Modification of Soybean Protein Hydrolysates by Alcalase-Catalyzed Plastein Reaction and the ACE-Inhibitory Activity of the Modified Product In Vitro

Soybean protein hydrolysates were prepared by hydrolyzing soybean protein isolates with a protease alcalase to a degree of hydrolysis of 16.6%, and then modified by alcalase-catalyzed plastein reaction to reveal the impact of plastein reaction on the ACE-inhibitory activity of the modified product in vitro. The suitable conditions of plastein reaction of soybean protein hydrolysates were selected based on the results of response surface methodology with the decreased amount of the free amino groups of the modified product as response. When reaction temperature was fixed at 30°C, the selected conditions were as follows: concentration of soybean protein hydrolysates of 45% (w/w), addition level of alcalase of 275 U/g peptides, and reaction time of 3 to 4 h. Soybean protein hydrolysates and eight modified products were evaluated for their ACE-inhibitory activities in vitro. The assay results highlighted that plastein reaction improved the ACE-inhibitory activity of the modified product. The IC₅₀ of the modified products ranged from 0.64 to 1.11 mg/mL, while that of soybean protein hydrolysates was 1.45 mg/mL. The decreased amount of the free amino groups of the modified product showed influence on the ACE-inhibitory activity in vitro. Analysis results from size exclusion chromatography confirmed that some plasteins with higher molecular weights were formed in the modified product. Our results showed that alcalase-catalyzed plastein reaction could be applied as a potential approach to enhance the ACE-inhibitory activity of soybean protein hydrolysates in vitro.     

Evaluation Of Antioxidant Properties In Vitro of Plastein-Reaction-Stressed Soybean Protein Hydrolysate

Alcalase was used in the present study to carry out an enzymatic hydrolysis of soybean protein isolate and a plastein reaction of the prepared hydrolysate in vitro, aiming to investigate the influence of the plastein reaction on the antioxidant properties of the modified hydrolysate. Soybean protein hydrolysate was prepared in a degree of hydrolysis of 14.0%, exhibited a scavenging activity of 43.6% on ABTS radical in vitro, and thus was used as the substrate of the plastein reaction to prepare the plastein-reaction-stressed hydrolysate. Response surface methodology was applied to select suitable reaction conditions as follows: enzyme addition level 1037 U/g peptides, substrate concentration 29.7% (w/v), reaction temperature 20.3°C. The stressed hydrolysate showed the highest scavenging activity on ABTS radical (about 47.9%) or maximal reaction extent when reaction time was 6 h. Three stressed hydrolysates with different reaction extents were prepared and evaluated for other antioxidant activities. Compared to the original hydrolysate, the stressed hydrolysate with lower reaction extent exhibited a similar (P > 0.05) scavenging activity on DPPH (or superoxide) radical and reducing power, but a significant higher activity (P < 0.05) on hydroxyl radical. The stressed hydrolysate with the highest reaction extent behaved as these investigated antioxidant properties were significantly higher (P < 0.05) than the original hydrolysate except for scavenging activity on DPPH radical. The results of the present study highlight that the alcalase-catalyzed plastein reaction appears to be capable of improving antioxidant properties of soybean protein hydrolysate.     

Antioxidative and ACE inhibitory activities in enzymatic hydrolysates of the cotton leafworm, Spodoptera littoralis

The larvae of the cotton leafworm, Spodoptera littoralis, were used as a source of food proteins exerting possible biological activities. A simulated gastrointestinal digestion (IC₅₀ = 320 μg/ml) and digestion by mucosal enzymes (IC₅₀ = 211 μg/ml) reveals a significantly higher in vitro ACE inhibitory activity compared to hydrolysis using thermolysin (IC₅₀ = 1392 μg/ml) and alcalase (IC₅₀ = 827 μg/ml) as pretreatment. This indicates that the choice of enzymes to generate ACE inhibitory peptides is important. All hydrolysates were also tested for antioxidant activity using two tests: a radical scavenging test using DPPH and the ferric reducing antioxidant power (FRAP) assay, and they showed a similar antioxidant activity which was relatively low compared to the standard antioxidants BHT and vitamin C. As a conclusion, the data obtained suggest that insect protein can be used to generate hydrolysates, exerting both ACE inhibitory and antioxidant activity, which might be incorporated as multifunctional ingredient into functional foods.     

Coupled Neutrase–Catalyzed Plastein Reaction Mediated the ACE-Inhibitory Activity In Vitro of Casein Hydrolysates Prepared by Alcalase

Casein hydrolysates with a degree of hydrolysis of 13.5% were prepared by hydrolyzing casein with an alkaline protease Alcalase, and showed ACE-inhibition in vitro with an IC₅₀ value of 45.2 μg/mL. The hydrolysates were modified by plastein reaction catalyzed by a neutral protease Neutrase to reveal the impact of the coupled Neutrase-catalyzed plastein reaction on the ACE-inhibition of the casein hydrolysates. The effects of addition level of Neutrase, substrate concentration, reaction temperature, and time on the plastein reaction of the casein hydrolysates were studied with the varying amount of free amino groups of the modified hydrolysates as index. The results illustrated that the amount of free amino groups of the modified hydrolysates increased in all occasions, and the addition level of Neutrase, substrate concentration, and reaction time had a clear impact on the plastein reaction. Six modified hydrolysates were prepared at a substrate concentration of 40% (by weight), Neutrase addition level of 3 kU/g peptides, reaction temperature of 35°C, and different reaction time. The assay results highlighted that the coupled Neutrase-catalyzed plastein reaction improved the ACE-inhibition of six modified hydrolysates with IC₅₀ values ranging from 15.6 to 20.0 μg/mL. Size exclusion chromatography analysis showed that some plasteins with a molecular weight of about 68 kDa existed in the modified hydrolysates. The results also demonstrated that it was the coupled Neutrase-catalyzed plastein reaction but not further hydrolysis of casein hydrolysates that enhanced the ACE-inhibition of the modified casein hydrolysates.     

In Vitro Angiotensin I-Converting Enzyme Inhibition of Casein Hydrolysate Responsible for Plastein Reaction in Ethanol-Water Medium,Solvent Fractionation,and Protease Digestion

A casein hydrolysate generated by Alcalase had in vitro ACE-inhibitory activity of 44.4%, and was treated by Alcalase-catalyzed plastein reaction in ethanol-water medium. Alcalase addition, ethanol, substrate concentration, and reaction temperature optimized from experimental design were 8.36 kU/g peptides, 56.8 (v/v), 56.8% (w/v), and 37.5°C, respectively, when reaction time was fixed at 6 h. Two treated casein hydrolysates, namely TCH4 and TCH8, were obtained with reaction time of 4 and 8 h, and exhibited the highest ACE-inhibitory activity of 62.5% or the greatest reaction extent but an activity of 35.6%, respectively. Fractionation of TCH4 and TCH8 by applying ethanol-water of 7:3 (v/v) conferred the obtained supernatant (precipitate) fractionates higher (lower) activity than the parent substrate, while applying ethanol-water of 3:7 (v/v) or water led to an opposite result in activity for the fractionates. In vitro digestion of TCH4, TCH8, and their fractionates revealed that they had resistance in activity towards the investigated four proteases, as the resulted 47 out of 48 digests had higher activities than casein hydrolysate. TCH8 exhibited better protease resistance than TCH4. It is concluded that the applied plastein reaction can enhance ACE inhibition and protease resistance of casein hydrolysate.     

Angiotensin I-converting enzyme inhibitory and hypocholesterolemic activities: Effects of protein hydrolysates prepared from Achatina fulica snail foot muscle

The angiotensin I-converting enzyme (ACE) inhibitory activity and hypocholesterolemic effect of Achatina fulica snail foot muscle protein hydrolysates (SFMPH) and its hydrolysates were studied. The SFMPHs were prepared at a temperature of 121°C for 60 min. To obtain the enzymatic hydrolysates, the SFMPHs were further hydrolysed with three proteases (papain, trypsin, or alcalase). Among all the hydrolysates, alcalase hydrolysate showed the highest degree of hydrolysis and was dominated by a small molecular size fraction (189–686 Da). The SFMPH treated by alcalase was effective in disintegrating intact cholesterol micelles. Furthermore, alcalase hydrolysate with a hydrolysis time of 60 min showed a strong ACE inhibitory activity in vitro with an IC₅₀ of 0.024 mg/mL. Therefore, alcalase hydrolysate may be a promising ingredient for the use in functional foods.     

酪蛋白水解物的类蛋白反应修饰及其产物ACE抑制活性特征

采用碱性蛋白酶水解酪蛋白,制备水解度为10.9%、IC50值为52.6μg/mL的酪蛋白水解物,并利用响应面法优化碱性蛋白酶催化的类蛋白反应修饰条件。修饰反应时间固定为6h时,适宜的条件为酶添加量3.1kU/g pro、底物质量浓度50g/100mL、反应温度25℃。制备9个修饰程度不同的修饰产物,结果显示：修饰产物ACE抑制活性均提高,并且活性最高的修饰产物的IC50降低至14.9μg/mL。该修饰产物离心分级后,上清液部分和沉淀部分的ACE抑制活性分别低于和高于修饰产物,表明沉淀部分是提高ACE抑制活性的主要原因;Tricine-SDS-PAGE电泳分析表明,修饰产物及沉淀部分有较大分子质量的肽分子生成;该修饰产物和上清液部分、沉淀部分的进一步酶水解处理则显示,酶水解会导致它们的ACE抑制活性降低,但是仍然高于最初的酪蛋白水解物。     

Protein hydrolysate from salmon frame debittered by plastein reaction: amino acid composition,characteristics and antioxidant activities

Impacts of plastein reaction on bitterness, physicochemical and antioxidant properties of salmon frame hydrolysate with the aid of various proteases (alcalase and papain) at different concentrations and varying reaction temperatures were investigated. Plastein was produced from hydrolysate by papain at 40°C, which had 30% degree of hydrolysis (30DHP). Rearrangement of peptides in hydrolysate was performed by 1% papain at 40°C for 10 h, yielding plastein namely ‘30DHP-P1’. It showed the lowest bitterness (P < 0.05) than other plasteins and hydrolysates. Surface hydrophobicity was not related well with bitterness. Therefore, the size of peptides also determines the bitterness. 30DHP-P1 had augmented solubility; however, its antioxidant activities (DPPH and ABTS radical scavenging activities and ferric reducing antioxidant power) were slightly lower (P < 0.05) than those of hydrolysates. Bitterness of hydrolysate was markedly debittered via plastein reaction under optimal condition. Plastein generally had lighter colour and still possessed antioxidant activity.     

Inhibition of the in vitro activities of α-amylase, α-glucosidase and pancreatic lipase by yellow field pea (Pisum sativum L.) protein hydrolysates

The aim of this work was to produce yellow field pea protein-derived peptides as inhibitors of α-amylase, α-glucosidase and pancreatic lipase activities. A pea protein concentrate was hydrolysed with alcalase, chymotrypsin, pepsin or trypsin and the hydrolysates separated into different fractions (<1, 1–3, 3–5, 5–10 kDa) by membrane ultrafiltration. Peptide sequence analysis showed that the alcalase hydrolysate had higher levels of di- and tripeptides when compared with the chymotrypsin, pepsin and trypsin hydrolysates. The peptide fractions inhibited α-amylase and α-glucosidase activities at levels that were similar to the unfractionated hydrolysates. The peptides were more active against α-amylase (inhibition at μg level) than α-glucosidase (mg level). In contrast, the fractionated peptides had reduced ability (IC₅₀ >4.2 mg mL⁻¹) when compared with the unfractionated hydrolysate (IC₅₀ <4.2 mg mL⁻¹) to inhibit lipase activity. Enzyme kinetic studies revealed that the peptides reduced α-amylase activity through competitive inhibition. However, inhibition of α-glucosidase activity was non-competitive.     

Effects of Heat Treatment on Total Phenolic Contents,Antioxidant and Anti-Inflammatory Activities of Pleurotus Sajor-Caju Extract

Untreated and heat treated Pleurotus sajor-caju methanolic extracts were examined for total phenolic content, antioxidant and anti-inflammatory activities. Total phenolic content was determined by using the Folin-Ciocalteu method, and results were expressed as mg gallic acid equivalent per g of sample extract. Antioxidant activity was measured by using the DPPH free radical scavenging method while anti-inflammatory activity was measured by using the Griess assay. Methanolic extracts of fresh fruiting bodies were heated at 100 and 121°C for 15 and 30 min. The results showed that total phenolic contents of heat-treated samples did not increase significantly when compared to the control sample. Total phenolic contents were within the range of 0.06 ± 0.05 to 0.08 ± 0.05 mg gallic acid/equivalents g of dry weight. The samples heated at 100°C for 15 and 30 min showed the highest radical scavenging activity at 500 μg/ml with the lowest IC₅₀ values of 10.4 ± 3.11 and 11.67 ± 2.75 μg/ml, respectively. The results suggested that the increased antioxidant activity of P. sajor-caju after the heat treatment might have increased the health beneficial effects to humans.     

酪蛋白类蛋白物的溶剂分级和酶水解对ACE抑制活性的影响

利用响应面法优化类蛋白反应条件修饰酪蛋白水解物制备酪蛋白类蛋白物.酪蛋白类蛋白物的ACE抑制活性高于酪蛋白水解物,IC50值从52.6 mg/L降低到14.9 mg/L.利用乙醇-水混合溶剂对酪蛋白水解物和类蛋白物进行分级,结果表明,极性最低的溶剂得到的上清液部分活性较高,而沉淀部分活性较低.4种蛋白酶水解酪蛋白类蛋白物的分级产物,导致活性下降,除碱性蛋白酶外,木瓜蛋白酶、胃蛋白酶和胰蛋白酶的水解产物ACE抑制活性为31.5％～46.8％,但是仍然高于酪蛋白水解物的ACE抑制活性(27.8％),表明类蛋白反应提高了酪蛋白水解物对一些蛋白酶的体外抵抗能力.     

Antioxidant Activity of Ethanol and Lipophilic Extracts of Common Fruity Vegetables in Bangladesh

The amount of phenolics, flavonoids, and anthocyanins in ethanol extracts and antioxidant activity of both ethanol and lipophilic extracts of common fruity vegetables in Bangladesh were studied. Among the ethanol extracts of 15 fruity vegetables, M. oleifera had the highest total polyphenol content (85.05 mg gallic acid equivalent/g extract) followed by L. acutangula (61.74 mg gallic acid equivalent/g extract) and A. esculentus (48.92 mg gallic acid equivalent/g extract). L. acutangula had high a content of flavonoids (14.46 mg (+)-catechin equivalent/g extract), which was almost similar to L. siceraria (13.67 mg catechin equivalent/g extract) followed by A. esculentus (11.95 mg catechin equivalent/g extract) and S. melongena (11.42 mg catechin equivalent/g extract). Highest anthocyanins content was in F. hispida (2.22 μmol/g extract) followed by S. melongena (1.04 μmol/g extract). Ethanol extracts of A. esculentus, F. hispida, L. acutangula, L. siceraria, and S. melongena exhibited high DPPH free radical scavenging activity with IC₅₀ of 70.4, 64.9, 70.4, 64.9, and 94.3 μg/mL respectively, whereas for the same lipophilic extracts of F. hispida and S. melongena showed lowest (37 μg/mL) IC₅₀ followed by M. oleifera (47.6 μg/mL), L. siceraria (57.5 μg/mL), and A. esculentus (63.3 μg/mL). These vegetables also showed high reducing powers, NO scavenging and total antioxidant capacity. Therefore, the top five potential fruity vegetables consist of both hydrophilic and lipophilic antioxidant(s), the order being F. hispida > M. oleifera > A. esculentus, L. acutangula > L. siceraria > and S. melongena.     

Antihypertensive effect of alcalase generated mung bean protein hydrolysates in spontaneously hypertensive rats

Mung bean protein isolates were hydrolyzed by alcalase and neutrase, respectively, and the angiotensin I-converting enzyme (ACE) inhibitory activities of the enzymatic hydrolysates were determined at different hydrolysis times. The highest ACE inhibitory activity recorded was for a hydrolysate generated by alcalase (IC₅₀: 0.64 mg protein/ml) after 2 h of hydrolysis. A significant decrease in systolic blood pressure was observed in spontaneously hypertensive rats following a single oral administration of this hydrolysate at a dose of 600 mg/kg of body weight.     

Production of ace inhibitory peptides by digestion of chickpea legumin with alcalase

Short peptides from different sources have proved to be very efficient inhibitors of the angiotensin I-converting enzyme, an enzyme with a major role in the regulation of blood pressure. These peptides are of therapeutic value, so that the possibility of obtaining such peptides by treatment of chickpea legumin with the protease alcalase has been explored. Legumin is the main storage protein in chickpea seeds. Treatment of legumin with alcalase yielded a hydrolysate that inhibited the angiotensin I-converting enzyme with an IC₅₀ of 0.18 mg/ml. Fractionation of this hydrolysate by reverse phase chromatography afforded six inhibitory peptides with IC₅₀ values ranging from 0.011 to 0.021 mg/ml. All these peptides contain the amino acid methionine and are also rich in other hydrophobic amino acids. These results demonstrate that hydrolysates of chickpea legumin obtained by treatment with alcalase are a good source of peptides with angiotensin I-converting enzyme inhibitory activity.     

The influence of heat treatment of chickpea seeds on antioxidant and fibroblast growth‐stimulating activity of peptide fractions obtained from proteins digested under simulated gastrointestinal conditions

This study presents the effect of heat treatment of chickpea seeds on biological activity of peptides obtained by in vitro gastrointestinal digestion. The most significant antiradical activity against ABTS^+? expressed as IC₅₀ value was observed for 3.5‐ to 7‐kDa peptide fraction from TC hydrolysate (41.01 μg mL^?1). In turn, peptide fraction of 3.5–7.0 kDa obtained from raw chickpea seeds hydrolysate showed the highest antiradical activity against DPPH^? and Fe²⁺ chelating activity with IC₅₀ value of 20.94 and 52.53 μg mL^?1, respectively. The highest Cu²⁺ chelating activity was observed for peptides obtained from TC hydrolysate (IC₅₀ = 56.60 μg mL^?1). Peptide fraction <3.5 kDa from TC hydrolysate demonstrated the most significant reducing power (0.362 A₇₀₀/μg mL^?1). The peptide fraction of 3.5–7 kDa from TC hydrolysate also showed the highest fibroblast growth‐stimulating activity. These results indicated that the heat treatment process has no significant effect on antiradical activity against DPPH^? and Fe²⁺ chelating ability of peptides.     

Angiotensin I-converting enzyme inhibitory activity of chickpea and pea protein hydrolysates

ACE inhibitory activity was studied for different hydrolysates obtained from protein concentrates of chickpea (kabuli and desi) and yellow pea (Golden) using in vitro gastrointestinal simulation, alcalase/flavourzyme, and papain. Protein/peptide profiles studied by SDS–PAGE and SE-HPLC, showed a rich composition of the hydrolysates in small peptides having MWs under 4 kDa. Papain hydrolysed yellow pea proteins showed the highest ACE inhibitory activity. In addition, chickpea desi proteins hydrolysed by in vitro gastrointestinal simulation showed higher ACE inhibition (IC₅₀ of 140 ± 1 μg/ml) compared to its digests obtained by alcalase/flavourzyme (IC₅₀ of 228 ± 3 μg/ml) or papain (IC₅₀ of 180 ± 1 μg/ml) and to chickpea kabuli hydrolysed by gastrointestinal simulation (IC₅₀ of 229 ± 1 μg/ml). The results demonstrate that enzymatic hydrolysates of chickpea and pea proteins contain bioactive ACE inhibitory peptides; furthermore, the type of enzyme used for hydrolysis affects the ACE inhibitory activity.     

Angiotensin converting enzyme inhibitory activity of proteolytic digests of peanut (Arachis hypogaea L.) flour

Defatted raw and roasted peanut flour were hydrolyzed with alcalase or sequentially with pepsin and pancreatin, and then the hydrolyzates were fractionated by RP-HPLC and tested for hypotensive potential. This research revealed that proteolytic peanut digests have an inhibitory effect on the activity of angiotensin converting enzyme (ACE). Three fractions from the hydrophobic end of the chromatogram of each hydrolyzate were the most potent for inhibiting ACE activity in comparison to seven other fractions. These potentially potent fractions were then assayed for IC₅₀. Fractions from the alcalase digestion of raw peanut exhibited IC₅₀ values of 8.7-122 μg/ml, and those from roasted flour exhibited values of 12-235 μg/ml. IC₅₀ values of 7.9-65.9 μg/ml, and 11-36 μg/ml for raw and roasted peanut, respectively, from the pepsin-pancreatin system were observed. These values compare to the IC₅₀ value of 0.36 μg/ml of a known commercial ACE inhibitor (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro).     

Preparation of alcalase hydrolysed rice bran protein concentrate and its inhibitory effect on soybean lipoxygenase activity

Rice bran protein concentrate (RBPC) was prepared from defatted rice bran and hydrolysed by alcalase at different hydrolysis times. As the hydrolysis times increased, the degree of hydrolysis (DHs) increased. RBPC hydrolysate obtained at 50 min (RBPCH‐50) had the highest inhibitory efficiency on soybean lipoxygenase (LOX) activity (66%). The inhibition kinetics of the reaction analysed by Lineweaver–Burk plots indicates that RBPCH‐50 is a competitive inhibitor. RBPCH‐50 inhibited soybean LOX with an IC₅₀ of 11.73 μg μL^?1 RBPCH‐50, and the obtained K_I was 4.59 μg μL^?1 RBPCH‐50. LOX inhibitory activity of RBPCH‐50 was significantly higher than that of 50 and 100 ppm butylated hydroxyanisole (BHA) and 50, 100, and 200 ppm butylated hydroxytoluene (BHT) (P ≤ 0.05); however, LOX inhibitory activity of RBPCH‐50 was similar to that of 200 ppm BHA (P > 0.05). Therefore, RBPCH might potentially be used as a natural LOX inhibitor.     

Separation of angiotensin I‐converting enzyme inhibitory peptides from bovine connective tissue and their stability towards temperature,pH and digestive enzymes

Bovine collagen was isolated from connective tissue, a by‐product in the meat processing industry and characterised by SDS‐PAGE. Alcalase and papain were employed to generate collagen hydrolysates with different degree of hydrolysis (DH). In vitro angiotensin I‐converting enzyme (ACE) inhibitory activities were evaluated and the two most potent hydrolysates from each enzyme were separated by two‐step purification. Both alcalase‐catalysed and papain‐catalysed hydrolysates exhibited strong ACE inhibitory capacities with IC₅₀ values of 0.17 and 0.35 mg mL^?1, respectively. Purification by ion‐exchange chromatography and gel filtration chromatography revealed higher ACE inhibitory activities in one fraction from each enzyme with IC₅₀ values of 3.95 and 7.29 μg mL^?1. These peptide fractions were characterised as 6‐12 amino acid residues by MALDI‐TOF/MS. The peptides retained their activity (>90%) after exposure to processing temperature and pH and in vitro simulated gastrointestinal digestion. The present results demonstrated that collagen peptides can be utilised for developing high value‐added ingredients, for example ACE inhibitory peptides.     

耦合Plastein反应的大豆蛋白降压肽酶法制备技术

利用碱性蛋白酶酶解大豆分离蛋白,制备出水解度为16.6% 的大豆蛋白水解物,随后对水解物进行Plastein反应修饰。利用响应面分析优化修饰反应条件,得到适宜参数：底物质量分数45%、酶添加量275U/g 蛋白质、反应时间3～4h、温度30℃。制备修饰反应程度不同的9 种修饰产物并评价其体外ACE 抑制活性,发现修饰产物的IC50 值为0.64～1.30mg/mL,均小于大豆蛋白水解物IC50 值(1.45mg/mL)。排阻色谱分析结果确认,修饰产物中有更多的高分子质量肽段存在。结果显示,大豆蛋白的酶解以及耦合Plastein 反应修饰,是一种制备高ACE抑制活性大豆蛋白降压肽的新技术。