Induction Of Viable But Non-culturable State In Lacticaseibacillus Casei Zhang And Proteomic Study

Lactic acid bacteria are inevitably subjected to various environmental pressures in the process of production and application.Some lactic acid bacteria will enter a "Viable but non-culturable state"(VBNC)under certain adverse conditions.At present,there are few reports on the induction condition,detection method,resuscitation condition and stress mechanism of VBNC state in lactic acid bacteria.In this study,a probiotic strain of Lacticaseibacillus casei Zhang(L.casei Zhang)with excellent fermentation characteristics was taken as the research object,and L.casei Zhang was placed in a variety of compound conditions(low temperature,low p H and low nutrition)to induce VBNC state,and samples were taken regularly.The cell activity was detected by plate counting method and non-culturable method,and the conditions for L.casei Zhang to enter VBNC state were determined,and the morphological changes of its cell surface were observed.The differential protein expression between L.casei Zhang VBNC state and normal state was analyzed by TMT proteomics technology.The main results are as follows:(1)The conditions for L.casei Zhang to enter VBNC state were detected by plate counting method and non-culturable method.The results showed that L.casei Zhang induced the VBNC state in MRS Liquid medium with lactic acid regulated p H4 at 4℃ for120 days.The VBNC state was also obtained after induction in 0.85% normal saline at 4℃for 90 days.(2)The cell morphology of L.casei Zhang in normal state and VBNC state was observed by scanning electron microscope(SEM).The results showed that the cell morphology of L.casei Zhang in VBNC state changed obviously,the cell surface became uneven,and the cells became thick and curved.(3)Proteomics technology was used to analyze the differential proteins between L.casei Zhang VBNC state(MRS liquid medium of p H4 at 4℃)and normal state.Compared with normal state,768 different proteins were identified in L.casei Zhang VBNC state.Among them,414 proteins were significantly up-regulated(FC >1.3,p<0.05),and 354 proteins were significantly down-regulated(FC≤1/1.3,p<0.05).Functional annotation and enrichment analysis of these proteins were conducted through GO,COG,KEGG and other databases,and significant enrichment analysis of differential proteins was further conducted.It was found that L.casei Zhang significantly up-regulated protein enrichment in phosphotransferase system(PTS),fructose and mannose-metabolism,pyruvate metabolism and other pathways under VBNC state.Among them,ABC transporter,and ribosome synthesis protein were highly upregulated.Down-regulated proteins were significantly enriched in purine metabolism,pyrimidine metabolism,alanine,aspartic acid and glutamic acid metabolism.Therefore,under the condition of low temperature and low p H,L.casei Zhang mainly provided energy for bacteria and repaired the damage caused by stress by up-regulating phosphotransferase system(PTS),pyruvate metabolism,DNA repair and other related proteins;meanwhile,weakly expressed amino acid metabolism,DNA replication and other related proteins reduced energy consumption caused by protein synthesis.This study provides certain data support for determining the induction conditions of L.casei Zhang VBNC state and preliminary analysis of the molecular mechanism of its entry into the VBNC state,and provides reference for the development and application of the VBNC state of lactic acid bacteria in the food industry and microbiology.