Identification Of Dominant Spoilage Flora And Its Phage Control During Freshwater Fish Refrigeration

Freshwater fish are prone to spoilage during storage due to their rich nutrition,high quality protein content and high moisture content.This is mainly because the growth and reproduction of a large number of spoilage bacteria carried by freshwater fish and the production of metabolic substances accelerate the spoilage rate of fish products.Therefore,this paper collected different kinds of freshwater fish samples,analyzed the bacterial flora of cold-resistant bacteria and bacterial phase changes during low-temperature storage,and studied the growth parameters between different bacteria genera and the corruption characteristics of dominant corrupt bacteria in the corruption process,so as to provide a theoretical basis for the targeted inhibition of corrupt bacteria in the refrigeration process of freshwater fish.The main research methods and results are as follows:(1)Two species of freshwater fish from Yancheng were used as experimental objects to screen the spoilage strains.By morphological observation,it was found that the colony morphology was round,bulgy,shiny on the surface,clear on the edge,and varied in color,which could be roughly divided into milky white,white and yellow.Physiological and biochemical analysis showed that among 302 cold-resistant strains isolated,150 strains were G-bacteria and 152 strains were G+bacteria.The observed thalli were mostly rod-shaped,spherical and club-shaped.The 16S rDNA sequencing was used to identify cold-tolerant strains,the results showed that the main cold-resistant strains were Psychrobacter,Shewanella,Acinetobacter,Aeromonas,Pseudomonas,Flavobacterium and Yeast.The highest proportion of Pseudomonas and Shewanella was found at the end of shelf life,and Rhodotorula graminis in the spoilage yeast group has the main spoilage effect on freshwater fish.(2)The pH value,TVB-N value and growth curve of three dominant strains(Pseudomonas,Shewanella,Rhodotorula graminis)were measured by simulated corruption experiment.The results showed that TVB-N and growth curve increased significantly with the prolongation of refrigeration time,while pH changed little,and protein concentration decreased with the prolongation of refrigeration time.During low temperature storage,the protein concentration of Pseudomonas showed a decreasing trend and TVB-N showed an increasing trend,both of which were larger than the other two strains,while the growth curve showed an opposite trend.According to the sensory evaluation results,the putrefaction parameters and ability of Pseudomonas are significantly larger than those of the other two strains,thus it can be determined that Pseudomonas is the dominant putrefaction bacteria.(3)Ten strains of Pseudomonas were selected as host bacteria and eight strains of Pseudomonas phages were isolated from sewage were named PspYZU01,PspYZU02,PspYZU03,PspYZU04,PspYZU05,PspYZU06,PspYZU07,PspYZU08.The pyrolysis spectrum experiment showed that 4 strains of Pseudomonas spp.could cleave 2 kinds of bacteria.PspYZU05 had the widest pyrolysis spectrum and could cleave 5 kinds of bacteria.Bacteriophages with bright lysing circle and broad lysing spectrum were selected for the determination of optimal infection complex and one-step growth curve,which provided candidate strains for the study of Pseudomonas bacteriophage.Thegenome-wide information of three Pseudomonas phages PspYZU01,PspYZU05 and PspYZU08 was further studied.PspYZU01 is a new bacteriophage by functional analysis of Pseudomonas phage gene.(4)The preservation effect of bacteriophage on silver carp was studied through the determination of pH value,TVB-N value,TAC value and sensory evaluation.Although TVB-N and TAC increased with the increase of storage time in the control and experimental groups,the total colonies of fish tubers added with bacteriophages were lower than those in the control group during the whole process,and the total colonies of fish tubers added with bacteriophages decreased by 2.2 lg(CFU/g)and 2.66 lg(CFU/g)on the 12th day at room temperature for 24 hours,respectively.The total number of colonies in the control group reached 7.5 lg(CFU/g)and 7.4 lg(CFU/g)at 24 hours and 8 days respectively,both exceeding 7 lg CFU/g.The total number of colonies in the experimental group only exceeded 7 lg(CFU/g)after 36 h and 10 d,respectively,which showed that the bacteriophage played a significant bacteriostatic role(P<0.05).