Combined effects of high-pressure and enzymatic treatments on the hydrolysis of chickpea protein isolates and antioxidant activity of the hydrolysates

A chickpea protein isolate (CPI) was pretreated before hydrolysis under a pressure that varied between 100 and 600MPa. The hydrolysis rate increased significantly with pressure above 300MPa. At 40min, the DH of the control was 15.3%, while the DH of the CPI treated at 300MPa was 18.5%, which reached 23.74% post treatment at 400MPa. The pretreatment of CPI above 300MPa enhanced the superoxide anion capturing rate of enzymatic hydrolysis. Pretreatment at 400MPa significantly reduced the hydrolysis time with the release of antioxidant peptides. While hydrolysis by Alcalase during treatment at high pressure (100-300MPa) significantly increased the degree of hydrolysis (DH), its maximum value peaked after hydrolysis at 200MPa for 30min. In addition, hydrolysates obtained at high pressure (100-300MPa) had a higher superoxide anion capturing rate. High-pressure treatment at 200MPa for 20min resulted in products with high antioxidative activity. The molecular-weight (MW) determination of the enzymatic hydrolysates indicated that hydrolysis at high pressure could significantly increase the amount of low-molecular-weight peptides.     

Proteinase and exopeptidase hydrolysis of whey protein: Comparison of the TNBS,OPA and pH stat methods for quantification of degree of hydrolysis

Whey protein hydrolysates were generated with Alcalase 2.4L and Debitrase HYW20 which are proteinase and exopeptidase enriched enzyme preparations, respectively. Degree of hydrolysis (DH) values were quantified with the TNBS, OPA and pH stat methods. Poor correlation was observed between the three methods for DH values in Debitrase HYW20 hydrolysates. For Alcalase 2.4L hydrolysates, the OPA method gave DH values that were approximately 15% lower than the pH stat, whereas TNBS DH values were similar to the pH stat method. As whey proteins are relatively rich in cysteine, a weak and unstable reaction between OPA and cysteine was thought to contribute to the under-estimation of DH in whey protein hydrolysates. Since TNBS reacts strongly with cysteine and TNBS DH values were unaffected by the type of enzyme preparation used to generate the hydrolysate, the TNBS method was deemed most suitable for the quantification of DH in whey protein hydrolysates.     

Assessment of the residual immunoreactivity of soybean whey hydrolysates obtained by combined enzymatic proteolysis and high pressure

Soybean (Glycine max) whey was hydrolyzed for 15 min using three food-grade proteases (Alcalase, Neutrase, Corolase PN-L) at atmospheric pressure (0.1 MPa) and under high pressure (HP) at 100, 200, and 300 MPa. All hydrolysates were analyzed by SDS-PAGE and their residual immunoreactivity was assessed by immunoblotting using the sera from children allergic to soybean. As shown in SDS-PAGE, Alcalase, Neutrase, and Corolase PN-L produced different patterns of hydrolysis. Each enzyme showed a similar proteolytic activity at atmospheric pressure and at 100 MPa, while an increased degree of hydrolysis was observed at 200 and 300 MPa. No residual antigenicity was observed in the hydrolysates obtained by Alcalase and Corolase PN-L in all considered conditions of hydrolysis. A positive reaction associated with a band having molecular weight of about 70 kDa was observed in the immunoblotting of the hydrolysates obtained with Neutrase at 0.1, 100, and 200 MPa, while no antigenicity was detected for those samples obtained under high pressure, at 300 MPa. These results suggest that high pressure combined with suitable enzymatic activity could be a useful tool for obtaining hydrolysates with low immunoreactivity to be used in special foods (hypoallergenic foods).     

Functional properties of hydrolysates from Phaseolus lunatus seeds

Use of low degree of hydrolysis (DH < 10%) with enzymatic treatment can produce protein hydrolysates with functional properties superior to the raw material. Suspensions of Phaseolus lunatus protein isolate (PPI) were treated with one of two commercial enzymes (Alcalase or Flavourzyme) at 50 °C and pH 8.0. DH with Alcalase was greater than Flavourzyme at 5 or 15 min of reaction. Alcalase-prepared hydrolysates had more peptides than those prepared with Flavourzyme. All the hydrolysates had higher solubility than the PPI, the highest being for the Alcalase-prepared hydrolysate at 15 min reaction time. Overall, the Alcalase-prepared hydrolysates had better solubility characteristics, whereas the Flavourzyme-prepared hydrolysates had better film properties (maximum emulsifying capacity and the highest foam formation values). This is probably because of the greater ease of movement toward the interface as shown by their high surface hydrophobicity values. The Alcalase-prepared hydrolysates had generally low or nonexistent film properties.     

酶法水解小麦面筋蛋白研究

分别采用Alcalase、Neutrase、Protamex、Flavorzyme、胃酶、木瓜蛋白酶对小麦面筋蛋白进行水解研究,结果表明,Alcalase能有效水解面筋蛋白,并确定其最佳水解条件:pH 9．0,温度60℃,底物浓度5％,酶浓度E/S为1％,即0．03AU/g蛋白,反应时间4hr左右,在此条件下,水解度可达 20％左右。     

罗非鱼皮明胶酶解物及其模拟消化产物的抗氧化活性

以罗非鱼皮为原料提取罗非鱼皮明胶,选用风味蛋白酶和胰蛋白酶制备罗非鱼皮明胶酶解物,采用ABTS自由基、DPPH自由基、羟基自由基及亚油酸过氧化体系,初步评价罗非鱼皮明胶酶解物的抗氧化活性,再通过模拟体外胃肠道消化实验,结合分子质量分布测定,进一步考察罗非鱼皮明胶酶解物的抗氧化活性。结果显示,在酶解过程中,风味蛋白酶及胰蛋白酶酶解的水解度逐渐升高,在3 h时达到最高,分别达到5.8%和25.36%。在酶解60 min时其TCA可溶性肽得率最高,风味蛋白酶、胰蛋白酶酶解物分别达56.82%和54.44%。通过比较半抑制浓度（IC₅0）,确定了酶解60 min时风味蛋白酶酶解物的清除DPPH自由基及抑制亚油酸过氧化能力较胰蛋白酶酶解物强。模拟体外胃肠道消化后,酶解物羟基自由基清除活性均显著提高（p<0.05）,亚油酸脂质过氧化活性明显降低,消化前后样品分子量分布范围均主要集中于3000⁵000 Da,消化后风味蛋白酶及胰蛋白酶酶解物3000⁵000 Da组分的含量分别提高了45%及13%。以上研究结果表明,罗非鱼皮明胶酶解后制备的明胶水解物具有一定的抗氧化能力,具有潜在的开发价值。      

Antioxidant activities and functional properties of grass carp (Ctenopharyngodon idellus) protein hydrolysates

BACKGROUND: Grass carp, with an annual production exceeding 4 × 10⁶ t in China in 2009, has not been developed into a high‐value product. In this study the antioxidant activities and functional properties of grass carp protein hydrolysates prepared with Alcalase 2.4L (HA) and papain (HP) were investigated. The hydrolysate with strongest radical‐scavenging activity and reducing power was assessed further for changes in its antioxidant activity during simulated gastrointestinal digestion. RESULTS: As the degree of hydrolysis (DH) increased, the metal‐chelating activity of both HA and HP increased while their reducing power and 1,1‐diphenyl‐2‐picrylhydrazyl radical (DPPH^?)‐scavenging activity decreased (P < 0.05). At the same DH, HP possessed higher DPPH^?‐scavenging activity and reducing power than HA (P < 0.05). The metal‐chelating activity of HP with 10% DH was significantly increased after in vitro gastrointestinal metabolism (P < 0.05). Regarding their functional properties, all hydrolysates were more than 81% soluble over a wide range of pH (3–8). At the same DH, HP showed higher emulsion activity index but lower solubility and foaming capacity than HA. CONCLUSION: Grass carp protein hydrolysates showed high solubility over a wide pH range and could be used as natural antioxidants in food systems. Copyright © 2011 Society of Chemical Industry     

鲑鱼皮明胶水解物的抗氧化活性及其对细胞氧化损伤的保护作用

采用碱性蛋白酶、木瓜蛋白酶和中性蛋白酶水解鲑鱼皮明胶,得到水解度为4.7%到13.5%的7个明胶水解物,分别评价明胶水解物对自由基的清除能力。明胶水解物(0.5、1、2mg/mL)预先作用BRL大鼠肝细胞2h后,通过H2O2(5mmol/L)诱导氧化损伤,分别测定细胞存活率、乳酸脱氢酶(LDH)、丙二醛(MDA)和谷胱甘肽(GSH)等细胞内抗氧化产物的含量变化。结果表明,明胶水解物的抗氧化活性随着水解度的增大呈增强趋势。明胶水解物对细胞具有保护作用,可显著提高细胞存活率,降低LDH渗出量和MDA生成量,且存在一定的剂量关系,但细胞中GSH含量变化不显著;7个明胶水解物对H2O2诱导肝细胞损伤保护时,细胞存活率与水解物的体外抗氧化活性大小显著正相关,而LDH渗出量和MDA生成量与水解物的体外抗氧化活性大小负相关。      

不同蛋白酶水解酪蛋白及其对产物功能性质的影响

采用Alcalase 2．4L和Protamex两种蛋白酶分别水解酪蛋白酸钠（蛋白质含量88．03％）至5％、10％、15％和20％等不同的水解度（DH），并对酪蛋白酸钠及填水解产物的各种功能性质进行了分析测定。结果表明：酪蛋白酸钠经水解后，蛋白质、水分和灰分含量发生变化，游离氨基量增加且增加与DH相关；水解产物中的多肽分子量较小，平均分子量小于8103D，并且分子量随DH的增大而减小，在DH为15％和20％的水解产物中多肽分子量均低于5043D：水解产物的溶解性随DH的增大而增强，在pH4．0～5．0、DH10％～20％的范围内产物溶解度84．8％～98％，说明在等电点条件下，酪蛋白酸钠水解后溶解性得到改善：与酪蛋白酸钠相比，水解产物的乳化性和起泡性减弱；不同水解产物的氨基酸组成差异不是很大，与酪蛋白酸钠也很接近。     

Effect of pretreatment on enzymatic hydrolysis of bovine collagen and formation of ACE-inhibitory peptides

Bovine collagen was pre-treated (boiled or high pressure (HP)-treated) and then hydrolysed by 6 proteases. The degree of hydrolysis (DH) and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates were measured. All enzymes used were able to partly degrade collagen and release ACE-inhibitory peptides. The highest ACE-inhibitory activity was obtained with Alcalase. Pretreatment significantly influenced the DH and ACE-inhibition. For most enzymes, boiling for 5 min resulted in a significantly higher DH and ACE-inhibitory activity. With Alcalase and collagenase, hydrolysis and release of ACE-inhibitory peptides occurred without any pretreatment, but HP-treatment significantly improved the DH and ACE-inhibitory activity. HP did not markedly affect the hydrolysis with the other enzymes. The major peptides obtained with Alcalase were identified; all were released from the triple helix structure of collagen. Many of these peptides had C-terminal sequences similar to known ACE-inhibitory peptides. The present results suggest that collagen-rich food materials are good substrates for the release of potent ACE-inhibitory peptides, when proper pre-treatment and enzymatic treatment is applied.     

鱼鳞明胶酶解制备超氧阴离子清除活性肽的研究

研究了Alcalase2.4L水解鱼鳞明胶的工艺条件及产物的超氧阴离子清除活性。以水解度为指标,在单因素实验的基础上,通过响应面实验优化得到最佳酶解条件为温度54.5℃,pH7.7,底物浓度1.8%(W/V),加酶量1%(W/W),在此条件下水解2h的水解度为13.02%。对酶解产物进行体外超氧阴离子清除活性分析,结果表明,不同水解度的酶解产物表现出不同的抗氧化活性。在优化条件下水解4h,水解产物的超氧阴离子清除活性最高,IC50为0.36mg/mL,此时水解度为14.11%。     

杏仁蛋白Alcalase水解工艺及其体外抗氧化活性的研究

为深入开发利用杏仁蛋白资源,采用Alcalase蛋白酶水解杏仁蛋白,以水解度为指标对酶解过程进行研究,在单因素试验基础上以水解度和DPPH自由基清除率为指标进行酶解正交试验.结果表明制备抗氧化能力较强的杏仁蛋白水解物的最佳条件为:pH 8.0,温度55 ℃,酶底比4%,底物浓度5%.在此条件下进行水解试验,水解度为21.02%,水解物对DPPH自由基的清除率为66.13%.     

Physicochemical and antioxidant properties of buckwheat (Fagopyrum esculentum Moench) protein hydrolysates

The enzymatic hydrolysis of common buckwheat (Fagopyrum esculentum Moench) protein isolate (BPI) by Alcalase and some physiochemical and antioxidant properties of the resulting hydrolysates were characterised. The hydrolysis resulted in remarkable decrease in the globulins or protein aggregates and concomitant increase in peptide fragments. The surface hydrophobicity of the hydrolysates decreased with increasing degree of hydrolysis (DH) and reached a minimum at DH 15%, but increased at further hydrolysis, whereas their amino acid compositions were unchanged. The polyphenol content of the hydrolysates gradually decreased with DH increasing from 0% to 15%, while it on the contrary increased upon further hydrolysis. The hydrolysates exhibited excellent antioxidant activities, including DPPH radical scavenging ability, reducing power and ability to inhibit linoleic acid peroxidation. The antioxidant activities of these hydrolysates were closely related to their polyphenol contents. The results indicated that polyphenol-rich buckwheat proteins are unique protein materials for the production of the hydrolysates with good nutritional and antioxidant properties.     

Preparation and Functional Properties of Enzymatically Deamidated Soy Proteins

Heat-denatured soy protein was hydrolyzed by Alcalase to 2.0% or 4.0% degree of hydrolysis (DH), heated again at 100°C and deamidated with B. circulans peptidoglutaminase. The extent of deamidation was 6.0% and 8.2% for 2.0 DH hydrolysates and 12.8% and 16.0% for 4.0 DH hydrolysates heated for 15 and 30 min, respectively. Deamidation increased protein solubility and substantially enhanced emulsifying activity under mildly acidic (pH 4–6) as well as alkaline conditions. Deamidation improved emulsion stability and foaming power of heat-denatured hydrolysed soy proteins. Enzymatically deamidated soy protein hydrolysates had improved functional properties compared to nondeamidated hydrolysates and the native soy protein.     

酶法制备不同水解度高温豆粕水解产物的理化特性研究

采用Alcalase酶和木瓜蛋白酶分别对高温大豆粕进行酶解,通过控制酶解反应得到水解度为5%、10%和15%的6种水解产物,研究两种酶对不同水解度的水解产物理化特性的影响。结果表明,Alcalase酶和木瓜蛋白酶均可产生6种不同分子量范围的水解产物,但各部分比例具有显著差异(P0.5),其平均分子量随水解度的增加逐渐减少,Alcalase酶的水解产物中小于2562 Da小分子量肽所占比例更高。豆粕蛋白的疏水基团在酶解反应中发生暴露与断裂的数量差,导致其表面疏水性随水解度增加呈现先下降再上升的变化,即水解度为10%的表面疏水性最低。zeta电势的绝对值随水解度不断上升,分子间的斥力增大,相同水解度下两种酶对zeta电势的影响并不显著。此外,在pH值为3、5、7和9时,水解产物的溶解性随着水解度的增加而逐渐增高,乳化活性和乳化稳定性则逐渐降低。     

Evaluation of the residual antigenicity of dairy whey hydrolysates obtained by combination of enzymatic hydrolysis and high-pressure treatment

Dairy whey was hydrolyzed for 15 min with five food-grade enzymes (Alcalase, Neutrase, Corolase 7089, Corolase PN-L, and Papain) at atmospheric pressure (0.1 MPa) and in combination with high pressure (HP) at 100, 200, and 300 MPa, applied prior to or during enzymatic digestion. The peptide profile of the hydrolysates obtained was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and their residual antigenicity was assessed by immuno-blotting with anti-beta-lactoglobulin monoclonal antibodies and the sera from pediatric patients allergic to cow's milk proteins. Moreover, to evaluate the presence of residual trace amounts of casein in bovine whey hydrolysates, immunoblotting with anti-cow's milk protein polyclonal antibodies was performed. SDS-PAGE analysis showed that HP treatment increased hydrolysis by the proteases assayed, especially when it was applied during the enzymatic digestion. Positive reactions at the band corresponding to beta-lactoglobulin were detected for Corolase PN-L and Corolase 7089 hydrolysates, except for those obtained under 300 MPa by the last protease. However, the immunochemical reaction was lower in the hydrolysis products obtained under HP than in those obtained at atmospheric pressure and after the HP treatment. On the contrary, no residual immunochemical reactivity associated with beta-lactoglobulin was observed in the hydrolysates obtained by Alcalase and Neutrase under HP, and none was observed in any of the hydrolysis products obtained by Papain. The presence of traces of casein was not significant. These results suggest that HP combined with selected food-grade proteases is a treatment that can remove the antigenicity of whey protein hydrolysates for their use as ingredients of hypoallergenic infant formulae.     

Improving antioxidant activities of whey protein hydrolysates obtained by thermal preheat treatment of pepsin,trypsin, alcalase and flavourzyme

Antioxidant activity of whey protein concentrate (WPC) hydrolysates was evaluated. Hydrolysates were obtained by pepsin, trypsin, alcalase and flavourzyme enzymatic reaction and preheat treatment of 95 °C for 5 or 10 min. The degree of hydrolysis (DH) was determined by 2,4,6‐trinitrobenzene sulphonic acid method, and antioxidant properties were determined by three spectrophotometric methods: ferricyanide method, ferric reducing/antioxidant power assay and diphenyl‐picryl hydrazinyl radical‐scavenging activity. For all the enzymes, briefly preheat treatment (95 °C/5 min) increased DH of WPC. Alcalase hydrolysates showed the highest antioxidant activity by three methods. The changes in antioxidant activity was coincidental with the changes in DH (R² = 0.988). Hydrolysates analysed by polyacrylamide gel electrophoresis and high performance liquid chromatography indicated that the α‐La was hydrolysed completely by pepsin, trypsin and alcalase and was resistant to flavourzyme to some extent; β‐lactoglobulin was only completely hydrolysed by trypsin and alcalase. Results indicated that antioxidant activity of hydrolysates was greatly related to the exposure of amino acid residues.     

鸡蛋清蛋白水解物的物化及功能性质的研究

蛋清蛋白质溶解性欠佳且容易起泡,使其应用受限。本文利用Alcalase酶解鸡蛋蛋清蛋白制取水解度为5~15%的酶解物并对酶解物的理化性质和功能性质进行了表征。测定性质包括乳化性、起泡性及稳定性以及溶解性、表面疏水性。所有酶解产物具有较低的表面疏水性,水解大大提高了溶解度,当水解度15%时最大值为89%,但乳化性有所降低,起泡性及稳定性也大约下降了40%。     

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.     

Solubility,functional properties,ACE‐I inhibitory and DPPH scavenging activities of Alcalase hydrolysed soy protein hydrolysates

Soy proteins are less soluble at acidic pH value, which impedes their utilisation in acidic beverages. Soy protein isolate (SPI) was hydrolysed using varying Alcalase concentrations (0.0001–2.0 U g^?1 protein) at different pHs (3.0–4.0). Degree of hydrolysis (DH) of soy protein hydrolysates (SPH) at pH 3.0, 3.5 and 4.0 were 5.0–10.7%, 2.3–6.1% and 0–5.4%, respectively, while solubilities ranged from 70.7 to 74.9%, 18.8 to 51.2% and 7.1 to 40.4%, respectively. The highest solubility (74.9%) was observed at pH 3.0 with 1.5 U Alcalase per g protein (DH = 9.2%). Emulsifying activities of SPHs at pH 3.0 and 4.0 ranged from 0.49 to 0.63 AU and 0.19 to 0.24 AU, respectively, while the emulsifying stabilities were 12.2–14.7 min and 18.7–56.0 min, respectively. The foaming capacity at pH 3.0 and 4.0 was 44.9–46.3 mL and 31.2–41.3 mL, respectively, whereas the foaming stability was 25.5–35.2 min and 12.8–15.1 min, respectively. However, hydrolysates had an insignificant effect on ACE‐I inhibitory and DPPH scavenging activities in comparison with SPI.