摘要
本课题以资源丰富、开发潜力巨大的鱼糜制品(梅鱼鱼丸)为研究对象,分析了其在冷藏条件下的菌相变化,分离鉴定了其优势微生物,评价了茶多酚对鱼糜制品(梅鱼鱼丸)的冷藏保鲜效果,并探讨了茶多酚对细菌的形态、生长特性、细菌膜的通透性、细菌总蛋白以及细菌膜蛋白的影响,从细菌细胞膜的损伤方面阐明茶多酚的抑菌机理。
本论文主要围绕以上几个方面开展工作,获得结果如下：
1.冷藏条件下茶多酚对鱼糜制品保鲜效果以及贮藏过程中生物菌相分析的研究结果表明,在0℃冷藏条件下,与空白对照组相比,添加茶多酚能明显降低梅鱼鱼丸的细菌总数,提高硬度、保持弹性,并使TBA值、TVB-N值、pH值均处于相对较低水平。实验结果表明茶多酚具有较好的抑菌、抗氧化能力,其中茶多酚添加量为0.20-0.30 g kg-1时其保鲜作用最为明显。
2.对照组贮藏初期假单胞菌、微球菌/葡球菌和肠杆菌为梅鱼鱼丸中的优势菌,由于真空包装条件下肠杆菌的生长速度比假单胞菌和微球菌/葡球菌的快,从第9天开始肠杆菌和假单胞菌一起组成梅鱼鱼丸中的优势菌。而添加茶多酚后从贮藏初期直至第17天,肠杆菌始终是优势腐败菌,其数量在第17天和细菌总数相当。
3.鱼糜制品中优势微生物分离鉴定研究结果显示,应用VRBGA和Pseudomonades琼脂培养基分离出8株优势菌株,分别为肠杆菌科中的沙雷氏菌属(液化沙雷菌、气味沙雷菌、液化沙雷菌、液化沙雷菌和粘质沙雷菌)和假单胞菌科中的假单胞菌属(荧光假单胞、门多萨假单胞菌和铜绿假单胞菌)。茶多酚对鱼糜制品中的优势菌有显著抑制作用。
4.茶多酚抑菌机理的研究结果显示,当茶多酚的浓度分别高于0.075%和0.0375%时,对铜绿假单胞菌和粘质沙雷氏菌有显著抑制作用(p<0.05)。对细菌生长曲线的研究发现茶多酚可以改变细菌预测模型的参数,减缓细菌生长速度,但是稳定期的细菌数量没有显著差异。茶多酚处理对细菌细胞形态结构造成明显的损伤,促使细胞膜扩散,不能可见明显的细菌拟核；茶多酚可以导致细菌膜通透性增加,体内小分子物质泄漏；体内ATP酶、AKP酶活性降低。应用SDS-PAGE方法研究发现茶多酚处理对细菌总蛋白的影响不显著,而对细菌膜蛋白的影响显著；进一步应用蛋白质组学方法,借助于双向电泳技术研究了茶多酚处理后细菌膜蛋白的差异蛋白,并应用MALDI-TOF-MS鉴定了差异蛋白点,发现主要的差异蛋白为细菌膜上的酶和伴侣蛋白,包括能量代谢相关蛋白,蛋白质、脂肪酸合成相关蛋白、伴侣蛋白和其他酶等。
Surimi products (Collichthys Fish Ball) having tremendous developing potentiality and most abundant resource was aimed in the study. Microbial profiles were analyzed, and the dominant bacteria species were isolated and identificated in storage time at 0℃. The effects of tea polyphenols (TP) on microbiological and biochemical quality of collichthys fish ball were studied. The effects of TP on the characters, membrane permeabilization, proteomics of membrane protein of bacteria were discussed. And a possible mechanism of the TP antibacterial ability was discussed through bacterial, membrane damage,
The main results obtained in this study were as follows:
1. TP served as the final bio-preservatives for fish surimi products (Collichthys Fish Ball). It was showed that adding TP could extend the shelf life of fish-ball, compared with the control group. Adding tea polyphenols could improve the texture of Collichthys fish ball, including increasing hardness and maintaining springiness. The freshness also could be kept at a good level, indicated by the relative low TBA, TVB-N and pH values. The 0.25 g kg﹣1 tea polyphenols addition in Collichthys fish ball VP kept good characteristics for a longer shelf life when stored under 0℃.
2 In the sample with addition of TP, the population of Enterobacteriacea was the dominant bacteria during the refrigerated storage period. The count levels of Pseudomonadaceae and Micrococcaceae increased, while the other members of the microbial association remained at lower levels during the entire storage period. For the control sample, the initial population of Pseudomonads was the dominant bacteria followed by Micrococcaceae and Enterobacteriacea. Enterobacteriacea increased more rapidly than Pseudomonads in the first 5 days, and became the dominant bacteria species in the control sample.
3. Five strains of typical single-colony bacteria isolated from VRBGA agar were Serratia liquefaciens, Serratia odorifera, Serratia liquefaciens, Serratia liquefaciens, and Serratia marcescen, belonging to the Serratia family. Three strains of typical single-colony bacteria isolated from Pseudomonads agar were Pseudomonas fluorescens, Pseudomonas mendocina, and Pseudomonas aeruginosa, belonging to the Pseudomonas family.
4. Research of growth characters of Pseudomonas aeruginosa and Serratia marcescens showed that the bacteriostasis circle formed of P. aeruginosa and S. marcescens were significant (p<0.05), when TP concentration was beyond 0.075% and 0.0375%, respectively. TP could change the bacterial prediction model parameters, and slowed bacterial growth rate, whereas the number of bacteria in stable phrase had no significant difference. Morphology changes of bacteria treated with TP were investigated by transmission electron microscopy, indicating that the primary inhibition action of TP was to damage bacterial cell membranes, and cause cell membrane diffusion, and inaddition, the nucleoid of the bacteria were not see clearly. TP also increased the permeability of the membranes of bacteria and disrupted the cell membrane with the release of small cellular molecules. Moreover, TP could make the ATP and AKP enzyme activity decreased. The effect of TP on the total bacterial proteins was not significant, but the change of the bacterial membrane protein was significant. Furthermore, a proteomics approach based on two-dimensional gel electrophoresis and MALDI-TOF/TOF MS analysis were used to study the differences in the membrane proteins of bacteria between the TP treatment and control samples. The results showed that the differential expressed proteins identified by MALDI-TOF/TOF MS were found to be enzymes, which might induce the metabolic disorder of the bacteria and resulted in the death of the bacteria.
引文
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