| Chilled beef is one of the most commonly consumed fresh meats in China,besides of delicious,it also has the advantage of high protein and low fat.However,chilled beef is susceptible to microbial contamination during processing,transportation and marketing.The oxidative decomposition of nutrients by cold-loving microorganisms can lead to spoilage of chilled beef.Pseudomonas and Brochothrix thermosphacta being the dominant spoilage bacteria that leading loose tissue structure and odor in beef.Photodynamic inactivation is a nontoxic,low-energy sterilization technique that is effective against both Gram-positive and Gramnegative bacteria.In order to reveal the microbial structure and evolution of chilled beef and the spoilage mechanisms of Pseudomonas and B.thermosphacta,the structural changes of the flora of cold beef from three different marketing routes were analyzed during storage at 4℃.The spoilage ability of the dominant spoilage bacteria,Pseudomonas and B.thermosphacta,were analyzed using comparative genomic assessment,and the spoilage ability of Pseudomonas and B.thermosphacta in minced beef was elucidated based on physicochemical indicators and HPLC-MS/MS metabolomes.The study further evaluated the effect of photodynamic inactivation on the quality improvement of minced beef during cold storage by using photodynamic combination with different packaging.The study provides a new theoretical basis for uncovering the microbial spoilage mechanisms of chilled beef and to develop highquality preservation technologies.The main results are as follows:Culture-independent and culture-dependent 16S rDNA high-throughput sequencing was used to analyze the microbial community structure and its succession patterns in cold beef from three sales routes:grocery market,supermarkets and e-commerce.The results showed that there were significant differences in microbial community species and abundance among the three sales sources of beef.The microbial diversity was higher in the early stage of beef from grocery market,and the abundance of each genus did not very much,while in the middle and late stages it was mainly dominated by Pseudomonas spp,Brochothrix spp and Acinetobacter spp.The microbial diversity of supermarket beef was low and dominated by Acinetobacter spp.,Photobacterium spp.,Pseudomonas spp.and Myroides spp.The microbial diversity of beef from e-commerce was highest and dominated by Pseudomonas spp.,Brochothrix spp.and Serratia spp.in the middle and late stages of storage.There were slightly more species in the culture-independent than in the culture-dependent by high-throughput sequencing,and the main dominant bacteria were basically similar.Pseudomonas spp.,Acinetobacter spp.,Brochothrix spp.,Myroides spp.and Serratia spp.were the dominated genus,while the abundance of some bacterial species changed significantly,especially Myroides spp.and Serratia spp.,which were much more abundant in the dependent culture than in the non-dependent culture.HS-SPMEGC-MS showed that a total of 30 volatile components were detected during beef refrigeration.The content of each component changing with longer storage periods,3-methyl-1-butanol and acetion positively correlated with Pseudomonas spp.and Brochothrix spp.The spoilage capacity of the isolates was evaluated by TVB-N,protease activity and acetion in beef juice at 4℃,and whole genome sequences of strong and weak spoilage strains were compared.Comparative genomics of BT27(strong spoilage strain)and BT25(weak spoilage strain)found that the strength of spoilage ability of the strains was related to the number of genes encoding carbohydrate metabolism.Comparative genomic analysis of Pseudomonas PF83(a strong spoilage strain)and PF57(a weak spoilage strain)showed that the genes encoding both strong and weak strains were mainly involved in amino acid metabolism and transport,and that PF83 encoded a higher number of genes than PF57,and that its spoilage potential was related to protein degradation and amino acid utilization.In view of the greater spoilage capacity of Pseudomonas lundensis compared to Pseudomonas fragi,P.lundensis and B.thermosphacta were used to study the spoilage characteristics of these two spoilage bacteria in mono/co-cultures in ground beef through physicochemical and metabolomic analyses.B.thermosphacta monocultures(BT)produced large amounts of ethylene coupling and organic acids in the beef.Metabolic pathways were screened,and it was found that BT caused beef spoilage mainly through sugar and nucleic acid metabolism.P.lundensis monocultures in(PL)beef caused protein degradation and a large accumulation of soluble peptides and TVB-N,mainly through amino acid metabolism to degrade proteins to produce large amounts of dipeptides and amines leading to spoilage of beef.The growth of P.lundensis in the co-culture(BP)system appeared to be unaffected by both,while the number of B.thermosphacta was slightly lower than in the BT group.In addition to this,the intensity of spoilage physicochemical indicators in the co-culture group was reduced to varying degrees compared to the monoculture.The metabolomic analysis revealed that new metabolites were generated in the co-culture group,with a total of 409 differential metabolites and four significant metabolic pathways screened.In addition,eight key metabolites of creatine,inosine,inosinic acid,uracil,alanine,glutamine,3-methylhistidine and 3-hydroxydecanoic acid were identified as potential biomarkers of spoilage.The effect of curcumin-mediated photodynamic inactivation on beef quality was investigated by analyzing changes in physicochemical parameters during storage.A singlefactor experiment was used to investigate the optimal inactivation conditions.60 μM curcumin concentration,0.5 mg/mL citric acid and 30 min of light were used as photodynamic treatment conditions.The number of microorganisms in the photodynamic treatment group was consistently lower than that in the control group throughout the storage period,and the rate of increase in TCA-soluble peptides,TVB-N and ΔE values was reduced,indicating that the photodynamic treatment inhibited the growth of microorganisms thereby delaying the degradation of beef proteins and achieving a preservation effect on beef.In summary,the microbial community structure of chilled beef and its succession pattern are related to its marketing route.The dominant spoilage bacteria during beef refrigeration at 4℃ including Pseudomonas,Acinetobacter,Photobacterium,Brochothrix,Serratia and Myroides.The differences between the strong and weak spoilage strains of Pseudomonas and B.thermosphacta are related to the number of genes coding for their metabolic functions.B.thermosphacta metabolised carbohydrates to form acetion,while Pseudomonas mainly metabolized amino acids.Both formed more spoilage metabolites in the co-inoculated system and showed competitive effects.Histidine metabolism involved in the spoilage metabolism of the two dominant bacteria.The curcumin-mediated photodynamic inactivation showed good bactericidal effect on the two dominant bacteria,and combined with different packaging methods,it could effectively reduce the growth of Pseudomonas and B.thermosphacta in beef,delay protein degradation and colour change,effectively prevent the beef from spoilage. |
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