大黄鱼生物保鲜技术及新鲜度指示蛋白研究
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摘要
新鲜度是鱼类等水产品品质评价最重要的因素,能够直接决定产品的价值,因此如何客观、快速、全面评价鱼类等水产品的新鲜度是水产研究领域亟待解决的课题。鱼肉中富含蛋白质,蛋白质是引起鱼类等水产品腐败的最重要因素,因此,鱼肉品质劣变与蛋白质的分解存在必然关联。然而,目前关于鱼类等水产品在低温贮藏过程中蛋白质变性及降解规律研究仍较少,以蛋白质为研究对象的鱼类新鲜度评价方法鲜有报道,利用蛋白质组学技术筛选鱼类新鲜度指示蛋白的研究尚不多见。
鉴于此,本课题以我国在国际市场上具有较高知名度和竞争力的大黄鱼为研究对象,(1)采用水产品评价指标(微生物、物理、化学及感官)系统评价大黄鱼冷藏过程中品质变化特性,建立有效表征大黄鱼品质变化的综合评价模型,并利用该模型筛选出适用于大黄鱼品质评价的相关指标,为后续生物保鲜以及肌肉品质变化提供依据和参考；(2)依据栅栏效应原理,以建立的大黄鱼新鲜度评价指标为依据,甄选出有效的生物保鲜剂,并探讨其对大黄鱼低温贮藏品质的影响；(3)从蛋白质变性及降解角度出发,研究大黄鱼在贮藏过程中肌肉蛋白质变化特性及降解规律,鉴别大黄鱼新鲜度指示蛋白,为品质劣变机理的探讨提供理论依据；(4)运用蛋白质组学技术研究冷藏大黄鱼肌肉的蛋白质变化规律,筛选并鉴定新鲜度指示蛋白,旨在从蛋白水平解析大黄鱼肌肉蛋白质的变化机制。主要研究结果如下：
1.建立了大黄鱼低温贮藏过程中的品质综合评价模型,确定了能够客观反映大黄鱼品质的几个主要评价指标：微生物指标(菌落总数、腐败希瓦式菌、假单胞菌、明亮发光杆菌、肠杆菌、乳酸菌)、化学指标(pH、挥发性盐基氮(TVB-N)、过氧化值(POV)、硫代巴比妥酸(TBA)、三磷酸腺苷(ATP)、二磷酸腺苷(ADP)、一磷酸腺苷(AMP)、肌苷酸(IMP)、次黄嘌呤核苷(HxR)、次黄嘌呤(Hx)、K值)、物理指标(弹性、回复力)以及感官评分。
2.以建立的大黄鱼新鲜度评价指标为依据,研究了不同浓度的茶多酚、迷迭香、壳聚糖及复合生物保鲜剂处理对冷藏大黄鱼品质的影响,表明经过壳聚糖茶多酚复合保鲜剂(Ch+TP)和壳聚糖迷迭香复合保鲜剂(Ch+R)处理的大黄鱼,其抑菌及抗氧化效果明显优于单一保鲜剂,能够有效延长大黄鱼的货架期8～10天。该结果充分体现了复合生物保鲜剂能够较好发挥各自的优势,并产生协同效应,从而有效减缓大黄鱼肉腐败变质,保持其良好的品质。
3.在冷藏过程中,大黄鱼肌肉蛋白质生化特性发生了明显变化,其中肌动球蛋白盐溶性、巯基含量以及Ca2+-ATPase活性等指标下降,而表面疏水性在贮藏初期阶段明显增加。肌肉组织微观结构也发生改变,表现为肌肉组织而积比例逐渐减少,肌节间隙面积比例增大,肌纤维结构变得疏松；巯基含量能够作为评价大黄鱼冷藏过程中新鲜度变化的潜在指标。
4.在对大黄鱼冷藏过程中肌肉蛋白质降解规律的研究中发现：总可溶蛋白中的16-17KDa条带、水溶性蛋白中的22-23KDa条带、低盐溶性蛋白中的35-36KDa条带以及高盐溶性蛋白中的MHC、肌动蛋白、原肌球蛋白、32～33KDa处条带随着贮藏时间的延长发生了明显的变化,因此这七个条带代表的蛋白或肽类可以作为大黄鱼冷藏过程中潜在的新鲜度指示蛋白；TCA可溶性肽的含量可以作为大黄鱼新鲜度评价指标。
5.采用蛋白质组学及质谱分析技术研究并筛选能够表征大黄鱼品质变化规律的差异蛋白质,经NCBI数据库比对分析寻找出8个潜在的大黄鱼新鲜度指示蛋白,他们分别为：T-肌钙蛋白同源蛋白(fast/white muscle troponin T embryonic isoform);丙酮酸激酶(pyruvate kinase);酪氨酸3/色氨酸5单加氧酶激活蛋白(tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein, theta polypeptide b); LDB3蛋白；肌球蛋白重链(myosin heavy chain);α-烯醇化酶同工酶(alpha-enolase-like isoform1);甘油醛-3-磷酸脱氢酶(glyceraldehyde3-phosphate dehydrogenase);叽肉型肌酸激酶(muscle-type creatine kinase)。这8种差异蛋白均有望作为大黄鱼冷藏过程中的新鲜度指示蛋白。
Freshness is an important factor in the aspect of evaluating the quality of aquatic products, which can determine the products value directly. Therefore, how to evaluate fish freshness objectively, rapidly and comprehensively has been a hot issue to be studied in aquatic products research field. It has been well known that protein is one of the most important ingredients of fish muscle, and it can have close association between aquatic quality deterioration and protein degradation. However, the freshness evaluation method based on muscle protein denaturation and degradation has received little attention, and little information is available on protein indicators of freshness based on the proteomics technology.
In view of above reasons, this paper investigated the quality indices as well as indicators of freshness based on muscle protein of large yellow croaker(Pseudosciaena Crocea) by the microbiological, physical, chemical and sensory methods, which provided the basis and reference for bio-preservation techniques. This issue researched the effects of biological preservatives on the quality of large yellow croaker during cold storage. Changes in biochemical characteristics as well as tissue microstructure of large yellow croaker muscle were studied from the aspect of the denaturation of protein, which provided relative theoretical basis. The proteomics technologies (2-DE and MS-MS) were applied to investigate the changes in muscle protein and researched the protein indicators. The work aims to give a new idea for the assessment of aquatic products freshness, to discuss the post-mortem change mechanisms of muscle change. The results were taken as follows:
1. The comprehensive models for evaluating large yellow croaker freshness and quality were established. The major quality indices that reflecting freshness of large yellow croaker during the storage at4℃,0℃and-2℃were microorganisms (total viable count, shewanella, pseudomonas, enterobacteriaceae, lactic acid bacteria, photobacterium phosphoreum), pH, TVB-N, PV, TBA, ATP, ADP, AMP, IMP, HXR, HX, K, springiness, resilience, sensory score.
2. Based on The comprehensive evaluating models, The effects of tea polyphenol (TP), rosemary extract (R), chitosan (Ch) respectively and compound bio-preservatives on the quality of large yellow croaker during refrigerated storage were evaluated. The results showed that the compound bio-preservatives could more effectively maintain the good quality and could extend the shelf life by8-10days compared with the control group.
3. The biochemical characteristics a of large yellow croaker muscle occured obvious changes during refrigerated storage. The salt-solubilities, SH contents, as well as Ca2+-ATPase activities of actomyosin obviously decreased with storage time, while surface hydrophobicity increased sharply in the intial phase of storage. The micro structure of the muscle was changed, including the significant decrease of the fiber area, and the increase of gaping and the gaping area. SH contents showed better correlations with storage time, and it may be considered a potential quality index of freshness.
4. The study of muscle protein degradation charactrestics on large yellow croaker were shown that the density of16-17KDa protein band in total soluble proteins,22-23KDa protein band in total soluble proteins,35-36KDa protein band in low-salt soluble proteins, MHC, actin, tropomysion,32-33KDa protein band in high-salt soluble proteins happened significantly change with the storage time. Therefore, this seven bands may be thought potential freshness indices poteins. The content of TCA soluble peptide could be considered as quality indices of large yellow croaker.
5. The proteomics and MS-MS technologies were applied to study the changes in muscle protein of large yellow croaker during refrigerated storage at4℃,0℃and-2℃. Eight differently expressed protein spots were obtained in2-DE maps of peptide mass fingerprinting and MS-MS. They were fast/white muscle troponin T embryonic isoform, pyruvate kinase, tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein, theta polypeptide b, LDB3protein, myosin heavy chain, alpha-enolase-like isoform1, glyceraldehyde3-phosphate dehydrogenase. The eight proteins could be considered as the indicators of freshness.
鉴于此,本课题以我国在国际市场上具有较高知名度和竞争力的大黄鱼为研究对象,(1)采用水产品评价指标(微生物、物理、化学及感官)系统评价大黄鱼冷藏过程中品质变化特性,建立有效表征大黄鱼品质变化的综合评价模型,并利用该模型筛选出适用于大黄鱼品质评价的相关指标,为后续生物保鲜以及肌肉品质变化提供依据和参考；(2)依据栅栏效应原理,以建立的大黄鱼新鲜度评价指标为依据,甄选出有效的生物保鲜剂,并探讨其对大黄鱼低温贮藏品质的影响；(3)从蛋白质变性及降解角度出发,研究大黄鱼在贮藏过程中肌肉蛋白质变化特性及降解规律,鉴别大黄鱼新鲜度指示蛋白,为品质劣变机理的探讨提供理论依据；(4)运用蛋白质组学技术研究冷藏大黄鱼肌肉的蛋白质变化规律,筛选并鉴定新鲜度指示蛋白,旨在从蛋白水平解析大黄鱼肌肉蛋白质的变化机制。主要研究结果如下：
1.建立了大黄鱼低温贮藏过程中的品质综合评价模型,确定了能够客观反映大黄鱼品质的几个主要评价指标：微生物指标(菌落总数、腐败希瓦式菌、假单胞菌、明亮发光杆菌、肠杆菌、乳酸菌)、化学指标(pH、挥发性盐基氮(TVB-N)、过氧化值(POV)、硫代巴比妥酸(TBA)、三磷酸腺苷(ATP)、二磷酸腺苷(ADP)、一磷酸腺苷(AMP)、肌苷酸(IMP)、次黄嘌呤核苷(HxR)、次黄嘌呤(Hx)、K值)、物理指标(弹性、回复力)以及感官评分。
2.以建立的大黄鱼新鲜度评价指标为依据,研究了不同浓度的茶多酚、迷迭香、壳聚糖及复合生物保鲜剂处理对冷藏大黄鱼品质的影响,表明经过壳聚糖茶多酚复合保鲜剂(Ch+TP)和壳聚糖迷迭香复合保鲜剂(Ch+R)处理的大黄鱼,其抑菌及抗氧化效果明显优于单一保鲜剂,能够有效延长大黄鱼的货架期8～10天。该结果充分体现了复合生物保鲜剂能够较好发挥各自的优势,并产生协同效应,从而有效减缓大黄鱼肉腐败变质,保持其良好的品质。
3.在冷藏过程中,大黄鱼肌肉蛋白质生化特性发生了明显变化,其中肌动球蛋白盐溶性、巯基含量以及Ca2+-ATPase活性等指标下降,而表面疏水性在贮藏初期阶段明显增加。肌肉组织微观结构也发生改变,表现为肌肉组织而积比例逐渐减少,肌节间隙面积比例增大,肌纤维结构变得疏松；巯基含量能够作为评价大黄鱼冷藏过程中新鲜度变化的潜在指标。
4.在对大黄鱼冷藏过程中肌肉蛋白质降解规律的研究中发现：总可溶蛋白中的16-17KDa条带、水溶性蛋白中的22-23KDa条带、低盐溶性蛋白中的35-36KDa条带以及高盐溶性蛋白中的MHC、肌动蛋白、原肌球蛋白、32～33KDa处条带随着贮藏时间的延长发生了明显的变化,因此这七个条带代表的蛋白或肽类可以作为大黄鱼冷藏过程中潜在的新鲜度指示蛋白；TCA可溶性肽的含量可以作为大黄鱼新鲜度评价指标。
5.采用蛋白质组学及质谱分析技术研究并筛选能够表征大黄鱼品质变化规律的差异蛋白质,经NCBI数据库比对分析寻找出8个潜在的大黄鱼新鲜度指示蛋白,他们分别为：T-肌钙蛋白同源蛋白(fast/white muscle troponin T embryonic isoform);丙酮酸激酶(pyruvate kinase);酪氨酸3/色氨酸5单加氧酶激活蛋白(tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein, theta polypeptide b); LDB3蛋白；肌球蛋白重链(myosin heavy chain);α-烯醇化酶同工酶(alpha-enolase-like isoform1);甘油醛-3-磷酸脱氢酶(glyceraldehyde3-phosphate dehydrogenase);叽肉型肌酸激酶(muscle-type creatine kinase)。这8种差异蛋白均有望作为大黄鱼冷藏过程中的新鲜度指示蛋白。
Freshness is an important factor in the aspect of evaluating the quality of aquatic products, which can determine the products value directly. Therefore, how to evaluate fish freshness objectively, rapidly and comprehensively has been a hot issue to be studied in aquatic products research field. It has been well known that protein is one of the most important ingredients of fish muscle, and it can have close association between aquatic quality deterioration and protein degradation. However, the freshness evaluation method based on muscle protein denaturation and degradation has received little attention, and little information is available on protein indicators of freshness based on the proteomics technology.
In view of above reasons, this paper investigated the quality indices as well as indicators of freshness based on muscle protein of large yellow croaker(Pseudosciaena Crocea) by the microbiological, physical, chemical and sensory methods, which provided the basis and reference for bio-preservation techniques. This issue researched the effects of biological preservatives on the quality of large yellow croaker during cold storage. Changes in biochemical characteristics as well as tissue microstructure of large yellow croaker muscle were studied from the aspect of the denaturation of protein, which provided relative theoretical basis. The proteomics technologies (2-DE and MS-MS) were applied to investigate the changes in muscle protein and researched the protein indicators. The work aims to give a new idea for the assessment of aquatic products freshness, to discuss the post-mortem change mechanisms of muscle change. The results were taken as follows:
1. The comprehensive models for evaluating large yellow croaker freshness and quality were established. The major quality indices that reflecting freshness of large yellow croaker during the storage at4℃,0℃and-2℃were microorganisms (total viable count, shewanella, pseudomonas, enterobacteriaceae, lactic acid bacteria, photobacterium phosphoreum), pH, TVB-N, PV, TBA, ATP, ADP, AMP, IMP, HXR, HX, K, springiness, resilience, sensory score.
2. Based on The comprehensive evaluating models, The effects of tea polyphenol (TP), rosemary extract (R), chitosan (Ch) respectively and compound bio-preservatives on the quality of large yellow croaker during refrigerated storage were evaluated. The results showed that the compound bio-preservatives could more effectively maintain the good quality and could extend the shelf life by8-10days compared with the control group.
3. The biochemical characteristics a of large yellow croaker muscle occured obvious changes during refrigerated storage. The salt-solubilities, SH contents, as well as Ca2+-ATPase activities of actomyosin obviously decreased with storage time, while surface hydrophobicity increased sharply in the intial phase of storage. The micro structure of the muscle was changed, including the significant decrease of the fiber area, and the increase of gaping and the gaping area. SH contents showed better correlations with storage time, and it may be considered a potential quality index of freshness.
4. The study of muscle protein degradation charactrestics on large yellow croaker were shown that the density of16-17KDa protein band in total soluble proteins,22-23KDa protein band in total soluble proteins,35-36KDa protein band in low-salt soluble proteins, MHC, actin, tropomysion,32-33KDa protein band in high-salt soluble proteins happened significantly change with the storage time. Therefore, this seven bands may be thought potential freshness indices poteins. The content of TCA soluble peptide could be considered as quality indices of large yellow croaker.
5. The proteomics and MS-MS technologies were applied to study the changes in muscle protein of large yellow croaker during refrigerated storage at4℃,0℃and-2℃. Eight differently expressed protein spots were obtained in2-DE maps of peptide mass fingerprinting and MS-MS. They were fast/white muscle troponin T embryonic isoform, pyruvate kinase, tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein, theta polypeptide b, LDB3protein, myosin heavy chain, alpha-enolase-like isoform1, glyceraldehyde3-phosphate dehydrogenase. The eight proteins could be considered as the indicators of freshness.
引文
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