The Cross-adaptation Mechanism Of Lactobacillus Rhamnosus Heat Stress And Oxidative Stress

As an important probiotic,human-derived Lactobacillus rhamnosus is one of the most widely studied and applied new lactic acid bacteria starters.However,the various stress conditions during the production and storage processes can cause serious loss of viable count which is essential to provide beneficial effects.A single stress can induce and enhance the tolerance of lactic acid bacteria to complex stress and form cross adaptation.Cross-adaptation involves different forms of stress,and provides more matching basis for transcriptome-phenotype matching,which is beneficial to reveal the mechanism of stress adaptation;In addition,cross-adaptation can also improve the flexibility and operability of pretreatment.The L.rhamnosus hsryfm 1301 isolated from the long-lived population in this article has significant probiotic functions and strong intestinal environmental adaptability.Preliminary studies have shown that L.rhamnosus hsryfin 1301 has cross-adaptation to stress,but its mechanism for stress cross-adaptation remains to be seen the study.In this study,transcriptome-phenotype matching and bioinformatics analysis methods are used to explore the key cross-adaptation genes,in order to reveal the cross-adaptation mechanism of L rhamnosus to heat stress and oxidative stress.The main research contents are as follows.(1)The results of genome sequencing of L.rhamnosus hsryfm 1301 showed that the genome of this strain was composed of a 3,022,021 bp circular chromosome with an average GC content of 46.75%and a 45,792 bp circular plasmid with an average GC content of 43.63%.The genome contains 3154 ORFs(open reading frame),15 rRNA,and 59 tRNA genes.Through COG(Orthologous Protein Cluster)functional analysis,there are 2049 ORFs(66.22%)on the chromosomes,which are divided into 21 functional categories.The functions of most genes were associated with important transport and metabolism pathways,replication,energy production and conversion pathways.On the plasmid,9 ORFs(39.13%of the classified ORFs)were classified into the replication,recombination,and repair functional categories,which contained 7 transposases.(2)Transcriptional analysis of gens specific to the heat stress(46?,1 h)and oxidative stress(0.5 mM H2O2,1hof L.rhamnosus hsryfm 1301 was performed.After treated with heat stress.562 genes were found to be differentially expressed,including 4 HSR genes(roEL,groES,clpL,clpX)of the protein repair system.After treated with oxidative stress,341 genes in L.rhamnosus hsryfm 1301 were found to be differentially expressed among which 2 OSR(oxidative shock response)genes(pgmB,yedF)were up-regulated.There are 154 overlapping DEGs after heat stress and oxidative stress treatment,150 of which are directionally regulated(accounting for 97.4%).and only 4 genes have been identified as differentially expressed between the heat group and the oxidative group,and 62.7%of the enriched overlapping DEGs are related to nitrogen transport and metabolism,all of which are down-regulated.This indicates that nitrogen source may play an important role in the cross adaptation of L.rhamnosus hsryfm 1301 to heat stress and oxidative stress(3)By explore the influence of nitrogen sources on the heat and oxygen tolerance of L rhamnosus,it was found that the peptone concentration had little effect on the strain's early growth,but the low peptone medium could significantly improve the heat and oxidation resistance of the strain.At the same time,it was found that the influence of low nitrogen source environment on the tolerance of strains was universal during the growth period.The low nitrogen source environment at the four growth stages of OD600 of 0.5,1.0,2.0,3.0 was beneficial to improve heat and oxidation resistance.Especially when the strain was cultivated to OD600 of 2.0,the survival rate of heat stress and oxidative stress was increased by 130 and 40 times,respectively.While OD600 is 4.0,this phenomenon of improving tolerance is weakened.6 strains of L rhamnosus and 4 other commonly used lactic acid bacteria strains were selected to verify the above rules.And it was found that the cultivation of 6 strains of L rhamnosus in a low nitrogen source environment can improve their heat resistance and oxidation resistance,while 4 other strains This phenomenon does not exist in lactic acid bacteria of the genus.After supplementing phenylalanine,isoleucine,glutamate,valine,histidine or tryptophan in tryptone-free MRS medium,the survival rate of strains under heat and oxidative stress decreased,indicating that the existence of these free amino acids is not conducive to the heat and oxidative stress tolerance of the strain.(4)The cross adaptation of L rhamnosus was investigated in spray drying.The results found that low nitrogen source concentration cultivation,heat stress and oxidative stress pretreatment can improve the survival rate of spray drying.The survival rate of spray drying of the untreated group is only 29%,while the survival rate of spray drying under low nitrogen source culture,heat stress or oxidative stress are 74%,76%,and 98%,which were increased by 2.55,2.6,and 3.37 times,respectively.At the same time,the three treatment methods have a good effect on the acid tolerance(pH 2.5)of the spray-dried bacterial powder.The acid-resistant survival rate of the untreated bacterial powder is 0.8%,while the bacterial powder under low nitrogen source culture,heat and oxidative stress of acid-resistant survival rate are 17%,76%,72%,which are increased by 22.5,95,and 90 times,respectively.Moreover,the bacterial powder obtained by heat and oxidative stress pretreatment have good storage ability in the environment of-20?.The bacterial powder obtained by heat stress pretreatment is stored at-20? for 3 months,and the cell viability is still greater than 90%.After the bacterial powder pretreated by oxidative stress is stored for 3 months,the survival rate is greater than 50%.