糜子蛋白水解物及酚类提取物的ACE抑制活性研究
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摘要
从天然食物中寻找更有效、更安全的控制血压升高的物质,减少高血压及降压药物给高血压患者带来的负面影响是医学家、营养学家和食品科学家面临的挑战。很多研究表明,食物蛋白质的一些水解物或者肽类具有控制血压升高的功效。谷物是人体能量的主要来源,吸引了很多科学家研究其对人体健康的意义。一些研究表明,食用全谷物可以降低患慢性病的风险。人口的增加、环境的变化、水资源的缺乏使全球农业和食品安全受到很大威胁。一些发展中国家,如印度、中国和非洲的一些国家开始重视抗旱谷物的种植。粟米是一种非常抗旱的作物,为这些国家数以万计的人们提供了能量。本着对膳食健康的重视,本文以糜子作为研究对象,分析其营养组分,评价其对抗高血压的潜在能力。研究结论如下：
营养成分组成和糜子蛋白质提取
糜子含有大量的碳水化合物、蛋白质和脂肪,以及促进人体健康的膳食纤维、必需氨基酸(如亮氨酸、苯丙氨酸和缬氨酸)和不饱和脂肪酸(如亚油酸,油酸和亚麻酸)。糜子也是矿物元素(如磷、钾、镁、钙、铁、锌)的良好来源。同时,糜子中含有大量的B族维生素,如尼克酸。糜子的营养组成可与小米和其他大宗谷物(如小麦、玉米和大米)相媲美。研究发现糜子蛋白质中含有61.89%的醇溶谷蛋白,之后是谷蛋白、白蛋白和球蛋白。提取糜子粗蛋白的最佳工艺是糜子粉：水为1：7,pH10,在40℃下浸提1小时,3000rpm离心15分钟,如此重复3次,收集上清液。在此条件下,每100g糜子粉中粗蛋白提取量为3.06g,其蛋白含量为56.43%。加入0.5%的亚硫酸钠作为还原剂,使碱溶蛋白质颜色变浅,此外在乙醇中加入0.5%的亚硫酸钠还可提高蛋白质提取率。
由碱性蛋白酶生产蛋白水解物的ACE抑制活性
本研究首先以水解产物产量和血管紧张素转换酶(ACE)抑制活性为指标,优化了碱性蛋白酶水解糜子蛋白质工艺参数。水解条件、制备工艺、ACE抑制活性的测定程序均影响水解产物的抑制活性。在最佳条件下,ACE抑制力ICso值粗蛋白为1.15+0.08mg蛋白/mL,白蛋白1.31+0.14mg蛋白/mL,球蛋白为1.56+0.21mg蛋白/mL,醇溶蛋白为1.23+0.19mg蛋白/mL,谷蛋白为0.84+0.11mg蛋白/mL。经体外模拟胃肠消化和加热处理后糜子蛋白质水解产物抑制活力仍表现出很好的稳定性。经过4次的冻融处理后,糜子蛋白质水解产物IC50值有所下降,而在3-7天的冷藏过程中糜子蛋白质水解产物ICso值有所上升。糜子蛋白质水解产物经Sephadex G-25色谱柱分离后,ACE抑制活力上升。糜子发芽5天后,水解产物的ACE抑制活力明显提高,而加热后ACE抑制活力明显下降。
由胃蛋白酶和胰蛋白酶生产蛋白质消化物的ACE抑制活性
本研究优化了胃蛋白酶、胰蛋白酶等人体消化酶消化糜子蛋白质的工艺参数。经胃蛋白酶酶解后,粗蛋白、醇溶蛋白和谷蛋白ICso值分别为1.67+0.20，1.35+0.16和0.71+14mg蛋白/mL；经胰蛋白酶酶解后,粗蛋白、醇溶蛋白和谷蛋白ICso值分别为1.92+0.15、1.48+0.24和1.2610.21mg蛋白/mL。采用胃蛋白酶和胰蛋白酶一起消化粗蛋白和醇溶蛋白,酶解物的ACE抑制活力高于用单一酶酶解后的值,ICso值低于用单一酶酶解后的值。经体外模拟胃肠消化糜子蛋白质酶解产物抑制活力仍表现出很好的稳定性。蛋白质酶解产物经凝胶过滤层析后,ACE抑制活力上升。糜子发芽5天后,用胃蛋白酶和胰蛋白酶酶解,其酶解产物的ACE抑制活力明显高于未处理的糜子粉或加热后的糜子粉。
生物活性提取物的ACE抑制活性
本研究以ACE抑制活性为指标,优化了糜子浸提物所用的提取溶剂。与水、乙醇或丙酮相比,糜子的甲醇提取物表现出高的ACE抑制活性。甲醇浓度为70%(体积/体积),在温度为30℃,浸提时间为1h,的条件下,ACE抑制活性为84.27%+1.1410mg粉/ml,ICso为2.64+0.12mg粉/mL。此外,糜子提取物的ACE抑制活性受提取工艺影响。糜子壳浸提物ACE抑制活性显著高于全粉或去壳粉浸提物的ACE抑制活性。糜子发芽3天后提取物的ACE抑制活性显著增加。加热会降低ACE抑制活性。总酚含量与提取物的ACE抑制活性之间没有明显的相关性,说明酚类化合物和其他组分有潜在的相关性。从脱脂糜子粉中获得的提取物经过体外胃肠液消化后ACE抑制活性显著提高。
从研究结果看,糜子富含人体必需的营养素,并可添加到高附加值食品和保健食品中食用。糜子蛋白经过碱性蛋白酶、胃蛋白酶和胰蛋白酶水解后产物,以及糜子的甲醇提取物具有潜在的抑制高血压的功能。今后还需要通过动物实验和人体实验进一步研究糜子的生物利用率、代谢情况及对健康的贡献率。开展临床试验,进一步认证糜子蛋白水解物和糜子提取物对抑制血压升高的影响。
Prevalence of hypertension and negative side effects of synthetic drugs, used for treatment of people suffering from high blood pressure, present a challenge to medical scientists, nutritionists, and food scientists to search for effective and safe antihypertensive components from natural sources. Food protein is one of the most components has been extensively studied for that purpose and many hydrolysates and peptides with antihypertensive properties have been isolated and identified. Cereal grains are most important source of energy to a majority of the world population and they have received more attention from researchers in terms of their health benefits during the last years. Several research studies found that consumption of whole cereal grains reduces risk of many chronic diseases. However, at present, increasing population, climate changes, and water scarcity are expected to generate a great threat to agriculture and food security worldwide. Therefore, interest in the development of policy statements about drought-tolerant grains is increasing in several developing countries, such as India, China, and some countries of Africa. Millet grains are one of the most drought-tolerant grains and constitutes as major source of energy for many million people in these countries. Therefore, and with the increasing healthy diet awareness, this study was carried out on proso millet grains for purpose of evaluating their nutritive value and potential health benefits focusing on inhibitory activity of their protein hydrolysates and bioactive etracts (phenolic extracts) against Angiotensin I-Converting Enzyme (ACE), which has a pivotal role in blood pressure regulation. The major findings of research processes could be summarized as follows:
Nutrients Composition and Extraction of Proso Millet Protein
Nutrients composition analysis showed that proso millet grains contain high contents of carbohydrate, protein, and fat. They also found to contain health-promoting components, including dietary fiber, essential amino acids, such as leucine, phenylalanine, and valine, and unsaturated fatty acids, such as linoleic, oleic, and linolenic. Proso millet grains can also be considered as a good source of minerals, such as phosphorus, potassium, magnesium, calcium, iron, and zinc. In addition, proso millet showed high contents of B vitamins, such as niacin. Nutrient contents in proso millet are comparable with those of foxtail millet and major cereal grains, such as wheat, corn, and rice. Extraction of protein fractions showed that prolamins are major proteins in proso millet with percentage of61.89%of the total proteins, followed by glutelin, albumin, and globulin. Optimal conditions for extracting of crude protein concentrate from proso millet were found to be1:7for flour/water ratio;10for the pH;40℃for temperature; and1h for extraction time;3,000rpm for15min centrifugal speed for separating of the extract from the residue; and up to3times repetitive extraction for same sample is recommended under same conditions. Under these optimal conditions, protein extract yield of3.06 g/100g flour with protein content of56.43%was obtained. Addition of0.5%sodium sulfite as reducing agent to the alkali solution resulted in protein extract with lighter colour. However, addition of0.5%sodium sulfite to ethyl alcohol resulted in high yield of protein extract.
ACE-inhibitory Activity of Protein Hydrolysates Produced by Alcalase
Hydrolysis conditions of proso millet protein by alcalase were optimized for production of hydrolysates with angiotensin converting enzyme (ACE) inhibitory activity. Hydrolysis conditions, preparation processes, and assay procedure for ACE-inhibitory activity were found significantly (p<0.05) affect inhibitory activity of the resulting hydrolysate. Under selected optimal conditions, ACE-inhibitory hydrolysates with IC5o value of1.15±0.08from crude protein concentrate,1.31±0.14from albumin,1.56±0.21from globulin,1.23±0.19from prolamin, and0.84±0.11mg protein/mL from glutelin were obtained. The inhibitory activity of hydrolysate showed good stability to simulated gastrointestinal digestion and heat treatment. However, the activity was reduced with the repetition of freeze/thaw treatment up to4cycles and with the increase in cold storage time from3up to7days. Filtration of hydrolysate on gel column chromatography, Sephadex G-25, resulted in fraction with higher ACE-inhibitory activity than that of the original hydrolysate. In addition, the inhibitory activity of hydrolysate obtaind from proso millet flour was increased with the increase of germination time up to5days; however, the activity was reduced with the increase in cooking time.
ACE-inhibitory Activity of Protein Digests Produced by Pepsin and Trypsin
Digestion process of proso millet protein by digestive enzymes similar to those of human, including pepsin and trypsin, was optimized. Under optimal conditions for digestion by Pepsin or Trypsin, ACE-inhibitory digests with IC50value of1.67±0.20or1.92±0.15,1.35±0.16or1.48±0.24, and0.71±14or1.26±0.21mg protein/mL were obtained from crude protein concentrate, prolamin, and glutelin; respectively. In addition, successive digestion of crude protein concentrate and prolamin by Pepsin and Trypsin resulted in digests with higher ACE-inhibitory activity, lower IC50value, than that of digests obtained by single enzyme, Pepsin or Trypsin. The inhibitory activity of digests showed a relatively good stability to simulated gastrointestinal digestion. Fractions with higher ACE-inhibitory activity were isolated from protein digests obtained by pepsin using gel filtration chromatography. On the other hand, digest produced from flour of5days germinated millet grains by successive digestion with Pepsin and Trypsin showed higher ACE-inhibitory activity compared with that of digest produced from flour of unprocessed or cooked grains.
ACE-inhibitory Activity of Phenolic Extracts
Solvent extraction conditions of proso millet grains were optimized and ACE-inhibitory activity of obtained extracts was evaluated. Methanolic extract showed high ACE-inhibitory activity compared to that of water, ethanol, or acetone extracts. Conditions of70%(v/v) for solvent concentration,1h for time, and30℃for temperature, were selected as optimal conditions for extraction by methanol. ACE-inhibitory activity for extract obtained under these conditions was found to be84.27±1.14%at extract concentration of10mg powder/mL with IC50value of2.64±0.12mg powder/mL. In addition, ACE-inhibitory activity of extract from proso millet grains was found to be influnced by processing. Extract from hull of the grains showed high inhibitory activity compared to that obtained from whole grains flour and dehulled grains flour. The inhibitory activity of the extract was increased by germination up to3days. However, the inhibitory activity was reduced by cooking process. On the other hand, no clear correlation was found between total phenolic content and ACE-inhibitory activity of extracts, indicating that synergistic effect of phenolics and other components is the most potential. ACE-inhibitory activity of extracts obtained by varying solvents from defatted flour of proso millet was significantly (p<0.05) increased after gastrointestinal digestion simulation.
Based on the obtained results, it can be concluded that presence of all the required nutrients in proso millet grains makes them suitable for large-scale utilization in the preparation of several value-added and health food-products. Proso millet protein hydrolysates and digests produced by alcalase, pepsin, and trypsin as well as methanol extract have the potential to be used as functional ingredients for production of functional foods with antihypertensive activity. Further research is needed to determine the bioavailability, metabolism, and health contribution of millet grains and their fractions in animal models and human subjects. Human clinical trials should be performed to obtain consistent evidence for the health effects of the ACE-inhibitory hydrolysates and extracts derived from proso millet grains.
营养成分组成和糜子蛋白质提取
糜子含有大量的碳水化合物、蛋白质和脂肪,以及促进人体健康的膳食纤维、必需氨基酸(如亮氨酸、苯丙氨酸和缬氨酸)和不饱和脂肪酸(如亚油酸,油酸和亚麻酸)。糜子也是矿物元素(如磷、钾、镁、钙、铁、锌)的良好来源。同时,糜子中含有大量的B族维生素,如尼克酸。糜子的营养组成可与小米和其他大宗谷物(如小麦、玉米和大米)相媲美。研究发现糜子蛋白质中含有61.89%的醇溶谷蛋白,之后是谷蛋白、白蛋白和球蛋白。提取糜子粗蛋白的最佳工艺是糜子粉：水为1：7,pH10,在40℃下浸提1小时,3000rpm离心15分钟,如此重复3次,收集上清液。在此条件下,每100g糜子粉中粗蛋白提取量为3.06g,其蛋白含量为56.43%。加入0.5%的亚硫酸钠作为还原剂,使碱溶蛋白质颜色变浅,此外在乙醇中加入0.5%的亚硫酸钠还可提高蛋白质提取率。
由碱性蛋白酶生产蛋白水解物的ACE抑制活性
本研究首先以水解产物产量和血管紧张素转换酶(ACE)抑制活性为指标,优化了碱性蛋白酶水解糜子蛋白质工艺参数。水解条件、制备工艺、ACE抑制活性的测定程序均影响水解产物的抑制活性。在最佳条件下,ACE抑制力ICso值粗蛋白为1.15+0.08mg蛋白/mL,白蛋白1.31+0.14mg蛋白/mL,球蛋白为1.56+0.21mg蛋白/mL,醇溶蛋白为1.23+0.19mg蛋白/mL,谷蛋白为0.84+0.11mg蛋白/mL。经体外模拟胃肠消化和加热处理后糜子蛋白质水解产物抑制活力仍表现出很好的稳定性。经过4次的冻融处理后,糜子蛋白质水解产物IC50值有所下降,而在3-7天的冷藏过程中糜子蛋白质水解产物ICso值有所上升。糜子蛋白质水解产物经Sephadex G-25色谱柱分离后,ACE抑制活力上升。糜子发芽5天后,水解产物的ACE抑制活力明显提高,而加热后ACE抑制活力明显下降。
由胃蛋白酶和胰蛋白酶生产蛋白质消化物的ACE抑制活性
本研究优化了胃蛋白酶、胰蛋白酶等人体消化酶消化糜子蛋白质的工艺参数。经胃蛋白酶酶解后,粗蛋白、醇溶蛋白和谷蛋白ICso值分别为1.67+0.20，1.35+0.16和0.71+14mg蛋白/mL；经胰蛋白酶酶解后,粗蛋白、醇溶蛋白和谷蛋白ICso值分别为1.92+0.15、1.48+0.24和1.2610.21mg蛋白/mL。采用胃蛋白酶和胰蛋白酶一起消化粗蛋白和醇溶蛋白,酶解物的ACE抑制活力高于用单一酶酶解后的值,ICso值低于用单一酶酶解后的值。经体外模拟胃肠消化糜子蛋白质酶解产物抑制活力仍表现出很好的稳定性。蛋白质酶解产物经凝胶过滤层析后,ACE抑制活力上升。糜子发芽5天后,用胃蛋白酶和胰蛋白酶酶解,其酶解产物的ACE抑制活力明显高于未处理的糜子粉或加热后的糜子粉。
生物活性提取物的ACE抑制活性
本研究以ACE抑制活性为指标,优化了糜子浸提物所用的提取溶剂。与水、乙醇或丙酮相比,糜子的甲醇提取物表现出高的ACE抑制活性。甲醇浓度为70%(体积/体积),在温度为30℃,浸提时间为1h,的条件下,ACE抑制活性为84.27%+1.1410mg粉/ml,ICso为2.64+0.12mg粉/mL。此外,糜子提取物的ACE抑制活性受提取工艺影响。糜子壳浸提物ACE抑制活性显著高于全粉或去壳粉浸提物的ACE抑制活性。糜子发芽3天后提取物的ACE抑制活性显著增加。加热会降低ACE抑制活性。总酚含量与提取物的ACE抑制活性之间没有明显的相关性,说明酚类化合物和其他组分有潜在的相关性。从脱脂糜子粉中获得的提取物经过体外胃肠液消化后ACE抑制活性显著提高。
从研究结果看,糜子富含人体必需的营养素,并可添加到高附加值食品和保健食品中食用。糜子蛋白经过碱性蛋白酶、胃蛋白酶和胰蛋白酶水解后产物,以及糜子的甲醇提取物具有潜在的抑制高血压的功能。今后还需要通过动物实验和人体实验进一步研究糜子的生物利用率、代谢情况及对健康的贡献率。开展临床试验,进一步认证糜子蛋白水解物和糜子提取物对抑制血压升高的影响。
Prevalence of hypertension and negative side effects of synthetic drugs, used for treatment of people suffering from high blood pressure, present a challenge to medical scientists, nutritionists, and food scientists to search for effective and safe antihypertensive components from natural sources. Food protein is one of the most components has been extensively studied for that purpose and many hydrolysates and peptides with antihypertensive properties have been isolated and identified. Cereal grains are most important source of energy to a majority of the world population and they have received more attention from researchers in terms of their health benefits during the last years. Several research studies found that consumption of whole cereal grains reduces risk of many chronic diseases. However, at present, increasing population, climate changes, and water scarcity are expected to generate a great threat to agriculture and food security worldwide. Therefore, interest in the development of policy statements about drought-tolerant grains is increasing in several developing countries, such as India, China, and some countries of Africa. Millet grains are one of the most drought-tolerant grains and constitutes as major source of energy for many million people in these countries. Therefore, and with the increasing healthy diet awareness, this study was carried out on proso millet grains for purpose of evaluating their nutritive value and potential health benefits focusing on inhibitory activity of their protein hydrolysates and bioactive etracts (phenolic extracts) against Angiotensin I-Converting Enzyme (ACE), which has a pivotal role in blood pressure regulation. The major findings of research processes could be summarized as follows:
Nutrients Composition and Extraction of Proso Millet Protein
Nutrients composition analysis showed that proso millet grains contain high contents of carbohydrate, protein, and fat. They also found to contain health-promoting components, including dietary fiber, essential amino acids, such as leucine, phenylalanine, and valine, and unsaturated fatty acids, such as linoleic, oleic, and linolenic. Proso millet grains can also be considered as a good source of minerals, such as phosphorus, potassium, magnesium, calcium, iron, and zinc. In addition, proso millet showed high contents of B vitamins, such as niacin. Nutrient contents in proso millet are comparable with those of foxtail millet and major cereal grains, such as wheat, corn, and rice. Extraction of protein fractions showed that prolamins are major proteins in proso millet with percentage of61.89%of the total proteins, followed by glutelin, albumin, and globulin. Optimal conditions for extracting of crude protein concentrate from proso millet were found to be1:7for flour/water ratio;10for the pH;40℃for temperature; and1h for extraction time;3,000rpm for15min centrifugal speed for separating of the extract from the residue; and up to3times repetitive extraction for same sample is recommended under same conditions. Under these optimal conditions, protein extract yield of3.06 g/100g flour with protein content of56.43%was obtained. Addition of0.5%sodium sulfite as reducing agent to the alkali solution resulted in protein extract with lighter colour. However, addition of0.5%sodium sulfite to ethyl alcohol resulted in high yield of protein extract.
ACE-inhibitory Activity of Protein Hydrolysates Produced by Alcalase
Hydrolysis conditions of proso millet protein by alcalase were optimized for production of hydrolysates with angiotensin converting enzyme (ACE) inhibitory activity. Hydrolysis conditions, preparation processes, and assay procedure for ACE-inhibitory activity were found significantly (p<0.05) affect inhibitory activity of the resulting hydrolysate. Under selected optimal conditions, ACE-inhibitory hydrolysates with IC5o value of1.15±0.08from crude protein concentrate,1.31±0.14from albumin,1.56±0.21from globulin,1.23±0.19from prolamin, and0.84±0.11mg protein/mL from glutelin were obtained. The inhibitory activity of hydrolysate showed good stability to simulated gastrointestinal digestion and heat treatment. However, the activity was reduced with the repetition of freeze/thaw treatment up to4cycles and with the increase in cold storage time from3up to7days. Filtration of hydrolysate on gel column chromatography, Sephadex G-25, resulted in fraction with higher ACE-inhibitory activity than that of the original hydrolysate. In addition, the inhibitory activity of hydrolysate obtaind from proso millet flour was increased with the increase of germination time up to5days; however, the activity was reduced with the increase in cooking time.
ACE-inhibitory Activity of Protein Digests Produced by Pepsin and Trypsin
Digestion process of proso millet protein by digestive enzymes similar to those of human, including pepsin and trypsin, was optimized. Under optimal conditions for digestion by Pepsin or Trypsin, ACE-inhibitory digests with IC50value of1.67±0.20or1.92±0.15,1.35±0.16or1.48±0.24, and0.71±14or1.26±0.21mg protein/mL were obtained from crude protein concentrate, prolamin, and glutelin; respectively. In addition, successive digestion of crude protein concentrate and prolamin by Pepsin and Trypsin resulted in digests with higher ACE-inhibitory activity, lower IC50value, than that of digests obtained by single enzyme, Pepsin or Trypsin. The inhibitory activity of digests showed a relatively good stability to simulated gastrointestinal digestion. Fractions with higher ACE-inhibitory activity were isolated from protein digests obtained by pepsin using gel filtration chromatography. On the other hand, digest produced from flour of5days germinated millet grains by successive digestion with Pepsin and Trypsin showed higher ACE-inhibitory activity compared with that of digest produced from flour of unprocessed or cooked grains.
ACE-inhibitory Activity of Phenolic Extracts
Solvent extraction conditions of proso millet grains were optimized and ACE-inhibitory activity of obtained extracts was evaluated. Methanolic extract showed high ACE-inhibitory activity compared to that of water, ethanol, or acetone extracts. Conditions of70%(v/v) for solvent concentration,1h for time, and30℃for temperature, were selected as optimal conditions for extraction by methanol. ACE-inhibitory activity for extract obtained under these conditions was found to be84.27±1.14%at extract concentration of10mg powder/mL with IC50value of2.64±0.12mg powder/mL. In addition, ACE-inhibitory activity of extract from proso millet grains was found to be influnced by processing. Extract from hull of the grains showed high inhibitory activity compared to that obtained from whole grains flour and dehulled grains flour. The inhibitory activity of the extract was increased by germination up to3days. However, the inhibitory activity was reduced by cooking process. On the other hand, no clear correlation was found between total phenolic content and ACE-inhibitory activity of extracts, indicating that synergistic effect of phenolics and other components is the most potential. ACE-inhibitory activity of extracts obtained by varying solvents from defatted flour of proso millet was significantly (p<0.05) increased after gastrointestinal digestion simulation.
Based on the obtained results, it can be concluded that presence of all the required nutrients in proso millet grains makes them suitable for large-scale utilization in the preparation of several value-added and health food-products. Proso millet protein hydrolysates and digests produced by alcalase, pepsin, and trypsin as well as methanol extract have the potential to be used as functional ingredients for production of functional foods with antihypertensive activity. Further research is needed to determine the bioavailability, metabolism, and health contribution of millet grains and their fractions in animal models and human subjects. Human clinical trials should be performed to obtain consistent evidence for the health effects of the ACE-inhibitory hydrolysates and extracts derived from proso millet grains.
引文
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