The Regulatory Mechanism Of AprD Of Pseudomonas Fragi In Chilled Chicken Spoilage

The consumption of chilled chicken has gradually increased worldwide due to its high nutritional value,relatively low fat content,and distinct flavor characteristics.Although chilled chicken is always produced and marketed under low-temperature conditions,the growth,metabolism,and spoilage effect of psychrotrophic bacteria that cause the spoilage of chilled chicken cannot be inhibited.Pseudomonas fragi is a Gramnegative aerobic psychrotrophic microorganisms and is frequently isolated from fresh and spoiled chilled meat.P.fragi plays a key role in inducing microbial spoilage of chilled chicken due to the fast growth rate and the secretion of extracellular proteases.Apr D is a well-established regulator controlling protease secretion in Pseudomonas spp.However,it is not clear how aprD affects the spoilage potential of P.fragi in chilled chicken.Therefore,P.fragi NMC25 and its aprD-mutant strain were used in the present study.The regulatory mechanism of aprD gene of P.fragi in the spoilage process of chilled chicken was investigated by determining the biological properties,assessing the in situ spoilage potential,identifying the characteristic metabolites and analyzing the effects on the structure and function of the microbial community of chilled meat.1.The Effects of aprD on the biological properties and in situ spoilage potential of P.fragi.The biochemical tests showed that the swimming motility and biofilm formation of ΔaprD were significantly different(P<0.05)from those of the wild-type strains,but the growth condition and swarming motility did not vary significantly.By comparing the architecture and composition of biofilms between NMC25 and ΔaprD,it was found that the spatial structure and extracellular matrix of ΔaprD biofilms on chilled meat changed significantly(P<0.05),but there was insignificant difference in the total viable counts between the NMC25 and ΔaprD biofilms.The quality characteristics tests indicated no significant differences in total viable counts of chicken samples between the groups.However,the total volatile basic nitrogen production of the mutant group was significantly lower(P<0.05)than that of the NMC25 group,and the odor and slime were slightly weaker after 4 days of storage at 8°C,while the p H value of the mutant group decreased significantly(P<0.05)from the 5th day.These results indicated that aprD has an important effect on the spoilage potential of P.fragi.2.The molecular mechanism of the aprD gene in regulating spoilage potential of P.fragi.The ultra-high performance liquid chromatography coupled with mass spectrometry was employed to investigate aprD-mediated alterations in the metabolic levels.The results revealed that the intracellular and extracellular metabolic profiles of the mutants had changed markedly.In the intracellular analysis,total of 80 differentially expressed metabolites were screened,including nicotinamide,and L-phenylalanine,etc.In the extracellular analysis,total of 92 discriminant metabolites were detected,including D-pipecolinic acid,and DL-tryptophan,etc.KEGG analysis illustrated that the differential metabolites were mainly related to β-alanine metabolism,histidine metabolism,and fatty acid elongation.By systematically analyzing all metabolic variations,it is found that the absence of aprD stimulated significant variations in a variety of metabolic pathways,especially amino acid and polypeptide metabolism.3.The effects of aprD of P.fragi on the microbial community structure and function of chilled chicken.The changes in biological properties between cocultures were compared,such as growth,swimming motility,and biofilm formation.The results revealed that the density of ΔaprD in the cocultures was lower than the inoculation ratio,and ΔaprD,1:1 and 1:100 cultures showed the strongest swimming motility,and the biofilm formation of the cocultured strains increased significantly(P<0.05),which further demonstrated that P.fragi community could regulate the ratio of each strain,swimming motility,and biofilm formation to facilitate its survival.16 S r RNA-based amplicon sequencing,in situ microbiological,physicochemical and sensory analyses were employed to show that the microbial diversity of samples contaminated with 1:1coculture was significantly lower(P<0.05)than that of NMC25 monoculture on day 3,and the inoculation of 1:1 and 100:1 coculture resulted in a certain extent of change in the spoilage characteristics of chicken samples under refrigerated conditions.However,the microbial community structure and spoilage characteristics of samples from each group were not significantly different at the 5th day of storage,suggesting that the aprD gene of P.fragi did not have an influence on the structure and function of total microbial communities at the end of microbial community development in chilled chicken.In conclusion,aprD gene could regulate the spoilage potential of P.fragi by affecting its physiological characteristics and metabolic pathways.However,the effect of aprD on community structure and function was limited when P.fragi was present in the microbial community of chilled chicken.