 297 300 350 388 Import 220 228 400 700 1,500 160 178 170 550 1,200 0 0 533 538 728 1078,0 1,916 (thousand ha) Harvest Area (thousand ha) Initial reserve Amount Domestic Import From US Import From EU Total supply Export 0 0 (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 Content by percentage by weight of dry matter Kind of beans Ash Đậu Hà Lan (Pisum sativum ) Đậu đỗ (Vigna radiata) Đậu ván (Lablab purpureus) Đậu nành Cellulose Sugars Starch Protein Lipid 3,0 6,5 50 28 2,0 4,1 5,2 55 28 2,4 3,2 4,4 56 29 2,0 4,0 3,7 19 - 42 20,0 (Glycine max) (Sources: USDA nutrient database) 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... structure of Glycinin 23 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