发酵大豆豆粕水提物分离纯化ACE抑制活性小肽

Isolation and purification of ACE inhibitory peptides from fermented soybean meal extracts

本文开发了一种利用枯草芽孢杆菌(Bacillus subtilis)发酵大豆豆粕并结合多级超滤、纳滤配合凝胶渗透色谱(GFC)和液相色谱(HPLC)分离纯化具有血管紧张素转换酶(ACE)抑制活性的小肽的方法。利用色谱法从发酵豆粕超滤提取液中分离纯化大豆小肽,用氨基酸序列分析仪PPSQ-21和基质辅助激光解吸/电离串联飞行时间质谱基质(MALDI-TOF-TOF/MS)定性,再用标准固相肽合成法(SPPS)合成小肽,用HPLC法测定其ACE抑制活性,并对小肽进行Sprague-Dawley Rat大鼠的心血管离体实验。结果表明,超滤后得到不同分子量滤液F1(1000~10000 Da)、F2(500~1000 Da)、F3(<500 Da)均具有不同程度的ACE抑制活性且分子量最小的F3(<500 Da)组分最强。从F3组分中分离纯化了两个ACE抑制活性小肽,HAGR和CGAAP,在相同浓度(2 mg/m L)下其抑制率分别为34.99%和77.79%,后者抑制活性高于F3组分。离体实验的结论同样证实,大豆提取小肽具有舒张已被收缩的血管环的活性,但活性强度与ACE抑制活性不直接相关。实验证明,枯草芽孢杆菌配合超滤的方式可以从大豆发酵物中制备具有血管生物活性的组分,其生物活性可能来源于这种组分中的部分小肽。

A method for isolation angiotensin-converting enzyme（ACE） inhibitory peptides from Bacillus subtilis SY fermented soybean meal hydrolyzate through uhrafiltration and nanofihration followed with liquid chromatography （HPLC）separation was established.The small peptides were separated and purified from the ultrafiltrated fermented soybean meal by chromatography. The peptides were characterized by PPSQ-21 amino acid sequencer and MALDI-TOF-TOF/MS and were synthesized by solid phase peptide synthesis （SPPS）. Their ACE inhibitory activities were determined by HPLC, and then their ex vivo cardiac performance of aorta ring of Sprague-Dawley Rats were also determined.The results showed that the filtrate of soybean meal after ultrafiltration was separated into 3 fractions F1 (1000-10000 Da） ,F2（500-1000 Da） ,F3 (〈 500 Da） and the fraction F3 (〈 500 Da)had the strongest inhibitory activity.Then two ACE inhibitory small peptides, His-Ala-Gly-Arg （HARG）and Cys-Gly-Ala-Ala-Pro（CGAAP）were isolated and purified from the F3, which showed the inhibition rates of 34.99% and 77.79% on the same concentration（2 mg/mL） ,respectively, and the CGAAP were higher than fraction F3.The vasorelaxation effect of the peptides were also confirmed by investigate the presence of the peptides with stimulated aorta rings of the Sprague-Dawley rats,but the activity intensity was not directly related to ACE inhibitory activity. Experiments have shown that the ultra-filtrate of fermented soy meal (fermented with Bacillus subtilis from Douchi） hydrolyzate could produce the vascular biologically active components,which provided a reference for further in-depth study of biologically active peptides derived from food.