Inhibition Mechanisms Of Map Against Chilled Chicken Spoilage Associated With Pseudomonas Fragi

Meat spoilage has caused great risk in the food supply chain.According to the statistics,the annual meat losses are as high as 263 million tons.Meat is the most perishable food due to its abundant nutrients and moisture content.It is a natural medium for the growth of microorganisms.Meanwhile,the process of meat slaughtering and processing is complexity and the microbial contamination caused by many external substances can result in serious cross-contamination.Controlling microbial contamination and meat spoilage have become a worldwide challenge nowadays.The use of modified atmosphere packaging(MAP,CO2/N230%/70%)has been proved to effectively inhibit the growth of microorganisms in chilled meat.On the base of considering the cost and effect,the application of modified atmosphere packaging is a general trend in the technological extension and industrial development of chilled meat.Understanding the detailed mechanism of the spoilage inhibition effect is essential for food administrators to develop effective strategies in meat preservation.Thus,this study isolated and identified the dominant spoilage bacteria,Pseudomonas fragi,from chilled chicken,chicken breast fillets were used as the growth substrate,the physiological and metabolic activity changes,gene expression patterns of the bacteria under air and modified atmosphere packaging were investigated.Moreover,we also use molecular biology techniques to study the function of the specific differentially expressed genes.The results are shown as follows:1.Evaluation of the spoilage potential of bacteria isolated from chilled chickenA total of 53 isolates,belonging to 7 species of 3 genera,were isolated using a selective medium based on the capacity to decompose chicken juice(Raw-chicken Juice Agar,RJA).Three isolates with large decompose zones,namely Aeromonas salmionicida 35,Pseudomonas fiuorescensH5,P.fragi NMC25 and one well-known spoilage bacterium Serratia liquefaciens 17,were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ.The results showed that A.salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins,followed by P.fragi NMC25.The major spoilage isolates in situ was P.fragi NMC25,which exhibited a fast growth rate,slime formation and increased pH and total volatile basic nitrogen(TVBN)on chicken breast fillets.The relative amounts of volatile organic compounds(VOCs)originating from the microorganisms,including alcohols,aldehydes,ketones and several sulfur compounds,increased during storage.In sum,this part of study demonstrated that P.fragi NMC25 showed the strongest spoilage potential in four isolates;RJA provides a simple and convenient method to assess spoilage bacteria during quality management.2.Spoilage properties of different P.fragi isolates response to modified atmosphere packagingAccording to the results of the previous chapter,7 different P.fragi isolates from the spoiled chicken were selected to investigate the effect of MAP on the physicochemical properties compared to the corresponding controls(air)under 28? in TSB.The results showed that:different isolates have different decompose zone areas in RJA,but none of them showed obvious decompose zone in MAP.MAP inhibited the growth of the isolates primarily in the exponential phase,with the prolonging of incubation periods,the inhibition effect disappeared in the strains with decompose zones.Isolates 2 and NMC25,which readily form biofilms,exhibited improved stationary phase growth in MAP compared to the control packaging.MAP weakened the isolate's ability to form biofilms.Changes in the surface properties,including reduced motility,increased aggregation and hydrophobicity were also induced by MAP.More cell aggregates were observed and complex 3D structures were formed via scanning electron microscopy.Significant differences were observed in cell phenotypic characterization between individual isolates tested,which indicated that the responses to MAP exposure were strain-dependent.These cell responses allowed P.fragi to adapt to MAP.MAP can inhibit the capacity to decompose chicken juice of all the P.fragi isolates,indicated that MAP may affect the metabolic activity of P.fragi,which will help further understanding the bacteriostatic mechanism of MAP.3.Cell metabolism changes of P.fragi under MAPThe isolate with strong spoilage potential,NMC25,was selected for preliminary investigate the mechanism by which MAP inllibits P fragi spoilage.The cell physiological changes were determined by measuring the characteristics of extracellular polymeric substances(EPS),membrane integrity,membrane potential,ATP level,and extracellular proteolytic activity.The results showed that:MAP inhibited the entire growth process of P.fragi,modified the amount,components,and surface properties of EPS.The samples stored under MAP retained cell membrane integrity,but loss significant transmembrane transport and ATP synthesis activity.The peptides issued from 2 structural proteins(myosin and actin)were mainly identified in air samples,indicating that these fragments result from bacterial proteolytic activity while MAP inhibited this activity.Overall,this part of study found that cell metabolism is actually influenced by MAP,nutrients transmembrane transport,intracellular ATP level,and extracellular protease activity may play important roles in the spoilage process of P.fragi.4.Gene expression changes of P.fragi under MAPThe complete genome information of the first meat-born P.fragi was obtained through whole-genome sequencing.Then the effects of MAP on P.fragi global gene expression patterns were determined to investigate the mechanism by which MAP inhibits P fragi spoilage.The results showed that:the gene expression patterns in different atmosphere showed great differences,a total of 559 differentially expressed genes(DEGs)were found by RNA-seq.MAP decreases the expression of genes involved in the electron transport chain(nuoAB),resulting in an inhibition of aerobic respiration.Meanwhile,MAP induced the downregulation of genes responsible for ATP-binding cassette transporters,flagellar and type I fimbrial proteins,and DNA replication and repair,which may further influence nutrient uptake,motility,and growth.In addition,NMC25 cells modified their pathways for energy production,amino acid synthesis,membrane lipid composition and other metabolic patterns to adapt to MAP.These data show that P.fragi regulates its gene expression patterns to survival under MAP,but reduces part of its metabolism related to its spoilage ability.5.Functional verification of spoilage related genes of P.fragiThe functions of two DEGs were determined by constructing P.fragi AaprD mutant and AnuoB mutant in this part of study.The effects on meat spoilage of these two mutants were also evaluated.The result showed that:compared the wild type(WT)strain,the ?aprD mutant strain has the lowest growth rate and maximum number in TSB followed by ?nuoB mutant,but no significant difference was observed between the growth conditions of ?aprD mutant and WT strain in situ.In RJA assay,?aprD mutant did not form a decomposition zone,indicating the aprD gene in P.fragi was crucial in protease secretion.However,the AnuoB mutant displayed a normal decomposition zone which was similar to the WT strain,contradicting the hypothesized effects.The swimming motility of AaprD mutant also showed an adverse tendency with the ?nuoB mutant.Moreover,there was no significant difference in the sensory changes associated with meat spoilage caused by WT and two mutants.This result indicated that the spoilage potential of P.fragi were coordinately regulated by several functional genes,the genes of aprD and nuoB played important roles in the meat spoilage process.