Research On Microbial Population Structure Analysis And Fermentation Strains Screening Of Fermented Meixiang Fish

The fermerntation process of traditional fermented Meixiang fish depended on the microorganisms which natural enrichment, the quality of products was not stable due to the effects of many factors and the unstable microbes of fermentation. In order to control the quality of the products effectively, the technologies of unculture and culture were used to analyze the microbial diversity and population structure of Guangdong fermented Meixiang fish, and isolate the dominant strains participate in natural fermentation. Then the preparation of the different fermentation agents to artificial simulate natural fermentation to ferment fish, and the quality of fermented fish products were evaluated.(1) The microbial population structure analysis of Meixiang fish under uncultured level: the DNA was exacted from Meixiang fish, after AGE(agarose gel electrophoresis), PCR amplification was taken by using primers of 16 S rDNA V4 area, and then the PCR amplicon was sequencinged by Illumina sequencing. According to the analysis of sequencing result, it showed that the microbial components involved in fermentation were abundant under the threshold of 0.03, and there were 8 kinds of phylum, they were Firmicutes(74.92%), Proteobacteria(18.11%), Actinobacteria(3.35%), Deferribacteres(1.31%), Acidobacteria(0.93%), Planctomycetes(0.16%), Verrucomicrobia(0.09%), and Tenericutes(0.04%), respectively. In the 8 phylum, the Firmicutes was the dominant phylum, and in Firmicutes, the highest abundance genus were Lactobacillus(25.36%), Staphylococcus(19.27%), and Tetragenococcus(14.62%), which could be considered as dominant genus.(2) The fermentation strains screening of Meixiang fish: the microorganisms of Meixiang fish were separated and purificated by using MRS, GAM and TSA medium, and 95 G+ strains were obtained with Gram staining. Then according to the requirement of the fermentation strains, that was catalase positive, glucose produce acid and don’t produce gas, tolerance of 20% NaCl, don’t produce histamine, amd with inhibitory effect of Escherichia coli and Staphylococcus aureus, 3 fermentation strains were obtained. Finally, the DNA of the 3 strains were exacted to PCR amplification by using 16 S rDNA primer, and sequencing with the PCR amplicon. After BLAST online, the 3 strains were Tetragenococcus halophilus, Staphylococcus xylosus and Staphylococcus saprophyticus, respectively.(3) The influence of different starter cultures on the quality of fermented fish: the fresh Sardine were cured in 10℃, and according to the antagonism test of the 3 strains, it showed that there was no antagonism among these strains, which could be used in fermentation together. Then the fermentation time and temperature,which were 44 h and 20℃, were determined with the growth curve of the 3 strains under 10、20、30℃. At last, under the fermentation time and temperature, the fish were fermented with different inoculation proportion of the 3 strains which designed by response surface, the inoculation proportion of Tetragenococcus halophilus(0, 1, 2), Staphylococcus xylosus(0, 1, 2) and Staphylococcus saprophyticus(0, 1, 2) were the influence factors, and the sensory score was response values. The result determined that the best inoculation ratio of strains were 1:1:1, and the results of physical and chemical index, sensory indexes and microbial indexes respectively showed that the inoculated fermented fish products were better than uninoculated fermented fish, and the inoculated fermented fish with best inoculation ratio were the best products, which with the best smell and taste.(4) The safety evaluation of fermented fish products: the 7 d test of gavage administrating to mice, the result showed that, about performance, there was no difference among these mice. About the mutagenicity of fermented fish to germ cells, there were no significant differences(p>0.05) of deformed sperm rate among the experimental groups and negative control group(distilled water), but there were significant differences(p<0.01) among the positive control group(cyclophosphamide), the experimental groups and negative control group(distilled water), it indicated that there was no genetic toxicity to the reproductive cells of fermented fish products. About the mutagenicity of fermented fish to somatic cell, there were also no significant differences(p>0.05) of the micronucleus rate among the experimental groups and negative control group(distilled water), but there were significant differences(p < 0.01) among the positive control group(cyclophosphamide), the experimental groups and negative control group(distilled water), the result indicated that there was also no genetic toxicity to somatic cell. So, the fermented fish products could be considered as safe food preliminary, and the starter cultures and fermentation process were also considered to be safe.