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.     

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。     

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.     

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。     

大豆蛋白水解物的酶法修饰及其亚铁和钙离子的螯合能力

利用碱性蛋白酶水解大豆分离蛋白制备出水解度为14.1%的水解物,并在醇-水介质中对其进行类蛋白反应修饰。固定底物质量分数30%,反应时间4 h,利用响应面分析法对类蛋白反应条件进行优化,得到最优条件为:酶添加量5.26 kU/g蛋白质,乙醇体积分数56.8%,温度33.1℃。在乙醇-水或甲醇-水介质中制备反应程度不同的修饰产物,并评价其对亚铁和钙离子的螯合能力。结果表明,亚铁离子螯合能力由39.8%提升至59.3%,钙离子螯合能力由62.1%提升至76.6%。大豆分离蛋白的酶解以及类蛋白反应修饰,是一个提高金属离子鳌合能力大豆蛋白螯合肽的新技术。     

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.     

三种氨基酸添加下酶法修饰酪蛋白水解物的ACE抑制活性

采用碱性蛋白酶水解酪蛋白,制备水解度为12.4%、IC50为42.19μg/mL的酪蛋白水解物。在添加外源氨基酸的情况下对水解物进行类蛋白反应修饰,并响应面法研究氨基酸添加量、酶添加量、反应温度及3种氨基酸的影响。结果表明:氨基酸添加量、反应温度、氨基酸种类对修饰反应影响显著,而酶添加量的影响不大;分别添加苯丙氨酸、亮氨酸、缬氨酸制备3个酪蛋白水解物修饰产物,其IC50降低至21.03～25.13μg/mL,表明添加外源氨基酸可提高修饰产物的体外ACE抑制活性,但添加不同氨基酸的影响不显著。     

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.     

酪蛋白水解物的酶法修饰与ACE抑制活性变化

利用枯草杆菌碱性蛋白酶水解酪蛋白制备酪蛋白水解物,其水解度为11.2%,IC50为47.1μg/mL。再应用相同的酶对酪蛋白水解物进行类蛋白反应修饰,考察底物浓度、温度和酶添加量对类蛋白反应的影响,并制备5个不同的修饰产物测定其ACE抑制活性和IC50值。结果表明,修饰产物的ACE抑制活性随修饰程度(游离氨基减少量)的增加而提高,并且都高于未经修饰的酪蛋白水解物。当游离氨基减少量为154.65μmol/g(蛋白)时,修饰产物的IC50值可降至0.6μg/mL。毛细管电泳分析结果显示类蛋白修饰后水解物的多肽组成情况发生明显变化。研究结果证明酪蛋白水解物的ACE抑制活性可以通过类蛋白反应的修饰作用而提高。     

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.     

大豆蛋白的酶水解及类蛋白反应对其抗氧化活性的影响

采用Alcalase 2.4L FG 蛋白酶水解大豆蛋白,筛选并制备出ABTS+·清除率最高的水解物,其水解度为14.0%,对ABTS+·清除率为43.6%。以此水解物为底物,以修饰产物的游离氨基减少量为指标,应用响应面分析得到类蛋白反应的优化条件为：酶添加量1037U/g pro、底物质量浓度30g/100mL、温度20℃。在此条件下反应6h,水解物的修饰反应程度和抗氧化活性均为最大。制备反应程度不等的3 个修饰产物,进一步抗氧化活性分析表明：大豆蛋白水解物及其修饰产物的抗氧化活性好于大豆蛋白;修饰产物与水解物的DPPH 自由基清除能力、还原力、超氧阴离子自由基(O2 － ·)清除率差别不显著,但是对羟自由基(·OH)清除率差别显著。     

Preparation of Alcalase-catalyzed casein plasteins in the presence of proline addition and the ACE-inhibitory activity of the plasteins in vitro

Casein hydrolysates were prepared by hydrolysis of casein with alkaline protease Alcalase for 6 h and showed the highest ACE-inhibitory activity in vitro with an IC₅₀ value of 47.1 μg mL⁻¹. Casein hydrolysates prepared were subjected to Alcalase-catalyzed plastein reaction in the presence or absence of proline addition to prepare casein plasteins. Some optimal reaction conditions of plastein reaction in the presence of proline addition were studied using response surface methodology with the decrease in free amino groups in the casein plasteins as response. When the concentration of casein hydrolysates was fixed at 35% (w w⁻¹) and reaction time at 6 h, the optimal conditions were reaction temperature 48 °C, addition level of proline 0.54 mol/mol free amino groups of casein hydrolysates and addition level of Alcalase 9.5 kU g⁻¹ proteins. With these conditions, the maximal decrease in free amino groups in casein plasteins was 195.7 μmol g⁻¹ proteins. The ACE-inhibitory activities of twelve casein plasteins in vitro, prepared in the presence or absence of proline addition with different reaction extents, were evaluated and compared. The results showed that the ACE-inhibitory activity of the casein plasteins prepared in the presence of proline addition changed irregularly, different to that of the casein plasteins prepared in the absence of proline addition, and might relate to the different linking of proline to the peptides in casein hydrolysates during plastein reaction. When the casein plasteins prepared in the presence of proline addition had a decrease in free amino groups 195.7 μmol g⁻¹ proteins, the IC₅₀ value of the casein plasteins was lowered to 0.2 μg mL⁻¹.     

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.     

Production of enzymatic hydrolysates with antioxidant and angiotensin-I converting enzyme inhibitory activity from pumpkin oil cake protein isolate

Protein isolate from pumpkin oil cake (PuOC PI) was hydrolysed by alcalase, flavourzyme and by sequential use of these enzymes, respectively, and the antioxidant properties and angiotensin-I converting enzyme (ACE) inhibitory activities of hydrolysates were evaluated. Under the same reaction conditions, alcalase hydrolysates showed a higher degree of hydrolysis (DH) than did flavourzyme hydrolysates. The highest DH’s by individual enzymes were 53.23 ± 0.7% and 37.17 ± 1.05%, respectively, both at 60 min. The increase of radical scavenging activity (RSA) in hydrolysates was positively correlated with the increase of DH, for both enzymes, though hydrolysates with flavourzyme showed two- or three-fold lower RSA than with alcalase. The highest bioactive potential was determined in the alcalase hydrolysate at 60 min, with RSA being 7.59 ± 0.081 mM TEAC/mg and ACE-inhibitory activity 71.05 ± 7.5% (IC₅₀ = 0.422 mg/ml). When this hydrolysate was further hydrolysed by flavourzyme, DH increased up to 69.29 ± 0.9%, but lower RSA (4.82 ± 0.21 mM TEAC/mg) and ACE-inhibitory activity (55.81 ± 6.196%) were determined in the final hydrolysate. This study suggested that the PuOC proteins could be converted into protein hydrolysates with antioxidant and ACE-inhibitory activities by enzymatic hydrolysis. Alcalase was shown as promising enzyme in further development of bioprocesses for the production of new bioactive food ingredients.     

Production of the Angiotensin-I-Converting Enzyme (ACE)-Inhibitory Peptide from Hydrolysates of Jellyfish (<Emphasis Type="Italic">Rhopilema esculentum</Emphasis>) Collagen

Collagen extracted from jellyfish (Rhopilema esculentum) was hydrolyzed with alcalase to prepare the ACE-inhibitory peptide. The optimal hydrolyzing conditions were determined using response surface methodology. The results showed that the optimal conditions were temperature of 52.7 °C, pH of 8.63 and enzyme-to-substrate ratio (E/S) of 3.46%, and the ACE-inhibitory activity of the obtained hydrolysates could reach 81.7%. Jellyfish collagen peptide, UF3-B2, was purified from the hydrolysates using ultrafiltration, ion-exchange chromatography and gel filtration. The IC₅₀ value of UF3-B2 was 43 μg/ml, and the yield accounted for 6.25% of the hydrolysates. The molecular weight distribution of UF3-B2 was from 200 to 600 Da. Amino acid analyses showed that UF3-B2 was rich in Gly, Pro, Glu, Ala, and Asp.     

Study on the Functional Properties of Soybean Protein Isolate Cross-Linked with Gelatin by Microbial Transglutaminase

A microbial transglutaminase was used to cross-link soybean protein isolates in the presence of gelatin to prepare a cross-linked composite soybean protein-gelatin. Cross-linking conditions, such as total proteins, the ratio of soybean protein isolates to gelatin, and original pH, were fixed at 5% (w/v), 4:1 (w/w), and 7.5, respectively. The 4-hydroxyproline content in the modified product was measured and used as an index to select suitable transglutaminase addition, reaction temperature, and time by single factor experiments, which were found to be 20 U/g proteins, 40°C, and 2 h, respectively. Under the identified conditions, a modified product with 4-hydroxyproline content of about 36 mg/g proteins was prepared, and its functional properties were evaluated. SDS-PAGE analysis showed that several protein polymers existed in the modified product. Compared to the original soybean protein isolates or a cross-linked soybean protein isolates, the modified product had a better emulsifying stability and water holding capacity but a poor enzymatic digestibility in vitro, emulsifying activity and oil binding capacity; meanwhile, the dispersions formed by the modified product exhibited the highest apparent viscosity, storage modulus, and viscous modulus.     

Angiotensin-I converting enzyme inhibitory and antioxidant activities of egg protein hydrolysates produced with gastrointestinal and nongastrointestinal enzymes

Egg is a well-known rich source of bioactive peptides. In this study, egg protein (egg white and egg yolk proteins) hydrolysates were produced with gastrointestinal enzymes (pepsin and pancreatin) or nongastrointestinal enzymes (thermolysin and alcalase), and fractionated by ultrafiltration and cation exchange chromatography. Angiotensin-I converting enzyme (ACE) inhibitory and antioxidant activities, amino acid composition and molecular weight distribution were studied, and the physicochemical properties were related with the bioactivities. Our results showed that egg protein hydrolysates produced with non-GI enzymes (thermolysin and alcalase) showed significantly higher ACE inhibitory activity, whereas similar or even lower antioxidative activities, than those of hydrolysates produced with GI enzymes. ACE-inhibitory activity significantly correlated with the amino acid composition, especially the proportion of positively charged amino acid, whereas antioxidant activities correlated with the proportion of low molecular weight peptides under 500 Da. Understanding the relationship between the bioactivities and physicochemical properties of the hydrolysates/fractions is important to facilitate the development technologies for preparing fractions with improved bioactivities.     

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。     

美拉德糖基化对玉米蛋白粉双酶水解产物抗氧化活性的影响

利用复合蛋白酶和碱性蛋白酶协同作用于玉米蛋白粉,将得到的双酶水解产物与壳寡糖进行美拉德糖基化反应,研究不同蛋白浓度下的酶解物和糖基化产物的抗氧化活性。结果表明,酶解物和糖基化产物都具有良好抗氧化活性,美拉德糖基化反应可以提高酶解物的抗氧化活性。当糖基化产物蛋白浓度为5.00 mg/mL时,其·OH清除率和DPPH·清除率分别达到了90.91%和61.75%,蛋白浓度为1.00mg/mL时,Fe~(2+)螯合能力为83.42%,比酶解物·OH清除率和DPPH·清除率分别提高了48.78%和32.26%,Fe~(2+)螯合能力提高了22.63%。