挤压协同酶法制备高粱蛋白ACE抑制肽及其稳定性

Preparation and Stability of Sorghum ACE Inhibitory Peptides by Extrusion-Enzyme Synergistic Method

【目的】利用挤压协同酶法制备高粱蛋白ACE抑制肽,为提高高粱蛋白资源的利用效率提供参考。【方法】以高粱粉为原料,先经挤压处理,再经淀粉酶酶解,最后通过碱性蛋白酶酶解,获得高粱蛋白ACE抑制肽。研究物料水分、挤压温度和淀粉酶活力对高粱蛋白酶解液的水解度和ACE抑制活性的影响,并探讨高粱蛋白ACE抑制肽的稳定性。【结果】随着物料水分含量和挤压温度的增加,挤压过程中单位机械能耗(SME)逐渐降低。在挤压环境下,高粱中淀粉和蛋白质的相互结合变得松散,淀粉-蛋白质包埋体系被破坏;同时高粱中球形蛋白质体被打破,提高所获得高粱蛋白的酶敏感性,在碱性蛋白酶的作用下生成更多具有抑制活性的短肽。挤压过程中物料水分含量和挤压温度以及α-淀粉酶活力对高粱蛋白酶解液的水解度和ACE抑制率有显著影响。随着物料水分的增加,蛋白质分子的聚合程度下降,使得高粱蛋白酶解液的水解度和ACE抑制率随之增加,当物料水分增加至19%后,挤压过程对蛋白质周围的淀粉分子的破坏作用降低,水解度和ACE抑制率的上升趋势趋于平缓;当挤压温度从120℃增加至180℃时,高粱内部的蛋白质-淀粉包埋体系破坏加剧,同时蛋白质的空间结构在高温作用下的变性程度加大,高粱蛋白酶解液的水解度由7.42%增加至11.06%,同时高粱蛋白ACE抑制肽的抑制率也由46.57%增加至53.41%;挤压后高粱粉经α-淀粉酶处理,进一步去除包裹在蛋白质周围的淀粉,发现随着α-淀粉酶活力的增加,高粱内部的蛋白质-淀粉包埋体系破坏程度加剧,为制备高粱蛋白ACE抑制肽提供更多原料,导致高粱蛋白酶解液的水解度和ACE抑制率随之增加,当α-淀粉酶活力增加至2.0 U·g-1时,淀粉酶与淀粉结合达到饱和状态,水解度和ACE抑制率趋于稳定。高粱蛋白ACE抑制肽经温度和酸碱处理后,ACE抑制活性在68.1%—71.31%,保持了良好的抑制活性;高粱蛋白ACE抑制肽在体外经胃肠道酶系消化酶解后,ACE抑制活性均高于73%,依然保持了较强的ACE抑制活性,说明挤压协同酶法制备的高粱蛋白ACE抑制肽具有长期保存有效性,同时能够在胃肠道消化后保持生物活性。【结论】采用挤压协同酶法可以显著提高高粱蛋白酶解液的水解度和ACE抑制肽的活性,同时制备的高粱蛋白ACE抑制肽具有良好的稳定性,为拓宽高粱的利用和制备功能性食品配料提供了一条新途径。

【Objective】Sorghum ACE inhibitory peptides were prepared by extrusion-enzyme synergistic method in order to provide a technical guidance to enhance the utilization efficiency of sorghum protein.【Method】Sorghum flour was introduced into an extruder, and then treated by α-amylase and alkaline protease to obtain the ACE inhibitory peptides. Effects of water content in sorghum flour, extrusion temperature and enzyme activity on the degree of hydrolysis and the activity and stability of ACE inhibitory peptides were investigated.【Result】With the increasing moisture content and extrusion temperature, the specific mechanical energy(SME) decreased. During extrusion, the interaction between starch and protein in sorghum became loose, which broke the starch-protein complex, and the spherical protein in sorghum was broken up, and thus the sensitivity of sorghum protease was enhanced and more ACE peptides were obtained after alkaline protease treatment. The moisture content, extrusion temperature and the activity of α-amylase presented a significant effect on the degree of hydrolysis and the inhibition rate of ACE inhibitory peptides. With the increasing moisture content, the assemble degree of protein decreased, which increased the degree of hydrolysis and the inhibition rate of ACE inhibitory peptides. When the moisture content reached to 19%, the damage degree of the starch around protein decreased, leading to the gentle increase trend of the hydrolysis degree and the inhibition rate of ACE inhibitory peptides. When the extrusion temperature increased from 120℃ to 180℃, the damage degree of starch-protein complex in sorghum increased. Meanwhile, the denaturation degree of protein also increased. The degree of hydrolysis increased from 7.42% to 11.06%, and the inhibition rate of sorghum protein ACE inhibitory peptides increased from 46.57% to 53.41%. The sorghum flour was treated by a-amylase to remove the starch around the protein after extrusion, and then it was found that when the activity of α-amylase increased, the damage degree of protein-starch complex in sorghum increased, which provided more raw materials for the preparation of sorghum protein ACE inhibitory peptides, leading to the higher the degree of hydrolysis and the activity of ACE inhibitory peptides. When the activity of α-amylase increased to 2.0 U·g-1, the binding of α-amylase to starch reached saturation, and the degree of hydrolysis and the inhibition rate of ACE inhibitory peptides tended to be stable. The activity of ACE inhibitory peptide fluctuated within 68.1%-71.31% after being treated by different storage temperatures and pH, suggesting a good inhibitory activity. After in vitro simulated gastrointestinal digestive enzymes digestion, the inhibitory activity of ACE inhibitory peptides was higher than 73%, which still maintained high value. The stability test indicated that sorghum ACE inhibitory peptides had good resistance to thermal, acid and alkaline treatment, and intestinal enzymes digestion.【Conclusion】The degree of hydrolysis and the inhibition rate of ACE inhibitory peptides all increased significantly by extrusion-enzyme synergistic method, while sorghum ACE inhibitory peptides had good stability, thus this work provided a new approach for the utilization of sorghum and the preparation of sorghum protein and ACE inhibitory peptides with potential use as functional food ingredients.