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。     

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.     

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)。     

海带多糖清除氧自由基的活性及机理

通过比较不同分子量和化学组成的海带硫酸多糖和褐藻胶的清除氧自由基的活性,探讨了海带多糖清除氧自由基活性的构效关系和作用机理。研究发现,分子量为6~15 ku、糖醛酸含量20.4%的低硫组分F-A2清除自由基活性羟基自由基和超氧阴离子自由基活性高于大分子量的组分F-A和F-B,而高硫低分子量组分清除氧自由基的活性非常低。酶降解得到的低分子量褐藻胶组分清除自由基活性随分子量的降低而升高,明显高于其他硫酸多糖,说明硫酸根对低分子量糖与自由基的反应有阻碍,而多糖中糖醛酸含量越高,清除自由基活性越好。大分子量的海带硫酸多糖经铜离子和H₂O₂反应产生的羟基自由基氧化降解以后,直接可以得到两个比较集中的低分子量岩藻聚糖硫酸酯Fa2(分子量为7 ku)和Fa1(分子量为1 ku),其化学组成和清除自由基活性的比较说明自由基首先降解糖醛酸含量高的硫酸糖片断,甘露糖、半乳糖和葡萄糖形成的糖苷键很容易被自由基氧化降解,而高硫高岩藻糖部分不易被自由基水解,研究结果说明,海带多糖的抗氧化活性不仅与分子量和硫酸根含量有关,糖醛酸、岩藻糖含量和糖链上中性糖的组成对多糖的清除自由基活性都有影响。     

鲢鱼鱼皮蛋白肽的制备与抗氧化活性评价

为制备抗氧化活性良好的鲢鱼鱼皮蛋白肽,采用胰蛋白酶、碱性蛋白酶、菠萝蛋白酶和木瓜蛋白酶等4种常见的商业酶对鲢鱼鱼皮进行酶解,测定酶解物的ABTS自由基清除力和Fe2+螯合力来评价其抗氧化活性,并用超滤及凝胶层析对酶解物进行分离,以期得到活性更好的酶解物分离组分。酶解后产物的抗氧化活性均有所提高,其中碱性蛋白酶酶解2 h产物活性较强。对此酶解物用截留分子量为10 k Da、5 k Da和3 k Da的中空纤维超滤膜进行超滤,得到的4个组分中,分子量越小的组分抗氧化活性越强。分子量小于3 k Da的组分经Sephadex G-15凝胶层析得到3个组分,其中分子量最大的组分活性较好,在0.51 mg/m L质量浓度下测定其ABTS自由基清除率和Fe~(2+)螯合力分别为(79.65±0.87)%和(93.40±0.20)%。该研究成果对鲢鱼鱼皮抗氧化肽的开发具有较好的指导作用。     

血管紧张素转换酶抑制肽的分离

采用截留分子量分别为30 ku、10 ku、6 ku、3 ku的中空纤维超滤膜对扇贝酶解产物进行分级分离,并结合凝胶柱层析分离技术测定了各分离组分的分子量分布,同时还测定了各分离组分对血管紧张素转换酶(ACE)的抑制活性。实验结果表明,不同截留分子量超滤膜可以实现酶解产物按其分子量大小的分级分离,不同分离组分的ACE抑制力活性差异较大,总趋势是截留分子量相对较小的超滤膜透过液的ACE抑制活性较强。其中,截留分子量为3 ku超滤膜的透过液具有最强的ACE抑制活性,其ACE抑制率I(%)=96.17%,半抑制浓度IC_(50)= 0.078 mg·mL~(-1)。通过对截留分子量为3 ku超滤膜的透过液依次经过Sephadex G-25及Sephadex G-15凝胶柱层析分离纯化后,分离出了ACE抑制活性最强的2个组分活性肽,其平均分子量分别为1 300 u和900 u左右,其IC_(50)分别为0.026 mg·mL~(-1)和0.012 mg·mL~(-1)。     

超声降解法制备可溶性鱿鱼墨黑色素及其抗氧化性

实验研究了一种在碱性条件下利用超声降解制备可溶性鱿鱼墨黑色素的新方法。将鱿鱼墨黑色素溶于NaOH后用超声细胞破碎仪处理1 h,超滤后分别获得不同分子量可溶性黑色素组分。结果显示,超声处理后黑色素平均粒度从17.34 μm下降至1.467 μm,紫外、红外光谱和核磁共振谱图从结构上说明超声作用主要破坏黑色素的高度聚合状态,而黑色素的主要化学结构并没有被破坏,只有少部分基团,尤其是较低分子量组分中的DHI和DHICA结构被氧化;体外抗氧化实验显示经过0.5 mol/L和1 mol/L的NaOH处理,分子量大于10 ku的组分具有很强的抗氧化性,这些组分清除超氧自由基能力(IC₅₀=19～80 μg/mL)远优于作为商品抗氧化剂的肌肽(IC₅₀=355 μg/mL);清除羟基自由基活性(IC₅₀=115～180 μg/mL)与肌肽(IC₅₀=110 μg/mL)相当。可溶性鱿鱼墨黑色素作为一种天然色素和新型自由基清除剂,其良好的可溶性大大提高了机体的吸收利用率。     

胰蛋白酶酶解制备孔鳐软骨蛋白寡肽及其抗氧化活性

以孔鳐软骨为材料,采用盐酸胍抽提、丙酮分级沉淀,制备孔鳐软骨蛋白;以DPPH·和HO·清除活性为导向,采用胰蛋白酶酶解、膜超滤、DEAE-52阴离子交换层析、Sephadex G-15凝胶层析和反相高效液相色谱(RP-HPLC)等技术,制备抗氧化肽,并对其活性进行系统评价。结果显示,孔鳐软骨蛋白经胰蛋白酶酶解和分离纯化得到2个抗氧化肽RCPE-A和RCPE-B,经氨基酸序列分析确定其序列分别为Gly-Glu-Glu-Gly-Pro-Arg-Gly (GEEGPRG)和Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu (GEEGTMGL),质谱(ESI-MS)测定其分子量分别为700.71和792.87 u。体外自由基清除实验结果显示,RCPE-A与RCPE-B对DPPH·(EC₅₀ 2.94和1.16 mg/mL)、HO·(EC₅₀ 0.34和0.54 mg/mL)、ABTS⁺·(EC₅₀ 0.34和0.10 mg/mL)和O₂^-·(EC₅₀ 0.11和0.03 mg/mL)具有良好的清除作用,RCPE-A与RCPE-B亦显示出较强的脂质过氧化抑制作用。研究表明,孔鳐软骨蛋白酶解物及制备多肽可用于抗氧化相关的功能食品开发,也可以用作抗氧化剂延长相关产品的货架期。     

鳄鱼血蛋白酶解产物抗氧化特性和血管紧张素转化酶抑制活性研究

研究了鳄鱼血蛋白酶解产物的抗氧化特性和对血管紧张素转化酶（ ACE）的抑制活性。利用木瓜蛋白酶酶解鳄鱼血浆蛋白和血球蛋白,用分光光度法测定了酶解产物的抗氧化能力和用高效液相色谱（ HPLC）测定其ACE的抑制率。结果显示：鳄鱼血浆和血球蛋白酶解产物的亚铁离子螯合能力差异性不显著（ P＞0．05）;在0～5 mg/mL的浓度范围内,血球蛋白酶解产物清除ABTS自由基的能力大于血浆蛋白酶解产物,且在浓度为1 mg/mL时,两者清除ABTS自由基的能力差异性极显著（ P＜0．01）;血浆蛋白酶解产物清除DPPH自由基的能力在0～5 mg/mL的浓度范围内随着蛋白浓度的增加而升高,血球蛋白酶解产物在蛋白浓度为4 mg/mL处达到最大清除率,之后下降;在0～20 mg/mL的浓度范围内,两种酶解产物的还原力随着蛋白浓度的提高显著升高,但两者还原力的差异性不显著（ P＞0．05）;鳄鱼血浆和血球蛋白酶解产物对ACE具有良好的抑制力,其最大抑制率可分别达到75．56％和86．42％。研究表明,鳄鱼血蛋白酶解产物在体外具有抗氧化和抑制ACE的活性。     

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。     

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.     

二种海胆性腺的脂质组成及其抗氧化活性

为分析马粪海胆和光棘球海胆性腺的脂质组成和抗氧化活性,采用核磁共振和气相色谱—质谱技术对2种海胆性腺油脂的脂质成分和脂肪酸组成进行分析,并通过DPPH自由基清除法、羟基自由基清除法和超氧阴离子自由基清除法对其脂质的抗氧化活性进行研究。结果显示,马粪海胆和光棘球海胆性腺脂质均以甘油三酯和磷脂为主,胆固醇、胆固醇酯和游离脂肪酸含量较低。马粪海胆和光棘球海胆性腺总脂富含C20:4n-6和C20:5n-3,且二者总量分别占脂肪酸含量的35.88%和34.98%;同时2种海胆性腺的中性脂和极性脂的脂肪酸组成存在较大差异,中性脂以C14:0和C16:0等饱和脂肪酸为主,而极性脂以C20:4n-6和C20:5n-3等多不饱和脂肪酸为主。马粪海胆和光棘球海胆性腺脂质对DPPH自由基、羟基自由基和超氧阴离子自由基均具有较好的清除能力,DPPH自由基IC₅₀分别为2.75和1.98 mg/mL,羟基自由基IC₅₀分别为0.33和0.29 mg/mL,超氧阴离子自由基IC₅₀分别为0.33和0.31 mg/mL。研究表明,马粪海胆和光棘球海胆性腺脂质具有较高的营养价值和抗氧化活性,可作为C20:4n-6、C20:5n-3和磷脂等功能性脂质因子的重要膳食来源。     

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.     

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.     

Assessment of Bioactive Potential of Aqueous Protein Extracts from Diatoms Nitzschia laevis,Spirulina platensis,and Chlorella vulgaris

The bioactivities of protein extracts from Nitzschia laevis, Spirulina platensis, and Chlorella vulgaris were evaluated in vitro. Free radical scavenging potential, reducing power, oxygen radical absorbance capacity, superoxide anion radical, xanthine oxidase inhibition, and potential to inhibit angiotensin 1-converting enzyme (ACE) and acetylcholinesterase enzyme (AChE) were studied. Nitzschia protein extracts showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities, reducing power, and ACE inhibitory activity. Spirulina’s proteins showed the highest 2,2′-Azino-bis (3 ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity, xanthine oxidase, and AChE inhibition activities. Aqueous protein extracts from Nitzschia, Spirulina, and Chlorella showed in vitro antioxidant, anti-ACE, and anti-AChE activities, suggesting possible new sources of bioactive proteins of different phyla with nutraceutical and pharmaceutical potentials.     

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.     

Chemical Compositions and Antioxidant Activities of a Specialty Aquatic Vegetable Nymphoides hydrophylla in Taiwan

Nymphoides hydrophylla (NH), a lesser known aquatic vegetable, is a popular dish among the Hakka community in Taiwan. This study aimed to examine the proximate and polyphenolic compositions of NH and evaluate its antioxidant activities using various antioxidant assays. Results showed that the moisture, crude protein, crude fat, carbohydrate (crude fiber), and ash contents of fresh NH were 89.46, 0.46, 0.47, 4.11 (3.65), and 5.50%, respectively. In the antioxidant studies, the NH ethanolic extract (NH-E) exhibited higher antioxidant activities than the aqueous extract (NH-H) in the antilipid peroxidation, reducing power, metal chelation, and 2,2?-azino-bis(3-ethylbenthiazoline-6-sulfonic acid) or ABTS radical scavenging assays, while NH-H possessed more potent activity in the superoxide and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. Compared with NH-H (6.86 mg/g for total flavonoids and 48.83 mg/g for total phenolics), NH-E showed a higher content of total flavonoids (197.21 mg/g) and total phenolics (79.43 mg/g). High-performance liquid chromatography (HPLC) analysis showed that the main difference between NH-H and NH-E polyphenolic contents was derived from the concentrations of kaempferol and quercetin.