Isolation, Identification, Biological Characteristics And Application Of Broad-spectrum Phages Of Pathogenic Vibrio

Vibrio pathogenic bacteria are a class of pathogenic Vibrio that can be shared by humans and aquatic organisms.Antibiotics or chemical preparations are generally used for the control of Vibrio,but with the emergence of drug-resistant strains,prevention and control become very difficult,and thus,the application of phage reappears in the public's field of vision.This study aims to find broad-spectrum phage with high-efficiency lyticity against a variety of pathogenic Vibrio,and to make a new bio-inhibitor,using these phage to prepare a new bio-inhibitor to control aquaculture and processing Reproduction and contamination of pathogenic Vibrio.(1)Separation and purification of phage from marine fish,freshwater fish and shellfish slaughtered blood samples,and 27 Vibrio phage which can be lysed by species were isolated.The phage morphology of the four strains of Vibrio bacteriophage was short-tailed phage and muscle phage.The phage VppYZU-Yl and bacteriophage OY-1 had the broadest cleavage spectrum;the phage had good stability under the condition of pH 5.0-12.0.Alkali resistant and acid resistant.The phage was subjected to the optimal multiplicity of infection and the one-step growth curve,and the phage with good stability and strong cleavage ability were selected.(2)The genetic information of phage was determined by whole genome sequencing.The genome-wide information of phage OY-1 did not find a homologous phage,and the phage was judged to be a new phage.The full-length genome of phage OY-1 was 43479 bp,and the total length of the coding gene was 38403 bp,the average length was 936.66 bp,accounting for 88.33%of the total length,the GC content was 49.26%,and there were 41 open reading frames(ORFs)..The coding gene is known to include a gene such as a DNA polymerase that regulates nucleic acid metabolism and expression,a structural protein gene such as a major capsid protein,and a gene involved in protease cleavage.No antibiotic resistance genes and virulence genes were found,which ensured the safety of phage.(3)Coating the normal growth curve of Vibrio and the growth curve after phage treatment,and comparing the inhibition effect of phage on Vibrio.The total number of colonies increased with time.The total number of colonies in the phage group was always lower than that in the control group.The maximum difference was between the 8 and 12 h and the control group.Two phage were used for the three different Vibrio and The inhibitory effect of mixed bacteria was better,and the effect of phage VppYZU-Y1 was generally better than that of phage OY-1.(4)Crystal violet 96-well plate method was used to determine the inhibitory effect of two broad-spectrum phage on host biofilm at different concentrations.The high-concentration phage solution was better.The phage VppYZU-Y1 decreased the OD600 nm value by about 90%after treatment with Vibrio parahaemolyticus CICC 21617 biofilm;the two phage and phage mixture at 25? and 37? Both the bacteria and the mixed Vibrio showed significant effects.It is indicated that Vibrio is difficult to form biofilm under the interference of these two phage,and can be effectively removed by phage after biofilm formation.In general,phage VppYZU-Y1 is more resistant to biofilm than phage OY-1.(5)Vibrio contamination has a great impact on product quality and safety in the processing of aquatic products.In particular,the carp family is susceptible to the safety of Vibrio to produce biogenic amines.Therefore,the use of phage combined with lactic acid bacteria to ferment squid improves fish quality.The microbial index,TVBN,TBARS?pH,biogenic amines of fermented fish meat were detected.It was found that lactic acid bacteria can improve the quality of fermented fish.However,when the fish pieces are contaminated by Vibrio,the growth of Vibrio is slow due to the slow growth of lactic acid bacteria in the early stage of fermentation.The speed is fast,so the quality of the product cannot be controlled in time.The isolated phage combined with lactic acid bacteria is applied to the squid contaminated by Vibrio.The results show that the phage has obvious inhibitory effect on Vibrio in the fermented fish system,and the pH value of the experimental group added with phage It was always lower than the control group;the TVBN value and the TBARS value showed that the lactic acid bacteria phage combination group was kept within the limit for a longer period of time than the other groups.The combination of lactic acid bacteria with the characteristics of high-efficiency pyrolysis of phage in the early stage can effectively improve the quality of fermented fish.