草鱼鱼鳞中活性胶原蛋白的制备与性能研究
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摘要
我国淡水鱼资源丰富,低值淡水鱼的深加工比例日益增加。目前,淡水鱼深加工的下脚料如鱼鳞、鱼骨等未得到充分利用,不仅浪费资源而且污染环境。研究表明,鱼鳞中含有丰富的优质胶原蛋白,在医药、食品等领域具有广泛的应用前景,充分利用这些胶原蛋白资源开发高附加值的产品不仅能扩大水产品加工利用途径,同时可以有效提高水产加工企业的综合经济效益。
本论文以草鱼鱼鳞为原料提取酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(PSC),重点讨论了两种胶原产品的制备工艺路线及其条件参数的优化。研究结果表明,在鱼鳞原料的前处理工序中,采用Na2CO3溶液浸泡的方法可有效脱除鱼鳞原料中的杂蛋白,其最佳工艺条件为:0.5M的Na2CO3溶液,料液比1:20,浸泡时间为6h ,重复三次即可达到最优的杂蛋白脱除效果;鱼鳞的脱钙工艺采用EDTA络合法,单因素试验表明,用EDTA溶液对鱼鳞进行脱钙的最佳条件为:EDTA浓度0.3M,料液比为1/60,脱钙时间为12h,脱钙三次即可达到最大脱钙率。
超声波辅助处理可有效提高鱼鳞中杂蛋白的脱除效果。超声波辅助脱除鱼鳞中杂蛋白的单因素试验表明,超声波处理的最佳频率是60Hz,最佳处理时间为10min,超声波辅助对鱼鳞中钙离子的脱除无显著性影响。
鱼鳞经前处理后,采用醋酸浸提的方法制取ASC产品,采用胃蛋白酶促溶结合醋酸提取的方法制取PSC产品。ASC提取实验表明,当醋酸浓度为0.8M,料液比为1/60,提取4h,可获得最大的提取率。PSC提取实验表明,醋酸浓度1.5M,料液比1/40,胃蛋白酶添加量为鱼鳞用量的2.5%,提取温度20℃,提取时间24h,可达到最大提取率。
分别采用SDS-PAGE凝胶电泳、红外光谱分析、氨基酸组成分析等方法对制备所得到的胶原蛋白产品进行初步的结构鉴定。结果表明,与猪皮胶原蛋白相比,ASC和PSC在红外光谱特征及亚基组成上与猪皮胶原蛋白基本一致,均为典型的Ⅰ型胶原蛋白;在氨基酸组成方面,ASC、PSC以及猪皮胶原蛋白均存在不同程度的差异,这种差异与原料来源及提取方法的差异有关。
在上述研究的基础之上,对草鱼鱼鳞胶原蛋白ASC和PSC的热变性温度、溶解性、乳化性等理化性质进行了系统研究,结果表明,ASC和PSC的热变性温度分别为30～35℃和28～35℃;PSC在溶解性能、乳化性能和乳化稳定性方面优于ASC,而在溶液黏度、吸水性能、吸油性能、起泡性以及泡沫稳定性方面,ASC明显好于PSC。
采用动态流变仪对ASC和PSC的溶液流体性能及其影响因素进行了深入研究,研究结果表明,在实验选择的剪切速率范围内,ASC和PSC溶液均表现为典型非牛顿流体的假塑性流动行为,即溶液黏度随剪切速率的增加而减小;在相同浓度条件下,溶液黏度的大小依次为ASC>PSC>明胶;剪切速率、温度、pH值等因素对ASC和PSC溶液流体性能均有显著影响;在0.6%浓度条件下,ASC和PSC溶液主要表现为流体行为,基本不具备凝胶的弹性行为;胶原蛋白发生热变性后,其流体的弹性行为增强而黏性行为下降。
The resource of freshwater fish of China is rich,and the proportion of freshwater fish further processing is increasing.Nowadays the waste materials left from the processing,such as the scales,the bones and so on haven't been utilized sufficiently,which result in the wast of resorce and the pollution of environment. The scales contain rich collagen,which has a bright future in the area of medicine,food and so on.
The full use of this resorce of collagen in the development of product with high additional value not only can help widen the path of the utilization of the aquatic products,but also can improve the comprehensive economic benefit of aquatic processing enterprises.
Aquatic collagen study of China is still at an initial stage in primary,and there is still little further study on extraction procedure and the physical and chemical properties,which has restricted the development of product from freshwater fish collagen.In this paper,the preparation of acid soluble collagen(ASC)and pepsin-solubilized collagen(PSC) and parameters optimization have been studied.
The results show that Na2CO3 can remove the waste protein of the scales effectively.The best condition can be abtained when the concentration of Na2CO3 is 0.5M,material to liquid ratio is 1/20,the processing time is 6h,and maximum removal rate can be achieved after 3 times of removement.The mineral matters are moved by EDTA complexing method.The single factor experiment show that when the concentration of EDTA is 0.3M ,the material to liquid ratio is 1/60,the time of processing is 12h,and the most ideal effect of decalcification can be abtained after 3 times of repetation.
Ultrasonic extraction can improve the effect of decalcification and the removement of other protein.The best effect of decalcification can be abtained when the processing time is 20min on the ultrasonic frequency of 40Hz,however, that of removement of other protein can be abtained when the processing time is 10min on the ultrasonic frequency of 60Hz.
The result of ASC extraction experiment show that the most optimum effect is abtained when the acetic acid concentration is 0.8M,the material to liquid ratio is 1/60,and the maximum extraction rate is received after 4h.The result of PSC extraction experiment show that the maximum extraction rate is abtained when the aceticacid concentration is 1.5M,material to liquid ratio is 1/40,the pepsin concentration is 3%,the extraction temperature is 20℃,the processing time is 24h.
The structural characteristics of the collagen samples preparared are identification by SDS-PAGE, infrared analysis,amino acid analysis.The result show that ASC and PSC are typical typeⅠcollagen,similar with pigskin collagen on infrared spectra and subunit composition;however,ASC,PSC and pigskin collagen are different on the amino acid composition in different extent,which result from the different source of material and the extraction method.
Based on the study above, the physical and chemical properties,such as thermal denaturation temperature, dissolve ability and emulsification property are studied systematically.The result show that the thermal denaturation temperature of ASC is 30～35℃,and of PSC is 28～35℃;PSC is better than ASC on solubility, emulsification property,but worse than ASC on solution viscosity, water absorption ability,oil-absorbing capacity and foamability.
The rheology properties of solution take an important part in the collagen chemical and physical properties,which is meaningful for the usetage of ASC and PSC in the areas of food and medicine.The result show that in the shear rates chosed, the solution of ASC and PSC perform as the pseudoplastic of typical non-newtonian fuild.In the same degree of concentration,the viscosity of the collagen solution ranged as ASC> PSC> gelatin; shear rate, the factors of temperature,pH and so on affect the properties of the solution of ASC and PSC significantly;when the concentration of the solution of collagen is 0.6%,ASC and PSC perform as fluid,not gel;the behavioral elasticity is enhanced and the viscosity behavior is weakened.
本论文以草鱼鱼鳞为原料提取酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(PSC),重点讨论了两种胶原产品的制备工艺路线及其条件参数的优化。研究结果表明,在鱼鳞原料的前处理工序中,采用Na2CO3溶液浸泡的方法可有效脱除鱼鳞原料中的杂蛋白,其最佳工艺条件为:0.5M的Na2CO3溶液,料液比1:20,浸泡时间为6h ,重复三次即可达到最优的杂蛋白脱除效果;鱼鳞的脱钙工艺采用EDTA络合法,单因素试验表明,用EDTA溶液对鱼鳞进行脱钙的最佳条件为:EDTA浓度0.3M,料液比为1/60,脱钙时间为12h,脱钙三次即可达到最大脱钙率。
超声波辅助处理可有效提高鱼鳞中杂蛋白的脱除效果。超声波辅助脱除鱼鳞中杂蛋白的单因素试验表明,超声波处理的最佳频率是60Hz,最佳处理时间为10min,超声波辅助对鱼鳞中钙离子的脱除无显著性影响。
鱼鳞经前处理后,采用醋酸浸提的方法制取ASC产品,采用胃蛋白酶促溶结合醋酸提取的方法制取PSC产品。ASC提取实验表明,当醋酸浓度为0.8M,料液比为1/60,提取4h,可获得最大的提取率。PSC提取实验表明,醋酸浓度1.5M,料液比1/40,胃蛋白酶添加量为鱼鳞用量的2.5%,提取温度20℃,提取时间24h,可达到最大提取率。
分别采用SDS-PAGE凝胶电泳、红外光谱分析、氨基酸组成分析等方法对制备所得到的胶原蛋白产品进行初步的结构鉴定。结果表明,与猪皮胶原蛋白相比,ASC和PSC在红外光谱特征及亚基组成上与猪皮胶原蛋白基本一致,均为典型的Ⅰ型胶原蛋白;在氨基酸组成方面,ASC、PSC以及猪皮胶原蛋白均存在不同程度的差异,这种差异与原料来源及提取方法的差异有关。
在上述研究的基础之上,对草鱼鱼鳞胶原蛋白ASC和PSC的热变性温度、溶解性、乳化性等理化性质进行了系统研究,结果表明,ASC和PSC的热变性温度分别为30～35℃和28～35℃;PSC在溶解性能、乳化性能和乳化稳定性方面优于ASC,而在溶液黏度、吸水性能、吸油性能、起泡性以及泡沫稳定性方面,ASC明显好于PSC。
采用动态流变仪对ASC和PSC的溶液流体性能及其影响因素进行了深入研究,研究结果表明,在实验选择的剪切速率范围内,ASC和PSC溶液均表现为典型非牛顿流体的假塑性流动行为,即溶液黏度随剪切速率的增加而减小;在相同浓度条件下,溶液黏度的大小依次为ASC>PSC>明胶;剪切速率、温度、pH值等因素对ASC和PSC溶液流体性能均有显著影响;在0.6%浓度条件下,ASC和PSC溶液主要表现为流体行为,基本不具备凝胶的弹性行为;胶原蛋白发生热变性后,其流体的弹性行为增强而黏性行为下降。
The resource of freshwater fish of China is rich,and the proportion of freshwater fish further processing is increasing.Nowadays the waste materials left from the processing,such as the scales,the bones and so on haven't been utilized sufficiently,which result in the wast of resorce and the pollution of environment. The scales contain rich collagen,which has a bright future in the area of medicine,food and so on.
The full use of this resorce of collagen in the development of product with high additional value not only can help widen the path of the utilization of the aquatic products,but also can improve the comprehensive economic benefit of aquatic processing enterprises.
Aquatic collagen study of China is still at an initial stage in primary,and there is still little further study on extraction procedure and the physical and chemical properties,which has restricted the development of product from freshwater fish collagen.In this paper,the preparation of acid soluble collagen(ASC)and pepsin-solubilized collagen(PSC) and parameters optimization have been studied.
The results show that Na2CO3 can remove the waste protein of the scales effectively.The best condition can be abtained when the concentration of Na2CO3 is 0.5M,material to liquid ratio is 1/20,the processing time is 6h,and maximum removal rate can be achieved after 3 times of removement.The mineral matters are moved by EDTA complexing method.The single factor experiment show that when the concentration of EDTA is 0.3M ,the material to liquid ratio is 1/60,the time of processing is 12h,and the most ideal effect of decalcification can be abtained after 3 times of repetation.
Ultrasonic extraction can improve the effect of decalcification and the removement of other protein.The best effect of decalcification can be abtained when the processing time is 20min on the ultrasonic frequency of 40Hz,however, that of removement of other protein can be abtained when the processing time is 10min on the ultrasonic frequency of 60Hz.
The result of ASC extraction experiment show that the most optimum effect is abtained when the acetic acid concentration is 0.8M,the material to liquid ratio is 1/60,and the maximum extraction rate is received after 4h.The result of PSC extraction experiment show that the maximum extraction rate is abtained when the aceticacid concentration is 1.5M,material to liquid ratio is 1/40,the pepsin concentration is 3%,the extraction temperature is 20℃,the processing time is 24h.
The structural characteristics of the collagen samples preparared are identification by SDS-PAGE, infrared analysis,amino acid analysis.The result show that ASC and PSC are typical typeⅠcollagen,similar with pigskin collagen on infrared spectra and subunit composition;however,ASC,PSC and pigskin collagen are different on the amino acid composition in different extent,which result from the different source of material and the extraction method.
Based on the study above, the physical and chemical properties,such as thermal denaturation temperature, dissolve ability and emulsification property are studied systematically.The result show that the thermal denaturation temperature of ASC is 30～35℃,and of PSC is 28～35℃;PSC is better than ASC on solubility, emulsification property,but worse than ASC on solution viscosity, water absorption ability,oil-absorbing capacity and foamability.
The rheology properties of solution take an important part in the collagen chemical and physical properties,which is meaningful for the usetage of ASC and PSC in the areas of food and medicine.The result show that in the shear rates chosed, the solution of ASC and PSC perform as the pseudoplastic of typical non-newtonian fuild.In the same degree of concentration,the viscosity of the collagen solution ranged as ASC> PSC> gelatin; shear rate, the factors of temperature,pH and so on affect the properties of the solution of ASC and PSC significantly;when the concentration of the solution of collagen is 0.6%,ASC and PSC perform as fluid,not gel;the behavioral elasticity is enhanced and the viscosity behavior is weakened.
引文
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