| Though the growth of most bacteria in chilled meat tends to be inhibited in lower temperature, some psychrotrophic bacteria still grow well even under the chilling conditions, leading to spoilage of the chilled meat. The composition of the bacterial flora in chilled meat and the development of the spoilage flora during storage have been extensively studied. However many studies were conducted by traditional cultivation methods, which failed to reveal the true micoflora. Recently, the molecular methods based on the 16S rRNA gene have been applied increasingly to study bacterial communities in food samples. The aims of this study were to investigate the changes in the bacterial communities of tray-packaged pork during chilled storage by DGGE and TRFLP, to investigate the changes in the bacterial communities of vacuum-packaged pork during chilled storage by DGGE and isolation and purification of lactic acid bacteria, to determine the predominant spoilage bacteria, and to further quantify these predominant spoilage bacteria using real-time PCR. The overall purpose was to provide a theory basis for understanding the changes of bacteria communities in chilled pork, thus to prevent meat contamination and ensure food safety. The contents and results are as follows:1. Influence of different chilled pork pre-treatment methods on bacteria DNA extraction and PCR-DGGEThe first step of microbiological research with molecular methods is DNA extraction. Different pre-treatment of samples may affect the extraction efficiency of DNA and the results of further PCR or other experiments. In this experiment, the influence of different chilled pork pre-treatment methods on bacteria DNA extraction and PCR-DGGE was investigated. After being stored at 4℃for four days, the chilled pork was pre-treated with four methods including freeze-thawing, ultrasonication, shaking and homogenation, and bacteria DNA extraction, dilution-PCR and DGGE were followed. The results showed that DNA yields were slightly different while there was no significant difference in DGGE profiles of V3 and V6-V8 regions of 16S rRNA gene among the four pre-treatment methods. These results indicated that the four pre-treatment methods could be applied according to actual condition. Sequence analysis of DGGE bands of the V3 region of the 16S rRNA gene showed that the predominant spoilage bacterium was Pseudomonas, others including Acinetobacter, Brochothrix, and Serratia.2. Changes in the bacterial communities of tray-packaged pork during chilled storagePCR-DGGE and TRFLP were used to investigate the changes in the composition of the bacterial population of tray-packaged pork during chilled storage and to determine the predominant spoilage bacteria in the end. Then, theΔΔCT method, one relative quantitative real-time PCR, was applied to evaluate the amount of this bacterium. The results are as follows:The results of selective cultivation showed that Pseudomonas increased rapidly and then became the main predominant bacteria, together with Brochothrix thermosphacta. Dilution-PCR of DNA directly extracted from chilled pork showed that the amount of bacteria in pork samples increased gradually, the result of this semi-quantitive method was consistent with that of culture enumeration. The DGGE of V3 and V6-V8 regions of 16S rRNA gene showed that the compositon of cultured bacteria varied during the whole storage, and the DGGE of DNA extracted from cultrued bacteria was different from that of DNA extracted directly from pork samples.The results of DGGE analysis of the V3 and V6-V8 regions of the 16S rRNA gene of DNA extracted directly from pork samples showed that the initial flora present in the fresh meat was complex, and Pseudomonas were the predominant bacterial species at the end of the monitoring period. Brochothrix thermosphacta became another dominant bacterium in the end obtained form the DGGE analysis of the V3 region, but not of the V6-V8 regions, which indicated that DGGE results may be different targeting different 16S variable regions. DGGE analysis of different variable regions of Pseudomonas clones obtained in this study also indicated that DGGE results may be different targeting different 16S variable regions.The results of PCR-TRFLP analysis of DNA extracted directly from pork samples, combined with the method of clones library building and sequencing, showed that 35 TRFs were observed in the TRFLP profiles, indicating that the composition of bacteria population in chilled pork was complex. The peak of the TRF corresponding to Pseudomonas varied significantly during the whole storage, and the relative peak area continued to increase from 5.7% at day 0 to 83.4% at the end of monitoring, which indicated that Pseudomonas became the predominant bacterial species in the end.The relative quantitative real-time PCR was applied to evaluate the amount of Pseudomonas in meat samples. The difference of standard curve slopes between the total bacterial 16S rRNA gene and Pseudomonas was 0.004 (less than 0.1), which indicated that their amplification efficiency was near and theΔΔCT method was valid for relative quantification of Pseudomonas. The average 2-ΔΔCT values continued to increase rapidly during storage from less than 0.001 at day 0 to 4.438 at the end of monitoring.In conclusion, PCR-DGGE and TRFLP were used to investigate the changes in the composition of the bacterial population of tray-packaged pork during chilled storage and Pseudomonas were observed to be the predominant bacteria in the end. Then, theΔΔCT method, one relative quantitative real-time PCR was applied and the relative amount of Pseudomonas in meat samples increased rapidly. The results obtained from these three methods all conformed that Pseudomonas was the predominant spoilage bacteria.3. Changes in the bacterial communities of vacuum-packaged pork during chilled storagePCR-DGGE was used to analysize the changes of lactic acid bacteria (LAB) isolated and purified from vacuum-packaged pork during chilled storage. Direct meat analysis of PCR-DGGE and Lactobacillus-specific PCR-DGGE of DNA derectly extracted from pork samples were conducted to investigate the changes of the bacterial communities of vacuum-packaged pork and to determine the predominant spoilage bacteria in the end. Then, theΔΔCT method, one relative quantitative real-time PCR, was applied to evaluate the amount of this bacterium. The results are as follows:The results of selective cultivation showed that LAB increased rapidly and the growth of other bacteria was inhibited. The shelflife of vacuum-packaged pork was 14 d stored in 4℃. Eight kinds of LAB were identified from the strains isolated and purified from MRS plates by PCR-DGGE and sequencing of 16S rRNA gene. The composition of the LAB community varied at different stages of storage, and Lb. sakei was the dominant species (71.4%) at the end of storage.The results of DGGE analysis of the V3 region of the 16S rRNA gene of DNA extracted directly from pork samples showed that LAB such as Carnobacterium, Lactobacillus sakei and Lactococcus, became the predominant bacteria at the end of the monitoring. The results of Lactobacillus-specific PCR and DGGE showed that different Lactobacillus-like populations increased gradually and Lactobacillus sakei became the predominant bacteria in the end.The relative quantitative real-time PCR was applied to evaluate the amount of Lactobacillus in meat samples. The difference of standard curve slopes between the total bacterial 16S rRNA gene and Lactobacillus was 0.096 (less than 0.1), which indicated that their amplification efficiency was near and theΔΔCt method was valid for relative quantification of Lactobacillus. The average 2-ΔΔCT values continued to increase rapidly during storage from less than 0.001 at day 0 to 3.696 at the end of monitoring.In conclusion, PCR-DGGE and Real-time PCR were used to investigate the changes in the composition of the bacterial communities of vacuum-packaged pork during chilled storage and the results conformed that Lactobacillus was the predominant spoilage bacteria. |
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