Selection Of Phage Resistant Strains Of Lactobacillus Delbrueckii And Identification Of Phage Resistance Mechanisms

Lactobacillus delbrueckii are one of the most important stainer contributing to fast lactic aciddevelopment, and also to flavor and texture modification in fermented milks.However, Lactobacill-us phages can result in seriously damage to dairy industries and cause huge economic loss.In ordeto control and to inquire theory evidence forstudying of phage-resistant stainer. We studied mainlyon Lactobacillus delbrueckii and Lactobacillus delbrueckii virulent phage phildb.The phage problem has promoted the adoption of various strategies.However, phage-resistantstarter cultures were traditionally obtained by recombinant DNA Technology or conjugal transferof plasmids conferring phage resistance, which holded Health potential dangers. Fortunately, theisolation of spontaneous phage-resistant. Mutants has been considered as a convenient, simpleand”natural”strategy to replace phage-sensitive strains. Lactobacillus delbrueckii subsp.bulgaricusKLDS1.1016and Lactobacillus delbrueckii subsp.bulgaricus KLDS1.1028were used as originalstrain.The bacteriophage-resistant mutants were isolated by secondary culture. Random amplifyi-cation of polymorphic DNA (RAPD-PCR) was used to confirm strain identity among phage resist-ant strains and their parent strain. Characteristics related to their phage-resistance capacities, EOP,phage-resistance stability, lysogeny were determined. The yogurt fermentation properties ofmutants were also studied for two strains of mutants (BIM02, BIM8). The study show thatsimilarity coefficients between hage-sensitive strains and all their derived mutant strains washigher than80%, the phage-resistant strains were isolated from original strains and were stable andnon-lysogen. They were not phage-resistant mutants which exhibited the ability to form visiblelysis plaques when they were infected with phage. The fermentation characteristic(viable count,acidifying)and texture characteristic(consistency, firmness)were superior to the origin strainregardless of the absence or presence of phage, so the two strains of mutants could be used foryogurt industrial production.The seventeen BIMs selection was proformed that the obvious decrease of the adsorption tothe same genus, and more probability in the same genus, or even in the same species between highDR homology. Similarly to other bacteria, the secondary structures of test strains DR displayedpalindromic structure. Rates exhibited by all mutants indicated the presence of adsorption interference duringphage infection. And the accessory polysaccharide-peptidoglycan complex was proved to beinvolved in the phage receptor sites.CRISPR (Clustered regularly interspaced short palindromic repeats) were detected in2Lactobacillus delbrueckii sensitive strains and17mutants by PCR method, but not because of theaddition of new spacers in CRISPR. Furthermore, they were investigated for the presence of R-Msystems. A type I R-M system were amplificated in L.bulgaricus KLDS1.1028and L.bulgaricusKLDS1.1016. The two sequences obtained from the amplified fragments of the L.bulgaricusKLDS1.1028and L.bulgaricus KLDS1.1016hsdR genes showed similarities higher than95%andhsdM genes85%with other hsdR and hsdM Lb. delbrueckii genes reported in GenBank.Conclusions were that the bacteriophage-resistant mutants were isolated. Rates exhibited byall mutants indicated the presence of adsorption interference during phage infection. And theaccessory polysaccharide-peptidoglycan complex was proved to be involved in the phage receptorsites. Phage resistance are not because of the addition of new spacers in CRISPR. They wereinvestigated for the presence of R-M systems.Innovative points:(1) The bacteriophage-resistant mutants of Lactobacillus delbrueckii subsp.bulgaricus KLDSBIM8and Lactobacillus delbrueckii subsp.bulgaricus KLDS BIM02were isolated, which establi-shed the basis for the development of antiphage strains within them.(2) The accessory polysaccharide-peptidoglycan complex was proved to be involved in thephage receptor sites.(3) Phage resistance are not because of the addition of new spacers in CRISPR.(4) A Type I R/M system were amplificated in L.bulgaricus KLDS1.1028and L.bulgaricusKLDS1.1016.