不同苦荞蛋白酶解产物抗氧化活性研究

以苦荞蛋白作为底物,采用碱性蛋白酶Alcalase 2.4 L、木瓜蛋白酶、胃蛋白酶、胰蛋白酶以及胃蛋白酶加胰蛋白酶模拟体内蛋白消化,制备苦荞麦蛋白水解物。采用DPPH及ABTS~+·法比较不同的蛋白水解物与水解前苦荞蛋白的体外抗氧化活性。结果表明:不同蛋白酶水解产物水解度由高到低的顺序为:碱性蛋白酶胃蛋白酶~胰蛋白酶胃蛋白酶木瓜蛋白酶胰蛋白酶,其中碱性蛋白酶水解苦荞蛋白水解度达29.95%。苦荞蛋白本身具有一定的抗氧化能力,其中DPPH清除率及ABTS~+·清除率最高分别达71.91%及11.25%,但均显著低于阳性对照Vc。随着水解程度的增加,苦荞蛋白水解产物抗氧化能力逐渐增强。其中,以碱性蛋白酶酶解产物抗氧化活性最高,其DPPH清除率及ABTS~+·清除率最高分别为91.65%(0.5 mg/mL)及16.67%(1 mg/mL),均显著高于原苦荞蛋白。其中,碱性蛋白酶水解产物的DPPH自由基清除率在高浓度(0.5mg/mL)条件下,与阳性对照Vc持平。同时碱性蛋白酶酶解产物抗氧化性(DPPH清除率及ABTS~+·清除率)显著优于其他蛋白酶解产物。因此,苦荞麦蛋白采用碱性蛋白酶解制备苦荞水解产物可作为天然的抗氧化剂。     

Improvement of the antioxidant properties of squid skin gelatin films by the addition of hydrolysates from squid gelatin

Gelatin hydrolysates (HG1 and HG2) were obtained from giant squid (Dosidicus gigas) gelatins (G1 and G2) by hydrolysis with Alcalase. Antioxidant properties of both gelatins were highly increased by hydrolysis, especially ABTS radical scavenging capacity, whereas no significant differences were found between HG1 and HG2. The amino acid composition of HG1 and HG2 closely resembled the amino acid composition of the parent proteins, gelatins G1 and G2. Both, HG1 and HG2 were composed by peptides below 30 kDa, although no clear protein bands were observed in HG2. Edible gelatin films with increasing percentages of HG1 (0–10%) were made from G1, giving rise to increasing values of FRAP and ABTS, as well as changes in mechanical properties (decrease puncture force and increase puncture deformation) and water vapour permeability (increase). HG1 gelatin hydrolysate showed lower antioxidant capacity in the gelatin films than in the free form at the same amount added into the filmogenic solution, probably due to interactions with protein matrix.     

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.     

海洋鱼蛋白低聚肽结构和抗氧化活性的体外消化稳定性

本研究通过体外模拟胃肠道消化海洋鱼蛋白低聚肽,运用高效凝胶过滤色谱、紫外全波长扫描、圆二色光谱表征其消化前后结构变化,测定其消化前后DPPH自由基清除率、ABTS自由基清除能力、铁离子还原能力(FRAP)及氧自由基吸收能力(ORAC)的变化,以探究模拟胃肠道消化对海洋鱼蛋白低聚肽结构及抗氧化活性的影响.分子量分布数据揭...     

Antioxidant and functional properties of gelatin hydrolysates obtained from skin of sole and squid

Antioxidant and functional properties were evaluated for gelatin hydrolysates obtained from sole and squid skin gelatin by Alcalase, with a degree of hydrolysis of ∼35% and ∼50%, respectively. Both hydrolysates mainly consisted of peptides below 6.5 kDa, together with peptidic material from around 16 to 6.5 kDa. Moreover, the squid hydrolysate showed a peptide band of around 26 kDa. Antioxidant properties of both gelatins were highly increased by hydrolysis, especially ABTS and metal chelating abilities. The squid hydrolysate showed the highest antioxidant capacity by FRAP, ABTS and metal chelating assays in spite of the lower content in hydrophobic amino acids. Both gelatin hydrolysates had a good solubility (over 95%). The emulsifying activity index (EAI) decreased with increasing concentration. Conversely, the foam expansion increased with increasing concentration. However, both foam and emulsion stabilities were not apparently affected by the concentration of hydrolysate. In the case of the sole hydrolysate, which showed a lower degree Pro and Lys hydroxylation, foam stability was very poor, and 50% of foam expansion was lost after 5 min at all concentrations.     

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。     

Antioxidant activity of protein hydrolysates derived from threadfin bream surimi byproducts

Antioxidant activities of protein hydrolysates from threadfin bream surimi wastes, including frame, bone and skin (FBS) and refiner discharge (RD), were investigated. FBS and RD were rich in Lys, Glu, Gly, Pro, Asp, Leu, His, Tyr and Phe. FBS was hydrolysed to a greater extent than RD regardless of proteinases tested (Virgibacillus sp. SK33 proteinase, Alcalase, pepsin and trypsin). Pepsin-hydrolysed FBS, at a 5% degree of hydrolysis (DH), showed the highest antioxidant activity based on 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) radical (0.455 ± 0.054 mg Trolox equivalents/mg leucine equivalents), ferric reducing antioxidant power (FRAP) (0.221 ± 0.005 mM Trolox equivalents) and inhibition of β-carotene bleaching assays. FBS hydrolysates showed higher antioxidant activity based on chemical assays than their RD counterparts. However, FBS and RD hydrolysates protected HepG2 cells against tert-butyl hydroperoxide-induced oxidative damage to a similar extent. Therefore, FBS and RD hydrolysates have a potential as antioxidative neutraceutical ingredients.     

模拟胃肠道消化对大豆低聚肽结构及抗氧化作用的影响

大豆低聚肽是一种有益健康的功能性配料,所呈现的健康益处高度依赖于肽结构。采用紫外全波长扫描法及圆二色光谱法分析其结构是如何受胃蛋白酶、胰蛋白酶以及先胃蛋白酶后胰蛋白酶处理的影响。为探讨消化处理前、后大豆低聚肽抗氧化能力的变化规律,分别计算消化前、后大豆低聚肽的ABTS自由基清除能力、DPPH自由基清除能力、氧自由基吸收能力(ORAC)和铁离子还原能力(FRAP)。结果显示:大豆低聚肽主要为分子质量<1000 u的组分,经胃蛋白酶、胰蛋白酶及先胃蛋白酶后胰蛋白酶处理后<1000 u的组分比例最大可达88.46%。在波长275 nm处均有最大吸收峰。二级结构中,4组大豆低聚肽中无规则卷曲所占比例均为30%左右,约占总二级结构组成的1/3,表明了大豆低聚肽无序性较高且结构疏松开放。大豆低聚肽的ABTS自由基清除能力和铁还原能力稳定性均较好。经胰蛋白酶消化后,DPPH自由基清除率有所降低,氧自由基吸收能力极显著提高(P<0.01)。     

大豆蛋白的酶水解及类蛋白反应对其抗氧化活性的影响

采用Alcalase 2.4L FG 蛋白酶水解大豆蛋白,筛选并制备出ABTS+·清除率最高的水解物,其水解度为14.0%,对ABTS+·清除率为43.6%。以此水解物为底物,以修饰产物的游离氨基减少量为指标,应用响应面分析得到类蛋白反应的优化条件为：酶添加量1037U/g pro、底物质量浓度30g/100mL、温度20℃。在此条件下反应6h,水解物的修饰反应程度和抗氧化活性均为最大。制备反应程度不等的3 个修饰产物,进一步抗氧化活性分析表明：大豆蛋白水解物及其修饰产物的抗氧化活性好于大豆蛋白;修饰产物与水解物的DPPH 自由基清除能力、还原力、超氧阴离子自由基(O2 － ·)清除率差别不显著,但是对羟自由基(·OH)清除率差别显著。     

Squid gelatin hydrolysates with antihypertensive, anticancer and antioxidant activity

Gelatin obtained from giant squid (Dosidicus gigas) inner and outer tunics was hydrolyzed by seven commercial proteases (Protamex, Trypsin, Neutrase, Savinase, NS37005, Esperase and Alcalase) to produce bioactive hydrolysates. The Alcalase hydrolysate was the most potent angiotensin-converting enzyme (ACE) inhibitor (IC₅₀ = 0.34 mg/mL) while the Esperase hydrolysate showed the highest cytotoxic effect on cancer cells, with IC₅₀ values of 0.13 and 0.10 mg/mL for MCF-7 (human breast carcinoma) and U87 (glioma) cell lines, respectively. The radical scavenging capacity of gelatin increased approximately 3-fold for Protamex, Neutrase and NS37005 hydrolysates and between 7 and 10-fold for Trypsin, Savinase, Esperase and Alcalase hydrolysates. Trypsin, Savinase, Esperase and Alcalase hydrolysates had a metal chelating capacity above 80% whereas Protamex, Neutrase and NS37005 hydrolysates registered less than 25%. The antioxidant activity measured by FRAP (ferric ion reducing power) was largely unaffected by the enzyme used, increasing approximately 2-fold for all hydrolysates. The most active hydrolysates (Alcalase and Esperase) were comprised mostly of peptides with molecular weights ranging from 500 to 1400 Da, however, a clear relationship between bioactive properties and molecular weight distribution of all the hydrolysates was not fully established.     

罗非鱼皮明胶酶解物及其模拟消化产物的抗氧化活性

以罗非鱼皮为原料提取罗非鱼皮明胶,选用风味蛋白酶和胰蛋白酶制备罗非鱼皮明胶酶解物,采用ABTS自由基、DPPH自由基、羟基自由基及亚油酸过氧化体系,初步评价罗非鱼皮明胶酶解物的抗氧化活性,再通过模拟体外胃肠道消化实验,结合分子质量分布测定,进一步考察罗非鱼皮明胶酶解物的抗氧化活性。结果显示,在酶解过程中,风味蛋白酶及胰蛋白酶酶解的水解度逐渐升高,在3 h时达到最高,分别达到5.8%和25.36%。在酶解60 min时其TCA可溶性肽得率最高,风味蛋白酶、胰蛋白酶酶解物分别达56.82%和54.44%。通过比较半抑制浓度（IC₅0）,确定了酶解60 min时风味蛋白酶酶解物的清除DPPH自由基及抑制亚油酸过氧化能力较胰蛋白酶酶解物强。模拟体外胃肠道消化后,酶解物羟基自由基清除活性均显著提高（p<0.05）,亚油酸脂质过氧化活性明显降低,消化前后样品分子量分布范围均主要集中于3000⁵000 Da,消化后风味蛋白酶及胰蛋白酶酶解物3000⁵000 Da组分的含量分别提高了45%及13%。以上研究结果表明,罗非鱼皮明胶酶解后制备的明胶水解物具有一定的抗氧化能力,具有潜在的开发价值。      

绿豆蛋白酶解物抗氧化活性与其结构、氨基酸组成的相关性

本试验以碱性蛋白酶、中性蛋白酶、胃蛋白酶及胰蛋白酶四种不同来源的蛋白酶酶解绿豆蛋白,测定酶解物的抗氧化能力,结合傅里叶红外光谱技术（FTIR）、聚丙烯酰胺凝胶电泳（SDS-PAGE）、圆二色谱（CD）以及氨基酸分析仪,分析酶解物的氨基酸组成以及抗氧化活性与结构变化的相关性。结果表明,绿豆蛋白4 h中性蛋白酶酶解物抗氧化能力最强,其DPPH自由基清除率、羟自由基清除率分别达到71.03%、51.94%,TBARS值达到0.4045 mg/L,Fe2+螯合率达到52.31%;结合SDS-PAGE、FTIR、CD等分析得出:绿豆蛋白酶解物的抗氧化能力与其分子量大小、二级结构构象及氨基酸组成变化紧密相关,与绿豆蛋白相比,绿豆蛋白酶解物的α-螺旋结构含量增加了4.12%、β-折叠结构含量降低了19.99%,而抗氧化活性与其α-螺旋含量的增加、β-折叠含量的降低密切相关;影响抗氧化活性的疏水氨基酸增加了0.208 g/100 g,芳香氨基酸增加了0.207 g/100 g,分子量低于10 kDa的小分子量酶解物具有更好的抗氧化性。综合以上研究结果证实绿豆蛋白酶解物活性与其α-螺旋、β-折叠的相关性较大。     

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。     

清酱肉体外模拟消化后粗肽的抗氧化活性和氨基酸分析

通过体外模拟胃、肠消化的方式分别对清酱肉和五花肉(对照组)进行酶解,比较其酶解物的抗氧化性,探究其氨基酸组成.结果表明,在相同浓度下,清酱肉酶解所得粗肽的抗氧化指标显著高于对照组五花肉粗肽的(P<0.05),当清酱肉酶解所得粗肽的质量浓度为5 mg/mL时,其DPPH·清除率最高达82.97％,Fe2+螯合率最高达30...     

Preparation and antioxidant activity of enzymatic hydrolysates from purple sea urchin (Strongylocentrotus nudus) gonad

Purple sea urchin (Strongylocentrotus nudus) gonad was treated separately with neutral protease, papain, pepsin and trypsin. The resultant hydrolysates were fractionated using a series of ultrafiltration membranes (molecular weight cut-offs of 10, 5, 3 and 1 kDa). Five fractions were prepared from each hydrolysate and the corresponding molecular weight ranges were below 10 kDa, 5-10 kDa, 3-5 kDa, 1-3 kDa and below 1 kDa. The peptide fractions were evaluated for antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and reducing power assay. Results indicated that all peptide fractions possessed DPPH radical scavenging capacity and reducing power in a dose-dependent manner. For all four hydrolysates, the below 1 kDa fractions exhibited the highest DPPH radical scavenging capacity. The below 1 kDa fractions prepared with neutral protease, papain and pepsin, and the 1-3 kDa fraction prepared with trypsin showed the highest reducing capacity among corresponding hydrolysates.     

Functionalities and antioxidant properties of protein hydrolysates from the muscle of ornate threadfin bream treated with pepsin from skipjack tuna

Functional properties and antioxidant activities of protein hydrolysates prepared from ornate threadfin bream (Nemipterus hexodon) muscle, using skipjack tuna pepsin, with different degrees of hydrolysis (DH: 10%, 20% and 30%), were evaluated. Emulsifying and foaming properties of hydrolysates were governed by their DH and concentrations used. Hydrolysates with 20% DH had the highest scavenging activities for ABTS and DPPH radicals. However, chelating activity of hydrolysates for ferrous ion increased as DH increased. Size exclusion chromatography of the hydrolysate with 20% DH using Sephadex G-25 revealed that antioxidative peptides with molecular weight of approximately 1.3 kDa exhibited the highest ABTS radical-scavenging activity. In vitro simulated gastrointestinal digestion indicated that ABTS radical-scavenging activity of the antioxidative peptides was not affected by pepsin hydrolysis, whilst further digestion by pancreatin enhanced the activity. Therefore, protein hydrolysate from the muscle of ornate threadfin bream produced by skipjack tuna pepsin can be used as a promising source of functional peptides with antioxidant properties.     

Antioxidant activities of enzymatic‐hydrolysed proteins of dromedary (Camelus dromedarius) colostrum

This work investigated the antioxidant activities of dromedary colostrum proteins before and after hydrolysis by pepsin, trypsin, α‐chymotrypsin, pancreatin and papain. The enzymatic hydrolysis affected the degrees of hydrolysis, electrophoretic profiles, molecular weight distribution and hydrophobic/hydrophilic properties of the generated peptides. The antioxidant activities were evaluated using four antioxidant assays, including 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) radical‐scavenging activities, ferric reducing power and ferrous ion chelating activity. Interestingly, the antioxidant activities of dromedary colostrum proteins were enhanced after enzymatic hydrolysis. The highest antioxidant potential was obtained by pancreatic hydrolysates (P ≤ 0.05). These results suggest that dromedary colostrum protein hydrolysates are an important source of natural antioxidant peptides.     

Antioxidant and metal chelating activities of peptide fractions from phaseolin and bean protein hydrolysates

Bean protein isolate and phaseolin were hydrolysed using pepsin and pancreatin, and the resulting hydrolysates were filtered through a 1kDa cut-off membrane and fractionated by size exclusion chromatography. Three fractions corresponding to MW 0.7-1.0kDa, 0.43-0.7kDa and <0.43kDa (A1, A2, and A3 for protein isolate fractions, and B1, B2, and B3 for phaseolin fractions) were assayed for antioxidant and metal chelating activity and they were also subjected to amino acid and SDS-PAGE analysis. Fractions A1 and B1 had the highest copper chelating activity (78% and 82%, respectively), while iron chelating activity was the highest in fractions A1 and B3 (36% and 16%, respectively). Fractions A2 and B3 had the highest antioxidant activity as determined by inhibition of reducing power and β-carotene bleaching, while the highest ABTS radical scavenging activity was found in A3 and B3. Thus, fractions coming from the isolate and phaseolin had similar activities except for iron chelation, suggesting that phaseolin is the major contributor to the antioxidant and copper chelating activities of the hydrolysed protein isolate.     

豆腐柴叶总黄酮不同溶剂级分抗氧化活性分析

采用1,1-二苯基-2-三硝基苯肼自由基、羟自由基、超氧阴离子、还原力、总抗等体外抗氧化活性模型和抗氧化活性综合评价指数,分析四川江油地区不同溶剂萃取的豆腐柴黄酮级分（乙醇、乙酸乙酯、正丁醇、氯仿、水）的抗氧化活性。结果显示,不同浓度乙醇提取总黄酮的1,1-二苯基-2-三硝基苯肼自由基清除活性最高。1,1-二苯基-2-三硝基苯肼自由基清除率、羟基自由基清除率、ABTS阳离子自由基清除率、总抗和还原力皆随乙醇浓度的升高而呈现先增强后减弱的趋势,超氧阴离子自由基清除率却随乙醇浓度增加一直降低。综合评价指数显示不同浓度乙醇抗氧化活性依次为75%醇提物>65%醇提物>55%醇提物>85%醇提物>95%醇提物。乙酸乙酯层萃取物的1,1-二苯基-2-三硝基苯肼自由基清除率、总抗、还原力及ABTS阳离子自由基清除率要优于其它各萃取层。正丁醇层萃取物的超氧阴离子自由基清除率要优于其它各萃取层。乙酸乙酯层抗氧化活性最佳。豆腐柴叶总黄酮有较好的抗氧化活性,可作为抗氧化剂或者健康食品原料开发。      

A study of the antioxidant capacity of oak wood used in wine ageing and the correlation with polyphenol composition

The antioxidant capacity of oak wood used in the ageing of wine was studied by four different methods: measurement of scavenging capacity against a given radical (ABTS, DPPH), oxygen radical absorbance capacity (ORAC) and the ferric reducing antioxidant power (FRAP). Although the four methods tested gave comparable results for the antioxidant capacity measured in oak wood extracts, the ORAC method gave results with some differences compared to the other methods. Non-toasted oak wood samples displayed more antioxidant power than toasted ones due to differences in the polyphenol composition. A correlation analysis revealed that ellagitannins were the compounds mainly responsible for the antioxidant capacity of oak wood. Some phenolic acids, mainly gallic acid, also showed a significant correlation with antioxidant capacity.