茶多酚对草鱼肉保鲜作用及机理研究
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摘要
淡水鱼是一种营养十分丰富、对人体健康具有重要意义的理想食物。据2006年统计,我国淡水鱼产量已达2350万吨,居世界第一位。然而,我国淡水鱼的保鲜和加工水平与养殖水平的发展是不相适应的,淡水鱼的加工率仅为6.9%,远远落后于世界水产品40%以上的加工率。茶多酚(Tea polyphenols,TP)是茶叶所含的一类多羟基类化合物的总称,具有抗氧化、抑菌、防癌、降低胆固醇等多种保健功能和药理效应。本文以草鱼(grass carp,Ctenopharyngodon idellus)为研究对象,采用食品化学、微生物学及仪器分析等方法研究茶多酚对淡水鱼类脂肪、蛋白质及鱼中微生物的影响,探讨茶多酚在淡水鱼保鲜中的应用前景。研究方法及结果如下:
1、通过测定盐溶性蛋白质的提取性、ATP酶活性、SDS-PAGE凝胶电泳、扫描电镜,分析4℃贮藏条件下的不同浓度TP处理的草鱼鱼肉蛋白质的生化特性的变化。结果显示:鱼肉盐溶性蛋白质的提取性能和ATP酶活性随时间延长而逐渐降低,添加TP后,其蛋白质的提取性和蛋白质的ATP酶活性下降明显减慢,TP浓度低于0.03%时,差异并不显著,当TP浓度超过0.05%时,出现较为明显的差异:鱼肉蛋白质的SDS-PAGE结果表明,用TP处理之后蛋白质的降解速度有一定程度的降低,并表现出剂量关系,而电镜检测肌肉蛋白质结构的变化也进一步证实了这一结果,表明在草鱼鱼肉中添加TP,对于蛋白质的生化特性具有明显的保护作用,其中添加量以0.05%～0.07%效果最好。
2、利用分光光度计、质构仪、流变仪对4℃贮藏的不同浓度TP处理的草鱼鱼肉蛋白质的乳化性、凝胶性、流变性进行了研究,结果表明:在冷藏草鱼鱼肉中添加TP 0.03%以上能显著延缓草鱼蛋白质乳化能力和乳化稳定性的降低,在试验范围内,浓度越高,效果越好;TP 0.03%以上可以明显提高鱼糜蛋白质的凝胶强度,但不具有剂量效应;TP 0.03%时,G′和G″最低,此时蛋白质变性交联程度最低。TP能改善蛋白质的乳化性和凝胶性。
3、利用GC-MS对草鱼鱼肉中的脂肪酸进行分析,鉴定出草鱼中含有棕榈酸、十六碳烯酸、油酸、亚油酸、亚麻酸、硬脂酸、花生四烯酸、DHA、EPA、十五烷酸、十七烷酸、十七碳三烯酸等十八种以上脂肪酸,除含有动植物中常见的偶数碳脂肪酸外,还含有一定量的奇数碳脂肪酸。其中饱和脂肪酸27.84%,单不饱和脂肪酸43.67%,多不饱和脂肪酸28.02%。
4、采用GC-MS测定了在4℃贮藏条件下,添加不同浓度TP的鱼肉脂肪中脂肪酸的变化,通过硫代巴比妥酸试验测定了脂肪酸氧化产物丙二醛的变化。结果表明:肉豆蔻酸、十五烷酸、十六烷酸、十七烷酸、十八烷酸、十六碳一烯酸、油酸、亚油酸、亚麻酸、十七碳三烯酸在贮藏5d以前的变化都不大,第10d检测到对照组各种脂肪酸的含量均明显下降,与添加TP试验组出现显著性差异(P＜0.05),而添加了TP的各试验组中上述脂肪酸在贮藏10d后相对含量没有发生很大变化。TBA试验结果显示,添加00.1%以上茶多酚对草鱼脂肪都有明显的抗氧化作用,随浓度增加TBA值增加,但不呈线性关系,0.03%～0.07%抗氧化效果比较接近,从经济、有效的角度考虑在实际应用中可采用0.03%的茶多酚。
5、从草鱼中分离出93株菌株,鉴定出以下几类主要腐败细菌:气单胞菌属、弧菌属、假单胞菌属、肠道杆菌科、黄杆菌属、邻单胞菌属、发光杆菌属、产碱杆菌属、短杆菌属、微球菌属、不动杆菌属、无色菌属。草鱼在贮藏过程中,前期(1～3d)以革兰氏阳性菌为主,占70%,多为短杆菌和微球菌;随时间延长,3d后微生物种类出现多样化且以革兰氏阴性菌为主,约占71%～82%,其中以假单胞菌、气单胞菌属、弧菌属与肠肝菌属为主,是导致鱼肉腐败的优势腐败菌。
6、通过滤纸圆片法和平板计数法,确定了茶多酚对鱼肉中的腐败菌即短杆菌、微球菌、气单胞菌、假单胞菌、弧菌、肠杆菌、产碱杆菌均具有很好的抑菌活性,且最小抑菌浓度不超过1.2g/L,对大多数的细菌来说,其最佳抑菌浓度为5g/L。
7、将山梨酸钾、Vc、茶多酚添加到鱼肉中比较保鲜效果,三组的保鲜效果均要好于对照组,其中以茶多酚保鲜效果最好,测得酸价、过氧化值最低,第11d测得的挥发性盐基氮和第9d测得的菌落总数均低于国家有关标准规定的一级鱼标准。
Fresh water fish is a kind of ideal food for people because it is rich in nutrients and good for health. In 2006, the product of fresh water in China was up to 23,500,000 tons and take the first place in the world. However, the technologies of fresh-keep and processing for fresh water fish in China didn't meet the development of acquaculture. The processing rate of fresh water fish was only 6.9% and far behind the worldwide average level of marine products processing (>40%). Tea polyphenols (TP), which is the generic terms of compounds with multiple hydroxyl groups in tea, has many functions of heath care and pharmacological effects, such as antioxidation, bacteriostasis, cancer-preventing, and cholesterol-lowering. In this project, the effects of TP on lipids, proteins, and microbe of fish meat from grass carp were studied to determine the possibility of TP applied in fresh-keeping of fresh water fish. The methodologies and results are described as following:
1. The changes of biochemical properties were studied by extractability and ATPase activity measurement, SDS-PAGE analysis, and ultrastructure observation of fish meat from grass carp treated with different concentration TP and stored at 4℃. The results revealed: the extractability and ATPase activity of protein decreased gradually with the time when the fish meat stored at 4℃, and these declines were retarded substantially by addition of TP. When the TP concentration was lower than 0.03% (w/w), the difference was insignificant (P>0.05), whereas the concentration was higher than 0.05%, the difference showed significant (P<0.05). The SDS-PAGE analysis showed the rate of protein degradation of fish meat was decelerated by applied of TP. The changes of ultrasturcture of fish surimi by scanning electro microscope checking confirmed the above observations. These results suggested that there were remarkable protecting effects of TP on biochemical properties of fish meat and the optimal concentration was about 0.05～0.07%.
2. The emulsion, gel property, and rheology of fish meat treated with different concentration of TP and stored at 4℃were studied using spectrophotometer, texture analyzer, and rheometer. The results indicated that the addition of more than 0.03% of TP could slow down the decrement of emulsion and emulsification stability of protein extracted from fish meat significantly and presented the does effects. In the meantime, 0.03% of TP increased the gel strength substantially and no does effect appeared. In rehological study, addition of TP reduced the storage and loss modulus (G' and G") compared to the negative control, and the G' and G" valves were the lowest when the TP was 0.03% suggesting that the extent of crosslink of proteins was the lowest.
3. The fatty acid composition of grass carp was analyzed using GS-MS and more than 18 fatty acids were identified, including hexadecanoic acid, 9-hexadecenoic acid, 9-octadecenoic acid, 6,9-octadecadienoic acid, 9,12,15-octadecatrienoic acid, octadecanoic acid, 5,8,11,14-eicosatetraenoic acid, 4,7,10,13,16,19-docosahexaenoic acid, 5,8,11,14,17-eicosapentaenoic acid, pentadecanoic acid, heptadecanoic acid, and 5,8,11-heptadecatrienoic acid. There were some fatty acids with odd carbon atoms besides the fatty acids with even carbon atoms common in animals and plants. Among the fatty acids, the saturated were taken 27.84%, the monounsaturated occupied 43.67%, and the polyunsaturated accounted for 28.02%.
4. The variations of fatty acids of fish meat treated with TP and stored at 4℃were assayed by GS-MS and the changes of malonaldehyde, the oxidative product of fatty acids, were measured by TBA experiment. The data indicated that there was no change in contents of fatty acids by 5 days after treatment, including tetradecanoate, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, 9-hexadecenoic acid, 9-octadecenoic acid, 6,9-octadecadienoic acid, 9,12,15-octadecatrienoic acid, and 5,8,11-heptadecatrienoic acid. There were obvious decreases in content of these fatty acids from fish meat without TP 10 days after treatment compared to the experimental groups (P<0.05) . TBA analysis showed 0.01% of TP had evident antioxidation on lipids from fish meat, and TBA increased with the TP concentration accretion presenting an unlinear relation. The effects of antioxidation of 0.03% and 0.07% of TP were close, thus, the best choice is 0.03% in practical application.
5. 93 bacteria strains were isolated and categorized into several spoilage bacteria classes: Aeromonas, Vibrio, Pseudomonas, Enterobacteriaceae, Flavobacterium, Acinetobacter, Photobacteriaceae, Brevibacterium, and Micrococcus. In the early stage of storage (1～3 days), Gram-positive bacteria were the major strains and accounted for 70%, most of them belonged to Brevibacterium and Micrococcus. Whereas the diversity was increased and Gram-negative bacteria became the dominant taking 71～82% after 3 days, the main strains such as Pseudomonas, Aeromonas, Vibrio and Enterobacteriaceae classed into spoilage bacteria attributing to the meat putrefaction.
6. The inhibition of TP on several spoilage bacteria from fish meat were determined by plate counting. TP presented potent inhibitory effect on Brevibacterium, Micrococcus, Aeromonas, Pseudomonas, Vibrio, and Enterobacteriaceae, and the minimum inhibition concentration (MIC) was lower than 1.2 g/L, whereas for most strains the optimal inhibition concentration was about 5 g/L.
7. The fresh-keeping effects of potassium sorbate, vitamine C, and TP were determined and showed good function compared to the negative control. Among three antioxidations, TP presented the best effect because the acid value, and peroxide value were the lowest, the total volatile basic nitrogen (TVBN) and total colonies number measured 11 days and 9 days after TP treatment respectively were lower than the first grade fish according to the GB.
1、通过测定盐溶性蛋白质的提取性、ATP酶活性、SDS-PAGE凝胶电泳、扫描电镜,分析4℃贮藏条件下的不同浓度TP处理的草鱼鱼肉蛋白质的生化特性的变化。结果显示:鱼肉盐溶性蛋白质的提取性能和ATP酶活性随时间延长而逐渐降低,添加TP后,其蛋白质的提取性和蛋白质的ATP酶活性下降明显减慢,TP浓度低于0.03%时,差异并不显著,当TP浓度超过0.05%时,出现较为明显的差异:鱼肉蛋白质的SDS-PAGE结果表明,用TP处理之后蛋白质的降解速度有一定程度的降低,并表现出剂量关系,而电镜检测肌肉蛋白质结构的变化也进一步证实了这一结果,表明在草鱼鱼肉中添加TP,对于蛋白质的生化特性具有明显的保护作用,其中添加量以0.05%～0.07%效果最好。
2、利用分光光度计、质构仪、流变仪对4℃贮藏的不同浓度TP处理的草鱼鱼肉蛋白质的乳化性、凝胶性、流变性进行了研究,结果表明:在冷藏草鱼鱼肉中添加TP 0.03%以上能显著延缓草鱼蛋白质乳化能力和乳化稳定性的降低,在试验范围内,浓度越高,效果越好;TP 0.03%以上可以明显提高鱼糜蛋白质的凝胶强度,但不具有剂量效应;TP 0.03%时,G′和G″最低,此时蛋白质变性交联程度最低。TP能改善蛋白质的乳化性和凝胶性。
3、利用GC-MS对草鱼鱼肉中的脂肪酸进行分析,鉴定出草鱼中含有棕榈酸、十六碳烯酸、油酸、亚油酸、亚麻酸、硬脂酸、花生四烯酸、DHA、EPA、十五烷酸、十七烷酸、十七碳三烯酸等十八种以上脂肪酸,除含有动植物中常见的偶数碳脂肪酸外,还含有一定量的奇数碳脂肪酸。其中饱和脂肪酸27.84%,单不饱和脂肪酸43.67%,多不饱和脂肪酸28.02%。
4、采用GC-MS测定了在4℃贮藏条件下,添加不同浓度TP的鱼肉脂肪中脂肪酸的变化,通过硫代巴比妥酸试验测定了脂肪酸氧化产物丙二醛的变化。结果表明:肉豆蔻酸、十五烷酸、十六烷酸、十七烷酸、十八烷酸、十六碳一烯酸、油酸、亚油酸、亚麻酸、十七碳三烯酸在贮藏5d以前的变化都不大,第10d检测到对照组各种脂肪酸的含量均明显下降,与添加TP试验组出现显著性差异(P＜0.05),而添加了TP的各试验组中上述脂肪酸在贮藏10d后相对含量没有发生很大变化。TBA试验结果显示,添加00.1%以上茶多酚对草鱼脂肪都有明显的抗氧化作用,随浓度增加TBA值增加,但不呈线性关系,0.03%～0.07%抗氧化效果比较接近,从经济、有效的角度考虑在实际应用中可采用0.03%的茶多酚。
5、从草鱼中分离出93株菌株,鉴定出以下几类主要腐败细菌:气单胞菌属、弧菌属、假单胞菌属、肠道杆菌科、黄杆菌属、邻单胞菌属、发光杆菌属、产碱杆菌属、短杆菌属、微球菌属、不动杆菌属、无色菌属。草鱼在贮藏过程中,前期(1～3d)以革兰氏阳性菌为主,占70%,多为短杆菌和微球菌;随时间延长,3d后微生物种类出现多样化且以革兰氏阴性菌为主,约占71%～82%,其中以假单胞菌、气单胞菌属、弧菌属与肠肝菌属为主,是导致鱼肉腐败的优势腐败菌。
6、通过滤纸圆片法和平板计数法,确定了茶多酚对鱼肉中的腐败菌即短杆菌、微球菌、气单胞菌、假单胞菌、弧菌、肠杆菌、产碱杆菌均具有很好的抑菌活性,且最小抑菌浓度不超过1.2g/L,对大多数的细菌来说,其最佳抑菌浓度为5g/L。
7、将山梨酸钾、Vc、茶多酚添加到鱼肉中比较保鲜效果,三组的保鲜效果均要好于对照组,其中以茶多酚保鲜效果最好,测得酸价、过氧化值最低,第11d测得的挥发性盐基氮和第9d测得的菌落总数均低于国家有关标准规定的一级鱼标准。
Fresh water fish is a kind of ideal food for people because it is rich in nutrients and good for health. In 2006, the product of fresh water in China was up to 23,500,000 tons and take the first place in the world. However, the technologies of fresh-keep and processing for fresh water fish in China didn't meet the development of acquaculture. The processing rate of fresh water fish was only 6.9% and far behind the worldwide average level of marine products processing (>40%). Tea polyphenols (TP), which is the generic terms of compounds with multiple hydroxyl groups in tea, has many functions of heath care and pharmacological effects, such as antioxidation, bacteriostasis, cancer-preventing, and cholesterol-lowering. In this project, the effects of TP on lipids, proteins, and microbe of fish meat from grass carp were studied to determine the possibility of TP applied in fresh-keeping of fresh water fish. The methodologies and results are described as following:
1. The changes of biochemical properties were studied by extractability and ATPase activity measurement, SDS-PAGE analysis, and ultrastructure observation of fish meat from grass carp treated with different concentration TP and stored at 4℃. The results revealed: the extractability and ATPase activity of protein decreased gradually with the time when the fish meat stored at 4℃, and these declines were retarded substantially by addition of TP. When the TP concentration was lower than 0.03% (w/w), the difference was insignificant (P>0.05), whereas the concentration was higher than 0.05%, the difference showed significant (P<0.05). The SDS-PAGE analysis showed the rate of protein degradation of fish meat was decelerated by applied of TP. The changes of ultrasturcture of fish surimi by scanning electro microscope checking confirmed the above observations. These results suggested that there were remarkable protecting effects of TP on biochemical properties of fish meat and the optimal concentration was about 0.05～0.07%.
2. The emulsion, gel property, and rheology of fish meat treated with different concentration of TP and stored at 4℃were studied using spectrophotometer, texture analyzer, and rheometer. The results indicated that the addition of more than 0.03% of TP could slow down the decrement of emulsion and emulsification stability of protein extracted from fish meat significantly and presented the does effects. In the meantime, 0.03% of TP increased the gel strength substantially and no does effect appeared. In rehological study, addition of TP reduced the storage and loss modulus (G' and G") compared to the negative control, and the G' and G" valves were the lowest when the TP was 0.03% suggesting that the extent of crosslink of proteins was the lowest.
3. The fatty acid composition of grass carp was analyzed using GS-MS and more than 18 fatty acids were identified, including hexadecanoic acid, 9-hexadecenoic acid, 9-octadecenoic acid, 6,9-octadecadienoic acid, 9,12,15-octadecatrienoic acid, octadecanoic acid, 5,8,11,14-eicosatetraenoic acid, 4,7,10,13,16,19-docosahexaenoic acid, 5,8,11,14,17-eicosapentaenoic acid, pentadecanoic acid, heptadecanoic acid, and 5,8,11-heptadecatrienoic acid. There were some fatty acids with odd carbon atoms besides the fatty acids with even carbon atoms common in animals and plants. Among the fatty acids, the saturated were taken 27.84%, the monounsaturated occupied 43.67%, and the polyunsaturated accounted for 28.02%.
4. The variations of fatty acids of fish meat treated with TP and stored at 4℃were assayed by GS-MS and the changes of malonaldehyde, the oxidative product of fatty acids, were measured by TBA experiment. The data indicated that there was no change in contents of fatty acids by 5 days after treatment, including tetradecanoate, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, 9-hexadecenoic acid, 9-octadecenoic acid, 6,9-octadecadienoic acid, 9,12,15-octadecatrienoic acid, and 5,8,11-heptadecatrienoic acid. There were obvious decreases in content of these fatty acids from fish meat without TP 10 days after treatment compared to the experimental groups (P<0.05) . TBA analysis showed 0.01% of TP had evident antioxidation on lipids from fish meat, and TBA increased with the TP concentration accretion presenting an unlinear relation. The effects of antioxidation of 0.03% and 0.07% of TP were close, thus, the best choice is 0.03% in practical application.
5. 93 bacteria strains were isolated and categorized into several spoilage bacteria classes: Aeromonas, Vibrio, Pseudomonas, Enterobacteriaceae, Flavobacterium, Acinetobacter, Photobacteriaceae, Brevibacterium, and Micrococcus. In the early stage of storage (1～3 days), Gram-positive bacteria were the major strains and accounted for 70%, most of them belonged to Brevibacterium and Micrococcus. Whereas the diversity was increased and Gram-negative bacteria became the dominant taking 71～82% after 3 days, the main strains such as Pseudomonas, Aeromonas, Vibrio and Enterobacteriaceae classed into spoilage bacteria attributing to the meat putrefaction.
6. The inhibition of TP on several spoilage bacteria from fish meat were determined by plate counting. TP presented potent inhibitory effect on Brevibacterium, Micrococcus, Aeromonas, Pseudomonas, Vibrio, and Enterobacteriaceae, and the minimum inhibition concentration (MIC) was lower than 1.2 g/L, whereas for most strains the optimal inhibition concentration was about 5 g/L.
7. The fresh-keeping effects of potassium sorbate, vitamine C, and TP were determined and showed good function compared to the negative control. Among three antioxidations, TP presented the best effect because the acid value, and peroxide value were the lowest, the total volatile basic nitrogen (TVBN) and total colonies number measured 11 days and 9 days after TP treatment respectively were lower than the first grade fish according to the GB.
引文
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