Compositions and antioxidant properties of protein hydrolysates from the skins of four carp species

Compositions and antioxidant properties of protein hydrolysates prepared from four carp skins: black carp, grass carp, silver carp and bighead carp, using pepsin, with a degree of hydrolysis (DH) of 6–15%, were investigated. The yield of freeze‐dried hydrolysates was in the range of 54–62 g/100 g (dry skin). The content of protein and ash in four freeze‐dried hydrolysates was 72–81% and 8–17%, respectively. All hydrolysates contained high amount of hydrophobic amino acid residues (389–480 residues/1000 residues). Meanwhile, their antioxidant properties were evaluated by in vitro assays. The results revealed that all hydrolysates possessed potent antioxidant activities and showed dose dependency as the activity increased with sample concentration, capable of scavenging 72–88% of DPPH and 61–69% of hydroxyl radicals, respectively, at the highest tested concentration. The hydrolysates exhibited high reducing power and β‐carotene–linoleic acid oxidation inhibition. Among the four hydrolysates, the hydrolysate derived from bighead carp skin was superior to others in terms of yield, DH and antioxidant activities.     

鲢鱼肉蛋白酶解产物功能特性的研究

以鲢鱼鱼肉为原料,利用响应面分析法对鲢鱼鱼肉酶解产物的酶解工艺进行优化,系统考察了木瓜蛋白酶、风味蛋白酶添加量和酶解时间对鱼肉酶解产物得率的影响,并对酶解产物的溶解性、热稳定性和感官评价进行了分析。结果表明,鱼肉酶解产物的较理想酶解工艺条件为：风味蛋白酶添加量0．6％、木瓜蛋白酶添加量0．6％和酶解时间55min,此时制备出的鲢鱼鱼肉酶解产物具有良好的溶解性、热稳定性和感官值。     

Effects of Hydrolysates from Silver Carp (Hypophthalmichthys molitrix) Scales on Rancidity Stability and Gel Properties of Fish Products

This study aimed to evaluate the effectiveness of hydrolysates, which were obtained from the scales of silver carp (Hypophthalmichthys molitrix) by papain, flavourzyme, and Alcalase 2.4 L, as natural antioxidants in silver carp mince and surimi gels during storage at 4 °C. The hydrolysates that possess greater in vitro antioxidant activities (DPPH radical-scavenging activity, Fe²⁺-chelating activity, and reducing power), including hydrolysates catalyzed by papain at 10 min (HP), flavourzyme at 5 min (HF), and Alcalase 2.4 L at 5 min (HA), were chosen as additives. Color, cooking loss, conjugated dienes (CDs), thiobarbituric acid reactive substances (TBARS), fatty acids, and sensory scores of mince were measured on days 0, 2, 4, 6, and 8 during 4 °C storage; additionally, whiteness, breaking force, deformation, gel strength, and sensory score of surimi gels were measured on days 1, 3, 5, 7, 9, and 11 during 4 °C storage. The results indicate that HA was conducive to lowering the cooking loss of mince and that HF significantly (P?<?0.05) reduced the CDs value of mince. For surimi gels, HF improved whiteness, deformation, and gel strength. Hence, HF could serve as a natural antioxidant during early oxidation and improve gel formation of silver carp products.     

Purification and identification of antioxidant peptides from grass carp muscle hydrolysates by consecutive chromatography and electrospray ionization-mass spectrometry

Grass carp muscles were hydrolyzed with various proteases (papain, bovine pancreatin 6.0, bromelain, neutrase 1.5MG and alcalase 2.4L) to extract antioxidant peptides. The hydrolysates were assessed using methods of hydroxyl radical scavenging ability and lipid peroxidation inhibition activity. Hydrolysate prepared with alcalase 2.4L was found to have the highest antioxidant activity. It was purified using ultrafiltration and consecutive chromatographic methods including ion-exchange chromatography, multilayer coil high-speed counter-current chromatography, and gel filtration chromatography. The purified peptide, as a potent antioxidant, was identified as Pro-Ser-Lys-Tyr-Glu-Pro-Phe-Val (966.3 Da) using RP-HPLC connected on-line to an electrospray ionization mass spectrometry. As well, it was found that basic peptides had greater capacity to scavenge hydroxyl radical than acidic or neutral peptides and that hydrophobic peptides contributed more to the antioxidant activities of hydrolysates than the hydrophilic peptides. In addition, the amino acid sequence of the peptide might play an important role on its antioxidant activity.     

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.     

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.     

仿刺参精低分子质量多肽的制备及抗氧化作用

本实验以仿刺参精为原料,制备出多肽并考察其体外和体内抗氧化活性。以清除羟自由基（•OH）能力为指标,考察木瓜蛋白酶、复合蛋白酶和风味蛋白酶的5 种酶解方式产生的酶解产物的活性,优化实验用酶。经超滤分级后,测定各级组分的分子质量分布、可溶性蛋白质含量、多肽含量和清除•OH能力。选择清除•OH能力较好的低分子质量多肽组分,测定氨基酸组成,设定低、中、高剂量组（100、200、600 mg/kg（以体质量计,下同））、模型对照组、和空白对照组进行42 d小鼠灌胃实验。实验完成后,检测小鼠血液中超氧化物歧化酶（superoxide dismutase,SOD）活力、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）活力、谷胱甘肽（glutathione,GSH）含量、丙二醛（malondialdehyde,MDA）含量和蛋白质羰基含量。结果表明,由木瓜蛋白酶水解,经超滤制备的＜1 000 u仿刺参精多肽具有较好的体外清除•OH能力,其半数抑制浓度为6.02 mg/mL,含量占比为58%,氨基酸组成中谷氨酸等酸性氨基酸和酪氨酸等疏水性氨基酸含量丰富。小鼠抗氧化模型实验表明,实验组生长正常,高剂量多肽组小鼠体内SOD活力显著提高（P＜0.05）,GSH的含量提高极显著（P＜0.01）、GSH-Px活力也呈剂量依赖性升高,但未达到差异显著（P＞0.05）。MDA和蛋白质羰基含量得到有效降低,后者达到差异显著（P＜0.05）。研究结果显示：＜1 000 u仿刺参精多肽具有提高小鼠抗氧化能力的效果,可以作为相关功能产品开发的原材料。     

Grass carp peptides hydrolysed by the combination of Alcalase and Neutrase: Angiotensin‐I converting enzyme (ACE) inhibitory activity,antioxidant activities and physicochemical profiles

In this study, grass carp peptides were prepared by enzymatic hydrolysis of grass carp protein using the combination of Alcalase and Neutrase, and angiotensin‐I converting enzyme (ACE) inhibitory activity in vitro, antihypertensive activity in vivo, antioxidant activities, and physicochemical properties of peptides achieved from grass carp protein were characterised after ultrafiltration and desalted processes using mixed ion exchange resins. The purified peptides exhibited strong ACE inhibitory activity (IC₅₀ = 105 μg mL^?1), antihypertensive activity with the maximal drop for systolic blood pressure (SBP) of 43 mmHg at a dosage of 100 mg per kg body weight in spontaneously hypertensive rat (SHR), and antioxidant activities indicated by thiobarbituric acid‐reactive substance values in a liposome‐oxidising system, radical‐scavenging activity and chelation of metal ions (Fe²⁺). The molecular weight of peptides was <1000 Da. Compared to grass carp protein, the peptides separated from enzymatic hydrolysates possessed similar amino acid compositions, but contained higher concentrations of essential amino acids. Moreover, the peptides exhibited excellent solubility at a wide range of pH values from 2 to 10, and lower apparent viscosity than the protein. The peptides separated from enzymatic hydrolysates might be used as a promising ingredient in antihypertensive functional foods and nutraceuticals.     

Isolation and characterisation of antibacterial peptides derived from the f(164–207) region of bovine αS2-casein

A chymosin digest of sodium caseinate, which showed antibacterial activity against Listeria innocua, was fractionated using reverse phase high performance liquid chromatography and the purified antibacterial peptides were characterised by mass spectrometry, N-terminal amino acid sequencing and comparison to peptide masses of theoretical enzymic digests of milk proteins. Five antibacterial peptides, Cr1, Cr3, Cr4, Cr5 and Cr7 corresponding to amino acid residues 181–207, 180–207, 175–207, 164–207 and 172–207 of bovine α_S2-casein, respectively, were isolated. The minimal inhibitory concentration of peptides Cr1, Cr4 and Cr5 was determined against a range of Gram-positive and Gram-negative bacteria and showed similar activities to those of the bacteriocin peptide, nisin and the antibacterial peptide, lactoferricin B against certain Gram-positive bacteria. A partially purified chymosin digest of sodium caseinate (Cr_MIX) was prepared and observed to be heat stable for up to 15 min on exposure to 121 °C. Although Cr_MIX showed bactericidal activity against Salmonella Typhimurium in 0.1% (w/v) peptone medium, no antibacterial activity was observed when tested in skim milk at the same concentration.     

白鲢鱼排酶解蛋白的制备及营养成分分析

以白鲢鱼排为实验材料,选用风味蛋白酶酶解制备蛋白水解液。考察起始pH值、酶解温度、料液比、加酶量、酶解时间对水解度的影响,采用Box—Behnken中心组合设计和响应面分析法,确定最佳工艺条件。结果表明：最佳工艺条件为起始pH7．59、酶解温度44．9℃、料液比30：100（g／mL）、加酶量4．02％、酶解时间5h,...     

超滤精制对芝麻蛋白水解液ACE抑制活性和抗氧化活性的影响

以芝麻饼粕为原料,采用超滤技术制备功能多肽,研究超滤对蛋白酶水解肽的氨基酸组成、肽谱、降血压和抗氧化功能特性等影响。结果表明:经截留分子质量为10 ku和3 ku的超滤膜过滤,所得到的3个组分(相对分子质量分布10 ku、3~10 ku和3 ku)的必需氨基酸质量分数分别为15.6%、14.9%、15.0%,显著高于未经超滤(12.9%),分子质量分布在3~10 ku组分的总氨基酸质量分数达53.1%,比未经超时提高了7.6%;其中分子质量分布为3~10 ku和3 ku酶解液的IC50值分别降低了6.6%和7.9%,血管紧张素转化酶(ACE)抑制活性显著提高;分子质量分布为3~10 ku和3 ku酶解液的DPPH·自由基清除率IC50值与未经超滤时均降低了8.6%,羟自由基清除率分别降低4.20%和26.57%,抗氧化活性显著提高。     

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.     

几种蛋白酶对文蛤肉的水解效果

在一定的酶解条件下,用Alcalase 2.4L、胰蛋白酶、复合风味酶、酸性蛋白酶、中性蛋白酶等几种蛋白酶对文蛤内进行水解,测定了这几种蛋白酶的水解度,以及其水解物的氨基态氮含量、可溶性蛋白含量和透明度,并用凝胶过滤色谱(Sephedex G-25)对水解物的分子质量分布进行了粗略分析。结果表明,Alcalase水解物分子质量的分布范围较广,适合制备具有一定肽链要求的短肽,酸性蛋白酶水解物分子质量较小,适合制备文蛤调味品,中性蛋白酶水解物分子质量主要集中在1 000 u左右与10 000u左右,胰蛋白酶和复合风味酶的水解物分子质量大部分在1 500 u以上。     

酸法和酶法水解海蚬蛋白的呈味作用研究

分别利用酸法和酶法水解海蚬蛋白制备出富肽和富氨基酸调味液，并通过成分分析和感官品评探讨肽的呈味作用。研究结果表明，海蚬酸法水解液的水解度为79．64％；酶法水解液的水解度为52．11％。酶法水解液的鲜甜味明显高于酸水解液，亲水性肽在海蚬酶法水解液的呈味中起到重要的作用。     

草鱼蛋白源抗氧化肽的分离及鉴定

利用超滤、离子交换色谱、高效逆流色谱以及凝胶过滤色谱等系列分离纯化技术从草鱼蛋白酶解产物中分离纯化抗氧化活性肽,分离过程中发现相对分子质量1～3kD 的组分抗氧化活性最强,且碱性肽组分的抗氧化活性强于酸性或中性肽组分、疏水性肽组分的抗氧化活性强于亲水性肽组分。最终借助反相高效液相色谱在线连接的电喷雾质谱结合氨基酸分析鉴定出一种抗氧化肽,一级结构序列为Pro-Ser-Lys-Tyr-Glu-Pro-Phe-Val,相对分子质量为966.3D。     

Isolation of antioxidative and ACE inhibitory peptides from protein hydrolysate of skipjack (Katsuwana pelamis) roe

Bioactive peptides from protein hydrolysate of defatted skipjack (Katsuwonus pelamis) roe with 5% degree of hydrolysis (DH) prepared by Alcalase digestion were isolated and characterised. Two active fractions with ABTS radical scavenging activity (973.01–1497.53 μmol TE/mg sample) and chelating activity (0.05–0.07 μmol EE/mg sample) from consecutive purification steps including ultrafiltration, cation exchange column chromatography and reverse phase high performance liquid chromatography (RP-HPLC), were subjected to analysis of amino acid sequence by LC–MS/MS. Seven dominant peptides with 6–11 amino acid residues were identified as DWMKGQ, MLVFAV, MCYPAST, FVSACSVAG, LADGVAAPA, YVNDAATLLPR and DLDLRKDLYAN. These peptides were synthesised and analysed for ACE-inhibitory activity and antioxidative activities. MLVFAV exhibited the highest ACE inhibitory activity (IC₅₀ = 3.07 μM) (p < 0.05) with no antioxidative property, whilst DLDLRKDLYAN showed the highest metal chelating activity, ABTS radical and singlet oxygen scavenging activities. Therefore, peptides prepared from skipjack roe could be further employed as a functional food ingredient.     

Physicochemical and sensory characteristics of soya protein isolate hydrolysates with added substrate‐like amino acids

Soya protein isolate (SPI) with or without added substrate‐like amino acid was subject to enzymatic hydrolysis catalysed by commercial proteases (Alcalase 2.4 L, flavourzyme and pancreatin). Addition of a small amount of amino acids (amino acid: SPI = 1: 2500, mol g^?1) during hydrolysis would cause a significantly (P < 0.05) reduced protein recovery, increased degree of hydrolysis, and altered amino acid composition and antioxidant activities of SPI hydrolysates. The SPI hydrolysates prepared with added Asp, Arg or Lys exhibited a higher antioxidant activity than the control. The bitterness of SPI hydrolysates was largely reduced upon addition of Met, Asp or Glu during hydrolysis, whilst the umami taste and mouthfeel‐liking were remarkably increased. Therefore, adding amino acid during hydrolysis is a feasible and beneficial approach to improve both the functional and sensory properties of SPI hydrolysate.     

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。     

分步酶解小麦面筋蛋白制备低苦味肽粉的研究

目的 研究分步酶解小麦面筋蛋白(wheat gluten, WG)制备低苦味肽粉的工艺。方法 选用中性蛋白酶、木瓜蛋白酶、胃蛋白酶水解WG至8%水解度,接着用风味蛋白酶对水解产物进行脱苦处理,对不同酶解产物中苦味肽的特性进行系统研究,探究苦味肽含量、氨基酸组成、分子量分布、表面疏水性等指标变化对WG酶解物苦味值的影响,对比风味蛋白酶对不同单酶酶解物的脱苦效果差异,分析风味蛋白酶对WG酶解物脱苦的内在机理,进而确定制备低苦味小麦蛋白肽粉的最佳酶解工艺。结果 中性蛋白酶的酶解产物经风味蛋白酶作用后,脱苦效果最显著,苦味肽苦味值从4.08降至2.25,酶解产物的苦味值可下降56.42%。木瓜蛋白酶的酶解产物经风味蛋白酶作用4 h后,酶解产物的苦味值最低,制备出苦味值为1.28的WG低苦味肽粉。结论 经分步酶解作用后,酶解产物中苦味肽的含量下降;疏水性氨基酸比例的下降和游离氨基酸含量的升高引起苦味肽苦味阈值的增大,共同导致酶解产物苦味值显著降低,该研究为酶解脱苦技术的快速发展和WG活性肽工业化生产提供新的参考。     

Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus)

Mackerel (Scomber austriasicus) hydrolysates were prepared by an autolytic process and accelerated hydrolysis with a commercial enzyme, Protease N. Changes in the levels and compositions of free amino acids and small peptides during hydrolysis were investigated to find out their relationships with antioxidant activities. Increased levels of free amino acids, anserine, carnosine and other peptides of the hydrolysates obtained with protease were much higher than those by autolysis. Different antioxidant measurements including the inhibition of linoleic acid autoxidation, the scavenging effect on ,-diphenyl-β-picrylhydrazyl free radical, and the reducing power showed that mackerel hydrolysates possessed noticeable antioxidant activities. A good correlation existed between the amount of peptides and antioxidant activity. Three peptide fractions were separated from the hydrolysate by size exclusion chromatography. Results revealed that the peptide with molecular weight of approximately 1400 Da possessed a stronger in vitro antioxidant activity than that of the 900 and 200 Da peptides.