不同酶水解菜籽蛋白的水解物的抗氧化活性研究

使用alcalase、protamex、flavourzyme、木瓜蛋白酶、中性蛋白酶和胰蛋白酶共6种蛋白酶水解菜籽蛋白,研究水解物清除DPPH自由基能力、还原能力和抑制亚油酸过氧化活性。结果表明,6种蛋白酶水解菜籽蛋白的水解物都具有抗氧化活性,但水解物的抗氧化活性与水解所用酶的种类和水解时间有关,胰蛋白酶和flavourzyme水解物显示了较强的清除DPPH自由基和还原能力,6种蛋白酶水解物抑制亚油酸过氧化活性高于VE,但略低于BHT。alcalase水解菜籽蛋白时,水解10~45 m in的水解物清除DPPH自由基和还原能力较强,延长水解时间并不能提高其抗氧化活性。     

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.     

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.     

Gelatin hydrolysates from sliver carp (Hypophthalmichthys molitrix) improve the antioxidant and cryoprotective properties of unwashed frozen fish mince

In this study, gelatin extracted from silver carp skin was hydrolysed by different enzymes (alcalase, papain and flavourzyme), aiming to improve the storage stability of unwashed fish mince during freeze-thaw cycles. Flavourzyme hydrolysates exhibited the highest DPPH• scavenging activity (1.2-fold of alcalase, 1.6-fold of papain) and reducing ability (1.2-fold of alcalase, 2.3-fold of papain), while the alcalase hydrolysates demonstrated the highest Fe²⁺ chelating ability. Moreover, fish mince treated with the mixture of flavourzyme hydrolysates and sucrose exhibited higher sulfhydryl and salt-soluble protein contents, greater Ca²⁺-ATPase activity and better water holding capacity. Hence, the gelatin hydrolysates of flavourzyme from silver carp skin are promising candidates as natural and high-performance antioxidant and cryoprotectants in fish mince processing.     

生物解离大豆蛋白酶解物体外模拟消化抗氧化活性变化

挤压膨化大豆在生物解离过程中,通过酶解（碱性蛋白酶、风味蛋白酶）产生的大豆蛋白酶解物（soybean protein hydrolysate,SPH）有良好的抗氧化活性,因此,需要探索模拟胃肠道（gastrointestinal,GI）消化对蛋白酶解物抗氧化活性的影响。分别以蛋白酶（碱性蛋白酶、风味蛋白酶）酶解得到的SPH和经过模拟GI消化后的产物作为研究对象,采用水解度、氨基酸组成、分子质量分布、氧化自由基吸收能力（oxidative radical absorption capacity,ORAC）、1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基清除能力及2,2’-联氨-双(3-乙基苯并噻唑啉-6-磺酸)（2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt,ABTS）阳离子自由基清除能力对样品抗氧化活性进行分析。结果显示：相对于风味蛋白酶酶解,采用碱性蛋白酶进行酶解时抗氧化活性更高;但是经过模拟GI消化后,风味蛋白酶酶解得到的SPH抗氧化活性更高,ORAC为69.15 μmol/mg、DPPH自由基清除能力为27.29%、ABTS阳离子自由基清除能力为56.21%,肽的相对含量更高,为75.86%,并且肽中具有抗氧化活性的氨基酸含量更高,分子质量小于1 kDa的肽相对含量最高,为17.35%。     

干酪乳清水解产物的抗氧化活性研究

选用新鲜干酪乳清为原料,研究碱性蛋白酶对乳清水解产物的抗氧化活性。以水解产物的亚铁还原能力、对卵磷脂脂质氧化体系的过氧化抑制作用、羟自由基清除能力和超氧阴离子自由基的清除能力为指标评价乳清水解产物的抗氧化能力。结果表明,乳清在水解前经过预热处理并不能增加其水解产物的抗氧化活性,1 h水解物的亚铁还原能力最高,2 h水解产物对卵磷脂脂质氧化体系的过氧化抑制作用最高,抑制率达到24.82%;2 h水解产物羟自由基清除率最高,达到70.28%;2 h水解产物超氧阴离子自由基清除率最高,达到21.4%。但是乳清水解产物的抗氧化能力与水解度没有线性关系。     

鳄鱼皮酶解产物功能特性及抗氧化活性

为了解鳄鱼皮酶解产物功能特性和抗氧化活性,采用2种商业蛋白酶(木瓜蛋白酶、碱性蛋白酶)在各自最适反应条件下分别酶解鳄鱼皮,研究水解度(DH)、酶种类及pH值对酶解产物功能特性及抗氧化活性的影响.结果显示:随着酶解时间延长,鳄鱼皮水解度逐渐增加,鳄鱼皮在碱性蛋白酶酶解作用下水解度较高,水解4h时可达12％;木瓜蛋白酶酶解产物与碱性蛋白酶酶解产物的溶解性差异不显著(P＞0.05).相同水解度下,碱性蛋白酶酶解产物的热稳定性在pH4时优于木瓜蛋白酶酶解产物.酶解时间在1h之内,木瓜蛋白酶酶解物亚铁离子螯合力明显增强;随着时间延长,酶解产物亚铁离子螯合能力变化不显著(P＞0.05).酶解3h后碱性蛋白酶酶解产物亚铁离子螯合能力高于木瓜蛋白酶酶解产物,但木瓜蛋白酶酶解产物具有较强的清除DPPH自由基能力.综上表明,碱性蛋白酶水解作用的鳄鱼皮水解度较高,其酶解产物乳化活性和热稳定性优于木瓜蛋白酶酶解产物;鳄鱼皮酶解产物抗氧化能力较强,有较高的开发利用价值.     

Improvement of the ACE‐inhibitory and DPPH radical scavenging activities of soya protein hydrolysates through pepsin pretreatment

This study aimed to investigate the effect of pepsin pretreatment on the ACE‐inhibitory and DPPH radical scavenging activities of soya protein hydrolysates prepared with alcalase and protamex. The protein recovery, TCA‐soluble peptide content, surface hydrophobicity, particle size and zeta potential were evaluated. Results showed that the hydrolysates exhibited varying ACE‐inhibitory (i.e. the highest value 72.6% by alcalase and 84.3% by protamex) and DPPH radical scavenging activities (i.e. the highest value 51.9% by alcalase and 51.7% by protamex). Pepsin pretreatment could make soya proteins more susceptible for hydrolysis, and the results showed that the ACE‐inhibitory and DPPH radical scavenging activities of the resultant hydrolysates were improved. A highly significant positive correlation between ACE‐inhibitory and DPPH radical scavenging activities was observed in the alcalase hydrolysates, while no significant correlation among other treatments. The physical properties of hydrolysates, that is surface hydrophobicity, particle size and zeta potential, could influence their ACE‐inhibitory and DPPH radical scavenging activities.     

鸭肉蛋白酶解产物的抗氧化特性

为了解酶解时间、蛋白酶种类对鸭肉蛋白酶解产物抗氧化特性的影响,分别用复合蛋白酶、风味蛋白酶和胰酶对鸭肉进行单酶酶解和双酶分步酶解（胰酶＋复合蛋白酶、胰酶＋风味蛋白酶）,制备不同时间段的酶解产物,并对其自由基清除能力（1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基、羟自由基（hydroxyl radical,·OH）和超氧阴离子自由基（superoxide radical,O2－·））和总还原力进行分析。结果表明：各鸭肉蛋白酶解产物的DPPH自由基清除率随着酶解时间的延长而增加,但·OH和O2－·清除率及总还原力随着酶解时间的延长先增加后降低（P＜0.05）。在5 种鸭肉蛋白酶解产物中,复合蛋白酶酶解物表现出最强的DPPH自由基清除能力（75.70±1.54）%、·OH清除能力（59.41±1.24）%和O2－·清除能力（98.50±4.51）%,但用双酶分步酶解得到的酶解产物表现出最强的总还原力（0.330±0.017）。因此鸭肉蛋白酶解产物的抗氧化特性受酶解时间和蛋白酶种类的影响,复合蛋白酶是制备鸭肉蛋白源抗氧化肽的最适蛋白酶。     

鳄鱼肉蛋白酶解产物抗氧化性的研究

为更加充分利用优质的鳄鱼肉蛋白资源,采用木瓜蛋白酶、风味蛋白酶单酶酶解及木瓜蛋白酶与风味蛋白酶复合酶解制备了鳄鱼肉蛋白多肽,并对其抗氧化活性（DPPH自由基清除能力、亚铁离子螯合能力和还原力）进行分析。结果显示,酶解时间为Q5h、1h和2h木瓜蛋白酶和风味蛋白酶复合酶解产物的DPPH自由基清除能力显著（P〈0．05）,高于木瓜蛋白酶和风味蛋白酶单酶酶解产物的DPPH自由基清除能力;除酶解时间为0．5、1的风味蛋白酶鳄鱼肉蛋白多肽外,其它鳄鱼肉蛋白多肽的亚铁离子螯合率均大于80％;随酶解时间延长,木瓜蛋白酶和风味蛋白酶复合酶解鳄鱼肉蛋白多肽的还原力显著降低（P〉0．05）。鳄鱼肉蛋白酶解产物具备开发为功能食品的潜力。     

Enzymatic hydrolysis of hemp (Cannabis sativa L.) protein isolate by various proteases and antioxidant properties of the resulting hydrolysates

Enzymatic hydrolysis of hemp protein isolate (HPI) by six proteases (alcalase, flavourzyme, neutrase, protamex, pepsin and trypsin) and antioxidant activities of the resulting hydrolysates, obtained for 2 and 4 h were investigated. The yield of trichloroacetic acid (TCA)-soluble peptides (Y_sp), protein composition and surface hydrophobicity (H_o) of the hydrolysates were evaluated. The results showed that the hydrolysates exhibited varying DPPH radical scavenging (with lowest IC₅₀, ∼2.3 mg/mL) and Fe²⁺⁺ chelating (with lowest IC₅₀ of 1.6–1.7 mg/mL) abilities and reducing power (with highest absorbance at 700 nm of 0.31–0.35), depending on their Y_sp and H_o values. The DPPH radical scavenging and Fe²⁺⁺ chelating abilities of the hydrolysates were positively correlated with their Y_sp or H_o values. The results suggest that enzymatic hydrolysis can be used as an effective technique to produce high value-added products of hemp proteins.     

Preparation and in vitro antioxidant activity of enzymatic hydrolysates from oyster (Crassostrea talienwhannensis) meat

Oyster is an abundant resource from ocean, which contains high content of protein. In the present study, oyster (Crassostrea talienwhannensis) meat was digested with three proteases including papain, neutrase and alcalase respectively and the derived hydrolysates were fractionated using a series of ultrafiltration membranes (molecular weight cut‐offs of 10, 5, 3 and 1 kDa). The resultant peptide fractions were evaluated for antioxidant activity using 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) assay and reducing power assay. Results indicated that oyster meat hydrolysates (OMHs) possessed DPPH radical scavenging capacity and reducing power in a dose‐dependent manner. The fractions below 1 kDa showed the strongest overall antioxidant activity. The present study demonstrated that the antioxidant potent of OMHs was strongly related to their concentration, size and amino acid composition.     

银鲳酶解物抗氧化活性研究

选用胃蛋白酶、胰蛋白酶、碱性蛋白酶和中性蛋白酶对银鲳蛋白进行酶解以制备蛋白酶解物,以羟基自由基清除活性为指标确定银鲳最佳水解酶。结果显示,碱性蛋白酶的水解物抗氧化活性最强。实验对碱性蛋白酶水解银鲳的酶解条件(时间、温度、pH、酶添加量和固液比)进行正交实验设计,并对最佳水解条件下所获得的酶解物进行抗氧化活性测试。结果表明,银鲳蛋白碱性蛋白酶水解物对DPPH自由基和羟基自由基具有清除作用,其自由基清除效果呈现剂量依赖性,而且银鲳蛋白水解物还具有明显还原能力。所有这些体外抗氧化数据说明,银鲳蛋白水解物有明显的抗氧化效力。     

Evaluation of free radical‐scavenging activities of sweet potato protein and its hydrolysates as affected by single and combination of enzyme systems

To evaluate the free radical‐scavenging activities of sweet potato protein (SPP) and its hydrolysates, single enzymes alone (alcalase, neutrase, protamex) or in combination with flavourzyme were employed. Compared with SPP, free radical‐scavenging activities of the resulting hydrolysates were all significantly increased (P < 0.05). Alcalase (ALC) hydrolysates exhibited the highest superoxide, hydroxyl and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging activities (P < 0.05), which was 18.71 ± 0.22, 27.13 ± 0.24 and 90.10 ± 0.15% respectively. Compared with SPP hydrolysates by single enzymes, the hydrolysates obtained by combination of enzyme systems exhibited higher degree of hydrolysis, but lower free radicals scavenging activities. In addition, the content of several antioxidant amino acid residues, such as His, Met, Tyr and Phe, in ALC hydrolysates was much higher compared with SPP and other hydrolysates using amino acids composition assay. The results suggested that peptides with free radical‐scavenging activity could be released from entire SPP chain via moderate enzymatic hydrolysis.     

不同蛋白酶水解棉籽蛋白制备抗氧化多肽的研究

利用6种蛋白酶对棉籽蛋白进行酶解,测定了各酶在水解过程中的水解度及其变化,对酶解产物的抗氧化活性进行了分析比较。研究表明,各蛋白酶在水解的前2h内,水解度迅速增加,2h之后水解曲线变得平缓。其中胃蛋白酶的水解能力最强,其4h水解产物水解度最大,为30.40%;胰蛋白酶的水解能力最差,最终水解产物的水解度为17.61%。中性蛋白酶水解产物的抗氧化活性较强,经测定其DPPH清除能力为54.95%,羟自由基清除能力为68.98%,超氧阴离子自由基清除能力为58.38%。     

鲤鱼鱼肉蛋白酶水解物抗氧化性及功能特性

采用碱性蛋白酶对鲤鱼鱼肉蛋白进行酶解,制备不同水解度的水解物。测定水解物的抗氧化活性以及不同pH值条件下水解物的功能特性。结果表明:随着水解度的逐渐升高,水解物的抑制脂质氧化能力、D PP H自由基清除能力、还原能力以及金属离子(Cu2+和Fe2+)螯合能力逐渐增加(P<0.05);同时,水解物的溶解性、乳化性和起泡性都在pH值为4.0(等电点)时达到最低,而后溶解性和乳化性随着pH值升高而增大(P<0.05),而起泡性随着pH值的升高先上升后又下降。因此,鲤鱼鱼肉蛋白碱性蛋白酶水解物可以提高蛋白质的抗氧化活性和溶解性,但是较高的水解度会在一定程度上降低其乳化性和起泡性。     

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.     

黑蚂蚁蛋白的酶解优化及抗氧化肽的超滤膜分离

本文研究了酶量、温度、pH和时间四个因素对黑蚂蚁蛋白的碱性酶法水解的影响。通过响应面设计,以各个抗氧化活性为指标,优化了超氧自由基清除率,DPPH自由基清除率,羟基自由基清除率,还原力和亚油酸自氧化抑制作用最强的酶解工艺条件。验证实验证实方程的准确率达95%以上。采用超滤膜及离子交换色谱对酶解产物进行了分离,发现5-10 kDa和1-3 kDa组分的抗氧化能力最强,碱性肽对超氧自由基清除能力、羟基自由基清除能力、亚油酸自氧化抑制作用贡献较大,酸性肽对DPPH自由基清除能力及还原力的贡献较大。     

猪血浆蛋白水解物抗氧化作用模式的研究

将猪血浆蛋白(4%浓度,W/W)分别用碱性蛋白酶水解0.5～6h。用pH-Stat方法测定其水解度;同时测定水解物还原能力、金属离子(Cu2+和Fe2+)鳌合能力以及对DPPH自由基清除能力来分析猪血浆蛋白水解物的抗氧化作用模式。结果表明,5h的猪血浆蛋白碱性蛋白酶水解物具有较高的还原能力、合成抗氧化剂所没有的金属鳌合能力以及较强的清除自由基的能力。通过比较不同浓度的水解物抗氧化性实验发现,随着水解物浓度的增高,其抗氧化活性显著增强(p＜0.05)。尽管未水解的猪血浆蛋白也有一定的抗氧化活性,但远远低于水解物的抗氧化活性(p＜0.05)。由于猪血浆蛋白水解物具有一定的抗氧化活性,所以可以作为金属离子鳌合剂、氢供体和自由基稳定剂用以抑制脂质氧化。     

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.