黄河鲤鱼鳞胶原蛋白的性质及胶原肽活性研究
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摘要
我国是淡水鱼养殖大国,黄河鲤鱼是淡水鱼中常见的一种。鱼鳞中胶原蛋白含量较为丰富,然而,鱼鳞多作为废弃物处理,不仅浪费资源,而且对环境造成污染。以鱼鳞为原料提取胶原蛋白和明胶,以及进一步生产具有生理功能的生物活性肽,既能减轻环境污染,又充分利用了鱼鳞资源,达到了淡水鱼的综合利用目的,同时还提高了鱼类产品的附加值。
本研究以黄河鲤鱼鳞为原料,对鱼鳞的预处理、胶原蛋白的提取技术、胶原蛋白的化学结构和理化性质、明胶的制备工艺和功能性质进行了研究,建立了黄河鲤鱼鳞胶原蛋白和明胶的提取技术。对鱼鳞明胶消化产物的抗氧化活性、酪氨酸酶抑制能力以及对小鼠B16黑素瘤细胞黑色素合成的影响进行了研究。
首先对黄河鲤鱼鳞的预处理工艺进行了研究。考察了不同工艺参数对鱼鳞矿物质脱除的影响。结果表明稀HCl溶液能够有效脱除鱼鳞中矿物质成分,得出最优工艺条件为：浸泡时间95min> HCl溶液浓度1.0M和料液比11mL/g。考察了碱液处理条件对鱼鳞杂蛋白脱除的影响。得出最优工艺条件为：浸泡时间26h、NaOH溶液浓度0.17M和料液比11mL/g。对鱼鳞预处理流程进行了比较研究。研究的结果表明：先脱除矿物质成分后更有利于鱼鳞中杂蛋白成分的去除。
研究了黄河鲤鱼鳞胶原蛋白的理化性质和结构。对鱼鳞胶原蛋白进行了电泳分析,研究结果显示鱼鳞胶原蛋白属于Ⅰ型胶原,分子构成可能为(α1)2α2型或α1α2α3型。红外光谱分析表明PSC与ASC的主体构型没有差异,胃蛋白酶不会造成胶原蛋白三螺旋结构的破坏。黄河鲤鱼鳞ASC和PSC的热变性温度分别为31.4℃和31.8℃,其热收缩温度分别为52.9℃和55.3℃。溶解性实验表明黄河鲤鱼鳞ASC和PSC的等电点都在pH7左右。
研究了黄河鲤鱼鳞明胶的提取工艺。工艺参数包括：提取温度、超声时间、超声功率和料液比,结果表明黄河鲤鱼鳞明胶的超声辅助热水提取最佳工艺参数为：提取温度70℃、超声处理时间100min、超声功率300W.料液比10mL/g。在此条件下制取的鱼鳞明胶粘度为4.8mPa-s,凝冻强度为186Bloom g,等离子点为pH6.2。
对黄河鲤鱼鳞明胶的模拟消化进行了研究。在37℃温度条件下,先采用胃蛋白酶对鱼鳞明胶进行2h酶解处理,随后采用胰蛋白酶消化2h研究鱼鳞明胶在胃肠道的消化特性。对鱼鳞明胶模拟消化的研究表明：胃蛋白酶对鱼鳞明胶的消化能力较弱,对胶原α-链和β-链不具有水解作用,而胰蛋白酶对鱼鳞明胶肽链有较强的酶解作用,鱼鳞明胶经消化后水解度可到达16.9%。对鱼鳞明胶消化产物的抗氧化活性和酪氨酸酶酶活性进行了研究,结果表明：消化产物的抗氧化性能和酪氨酸酶抑制活力与其消化过程中水解度有较强的相关性。
应用超滤技术将黄河鲤鱼鳞明胶消化产物按相对分子质量的不同分为3个组分：JCP1(Mw>3000Da)、JCP2(1000Da 研究了黄河鲤鱼鳞明胶消化产物中不同相对分子质量组分的酪氨酸酶抑制活性,研究结果表明,JCP3组分对酪氨酸酶单酚酶稳定态活力的抑制作用最强,其IC50值为75.24μg/mL。不同相对分子质量的鱼鳞明胶肽均能对酪氨酸酶的二酚酶活性产生抑制作用,且具有明显的量效关系,其中JCP3组分具有最强的酪氨酸酶二酚酶活性抑制作用,其IC50值为51.11μg/mL。
研究了黄河鲤鱼鳞明胶消化产物对小鼠B16黑素瘤细胞的药效学效果,实验结果表明：黄河鲤鱼鳞明胶模拟消化产物能够促进小鼠B16黑素瘤细胞的增殖,对小鼠B16黑素瘤细胞无细胞毒性。研究了鱼鳞明胶消化产物中JCP3组分对小鼠B16黑素瘤细胞黑色素生物合成的影响,研究结果表明：JCP3组分能够减少小鼠B16黑素瘤细胞中黑色素的产生量,能够降低小鼠B16黑素瘤细胞中酪氨酸酶的活力,能够增加小鼠B16黑素瘤细胞中GSH的含量和降低其GSSG的含量,能够降低小鼠B16黑素瘤细胞中cAPM的含量。
China is the largest country in production freshwater fish. And the Cyprinus carpio haematopterus is a common kind of freshwater fish. Fish scale, which are relatively rich in collagens, are directly discarded into the environment, which not only wastes resources but also pollutes the environment. Collagen and gelatin, as well as bioactive peptide can be obtained from Cyprinus carpio haematopterus scale. It can be not only reducing environment pollution but also making full use and comprehensive utilization of fish scales. At the same time, the new product based on fish scale may improve fish value.
This study investigated the pre-treatment technology, collagen extraction technology, collagen structure, properties and gelatin extraction technology of Cyprinus carpio haematopterus scale. And the antioxidant activities, the tyrosinase inhibition activities and the effects on melanin content in B16melanoma cell of Cyprinus carpio haematopterus scale gelatin digestion.
First, it investigated the pre-treatment technology of Cyprinus carpio haematopterus scale. It investigated the effects of various processing parameters on demineralization of Cyprinus carpio haematopterus scale. It was observed that the HCl solution can Effectively removing the minerals of fish scale. It was concluded that the optimum conditions were as follows:extraction time of95min, concentration of HCl of1.0M, and ratio of material to solution of11mL/g. It investigated the effects of NaOH on deproteinization of Cyprinus carpio haematopterus scale. And the optimum conditions were as follows:extraction time of26h, concentration of NaOH of1.0M, and ratio of material to solution of11mL/g.
The properties and structure of Cyprinus carpio haematopterus scale collagen were studied. The results suggested that the collagen of Cyprinus carpio haematopterus scale classified as type I collagen. Combined with the results of SDS-PAGE, the collagen was (al)2α2or ala2a3trimers. FTIR investigations showed that the existence three helical conformation of PSC. There is no difference of major structure of PSC and ASC indicates that the structure of Cyprinus carpio haematopterus scale collagen could not be destroyed by pepsin. The denaturation temperature of ASC and PSC of Cyprinus carpio haematopterus scale were31.4℃and31.8℃respectively, and the shrinkage temperature were52.9℃and55.3℃respectively. Futhermore, the solubility of fish scale collagen were affected by pH value and salt concentration. And the isoelectric point of ASC and PSC were at about pH7.
The gelatin extraction technology of Cyprinus carpio haematopterus scales were studied. The investigated parameters were extraction temperature, ultrasonic time, ultrasonic power and ratio of solvent to solid. The optimum extraction conditions were found to be:optimized extraction temperature70℃, ultrasonic time100min, ultrasonic power300W and ratio of solvent to solid10mL/g.
It investigated the simulated gastrointestinal (GI) digestion of Cyprinus carpio haematopterus scale gelatin. A two-stage in vitro digestion model system (a pepsin treatment for2h followed by a pancreatin treatment for2h, both at37℃) was used to simulate the process of human gastrointestinal (GI) digestion to determine the digestibility of fish scale gelatin. The results showed that there is very low digestive power of pepsin on a chain and β chain of scale collagen compared with pancreatin. And the final degree of hydrolysis of GI digests was16.9%. The changes in antioxidant activities and tyrosinase inhibition activities of fish scale gelatin during simulated gastrointestinal (GI) digestion were studied. The results suggested that the antioxidant activities and tyrosinase inhibition activities of fish scale gelatin correlates with the degree of hydrolysis.
The digests Cyprinus carpio haematopterus scale gelatin were fractionated into three ranges of molecular weight (JCP1>3000Da,1000Da The tyrosinase inhibition activities of fish scale gelatin digests were studied. The results indicated that JCP3has the strongest inhibitory ability on the steady-state monophenolase activiey of tyrosinase, and the IC50to monophenolase activiey of tyrosinase was75.24μg/mL. The different molecular weight fish scale gelatin digests showed inhibitory ability on the diphenolase activiey of tyrosinase, and the tyrosinase inhibition activities were related to their concentrations use. JCP3showed the strongest inhibitory ability on the diphenolase activiey of tyrosinase, and the IC50to diphenolase activiey of tyrosinase was51.11μg/mL.
The pharmacodynamics effect of Cyprinus carpio haematopterus scale gelatin digests on the mouse B16melanoma cells was studied. The results showed that the digests could markedly improve the proliferation of the mouse B16melanoma cells and had no cytotoxic activity. The effect of JCP3on melanin biosynthesis of the mouse B16melanoma cells and the molecular mechanism were investigated from the aspects of antioxidant. The results indicated that Cyprinus carpio haematopterus scale gelatin digests exerted marked inhibition on productions of melanin and the activity of tyrosinase. And the Cyprinus carpio haematopterus scale gelatin digests exerted marked inhibition on productions of melanin and the activity of tyrosinase. And the pharmacodynamics effect of Cyprinus carpio haematopterus scale gelatin digests could maintain the GSH levels of cells. Besides, it could decrease intracellular levels of GSSG and cAMP significantly.
本研究以黄河鲤鱼鳞为原料,对鱼鳞的预处理、胶原蛋白的提取技术、胶原蛋白的化学结构和理化性质、明胶的制备工艺和功能性质进行了研究,建立了黄河鲤鱼鳞胶原蛋白和明胶的提取技术。对鱼鳞明胶消化产物的抗氧化活性、酪氨酸酶抑制能力以及对小鼠B16黑素瘤细胞黑色素合成的影响进行了研究。
首先对黄河鲤鱼鳞的预处理工艺进行了研究。考察了不同工艺参数对鱼鳞矿物质脱除的影响。结果表明稀HCl溶液能够有效脱除鱼鳞中矿物质成分,得出最优工艺条件为：浸泡时间95min> HCl溶液浓度1.0M和料液比11mL/g。考察了碱液处理条件对鱼鳞杂蛋白脱除的影响。得出最优工艺条件为：浸泡时间26h、NaOH溶液浓度0.17M和料液比11mL/g。对鱼鳞预处理流程进行了比较研究。研究的结果表明：先脱除矿物质成分后更有利于鱼鳞中杂蛋白成分的去除。
研究了黄河鲤鱼鳞胶原蛋白的理化性质和结构。对鱼鳞胶原蛋白进行了电泳分析,研究结果显示鱼鳞胶原蛋白属于Ⅰ型胶原,分子构成可能为(α1)2α2型或α1α2α3型。红外光谱分析表明PSC与ASC的主体构型没有差异,胃蛋白酶不会造成胶原蛋白三螺旋结构的破坏。黄河鲤鱼鳞ASC和PSC的热变性温度分别为31.4℃和31.8℃,其热收缩温度分别为52.9℃和55.3℃。溶解性实验表明黄河鲤鱼鳞ASC和PSC的等电点都在pH7左右。
研究了黄河鲤鱼鳞明胶的提取工艺。工艺参数包括：提取温度、超声时间、超声功率和料液比,结果表明黄河鲤鱼鳞明胶的超声辅助热水提取最佳工艺参数为：提取温度70℃、超声处理时间100min、超声功率300W.料液比10mL/g。在此条件下制取的鱼鳞明胶粘度为4.8mPa-s,凝冻强度为186Bloom g,等离子点为pH6.2。
对黄河鲤鱼鳞明胶的模拟消化进行了研究。在37℃温度条件下,先采用胃蛋白酶对鱼鳞明胶进行2h酶解处理,随后采用胰蛋白酶消化2h研究鱼鳞明胶在胃肠道的消化特性。对鱼鳞明胶模拟消化的研究表明：胃蛋白酶对鱼鳞明胶的消化能力较弱,对胶原α-链和β-链不具有水解作用,而胰蛋白酶对鱼鳞明胶肽链有较强的酶解作用,鱼鳞明胶经消化后水解度可到达16.9%。对鱼鳞明胶消化产物的抗氧化活性和酪氨酸酶酶活性进行了研究,结果表明：消化产物的抗氧化性能和酪氨酸酶抑制活力与其消化过程中水解度有较强的相关性。
应用超滤技术将黄河鲤鱼鳞明胶消化产物按相对分子质量的不同分为3个组分：JCP1(Mw>3000Da)、JCP2(1000Da
研究了黄河鲤鱼鳞明胶消化产物对小鼠B16黑素瘤细胞的药效学效果,实验结果表明：黄河鲤鱼鳞明胶模拟消化产物能够促进小鼠B16黑素瘤细胞的增殖,对小鼠B16黑素瘤细胞无细胞毒性。研究了鱼鳞明胶消化产物中JCP3组分对小鼠B16黑素瘤细胞黑色素生物合成的影响,研究结果表明：JCP3组分能够减少小鼠B16黑素瘤细胞中黑色素的产生量,能够降低小鼠B16黑素瘤细胞中酪氨酸酶的活力,能够增加小鼠B16黑素瘤细胞中GSH的含量和降低其GSSG的含量,能够降低小鼠B16黑素瘤细胞中cAPM的含量。
China is the largest country in production freshwater fish. And the Cyprinus carpio haematopterus is a common kind of freshwater fish. Fish scale, which are relatively rich in collagens, are directly discarded into the environment, which not only wastes resources but also pollutes the environment. Collagen and gelatin, as well as bioactive peptide can be obtained from Cyprinus carpio haematopterus scale. It can be not only reducing environment pollution but also making full use and comprehensive utilization of fish scales. At the same time, the new product based on fish scale may improve fish value.
This study investigated the pre-treatment technology, collagen extraction technology, collagen structure, properties and gelatin extraction technology of Cyprinus carpio haematopterus scale. And the antioxidant activities, the tyrosinase inhibition activities and the effects on melanin content in B16melanoma cell of Cyprinus carpio haematopterus scale gelatin digestion.
First, it investigated the pre-treatment technology of Cyprinus carpio haematopterus scale. It investigated the effects of various processing parameters on demineralization of Cyprinus carpio haematopterus scale. It was observed that the HCl solution can Effectively removing the minerals of fish scale. It was concluded that the optimum conditions were as follows:extraction time of95min, concentration of HCl of1.0M, and ratio of material to solution of11mL/g. It investigated the effects of NaOH on deproteinization of Cyprinus carpio haematopterus scale. And the optimum conditions were as follows:extraction time of26h, concentration of NaOH of1.0M, and ratio of material to solution of11mL/g.
The properties and structure of Cyprinus carpio haematopterus scale collagen were studied. The results suggested that the collagen of Cyprinus carpio haematopterus scale classified as type I collagen. Combined with the results of SDS-PAGE, the collagen was (al)2α2or ala2a3trimers. FTIR investigations showed that the existence three helical conformation of PSC. There is no difference of major structure of PSC and ASC indicates that the structure of Cyprinus carpio haematopterus scale collagen could not be destroyed by pepsin. The denaturation temperature of ASC and PSC of Cyprinus carpio haematopterus scale were31.4℃and31.8℃respectively, and the shrinkage temperature were52.9℃and55.3℃respectively. Futhermore, the solubility of fish scale collagen were affected by pH value and salt concentration. And the isoelectric point of ASC and PSC were at about pH7.
The gelatin extraction technology of Cyprinus carpio haematopterus scales were studied. The investigated parameters were extraction temperature, ultrasonic time, ultrasonic power and ratio of solvent to solid. The optimum extraction conditions were found to be:optimized extraction temperature70℃, ultrasonic time100min, ultrasonic power300W and ratio of solvent to solid10mL/g.
It investigated the simulated gastrointestinal (GI) digestion of Cyprinus carpio haematopterus scale gelatin. A two-stage in vitro digestion model system (a pepsin treatment for2h followed by a pancreatin treatment for2h, both at37℃) was used to simulate the process of human gastrointestinal (GI) digestion to determine the digestibility of fish scale gelatin. The results showed that there is very low digestive power of pepsin on a chain and β chain of scale collagen compared with pancreatin. And the final degree of hydrolysis of GI digests was16.9%. The changes in antioxidant activities and tyrosinase inhibition activities of fish scale gelatin during simulated gastrointestinal (GI) digestion were studied. The results suggested that the antioxidant activities and tyrosinase inhibition activities of fish scale gelatin correlates with the degree of hydrolysis.
The digests Cyprinus carpio haematopterus scale gelatin were fractionated into three ranges of molecular weight (JCP1>3000Da,1000Da
The pharmacodynamics effect of Cyprinus carpio haematopterus scale gelatin digests on the mouse B16melanoma cells was studied. The results showed that the digests could markedly improve the proliferation of the mouse B16melanoma cells and had no cytotoxic activity. The effect of JCP3on melanin biosynthesis of the mouse B16melanoma cells and the molecular mechanism were investigated from the aspects of antioxidant. The results indicated that Cyprinus carpio haematopterus scale gelatin digests exerted marked inhibition on productions of melanin and the activity of tyrosinase. And the Cyprinus carpio haematopterus scale gelatin digests exerted marked inhibition on productions of melanin and the activity of tyrosinase. And the pharmacodynamics effect of Cyprinus carpio haematopterus scale gelatin digests could maintain the GSH levels of cells. Besides, it could decrease intracellular levels of GSSG and cAMP significantly.
引文
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