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.     

高活性酪蛋白抗氧化肽的制备方法研究

采用木瓜蛋白酶对酪蛋白进行水解,得到抗氧化活性较好的酪蛋白水解物,并且水解物在木瓜蛋白酶作用下进行类蛋白反应制备出高活性酪蛋白抗氧化肽。第一步制备酪蛋白水解时酶添加量为500 U/g酪蛋白、温度45℃、底物浓度5%、反应时间2 h。第二步类蛋白反应的最优条件为：酶添加量为500 U/g水解物、温度30℃,底物浓度50%、作用时间5.5 h。毛细管电泳结果确认,类蛋白反应修饰后抗氧化肽的组成情况发生变化。抗氧化活性分析结果表明,类蛋白反应修饰后的酪蛋白抗氧化肽对两种自由基的清除能力显著提高。     

实用类蛋白反应的作用机制及其对海洋源蛋白修饰的研究进展

类蛋白反应作为一种可以修饰生物活性肽的新方法,已经成为蛋白质食品研究的热点。海洋生物含有丰富的生物活性成分,其含有的蛋白肽具有多种人体代谢和生理调节功能。然而蛋白肽存在生物利用度有限、酶解液味苦等问题。本文在阐述类蛋白反应过程和机制的基础上,对类蛋白反应修饰提高海洋源活性蛋白肽的生物活性、改善蛋白的加工特性及减少蛋白酶解液苦味等研究进展进行了详细的介绍,以期为海洋源蛋白的深度利用以及高值化研究提供理论参考。     

Physicochemical and bitterness properties of enzymatic pea protein hydrolysates

ABSTRACT:  The effects of different proteolytic treatments on the physiochemical and bitterness properties of pea protein hydrolysates were investigated. A commercial pea protein isolate was digested using each of 5 different proteases to produce protein hydrolysates with varying properties. After 4 h of enzyme digestion, samples were clarified by centrifugation followed by desalting of the supernatant with a 1000 Da membrane; the retentates were then freeze-dried. Alcalase and Flavourzyme™ produced protein hydrolysates with significantly higher ( P < 0.05) degree of hydrolysis when compared to the other proteases. Flavourzyme, papain, and alcalase produced hydrolysates that contained the highest levels of aromatic amino acids, while trypsin hydrolysate had the highest levels of lysine and arginine. Papain hydrolysate contained high molecular weight peptides (10 to 178 kDa) while hydrolysates from the other 4 proteases contained predominantly low molecular weight peptides (≤ 23 kDa). DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity of the Flavourzyme hydrolysate was significantly ( P < 0.05) the highest while alcalase and trypsin hydrolysates were the lowest. Inhibition of angiotensin converting enzyme (ACE) activity was significantly higher ( P < 0.05) for papain hydrolysate while Flavourzyme hydrolysate had the least inhibitory activity. Sensory analysis showed that the alcalase hydrolysate was the most bitter while papain and α-chymotrypsin hydrolysates were the least. Among the 5 enzymes used in this study, papain and α-chymotrypsin appear to be the most desirable for producing high quality pea protein hydrolysates because of the low bitterness scores combined with a high level of angiotensin converting enzyme inhibition and moderate free radical scavenging activity.     

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.     

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.     

醋蛋水解物的类蛋白反应及对抗氧化活性的影响

先利用胃蛋白酶水解醋蛋液,再添加胃蛋白酶和氨基酸对其进行类蛋白反应,研究反应条件对类蛋白产率的影响和产物抗氧化活性的变化。结果表明,优化类蛋白反应条件为:氨基酸选用色氨酸、色氨酸添加量为1mol/mol水解物游离氨基、底物浓度40%(质量分数)、反应温度65℃、pH6.0、反应时间6h。通过类蛋白反应,醋蛋水解物的抗氧化活性得到很大改善。聚丙烯酰胺凝胶电泳结果表明,反应前后醋蛋水解物的组成发生变化,有较大分子质量的肽生成。     

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 antioxidant activities of hydrolysates obtained from Iranian wild almond (Amygdalus scoparia) protein by several enzymes

A protein extract from wild almond was hydrolysed using five different enzymes (pepsin, trypsin, chymotrypsin, alcalase and flavourzyme). The hydrolysates were then assayed for their antioxidant activities. The highest extent of proteolysis was obtained with alcalase (0.35; determined as the change in the absorbance at 340 nm, ΔA₃₄₀) and the lowest was with pepsin (ΔA₃₄₀ = 0.12). Radical scavenging activities obtained by 2, 2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulphonic acid) and ferric‐reducing abilities of the hydrolysates demonstrated that the hydrolysate from alcalase had significantly (P < 0.05) greater antioxidant activity. Analysis of the molecular weight distributions showed that peptides produced by alcalase were smaller than those produced by chymotrypsin, trypsin and flavourzyme. Based on the current study, the hydrolysates produced by alcalase can be suggested as potential antioxidant agents in food industry and for use in functional foods.     

分步酶解小麦面筋蛋白制备低苦味肽粉的研究

目的 研究分步酶解小麦面筋蛋白(wheat gluten, WG)制备低苦味肽粉的工艺。方法 选用中性蛋白酶、木瓜蛋白酶、胃蛋白酶水解WG至8%水解度,接着用风味蛋白酶对水解产物进行脱苦处理,对不同酶解产物中苦味肽的特性进行系统研究,探究苦味肽含量、氨基酸组成、分子量分布、表面疏水性等指标变化对WG酶解物苦味值的影响,对比风味蛋白酶对不同单酶酶解物的脱苦效果差异,分析风味蛋白酶对WG酶解物脱苦的内在机理,进而确定制备低苦味小麦蛋白肽粉的最佳酶解工艺。结果 中性蛋白酶的酶解产物经风味蛋白酶作用后,脱苦效果最显著,苦味肽苦味值从4.08降至2.25,酶解产物的苦味值可下降56.42%。木瓜蛋白酶的酶解产物经风味蛋白酶作用4 h后,酶解产物的苦味值最低,制备出苦味值为1.28的WG低苦味肽粉。结论 经分步酶解作用后,酶解产物中苦味肽的含量下降;疏水性氨基酸比例的下降和游离氨基酸含量的升高引起苦味肽苦味阈值的增大,共同导致酶解产物苦味值显著降低,该研究为酶解脱苦技术的快速发展和WG活性肽工业化生产提供新的参考。     

Effect of substrate ratios and temperatures on development of Maillard reaction and antioxidant activity of silver carp (Hypophthalmichthys molitrix) protein hydrolysate–glucose system

The study of antioxidant activity of the hydrolysates is necessary during its processing in which Maillard reaction would often occur. To understand the effect of Maillard reaction on antioxidant activity of silver carp protein hydrolysates (SPH), the Maillard reaction products (MRPs) were prepared at different ratios between SPH and glucose by Maillard reaction in powdered state, respectively. MRPs possessed a strong 2, 2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging activity and reducing power (P < 0.05). The hydrolysate and glucose heated with the ratio of 2:1 at 60 °C showed high browning intensity and good antioxidant properties (P < 0.05). According to the correlation coefficients of variables included in the hydrolysate–glucose system, good correlations were observed among the antioxidant activities, the absorbance at 294 nm and the loss of free amino groups. The results suggested that Maillard reaction has a good potential to improve the antioxidant activity of SPH.     

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.     

类蛋白反应及其在肉类中的应用

类蛋白反应是食品科学领域涉及蛋白质的一个重要反应,可以通过肽键结合的方式对蛋白质中的必需氨基酸进行补充,一般认为是蛋白质水解的逆反应,可以提高肽的生物活性、改善蛋白的加工特性、减少酶解液苦味。到目前为止,类蛋白反应已应用到鱼蛋白、大豆蛋白、乳蛋白等多个领域,提高了食品的营养价值,很好地解决了蛋白肽存在的生物利用率有限、酶解液味苦等问题。我国是肉类消费大国,肉类消费量占全球消费量的1/4,类蛋白反应应用于肉类工业可以为人们提供更加优质的蛋白资源,具有很重要的研究价值。该文综述了类蛋白反应的3种作用机制,影响类蛋白反应的因素以及类蛋白反应在肉类中的应用和发展前景,以期为蛋白质的深度利用以及高值化研究提供理论参考。     

Original article: Thermal pretreatment and chemical modifications as a means to alter hydrolytic characteristics and prevent bitterness in hydrolysates of fishery by‐catch (Decapterus maruadsi) protein

In this study, fishery by‐catch protein (Decapterus maruadsi) was first thermal treated (60–100 °C), phosphorylated (0.5–4.0 g g^?1 protein) or succinylated (0.05–0.3 g g^?1 protein) before hydrolysis to prevent the bitterness of the resulting hydrolysate. Hydrolysis was performed at 50 °C, 10 h, pH 8.0 by Alcalase. Results showed that thermal treatment upon 80 °C, succinylation and phosphorylation resulted in significant decrease in amino acid content and increase in larger peptide content (>1450 Da). Succinylation did not reduce the bitterness as a result of higher content of small peptides (<550 Da), which was found to be correlated positively with the bitterness of hydrolysates. However, thermal treatment, especially phosphorylation, could reduce the bitterness to discernible level, indicating that phosphorylation before hydrolysis could be a potential means to prevent the evolution of bitter flavour which restricts the practical uses of the hydrolysates.     

Biochemical properties and antioxidant activity of myofibrillar protein hydrolysates obtained from patin (Pangasius sutchi)

Antioxidant activities of myofibrillar protein hydrolysates (MPH) prepared from patin (Pangasius sutchi) using papain and Alcalase^® 2.4 L with different degrees of hydrolysis (DH) were investigated. With a DH of 65.83%, the hydrolysate prepared with papain exhibited the maximum of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging activity (71.14%) with a reducing power of 0.310. At a concentration of 1 mg mL^?1, the papain‐MPH exhibited a Trolox equivalent antioxidant capacity (TEAC) of 70.50 ± 1.22 μmol g^?1 protein. With a DH of 83.6%, the Alcalase‐MPH had the highest metal‐chelating activity. Low molecular weight peptides showed higher antioxidant activities than high molecular weight peptides. Both papain‐MPH and Alcalase‐MPH contained high amounts of the essential amino acids (48.71% and 48.10%, respectively) with glutamic acid, aspartic acid and lysine as the dominant amino acids. These results suggest that the protein hydrolysates derived from patin may be used as an antioxidative ingredient in both functional food and nutraceutical applications.     

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%。     

Fractionation and purification of antioxidant peptides from abalone viscera by a combination of Sephadex G-15 and Toyopearl HW-40F chromatography

Abalone viscera, a protein-rich by-products from the abalone processing industry, are normally discarded as wastes. In the present study, four different proteases were used to hydrolyse abalone viscera to prepare high-activity antioxidant peptides, and their hydrolysis effects were compared. Although the hydrolytic abilities of papain and trypsin were inferior to the alcalase and neutrase, their use resulted in significantly (P < 0.05) higher scavenging activities for DPPH. The hydrolysates mainly consisted of peptides with a molecular weight <3 kDa, and the fractionation was achieved using a combination of Sephadex G-15 and Toyopearl HW-40F gel filtration chromatography, which overcomes the disadvantages of traditional membrane separation technology in fractionating peptides with relatively similar molecular weight distribution. The DPPH scavenging activities of the components containing peptides <1 kDa (fractions E) were significantly (P < 0.01) higher than those containing peptides >1 kDa. The different fractions E were further purified by reversed-phase high-performance liquid chromatography, and a total of 16 target peptides, containing 4-8 amino acids, enriched in hydrophobic amino acids and C-terminal Arg, with molecular weights ranging from 500 to 850 Da, were identified.     

Antioxidant activities of enzymatic‐hydrolysed proteins of dromedary (Camelus dromedarius) colostrum

This work investigated the antioxidant activities of dromedary colostrum proteins before and after hydrolysis by pepsin, trypsin, α‐chymotrypsin, pancreatin and papain. The enzymatic hydrolysis affected the degrees of hydrolysis, electrophoretic profiles, molecular weight distribution and hydrophobic/hydrophilic properties of the generated peptides. The antioxidant activities were evaluated using four antioxidant assays, including 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) radical‐scavenging activities, ferric reducing power and ferrous ion chelating activity. Interestingly, the antioxidant activities of dromedary colostrum proteins were enhanced after enzymatic hydrolysis. The highest antioxidant potential was obtained by pancreatic hydrolysates (P ≤ 0.05). These results suggest that dromedary colostrum protein hydrolysates are an important source of natural antioxidant peptides.     

Valorisation of protein hydrolysates from animal by-products: perspectives on bitter taste and debittering methods: a review

Conversion of animal by-products to high value-added food ingredients is one of the top trends in the slaughter industry. Enzymatic hydrolysis of animal by-products can generate protein hydrolysates, which provides an opportunity for effective utilisation. However, bitterness of protein hydrolysate is a major undesirable aspect for various applications. In this review, the current knowledge on protein hydrolysates from animal by-products is briefly reviewed. The structural features of bitter peptides and bitter taste receptors are summarised. Moreover, the potential approaches for debittering protein hydrolysates are highlighted, including exopeptidase treatment, Maillard reaction, plastein reaction and encapsulation. In addition, the current debittering strategies and challenges are also discussed. This article presents some opportunities to utilise protein hydrolysates from animal by-products and their debittering methods.     

Generation,Fractionation, and Characterization of Iron‐Chelating Protein Hydrolysate from Palm Kernel Cake Proteins

Palm kernel cake protein was hydrolyzed with different proteases namely papain, bromelain, subtilisin, flavourzyme, trypsin, chymotrypsin, and pepsin to generate different protein hydrolysates. Peptide content and iron‐chelating activity of each hydrolysate were evaluated using O‐phthaldialdehyde‐based spectrophotometric method and ferrozine‐based colorimetric assay, respectively. The results revealed a positive correlation between peptide contents and iron‐chelating activities of the protein hydrolysates. Protein hydrolysate generated by papain exhibited the highest peptide content of 10.5 mM and highest iron‐chelating activity of 64.8% compared with the other hydrolysates. Profiling of the papain‐generated hydrolysate by reverse phase high performance liquid chromatography fractionation indicated a direct association between peptide content and iron‐chelating activity in most of the fractions. Further fractionation using isoelectric focusing also revealed that protein hydrolysate with basic and neutral isoelectric point (pI) had the highest iron‐chelating activity, although a few fractions in the acidic range also exhibited good metal chelating potential. After identification and synthesis of papain‐generated peptides, GGIF and YLLLK showed among the highest iron‐chelating activities of 56% and 53%, whereas their IC₅₀ were 1.4 and 0.2 μM, respectively.