| Shrimp(Litopenaeus vannamei)are one of the most important aquatic products,which has important social and economic value and nutritional value.At the same time,shrimp are also the best place for growth and reproduction of all kinds of spoilage and pathogenic microorganisms.It was a serious challenge to the food safety and public health and safety that the aquatic products are caused by microbial activity.Therefore,we need to develop effective,non-toxic sterilization and preservation technology and the establishment of efficient and accurate microb iological monitoring technology so as to meet the growing global demand for fresh,safe,high quality water product requirements.Acidic electrolyzed water(AEW)sterilization technology is a new type of non thermal sterilization technology developed in recent years.Because of the low price of raw materials,production safety,environmental protection and high efficiency,fast and broad-spectrum sterilization,so it has been widely used in agriculture,animal husbandry,disinfection process equipment,pharmaceutical industry and food industry.In the past period of time,the researchers introduced a nucleic acid dye-propidium monoazide(PMA),which could couple with the DNA before PCR reaction.This dye can penetrate the cell which membrane is not complete,then by the crosslinking reaction with DNA in exposed to visible light,so that the damaged membrane cells DNA could not amplified in subsequent PCR reactions,thereby shielding the dead bacteria in bacterial detection.Therefore,this paper takes shrimp as the research object(1)Explore the effect of PMA on microbial diversity by PMA combined with PCR-DGGE fingerprinting and high throughput sequencing technology to establish the diversity analysis of living bacteria community.(2)The application of live bacteria diversity analysis technique to evaluate the bactericidal effect of different concentrations of AEW.(3)Study on the changes of microbial diversity of shrimp during storage after TW and AEW treatment by using the technique of living bacteria diversity analysis.The effect of AEW treatment on the changes of microbial community diversity during the storage period was analyzed,so as to provide theoretical basis for the preservation of aquatic products during cold storage.The main research contents and results are as follows:1.The technique that pre-PCR PMA treatment coupled with DGGE fingerprinting was successfully used to assess the effects of PMA treatment on microbial community in raw shrimp stored at 4 °C and 25 °C.When samples stored at 25 °C,PMA treatment has little influence on banding patterns in DGGE profiles.During storage at 4 °C,Shannon-Wiener index(H?)and PCoA analysis indicated there were significant differences between bacterial diversity in samples treated with and without PMA.At the level of species,compared with samples without PMA treatment,Vibrio cholera,Exiguobacterium acetylicum,Aeromonas enteropelogenes,Eubacterium multiforme,Vibrio mimicus in PMA-treated samples disappeared during storage.In 4 °C storage conditions,Lactococcus,Exiguobacterium,Kurthia spp.and Enterococcus after PMA treatment were significantly lower relative content by high-throughput sequencing analysis.The results of the analysis at 4 °C storage were verified by high throughput sequencing technique,the results showed that the H? value of the sample after PMA treatment was lower than that of the non PMA treated sample,and the relative contents of Lactococcus,Exiguobacterium,Kurthia and Enterococcusin in PMA treated samples were significantly lower,which were same as to the results of PCR-DGGE.In conclusion,this study provides an effective tool for precisely monitoring the diversity of viable bacteria in food samples,especially at low temperature.2.The application of live bacteria diversity ana lysis technique to evaluate the bactericidal effect of different concentrations of AEW.Shrimp were treated 2min and 5min by TW and AEW(0.05%NaCl,0.15%NaCl,0.30%NaCl),respectively.UPGMA cluster analysis showed that when PMA was treated with high conce ntration of AEW(0.15%NaCl,0.30%NaCl),the distance between PMA and non PMA treated samples was low and the similarity was low,which indicated that the diversity of microbial community changed greatly after the PMA treatment.Furthermore,when the concentration of 0.15% AEW was treated with 5 min has the best bactericidal effect.Under this condition,the H? value and S value of PMA treated samples were decreased by 44% and 66%,respectively,compared with those of TW treated samples,which were decreased by 37% and 38%,respectively,compared with those of non-PMA treated samples.The above results indicated that the diversity analysis of viable bacteria could accurately reflect the germicidal efficacy of AEW and the actual situation of the residual microorganisms in the samples.3.Study on the changes of microbial diversity of shrimp during storage after TW and AEW treatment by using the technique of living bacteria diversity analysis.After the sample was treated with PMA,the H? value based on DGGE and high throughput sequencing was significantly lower than that of untreated samples.And heatmap shows that in PMA treated samples 11 genera on behalf of the Heatmap color in bacteria by white or red into white or blue light,that reduce the content of these spp.after PMA treatment.Furthermore,the DGGE spectra showed that the brightness of AEW treated samples was lower than that of TW treated samples in the same storage days,and the H? value of AEW treated samples was lower than that of TW treated samples.According to PCoA analysis,TW treatment did not significantly change the microbial species during storage.However,the sample of the AEW treatment group was far away from each other,and the relative dispersion of the samples showed that there were significant differences in the microbial species of the samples at different storage time.Although Fusobacteria was disappear in TW treatment group and AEW treatment group at the end of storage,the Fusobacteria have disappeared 4t h d of storage in AEW treatment group,but it disappeared in the TW group until 14 t h d of storage.AEW treatment could effectively inhibit the proliferation of Aeromonas sp.and Psychrobacter during storage time.However,the contents of these two kinds of bacteria were higher in TW treated samples or increased with storage time.We hope that through this observation of the true microbial change process to further confirm the bactericidal effect of AEW and the microbial community changes during storage timeafter AEW treatment,and to provide theoretical basis for the preservation of aquatic products and the prevention and control of pathogenic microorganisms.This study can provide technical support and theoretical basis for the further application of viable bacteria detection technology,and can promote the analysis of the diversity of living bacteria in aquatic products,monitor the changes of the real microbial community in the storage process,and clarify the bactericidal effect of AEW,achieveing the purpose of ensuring the quality and safety of food products and improving public health. |
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