Isolation And Identification Of Two Lactobacillus Phages And Screening Of Phage-resisted Strains

Lactobacilli are important strains used in feed, meat, vegetables and dairy fermentation. During the fermentation, it is mainly responsible for the rapid accumulation of lactic acid, the producing of flavor substances as well as improving texture. However, infection of Lactobacillus phages would result in delaying of fermentation process or completely terminated, thereby making the product quality reduced. To control Lactobacillus Phages effectively, lactobacilli was choosed as host bacteria, phages were isolated from kimchi, its characteristics were studied and phage-resisted Strains were screened. Main research contents and results are as follows:In this study, double-layer agar plate was used to isolate and purificate phage and two virulent Lactobacillus phages have been isolated: one strong-lysis L.brevis ATCC 367 phage named as Lb and one strong-lysis L.plantarum WCFS1 phage named as Lpla, and studied their major characteristics. Electron microscopy revealed that the phage Lb was composed of polyhedral head and contractile tail, the phage Lpla was composed of polyhedral head and non-contractile tail. According to their morphology, Lb was classified as Myoviridae Branch, A1 Class; Lpla was classified as Siphoviridae Branch, B1 Class. The genome size of Lb was about 40 kb with cohesive ends, so it belonged to the cos-type; The genome size of Lpla was about 53 kb with non-cohesive ends, so it was packaged in a headful mechanism, belonged to the pac-type. One-step growth curve of phages showed the latency and burst period of Lb was 165 min and 105 min, respectively, and the burst size was 62 PFU/cell; the latency and burst period of Lpla was 15 min and 180 min, respectively, and the burst size was 43 PFU/cell. The presence of Ca2+ and/or Mg2+ was indispensable to gain complete lysis and improve plague formation. The detection results of phage stability showed that the phages Lb and Lpla maintained their viability within the range of p H3-12; they had a lower tolerance to ethanol and isopropanol, less than a live phage could be detected when Lb and Lpla treated with 35% and 25% ethanol(isopropanol) for 40min; a lower tolerance to UV, 98% Lpla and 77% Lb were inactivated when Lb and Lpla treated with UV for 20min; it also showed a lower tolerance to thermal, the phage Lb can be inactivated when treated at 56℃ for 10 min, the phage Lpla can be inactivated when treated at 56℃ for 2 min. The detection results of influence of physicochemical agents on phage adsorption showed that the highest and lowest adsorption rates of Lb were observed at 32℃(96.75%) and at 50℃(83%), respectively; the highest and lowest adsorption rates of Lpla were observed at 4℃(98.3%) and at 45℃(1.5%), respectively; the adsorption of phages to the host strain could be improved with addition of Ca2+ and/or Mg2+; phages Lb and Lpla could absorb to thermally killed cells, but the adsorption rate decreased by 50% and 20%, respectively. A partial sequence of the phage Lb showed 71% similarity to phage Lv-1; partial sequence of the phage Lpla showed 74% similarity to Lactobacillus plantarum bacteriophage LP65. Phenotypic and genotypic characteristics of phages Lb and Lpla showed that they are novel Lactobacillus phages.The results of research on storage methods of phages showed that stabilizers glycerol and dimethyl sulfoxide have similar effects. Storage for a short time(within two months), no significant difference between different storage temperatures; Storage for a long time, the best storage temperature was-80 ℃ and secondly-40 ℃. Similar effect was observed between filter-sterilized phages and un-filter sterilized phage. Therefore, phages suspensions storage with glycerol or DMSO at-80℃ or-40℃ are most simple and efficient method for laboratory preservation. The results of phage receptor analysis showed that receptors of Lb and Lpla were sensitive to heat, the receptor of Lb was related with peptidoglycan, rhamnose and galactose; the receptor of Lpla was related with cell wall polymers coupled with peptidoglycan and a variety of sugars.To control Lactobacillus phages effectively, phage-resisted strains against Lb and Lpla were obtained by secondary infection methods, including eight nonlysogenic strains had stable resistance to phage Lb; six nonlysogenic strains had stable resistance to phage Lpla. Resistance was hereditary stability. The resistance mechanisms are concerned with interference adsorption, and there are mutations on Lb-resistant bacteria.In this study, two novel lactobacilli phages and phage-resistant strains were isolated; the study of their main characteristics provides some data to prevent commercially valuable bacteria from being attacked by phage, which also leads to a better understanding of phage-host interactions.