酶法制备鲢鱼蛋白抗氧化肽研究

为制备具有良好抗氧化活性的鲢鱼蛋白肽段,选择了5种蛋白酶在相同酶活力下水解鲢鱼鱼肉蛋白,通过三硝基苯磺酸法(TNBS法)和分光光度法分别测定了鲢鱼鱼肉蛋白的水解度及酶解产物的抗氧化活性,并分析了水解度与抗氧化各指标间的相关性.结果表明:鲢鱼鱼肉蛋白的5种酶解产物中,碱性蛋白酶酶解鲢鱼鱼肉蛋白5 h后的水解度最高;清除DPPH自由基能力和还原力较强的是胃蛋白酶酶解产物,其清除DPPH自由基能力最高达到了61%,还原力最高达到了1.6;酶解产物的水解度和还原力分别与亚铁离子螯合力具有显著的相关性(P﹤0.05).酶的种类对鲢鱼蛋白的水解度和酶解产物的抗氧化活性有较大的影响.     

牡蛎酶解液的抗氧化活性

检测了牡蛎(Crassotera gigas)木瓜蛋白酶和中性蛋白酶酶解液的抗氧化活性。由Sephadex G-15葡聚糖凝胶柱层析分析牡蛎酶解液,得到具抗氧化活性组分的分子量分布。分子量为1191 D和826 D左右的木瓜蛋白酶酶解活性肽组分的自由羟基清除活性最强,当其质量浓度分别为0.184 mg/mL和0.673 mg/mL时,体外自由羟基清除活性分别为53.6%和66.5%;分子量分别为1074 D和735 D左右的中性蛋白酶酶解活性肽的自由羟基清除活性最强,当其质量浓度分别为0.166 mg/mL和0.830 mg/mL时,体外自由羟基清除活性分别57.6%和70.5%。HPLC分析结果表明,木瓜蛋白酶与中性蛋白酶酶解的活性肽组分分别由几种抗氧化肽组成。抗氧化活性最强的木瓜蛋白酶与中性蛋白酶酶解的肽组分在质量浓度为2.5 mg/mL时,活性分别可达83.6%和80.8%。用牡蛎原浆与各酶解液灌胃昆明小白鼠。研究结果表明,用木瓜蛋白酶与中性蛋白酶酶解原液及经超滤纯化的酶解液对小白鼠实施灌胃,其肝脏组织的SOD活力都显著提高(P<0.05);灌胃经超滤纯化的木瓜蛋白酶和中性蛋白酶酶解液,小白鼠肝脏组织GSH-PX活力、GSH含量显著提高(P<0.05),MDA含量显著降低(P<0.05)。     

Optimization of the preparation of bioactive peptides from Sinonovacula constricta and its hydroxyl free radical scavenging activity

以缢蛏为试验材料,采用羟自由基清除率为评价指标,从胰蛋白酶、胃蛋白酶、木瓜蛋白酶、碱性蛋白酶和中性蛋白酶5种蛋白酶中筛选最适水解用酶,在单因素试验基础上使用响应面法优化酶解工艺条件,考察缢蛏蛋白肽的羟自由基清除能力并通过SephadexG-15凝胶层析测定其分子质量分布.结果表明,碱性蛋白酶酶解制备的缢蛏蛋白肽清除羟自由基能力明显强于其他蛋白酶,优化后的碱性蛋白酶水解缢蛏蛋白工艺条件为底物浓度6 mg/ml、加酶量3％、pH8.0、温度55℃、酶解时间4h,蛋白肽的羟自由基清除率为76.60％,IC50值为1.89 mg/ml,蛋白肽中80％以上是分子质量小于1500 Da的小分子肽.     

缢蛏蛋白肽制备工艺优化及其清除羟自由基作用

以缢蛏为试验材料,采用羟自由基清除率为评价指标,从胰蛋白酶、胃蛋白酶、木瓜蛋白酶、碱性蛋白酶和中性蛋白酶5种蛋白酶中筛选最适水解用酶,在单因素试验基础上使用响应面法优化酶解工艺条件,考察缢蛏蛋白肽的羟自由基清除能力并通过Sephadex G-15凝胶层析测定其分子质量分布。结果表明,碱性蛋白酶酶解制备的缢蛏蛋白肽清除羟自由基能力明显强于其他蛋白酶,优化后的碱性蛋白酶水解缢蛏蛋白工艺条件为底物浓度6mg/ml、加酶量3%、pH8.0、温度55℃、酶解时间4h,蛋白肽的羟自由基清除率为76.60%,IC50值为1.89mg/ml,蛋白肽中80%以上是分子质量小于1500Da的小分子肽。     

尼罗罗非鱼鱼皮胶原蛋白中游离基清除肽的制备及其抗氧化活性

采用菠萝蛋白酶和Alcalase酶依次对尼罗罗非鱼皮胶原进行复合酶解.研究了该酶解产物的体外抗氧化活性.实验表明,该酶解产物具有较强的超氧阴离子/羟基自由基清除活性和还原能力.采用不同截留分子量的超滤膜将该酶解物分离成5个组分,即TGH-Ⅰ(＞10 ku),TGH-Ⅱ(10～5 ku),TGH-Ⅲ(5～3 ku),TGH-Ⅳ(3～1 ku)和TGH-Ⅴ(10 ku).其中TGH-Ⅴ组分显示出最强的超氧阴离子自由基清除活性,因此采用凝胶过滤、离子交换和反向高压液相色谱技术对该组分进一步分离纯化.纯化得到的肽具有很强的超氧阴离子自由基清除活性,其IC50值为4.6 μg·mL-1.通过质谱分析可知,该肽的分子量位于311.3～932.8 u之间.     

金枪鱼鱼骨胶原肽的制备及抗氧化活性研究

为制备金枪鱼鱼骨胶原肽,并对其抗氧化活性进行研究,利用酶解、超滤、凝胶色谱和反相高效液相色谱制备抗氧化胶原肽,采用氨基酸序列分析仪测定其氨基酸序列,利用质谱(ESIMS)确定其分子量,采用羟自由基、DPPH自由基、ABTS自由基和超氧阴离子自由基清除实验和脂质过氧化抑制实验对胶原肽抗氧化能力进行评价。结果显示,金枪鱼鱼骨胶原蛋白经胃蛋白酶和胰蛋白酶2步酶解和分离纯化得到1个十肽(TFCH-P2),经氨基酸序列分析和质谱(ESIMS)确定其氨基酸序列为Gly-Pro-Ala-Gly-Pro-Ala-Gly-Glu-Gln-Gly(GPAGPAGQEG),分子量为839.87 u([M+H]+840.68 u)。体外抗氧化实验结果表明,GPAGPAGQEG对羟自由基(EC500.18 mg/mL)、DPPH自由基(EC500.97 mg/mL)、ABTS自由基(EC500.52 mg/mL)和超氧阴离子自由基(EC500.38 mg/mL)具有良好的清除作用;GPAGPAGQEG亦显示出良好的脂质过氧化抑制作用。研究表明,胶原肽GPAGPAGQEG抗氧化活性良好,可以用于抗氧化相关的功能食品、药物或者食品添加剂。     

鲣鳔蛋白抗氧化酶解物制备工艺

为有效提高鲣鳔蛋白的附加值,研究以DPPH自由基清除率为抗氧化活性评价指标,采用蛋白酶酶解制备活性多肽的工艺,选用菠萝蛋白酶、复合蛋白酶、碱性蛋白酶、木瓜蛋白酶、胃蛋白酶、胰蛋白酶、中性蛋白酶7种酶在各自最适的条件下酶解,筛选出复合蛋白酶为最适用酶,通过单因素实验分别研究加酶量、溶液初始p H、酶解温度和时间对酶解物抗氧化活性的影响,在此基础上,根据响应面法优化鲣鳔抗氧化酶解物的制备工艺。结果显示,最佳酶解工艺条件为加酶量8.53 U/mg,p H 5.54,温度50.03°C,时间5.07 h。此外,利用超滤法对最佳条件下制备的酶解物进行初步分级,得到分子质量分别为大于10 000 u、3000~10 000 u和小于3000 u的3段组分,且这3段组分对DPPH自由基的半抑制浓度IC50值分别为0.64、0.52和0.37 mg/m L。研究表明,最优条件下制备的酶解物的DPPH清除率达72.00%,与模型预测值71.60%接近,且其中小于3000 u的组分具有较强的DPPH自由基清除活性。     

鲢酶解物对羟自由基的清除作用

通过测定酶解物对Fenton体系产生的羟自由基的清除效果，从胰蛋白酶、木瓜蛋白酶、胃蛋白酶、枯草杆菌蛋白酶和复合蛋白酶5种酶中，筛选出木瓜蛋白酶和胰蛋白酶作为酶解鲢制备具有较高清除羟自由基活性酶解物的理想水解酶；用正交试验L9(3^4)对两种酶的水解条件进行了优化，并对最佳酶解条件下得到的酶解物进行Sephadex G-25凝胶柱分离，洗脱液分别在波长280nm处比色，测定酶解物中主要抗氧化活性肽的分子量分布。结果表明，木瓜蛋白酶在温度50℃、酶解时间15min、pH=6．5、酶质量分数1．50％、底物：水=1：2的水解条件下，酶解物对羟自由基清除效果较好，清除率为88．2％；胰蛋白酶在温度55℃、酶解时间60min、pH=8．0、酶质量分数0．25％、底物：水=1：2的水解条件下，酶解物对羟自由基清除效果较好，清除率为84．2％。木瓜蛋白酶酶解物在最大洗脱峰时有最大羟自由基清除率峰，清除率为95．1％，在最大峰处酶解物中活性肽的分子量为2．2kDa；胰蛋白酶酶解物在最大洗脱峰时也有最大羟自由基清除率峰，其清除率为89．6％，该峰处活性肽的分子量为14．2kDa。     

罗非鱼活性肽分离及抗氧化能力研究

利用正交试验L9(34),以清除超氧自由基能力为指标,分别对木瓜蛋白酶、中性蛋白酶水解罗非鱼肉的水解条件进行优化,并对最佳清除率下的两种酶解物进行Sephadex G-50凝胶柱分离,检测了各组分对超氧自由基清除率及活性肽分子量分布情况.结果表明:中性蛋白酶在45 ℃、酶的质量分数2.0%、水解105 min及肉水比1∶3的水解条件下对超氧自由基有较好清除作用;木瓜蛋白酶在60 ℃、酶的质量分数2.0%、时间150 min及肉水比1∶2的水解条件下对超氧自由基有较好清除效果.木瓜蛋白酶酶解物在分子量为660 Da的多肽洗脱峰具有最大超氧自由基清除率,中性蛋白酶酶产物在分子量为1320 Da多肽洗脱峰具有最大超氧自由基清除率.     

虾头自溶产物中ACE抑制肽的分离鉴定

虾头在一定的条件下发生自溶作用,其所含蛋白质以肽和氨基酸等形式释放出来,有些肽产物具有ACE抑制活性.实验采用8000、5000和3000 u的超滤膜分级分离虾头自溶产物,活性检测结果表明,ACE抑制肽主要分布在3000 u超滤组分中;3000 u超滤组分进一步经Sephadex G-25葡聚糖凝胶层析、SP Sephadex C-25离子交换层析及Sephadex G-15葡聚糖凝胶层析纯化,ACE抑制活性提高将近8倍(IC50 =0.19 mg/mL);Sephadex G-15葡聚糖凝胶层析收集的高活性成分再经两次RP-HPLC纯化,分离纯化得到两条ACE抑制肽,质谱分析推测其氨基酸序列为Tyr-Pro和Leu-Pro/Ile-Pro,分子量分别为279和229 u.     

Assessing the Antioxidant Activity of the Ultrafiltration Fractions From Silver Carp Protein Hydrolysate by Different Antioxidant Methods

Silver carp protein hydrolysate (SH) was fractionated by 5- and 1-kDa cutoff ultrafiltration (UF) membrane, and three ultrafiltrates were obtained: SHI (>5 kDa), SHII (1–5 kDa), and SHIII (<1 kDa) which were screened for antioxidant activity by five in vitro assays. The SHIII showed higher 2, 2-azobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC) values, inhibiting effects for liposome oxidation induced by Fe³⁺/V_C. The SHII showed higher ferric reducing power (FRAP) and inhibiting effects for liposome oxidation induced by 2, 2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH). However, the SHI showed the highest 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging activity. Therefore, the antioxidant activity of silver carp protein hydrolysates was mainly associated with their molecular weight distribution, and the antioxidant assays used to test different samples gave comparable results.     

Evaluation of the Antioxidant and Antimicrobial Activity of Protein Hydrolysates and Peptide Fractions Derived from Colossoma macropomum and Their Effect on Ground Beef Lipid Oxidation

The objective of this study was to obtain protein hydrolysate from the mechanically separated meat of blackfin pacu to evaluate the influence by ultrafiltration in the antioxidant and antimicrobial activities of the peptide fractions obtained and to apply in ground beef to evaluate the lipid stability. The enzymatic hydrolysis was performed using the enzyme Protamex (pH 7.0, 60°C) for 240 min. The protein hydrolysate was fractionated by ultrafiltration. Then, the antioxidant capacity of the protein hydrolysate and the peptide fractions were evaluated in vitro by the methods of 2,2’-azinobis (3-ethylbenzothiazoline sulfonic acid) radical capture, 2,2-diphenyl-1-picrylhydrazyl radical-scavenging assay, reducing power, and hydroxyl radical scavenging activity. The antimicrobial activity of the samples was evaluated by disc-diffusion against Staphylococcus aureus and Escherichia coli. After evaluation, the peptide fractions did not present higher bioactivities than that shown for the hydrolysate. The protein hydrolysate was applied to ground beef, where the substances reactive to thiobarbituric acid and color were evaluated during 7 days of storage at 4°C. Lipid oxidation was reduced up to 60.9% and there was no modification of the natural coloration. Thus, the protein hydrolysate can be used as an alternative source of antioxidant for the preservation of refrigerated meats.     

Antioxidant and ACE Inhibitory Activities of Kawakawa (Euthynnus affinis) Protein Hydrolysate Produced by Skipjack Tuna Pepsin

ABSTRACT

Pepsin enzyme from skipjack tuna was extracted for the production of kawakawa (Euthynnus affinis) fish protein hydrolysate. Using ultra-fractionation, Kawakawa protein hydrolysates were separated into four different fractions, including fractioned protein hydrolysate I (FPH I) (< 1 kDa), FPH-II (1–3 kDa), FPH-III (3–10 kDa), and FPH-IV (> 10 kDa). The antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, inhibition of linoleic acid oxidation, reducing power tests, and chelating activity of metal ions. Results indicated that FPH II fraction peptides had higher antioxidant activity in comparison with the other fractions, followed by FPH I. Further, the fractions were evaluated for angiotensin converting enzyme (ACE) inhibition, and IC₅₀ value ranged from 0.45 to 1.86 mg/ml with higher activity in FPH I (IC₅₀ 0.45). Finally, the amino acid profile of different fractions was analyzed. The fractions exhibited significant amounts of hydrophobic amino acids, which could perform as hydrogen donors, frustrate the free radicals, and inhibit the ACE. The recovered pepsin from the viscera was used to produce hydrolysates with good biological activities. Peptides lower than 3 KDa had antioxidant activity as positive controls and significant ACE activity. These are very important findings that could be used to conduct further research in a preclinical study of these peptides.     

Purification and Characterization of a New DPPH Radical Scavenging Peptide from Shrimp Processing By-products Hydrolysate

A peptide with high antioxidant activity was isolated and identified from shrimp processing by-products hydrolysate. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity was used to evaluate the antioxidant activity of fractions. The purified antioxidant peptide was identified as Ser-Val-Ala-Met-Leu-Phe-His (804.4 Da) by electrospray ionization tandem mass spectrometry. The purified peptide at 50 μg/mL showed antioxidative activity of 65.7 ± 0.9%, which was 3.18-fold higher compared with the first step separation by ion-exchange chromatography. It is possible to produce natural antioxidative peptides from shrimp processing by-products hydrolysate. The high antioxidant activity may be due to the presence of Phe-His segment at the C-terminus of the peptide.     

Antioxidative Activitiy of Protein Hydrolysate from the Muscle of Common Kilka (Clupeonella cultriventris caspia) Prepared Using the Purified Trypsin from Common Kilka Intestine

Trypsin from the intestine of common kilka (Clupeonella cultriventris caspia) was purified using ammonium sulfate precipitation (30–50% saturation), Sephadex G-75, and DEAE-cellulose chromatography with the purity of 30-fold and the yield of 12%. The molecular weight of trypsin was estimated to be 23.2 kDa based on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The trypsin had optimal activity at pH 8.0 and 60°C using N-α-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) as a substrate and showed high stability in the pH range of 7.0–10.0. It was stable up to 50°C. Soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysine-chloromethylketone (TLCK) significantly inhibited trypsin activity (p < 0.05). Protein hydrolysate from common kilka muscle with different degrees of hydrolysis (DHs; 20, 30, and 40%) was prepared using the purified trypsin, and antioxidative activities were determined. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities, ferric reducing antioxidant power, and ferrous chelating activity of hydrolysate increased with increasing DH up to 40% (p < 0.05). Therefore, trypsin from intestine of common kilka could be used as a processing aid for production of fish protein hydrolysate with antioxidative activity.     

大眼金枪鱼头蛋白酶解物1 ku超滤组分的抗氧化活性及其理化性质

研究大眼金枪鱼头蛋白酶解物1 ku超滤组分体外的还原力、自由基清除能力及对衰老小鼠体内抗氧化能力的影响,分析1 ku超滤组分的一般成分、氨基酸组成及分子量分布,为进一步分离纯化金枪鱼头抗氧化肽提供基础。体外结果显示,1 ku超滤组分对羟基自由基、超氧阴离子和DPPH自由基的清除活性随浓度的增加而增强,IC50分别为1.38、0.73与0.93mg/mL,还原力也随浓度的增加而增大,在浓度为12.5 mg/mL时为0.763;体内结果显示,灌胃30 mg/kg的1 ku超滤组分连续42 d,D-半乳糖致衰老小鼠肝组织的超氧化物歧化酶(SOD)活性、肝组织和血清的谷胱甘肽过氧化物酶(GSH-PX)活性显著提高(P<0.05),血清丙二醛(MDA)含量显著降低(P<0.01);理化分析结果显示,1 ku超滤组分(干基计)蛋白质含量为96.40%,脂肪0.11%,灰分4.86%,疏水性氨基酸占氨基酸总量的35.8%,活性组分分子量在1 802~2 519 u和422~922 u。     

Antioxidant Properties of Protein Hydrolysate Obtained from Oyster Saccostrea cucullata (Born, 1778)

The antioxidant activities of enzymatically hydrolyzed (protease from Bacillus cereus SU12) oyster (Saccostrea cucullata) protein were studied. The hydrolysate exhibited a strong antioxidant potential in 1, 1-diphenyl-2-picrylhydrazyl (DPPH, 85.7 ± 0.37%), followed by hydrogen peroxide radical scavenging activity (81.6 ± 0.3%), hydroxyl radical scavenging activity (79.32 ± 0.6%), and reducing power assay (2.63 ± 0.2 OD at 700 nm) at a concentration of 1 mg/mL. Due to the high antioxidant potential, the hydrolysate was purified in Sephadex G-25 gel filtration chromatography. The active peptide fraction was identified by DPPH and reducing power assay. The amino acid content of the purified active peptide fraction was analyzed by high performance liquid chromatography. The active fraction contained a good quantity of both essential and nonessential amino acids. The present study revealed that oyster (S. cucullata) protein hydrolysate is a potential source for natural antioxidants.     

Protein Hydrolysates and Ultrafiltration Fractions Obtained from Krill (Euphausia superba): Nutritional,Functional, Antioxidant,and ACE-Inhibitory Characterization

ABSTRACT

Krill (Euphausia superba) was hydrolyzed by proteolytic enzymes in order to produce multifunctional bioactive peptides, and their functional properties were evaluated. Krill protein hydrolysate (KPH) by pepsin with 4-h hydrolysis showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and angiotensin I converting enzyme (ACE) inhibitory activities. The solubility and foaming properties of KPH were higher than those of the unhydrolyzed krill protein at a wide range of pHs. KPH was further fractionated based on molecular weight. The 1- to 3-kDa peptide fraction exhibited the highest DPPH scavenging activity (IC₅₀ value of 0.5 mg/mL), oxygen radical absorbance capacity (497.39 ± 4.31 µM TE/mg fraction), 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid cation radical scavenging activity (48.41 ± 0.23 µM TE/mg fraction), and reducing power (110.40 ± 2.07 µM TE/mg fraction). However, the < 1-kDa peptide fraction exhibited a higher ACE inhibitory activity than that of other fractions. The 1- to 3- and < 1-kDa peptide fractions are rich in aromatic and hydrophobic amino acids, respectively.     

Antioxidant Properties and Arsenic Speciation of Ultrafiltration and Nanofiltration Derived Abalone Viscera Hydrolysate Fraction

A fraction with molecular weight of 0.2–1.0 kDa was prepared from abalone viscera hydrolysate by ultrafiltration and nanofiltration. The contents of protein, carbohydrate, and fat in abalone viscera were 45.75%, 18.19%, and 13.60%, respectively. The protein in abalone viscera hydrolysate faction (AVHF) reached up to 75.73%, while carbohydrate and fat fell to 1.80% and 0.20%, respectively. The leucine (Leu) content was the highest in the AVHF. On the other hand, the hydroxyl and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities and reducing power increased with increased concentration of AVHF. The inhibiting ability of AVHF on lipid peroxidation was higher than that of ascorbic acid. The total content of K, Na, and P in AVHF was 9.78 g/100 g, and total arsenic content reached 16.23 mg/kg. The proportion of arsenobetaine and arsenocholine accounted for more than 96%, while the proportion of arsenate was 0.47%, and arsenite was not detectable. It is concluded that the AVHF from abalone viscera is a safe natural antioxidant.