| Shrimp(Litopenaeus vannamei) has been favored by consumers in China and other parts of Asia due to its delicious taste, rich nutrition. However, due to its abundant nutrients, it become a best place to gather and live for microbial. Vibrio parahaemolyticus and Salmonella spp. are the two important pathogens in shrimp products, which will be a main hazards factors among food poisoning incidents. In addition, food deterioration caused by microbial activity also bring serious challenge for food quality safety and life property safety, which may hinder relative industries development and produce losses of national economy in a certain degree. In order to solve these potential threats, developing an effective microbial monitoring technology has become the focus work in the food industry. Propidium monoazide(PMA) is a kind of nucleic acid with high affinity for photosensitive reactive dyes. It can selectively permeate through the damaged cell membrane of dead cells and can be covalently cross-linked under exposure to light, which strongly inhibits DNA amplification resulting in selective prevention of DNA amplification from dead cells. Thus, this study aimed to(1) develop a rapid quantification method for detecting viable Salmonella spp. in raw shrimp;(2) develop a high throughput method to simultaneously quantify viable V. parahaemolyticus and Salmonella spp. in shrimp and its application in inactivation model for non-thermal sterilization(Ultrahigh pressure and acid electrolysis of water);(3) develop a viable treatment combined with high-throughput sequencing method to describe real microbial diversity changes for postharvested shrimp during storage.1. PMA is applied to develop a quantification method of viable Salmonella spp. in fresh shrimp In this study, we develop a PMAcombined with real-time PCR method to quantify viable Salmonella spp. in fresh shrimp. This method has high specificity by using 60 strains belonging to 23 species. The optimization of the PMA concentration showed that 100 μM was considered optimal to effectively inhibit 106 CFU/m L dead cells, while only 103 ~ 104 CFU/m L dead cells could be inhibited in previous reports. This assay could detect viable Salmonella spp. at as low as 36 CFU/m L in pure culture and 100 CFU/g in raw shrimp. By comparing with PMA-q PCR, q PCR and plate counting for quantifying Salmonella in samples, this PMA-q PCR was obviously superior to q PCR and had good agreement with plate counting. In conclusion, this effective method can be used as an available tool to quantify viable Salmonella spp. in raw shrimp.2. PMA is applied to develop a high throughput method for viable V. parahaemolyticus and Salmonella spp. in fresh shrimp simultaneously In this work, a real-time multiplex PCR combined with propidium monoazide was developed for quantifying viable V. parahemolyticus and Salmonella spp. simultaneously in raw shrimp. The optimization protocol of PMA treatment showed that 15 min of dark incubation duration and 100 μM of PMA concentration could effectively inhibit 106 CFU/m L of each dead strains. The high specificity was confirmed on assay using 68 target and non-target strains. Besides, the novel method could detect as low as 2 Log10 CFU/g of both strains in raw shrimp. To assess whether this novel method is a suitable approach to inhibit DNA amplification from target pathogens in shrimp upon ultra-high pressure(UHP) and acidic electrolyzed water(AEW) treatment, the inactivation models of pathogens were developed based on the counting by plate counting, PMA combined with multiplex-q PCR and multiplex-q PCR. The results indicated that inactivation rate(D10) from PMAcombied with multiplex q PCR and plate counting were statistically similar, which were obviously superior to multiplex q PCR. In conclusion, this PMA-multiplex-q PCR method is a rapid and effective diagnostic tool for the monitoring of viable V. parahemolyticus and Salmonella spp. in seafood, which also has a potentially usefulness for evaluating the bactericidal effect of non-thermal sterilization.3. The microbial diversity of postharvest shrimp during storage at different temperature was analysed by PMA treatment combined with high throughput sequencing This study analyzes the microbial diversity of postharvest shrimp during storage at different temperatures(4, 7, 15, 30 oC). PMA pretreatment can move dead bacteria of sample to obtain DNA of viable cells, and it was applied to sequence through the high-throughput sequencing technologies(Illumina Hi Seq2000/2500/4000) based on 16 s r DNA. The figures of Community Composition, Principal Component Analysis(PCA), Heatmap and Venn were made based on different analysis. The results showed, compared to sample without PMA pretreatment, sample treated by PMA was different in the microbial community structure including microbial species and species abundance. In addition,the temperature was higher the difference was littler, thus the microbial diversity in refrigerated shrimp can be modified by PMA treatment. In order to explore the reason that why shrimp could corrupt during storage, this study analyzed main microbial species and abundance for each sample. The results indicated that Exiguobacterium、Vibrio、Lactococcus、Aeromonas、Shewanella、Kurthia、Enterococcus were the prominent family. This method of PMA pretreatment combining with high throughput sequencing not only can provide reference for the precise detection of microbiota in cold chain, but also can obtain data base to study the mechanism of deterioration for shrimp during storage.We believe that this research can promote the developing of detecting viable cells based on PMA pretreatment, microbial diversity of aquatic products during storage and the other related researches, it may provide research data in the field of viable cells. |
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