Screening And Functional Verification Of Aeortolerant Bifidobacterium Animals BAA6 In Space Mutation

Bifidobacterium animals BAA6 is isolated from the feces of health longevity elderly in Bama,Guangxi,and has the efficacy of preventing or improving diarrhea or constipation,improving immunity,regulating intestinal flora,and delaying aging.In this study,the Bifidobacterium animalis strains carrying the Shenzhou XI return spacecraft were isolated and purified,and 10 strains of mutant strains were obtained.The strains were screened using the oxygen tolerance coefficient as an evaluation index,and the genetic stability of the wild strain BAA6 and the mutant strain was also determined.The number of viable cells under the conditions of sex,growth curve,and different oxygen concentration?Oxygen concentration of0%,5%,10%,15%,and 21%?were measured,and the colony morphology and microscopic morphology of the strain were observed.Genome-wide re-sequencing method was used to obtain gene mutation information.Through the whole genome comparison,the mutant gene was obtained and the mutation was verified by using fluorescence quantitative PCR technology and the mutation gene's survival rate and mutation enzyme activity at 10%oxygen concentration.The results provide new evidence for the oxygen tolerance mechanism of Bifidobacterium animalis and provide new ideas for the selection of good oxygen tolerance probiotics.The main research conclusions are summarized as follows:?1?After B.animalis BAA6 was returned to the ground after space mutagenesis,10strains were obtained,of which 5 strains were significantly higher in oxygen tolerance than wild strains?p<0.05?,and 10 strains exhibited an oxygen tolerance after 30 passages.Stable and always significantly higher than the wild strain BAA6;growth curves showed that the growth trend of the resistant mutant was similar to that of the wild strain,but higher than that of the wild strain BAA6;at an oxygen concentration of 5%,the mutant strain was resistant to oxygen.The OD₆₀₀ value and viable cell count were higher than those of the wild-type strain BAA6.However,after scanning electron microscopy and morphological observation,it was found that there was no difference in apparent morphology between the wild-type strain BAA6 and the oxygen-tolerant mutant strains;meanwhile,the acid resistance and resistance of the oxygen-tolerant mutant strains.Bile salt performance has not changed.The surface hydrophobicity of the mutant strain was significantly higher than that of the wild strain BA A6.?2?A total of 19 mutation sites were detected by whole genome re-sequencing,and a total of four genes were identified for mutation,namely BAA6_RS04435,BAA6_RS05735,BAA6_RS06425,and BAA6_RS08120.The protein encoded by BAA6_RS04435 is a membrane protein,which is predicted to be the structural basis of the conductance cation channel and its spatial structure is changed.BAA6_RS05735 gene encodes an MFS transporter,which mainly promotes the glucose transporter,and its spatial structure has not changed;BAA6_RS06425 and BAA6_RS08120 are assumed Protein,its function is still not clear,and the spatial structure has not changed.Therefore,the ability of BAA6_RS04435 to encode protein changes is very high?3?According to the heterologous gene overexpression analysis,the relative expression level of gene BAA6_RS04435 was higher than that of the no-load control vector,which was4.9 times higher.At the same time,through functional verification,the survival rate of the gene at the 2h at 10%oxygen concentration Begin significantly higher than the no-load control group.Therefore,space mutagenesis caused mutations in the BAA6_RS04435 gene of Bifidobacterium animalis BAA6,which may have altered the oxygen tolerance of strains.