柴鱼蛋白肽的酶法制备、分离纯化及结构鉴定

Enzymatic Preparation, Separation, and Structural Identification of Bonito Protein Hydrolysates

以柴鱼为原料,酶解制备柴鱼蛋白肽,并以抗氧化活性和蛋白质回收率为指标对柴鱼蛋白肽酶解工艺进行了优化研究,用离子交换层析法和凝胶层析法对酶解液进行分离纯化,最后用基质辅助激光解吸电离飞行时间(MALDI-TOF-MS)串联质谱法和反相高效液相色谱法(RP-HPLC)对分离组分进行结构鉴定。研究显示,1、最佳用酶为木瓜蛋白酶;最适加酶量为1.0%;最佳预处理条件为100℃、5 min;最佳酶解时间为4 h。2、经过DEAE-52纤维素层析后,分离出A、B、C三个组分,其中A组分的抗氧化活性最好,后对A组分用Sephadex G-15葡聚糖凝胶进一步分离纯化,分离出A1、A2两个成分,其中A1的抗氧化活性最好。3、用MALDI-TOF-MS质谱对A1、A2进行分子量鉴定,检测发现,A1、A2组分的分子量主要集中在378.830 Da,进一步做氨基酸分析后,推测其可能含有的氨基酸组成为Lys、Leu、Pro。

Bonito protein hydrolysates were prepared by enzymatic hydrolysis using bonito fish as the raw material. Enzymatic hydrolysis processing was optimized using antioxidant activity and protein recovery rate as indicators. The protein hydrolysates were then separated and purified by means of ion exchange chromatography and gel chromatography. Finally, matrix-assisted laser desorption/ionization-time of flight-mass spectra(MALDI-TOF-MS), in tandem with reversed-phase high-performance liquid chromatography(RP-HPLC) was applied for structural identification of fraction composition. The optimum For the hydrolysis reaction, the best hydrolytic enzyme and its concentration were papain; and 1.0%, respectively. The optimum pretreatment conditions were 100 ℃ for 5 minutes After 4-hour hydrolysis, three fractions, labeled A, B, and C, were obtained from the hydrolyzate through DEAE-52 cellulose column chromatography. Fraction A had the highest antioxidant activity, which was further separated by gel chromatography on Sephadex G-15 column to give two fractions. The fraction with the highest antioxidant activity was labeled A1 and the other was labeled A2. Using MALDI-TOF-MS, the molecular weights of A1 and A2 were found to be around 378.830 Da. Further amino acid analysis revealed that the short-chain peptide had a possible amino acid composition of Lys, Leu, and Pro.