| Pseudomonas fluorescens belongs to the genus Pseudomonas,which is aerobic or facultative anaerobic gram-negative bacteria.P.fluorescens is often related to the deterioration of many foods.It is a common contaminating bacterium in milk and its products.As an important preservation method in the food industry,freezing can reduce the biological activity of microorganisms to a certain extent and extend the shelf life.However,once the cold chain is interrupted or the temperature is out of control,the remaining bacteria will quickly restore their growth,reproduction and pathogenic ability.So,sustained low temperature cannot reduce the pathogenicity of microorganisms.It can still cause viral infection to eat frozen food contaminated with sublethal bacteria,and the risk of disease may even increase,which brings great harm to food safety.Therefore,this article studied the changes in pathogenicity and spoilage potential of P.fluorescens and its mechanism during the temperature recovery process after freezing stress.In this study,a one-step numerical analysis method was used to integrate the growth and survival of P.fluorescens in fish juice broth under both dynamic and static temperature conditions.Then a growth predictive model of P.fluorescens in fish juice broth at different temperatures was constructed.The estimated values of related parameters a,Y max and T min are 1.386×10-3,8.431 Log CFU/m L and-4.68?respectively.According to the residual error(?)value obtained,the model constructed in this study can accurately predict the growth of P.fluorescence in contaminated fish juice broth during processing,storage and retail.Secondly,this article studied the effect of freezing stress on the pathogenicity of P.fluorescens.By drawing the growth curve,it is found that the growth ability of P.fluorescens will be significantly enhanced after 2 or 4 hours of recovery at the appropriate temperature.In addition,the results of crystal violet adhesion experiments showed that P.fluorescens had an enhanced ability to form biofilms after 3,6,and 9days of freezing stress.The biofilm formation ability of P.fluorescens at the appropriate temperature was restored for 3-5 hours.The results of agarose gel electrophoresis showed that the secretion of P.fluorescens eDNA increased by 76.4%and 57.8%after freezing stress for 6 and 9 days,respectively.Dot Blot results showed that PIA synthesis increased by 14%to 17%after freezing stress for 6 and 9 days.By studying the hemolytic effect of P.fluorescens on rabbit blood,it was found that the hemolytic activity of P.fluorescens was enhanced during the recovery process at the appropriate temperature after a certain number of days of freezing stress.The survival of frozen P.fluorescens during the temperature recovery process was investigated by researching in vitro gastrointestinal simulation methods.The results showed that the number of surviving colonies of frozen P.fluorescens increased after passing through the gastrointestinal tract.The adaptability of the gastrointestinal tract is enhanced,which may be more pathogenic to the human body.Finally,this article studied the effect of freezing stress on the rot of P.fluorescens and its mechanism.The experimental results of skimmed milk agar medium and semi-solid medium showed that the extracellular protease production,clustering and mobility of P.fluorescens were enhanced after freezing stress.The q RT-PCR method was used to investigate the regulation effect of freezing stress on protein related genes.The results showed that the expression of the gene fur of P.fluorescens increased by1.77,19.16,and 19.16 times the original P.fluorescens after 3,6,and 9 hours after freezing for 3,6,and 9 days,respectively.The expression level of the gene clp P increased by 3.94 and 2.06 times after 6 hours of recovery of the optimal temperature.These results indicate that freezing stress may promote the spoilage ability of P.fluorescens by up-regulating these genes.Finally,the reporter strains CV026 and GC-MS were used to qualitatively and quantitatively detect the changes in the production of QS signal molecules AHLs.It was found that the output of AHLs signal molecules after cold shock was higher than that of those without cold shock.The result of C14-HSL was particularly significant,with an increase of 197.4%,indicating that freezing stimulation can promote the production of P.fluorescens QS signal molecules.The above experimental results showed that P.fluorescens has increased decay and pathogenicity during the process of temperature recovery after freezing stress for a certain number of days.This research laid the foundation for further research on related mechanisms and screening low-temperature antibacterial agents of P.fluorescens by regarding related proteins as targets. |
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