The High-efficiency Isolation Technology Of Bifidobacterium Using Omics And Its ?-Galactosidase Mining

Bifidobacterium is one of the important bacterials of gastrointestinal microorganisms,and it is also a kind of probiotic that is beneficial to human health.However,it is difficult to separate from sampes which including complex microbial communities,due to its own harsh growth conditions.With the rapid development of the omics technology,it is possible to study bifidobacteria from the genetic level and explore conditions that conducive to the growth of bifidobacteria.There are a large number of carbohydrate metabolizing enzymes in the genome of bifidobacteria,so changing the carbon source of the medium is a way to improve the efficiency of screening and separating bifidobacteria.At the same time,there is still a lack of glycoside hydrolases(GHs)with high activity and excellent properties in the market,and many commercial enzymes cannot be directly used in the food industry.The GH from food-grade probiotic is safe.Therefore,it is feasible to use bifidobacteria to mine GH with high activity and excellent properties.In this study,metagenomics,pan-genomics and enzymology were used to determine the main GH in bifidobacteria and obtained selective media for the isolation of bifidobacteria.On the basis of the obtaining of bifidobacteria,the genome of the bifidobacteria was further analyzed to mine the functional?-galactosidase(GH2/42)in bifidobacteria.The main conclusions were as follows:(1)The GH13,GH3,GH42 and GH43 families are universal in Bifidobacterium.The pan-genomic analysis of the whole genome of 144 Bifidobacterium spp.showed that GH13,GH3,GH42 and GH43 families were common in bifidobacteria.Further pan-genomic analysis(selecting five intestinal microbial genera(Alistipes,Anaerostipes,Barnesiella,Blautia and Collinsella)as a reference),the results showed that?-galactosidase,?-glucosidase,oligo-1,6-glucosidase,sucrose phosphorylase,trehalose-6-phosphate hydrolase,pullulanase and?-xylosidase of the four GH families had more preference for bifidobacteria.At the same time,the metagenomic sequencing analysis of stool samples showed that Bifidobacterium longum occupied a dominant position in the Bifidobacterium,and there were differences in the content of bifidobacteria between different samples.And the number of?-galactosidase genes was the first.Further through the SWISS-MODEL structure prediction,the results showed that these 7GHs in the metagenomic group had the typical structure of the GH3,GH13,GH42 and GH43families,that is,the 7 GHs existing in the metagenomic group had the potential to hydrolyze the corresponding substrates.(2)Using oligosaccharides corresponding to GHs,a preference for bifidobacteria,as the main carbon source of the selective medium,which could improve the separation and culture efficiency of bifidobacteria.According to the results of pan-genomic and metagenomic analysis,raffinose,trehalose,cellobiose,melibiose,lactulose,lactose,sucrose,resistant starch,pullulan,xylan and dextran were used as the main carbon source of the selectivity medium.In the medium with lactose,raffinose and xylan as the main carbon sources,the percentages of cultivable bifidobacteria to the total number of cultivable microorganisms were 89.39%±2.50%,71.45%±0.99%and 53.95%±1.22%,respectively,while the ordinary GAM(Gifu anaerobic medium)medium was only 17.90%±0.58%,and xylan might be a prebiotic beneficial to the host health.Among the 76 strains,there were 28 bifidobacteria,accounting for 37%of the total.Mupirocin and rifampicin resistance genes were common in the genomes of 22 bifidobacteria sequenced by whole-genome sequencing,but virulence genes were not present.(3)The?-galactosidase B17?2 from Bifidobacterium had more potential for commercial application.Two?-galactosidase B17?2 and B26?3 from Bifidobacterium were successfully obtained with sizes of 719 and 697 amino acids respectively,which belonged to the GH42family.The optimum pH of both enzymes was 5.5 and 6.0,respectively,and the optimum temperature of both was 45?.After low temperature and short time(35?40?,10?30 min)treatment,the relative activity of enzyme B26?3 greatly increased and reached up to 300%.The enzyme B17?2 was not suitable for any temperature treatment,while it showed good acid-base tolerance,especially in the pH range of 4.0?8.0.The V_max of enzymes B17?2 and B26?3 were3.83±0.14 mmol·(min·g)^-1 and(1.11±0.063)×10^-3 mmol·(min·g)^-1,respectively;K_m were1.75±0.2 m M and 1.7±0.27 m M,respectively;K_cat were 5127±183 s^-1 and 1.47±0.084 s^-1,respectively.