珠蚌抗氧化肽的加工工艺研究
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摘要
我国是珍珠生产大国,采珠后大量珠蚌被丢弃,利用率低,污染环境。而研究表明,珠蚌肉质是很好的蛋白原料。本论文将采用取珠后珠蚌肉为原料,选用两种方法,并优化、比较两种方法制备的多肽的区别。以此为基础,设计一个生产珠蚌多肽粉工厂。主要研究结果如下：
1、考察酶解液抗氧化性及水解度,对水解珠蚌粗蛋白的酶种类进行选择,最终确定木瓜蛋白酶为最适水解蛋白酶。然后,以DPPH抑制率、·OH清除率及蛋白水解度作为评价指标,研究珠蚌抗氧化肽的制备工艺。在单因素基础上,通过三个指标的综合考虑,设计L18(37)多指标正交试验,对制备工艺进行优化,得出最佳工艺条件。结果表明,最佳条件为：酶解温度60℃、酶解时间5h、料液比3：10(w/v)、酶解pH6.5、加酶量6000U/g,制得的多肽DPPH-抑制率78.79%,·OH清除率49.7%,水解度23.09%。
2、采用超声波-酶法制备珠蚌抗氧化肽,先以抗氧化性为指标,得到最佳制备工艺,即先超声波处理,再使用木瓜蛋白酶水解。经过工艺优化,得到最佳超声波-酶解条件为：超声时间40min、超声温度50℃、超声功率100W、料液比3：20(w/v)。超声后进行酶解,酶解条件为温度60℃、时间5h、酶解pH6.5、加酶量6000U/g,在此超声条件下处理的珠蚌制备的抗氧化肽,DPPH抑制率为73.84%,·OH清除率为63.14%。在此基础上研究超声波对酶水解的影响,发现经过超声作用后,酶水解时间缩短1小时,料液比也由原来的3：10缩小为3：20(v/w)。
3、在得到两种方法的最佳制备工艺后,采用5种不同方法分别对酶法、超声波-酶法制备的珠蚌抗氧化肽进行比较。从电泳、柱层析结果比较发现两种分子量分布大致相同,说明两种方法得到的多肽在一定程度上是相同的。而从真蛋白含量、产品回收率、EC5o的测定结果得出,单位质量中,超声波-酶法制备的珠蚌抗氧化肽量较酶法制备的大,推断出超声波能促进珠蚌蛋白的溶解,增加蛋白与木瓜蛋白酶的接触概率,促进蛋白的水解,从而提高产量。
4、在实验结果的基础上,结合鄱阳湖当地资源、地理位置、政策等条件,设计一年产300吨珠蚌多肽粉的工厂。本设计主要从工厂总平面图、车间平面图、工厂工艺、物料衡算、设备选型等进行研究设计,并对本设计方案进行技术经济分析。设计结果表明,该珠蚌多肽粉厂固定资产投资2390.04万元,年利润达到1826.049万元,经营安全率达到89.65%,投资回收期为1.3年。
Our country is a big country with abundance production of pearl mussel, a large number of pearl mussel had been discarded after extraction of pearl, which has a low utilization rate and a environmental pollution. But research shows that, the mussel meat is also an excellent material of protein. This subject use pearl mussel as raw materials, generally the pearl mussel which take out pearl always used as waste to be discarded. The preparation of antioxidant peptides is depended on two kinds of methods, adopted papain to enzymolysis pearl mussel, and the two methods are optimized. Compared the distinction of two methods to prepare polypeptide, and design a factory of mussel polypeptide powder production depended on this.
1、Adopting the method of enzymatic to prepare antioxidant peptide of mussel, using DPPH free radical scavenging rate、OH clearance rate and mussel protein degree of hydrolysis as evaluation indexes, then research on the preparation technology of antioxidant peptide. The designation of L18(37) orthogonal test is based on single factor and through comprehensive consideration on these three indicators, then optimization of the preparation technology. The results showed that, the optimal conditions for enzymatic hydrolysis:enzymolysis temperature60℃, enzymolysis time5h, solid-liquid ratio3:10(w/V), enzymolysis pH6.5,, enzyme concentration6000U/g, the DPPH free radical scavenging rate of prepared polypeptide is85.5%, OH removal rate is50.7%and the hydrolysis degree is23.09%.
2、To prepare the mussel antioxidant peptides with ultrasonic-enzymatic method, first we get the best preparation process with antioxidant activity as an index, namely first we treated the mussel with ultrasonic microwave, and then hydrolyzed with papain. After the optimization of the process, the optimum of ultrasonic-enzymolysis conditions we get is:100W of ultrasonic power for40min at40℃,3:20of the ration of material and water。 Based on these experiments, we study the effect of ultrasonic treatment on the enzymolysis, it's found that the time of enzymolysis can be reduced to1h, the ration of material and water can be shortened to3:20from the initially3:10.
3、After getting the optimum preparation process of the two method, we compare the two methods, namely the enzymatic and ultrasonic-enzymolysis which both used for the prepared of mussel's antioxidant peptide, by five different methods. The result is that:the EC50and the distribution of Molecular weight of the two method are much the same, which shows that the polypeptide prepared by the two methods are the same; compared the content of the water-soluble protein and recovery rate of the product, we find that the amount of mussel's antioxidant peptide prepared by the ultrasonic-enzymolysis is lager than the enzymatic method. From these experiments, we can deduced that the ultrasonic wave can promote the solubility of the mussel protein, increase the contact between the protein and the papain, promote the hydrolysis of the protein, and then result in the increase of the product.
Finally, on the basis of the experimental results, combined with the local resources, geographical location and policy conditions of Poyang Lake, we design an plant with a annual output of300tons of mussel polypeptide powder. This design are carried out in the following aspects like the factory's total plane chart, workshop floor plan, the plant's process, material balance, equipment selection and so on. In addition, the economic analysis is conducted with this design. The results of design shows that the fixed assets investment of the mussel polypeptide powder plant is2.39004million RMB, the annual profit reach up to18.26049million RMB, the safety rate of the management is89.65%, and the recovery period of investment is1.3Years.
1、考察酶解液抗氧化性及水解度,对水解珠蚌粗蛋白的酶种类进行选择,最终确定木瓜蛋白酶为最适水解蛋白酶。然后,以DPPH抑制率、·OH清除率及蛋白水解度作为评价指标,研究珠蚌抗氧化肽的制备工艺。在单因素基础上,通过三个指标的综合考虑,设计L18(37)多指标正交试验,对制备工艺进行优化,得出最佳工艺条件。结果表明,最佳条件为：酶解温度60℃、酶解时间5h、料液比3：10(w/v)、酶解pH6.5、加酶量6000U/g,制得的多肽DPPH-抑制率78.79%,·OH清除率49.7%,水解度23.09%。
2、采用超声波-酶法制备珠蚌抗氧化肽,先以抗氧化性为指标,得到最佳制备工艺,即先超声波处理,再使用木瓜蛋白酶水解。经过工艺优化,得到最佳超声波-酶解条件为：超声时间40min、超声温度50℃、超声功率100W、料液比3：20(w/v)。超声后进行酶解,酶解条件为温度60℃、时间5h、酶解pH6.5、加酶量6000U/g,在此超声条件下处理的珠蚌制备的抗氧化肽,DPPH抑制率为73.84%,·OH清除率为63.14%。在此基础上研究超声波对酶水解的影响,发现经过超声作用后,酶水解时间缩短1小时,料液比也由原来的3：10缩小为3：20(v/w)。
3、在得到两种方法的最佳制备工艺后,采用5种不同方法分别对酶法、超声波-酶法制备的珠蚌抗氧化肽进行比较。从电泳、柱层析结果比较发现两种分子量分布大致相同,说明两种方法得到的多肽在一定程度上是相同的。而从真蛋白含量、产品回收率、EC5o的测定结果得出,单位质量中,超声波-酶法制备的珠蚌抗氧化肽量较酶法制备的大,推断出超声波能促进珠蚌蛋白的溶解,增加蛋白与木瓜蛋白酶的接触概率,促进蛋白的水解,从而提高产量。
4、在实验结果的基础上,结合鄱阳湖当地资源、地理位置、政策等条件,设计一年产300吨珠蚌多肽粉的工厂。本设计主要从工厂总平面图、车间平面图、工厂工艺、物料衡算、设备选型等进行研究设计,并对本设计方案进行技术经济分析。设计结果表明,该珠蚌多肽粉厂固定资产投资2390.04万元,年利润达到1826.049万元,经营安全率达到89.65%,投资回收期为1.3年。
Our country is a big country with abundance production of pearl mussel, a large number of pearl mussel had been discarded after extraction of pearl, which has a low utilization rate and a environmental pollution. But research shows that, the mussel meat is also an excellent material of protein. This subject use pearl mussel as raw materials, generally the pearl mussel which take out pearl always used as waste to be discarded. The preparation of antioxidant peptides is depended on two kinds of methods, adopted papain to enzymolysis pearl mussel, and the two methods are optimized. Compared the distinction of two methods to prepare polypeptide, and design a factory of mussel polypeptide powder production depended on this.
1、Adopting the method of enzymatic to prepare antioxidant peptide of mussel, using DPPH free radical scavenging rate、OH clearance rate and mussel protein degree of hydrolysis as evaluation indexes, then research on the preparation technology of antioxidant peptide. The designation of L18(37) orthogonal test is based on single factor and through comprehensive consideration on these three indicators, then optimization of the preparation technology. The results showed that, the optimal conditions for enzymatic hydrolysis:enzymolysis temperature60℃, enzymolysis time5h, solid-liquid ratio3:10(w/V), enzymolysis pH6.5,, enzyme concentration6000U/g, the DPPH free radical scavenging rate of prepared polypeptide is85.5%, OH removal rate is50.7%and the hydrolysis degree is23.09%.
2、To prepare the mussel antioxidant peptides with ultrasonic-enzymatic method, first we get the best preparation process with antioxidant activity as an index, namely first we treated the mussel with ultrasonic microwave, and then hydrolyzed with papain. After the optimization of the process, the optimum of ultrasonic-enzymolysis conditions we get is:100W of ultrasonic power for40min at40℃,3:20of the ration of material and water。 Based on these experiments, we study the effect of ultrasonic treatment on the enzymolysis, it's found that the time of enzymolysis can be reduced to1h, the ration of material and water can be shortened to3:20from the initially3:10.
3、After getting the optimum preparation process of the two method, we compare the two methods, namely the enzymatic and ultrasonic-enzymolysis which both used for the prepared of mussel's antioxidant peptide, by five different methods. The result is that:the EC50and the distribution of Molecular weight of the two method are much the same, which shows that the polypeptide prepared by the two methods are the same; compared the content of the water-soluble protein and recovery rate of the product, we find that the amount of mussel's antioxidant peptide prepared by the ultrasonic-enzymolysis is lager than the enzymatic method. From these experiments, we can deduced that the ultrasonic wave can promote the solubility of the mussel protein, increase the contact between the protein and the papain, promote the hydrolysis of the protein, and then result in the increase of the product.
Finally, on the basis of the experimental results, combined with the local resources, geographical location and policy conditions of Poyang Lake, we design an plant with a annual output of300tons of mussel polypeptide powder. This design are carried out in the following aspects like the factory's total plane chart, workshop floor plan, the plant's process, material balance, equipment selection and so on. In addition, the economic analysis is conducted with this design. The results of design shows that the fixed assets investment of the mussel polypeptide powder plant is2.39004million RMB, the annual profit reach up to18.26049million RMB, the safety rate of the management is89.65%, and the recovery period of investment is1.3Years.
引文
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