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.     

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.     

An approach to improve ACE-inhibitory activity of casein hydrolysates with plastein reaction catalyzed by Alcalase

The preparation method of casein hydrolysates with high ACE-inhibitory activity was studied by Alcalase-catalyzed hydrolysis coupled with plastein reaction. Casein hydrolysates with an IC₅₀ value of about 47 μg mL⁻¹ were first prepared by hydrolysis of casein with Alcalase and then modified with plastein reaction catalyzed by the same enzyme. The impacts of four reaction conditions on plastein reaction of casein hydrolysates were studied, and then optimal conditions were determined using response surface methodology with the decrease of free amino groups in the reaction mixture as response. When the concentration of casein hydrolysates was fixed at 35% by weight, the maximum decrease of free amino groups in the reaction mixture of 181.8 μmol g⁻¹ proteins was obtained. The optimum conditions for the above decrease were found to be an E/S ratio of 7.7 kU g⁻¹ proteins, reaction temperature of 42.7 °C and reaction time of 6 h. Analysis results showed that ACE-inhibitory activity of casein hydrolysates prepared could be improved significantly by plastein reaction. When casein hydrolysates were modified by plastein reaction, with a decrease of free amino groups in the mixture of about 154.7 μmol g⁻¹ proteins and 181.8 μmol g⁻¹ proteins, their IC₅₀ values could be decreased to 0.6 and 0.5 μg mL⁻¹.     

酶法联合Plastein反应制备海参肠调味料

为制备风味良好的海参肠调味料产品,该研究以海参肠为原料,采用酶解方法和Plastein反应修饰的方法,获得味道鲜美的调味料。以感官评分为指标,对比中性蛋白酶、风味蛋白酶、碱性蛋白酶、木瓜蛋白酶、胰蛋白酶这5种蛋白酶对海参肠的酶解效果,通过单因素探究料液比、加酶量、酶解温度的最适条件。采用Plastein反应对酶解产物进行脱腥处理,以游离氨基酸减少量和脱腥率为指标,考察Plastein反应过程中酶的种类、底物质量分数、加酶量、时间和温度对脱腥结果产生的影响。经过单因素和响应面实验优化后,获得Plastein反应最佳脱腥工艺。最后对最终产品进行游离氨基酸成分分析。结果表明,风味蛋白酶的最佳酶解工艺为:料液比1∶8(g/mL),加酶量0.5%,温度50℃,时间4 h,此条件下酶解产物鲜味浓郁,但略有腥味。最佳脱腥工艺为中性蛋白酶加酶量3.40%,底物浓度20%,温度57℃,时间100 min,所得调味料味道鲜美,无腥臭味道。游离氨基酸组成分析显示,经过Plastein反应后,必需氨基酸从反应前的45.67%增加到46.88%,6种呈味氨基酸(Gly、Ala、Asp、Thr、Ser和Glu)占总氨基酸含量的35.77%,脱腥后的修饰物有一定的海鲜风味。     

Ace Inhibition and Enzymatic Resistance In Vitro of a Casein Hydrolysate Subjected to Plastein Reaction in the Presence of Extrinsic Proline and Ethanol- or Methanol-Water Fractionation

Casein was hydrolyzed by alcalase to a degree of hydrolysis of 10.9% to obtain a hydrolysate having ACE-inhibition in vitro with an IC₅₀ value of 52.6 μg/mL. The prepared hydrolysate was modified by alcalase-catalyzed plastein reaction with extrinsic proline added at 0.4 mol/mol free amino groups (on the basis of the hydrolysate), and fractionated by ethanol- or methanol-water solvents in proportions of 3:7, 5:5, or 7:3 (v/v), respectively. With the decrease of free amino groups of the modified hydrolysate as the response, the optimized plastein reaction conditions were alcalase addition of 3.1 kU/g peptides, substrate concentration of 50% (w/v), and reaction temperature of 25°C. Four modified hydrolysates prepared with different reaction times exhibited higher ACE-inhibitory activities than the original hydrolysate. The evaluation results showed that solvent fractionation of the modified hydrolysate with the maximum activity (IC₅₀ = 13.0 μg/mL) yielded the separated soluble fraction's higher activity but the precipitate fraction's lower one. Further enzymatic digestion of the modified hydrolysate with the maximum activity and its two fractionated products by four proteases in vitro caused damage to the activities, but the residual activities of the final digests were higher than that of the original hydrolysate, indicating that the plastein reaction could confer casein hydrolysate protease resistance.     

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.     

蛋白质酶法修饰的初步探讨──大豆蛋白和芝麻蛋白的合成类蛋白质研究

本文报道了以大豆蛋白和芝麻蛋白的胰蛋白酶水解物为原料，以胃蛋白酶为催化剂，通过合成类蛋白反应制取合成类蛋白质。结果表明，在上述反应中，胃蛋白酶是一种适宜的催化剂。底物水解度为８０％时效果最佳。大部分底物的分子量在１０００以下。底物最佳浓度为３０％，最佳ｐＨ为５．０。合成类蛋白质分子量约为１０，０００。     

酶法制备联合Plastein反应修饰牡蛎ACE抑制肽工艺优化

以牡蛎为原料,采用酶解联合Plastein反应修饰的方法,获得高活性血管紧张素转换酶(angiotensin converting enzyme,ACE)抑制肽。以ACE抑制率和水解度为指标,对比胃蛋白酶、木瓜蛋白酶、碱性蛋白酶、中性蛋白酶、胰蛋白酶这5种蛋白酶对牡蛎肉的酶解效果,筛选出木瓜蛋白酶最佳。通过单因素试验和正交试验对酶解工艺进行优化,得到最佳酶解工艺为料液比1∶8(g/m L)、加酶量2.0%、温度65℃、时间1.0 h、pH6.0,此条件下酶解产物的ACE抑制率可达到63.30%,在此基础上采用Plastein反应对酶解产物进行修饰,以游离氨基酸减少量和ACE抑制率为指标,考察反应过程中酶种类、底物质量分数、加酶量、时间和温度对修饰结果产生的影响。通过该反应的修饰,得到选用中性蛋白酶、底物质量分数40%、加酶量1.0%、温度30℃、时间2.5h、pH7.0时,ACE抑制率最高可达82.31%,比修饰前提高了19%。     

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.