Study On Microbial Diversity And Molecular Detection Technology For Dominant Spoilage Bacteria In Refrigerated Pork

At present,the problem of food spoilage caused by microorganisms has became a major concern in the food industry.Pork is the most commonly consumed meat in China.It provides abundant substrates for the growth of microorganisms owing to its rich nutritional components.Pork is inevitably contaminated by microorganisms when the pork is produced,processed,transported and so on.So,the pork is highly easy to spoilage.The spoilage of pork results in issues with economic losses and even meat safety problems.The objective of this study was to analyze the freshness change of the refrigerated pork with storage time.During the storage time,the microorganism diversity and dominant spoilage bacteria of the refrigerated pork were analyzed based on the high-throughput sequencing technology,and the dominant microorganisms were confirmed.Meanwhile,the fluorescent quantitative PCR（qPCR）and real-time fluorescence loop-mediated isothermal amplification techniques（LAMP）were established to detect the dominant spoilage bacteria at genus level.In this study,the total viable count（TVC）,sensory scores,pH,total volatile basic nitrogen（TVB-N）and microbial diversity of refrigerated pork in supermarkets were investigated.A total of 259 bacterial genera belonging to 21 phyla were identified based on V3-V4 region of the 16S rDNA using high-throughput sequencing analysis.The bacterial community composition and bacterial diversity changed significantly along with the time passage.After 5 days,Pseudomonas,Acinetobacter and Photobacterium were dominant in refrigerated pork,especially Photobacterium,which was rarely reported to be associated with meat spoilage in previous studies.The results suggested that the three taxa contributed to refrigerated pork spoilage.The changes of pH and TVB-N showed similar trends during storage.In addition,TVC increased steadily and sensory score of refrigerated pork decreased.The changes of TVC,pH,TVB-N and sensory scores indicated that the refrigerated pork had not been fresh meat on day 5.The new genus-specific sucD gene of Pseudomonas spp.,which was dominated spoilage bacteria,was screened by comparative genomic analysis.The gene of sucD is a coding gene of succinyl coenzyme A synthease alpha-subunit.Specific primers were designed for the sucD gene.Then the specificity of primers was verified,and the annealing temperature of quantitative real-time PCR was optimized.A standard curve of qPCR was established using genomic DNA of pure bacterial cultures as standards.The results showed that the DNA fragment of sucD gene was successfully amplified in 12 Pseudomonas spp.strains and no amplified product was observed in 22 non-Pseudomonas spp.strains,suggesting the primers of qPCR were specific.The optimized annealing temperature of qPCR was 60°C.The standard curve of y=-3.2034x+40.3640（R²=0.9956）was generated for qPCR,and the amplification efficiency was 105%.The sensitivity for Pseudomonas was 8.1×10² CFU/mL in pure cultures.The detection limit of qPCR in artificially contaminated samples was 7.8×10³CFU/g.In addition,a good correlation coefficient（0.9980）was observed between the qPCR detection results and the plate count method in artificially contaminated samples.Based on the 16S rDNA sequence of Pseudomonas,the primers of the real-time fluorescence LAMP were designed.The saturated nucleic acid dye Eva Green was added to the reaction system.The real-time fluorescence LAMP was established and optimized based on the real-time fluorescence platforms.The specificity of the primers was tested by 12Pseudomonas spp.strains and 23 non-Pseudomonas spp.strains.And the sensitivity difference between real-time fluorescence LAMP and ordinary LAMP was compared.Meanwhile,the detection limit of artificially contaminated pork was also determined.The results showed that the result of amplification was positive in 12 Pseudomonas spp.strains and negative in 23 non-Pseudomonas spp.,indicating that the primers of LAMP were specific.The results showed that the optimal conditions for LAMP amplification were:6 mmol/L Mg²⁺,1.0 mmol/L dNTPs,0.30 mol/L betaine,F3/B3:LF:FIP/BIP=1:4:8（1=0.2μmol/L）.The annealing temperature was 65°C.The sensitivity of real-time fluorescence LAMP for Pseudomonas was 3.2×10¹ CFU/mL in pure cultures,which was 10-fold more sensitive than the ordinary LAMP.The detection limit of real-time fluorescence LAMP in artificially contaminated samples was 1.7×10³ CFU/g.Pseudomonas in fresh pork could be detected within 2.5 hours by real-time fluorescence LAMP.