| Oyster is a kind of bivalve molluscs, which is one of Chinese four major farming shellfish and the world’s Number one farming shellfish with high nutritive value. Oyster’s meat with important edible value and medicinal value contains abundant of nutrients and active ingredients, so it is very promising of the processing development of the oyster’s meat. There are two types of feeding mechanism in oyster, however, with the nonselective feeding process, the microbes which exist in water will be accumulated and enriched into the oyster’s body. The security problems of oyster product are getting more and more attention with the popularity of fresh instant oyster. Now, the exsiting non-thermal sterilization technology of the oyster processing has shortcomings and deficiencies that barricade its large-scale practical application. Photodynamic inactivation has a pronounced application in the medical field, which usually used to kill malignant tumor cells and pathogenic microorganisms in human body. It was reported that photodynamic sterilization was applied in liquid food for the inactivation of Staphylococcus Aureus. However, the application for inactivation of E.coli and norovirus in oysters has not been reported. The main purposes of this study were to detect the E.coli and norovirus in raw oysters on Qingdao market, and further study the inactivation effect of photodynamic non-thermal sterilization on E.coli and norovirus in oyster’s intestine. Finally, the oyster edible safety was evaluated after its photodynamic treatment by curcumin, thus lays the foundation for the further utilization and application of this technology in shellfish processing.This thesis consists of three main parts:1. Detection of the E.coli and norovirus in raw oysters on Qingdao market. E.coli which is the common pathogenic microorganism in oyster, was tested using a rapid detection method of 3M test paper. Then, correlation between the quntities of E.coli and week" average temperatures variance was analyzed using Spearman’s rank correlation coefficient caculation. Results showed that the positive rate of E.coli was 5%, detected by the 3M test paper. The detected rate was low, but the level was 700 cfu/g, which was much higher than the directive requirement of the Ec council. There is a positive correlation relationship (p=0.639, P=0.001) and (p=0.317, P=0.131) between the level of Coliforms/E.coli and week’average temperatures according to the spearman rank correlation analysis, respectively. Therefore, this article suggests high attention should be paid to control the storage, transport, and quality of the oyster products.Relative abundances of type of GI and G11 norovirus were tested using real-time quantitative PCR. Due to the high sensitivity of this assay, it is easy to get false positive results. Our cooperative laboratory in University of Ghent that established a sequence alignment method to exclude false positive norovirus results after Q-PCR detection. First of all, this study was carried out on 48 samples of fresh oysters by Q-PCR detection, and then randomly selected 8 samples from the 20 GⅡ norovirus positive samples to identify true or false. The results showed that in the 8 samples,2 samples were true positive,4 were false positive, and the remaining 2 may be norovirus variants. Thus, the sequence alignment method was feasible to exclude false positive results, and worthy of further promotion. It also hints that experimental operation process should be very careful to avoid man-made pollution.2. Research of the inactivation effect of photodynamic non-thermal sterilization on E.coli and norovirus in raw oyster’s intestine. Firstly, the cultured DH5a and DH5a which was bio-accumulated in the gut of oyster were inactivated by curcumin-mediated photodynamic non-thermal sterilization respectively. After that, the inactivation efficiency of DH5a was evaluated regarding membrane proteins, membrane permeability and the integrity of nucleic acid using membrane protein extraction and SDS-PAGE electrophoresis, bacterial membrane permeability test, nucleic acid extraction and electrophoresis method. The results showed that the viability of the cultured DH5a could be reduced 2-3 log (CFU/mL), and the viability of DH5a which was bio-accumulated in the gut of oyster could be reduced 1-2 log (CFU/mL). The mechanism of photodynamic damage to DH5a firstly destroyed the senior structure of membrane protein leading to the increase of membrane permeability, and then the inorganic salt substances divulged from the bacteria inside, at the same time, nucleic acids were destroyed, causing the death of bacteria.Both cultured MNV-1 and bio-accumulated MNV-1 in the gut of oyster were inactivated by curcumin-mediated photodynamic non-thermal sterilization. After that, changes of the ultrastructure, integrity of RNA and the stability of viral capsid proteins of MNV-1 in oysters were detected using transmission electron microscopy, RT-PCR and enzyme treatment-RT-PCR methods respectively. Results showed that, the virus titer of the cultured MNV-1 could be reduced 2-3 log (PFU/mL), and the virus titer of MNV-1 which was bio-accumulated in the gut of oyster could be reduced 1-2 log (PFU/mL). The mechanism of photodynamic damage to MNV-1 could destroy the ultrastructural morphology, the integrity of the viral nucleic acid and the stability of viral capsid proteins.3. Preliminary evaluation of the edible security of raw oysters after photodynamic non-thermal sterilization. We preliminarily evaluated the changes of curcumin and oysters which enriched curcumin after photodynamic treatment on L929 cell morphology, toxicity and apoptosis based on cell morphology observation, MTT experiment, DNA fragmentation analysis and Hoechst staining assays. The results showed photodynamic treated oyster enriched curcumin did not affect the cell morphology, and had no cytotoxicity, with the toxicity grade "0" or "1". It did not cause apoptosis. This preliminarily confirmed the edible security of the oyster after photodynamic treatment, but it also remained to be verified in vivo. These results will provide the theory basis for popularization and application of curcumin-mediated photodynamic non-thermal sterilization technology in the oyster’s pathogenic microorganism inactivation.In summary, there is a positive correlation between the E.coli level and week average temperature changes. Sequence alignment method is worthy of promotion to eliminate the false positive results. Curcumin mediated photodynamic non-thermal sterilization could achieve a good inactivated effect on E.coli and norovirus in oysters’ intestine inside a short time of 1 min, and the optimal curcumin treatment concentration is 10μM Moreover, it was preliminarily confirmed the edible security of the oyster based on scarce cell toxicology after photodynamic treatment. After optimizing the light source and photosensitizer, the inactivation will achieve a better effect in raw oysters’intestine, which would make the non-thermal sterilization more plausible and more advantageous than any other microorganism inactivated technologies. |
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