Analysis Of Microbial Diversity,Metabolites And Gene Sequence Of Functional Lactobacillus In Traditional Fermented Meat Products

Traditional fermented meat products are mostly natural fermentation,and the microorganisms involved in fermentation mainly come from raw materials and production environment.Different types and metabolic characteristics of bacteria can be screened and enriched by environmental conditions,and regional differences.Influenced by climate,artificial operation and other factors,the microbial community structure and probiotic abundance of fermented meat products are often significantly different,resulting in the microbial diversity in fermented meat products,and also lead to differences in product quality.The purpose of this paper is to explore the influence of microbial community structure and distribution of dominant bacteria on product quality in traditional fermented meat products.And the metabolic characteristics,genome sequence and gene function of functional strains were studied to provide basis for the development and safe application of meat starter.Fourteen representative fermented meat products were collected from Inner Mongolia,Tibet and Xinjiang.According to regional differences and different fermentation modes,these samples were divided into four groups:A,B,C and D.Illumina MiSeq sequencing technology was used to high-throughput sequencing and sequence 16S rRNA gene of bacteria in samples.The results showed that there were significant differences in diversity of microbial communities between the 2 groups of natural fermented called A,B and 2 regulated called C,D samples from different regions?P<0.01?.Firmicutes,Proteobacteria and Bacteroidetes were the dominant bacteria in group A and B,accounting for more than 10%.Firmicutes were the dominant bacteria in C and D groups,accounting for 92%.The species with high microbial abundance were mainly Lactobacillus of Firmicutes and Pseudomonas of Proeobacteria.Lactobacillus is a common probiotic lactic acid bacteria in food,while Pseudomonas includes Escherichia coli,Salmonella,and other pathogenic bacteria.It can be seen that the dominant microflora of naturally fermented meat products is abundant,but there are potential safety hazards due to the strains containing Proteobacteria.Lactobacillus of the Firmicutes,the only dominant microflora in regulated meat products,has been widely used in fermented foods.Therefore,it is imperative to use probiotic lactobacillus to control the production of traditional fermented meat products artificially.101 strains of lactic acid bacteria were isolated from naturally fermented meat products by isolation,purification and identification of lactic acid bacteria.By 16S rDNA sequencing and NCBI homologous sequence analysis,these strains were identified from 7 genera,14 species.Thirty strains,producing biogenic amine negative,were screened through biogenic amines test,salt tolerance,nitrite tolerance,bacteriostatic effect,cholesterol-lowering ability and Lipase-producing ability.Ten of them were salt-tolerant,bacteriostatic and cholesterol-degrading.And Lactobacillus of TR13 and ZF22 also had strong Lipase-producing ability.Fermented sausage was prepared by using TR13 and ZF22 as culture starter,respectively.Method with natural fermentation ZR group,adding lipase M group,adding strains TR14 with weak lipase production ability and commercial culture starter 302 group as control,the effect of strains TR13 and ZF22 on amino acids and fatty acids in fermented meat products were analyzed.The results showed that the content of essential amino acids?EAA?in M,TR13 and ZF22 groups began to increase on the 12th day?P<0.05?compared with the 0th day.EAA content in ZR and TR14 groups increased on the 24th day?P<0.05?.It was obvious that strains TR13 and ZF22 with high lipase production could promote the production of EAA.During fermentation and cryopreservation at 4?,the contents of MUFA and PUFA in TR13 group on 12d increased by 640.37%and 54.17%,which were significantly different from those of other groups?P<0.05?.The effects of TR13 on the metabolites of fermented meat products showed that under pos and neg modes,the results of secondary identification of metabolites were 537 and 336,respectively.LC-MS chromatogram and PCA showed that there were significant differences in metabolites among components?P<0.05?.It showed that raw meat after microbial fermentationt,the abundance of metabolites changed significantly,and significantly affected by microbial species.Compared with the naturally fermented meat products,the content of 75 substances in TR13 group increased significantly?P<0.05?.Among them,The average content of 28 kinds of peptide substances,8 kinds of acyl carnitine,lecithin and glycerol phosphate choline increased by 4.79,2.38,2.97 and 2.68 times,respectively.The genome sequencing and annotation of gene function for TR13 were carried out by PacBio and Illumina Hiseq sequencing platform.The results showed that the genome of TR13 was a ring molecule with one chromosome,the total length of the sequence was 217224 bp,without plasmid gene,and had an average GC content of 36.82%.There were 2356 coding genes?CDS No?,the total length of which was 188 3532 bp,accounting for 86.71%of the genome.The gene contained 15 rRNA operons,63 tRNAs,202 promoters,17 horizontal transfer element gene island and 36 repeats.It contained a prophage,starting at 1798 554 bp and terminating at 1828 331 bp.The possible phage was NC₀06565.The number of genes that can annotate GO information was 1792,which contained 40 functional characteristics,accounting for 76.1%of the coding genes.The number of genes that can annotate COG information was 1942,accounting for 82.43%of the coding genes.In the KEGG database,1144 genes were annotated on 35 pathways of six major functions:metabolism,genetic information processing,environmental information processing,cellular processes,organismal systems and human diseases.The application test of TR13 in fermented sausage showed that the co-culture of TR13 and Staphylococcus xylose could promote the reduction of the pH value and Aw,effectively reduce the content of nitrite and saturated fatty acid in sausage,and play a role in the release of PUFA in the later drying stage.In summary,Lactobacillus of Firmicutes was the dominant bacteria in traditional fermented meat products.However,the samples collected contain Pseudomonas with high abundance of Proteobacteria,and had potential safety hazards.It is necessary to regulate the microbial community structure during fermentation.The dominant strain TR13 had the function of bacteriostasis and lipase production,and its fermentation performance was good.And the research of TR13 contained metabolomics,whole gene sequence and functional annotation of coding genes can provide theoretical basis for the industrial application of the strain.