【作者】 李学鹏；

【导师】 励建荣；

【作者基本信息】 浙江工商大学 ， 食品科学， 2012， 博士

【摘要】 新鲜度对水产品品质及原料的加工适性具有较大影响,因此新鲜度评价对水产品安全、运输、仓储及加工具有重要意义,而如何快速、客观评价水产品新鲜度一直是水产品加工工业亟待解决和水产学者不断探索的课题。水产品在贮藏过程中,新鲜度下降和腐败是一个复杂的物理、化学、生化及微生物生长代谢的过程。其中,蛋白质是引起水产品新鲜度下降和品质劣变的最主要因素,但目前对水产品贮藏过程中蛋白质变化的研究报道仍较少,同时,基于二蛋白质的新鲜度评价方法和新鲜度指示蛋白的研究也鲜见报道。鉴于此,本文以我国特色水产品——中国对虾为研究对象,(1)采用感官评价、化学评价、物理评价和微生物评价等方法,对不同冷藏温度条件下中国对虾的品质变化进行综合评价,筛选出适用于中国对虾的新鲜度评价指标；(2)从蛋白质变性的角度出发,研究中国对虾在不同温度冷藏条件下蛋白质的生化特性和组织结构的变化,筛选出中国对虾的新鲜度评价指标,同时为中国对虾在贮藏过程中品质变化规律和劣变机制的解析提供理论依据和参考；(3)从蛋白质降解的角度出发,研究中国对虾在不同温度冷藏过程中肌肉蛋白质的降解规律,筛选出中国对虾的新鲜度指示蛋白或新鲜度指标,同时为研究中国对虾在冷藏过程中品质变化机制的解析提供理论依据；(4)建立适用于中国对虾肌肉组织的蛋白质双向电泳技术,运用蛋白质组学技术研究肌肉蛋白质在冷藏过程中的变化,寻找并鉴定新鲜度指示蛋白。本研究旨在筛选适合用于评价中国对虾的新鲜度指标和新鲜度指示蛋白,为水产品新鲜度评价提供新的研究思路,同时对中国对虾冷藏过程中肌肉品质的变化机制进行探讨,进一步闸述导致水产品品质劣变的根本原因。主要研究结果如下1.中国对虾在4℃、0℃、-2℃贮藏过程中反映品质变化的主要指标依次是感官评分、化学指标、质构特性(硬度、弹性、凝聚性、回复性、胶性、咀嚼性)、色差属性及微生物指标；其中,感官评分、TVB-N、HxR、Hx、 K值、硬度、弹性、凝聚性、胶性、咀嚼性可以作为中国对虾的新鲜度指标,pH值可以作为一个参考指标,而TBA值、回复性、色差属性(L*、a*、b*、C*ab/H0ab)则不适合；同时,感官评分、TVB-N值、Hx值可以进一步作为判定中国对虾腐败的品质指标；建立了中国对虾在不同温度冷藏过程中的品质综合评价模型,为全面分析其品质状况和质量评级提供了参考和依据；中国对虾真空包装条件下在4℃、0℃、-2℃冷藏时的货架期分别为5.5～5.7天、7.9-8.0天和16.0-16.8天,冰温贮藏(-2℃)能够显著延长中国对虾的货架期。2.在4℃、0℃、-2℃贮藏过程中,中国对虾的肌肉蛋白质生化特性和肌肉组织结构发生了显著变化,表现为肌动球蛋白的盐溶性、巯基含量和Ca2+-ATPase活性均显著下降,表面疏水性显著增加,肌纤维发生持续的断裂和小片化,肌纤维面积比例显著减少,肌节间隙和间隙面积比例明显增大；Ca2+-ATPase活性和巯基含量可以作为评价中国对虾新鲜度的潜在指标；冰温贮藏(-2℃)对中国对虾的肌肉蛋白质变性和组织结构劣变具有明显的抑制作用。3.不同温度(4℃、0℃、-2℃)贮藏过程中,中国对虾的肌原纤维蛋白、肌基质蛋白含量随着贮藏时间的延长均显著下降,肌浆蛋白含量略有减低,碱溶性蛋白含量则呈增加趋势；MHC(肌动球蛋白重链)、肌动蛋白和原肌球蛋白、肌钙蛋白I、肌钙蛋白C和分子量分别为30～31KDa、6.5KDa的蛋白均发生了不同程度的降解；总可溶蛋白中23～25KDa条带、水溶性蛋白中176-178KDa条带、低盐溶性蛋白中67～68KDa条带,以及酸溶性氮中的组分I和组分V可以作为中国对虾潜在的新鲜度指示蛋白；TCA(三氯乙酸)可溶性肽和色氨酸的含量可以作为中国对虾的新鲜度指标。4.中国对虾肌肉蛋白质的等电点主要位于4-7之间,裂解液中添加Tris(三羟甲基氨基甲烷)和TBP(三丁基膦),可以增加对虾肌肉蛋白质的提取率；采用17cm,pH4～7的中型IPG胶条,主动水化上样120μg,适当延长除盐时间和等电聚焦时间,搭建盐桥,采用12%浓度的二向分离胶和硝酸银染色,能有效提高双向电泳(2-DE)图谱中蛋白点的分离度和分辨率；建立的中国对虾肌肉蛋白质双向电泳图谱具有良好的重复性和稳定性,为进一步探索中国对虾肌肉品质变化机制及其肌肉蛋白质组学研究奠定了基础。5.采用蛋白质组学技术研究了中国对虾在4℃和-2℃冷藏过程中肌肉蛋白质的变化。4℃组样品的2-DE图谱中筛选出6个具有统计学意义和规律性变化的差异蛋白点,其中差异蛋白点1501的丰度变化与贮藏时间这两者呈线性正相关(相关系数达0.949)；-2℃组样品的2-DE图谱中筛选出11个规律性变化的差异蛋白点,其中蛋白点1505与4℃组中1501为同一蛋白点,蛋白点7202和7205的丰度变化与贮藏时间呈线性负相关(相关系数达0.901～0.905)；蛋白点1405和1304的丰度变化与贮藏时间呈线性正相关(相关系数达0.937～0.940),因此这5个差异蛋白点均有望作为冷藏中国对虾的新鲜度指示蛋白；蛋白点1501、1405和1304可能为蛋白质的降解产物,蛋白点7202和7205可能为对虾肌肉中固有蛋白质。采用MALDI-TOF-MS得到11个差异蛋白点(包括5个可作为新鲜度指示蛋白的蛋白点和其他6个差异蛋白点)的肽指纹图谱,经数据库比对分析,蛋白点6001预测为酪氨酸酶活性相关蛋白(SH2domain-containing protein6),该蛋白与虾体黑变具有密切关系；蛋白点7205预测为腺苷钻啉胺酸合成酶(adenosylcobyric acid synthase),其余9个差异蛋白点的功能未知。腺苷钴啉胺酸合成酶(即蛋白点7205)的丰度变化与贮藏时间呈良好线性关系,可作为中国对虾冷藏过程中的新鲜度指示蛋白。更多还原

【Abstract】 Freshness makes a major contribution to the quality and processing suitability of aquatic products. Freshness assessment is essential for the safety, transportation, storage, and processing of aquatic products. Thus, how to quickly and objectively evaluate fish freshness has been a hot issue to be studied in aquatic products processing industiy and researchers. Protein is one of the most important substrates of spoilage for fish products. However, the evaluation method of freshness based on muscle protein has received little attention, and little information is available on the quality indices and protein indicators of freshness.In this paper, to search the quality indices and indicators of freshness based on muscle protein of Chinese shrimp (Fenneropenaeus chinensis), freshness of the shrimp stored under vacuum-packing at4℃,0℃and -2℃was assessed by physicochemical, sensory and microbiological methods. Changes in biochemical characteristics and tissue microstructure of Chinese shrimp muscle were studied based on the denaturation of protein. Changes in protein composition and patterns were studied based on the degradation of muscle protein. Two-dimensional gel electrophoresis (2-DE) related techniques of muscle proteome of Chinese shrimp were constructed and optimized. The proteomics technologies (2-DE and MS) were applied to study the changes in muscle protein and research the indicators of freshness based on muscle protein. The work aims to provide a new thought and reference for the assessment of seafood freshness, to discuss the post-mortem change mechanisms of the quality, and to give a further explanation of the basic reason for the spoilage of the muscle. The results are showed as follows:1. The major quality indices that reflecting freshness of Chinese Shrimp during the refrigerated storage at4℃,0℃and-2℃were sensory score, chemical indices, texture and color attributes and microorganisms. Sensory score, TVB-N, HxR, Hx, K value, hardness, springiness, cohesiveness, Gumminess, and chewiness displayed strong correlations with storage time, and they may be considered suitable indicators for evaluating freshness. Sensory score, TVB-N, Hx could also be indices for evaluating spoilage. The comprehensive models for assessing shrimp freshness and quality were constructed by Principal Component Analysis, which provided a reference for quality assessment. The shelf-lives of vacuum package Chinese shrimp stored at4℃,0℃and-2℃were5.5-5.7,7.9-8.0and16.0-16.8days respectively. Superchilled storage could significantly extend the shelf-life of the shrimp.2. The biochemical characteristics and tissue microstructure of Chinese shrimp muscle suffered obvious changes during refrigerated storage. The salt-solubilities, SH contents and Ca2+-ATPase activities of actomyosin significantly decreased with storage time, while the surface hydrophobicity increased. The microstructure of the muscle was disrupted, including the fracture and fragmentation of the muscle fiber, the significant decrease of the fiber area, and the increase of gaping and the gaping area. SH contents and Ca2+-ATPase activities displayed strong correlations with storage time, and they may be considered potential quality indices of freshness. Superchilled storage could inhibit the denaturation of protein and disruption of the muscle fiber.3. Changes in protein composition and patterns indicated the degradation of the shrimp muscle protein during refrigerated storage. The contents of myofibrillar protein and stroma significant decreased with storage, and the contents of sarcoplasmic protein slight decreased, while the contents of alkali-soluble protein increased during the storage. MHC actin, tropomysion, troponin I, troponin C and the proteins of30-31KDa、6.5KDa showed a different degradation during the storage. The density of23-25KDa protein band in total soluble proteins,176-178KDa protein band in water soluble proteins,67-68KDa protein band in low-salt soluble proteins, and the relative contents of fraction I and fraction V in acid soluble nitrogen displayed strong correlations with storage time, so they may be the potential protein indicators of freshness. The contents of TCA soluble peptide and tryptophan could be considered as quality indices of Chinese shrimp.4. Most of shrimp muscle protein isoelectric points were between4and7. The2-DE related techniques was constructed and optimized in muscle proteome of Chinese shrimp by comparative tests on different extraction methods, IPG gel strips, isoelectric focusing programs, salt bridge, and sample volume, etc. The results showed that the resolution and reproducibility of2-DE profiles was significantly improved by adding Tris and TBP in lysis buffer, active rehydrating of17cm (pH4-7) IPG gel strips, loading the sample120μ,g, prolonging the time of desalting, increasing the voltage and power of isoelectric focusing, employed the salt bridge, preparing12.5%SDS-PAFE gel, and were dying the gels by the silver stained. This work provided a technical basis for the further study on mechanism of muscle quality changes and differential proteomics on muscle tissue of Chinese shrimp. 5. The proteomics technologies were used to study the changes in muscle protein of Chinese shrimp during refrigerated storage at4℃and-2℃. Six differently expressed protein spots were found in2-DE maps of samples stored at4℃, while11different spots were found in the samples stored at-2℃. The density of spot1501,1304and1405correlated positively with storage time (correlation coefficients were0.949,0.947and0.940, respectively), while that of7202and7205correlated negatively with storage time (correlation coefficients were0.901and, respectively). Thus those five proteins could consider as the indicators of freshness. The peptide mass fingerprinting were obtained by MALDI-TOF-MS. Spot6001and7205was identified to be SH2domain-containing protein6and adenosylcobyric acid synthase, while other9spots were uncharacterized. SH2domain-containing protein6might be a key protein related to tyrosinase activity, which make great contribution to shrimp melanosis. Adenosylcobyric acid synthase (Spot7205) could be a potential protein indicator of freshness, because of a strong correlation between the density and storage time.更多还原