Study On Mechanism Of Acid Stress Responses In Bifidobacterium Animalis Subsp.lactis A6

Bifidobacteria are important probiotics and widely used in the food industry.But the acid stress during the manufacturing process as well as digestion threatens the survival of bifidobacteria.Bifidobacterium animalis is the most acid-tolerant species in bifidobacteria.Howerver,the aicd tolerance mechanisms of B.animalis are still not clear.In this study,we took B.animalis subsp.lactis A6(CGMCC No.9273)as the research object.The comparative genome analysis,transcriptomic analysis and the gene overexpressions were used to investigate the acid tolerance mechanisms of B.animalis.The results of this study would offer important theory support for the acid tolerance mechanism in B.animalis and the acid tolerance improving of Bifidobacterium in industrial application.The conclusion was summarized as follows:(1)Evaluation of the acid resistance ability of B.animalis subsp.lactis A6.The viable count of B.animalis subsp.lactis A6 before acid treatment was 9.42±0.06 log10 cfu/ml,and it did not decline significantly after exposed to pH2.5 condition for 2h(9.22±0.17 log10 cfu/ml).The viable count of all the B.animalis strains did not decline after exposed to pH2.5 condition for 2h while the B.longum subsp.longum,B.longum subsp.infaintis,B.bifidum,B.breve,B.adolescentis strains died completely.These results indicated that B.animalis subsp.lactis A6 has high acid resistance ability,and the high acid resistance ability was species specific.(2)Identification of B.aniamlis unique-gene dataset.The whole genome sequences of B.animalis subsp.lactis A6 were determined by PacBio sequencing technology.The complete genome of A6 consists of one 1,958,651bp circular chromosome,encoding 1622 CDSs,52 tRNA genes and 16 rRNA genes.98 unique genes in B.animalis were identified by comparative genome analysis of 51 Bifidobacterium genomes.(3)Identification of the acid stress response genes in B.animalis A6.53 1 differentially expressed genes were identified by RNA-seq transcriptomic analysis.The function analysis of the differentially expressed genes showed that the integrity and impermeability of the B.animalis A6 cell membrane was increased by enhancing the synthesis of fatty acid and phage shock protein A to block HT ions;di/oligo/polysaccharide uptake,utilization of ribose as a carbon source,and the bifid shunt pathway were enhanced to increase energy production;the DnaK system,GroES/EL system,and ClpB were induced to protect proteins;direct repair and the base-excision repair system was initiated to repair DNA damage;the phosphorylation/dephosphorylation and quorum-sensing signaling system were initiated to transduce the acid stress signal,and triggered the transcription alteration and the decreasing of translation.These findings allowed us to propose a model for the acid stress-response mechanism in B.animalis.(4)Verification of the function of the key stress response genes.The overexpression of BAA6?RS00480,BAA6?RS06240,BAA6?RS03885,BAA6?RS05205 in B.animalis A6 could improve the survival rate of A6 at lethal acid treatment by 22.43 folds,9.58 folds,9.62 folds and 11.58 folds,respectively.This result indicated that BAA6?RS00480,BAA6?RS06240,BAA6?RS03885,BAA6?RS05205 played an important role in the acid stress responses of B.animalis A6.