【作者】 黄晓毅；

【导师】 韩剑众；

【作者基本信息】 浙江工商大学 ， 食品科学， 2010， 硕士

【摘要】 现代肉品的食用品质大幅下降,引发了人们对现代肉品品质及其评价标准的深入思考,寻找一种有效的肉品品质评价及等级标注方法已成为国内外研究的热点。肉品品质的形成机理与肉质性状有高度的相关性,而几乎所有的肉质性状都受到复杂的基因和蛋白质控制,已有的肉类研究方法未能从肉品品质形成的生理生化机制出发对肉品品质进行深入有效的探究。以双向电泳分离技术(2-DE)、质谱鉴定技术(MS)和生物信息学(bioinformatics)三大技术为核心的差异蛋白质组学是研究生物蛋白质最有效最直接的方法,为生鲜猪肉品质评价及形成机理研究提供了新的思路。借助差异蛋白质组学寻找、筛选并鉴定与肉质性状相关的标记蛋白,不仅可以探究猪肉品质形成和遗传调控的生理生化基础,通过cDNA库找到品质控制基因,而且可以为肉质评价和等级标注提供相关依据。本研究通过对猪骨骼肌双向电泳条件相关技术参数进行比较和优化,在建立猪骨骼肌双向电泳技术的基础上,以浙江优质地方品种金华“两头乌”猪和目前国内普遍养殖的三元杂交品种“杜长大”猪为研究对象,对宰后肉质的变化进行了差异蛋白质组学研究。主要试验结果如下：1、通过对双向电泳的技术参数(胶条、加样量和等电聚焦升压方法等)进行控制与优化研究,建立了适于猪肉骨骼肌的双向电泳技术。猪肉骨骼肌蛋白质等电点在5-8之间,分子量在10 KDa～200 KDa之间。蛋白质加样量150μg,加样体积为300μL,采用在5-8的17 cm线性干制胶条,可得到具有较高的分离度和分辨率的双向电泳图谱。2、对宰后不同时间点的杜长大猪和金华猪背最长肌的热鲜肉蛋白质降解变化进行了比较研究,发现：杜长大猪宰后6 h,果糖二磷酸激酶、锌指蛋白和高速泳动蛋白开始降解；宰后24 h肌动蛋白和肌间线蛋白开始降解。宰后48 h肌动蛋白大量降解并发现一个未知蛋白也发生显著性上调。金华猪宰后6 h,热休克蛋白、线粒体蛋白、RWD domain-containing和一个未知蛋白开始降解；宰后24 h,肌动蛋白、CGRP type 1 receptor和分子黏连蛋白开始降解；宰后48 h到72 h的时间内,发现肌动蛋白的显著性高表达,并发现两个未知蛋白。3、对宰后不同时间点的杜长大猪和金华猪背最长肌的冷鲜肉蛋白质降解变化进行了比较研究,发现：宰后6 h杜长大冷鲜肉中肌间线蛋白、Defensin-1和一个未知蛋白开始降解；宰后24h,肌钙蛋白和一个未知蛋白开始降解；宰后48 h,抗氧化蛋白、超氧化物歧化酶和细胞膜糖蛋白开始降解。宰后96 h到144 h的时间内肌球蛋白大量降解,同时也发现一个未知蛋白。金华猪宰后6 h,热休克蛋白、肌间线蛋白和一个未知蛋白发生降解；宰后24 h开始,肌钙蛋白T、水通道蛋白-11和锌指蛋白开始降解；宰后48 h,肌动蛋白和抗氧化蛋白开始降解。宰后96 h到144 h时间内肌球蛋白也发生了降解,同时还发现SHC-transforming protein 3和一个未知蛋白。4、对相同条件下杜长大和金华猪背最长肌的冷鲜肉进行了品种间的比较研究,质谱鉴定的12个差异蛋白点初步确定为热休克蛋白,肌间线蛋白,烯醇化酶,丙酮酸激酶、腺苷酸激酶、锌指蛋白、糖原磷酸化酶、26S蛋白酶体、Sarcolumenin、NADH-ubiquinone oxidoreductase、Leucine rich repea、ATP phosphoribosyltransferase,这些蛋白质与猪肉持水性和嫩度的形成密切相关。由于猪肉可供检索的蛋白质组学数据库并不全面,提示宰后不同品种的猪背最长肌品质的差异可能是由于这些蛋白质量的变化即斑点的上调或者下调引起。5、以杜长大和金华猪背最长肌为实验材料,对宰后不同时间的肌肉氨基酸含量变化分别进行了跟踪测定。随着宰后储藏时间的增加,无论是热鲜肉还是冷鲜肉中氨基酸含量并没有显著性的变化,在所测定的17种氨基酸中动态变化情况与肉质变化没有相关性。更多还原

【Abstract】 The quality of fresh pork is a complicated and multipleunit character. Studies on its intrinsic mechanism, evaluation and management are always the hottest research fields of the meat science. So far, such kinds of researches have been generally focused on lean rates and differences in the quality between normal and poor meat. However, methods for effectively evaluating the quality of the fresh pork and its intrinsic mechanism are still not well established as far as we know. Two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS) and bioinformatics are the three critical technologies of proteomics, which provide us with the new thought to study the intrinsic mechanism of fresh pork quality. With the power of proteomics, the intrinsic mechanism of fresh meat quality and the physiological and biochemical basis of genetic regulation were investigated by searching for and identifying the protein markers of meat quality traits. Thus proteomics offers a related foundation for finding quality genes through cDNA, also evaluation and grade mark of meat quality under different conditions.Therefore, this study was conducted to evaluate the quality of fresh pork of two different species (Jinhua pigs termed as JH, and hybrid pigs of Duroc×Landrace×Yorkshire, termed as DLY) at different postmortem time by using technologies of differential proteomics. We have established the pork muscle proteomic methods of 2-DE conditions through comparing and optimizing the relevant technical parameters, as well as the amino contents was detected. The main results of the present study were as follows:1.The 2-DE techniques of fresh pork musle were established with the relative technique parameters including gel selection, loding amount of protein and program of the isoelectric focusing (IEF). It has been found that the pI of the pork muscle protein was 5-8, while their mass was about 10 KDa-200KDa. The optimized method is that the loding amount of protein is 150μg, and the volume is 300μL. With this mothod, the 2-DE profile was of good resolution and good repetition.2.The comparison study was conducted to analyze the degradation of the proteins at different postmortem time, using the JH and DLY pig muscles (stored at room temperature) as materials. The results showed that High mobility group protein, Zinc finger protein and Fructose-bisphosphate aldolase A of DLY pig fresh were degraded in postmortem 6 h. Acin and desmin were degraded in postmortem 24 h. After 48 h, Acin were degraded significantly, and an uncharacterized protein was found. Also results in JH pig showed that heat shock protein, mitochondrial protein, RWD-domain-containing and an uncharacterized protein were found to be degraded in postmortem 6 h. Actin, CGRP type 1 receptor and Intercellular adhesion molecule were degraded at postmortem 24 h. Actin and other two uncharacterized proteins were significantly up-regulated between postmortem 48 h and 72 h.3.The comparison study was conducted to analyze the degradation of the proteins at different postmortem time, using the JH and DLY pig muscles (stored at 2℃) as materials. The results showed that desmin, Defensin-1 and uncharacterized protein of DLY meat were degraded at postmortem 6 h. Troponin T and uncharacterized protein were degraded at postmortem 24 h. After 48 h, Thy-1 membrane glycoprotein, Peroxiredoxin and SOD were degraded. Actin was significantly degraded from postmortem 96 h to 144 h, and a uncharacterized protein was found Also results in JH pig showed that heat shock protein, desmin and an uncharacterized protein were found to be degraded in postmortem 6 h. Troponin T, Aquaporin-11 and Zinc finger protein were degraded in postmortem 24 h. Actin and Peroxiredoxin-4 proteins were significantly up-regulated at postmortem 48 h. Myosin heavy chain, SHC-transforming protein 3 and uncharacterized protein were found to be degraded from postmortem 96 h to 144 h.4.The comparison study was conducted to analyze the differentially expressed proteins between two breeds (JH pig and DLY pig). The results obtained from the samples prepared at 0 h indicated that there were three different proteins(Sarcolumenin; Leucine rich repeat and heat shock protein) between two species of pig. As for the samples at 24 h, three differential proteins(Adenylate kinase; ATP phosphoribosyltransferase and 26S protease regulatory protein) were observed. The differential proteins obtained from the samples prepared at 48 h indicated that there were three different proteins (Zinc fingering protein; (3-enolase and desmin) were significantly up-regulated. According to the postmortem 72 h samples, Pyruvate kinase, NADH-ubiquinone oxidoreductase, Glycogen phosphorylase were differentially expressed in different species of pig. These results suggested that meat quality of porcine longissimus dorsi might be associated with these differential proteins.5.The amino contents were detected according different postmortem time, using the JH and DLY meat as materials. Results showed that none of the 17 kinds of Amino have any pertinence with the meat quality.更多还原