Isolation And Identification Of Potent Bacteriophages Of Spoilage In Soybean Products And Their Antibacterial Effects

Soy is rich in nutrients and contains various physiologically active substances such as isoflavones,saponins,alcohols,and phospholipids.Soy products are rich in high-quality soy protein and have become one of the most popular foods.However,most soy products are rich in protein,fat,carbohydrates and water,so they are easy to breed microorganisms,causing acidosis,sticky and foul odor,which greatly shortens the shelf life of soy products and also affects the health of consumers.Therefore,further study on the composition of the spoilage bacterium of soy products,screening safe and efficient bio-preservatives is particularly important to extend the shelf life and sales of soy products.In this study,the bean products(tofu)made by Yangzhou University canteen were used as raw materials,and forced spoilage was carried out by constant temperature at 37 C.High-throughput sequencing and conventional colony separation and identification were used to analyze the composition of spoilage bacteria.Subsequently,a high-efficiency lytic phage was isolated from environmental samples by a double-layer plate method using a dominant spoilage bacteria,Enterobacter cloacae,as a host strain,and the biological characteristics of the phage(cleavage spectrum,the optimal infection number and one-step growth curve),the antibacterial effect and the whole genome information were measured and analyzed,and the inhibition effect of the phage on the spoilage bacteria of soybean products was explored,which laid a theoretical foundation for the development of safe and effective phage biological preservatives.The main research contents of this paper are as follows:(1)Fresh tofu was placed in a 37? incubator at a constant temperature for forced spoilage.From 0 h to 36 h,samples were taken every 2 h for sensory analysis and colony counts.The sensory evaluation score was found dropped to 4.0 when forced spoilage for 14 h,which means the soy products began to corrupt;when forced spoilage for 24 h,the total score of sensory evaluation fell to 2.0,which means the soy products were severely corrupt and inedible;when forced spoilage was 32 h,the total number of colonies reached a maximum of 7.5 log CFU/g;From 0 h to 36 h,colony isolation and 16S rDNA identification were performed every 12 h,and the Enterobacter cloacae was found to be one of the dominant spoilage organisms of soy products.(2)The total number of colonies in soy products reached the maximum due to forced spoilage for 32 h.Therefore,we used high-throughput sequencing technology to sequence the 16S rDNA and analysis the microbial structure of bean samples at 32 h,and found that the most dominant spoilage organism was Moraxellaceae,which accounted for 87.3%of the total number of bacteria;the second one is Enterobacteriaceae,which accounts for 6.5%of the total bacterial population;then Pseudomonadaceae and Bacillaceae,which account for 2.9%and 2.1%of the total bacterial population,respectively.The sequencing and analysis results also confirmed that Enterobacter cloacae is one of the dominant spoilage bacteria in soy products.(3)Using Enterobacter cloacae EcYZU E36 as the host strain,a phage was successfully isolated from the farmer's market and the sewage of major supermarkets and named as Ecp 01.Subsequently,it was confirmed by transmission electron microscopy that the phage belonged to the Myophage family;after biological characteristics analysis,it was found that the phage 01 has a wide range of lysis(can lyse all the 11 Enterobacter cloacae in the test strain),incubation period is short(20 min),and the outbreak The duration(50 min),the inhibition of Enterobacter cloacae and biofilm formation were significant,indicating that Ecp 01 is an ideal potent phage with good application prospects.(4)Whole-genome sequencing and bioinformatics analysis of phage Ecp 01 were analy using second-generation sequencing technology.The results showed that the Ecp 01 genome 39,636 bp in length,of which the coding gene was 28,938 bp in length and the GC content 57.9%.Functional annotation showed that the genome contained 41 ORFs,26 of which had c functions;in addition,the phage genome does not carry any virulence genes and druggenes,which is safe and reliable.