毛蚶酶解液营养评价及其生理功效的研究
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摘要
毛蚶属于我国沿海重要的养殖经济贝类之一,其体内富含蛋白却含较低的脂肪,具有一定的药用价值,口感鲜美,深受大众喜爱。本课题以毛蚶为研究对象,研究酶解工艺条件对毛蚶水解效果的影响、毛蚶酶解液制备的工艺优化、酶解液中肽类物质的功能和特性,为开发利用毛蚶酶解液奠定基础。主要研究内容及结果如下：
(1)研究了不同酶解条件对蛋白酶水解毛蚶效果的影响。选取加酶量、温度、pH值和反应时间四个因素对蛋白酶酶解效果进行正交试验。
中性蛋白酶酶解工艺最佳条件为：加酶量0.8%,水解温度50℃,pH7.5的条件下反应7h；木瓜蛋白酶酶解工艺最佳条件为：加酶量1.0%,水解温度45℃,pH7.5的条件下反应8h。
(2)研究不同处理条件下蛋白质变性程度对酶解特性的影响。实验结果表明蛋白质变性程度增大,不利于酶解,水解度越低。
(3)通过SDS-PAGE电泳测定酶解液的相对分子量范围,发现酶解液中多为分子量14400Da左右的多肽。
(4)以酪蛋白组为对照组,分别用毛蚶原浆粉和两种蛋白酶水解后的酶解液粉状物作为饲料的蛋白质来源,配制人工半合成饲料,采用对喂法,进行大鼠营养生理功能实验。酶解组的蛋白质消化率和净利用率都比原浆组高。
在血脂方面,中性蛋白酶酶解组大鼠血清的TC、TG、LDL-C较原浆组大鼠低,HDL-C较原浆组大鼠高,证明这类酶解后的肽类物质对血脂调节具有显著地作用。
在抗氧化方面,中性蛋白酶酶解组大鼠血清及肝组织中的SOD、GSH-Px酶活性较原浆组大鼠的酶活性高,而MDA含量较原浆组大鼠的含量低,证实该酶解液中所含肽类物质可起到较好的抗氧化功效。
(5)将70只小鼠随机分成7组。以普通饲料喂食正常对照组小鼠,高脂饲料喂食其他各组。正常对照组和高脂模型组每天灌胃相应体积蒸馏水；Vc组和辛伐他汀组阳性药物组分别给予20mg/(kg·d)Vc、0.5mg/(kg·d)辛伐他汀；中性蛋白酶酶解高、中、低剂量组,分别以1500mg/(kg·d)、1000mg/(kg·d)、500mg/(kg·d)中性蛋白酶酶解液灌胃,连续给药6周后检测小鼠血清和组织各项指标。
实验结果证明中毛蚶酶解液对食源性高脂模型组小鼠的脂代谢具有较强的调节作用,可以显著地降低血清中的TC、TG、LDL-C,并升高血清中的HDL-C。
灌胃毛蚶酶解液组的小鼠肝、肾组织中的SOD及GSH-Px酶活性显著升高,MDA含量显著降低,证实该类酶解液在体内通过调节抗氧化物酶活力降低脂质过氧化的程度,而促进降脂。
Scapharca subcrenata is a significant economic shellfish widely bred in littoral of China. People like it for its peculiarity of high contents of protein, low fat content and its remedy effects. Scapharca subcrenata is object of study in this dissertation. In order to offer foundation for preparing peptide from Scapharca subcrenata,the aim of this experiment was to study the effects of hydrolyzing conditions on the hydrolysates of Scapharca subcrenata, the optimal technological of hydrolyzing Scapharca subcrenata with protease, and the characterization of peptide. The main results were as follows:
1. The effects of hydrolyzing conditions on the hydrolysates of Scapharca subcrenata. Enzyme quantity, temperature, pH and hydrolysis time were selected as four orthogonal factors. The results by using neutral protease to hydrolyze were as follows:enzyme quantity of0.8%, temperature50℃, pH7.5and time7h. Otherwise,the results by using papain were as follows:enzyme quantity of1.0%, temperature45℃, pH7.5and time8h.
2. Study on the denaturation level in different treatment and the effect of enzymatic hydrolysis. The result show that denaturation level rose while disadvantageous enzymatic hydrolyzing, the DH was reducing.
3. Used by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the result with molecular weight demonstrate show that lots of peptides from Scapharca subcrenata hydrolysate was about14400Da.
4. The diets were prepared with the crude Scapharca subcrenata meat or two kinds of protease hydrolysates and compared with casein. In order to study the nutritional values and physiological function of Scapharca subcrenata hydrolysates used pair fed method. The findings in antitumor test shown that neutral protease hydrolysates groups could improved the digestibility and protein efficiency ratio compared with crude group.
On aspect of blood lipids, neutral protease hydrolysates group diet could depress the serum cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C), while also can increase high-density lipoprotein cholesterol (HDL-C). So hydrolysis of Sinonovacula Constricta Lamarck obtain peptides had functions on blood lipids metabolism.
On the antioxidation aspect, neutral protease hydrolysates group diet can improve the activities of SOD and GSH-Px, while also can reduce the concentration of MDA in serum and liver.
5. The70mice were randomly divided into seven groups, the normal control group was fed by common feeds stuffs, while other groups fed by high-fat diet. Normal and model control groups were offered by corresponding volume of distilled water, two positive drug groups were offered by Vitam in C with20mg/(kg'd) and simvastatin with0.5mg/(kg'd) respectively. Large, medium and small dose groups were taken medicine by neutral protease hydrolysates with1500,1000and500mg/(kg·d) dose.The serum and tissue of rats in treatment groups was determined6weeks after treatment.
The protease hydrolysates of Scapharca subcrenata had potent effects on regulating blood lipids in hyperlipidemic rats. Protease hydrolysates could significantly reduce the TC,TG,LDL-C and increase HDL-C level in serum.
The activity of SOD and GSH-Px had raised, content of MDA had degraded, which proved that protease hydrolysates of Scapharca subcrenata regulated the antioxidation enzyme activity and then reduced the lipid peroxidization.
(1)研究了不同酶解条件对蛋白酶水解毛蚶效果的影响。选取加酶量、温度、pH值和反应时间四个因素对蛋白酶酶解效果进行正交试验。
中性蛋白酶酶解工艺最佳条件为：加酶量0.8%,水解温度50℃,pH7.5的条件下反应7h；木瓜蛋白酶酶解工艺最佳条件为：加酶量1.0%,水解温度45℃,pH7.5的条件下反应8h。
(2)研究不同处理条件下蛋白质变性程度对酶解特性的影响。实验结果表明蛋白质变性程度增大,不利于酶解,水解度越低。
(3)通过SDS-PAGE电泳测定酶解液的相对分子量范围,发现酶解液中多为分子量14400Da左右的多肽。
(4)以酪蛋白组为对照组,分别用毛蚶原浆粉和两种蛋白酶水解后的酶解液粉状物作为饲料的蛋白质来源,配制人工半合成饲料,采用对喂法,进行大鼠营养生理功能实验。酶解组的蛋白质消化率和净利用率都比原浆组高。
在血脂方面,中性蛋白酶酶解组大鼠血清的TC、TG、LDL-C较原浆组大鼠低,HDL-C较原浆组大鼠高,证明这类酶解后的肽类物质对血脂调节具有显著地作用。
在抗氧化方面,中性蛋白酶酶解组大鼠血清及肝组织中的SOD、GSH-Px酶活性较原浆组大鼠的酶活性高,而MDA含量较原浆组大鼠的含量低,证实该酶解液中所含肽类物质可起到较好的抗氧化功效。
(5)将70只小鼠随机分成7组。以普通饲料喂食正常对照组小鼠,高脂饲料喂食其他各组。正常对照组和高脂模型组每天灌胃相应体积蒸馏水；Vc组和辛伐他汀组阳性药物组分别给予20mg/(kg·d)Vc、0.5mg/(kg·d)辛伐他汀；中性蛋白酶酶解高、中、低剂量组,分别以1500mg/(kg·d)、1000mg/(kg·d)、500mg/(kg·d)中性蛋白酶酶解液灌胃,连续给药6周后检测小鼠血清和组织各项指标。
实验结果证明中毛蚶酶解液对食源性高脂模型组小鼠的脂代谢具有较强的调节作用,可以显著地降低血清中的TC、TG、LDL-C,并升高血清中的HDL-C。
灌胃毛蚶酶解液组的小鼠肝、肾组织中的SOD及GSH-Px酶活性显著升高,MDA含量显著降低,证实该类酶解液在体内通过调节抗氧化物酶活力降低脂质过氧化的程度,而促进降脂。
Scapharca subcrenata is a significant economic shellfish widely bred in littoral of China. People like it for its peculiarity of high contents of protein, low fat content and its remedy effects. Scapharca subcrenata is object of study in this dissertation. In order to offer foundation for preparing peptide from Scapharca subcrenata,the aim of this experiment was to study the effects of hydrolyzing conditions on the hydrolysates of Scapharca subcrenata, the optimal technological of hydrolyzing Scapharca subcrenata with protease, and the characterization of peptide. The main results were as follows:
1. The effects of hydrolyzing conditions on the hydrolysates of Scapharca subcrenata. Enzyme quantity, temperature, pH and hydrolysis time were selected as four orthogonal factors. The results by using neutral protease to hydrolyze were as follows:enzyme quantity of0.8%, temperature50℃, pH7.5and time7h. Otherwise,the results by using papain were as follows:enzyme quantity of1.0%, temperature45℃, pH7.5and time8h.
2. Study on the denaturation level in different treatment and the effect of enzymatic hydrolysis. The result show that denaturation level rose while disadvantageous enzymatic hydrolyzing, the DH was reducing.
3. Used by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the result with molecular weight demonstrate show that lots of peptides from Scapharca subcrenata hydrolysate was about14400Da.
4. The diets were prepared with the crude Scapharca subcrenata meat or two kinds of protease hydrolysates and compared with casein. In order to study the nutritional values and physiological function of Scapharca subcrenata hydrolysates used pair fed method. The findings in antitumor test shown that neutral protease hydrolysates groups could improved the digestibility and protein efficiency ratio compared with crude group.
On aspect of blood lipids, neutral protease hydrolysates group diet could depress the serum cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C), while also can increase high-density lipoprotein cholesterol (HDL-C). So hydrolysis of Sinonovacula Constricta Lamarck obtain peptides had functions on blood lipids metabolism.
On the antioxidation aspect, neutral protease hydrolysates group diet can improve the activities of SOD and GSH-Px, while also can reduce the concentration of MDA in serum and liver.
5. The70mice were randomly divided into seven groups, the normal control group was fed by common feeds stuffs, while other groups fed by high-fat diet. Normal and model control groups were offered by corresponding volume of distilled water, two positive drug groups were offered by Vitam in C with20mg/(kg'd) and simvastatin with0.5mg/(kg'd) respectively. Large, medium and small dose groups were taken medicine by neutral protease hydrolysates with1500,1000and500mg/(kg·d) dose.The serum and tissue of rats in treatment groups was determined6weeks after treatment.
The protease hydrolysates of Scapharca subcrenata had potent effects on regulating blood lipids in hyperlipidemic rats. Protease hydrolysates could significantly reduce the TC,TG,LDL-C and increase HDL-C level in serum.
The activity of SOD and GSH-Px had raised, content of MDA had degraded, which proved that protease hydrolysates of Scapharca subcrenata regulated the antioxidation enzyme activity and then reduced the lipid peroxidization.
引文
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