藜麦源ACE抑制糖肽制备、结构表征及体外稳定性研究

Preparation,Structure Identification of ACE Inhibitory Glycopeptide from Quinoa and Its Stability in Vitro

利用毛霉和米根霉混合发酵藜麦制备藜麦源血管紧张素转化酶(angiotensin-I converting enzyme,ACE)抑制糖肽。通过单因素实验和响应面试验优化发酵工艺条件,发酵液经真空浓缩、醇沉、葡聚糖凝胶G-15和反相高效液相色谱分离纯化,利用傅里叶红外光谱法判断官能团构成,β-消除法判断糖肽键链接方式,并分析温度、p H、金属离子以及体外模拟消化对活性的影响。结果表明:在料液比1:18 g/m L、毛霉与米根霉接种量比1:1、总接种量2.8%(φ)、发酵时间8.7 d时,得到ACE抑制率为64.22%±4.57%的藜麦发酵液,分离纯化后获得单一组分F_(3)b。利用傅里叶红外光谱检测,发现F_(3)b具有多肽和多糖的特征吸收,β-消除法确定其存在O-糖肽键。体外稳定性研究结果表明,温度(25~55°C)对F3b活性影响较小,100℃处理后ACE抑制率为25℃时的86.23%±3.47%;不同p H条件(pH2~12)对其活性无显著影响(P>0.05);在Zn2+和Fe3+浓度为4 mmol/L时,ACE抑制率分别为对照的109.91%±8.12%和117.43%±6.78%,增强其活性;相同浓度的K+和Ca2+减弱其活性,ACE抑制率分别为对照的78.94%±2.18%和85.31%±4.99%;模拟胃肠消化过程中,F3b活性略有降低,ACE抑制率为对照的70.00%±3.30%。该研究丰富了ACE抑制肽的种类,为藜麦资源高值化利用提供理论和数据技术参考。

The aim of this study was to purify angiotensin I-converting enzyme(ACE)inhibitory glycopeptide from quinoa fermented by Mucor wutungkiao and Rhizopus oryzae.The fermentation process conditions were optimized by a single factor experiment and a response surface experiment.The fermentation liquid was separated and purified by vacuum concentration,alcohol precipitation,Sephadex G-15 and reverse-phase high performance liquid chromatography.Fourier infrared spectroscopy was used to determine the composition of functional groups and β-elimination method to determine the glycopeptide bonding type.The effects of temperature,pH,metal ions,and in vitro simulated digestion on the activity were analyzed.The results showed that the optimal fermentation parameters were as follows:8.7 d of fermentation time,2.8%(φ)of total inoculum size containing Mucor wutungkiao to Rhizopus oryzae in a volume ratio of 1:1,and 1:18 g/mL of a solid-liquid ratio.The ACE inhibition rate was 64.22%±4.57%.A novel glycopeptides F_(3)b,was isolated and purified by ethanol precipitation,Sephadex G-15,and reverse-phase high performance liquid chromatography.Fourier-transform infrared spectroscopy proved the presence of polypeptides and polysaccharides in F_(3)b,andβ-elimination reaction further demonstrated that the glycoprotein was an O-linked type.In vitro stability results indicate that the ACE inhibitory activity of glycopeptides was hardly changed under heating conditions from 25 to 55℃,and was 86.23%±3.47%of the original activity at 100°C.Conversely,pH(from 2 to 12)had no significant effect(P>0.05)on the ACE inhibitory activity of glycopeptides.4 mmol/L of Zn2+and Fe3+increased the ACE inhibitory activity of F3b to 109.91%±8.12%and 117.43%±6.78%of the control,respectively.While same concentration of K+and Ca2+reduced the ACE inhibitory activity to 78.94%±2.18%and 85.31%±4.99%of the control,respectively.After simulated gastrointestinal digestion,the ACE inhibitory activity of F3b decreased to 70.00%±3.30%of the control.In summary,this study could enrich the variety of ACE inhibitory peptides and provide technical reference for high-value utilization of quinoa.