| Objective This experiment,with the metagenome technology,determined the microflora in the Douzhi and its dominant functioning bacteria,from which it separated the dominant bacteria suitable for the fermentation of soy-bean milk,and inoculated the Douzhi with these bacteria,to optimize its production process,improve the safety and the stability of the product.The taste is generally accepted by the vast consumers,and it provides theoretical basis and technological references for the industrialized production for Douzhi.Methods With the metagenome technology,it carried out tests on the diversification of OUT and Alpha in the Douzhi and the Cooking Starch Residue,and performed analysis on its abundance and evenness,therefore analyzing the microflora in the Douzhi and its dominant functioning bacteria.With the methods as dilution and plate application,it made primary selection for the bacteria with the flocculation rate of above 40% and the ones with strong acid-producing abilities in the Douzhi.By the sensory evaluation,it made a second selection to select a bacterium with the best fermentation effects.Then,based on the individual's form,bacteria forms,the physiological and biochemical experiments and the 16 S r DNA sequencing methods,it made verifications over this bacterium.With the single-factor experiment and the orthogonal experiment,it made optimization for the culture conditions of the fermentation bacteria of Douzhi,and designed and optimized the cultural medium formula with the uniform experiment.Through the sensory evaluation,and with the single-factor experiment and the orthogonal experiment,it made optimizations over the fermentation conditions for pure soy-bean fermented beverages,and determined the optimized production process.And,with the evaluation standards as in PH value,the number of bacteria and sensory scoring,it carried out sensory scoring,physical and chemical indexes and microbial indexes tests on the products produced,with which to determine the validity period of the product.Results The analysis on the microflora and its dominant bacteria in Douzhi with metagenome technology: this research collected 12 Douzhi samples(group D)and cooking starch residue(Group M)from three manufacturers.By analyzing the composition and diversification of the bacteria with high-throughput sequencing analysis,it discovered the similar OUT quantity in the samples of Douzhi samples and cooking starch residue.However,the Douzhi from different manufacturers maintain more special OUT quantity than the cooking starch residue.There are no significant changes(P> 0.05)in Chao,Ace and Shannon in the Douzhi.M-LCQK's Shannon indexes are significantly higher than the group M-DZ and Group M-LFS(P<0.05).The 12 samples can be classified into 6 bacteroidetes.Among the Douzhi,the dominant genera include Lactococcus,Lactobacillus,Streptococcus and Klebsiella spp.While the cooking starch residue from three manufacturers maintains the lactobacillus of above 10%.The selection and identification for the dominant bacteria in Douzhi: with the indexes as the acid-generating ability and the flocculation rate,it primarily selected 8 bacteria with the PH value of below 4.0 and the flocculation rate of above 40%.By fermenting the Douzhi with these 8 bacteria,and with the sensory evaluation,it made the second selection and obtained the bacterium D-7 which maintains the highest score.For the bacterium D-7,which was selected,separated and purified from the Douzhi,combining with the individual form characteristics,bacteria form characteristics,the physiological and biochemical experiment and 16 S.r DNA sequencing,it made the final determination that this bacterium is Lactococcus lactis subsp.lactis,and named it Lactococcus lactis subsp.lactis LLY003.The optimization for the culture conditions of fermenting bacteria of Douzhi: with the single-factor experiment and orthogonal experiment on the culture conditions,it concluded that the best culture condition is under the culture time of 24 hours,the culture temperature at 30? and PH value of 6.5.With the uniform experiment design and the Quadratic Polynomial Stepwise Regression Analysis,it concluded the best culture medium would be glucose 3.37%,yeast extract 0.80%,hydrogen phosphate two,potassium 0.35%,lactose 0.05%,sodium acetate 0.73%.The development of soy-bean milk fermented beverages: with the single-factor experiment and orthogonal experiment on the fermentation conditions,it concluded that the best fermentation conditions are the fermentation time at 6 hours,temperature at 39?,the sucrose content of 6.5%,the inoculation amount of 10%.The validity period for the soy-bean milk fermented beverage produced with this process is 20 days(4?).Conclusions In the Douzhi,the thick walled phylum is the dominant bacteroidete and the lactococcus is the dominant genus.In the cooking starch residue,the proteus and thick walled phylum are the dominant bacteroidetes and the acetobacter is the dominant genus.In the samples of Douzhi and cooking starch residue,with the indexes as the acid-generating ability and the flocculation rate,it primarily selected 8 bacteria.By fermenting the Douzhi with these 8 bacteria,and with the sensory evaluation,it made the second selection and obtained the bacterium which maintains the highest score.It made the final determination that this bacterium is Lactococcus lactis subsp.lactis,and named it Lactococcus lactis subsp.lactis LLY003.The best culture conditions for Lactococcus lactis subsp.lactis LLY003: the culture time of 24 hours,the culture temperature at 30? and PH value of 6.5.The best culture medium would be glucose 3.37%,yeast extract 0.80%,hydrogen phosphate two,potassium 0.35%,lactose 0.05%,sodium acetate 0.73%.The best fermentation conditions for the pure-bacteria soy-bean milk fermented beverage is: fermentation time of 6 hours,the fermentation temperature at 39?,the sucrose content of 6.5% and inoculation amount of 10%.The sensory scoring for the products produced under these conditions is 94 scores,and the validity period for this product is 20 days(4?). |
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