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).     

High pressure can reduce the antigenicity of bovine whey protein hydrolysates

The combined effect of high pressure (HP) and enzymatic treatments on the proteolysis and antigenicity of hydrolysates from bovine whey proteins (WP) was studied. Four food grade protease preparations (Alcalase, Neutrase, Corolase 7089 and Corolase PN-L) were used. Hydrolysis was performed at 40 °C for Corolase PN-L and 50 °C for the other enzymes, for 15 min, after or during HP treatment. The degree of hydrolysis and RP-HPLC peptide profile were evaluated. An indirect ELISA test using polyclonal rat anti β-lactoglobulin antibodies was used to determine the residual antigenicity. The results showed that HP treatment enhanced the hydrolysis of bovine WP. For most enzymes, the best results were obtained at a pressure of 300 MPa. For two enzymes, Corolase PN-L and Neutrase, differences in peptide profiles were obtained due to the pressure applied during the enzymatic hydrolysis. Based on these differences, the residual antigenicity of these preparations were determined. An important reduction was found in the antigenicity of the hydrolysates obtained with Corolase PN-L and Neutrase in combination with HP treatment (300 MPa), prior to or during enzymatic hydrolysis, respectively. These results suggest that HP can enhance the WP hydrolysis, and, depending on the choice of enzymes, reduce the residual antigenicity of the hydrolysates. The reduced antigenicity of hydrolysates obtained by the combined treatments could have a relevant application in the development of hypoallergenic infant formulae.     

Enzymatic proteolysis,under high pressure of soybean whey: Analysis of peptides and the allergen Gly m 1 in the hydrolysates

Soybean (Glycine max) whey was hydrolyzed with Alcalase, Neutrase, Corolase 7089 and Corolase PNL during high pressure (HP) treatment at 100, 200 and 300 MPa and at atmospheric pressure for 15 min. The protein content and the degree of hydrolysis were determined. Furthermore, the allergen Gly m 1 in the treated soybean whey and the hydrolysates was detected. The results showed that HP treatments increased the hydrolysis by the four proteases used. Pressure at 200 and 300 MPa proved to be better pressures to enhance the proteolysis. The immunochemical response of soybean whey to anti-Gly m 1 monoclonal antibodies decreased after the HP treatments and this decrease was greater after the combined treatment of high pressure and enzymatic hydrolysis. Soybean whey proteins hydrolysed at high pressure could be used as sources of peptides with low antigenicity when incorporated as food ingredients.     

Effects of Enzymatic Hydrolysis Assisted by High Hydrostatic Pressure Processing on the Hydrolysis and Allergenicity of Proteins from Ginkgo Seeds

The purpose of this work was to study the combined effect of high hydrostatic pressure (HHP) and enzymatic hydrolysis treatment on the hydrolysis and allergenicity of ginkgo seed proteins (GSPs). Four food-grade proteases (papain, alcalase, pepsin, and neutrase) were used, and HHP (200, 300, and 400 MPa separately) was applied prior to hydrolysis. The extent of hydrolysis was measured with the o-phthaldialdehyde method, SDS-PAGE, and MALDI-TOF-MS, and the allergenicity was assessed with a Western blot and enzyme-linked immunosorbent assay (ELISA). The results showed that HHP could significantly improve the extent of proteolysis by papain, alcalase, or pepsin and reduce the antigenicity of GSP, whereas neutrase showed poor effects at any pressure. Papain and alcalase showed the highest proteolysis at 300 MPa, followed by pepsin at 400 MPa, and all of the obtained hydrolysates showed molecular weights lower than 10 kDa; furthermore, papain or alcalase at 300 MPa as well as pepsin at 400 MPa reduced antigenicity by more than 95 %, and all of the immunoreactive bands disappeared in the obtained hydrolysates. These results suggest that HHP can enhance the hydrolysis of GSP by certain enzymes and reduce the residual antigenicity of the hydrolysates. The obtained hypoallergenic hydrolysates could be used as a source of peptides for food ingredients.     

Reduction of immunoreactivity of bovine beta-lactoglobulin upon combined physical and proteolytic treatment

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6.8 for 10 min at 30, 37 and 44 degrees C. The extent of beta-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated beta-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact beta-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of beta-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.     

制备芝麻抗氧化肽的蛋白酶筛选

研究了菠萝蛋白酶、木瓜蛋白酶、中性蛋白酶、胰蛋白酶、碱性蛋白酶2709和Alcalase蛋白酶分别酶解芝麻蛋白制备芝麻抗氧化肽,测定多肽产率、水解度并评价抗氧化活性,以多肽产率和抗氧化活性为参数采用逼近理想解排序法对酶解物进行排序,确定适宜制备芝麻抗氧化肽的蛋白酶。结果表明:蛋白酶品种差异会显著影响芝麻抗氧化肽的制备效果;在加酶量相同时,碱性蛋白酶2709、Alcalase蛋白酶、胰蛋白酶酶解芝麻蛋白能力强,多肽产率高,且抗氧化活性强;根据与最优向量距离总和,用于制备芝麻抗氧化肽蛋白酶的适宜性由高到低依次为碱性蛋白酶2709、Alcalase蛋白酶、胰蛋白酶、中性蛋白酶、菠萝蛋白酶、木瓜蛋白酶。     

杏仁蛋白水解物对血管紧张素转化酶抑制作用的研究

分别采用Protamex、Alcalase、Neutrase、Flavourzyme、Proleather FG-F、木瓜蛋白酶水解杏仁蛋白,利用高效液相色谱法测定水解物对血管紧张素转化酶(ACE)的抑制活性,以水解度(DH)和水解产物对ACE的抑制率为指标对酶解过程进行分析,并研究水解物的体外消化稳定性。结果表明,Proleather FG-F 和Alcalase 对杏仁蛋白有较好的水解效果,其水解物对ACE 抑制率较高,IC50 分别为1.24mg/ml 和0.98mg/ml。模拟胃肠消化实验结果表明,在消化酶的作用下杏仁蛋白水解物仍具有较强的ACE 抑制活性。     

不同蛋白酶对小麦蛋白酶解物抗氧化活性的影响

小麦蛋白是小麦淀粉加工的副产物,酶解是提高小麦蛋白溶解性和功能性的有效方式,而酶解用酶种类可能对酶解产物的功能性如抗氧化活性有一定影响。采用碱性蛋白酶、中性蛋白酶、胃蛋白酶、风味蛋白酶、胰蛋白酶、木瓜蛋白酶6种常用的蛋白酶分别对小麦蛋白进行酶解,并对酶解4 h后酶解物的多肽得率、分子质量分布、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除率、超氧阴离子自由基(O_2~-·)清除率、羟自由基(·OH)清除率等反映水解程度和抗氧化能力的主要指标进行评价。结果表明,风味蛋白酶酶解物中多肽得率最高,达91.44%,且分子质量小于3 000 D的多肽含量达76.9%;酶解物质量浓度为3 mg/m L时,木瓜蛋白酶酶解物对DPPH自由基清除作用最好,清除率为65.12%(P0.01),其次是风味蛋白酶(58.43%)和碱性蛋白酶(55.29%);碱性蛋白酶酶解物对O_2~-·清除率效果最好,清除率为58.68%(P0.01),其次是风味蛋白酶(49.25%);碱性蛋白酶和木瓜蛋白酶酶解物对·OH清除效果最佳,清除率分别为59.23%和58.16%。结果说明,蛋白酶种类对小麦蛋白酶解物抗氧化活性影响显著,风味蛋白酶对提高蛋白水解程度和生成小分子质量多肽的作用明显,而碱性蛋白酶、木瓜蛋白酶和风味蛋白酶对提高酶解产物抗氧化活性效果较好。     

Whey Protein Antigenicity Reduction by Fungal Proteinases and a Pepsin/Pancreatin Combination

Whey protein components were hydrolyzed with Corolase 7092? (peptidases from Aspergillus strains), pepsin and Corolase PP? (a mixture of pancreatic enzymes), either individually or in combination, in trials to eliminate protein allergenicity. The hydrolysates were characterized by physico-chemical and by immunological techniques using sera from patients allergic to milk proteins. Enzyme specificity rather than degree of hydrolysis or molecular mass distribution of hydrolysates determined the residual antigenicity of the whey proteins. Ultrafiltration was a prerequisite for obtaining hypoallergenic whey protein hydrolysates.     

Bitterness in Bacillus proteinase hydrolysates of whey proteins

Whey protein concentrate (WPC) hydrolysates were generated with three commercially available Bacillus proteinase preparations (pH 7.0, 50 °C, 20% (w/v) WPC). Alcalase 2.4L hydrolysates were more bitter than Prolyve 1000 and Corolase 7089 hydrolysates when the proteinase activities were included at equivalent high and low addition levels. A glutamyl endopeptidase (GE) activity present in Alcalase was not detected in the Prolyve and Corolase preparations. Hydrolysate bitterness significantly increased when GE activity was included during Prolyve hydrolysis of WPC, indicating that inclusion of the GE activity was linked with the higher bitterness in Alcalase hydrolysates. A peptide present at higher levels in Prolyve compared to Alcalase hydrolysates was identified by mass spectrometry as β-lactoglobulin f(43–57). Hydrolysis of this peptide by GE was shown to release fragments with increased average hydrophobicity (Q-value). This may, in part, explain the higher level of bitterness associated with Alcalase compared to Prolyve hydrolysates of WPC.     

高静压协同酶法处理对白果蛋白抗原性的影响

本文研究了高静压结合酶解处理对白果蛋白抗原性的影响,分别采用4种蛋白酶水解白果蛋白,水解前分别采用不同压力的高静压对白果蛋白进行预处理,酶解产物水解率和分子量采用OPA法和SDS-PAGE测定,致敏性采用western-blotting和ELISA法测定。结果表明,木瓜蛋白酶,碱性蛋白酶或胃蛋白酶为水解酶时,高静压能显著提高白果蛋白的水解率和降低其致敏性;而中性蛋白酶为水解酶时,白果蛋白的水解和脱敏效果很差,即使高压处理也未见明显提高。木瓜蛋白酶或碱性蛋白酶在处理压力为300 MPa时,而胃蛋白酶在400 MPa时,其水解和脱敏效果最好,在此条件下白果蛋白能被水解为分子量小于15 ku的多肽,95%以上的白果蛋白致敏性能被消除,酶解产物中致敏蛋白条带全部消失。因此,高静压处理能明显提高蛋白酶对白果蛋白的水解效率和脱敏效果,但是取决于选择的蛋白酶种类和处理压力的大小。     

Yak milk casein as a functional ingredient: preparation and identification of angiotensin-I-converting enzyme inhibitory peptides

Yak milk casein derived from Qula, a traditional Tibetan acid curd cheese, was hydrolyzed by six commercially available proteases (Trypsin, Pepsin, Alcalase, Flavourzyme, Papain and Neutrase). These hydrolysates were assayed for their inhibitory activity of Angiotensin-I-converting enzyme (ACE). The hydrolysates obtained by Neutrase from Bacillus amyloliquefaciens showed the highest ACE inhibitory activity. The IC50 value of Neutrase-hydrolysate was 0.38 mg/ml. The hydrolysate obtained by Neutrase was further separated by consecutive ultra-filtration with 10 kDa and then with 6 kDa molecular weight cut-offs into different permeated parts and fractionated by gel filtration chromatography with a Sephadex G-25 column. The active fraction was subjected to RP-HPLC, in which five peaks were purified and identified. Amino acid sequence analysis confirmed that the peptides and origins were as follows: YQKFPQY (alphas2-CN; f89-95), LPQNIPPL (beta-CN; f70-77), SKVLPVPQK (beta-CN; f168-176), LPYPYY (kappa-CN; f56-61) and FLPYPYY (kappa-CN; f55-61). Their amino acid sequences matched well with those of known bioactive peptides from bovine casein. The results indicated that yak milk casein could be a resource to generate antihypertensive peptides and be used as multifunctional active ingredients for many value-added functional foods as well as a traditional food protein.     

Effects of combined high pressure and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins

The effect of high-pressure (HP) treatment on the hydrolysis of dairy whey proteins by trypsin, chymotrypsin and pepsin was analysed. Isostatic pressure (100–300 MPa for 15 min at 37 °C) was applied to the protein substrate prior to its enzymatic hydrolysis. Digestion was also conducted at atmospheric pressure (0.1 MPa) and under high pressure. The extent of hydrolysis was measured by the o-phthaldialdehyde method, the peptide profile was analysed by reverse-phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the residual immunochemical reactivity was assessed by an ELISA test using a pool of seven sera from children allergic to bovine milk, an individual serum also positive (positive control) and two sera from non-allergic children (negative controls). The high pressure increased the degree of hydrolysis by the three enzymes used. Chymotrypsin and trypsin showed the highest proteolysis at 100 and 200 MPa followed by pepsin at 300 MPa. The β-lactoglobulin was hydrolysed by trypsin and chymotrypsin at atmospheric and at high pressures, whereas the pepsin only hydrolysed this protein under high pressure. Pepsin and trypsin hydrolysed α-lactalbumin in all cases. In contrast, this protein was not digested by chymotrypsin, irrespective of the pressure applied. An important decrease of immunochemical reactivity was found for pepsin and trypsin hydrolysates obtained under high pressure. The pool of seven sera detected immunoreactivity in the products of chymotrypsin hydrolysis under high pressure, which was not detected when the serum of one patient was used. The results suggest that dairy whey hydrolysates obtained by pepsin and trypsin in combination with HP treatment could be used as a source of peptides in hypo-allergenic infant formulae.     

小麦蛋白酶解物中抗氧化肽的纯化与鉴定

以自由基清除能力为指标,分别采用4种蛋白酶酶解小麦蛋白,其中3 h复合蛋白酶酶解物显示出最高抗氧化活性。用超滤、凝胶过滤色谱和RP-HPLC的方法纯化抗氧化活性肽,并对抗氧化活性高的多肽进行质谱分析。结果显示,分子质量Mr1 ku组分的抗氧化活性较高(5 mg/m L时达到76.08%),对该组分进行纯化后,经ESI-TOF-MS质谱分析可知其荷质比m/z为730.83,氨基酸序列为Gln-Gln-Gln-ProArg(QQQPR)。     

Enzymatic hydrolysis of wheat gluten by proteases and properties of the resulting hydrolysates

The insolubility of gluten in aqueous solutions is one of the major limitations for its more extensive use in food processing. Wheat gluten was enzymatically hydrolyzed by several commercially available proteases (Alcalase 2.4L, PTN 6.0S, Pepsin, Pancreatin, Neutrase and Protamex™) with protein recovery of 81.3%, 42.5%, 53.3%, 61.6%, 46.3% and 43.8%, respectively. The hydrolytic efficiency of these proteases on wheat gluten was also compared. Alcalase served best for the preparation of wheat gluten hydrolysates with the maximum degree of hydrolysis (DH) 15.8%. Subsequently, the solubility of wheat gluten hydrolysates (WGHs) obtained with those enzymes was comparably evaluated. The products had excellent solubility (>60%) over a pH range of 2–12. The molecular weight distribution of WGHs was further determined by SDS-PAGE and size exclusion chromatography on Sephadex G-15. The results showed that with the increasing of DH values, there occurred a large amount of smaller polypeptides.     

花生分离蛋白碱性蛋白酶Alcalase水解物具有血管紧张素转化酶抑制活性

分别以碱性蛋白酶Alcalase和中性蛋白酶Neutrase对花生分离蛋白进行水解，制备花生分离蛋白水解物，并测定不同水解时间所得产物对血管紧张素转化酶(ACE)的抑制活性。未水解的花生分离蛋白没有ACE抑制活性，用中性蛋白酶Neutrase水解所得的水解物显示弱ACE抑制活性。然而，碱性蛋白酶Alcalase水解物具有很强的ACE抑制活性，水解0．5h时水解物活性最高，其半抑制浓度为(IC50)0．56mg／ml。本研究表明，当用碱性蛋白酶Alcalase水解时，花生分离蛋白是生产ACE抑制肽的良好蛋白质来源，花生分离蛋白碱性蛋白酶Alcalase水解物可作为具有降压功能的功能食品添料。     

鳄鱼骨双酶酶解产物的功能特性及其抗氧化活性

为了全面了解酶解时间、蛋白酶种类对鳄鱼骨蛋白酶解产物抗氧化性和功能特性的影响,采用木瓜蛋白酶、碱性蛋白酶及双酶(先加入木瓜蛋白酶,后加入碱性蛋白酶)在各自最适条件下对其进行酶解,制备了不同水解度的酶解产物,并对其功能特性及抗氧化性进行分析。结果表明:随着酶解时间的延长,酶解产物的亚铁离子螯合能力和还原力均有所增强。在酶解0.25 h时,酶解产物具有较强的清除DPPH自由基的能力,随着酶解时间的延长,木瓜蛋白酶酶解产物清除DPPH自由基的能力不断下降,碱性蛋白酶先下降后上升,而双酶酶解产物则没有显著变化。在2～4 h内相同酶解时间下,与单酶酶解产物相比,双酶酶解产物具有较强的亚铁离子螯合能力、还原力及清除DPPH自由基的能力(P<0.05)。在酶解产物的功能性质方面,随着酶解时间的延长,双酶酶解产物较单一酶酶解产物具有更好的溶解性、热稳定性及乳化性。结果表明,双酶酶解较单一酶酶解得到的产物具有较强的抗氧化性。     

Effect of combined treatment of hydrolysis and polymerization with transglutaminase on β-lactoglobulin antigenicity

The effect of combined treatments of hydrolysis with different proteases, and subsequent polymerization with transglutaminase on the antigenic activity of β-Lg was studied. For the hydrolysis of β-Lg using Alcalase, Neutrase or bromelain, the reaction conditions were 3?% β-Lg and enzyme:substrate 25?U?g^?1 of protein, as was defined using factorial study. Under these conditions, the degree of hydrolysis (DH) of the hydrolysates was 12.6?% when obtained with Alcalase and approximately 4?% with Neutrase or bromelain. Post-hydrolysis polymerization did not result in an increase in molecular mass of the protein, but these samples presented a lower DH, determined by trinitrobenzenosulfonic acid (TNBS) method, suggesting that polymerization had occurred. Hydrolysis with the three enzymes reduced the β-Lg antigenicity, as evaluated by ELISA and immunoblotting analyses. The IgE-binding responses were practically null (<9?μg?mL^?1), 22.82 and 55.73?μg?mL^?1 towards the hydrolysates obtained with Alcalase, bromelain, and Neutrase, respectively. The post-hydrolysis polymerization increased or had no significant effect (P?≥?0.05) on the antigenic response of the hydrolysates.     

Antioxidant properties of whey protein hydrolysates as measured by three methods

Four microbial proteases (Alcalase, Flavourzyme, Neutrase and Protamex) were used for the preparation of whey protein hydrolysates. The aim of this research was to find out whether these hydrolysates can be used as a source of whey derived antioxidants. Hydrolyzed samples, including their unhydrolyzed protein solutions were tested by the ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) decolorization assay, by the total radical-trapping potential method and by the assay of liposomes peroxidation (fluorescence photometry). Antioxidant properties were enhanced by hydrolysis in most of cases. Alcalase hydrolysates were found as the most effective antioxidants as determined by ABTS assay (~50% of antioxidant activity at 0.1 mg ml⁻¹ of hydrolysate in reaction) and fluorescence photometry. Liposomes were oxidized ~50% less (1.1 μM of α-tocopherol equivalent) with Alcalase hydrolysates additive (at 5.85 mg ml⁻¹ of hydrolysate in reaction). Hydrolysates did not inhibit the oxidation of liposomes at concentrations below 1.0 mg ml⁻¹ in reaction. On the contrary, results of total trapping potential method did not agree with findings observed in other tests. In this assay, Neutrase hydrolysates showed the best antioxidant properties. Pro-oxidant properties were observed in solutions containing (prior to the enzyme Protamex addition only) intact whey protein as determined by the measurement of the liposome peroxidation. The ABTS assay was optimized for the evaluation of the antioxidant activity in whey protein hydrolysates. The reaction time should be prolonged to avoid underestimation of the antioxidant activity.     

花生蛋白水解产物ACE抑制活性的研究

采用胰蛋白酶、碱性蛋白酶、中性蛋白酶水解花生蛋白,研究了水解过程中水解度的变化,并对水解产物的ACE抑制活性进行了探讨。得出三种酶对花生蛋白的水解作用:碱性蛋白酶>胰蛋白酶>中性蛋白酶。碱性蛋白酶水解产物ACE抑制活性明显高于胰蛋白酶和中性蛋白酶,水解产物的ACE抑制活性高达89.73%,中性蛋白酶水解产物ACE抑制率仅为27.24%。