The Key Molecular Mechanism For Osmotic Tolerance Of Bifidobacterium Bifidum And Its Manufacturing Process

Bifidobacterium bifidum CCFM16 has the efficacy of regulating intestinal homeostasis,relieving constipation and improving depression.It is widely used in the development of probiotic formulations because of its high safety.Osmotic stress is the main factor limiting the proliferation of Bifidobacterium bifidum during high-density culture,and the poor ability of B.bifidum CCFM16 to tolerate osmotic stress has limited its industrial application.In this study,laboratory adaptive evolution was applied to B.bifidum CCFM16,and mutant strains with high permeability resistance were obtained by screening.Second,key mutant genes were identified through genome-wide comparison,candidate gene stress expression,and three-dimensional protein structure modeling.Then,the metabolites of the strains at different stages of domestication were compared to find out the key difference substances,and the possible mechanism of the osmotic tolerance of B.bifidum CCFM16 was proposed.Based on this theoretical basis,the culture of the strain was optimized to improve its growth density.This study revealed the key molecular mechanism of the increased osmotic tolerance of B.bifidum.And it provided a new idea for achieving the growth of B.bifidum under high osmotic stress.The main results of the paper are as follows.（1）The osmotic-resistant mutant strain of B.bifidum CCFM16 was obtained by adaptive evolution.The osmotic stress-resistant mutants CCFM16-700,CCFM16-1000,and CCFM16-1300 were obtained by screening on 700 m Osm/kg,1000 m Osm/kg,and 1300 m Osm/kg solid plates by continuously increasing the osmotic stress of the liquid medium.Permeability analysis was carried out on them.The results showed that the growth rate of CCFM16-1300 in the medium with osmotic stress of 1300 m Osm/kg（3.0%Na Cl）was 3 times that of the original strain CCFM16,and the osmotic stress resistance could be inherited stably.（2）Screening mutant genes related to osmotic stress tolerance in mutant strains by whole-genome resequencing technology.The three mutant strains that tolerated different osmotic stress were resequenced by the Illumina Hi Seq platform,and their genomes were compared individually.A total of 20 SNPs were annotated on the genome,of which 13 genes encoded amino acid sequences that were changed,and 6 mutated amino acids were in the functional domain.The expression of genes ccfm16＿290,ccfm16＿1673,and ccfm16＿1898 showed high consistency with the osmotic environment,and the encoded proteins were related to glutamine metabolism,glutamate transport,and glutamate synthesis.3D protein structure simulation prediction of genes before and after mutation found that no structural damage was caused.（3）The key molecular regulation mechanism of osmotic resistance was further identified by metabolomics.Metabolomic analysis of mutant strains at different stages of domestication and comparison of metabolites.The results showed that the differential metabolites were mainly sugars and amino acids.And with the progress of acclimation,the accumulation of intracellular glucose,glucose-6-phosphate,fructose and xylose decreased to varying degrees.However,the key enzyme activities of the"Bifidus Pathway"increased with the weakening of growth.On the contrary,except for the decrease of citrulline,the contents of other key differential amino acids showed an increasing trend,among which the increase of proline was the most significant.Furthermore,when osmotic stress increased to a certain level,glutamate accumulation began to decrease.The key mutant genes were all related to glutamate synthesis or transport,indicating that under long-term hyperosmotic stress,mutant strains have evolved an osmotic protection system that synthesized proline from glutamate,and provided energy by sacrificing basal metabolism.（4）The effect of osmotic stress regulation pathway activation on strain growth was verified.The genome analysis of B.bifidum CCFM16 shows that it does not have a proline transport system,but has a relatively complete peptide transport and hydrolysis system.Proline-containing dipeptide,tripeptide and nonapeptide were synthesized,and it was found that the addition of dipeptide FP（Phe-Pro）could increase the growth density and freeze-drying survival rate of B.bifidum CCFM16 by 15.74%and 32.48%.However,the addition of the tripeptide VPQ（Val-Pro-Gln）had no obvious effect.It indicated that the proline-containing polypeptide was helpful for the growth of the strain but was selective.（5）The optimum culture process of B.bifidum CCFM16.By comparing the effects of different nitrogen sources and trace elements on the proliferation efficiency of the strain,the optimal substrates and limiting trace elements were optimized.The substrate concentration,ratio and cultivation process conditions were optimized based on growth inhibitory factors.The optimization results were:30 g/L glucose,26 g/L tryptone,0.26 g/L Mg SO₄·7H₂O,1 m L/L Tween 80 and 1 g/L cysteine hydrochloride.The highest viable bacterial count was（3.70±0.61）×10⁹ CFU/m L under constant p H 6.5 culture for 18 h,which was 3 times that of MRS medium constant p H batch culture.The proline-rich polypeptide mixture was obtained by enzymatic hydrolysis of casein,which was added at 5 g/L after 8 h of culture.The highest number of viable bacteria in the fermentation broth could reach（5.75±0.13）×10⁹ CFU/m L,and the lyophilization survival rate could reach 59.77%,which were 55.40%and 27.38%higher than those without addition.