Analysis Of Microbial Community In Naturally Fermented Tofu Whey And Investigation On The Metabolic Characteristics Of The Key Lactic Acid Bacteria

Tofu is a traditional food in China,and large amount of tofu whey generated in the process of tofu production.Tofu whey contained aboudant nutrient substances,hence a lot of microrganism from the environment could grow in tofu whey,promoting the acidification of tofu whey.Tofu coagulated with acidic whey was more acceptable than that coagulated with CaSO₄ or MgCl₂ in terms of flavor,texture and so on.The microbial community determined the quality of acidic tofu whey,further affecting the quality of tofu and its derivative products.Except the small part of tofu whey used as coagulator,most of tofu whey was discharged into environment,causing environmental pollution and wasting of resources.Therefore,this study aimed at analyzing microbial community in naturally fermented tofu whey and investigating on the metabolic characteristics of the key lactic acid bacteria.High-throughput sequencing was firstly applied to study the microbial diversity and its effect on the chemical changes in the acidification process of tofu whey.The results indicated that Lactobacillus with 95.31%was the predominant genus in the microbial community of acidic tofu whey while Picha,Enterococcus,Bacillus and Acetobacter occupied about only0.90%,0.04%,0.02%and 0.09%,respectively.Besides,Lactobacillus amylolyticus were determined to be one of the dominated species with metagenomic analysis and culture method.Lactobacillus with?-galactosidase activities played leading role in metabolizing the soybean oligosaccharides of TW to produce lactic acid.And acetic acid produced by genus of Acetobacter was another main organic acid attributed to the acidification of TW except lactic acid.Meanwhile,the bioconversion of isoflavone glucosides into aglycones could also be promoted by Lactobacillus with the help of?-glucosidase activity.Moreover,the production of equol in NFTW was confirmed,which might be jointly converted from daidzein by several strains.Therefore,our results indicated that Lactobacillus was the dominated microorganism and mainly affected the chemical changes of NFTW.This study help provide basic theory and technical references for the production of tofu and its derivative products?like sufu?with NFTW as coagulator.Then we conducted a preliminary exploration on new isolated Lactobacillus amylolyticus L5 and L6 from acidic tofu whey to be the candidate probiotic based on the whole genome sequence and corresponding phenotypes.L.amylolyticus L5 and L6 were confirmed to be different strains with typing of RAPD-PCR.Their susceptibility to antibiotics,toxic metabolites such as biogenic amine and haemolysin were tested and it met the requirements established by European Food Safety Authority.Meanwhile the genes in terms of antibiotic resistance and toxic metabolites were not found in their genome.With Lactobacillus acidophilus NCFM as positive control,several established experiments about evaluating potential probiotics were performed on L.amylolyticus L5 and L6.The result indicated that L.amylolyticus L5 and L6 could survive in human gastrointestinal?GI?tract,inhibit the growth of pathogenic bacteria,adhere to Caco2 cells,metabolize raffinose,stachyose?flatulence factors?and partial starch.Besides,the mechanism of probiotic properties of L.amylolyticus L6 were further illustrated with identification of related genes through analysis of genome sequence.We proposed that L.amylolyticus L5 and L6 have the potential to be used as probiotics from phenotypes to genotypes.Besides,comparative genome and phenotype analysis indicated that L5 and L6 have a rather close consanguinity in the process of evolution,but they displayed difference in terms of morphology,acid tolerance and?-galactosidase.The previous study indicated that L.amylolyticus L6 had strong ability of metabolizing stachyose.Therefore,the?-galactosidase gene from this stain was cloned and expressed,and enzymatic properties and transgalactosylating activity were further investigated.The result showed that the optimal temperature was 37°Cand the optimal pH was 6.0.Meanwhile,the temperature stability,pH stability and the effect of metal ion on?-galactosidase were further studied.Besides,the HPLC and HPLC-MS were used to study the transgalactosylating activity of this enzyme,finding that it could catalyze the substrate melibiose to produce galactooligosaccharides.The study provided basic theory for industrialization of?-galactosidase.Finally,the isolated lactobacilli from acidic tofu whey were combined with Streptococcus thermophilus to ferment tofu whey added with pineapple juice.The acidity value,ability of converting isoflavone glucosides and sensory evaluation of the fermented tofu whey were assessed.Meanwhile,GC-MS was applied to determine the change of flavor substance after fermention of tofu whey.The results indicated that the pineapple tofu whey fermented by C1+ST3 and L6+ST3 more delicious and functional than the samples fermented by other strain combination.This study provides new way for the development and utilization of tofu whey.This study analyzed microbial community in naturally fermented tofu whey and investigating on the metabolic characteristics of the key lactic acid bacteria,providing theoretical basis for the production of acidic whey tofu and application of tofu whey.