【作者】 扈麟；

【导师】 李宗军；

【作者基本信息】 湖南农业大学 ， 营养与食品卫生学， 2010， 硕士

【摘要】 我国是猪肉的生产消费大国,其最主要的消费形式是生鲜肉。冷却猪肉以其营养、安全、卫生等特点,已成为我国乃至世界生肉销售的主要发展方向。虽然冷却猪肉从生产到销售始终处于0-4℃的低温环境,但仍旧不能完全抑制嗜冷微生物的繁殖,从而导致其货架期不长,这严重制约了我国冷却猪肉生产和国内外销售市场的发展。冷却猪肉腐败是特定腐败微生物共同作用的结果。假单胞菌是一种革兰氏阴性菌,其是导致高蛋白食品腐败的嗜冷性微生物,它在冷却猪肉腐败中起到至关重要的作用。近几年对食品腐败的研究发现,引起食品腐败的微生物主要是革兰氏阴性菌,且在腐败食品中都检测到了酰基高丝氨酸内酯(AHLs),因此,本研究利用根瘤农杆菌(Agrobacterium tumefaciens)A136(pCF218/pCF372)报告平板筛选的方法,从腐败的冷却猪肉中筛选出产AHLs信号分子的假单胞菌,并提取其产生的AHLs,模拟导致冷却肉腐败的不同微生物群系,探讨肉源假单胞菌及其群体感应干预猪肉蛋白降解的情况,为进一步明确群体感应在肉制品腐败中的作用,以及基于干扰腐败细菌群体感应为靶点的冷却肉防腐保鲜策略提供研究基础。试验得到主要结论如下：1、首先采用选择性培养基对托盘包装冷却猪肉的初始菌相进行了分析,并研究了在4℃储藏温度下托盘包装冷却猪肉微生物的相互作用和消长变化规律。结果表明：假单胞菌和乳酸菌是构成有氧条件下冷却猪肉初始菌相的主要微生物。在4℃储藏温度下,细菌总数都在增长；随着贮藏时间的延长,假单胞菌增长速度最快,菌数增长最多,肠杆菌和葡萄球菌也有不同程度的增长,在试验末期也达到了较高的对数值,而乳酸菌增长速度最慢,其比例不断下降,在试验末期,已失去优势菌的地位。假单胞菌与乳酸菌呈负相关关系而与肠杆菌和葡萄球菌呈正相关关系。2、采用假单胞菌分离培养基,丛低温贮藏冷却猪肉和腐败的冷却猪肉肉样中分离到约66株肉源假单胞菌,再采用根瘤农杆菌(Agrobacterium tumefaciens)A136(pCF218/pCF372)的报告平板对其进行复筛得到15株产AHLs的假单胞菌,并采用16S rRNA分子生物学鉴定结合细菌的常规形态学和生理生化方法对其中产量最高的1株菌株C2-7-2进行鉴定,鉴定其为铜绿假单胞菌(Pseudomonas aeruginosa),并命名为铜绿假单胞菌C272。从GenBank数据库中选取代表性假单胞菌的16SrRNA序列,利用DNAMAN软件构建系统发育树,同时对该菌的蛋白水解活性和生物膜形成能力等与食品腐败相关特性进行检测,发现其具有很强的蛋白水解能力和生物被膜形成能力。3、提取铜绿假单胞菌C272合成的AHLs,与冷却猪肉主要的几种致腐微生物共同添加到猪肉蛋白提取物中,采用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)方法,研究了AHLs介导冷却肉主要致腐微生物降解猪肉蛋白提取物的情况。结果发现,铜绿假单胞菌C272释放的AHLs信号分子可以促进其本身、金黄色葡萄球菌、大肠杆菌、植物乳杆菌对肌浆蛋白的降解,尤其对于金黄色葡萄球菌的促进作用最强,其次是其本身和植物乳杆菌,而对于大肠杆菌降解肌浆蛋白促进作用不显著。由图谱观察可知,该信号分子主要促进分子量约40kDa肌浆蛋白的降解。然而铜绿假单胞菌C272释放的AHLs调控各菌降解肌原纤维蛋白的情况有所不同。铜绿假单胞菌C272释放的AHLs可促进其本身降解肌原纤维蛋白,也可促进大肠杆菌降解分子量约为105和35kDa的蛋白,而对于金黄色葡萄球菌和植物乳杆菌,AHLs却抑制它们对肌原纤维蛋白的降解。4、模拟导致冷却猪肉腐败的不同微生物群系,采用SDS-PAGE方法,研究了不同微生物群系对猪肉蛋白降解的影响,结果发现,铜绿假单胞菌C272与其他主要腐败微生物组合的不同微生物群系,都可降解肌浆蛋白和肌原纤维蛋白。但不同微生物群系对猪肉肌浆蛋白的降解作用与单独微生物降解情况基本相同,不同微生物群系间降解肌浆蛋白情况也无明显差异。各微生物群系降解肌原纤维蛋白情况较为复杂,但从图谱可以看出,有铜绿假单胞菌C272参与的微生物群系,肌原纤维蛋白降解都受到不同程度的抑制,且四株菌共存的微生物群系,肌原纤维蛋白降解程度最小,可能原因是四种菌的竞争拮抗作用,抑制彼此降解蛋白酶的活力。更多还原

【Abstract】 China is a big country of pig breeding and pork consumption in which the main consumption pattern is fresh meat. Due to the characteristics of nutritious, safe and healthful, chilled pork has become the leading developing direction of fresh meat.Though the temperature environment remains 0-4℃through production to marketing, it cannot inhibit the propagation of psychrophilic microorganisms which leads to a short shelf life of chilled pork. Based on that, China has been severely restricted in the development of production and domestic sales market of chilled pork.The corruption of chilled pork results from the interaction of specific spoilage microorganisms.Pseudomonas aeruginosa is a kind of Gram-negative bacterium,a psychrophilic microorganism which causes the corruption of high-protein food,and it plays an important role in spoilage of chilled pork. It is the Gram-negative bacterium that give rise to food spoilage based on the research of recent years. And N-3-acyl-homoserine lactones (AHLs) have been detected in corruption food. Therefore this assay is aimed to select P.aeruginosa which produce signal molecules of AHLs in putrid chilled pork, and extract the AHLs using an Agrobacterium tumefaciens A136(pCF218/pCF372) plate screening model. Then the researcher simulated the microenvironment of putrid chilled pork, investigated the intervention of Pseudomonas and its quorum sensing(QS)in degradation of pork protein extracts in chilled pork. It provides research basis of the role that QS plays in meat corruption and antisepsis and preservation tactics which base on the interference with corruption bacterial quorum sensing as a target of chilled meat. Main conclusions of the tests are as follows:1、Selective medium were used to study the initial microflora of pallet packaging chilled pork, then the interaction and changes of growth and decline of microorganisms in pallet packaging chilled pork were investigated under the storage temperature of 4℃. The results showed:the initial microflora of main microorganisms in chilled pork was Pseudomonas and lactobacillus under aerobic condition. Total bacterial count increased under the storage temperature of 4℃; as the storage time extended, the growing speed of Pseudomonas was the highest, and the bacterial count was the most. The number of Escherichia Coli and Staphylococcus grew at different degrees, reached a higher logarithm value at the end of the experiment. During the experiment, growing speed of lactobacillus was the slowest; its proportion kept falling during the period and it lost the status of dominant bacteria at the end of the experiment. Pseudomonas had a negative correlation with lactobacillus while Escherichia Coli had a positive correlation with Staphylococcus.2、66 strains Pseudomonas were gained from chilled pork under low temperature storage and corrupted chilled pork sample using Pseudomonas isolation medium 15 strains Pseudomonas that produce AHLs were gained using A.tumefaciens A136 (pCF218/pCF372) reported plates for secondary screening. Then one Pseudomonas strain that gained the highest yield of AHLs was identified by combining 16S rRNA molecular biology identification with conventional bacterial morphological and biochemical methods. Finally the one was identified as Pseudomonas aeruginosa.The representative 16S rRNA sequence of Pseudomonas was selected from GeeBank. DNAMAN software was used to structure Phylogenetic tree. Properties that related to food spoliage, such as proteolytic activity and biofilm formation ability of the P. aeruginosa, were analyzed at the same time.And study shows that it can generate biofilm and has a strong capacity of protein forming.3、AHLs that were synthesis by P.aeruginosa C272, together with microorganisms that caused spoilage of chilled pork,were added to the extracts from pork protein.Using SDS-polyacrylamide gel electrophoresis (SDS-PAGE),AHLs were found to mediate the biodegradation process of chilled pork protein extracts that were caused spoilage by microorganisms. The results showed that AHLs extracted from P.aeruginosa C272 has a promote effect on sarcoplasmic protein degradation of itself,Staphylococcus aureus, Escherichia coli,Lactobacillus plantarum.Especially AHLs had a most promote effect on S. aureus and a secondary effect on P.aeruginosa C272 itself and L.plantarum while the effect on E.coli was not so significant. According to the atlas, AHLs were found to help degradation of protein whose molecular weight is about30-40kDa. However, AHLs from P.aeruginosa. C272 had different regulations of myofibrillar protein degradation.They stimulated myofibrillar protein degradation of P.aeruginosa C272, and had a certain promotion of E.coli on degradation of myofibrillar protein whose molecular weight is about 105kDa and 34kDa, while they helped S.aureus and L.plantarum restrict degradation of myofibrillar protein. 4、A research about the effects of different microbiota on the degradation of pork protein was carried out by mimicking various pork spoilage microbiota with SDS-PAGE. The results indicated that both sarcoplasmic protein and myofibrillar protein would be degraded under various microbiota containing P.aeruginosa C272 and other main spoilage organism. The degradation extent of swine sarcoplasmic protein under different microbiota and single species of microbiota were almost without differences, and the situation was the same between different microbiota.The degradation conditions were complex under various microbiota,but the atlas indicated that myofibrillar protein degradation was suppressed to different extent under the microbiota with Pseudomonas and with four strains the degradation was minimum.Antagonism of the four strains to suppress each other’s degradation activity may contribute to the situation.更多还原