超声辅助竹节虾头酶解及抗氧化肽分离研究

Study on Ultrasound-assisted Enzymatic Hydrolysis of Penaeus japonicus Head and Separation of the Antioxidant Peptides

为了实现竹节虾加工副产物的高值化利用,以竹节虾加工废弃物中的虾头副产物为原料,以水解度和DPPH清除率作为评价指标,采用中性蛋白酶酶解,通过响应面法优化超声辅助酶解工艺,并依次通过超滤、凝胶层析色谱和反相高效液相色谱等分离方法,从竹节虾虾头酶解产物中分离制备抗氧化肽,采用超高压液相色谱串联质谱联用技术对肽的结构进行表征。结果表明,在中性蛋白酶添加量为3 000 U·g^(-1)、p H值7.0条件下,最佳超声辅助酶解工艺参数为超声时间41 min,超声温度55℃,超声频率22 k Hz,料液比1∶9(w/v),在此条件下获得的酶解产物DPPH清除率达69.50%。当水解时间为0.5~2.5 h时,超声辅助酶解的酶解产物水解度和DPPH清除率比非超声辅助酶解工艺分别高17.95%和18.83%,该工艺缩短了酶解时间,节约了能耗。酶解产物经超滤初步分离发现,相对分子质量在<3k Da(SHP4)的组分具有显著的抗氧化活性。用凝胶层析法进一步分离纯化SHP4组分后得到4个峰,其中SHP4-II的DPPH清除率最高;SHP4-II通过反相高效液相纯化后也得到4个主要肽峰,在多肽含量为1.5 mg·m L^(-1)时,SHP4-II-4的DPPH清除率最高,达到85.69%,并且具有较好的分离度,质谱分析发现,该抗氧化肽的结构为Gly-Asn-Gly-Leu-Pro(455.99 Da)。本研究结果为虾肽抗氧化保健食品的研发提供了一定的科学依据。

In this paper,the head of Penaeus japonicus was used as raw material for further development of offal resource. Antioxidant peptides were prepared by ultrasonic assisted hydrolytic method. Based on the degree of hydro-lysates（ DH） and DPPH scavenging,a series of enzyme-screening pre-experiments were carried out. The neutral protease was chose as hydrolysis, and we optimized the conditions of hydrolysis by single-factor test and respon-se surface methodology. Moreover,the purified antioxidant peptides were prepared from hydrolysates by ultrafiltr-ation（ UF）membrane system,gel filtration chromatography and reverse phase high-performance liquid chromatog-raphy（ RP-HPLC）in turn. Their structures were further identified by ultra performance liquid chromatography/time-of-flight mass spectrometry（ UPLC-TOF-MS/MS）. The results showed that the optimal ultrasonic-assisted hydrolytic conditions for the neutral protease with 3 000 U·g^-1 enzymatic activity and pH 7. 0 were 41 minutes of ultrasonic time,55 ℃ of ultrasonic temperature,22 k Hz of ultrasonic power and 1 ∶ 9 of ratio of solid to liquid. The DPPH scavengingrate increased to 69. 50% under these conditions. Compared with the non-ultrasonic assisted enzymatic method,the DH and DPPH scavenging rate increased by 17. 95%,18. 83% within 0. 5 to 2. 5 h. The improved technique showed good results in reducing the reaction time and saving energy. Hydrolysates with 3 k Da（ SHP4） relative molecul-ear mass that were purified by UF showed significant antioxidant activity. Four components were then separated by Sephadex G-25 gel chromatography. Among the four components,SHP4-II has the highest DPPH scavenging. Four components were also separated from SHP4-II by RP-HPLC. SHP4-II-4 has the highest DPPH scavenging with the value of 85. 69%.One peptide with strong antioxidant activity was purified from the hydrolysates. Of which the structure was Gly-Asn-GlyLeu-Pro（ MW： 455. 99 Da）. The results of my research provided a certain scientific basis for the development of the new health foods with antioxidant activity.