| Food spoilage is a serious threat to food safety which became a safety issues on food industry. Mutton is easy to promoting microbial growth because it rich in protein,fat, water and other substances. Therefore, microbial contamination is considered to be the most important factor to causing mutton corruption. So, the control of microbial contamination has become the main way to ensure the safety of mutton. the low price of synthetic preservatives make it use widely in food preservation previously. But with the quality of life and the demand increase, people have realized that there are defects in artificial preservatives like affecting the food flavor and safety,so natural preservatives as harmless and extensive sources have attracted great attention.In this paper, through the detection and identification of the slaughter environment and cooked products processing workshop of slaughterhouse, three dominant spoilage bacteria were selected to compare the spoilage ability on cooked mutton. In order to inhibit the growth of spoilage bacteria in mutton, we used the strain Lactobacillus crispatus K313 which produces bacteriocin to amplify the gene of bacteriocin Helveticin-M by PCR. The expression vector pCNCH was constructed and transformed into Escherichia coli BL21. The purified bacteriocin Helveticin-M acted on the main spoilage bacteria which in the mutton, then studied the action mode of bacteriocin by observing the surface morphology and internal structure of indicates spoilage bacteria. Finally, the spores were separated by ultrasonic assisted bacteriostatic agent in order to control the growth of spoilage bacteria in mutton. The research contents and results are as follows:1) Microbiological detection was performed in the representative mutton slaughter and processing plant in Suzhou, Jiangsu province, China. This study mainly focused on the processing of cooked products, including the contact surface of cooking room, the air of each processing room, raw meat before and after washing and cooked meat to determine the distribution of contaminating bacteria in cooked products. The contaminated spoilage bacteria were mainly Bacillus, Staphylococcus saprophyticus, Proteobacterium, Chryseobacterium and Microbacterium.Subsequently, three dominant spoilage bacteria Bacillus sp.MI, Staphylococcus saprophyticus M7 and Bacillus sp.M9 were selected to evaluate the putrefaction potential to cooked mutton by detecting the sensory scores, pH value, colonies numbers, nitrogen value of volatile salt values and corruption metabolites yield factors. The results suggested that Bacillus sp.Ml displayed the strongest putrefaction potential than Staphylococcus saprophyticus M7 and Bacillus sp.M9.2) Previous study found that the fermentation broth of L. crispatus K313 had a strong inhibitory effect on S. saprophyticus M7, speculated that L. crispatus K313 may synthesize a bacteriocin to kill S. saprophyticus M7. The sequence analysis showed the sequence of L. crispatus K313 could encode Helveticin-like bacteriocin,Therefore, the conserved region of Helveticin-like was amplified by degenerate primers. Then we used Tail-PCR to amplify the upstream and downstream sequence of the conserved region to obtain the full length of the gene, named Helveticin-M. The expression of Helveticin-M gene was transferred into E.coli BL21 by expression vector pCNCH. The recombinant protein was obtained after 18℃ induced by 1 mmol/L IPTG. After purification of the fusion protein by nickel column, the fusion protein with a single component can be collected by elution of 500 mmol/L imidazole.Electrophoresis showed that its purity was higher.3) We have observed the effects of bacteriocin Helveticin-M to external form and internal structure of S.saprophyticus M7, Staphylococcus aureus J1 and Enterobacter cloacae C5 bacterial by SEM and TEM, observed the influence of Helveticin-M on membrane permeability through the determination of extracellular ATP, UV absorbing substances and confocal scanning electron microscopic. Then the cells death were detected by flow cytometry. Finally, the changes of membrane potential were detected to further explore the mechanism of Helveticin-M. The results showed that bacteriocin increased the permeability of indicator bacteria to cause cell death, the permeability change led to the release of ATP and UV absorption material and the dissipation membbranepotential. The inhibition effect of bacteriocins on indicator bacteria varied a lot, followed the order of S. aureus J1 >S. saprophyticus M7>E.cloacae C5.Based on the observed results of transmission electron microscopy, the reason of the weakness inhibition effect on S.saprophyticus M7 and E.cloacae C5 may be accused o f the thick cell wall of S.saprophyticus M7 wheareas E.cloacae C5 had an extracellula r layer that caused a barrier to enter the cell difficultly.4) Ultrasonic treatment of spores in mutton, the best effect of Bacillus subtilis spores was 17.9 W/g power and 90min during processing. So this condition was selected as the optimal power and time. According to the small inhibitory concentration of antibacterial agents, 0.64 mg/mL phenyl lactic acid and 10 mg/mL poly lysine were added in the case of optimal power and time. The effect of ultrasonic assisted bacteriostatic agent on killing spores was higher than that ultrasonic treatment alone, but it could not kill spores effectively. Therefore, the germination agent was used to treat the spores to become vegetative bodies. The results showed ultrasonic assisted bacteriostatic agent was more effective in killing spores after the addition of the germination agent... |
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