| 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 ProteinNutrients 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 AlcalaseHydrolysis 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 TrypsinDigestion 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 ExtractsSolvent 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. |
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