以鹿茸中下段胶原蛋白为酶解底物，用木瓜蛋白酶酶解制备小分子抗氧化肽，以清除1,1-二苯基-2-苦基肼基(DPPH?)自由基的能力为指标，采用响应面法优化酶水解条件。结果表明，最优实验条件为时间56 min，酶添加量1.40wt%，pH=5.60，温度60℃。该条件下所得抗氧化肽对DPPH?自由基的清除率为83.09%。用超滤膜、半制备色谱柱和超高效液相色谱?质谱联用仪分级分离获得分子量0.2?0.6 kDa的具有最高抗氧化活性的多肽，其具有与头段类似的保健功效，更易被人体吸收，且易进一步加工和储存。
It is believed that the velvet antler head has the best health benefits in traditional Chinese medicine. But its output is small, and the price is high. The first section of velvet antler is used frequently, and its protein/peptide content is highest among all sections. The polypeptide molecular weight is about 5 kDa. In this work, in order to make full use of the velvet antler resources, the water extract of the middle and low sections collagen of velvet antler was used as an enzymatic substrate to prepare an antioxidant peptide using papain. Enzymatic hydrolysis conditions (time, enzyme addition ratio, pH and temperature) were optimized by response surface methodology (RSM). The 1,1-diphenyl-2-picrylhydrazyl (DPPH?) radical-scavenging activity rate was used as an index. The target antioxidant peptide (molecular weight less than 5 kDa) obtained by separation through an ultrafiltration membrane was further separated by reversed-phase high-performance liquid chromatography. And 10 components, S1?S10, were obtained respectively and the components were lyophilized. Component S3 was further subjected by reversed-phase ultra-high performance liquid chromatography (UPLC) on-line electrospray ionization mass spectrometry (ESI-MS) for molecular weight determination. The polypeptide with the highest antioxidant activity was fractionated by ultrafiltration membranes of molecular weight cut off of 50, 10 and 5 kDa respectively. The results showed that the optimal experimental conditions were time of 56 min, enzyme addition of 1.40wt%, pH of 5.60, temperature of 60℃, DPPH? radical-scavenging activity rate was 83.09%. The molecular weight of polypeptide was 0.2?0.6 kDa. The resulting small molecule peptide has similar health benefits as the head section, is more easily absorbed by the body, and is easier to further process and store.
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