Antioxidant Activity of Soy Protein Hydrolysates in a Liposomal System

ABSTRACT: Native and heated soy protein isolate was hydrolyzed with 3 purified (pepsin, papain, and chymotrypsin) and 3 crude (Alcalase®, Protamex^TM, and Flavourzyme^TM) proteases. The hydrolysates were incubated (37 °C, 1 h) with a liposome-oxidizing system (50 μM FeCl₃/0.1 mM ascorbate, pH 7.0) to test antioxidant activities by determining the concentrations of TBARS. Degree of hydrolysis of SPI hydrolysates ranged from 1.7 to 20.6%. Both hydrolyzed and nonhydrolyzed SPI decreased TBARS (by 28 to 65%), except for papain-hydrolyzed samples. Samples of chymotrypsin- and Flavourzyme-hydrolyzed (0.5 h) preheated SPI had the greatest inhibitory effect on lipid oxidation.     

Whey and soy protein hydrolysates inhibit lipid oxidation in cooked pork patties

Pork patties containing 1.5% NaCl and 2% hydrolyzed whey protein isolate (WPI, 1 h with flavourzyme or 6 h with protamex) or soy protein isolate (SPI, 0.5 h with chymotrypsin or flavourzyme) were cooked to 70?°C and subsequently stored at 4?°C up to 7 days. Lipid oxidation in patties during storage was analyzed by measuring the concentration of conjugated dienes (CD) and thiobarbituric acid-reactive substances (TBARS). Intact WPI and SPI, and their hydrolysates, were all inhibitory of oxidation (P <0.05) in cooked patties, with SPI being slightly more effective than WPI. Hydrolysis with protamex augmented the antioxidative activity (CD, TBARS) of WPI. Hydrolysis with either chymotrypsin or flavourzyme improved the ability of SPI to retard CD formation but did not delay the production of TBARS in stored pork patties.     

两步酶解法制备低致敏性乳清蛋白及其致敏性评价

目的 探究两步复合酶解工艺处理对牛乳清蛋白(whey protein isolate, WPI)结构和致敏性的影响。方法 实验采用邻苯二甲醛法、体积排阻色谱法分析WPI水解物在不同酶解时间下的水解程度,同时采用酶联免疫吸附法(enzyme-linked immunoassay, ELISA)、细胞模型和小鼠模型对水解物进行体外和体内的致敏性评估,并利用超高效液相色谱-串联质谱法(ultraperformanceliquidchromatography-tandemmassspectrometry,UPLC-MS/MS)分析水解物中的残留过敏原表位。结果 WPI经碱性蛋白酶和木瓜蛋白酶顺序酶解3.0h后水解度达到14.19%,在水解2.0～3.0 h期间, WPI水解物主要由小肽(<5.0 kDa)组成。水解3.0 h后,水解产物与特异性抗体的结合能力降低了97.83%,细胞模型和小鼠模型结果证实,酶解产物的体外、体内致敏性显著降低,与空白对照组无显著性差异。UPLC-MS/MS结果表明WPI水解3.0 h产物中70.59%的肽段不含过敏原表位。结论 复合酶解工艺有效降低了WPI致...     

乳清分离蛋白水解物对猪肉糜抗氧化作用的研究

研究乳清分离蛋白(WPI)的碱性蛋白酶(alcalase)水解物的抗氧化能力,并将其应用于生肉糜中研究其抗氧化效果。实验分为6组,第1组为对照组,第2组加入2.0%的WPI未水解物,第3～5组中分别加入1.0%、1.5%、2.0%的水解物冻干粉(5h),第6组中加入0.02%的BHA,在冷藏过程中测定肉糜的红度值(a*)、硫代巴比妥酸值(TBARS)值、pH值、高铁肌红蛋白(MetMb)含量,并对产品的感官指标进行评定。结果表明,在贮藏7d内,与对照组相比,添加WPI水解物处理组能够显著抑制生肉糜脂肪的氧化(P<0.05),其中2%WPI水解物处理组效果最明显,能显著降低TBARS值、增加肉糜的红度值(a*)(P<0.05),且其高铁肌红蛋白(MetMb)含量仅为对照组的79%,与添加BHA处理组的水平相当。同时,WPI水解物处理组抑制脂肪的氧化效果比WPI未水解组好。因此,WPI水解物可以有效的抑制食物中脂肪氧化,且其抗氧化效果与水解物的使用量相关。     

Fractionation and characterisation for antioxidant activity of hydrolysed whey protein

Whey protein isolate (WPI) was hydrolysed for 1 h using Alcalase, Protamex and Flavourzyme. Native WPI, hydrolysed WPI and two commercial WPI hydrolysates were subjected to fractionation by size exclusion chromatography. Antioxidant activity of WPI fractions was measured with a liposome‐oxidising system (50 µM FeCl₃/0.1 µM ascorbate, pH 7.0). Lipid oxidation was measured as thiobarbituric acid‐reactive substances (TBARS). Gel electrophoresis and amino acid analysis were run to identify the peptide composition. The influence of amino acid composition on antioxidant activity was evaluated using multivariate analysis methods (correlation analysis, principal component analysis, multiple linear regression and discriminant analysis). TBARS assays indicated the presence of antioxidant activity in all protein fractions, including non‐hydrolysed WPI. For native and hydrolysed WPI samples the first fraction (> 45 kDa) showed a higher TBARS inhibition effect (24–27%) when compared with lower‐molecular‐weight fractions and hydrolysate mixtures. In contrast, for commercial WPI hydrolysates a higher inhibitory effect was found in most of the lower‐molecular‐weight fractions (30–55%). The ability of WPI fractions to delay lipid oxidation was found to be related to the prevalence of histidine and hydrophobic amino acids. Copyright © 2004 Society of Chemical Industry     

乳清分离蛋白酶解制备抗氧化和ACE抑制活性产物

以乳清分离蛋白为原料,通过对肽浓度、游离氨基酸含量及水解度的研究,探讨了碱性蛋白酶Alkaline、中性蛋白酶NPU、肽酶AX和风味蛋白酶Flavourzyme对乳清蛋白酶解的影响。在优化碱性蛋白酶Alkaline水解条件的基础上,考察了中性蛋白酶NPU、风味酶Flavourzyme添加时间,获得了复合酶分步酶解工艺:酶解温度55℃,加入1.5% Alkaline([E/S]),维持pH8.5酶解10 min,待pH7.2时加入1% NPU([E/S]),酶解240 min,加入1% Flavourzyme([E/S])继续酶解20 min。在此条件下,肽浓度达到60 g/L以上,游离氨基酸含量为4348 mg/kg。获得的酶解产物基本无苦味。酶解物分子量主要分布在<1 kDa(69.62%)。酶解产物具有ABTS自由基清除和ACE抑制活性,IC₅₀值分别为1.02(0.377 μmol Trolox当量/mg)和1.38 mg/mL。综上,采用复合酶酶解乳清分离蛋白可获得具有抗氧化活性和ACE抑制活性产物,为乳清酶解物在功能食品或保健品领域的运用提供科学参考。     

顶青霉木聚糖酶的水解动力学与水解模式

从顶青霉（Penicillium corylophilum）培养液中分离纯化得到的3种木聚糖酶组分(Part A,Part B和Part C),对其水解动力学和水解模式的研究结果表明,3种木聚糖酶的动力学常数（桦木木聚糖为底物）分别为Part A,K     

麦胚蛋白水解物抗氧化活性及其作用模式

采用中性蛋白酶水解麦胚蛋白0.5～6 h,测定水解物的DH(degree of hydrolysis)、硫代巴比妥酸值(TBARS值)、POV值(Prroxide value)、Cu2+螯合能力、二苯代苦味肼基自由基清除能力(DPPH自由基)。结果表明,麦胚蛋白水解度随着水解时间的延长而增加;水解物5 h与未水解的蛋白及0.02%VC相比,在大豆卵磷脂脂质氧化体系(Liposome体系)中具有较强的抑制脂质氧化的能力,还具有较强的Cu2+螯合能力和清除自由基能力,未水解的蛋白具有较弱的抗氧化能力;通过对未水解的麦胚蛋白及不同浓度蛋白水解物的抗氧化指标测定和分析,认为水解物的抗氧化活性与蛋白本身的结构、肽链长短及氨基酸残基有关。     

仔乌蛋白酶解物抗氧化性研究

通过测定仔乌酶解物对Fenton反应体系产生的羟基自由基的清除效果,筛选出胰蛋白酶和中性蛋白酶作为仔乌水解理想酶。两种酶水解仔乌所获得到酶解液对羟基自由基具有较好清除效果。采用正交试验对胰蛋白酶和中性蛋白酶的水解条件进行优化。其中,胰蛋白酶酶解工艺条件为温度45℃、时间3h、酶添加量900U／g、pH6．5;中性蛋白酶则为温度50℃、时间2h、pH6．5、加酶量为600U／g。二者料液质量比均为1：4。     

Combined biocatalytic conversion of smooth hound viscera: Protein hydrolysates elaboration and assessment of their antioxidant,anti-ACE and antibacterial activities

The aim of this study was to investigate the antioxidant, anti-ACE and antibacterial activities of smooth hound viscera hydrolysates (SHVHs). Viscera, as by-product, were hydrolyzed with crude alkaline protease extract from the same species, three commercial proteases and a combination endogenous and exogenous preparations. Hydrolysates showed different degrees of hydrolysis and the digestion with endogenous enzymes in combination with commercial proteases was found to enhance protein hydrolysis. The resulting SHVHs had high protein content (70.89%–89.06%) and they were mainly constituted by Gly, Glu and Gln, while Tau was the major free amino acid. In addition, they contained high amounts of UMP, Uridine, GMP and Guanosine, while undigested proteins were mainly rich in IMP and Xanthine. Furthermore, SHVHs showed different molecular mass distribution and RP-HPLC profiles proving their molecular mass and hydrophilic/hydrophobic peptide heterogeneity. All the SHVHs exhibited antioxidant activity, in terms of radical-scavenging activity, reducing power, metal chelating activity, β-carotene protection, lipid peroxidation inhibition and DNA breakage assay, and it was found that a medium degree of hydrolysis was appropriate to obtain hydrolysates with good antioxidant capacity. In addition, the SHVHs antioxidant activity was improved after simulated gastrointestinal digestion. Besides their antibacterial effect against several Gram-positive and Gram-negative bacteria, all the hydrolysates showed varying degrees of ACE inhibitory activities and the highest one was achieved by the Purafect hydrolysate (IC₅₀ = 75 μg/mL). The overall data suggested that the SHVHs could be used as potential source of natural antioxidant, antimicrobial and anti-ACE peptides to formulate functional foods.     

Optimization of enzymatic hydrolysis of Alaska pollock frame for preparing protein hydrolysates with low-bitterness

To obtain hydrolysates with high nitrogen recovery (NR) and low-bitterness, Alaska pollock frame (APF) was treated by high-pressure cooking at 120 °C for 30 min and hydrolyzed with ten different commercial proteases. MEAP (Mixed enzymes for animal proteolysis) hydrolysates exhibited the best characteristics (high NR and low bitter taste), which had high content of oligopeptides with molecular mass lower than 1355 Da. The optimum hydrolysis conditions using MEAP were determined as follows: temperature 45 °C, hydrolysis time 300 min, enzyme to substrate ratio (E/S) of 1.2 g/100 g, liquid-solid ratio 6:1, and initial pH 8.0. The NR and DH (degree of hydrolysis) of the hydrolysates were 76.89% and 18.09%, whereas the bitterness value was lower than that of 0.1 g/L tea alkaloid. The essential amino acid chemical scores (CS) were greater than 0.8 (0.8-1.4) in pollock frame hydrolysates (PFH) which exhibited high nutritional value. It showed that APF and PFH as high-quality protein source may have a wide application.     

乳清蛋白水解物抗氧化活性的研究

本实验主要研究了乳清蛋白的碱性蛋白酶水解产物在不同体系中的抗氧化活性。通过测定水解物对卵磷脂脂质氧化体系的硫代巴比妥酸值(TBARS)抑制作用、过氧化值(PV)的抑制作用、亚铁还原能力、金属离子螯合能力、DPPH 自由基清除能力,研究乳清多肽的抗氧化活性。结果表明,底物浓度为5%、蛋白酶量的添加量为2%、水解时间为5h 时乳清蛋白的水解物具有最强的抗氧化能力;碱性蛋白酶制备的乳清蛋白水解物的抗氧化能力与底物浓度、水解时间、溶解度等有关。     

Peptides released from acid goat whey by a yeast-lactobacillus association isolated from cheese microflora

Seven lactobacilli and a variety of microflora extracted from twenty five commercial cheeses were grown on unsupplemented acid goat whey and screened for their capacity to hydrolyse whey proteins [alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg)] and to generate peptides. Fermentations were performed aerobically or anaerobically at 37 degrees C using crude or pre-heated whey (10 min at 65, 75 or 85 degrees C). Under aerobic conditions, growth of lactobacilli was poor and protein hydrolysis did not occur. Anaerobic conditions slightly increased lactobacilli growth but neither beta-lg hydrolysis nor peptide generation were observed. More than 50% of alpha-la was digested into a truncated form of alpha-la (+/- 12 kDa) in crude whey and whey pre-heated at 65 degrees C. Twenty-five microflora extracted from raw milk cheeses were screened for their proteolytic activities on acid goat whey under the conditions previously described. Eight of them were able to hydrolyse up to 50% of alpha-la mainly during aerobic growth on crude or pre-heated whey. The corresponding hydrolysates were enriched in peptides. The hydrolysate involving microflora extracted from Comté cheese after or at 18 months ripening was the only one to exhibit hydrolysis of both alpha-la and beta-lg. Microbiological analysis showed that microorganisms originating from Comté cheese and capable of growth on unsupplemented whey consisted of Candida parapsilosis and Lactobacillus paracasei. Fermentation kinetic profiles suggested that peptides were released from alpha-la hydrolysis. The co-culture of both microorganisms was required for alpha-la hydrolysis that occurred concomitantly with the pH decrease. During whey fermentation, Cand. parapsilosis excrete at least one protease responsible for alpha-la hydrolysis, and Lb. paracasei is responsible for medium acidification that is required for protease activation.     

Effect of heat and enzymatic treatment on the antihypertensive activity of whey protein hydrolysates

The influence of heat and enzymatic treatments on the hypotensive activity of hydrolysates derived from whey protein isolate was examined. The whey protein isolate (WPI) was previously denatured at 65 or 95 °C and hydrolyzed using the enzymes Alcalase, α-chymotrypsin or Proteomix. The hydrolysates thus obtained were characterized and studied with regard to their angiotensin converting enzyme (ACE) inhibitory activity and hypotensive activity in spontaneously hypertensive rats (SHR). The enzyme α-chymotrypsin was found to produce hydrolysates with the highest ACE inhibitory activity. The hydrolysate that most effectively reduced blood pressure in SHR was obtained from WPI previously denatured at 65 °C and treated with the enzyme Alcalase. The hydrolysate with the highest ACE inhibitory activity was able to reduce the arterial blood pressure of the animals only after intraperitoneal administration, suggesting an interference of gastrointestinal enzymes in the absorption of active peptides from this hydrolysate.     

Bioactive properties of Kilka (<Emphasis Type="Italic">Clupeonella cultriventris caspi</Emphasis>) fish protein hydrolysates

Whole common Kilka fish was hydrolyzed separately using four commercial enzymes, Alcalase, Neutrase, Protamex at 50 °C and Pepsin at 37 °C for 30, 60 and 90 min. Degree of hydrolysis, angiotensin-I-converting enzyme (ACE) inhibitory activity and antimicrobial activity of each hydrolysate against Gram-negative (Escherichia coli, Salmonella enteritidis) and Gram-positive (Staphylococcus aureus, Listeria innocua) bacteria were studied. Results showed that the degree of hydrolysis for all enzymes was in the range of 2.63–3.36%. Electrophoresis profiles of the Kilka protein hydrolysates showed that most of produced peptides were in the range of 30 D but Alcalase and Neutrase had a better performance in the production of low molecular weight peptides in the range of 10 D. This led to increase the antimicrobial activity against the examined bacteria at the concentration of 200 µg/mL peptide solution. The Neutrase enzyme produced hydrolysate with the highest ACE inhibitory activity (53%?±?1.8 at 500 µg/mL). Antimicrobial activity of Kilka protein hydrolysates using Protamex and Pepsin was lower than the others due to lack of considerable amount of small peptides. The current research has demonstrated that the peptides derived from the enzymatic hydrolysis of Kilka fish protein in optimum conditions are capable of being converted to antimicrobial and antihypertensive agents to be used in functional foods.     

酶法制备火麻肽及其血管紧张素转化酶抑制活性研究

以火麻蛋白为原料,在碱性蛋白酶、中性蛋白酶、风味酶和木瓜蛋白酶4种单酶酶解火麻蛋白的基础上,再优选碱性+中性蛋白酶、碱性+风味酶、碱性+木瓜蛋白酶双酶分步对火麻蛋白进行酶解,酶解物(HPH)及其超滤组分的体外血管紧张素转化酶(ACE)抑制活性采用高效液相检测法(HPLC)进行测定。结果得到火麻蛋白最佳酶解组合为碱性+中性蛋白酶,最佳工艺条件为:碱性蛋白酶加酶量8000 U/g,pH10.0,酶解温度50℃,酶解时间4 h;中性蛋白酶加酶量8000 U/g,pH7.0,酶解温度45℃,酶解时间4 h,分步酶解物水解度(DH)和ACE抑制活性分别达74.52%和82.14%,但其与超滤各组分对ACE抑制活性差异并不显著。该研究为产业化制备火麻降血压肽提供理论依据。     

大豆粕蛋白酶酶解条件和产物分析研究

以水解度为衡量指标,通过正交试验设计,对木瓜蛋白酶、胰蛋白酶和As1.398蛋白酶酶解大豆粕的最佳水解条件进行了筛选.试验结果表明:木瓜蛋白酶水解大豆粕的最佳条件为pH7.0、温度60℃、时间5 h、酶浓度5%.胰蛋白酶水解大豆粕的最佳条件为pH7.0、温度50℃、时间7 h、酶浓度5%;As1.398蛋白酶水解大豆粕的最佳条件为pH6.5、温度50℃、时间7 h、酶浓度5%.对酶解产物进行超滤和SDS-PAGE电泳分析表明,因酶解条件不同,大豆粕酶解产物中蛋白质和肽的组成及其数量也不同;酶的种类不同,大豆粕酶解产物组成也不同;大豆粕水解度越高,其酶解产物中小分子肽数量越多.     

海参皂苷Echinoside A的酶解及其产物结构鉴定与溶血毒性

利用商业酶将海参皂苷Echinoside A(EA)酶解成低溶血毒性的次级苷。为了得到溶血毒性更低的稀有次级海参皂苷,以海参皂苷Echinoside A为底物,选用5种商品糖苷酶,对其进行酶解。利用TLC、高效液相色谱-质谱法(HPLC-MS)和电喷雾串联质谱法(ESI-MS/MS)筛选出具有能将海参皂苷EA降解生成新物质的商品糖苷酶,并进行其酶解产物结构鉴定和溶血毒性研究。结果表明,果胶酶、果胶酶Ultra SP-L和纤维素酶有酶解效果,其中果胶酶酶解生成二糖基次级海参皂苷(化合物1)和一糖基次级海参皂苷(化合物2);果胶酶Ultra SP-L和纤维素酶酶解生成化合物1,其他两种酶对海参皂苷EA没有酶解作用。溶血实验证明酶解产物的溶血毒性相对于海参皂苷EA的溶血毒性有明显地降低,其中化合物2的溶血毒性降低到30%以下,效果显著。     

自然酸度下乳蛋白酶解动态分析及其物化特性研究

通过在自然变化pH值和人工调控恒定pH值两种酸度下,四种蛋白酶水解乳蛋白的动态分析及测定前种方法的酶解产物的主要理化特性,特别是苦味特性,以期筛选出低苦味水解酶及水解工艺.以蛋白水解度(DH)和10%三氯乙酸可溶性氮含量(TCA-SN)为指标,比较四种蛋白酶在两种工艺下水解的情况.以溶解度、黏度、苦味和蛋白水解电泳图谱指标研究了自然pH值条件下四种酶解物的理化特性.结果表明:四种蛋白酶分别在两种处理条件下的水解度和可溶性氮含量差异不大;pH未恒定条件下得到的酶解物与乳蛋白相比,在酸性范围内有较大的溶解度;蛋白浓度升高时,黏度变化不大;四种酶解物中Neutrase和木瓜蛋白酶的苦味最强,Protamex的苦味最小;SDS-PAGE电泳表明,两种中性蛋白酶主要水解乳中的酪蛋白.因此可用本研究选用的四种蛋白酶在pH值自然变化条件下酶解乳蛋白,从而简化酶解工艺,并且该酶解物都有较好的理化特性.     

Peptide–peptide and protein–peptide interactions in mixtures of whey protein isolate and whey protein isolate hydrolysates

The extent of aggregation in whey protein isolate (WPI) hydrolysates induced by Bacillus licheniformis protease was quantified as a function of degree of hydrolysis (DH), temperature and ionic strength. The capacity of the hydrolysates to aggregate added intact protein was also studied. The amount of aggregated material and the size of the aggregated peptides were measured by nitrogen content and size exclusion chromatography, respectively. Aggregation increased with DH up to the practical end point of hydrolysis (DH 6.8%). The aggregates formed under the various conditions studied consisted of peptides with masses ranging from 1.4 to 7.5 kDa. The hydrolysates were also able to aggregate added WPI. The additional amount of aggregated material increased with increasing DH. Peptides involved in peptide–peptide interactions were also involved in protein–peptide interactions. It is hypothesized that hydrophobic interactions dominated peptide–peptide interactions, while protein–peptide interactions depended on the balance between hydrophobic attraction and electrostatic repulsion.