甲壳低聚糖酶法制备工艺优化及生理活性研究
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摘要
甲壳低聚糖(Chito-oligosaccharides)是壳聚糖降解后的产物。当壳聚糖降解为甲壳低聚糖后,单体分子结构并未发生变化。由于其聚合度低、分子量小,有良好的水溶性。因此,比壳聚糖具有更广泛的应用空间。
我国海产品资源丰富,虾壳、蟹壳作为水产品加工后剩余的下脚料,不仅造成浪费,更是产生大量的垃圾,污染环境。本实验重点探讨了制备甲壳低聚糖的最佳工艺条件,并针对MW<10kDa(分子量小于10000道尔顿)甲壳低聚糖的抗氧化、降血糖、降血脂以及吸附重金属等生理活性进行研究。研究结果如下:⑴选用成本低、降解效果好的纤维素酶对壳聚糖进行降解。测定不同酶解时间、酶浓度、pH值、温度条件下的降解效果,通过单因素实验和正交实验得出最佳制备甲壳低聚糖的工艺组合为:酶浓度1600U/g、酶解时间7 h、pH=5.4、温度55℃。以此工艺组合制备MW<10kDa甲壳低聚糖得率为69.51%。在影响甲壳低聚糖产率的4个因素中,pH对甲壳低聚糖的产率影响最显著(P<0.05),温度则影响最不显著(P>0.05)。
⑵通过甲壳低聚糖对(·OH)和(O2?·)的清除实验看出,甲壳低聚糖浓度为10 mg/ml时,对(·OH)和(O2?·)的清除率均能达到80%左右,浓度为30 mg/ml时,对(·OH)和(O2 ?·)的清除率均能接近100%。用甲壳低聚糖对小鼠灌胃30 d后测定小鼠体内的MDA、SOD和T-AOC指标,结果表明:中剂量组(400 mg/kg)和高剂量组(800 mg/kg)小鼠的血清和肝脏中SOD、T-AOC活力显著高于模型对照组(P<0.05),MDA含量显著低于模型对照组(P<0.05)。⑶对注射60 mg/kg四氧嘧啶诱导糖尿病的小鼠,甲壳低聚糖具有一定的降血糖功效,结果表明:灌胃甲壳低聚糖的小鼠血糖值与模型对照组有显著性差异(P<0.05),说明甲壳低聚糖能明显降低糖尿病小鼠的血糖值,但还未能完全降到正常血糖值水平。
⑷对不同分子量的甲壳低聚糖进行体外降脂实验,结果表明:MW>2kDa的甲壳低聚糖对油脂、胆酸盐和胆固醇具有较好的吸附性。动物实验则也表明:MW>2kDa甲壳低聚糖具备减轻体重、降低血脂、保护肝脏的作用,其中小鼠灌胃高剂量(400mg/kg)甲壳低聚糖效果最为明显。
⑸在甲壳低聚糖吸附贝类中重金属的研究中,将花蛤放在富含重金属的海水中饲养,得到富集重金属的花蛤模型,再将其放入加有甲壳低聚糖的海水中养殖,5d后通过原子吸收检测贝类体内的重金属残留量的差异。结果表明:甲壳低聚糖能促进花蛤对重金属离子的代谢,其中对Pb、Cu的效果较好,当甲壳低聚糖添加量为0.4g/L时,对Pb、Cu的代谢率分别能达到26.00%和21.47%,高于模型组的代谢率10.64%和11.83%;Zn次之,为8.40%,略高于模型组的7.41%;对Cd则无明显的效果。
Chito-oligosaccharides is the degradation product of Chitosan. The molecular structure of Chito-oligosaccharides is similar with Chitosan. Because of low polymerization degree, small molecular weight and good water-soluble, Chito-oligosaccharides have more extensive application space than Chitosan.
In our country there are abundant sea foods. Shrimp shell and crab shell which are regarded as residual leftover after aquatic product processing, not only produce lots of waste but also pollute environment. This experiment explores optimum technological conditions of preparing Chito-oligosaccharides, and studies on physiological activities of MW<10kDa(molecular weight is less than 10000 Dalton) Chito-oligosaccharides such as antioxidant, hypoglycemia, reducing blood lipid and biosorption of heavy metal. The research results as follows:
(1) This experiment selects cellulose which is low cost and has good degradation effect degrading Chitosan, and determines degradation effect in different enzymolysis time, enzyme concentration,pH and temperature. Through single factor experiment and orthogonal experiment, we obtain the best technological combination for producing Chito-oligosaccharides which the enzyme concentration is 1600U/g、enzymolysis time 7h, pH 5.4 and temperature 55℃. In four factors of influencing Chito-oligosaccharides production, pH has the most significant influence (P<0.05), and temperature has the least significant influence (P>0.05). On this technological combination of preparing MW<10kDa, the production rate of Chito-oligosaccharides is 69.51%.
(2) Through the experiment of Chito-oligosaccharides scavenging(·OH)and(O2?·)we can know when the Chito-oligosaccharides concentration is 10 mg/ml, the clearance to(·OH)and(O2 ?·)can reach about 80%, Chito-oligosaccharides concentration is 30 mg/ml, the clearance to(·OH)and(O2 ?·)can reach close to 100%.After the mice given Chito-oligosaccharides by gavage 30d I determine the MDA, SOD and T-AOC indexs of the mice. The results showed that the SOD, T-AOC activity in liver and serum of middle dose group (400 mg/kg) and high dose group (800 mg/kg)mice is higher than high fat model group mice significantly (P<0.05), MDA content is lower than high fat model group significantly (P<0.05).
(3) Chito-oligosaccharides have hypoglycemic function to diabetic mice induced by 60mg/kg alloxan. The results showed that the blood glucose value of mice given Chito-oligosaccharides by gavage have significant difference with the model control group (P<0.05). So the experiment illustrates Chito-oligosaccharides can decrease blood glucose value of diabetic mice obviously, but it can’t reduced to the normal blood glucose value completely.
(4) We choose the Chito-oligosaccharides of different molecular weigh to do descending blood lipid experiment in vitro. The result showed that the MW>2kDa Chito-oligosaccharides have good absorbability to oil, bile salts and cholesterol. Animal experiment also showed that MW>2kDa Chito-oligosaccharides have the function of reducing weight, decreasing blood lipid and protecting liver. Among which the high dose group has the most significant effect.
(5) In the experiment of adsorbing heavy metal in shellfish, I feed clam in the seawater rich with heavy metal to make shellfish model rich with heavy metal. Then I took the clam rich with heavy metal into seawater containing Chito-oligosaccharides. After 5d I detect the difference of heavy metal residue in shellfish by atomic absorption. The result showed that Chito-oligosaccharides can promote the clam’s metabolism of heavy metal. The effect of metabolism of Pb and Cu is better, when Chito-oligosaccharides concentration in seawater is 0.4g/L, the metabolic rate of Pb and Cu can reach to 26.00% and 21.47%, higher than model group which is 10.64% and 11.83%; the metabolic rate of Zn is 8.40%, in the second place, higher than model group which is 7.41% and to Cd it has not significant effect.
我国海产品资源丰富,虾壳、蟹壳作为水产品加工后剩余的下脚料,不仅造成浪费,更是产生大量的垃圾,污染环境。本实验重点探讨了制备甲壳低聚糖的最佳工艺条件,并针对MW<10kDa(分子量小于10000道尔顿)甲壳低聚糖的抗氧化、降血糖、降血脂以及吸附重金属等生理活性进行研究。研究结果如下:⑴选用成本低、降解效果好的纤维素酶对壳聚糖进行降解。测定不同酶解时间、酶浓度、pH值、温度条件下的降解效果,通过单因素实验和正交实验得出最佳制备甲壳低聚糖的工艺组合为:酶浓度1600U/g、酶解时间7 h、pH=5.4、温度55℃。以此工艺组合制备MW<10kDa甲壳低聚糖得率为69.51%。在影响甲壳低聚糖产率的4个因素中,pH对甲壳低聚糖的产率影响最显著(P<0.05),温度则影响最不显著(P>0.05)。
⑵通过甲壳低聚糖对(·OH)和(O2?·)的清除实验看出,甲壳低聚糖浓度为10 mg/ml时,对(·OH)和(O2?·)的清除率均能达到80%左右,浓度为30 mg/ml时,对(·OH)和(O2 ?·)的清除率均能接近100%。用甲壳低聚糖对小鼠灌胃30 d后测定小鼠体内的MDA、SOD和T-AOC指标,结果表明:中剂量组(400 mg/kg)和高剂量组(800 mg/kg)小鼠的血清和肝脏中SOD、T-AOC活力显著高于模型对照组(P<0.05),MDA含量显著低于模型对照组(P<0.05)。⑶对注射60 mg/kg四氧嘧啶诱导糖尿病的小鼠,甲壳低聚糖具有一定的降血糖功效,结果表明:灌胃甲壳低聚糖的小鼠血糖值与模型对照组有显著性差异(P<0.05),说明甲壳低聚糖能明显降低糖尿病小鼠的血糖值,但还未能完全降到正常血糖值水平。
⑷对不同分子量的甲壳低聚糖进行体外降脂实验,结果表明:MW>2kDa的甲壳低聚糖对油脂、胆酸盐和胆固醇具有较好的吸附性。动物实验则也表明:MW>2kDa甲壳低聚糖具备减轻体重、降低血脂、保护肝脏的作用,其中小鼠灌胃高剂量(400mg/kg)甲壳低聚糖效果最为明显。
⑸在甲壳低聚糖吸附贝类中重金属的研究中,将花蛤放在富含重金属的海水中饲养,得到富集重金属的花蛤模型,再将其放入加有甲壳低聚糖的海水中养殖,5d后通过原子吸收检测贝类体内的重金属残留量的差异。结果表明:甲壳低聚糖能促进花蛤对重金属离子的代谢,其中对Pb、Cu的效果较好,当甲壳低聚糖添加量为0.4g/L时,对Pb、Cu的代谢率分别能达到26.00%和21.47%,高于模型组的代谢率10.64%和11.83%;Zn次之,为8.40%,略高于模型组的7.41%;对Cd则无明显的效果。
Chito-oligosaccharides is the degradation product of Chitosan. The molecular structure of Chito-oligosaccharides is similar with Chitosan. Because of low polymerization degree, small molecular weight and good water-soluble, Chito-oligosaccharides have more extensive application space than Chitosan.
In our country there are abundant sea foods. Shrimp shell and crab shell which are regarded as residual leftover after aquatic product processing, not only produce lots of waste but also pollute environment. This experiment explores optimum technological conditions of preparing Chito-oligosaccharides, and studies on physiological activities of MW<10kDa(molecular weight is less than 10000 Dalton) Chito-oligosaccharides such as antioxidant, hypoglycemia, reducing blood lipid and biosorption of heavy metal. The research results as follows:
(1) This experiment selects cellulose which is low cost and has good degradation effect degrading Chitosan, and determines degradation effect in different enzymolysis time, enzyme concentration,pH and temperature. Through single factor experiment and orthogonal experiment, we obtain the best technological combination for producing Chito-oligosaccharides which the enzyme concentration is 1600U/g、enzymolysis time 7h, pH 5.4 and temperature 55℃. In four factors of influencing Chito-oligosaccharides production, pH has the most significant influence (P<0.05), and temperature has the least significant influence (P>0.05). On this technological combination of preparing MW<10kDa, the production rate of Chito-oligosaccharides is 69.51%.
(2) Through the experiment of Chito-oligosaccharides scavenging(·OH)and(O2?·)we can know when the Chito-oligosaccharides concentration is 10 mg/ml, the clearance to(·OH)and(O2 ?·)can reach about 80%, Chito-oligosaccharides concentration is 30 mg/ml, the clearance to(·OH)and(O2 ?·)can reach close to 100%.After the mice given Chito-oligosaccharides by gavage 30d I determine the MDA, SOD and T-AOC indexs of the mice. The results showed that the SOD, T-AOC activity in liver and serum of middle dose group (400 mg/kg) and high dose group (800 mg/kg)mice is higher than high fat model group mice significantly (P<0.05), MDA content is lower than high fat model group significantly (P<0.05).
(3) Chito-oligosaccharides have hypoglycemic function to diabetic mice induced by 60mg/kg alloxan. The results showed that the blood glucose value of mice given Chito-oligosaccharides by gavage have significant difference with the model control group (P<0.05). So the experiment illustrates Chito-oligosaccharides can decrease blood glucose value of diabetic mice obviously, but it can’t reduced to the normal blood glucose value completely.
(4) We choose the Chito-oligosaccharides of different molecular weigh to do descending blood lipid experiment in vitro. The result showed that the MW>2kDa Chito-oligosaccharides have good absorbability to oil, bile salts and cholesterol. Animal experiment also showed that MW>2kDa Chito-oligosaccharides have the function of reducing weight, decreasing blood lipid and protecting liver. Among which the high dose group has the most significant effect.
(5) In the experiment of adsorbing heavy metal in shellfish, I feed clam in the seawater rich with heavy metal to make shellfish model rich with heavy metal. Then I took the clam rich with heavy metal into seawater containing Chito-oligosaccharides. After 5d I detect the difference of heavy metal residue in shellfish by atomic absorption. The result showed that Chito-oligosaccharides can promote the clam’s metabolism of heavy metal. The effect of metabolism of Pb and Cu is better, when Chito-oligosaccharides concentration in seawater is 0.4g/L, the metabolic rate of Pb and Cu can reach to 26.00% and 21.47%, higher than model group which is 10.64% and 11.83%; the metabolic rate of Zn is 8.40%, in the second place, higher than model group which is 7.41% and to Cd it has not significant effect.
引文
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