Screening And Breeding Of Phage-resistant Strain Of Lactiplantibacillus Plantarum And Optimization Of Culture Conditions

Lactiplantibacillus plantarum is widely used and closely related to human life,it is considered as one of the most common used starter cultures.However,if the fermentation process is infected by Lactiplantibacillus phage,it may lead to cell lysis,resulting in industrial losses.Therefore,it is of theoretical and practical importance to use phage-resistant and genetically stable strains of Lactiplantibacillus plantarum instead of wild strains for the industrial production of fermented products to improve product stability.In this study,Lactiplantibacillus plantarum IMAU10120 was used as the researching object to screen spontaneously mutated phage-resistant strain by secondary infection method,and its mutated genes were identified by whole genome sequencing,and then the culture conditions of the mutant strain were optimized to analyze the effect of the mutated loci on their nutritional requirements,so as to provide data and theoretical support for the replacement of wild strains by mutant strains for industrial production.The main results are as follows.（1）A stable and heritable phage-resistant mutant strain of Lactiplantibacillus plantarum was successfully obtained by secondary infection method,named as IMAU BIM03.（2）The genomic sequencing and comparative analysis showed that 84 loci of 79genes were mutated in the phage-resistant mutant strain IMAU BIM03,involving glycerophospholipid metabolism,fructose and mannose metabolism,and purine metabolism.（3）In compared with the wild strain,the mutant strain showed different uptake ability for the nutrients like Fe SO₄·7H₂O and V_B6,which might be related to the mutation of GTP pyrophosphokinase and holo-ACP synthase genes.（4）The optimal medium culture for the phage-resistant mutant strain IMAU BIM03was:17.90 g/L glucose,9.30 g/L yeast peptone,27.90 g/L yeast extract,1.20 g/L citric acid,10.50 g/L sodium citrate,0.12 g/L Mn SO₄·7H₂O,0.30 g/L histidine,and 1.00 g/L Tween-80.（5）The optimal static culture conditions for the phage-resistant mutant strain IMAU BIM03 were:initial p H of 6.5,temperature of 37℃,and inoculum of 4%.Using the optimized medium formulation and optimal static culture conditions,the viable count could increased to 8.45×10⁹CFU/m L,which was 4.26 times higher than that in MRS medium.