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。     

不同分子质量鲍鱼外套膜酶解物抗氧化活性研究

研究鲍鱼外套膜酶解物(AMH)及其不同分子质量组分的抗氧化活性。采用中性蛋白酶水解鲍鱼外套膜制得AMH,通过超滤得到分子质量分别为小于1kDa和1～3,3～5,5～10kDa的4种酶解物组分;考察AMH和不同分子质量组分的体外抗氧化活性。结果表明:AMH清除DPPH自由基、OH自由基和亚铁离子螯合能力的IC50值分别为11.76,12.07,4.04mg/mL;经过超滤分离后,其抗氧化活性明显增强,且1～3kDa分子质量范围组分的清除DPPH自由基和OH自由基能力最强,3～5kDa分子质量范围组分的亚铁离子螯合能力最强。     

复合蛋白酶水解玉米谷蛋白产物的抗氧化活性研究

以玉米谷蛋白为原料,利用复合蛋白酶对其进行限制水解,探讨不同水解时间所获得的谷蛋白酶解物的分子量分布和抗氧化活性。结果表明:不同水解时间获得的酶解物分子量分布差异较大。水解时间为120min的酶解物中,分子量分布为6 511.51~307.32Da的肽段占94.36%,此时获得的酶解物的抗氧化活性最高,其对DPPH自由基、O-2·、·OH的清除率分别为58.86%,82.64%,37.21%;还原力为0.236;与亚铁离子的螯合能力为29.92%。     

低苦味花生多肽的制备

以花生分离蛋白为原料,研究复合酶协同酶解法制备低苦味花生多肽的最佳条件,并分析其体外抗氧化活性。根据单因素实验的结果,筛选出低苦味花生多肽的最佳水解条件,并通过测定清除DPPH、ABTS⁺自由基的能力来评价其抗氧化活性。结果表明,复合蛋白酶协同酶解花生分离蛋白的最佳反应条件为:使用碱性蛋白酶(pH8.5,温度55℃,时间4 h)、蛋白酶P(pH7.0,温度50℃,时间3 h)和风味酶(pH7.0,温度50℃,时间2 h)依次分步酶解花生分离蛋白,三种酶添加量分别为0.8%、0.4%和0.2%。此工艺下,酶解液苦味值为1.3,多肽得率为86.75%,相对分子质量低于1 kDa的多肽含量达85.93%。酶解液质量浓度为4 mg/mL时,对DPPH自由基和ABTS⁺自由基清除率分别为80.70%和87.92%,表明花生粗多肽抗氧化活性较好。     

Antioxidative properties of protein hydrolysate from defatted peanut kernels treated with esperase

The aim of this study was to facilitate development of natural antioxidants from defatted peanut kernels. Protein hydrolysates obtained from defatted peanut kernels with esperase treatment for 2 h exhibited higher antioxidative activity toward linolenic acid peroxidation than other proteases (including neutrase, pepsin, protease A and protease N). The esperase hydrolysate of peanut protein (EHPP) was further separated with 3 and 5 kDa molecular cut-off membranes to determine the influence of molecular weight. EHPP with molecular weight 3∼5 kDa showed higher relative antioxidative activity, DPPH radical scavenging activity and metal chelating activity than that with molecular weight lower than 3 kDa or higher than 5 kDa. The fraction was further purified by ion exchange chromatography and high-performance liquid chromatography; the final peanut peptides exhibited higher relative antioxidative activity (27.5) than ascorbic acid (9.5). This study reported for the first time that protein hydrolysates from defatted peanut kernels possess antioxidative activity.     

Reducing,Radical Scavenging,and Chelation Properties of Fermented Soy Protein Meal Hydrolysate by Lactobacillus plantarum LP6

Fermented soybean protein meal hydrolysate (FSPMH) was fractionated by gel filtration on Sephadex G-15. The fractions obtained were subjected to various antioxidant assays, amino acid and molecular weight determinations. Among the seven fractions, the highest antioxidant activity was found in fraction F2, with significant differences (P < 0.01) 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, hydroxyl radicals (^.OH) scavenging and Cu²⁺ chelating activity. Fraction F2 exhibited scavenging of DPPH (59.43%), ^.OH (72.80%) and 44.47% Cu²⁺ chelating activity. All other fractions showed variable activities in different assays. Amino acid analyses of F2 fraction with the strongest antioxidant activity also had the highest percentage of related antioxidative amino acids content (Histidine 3.46, Serine 5.78, Valine 4.08 and Lysine 11.49 g/100 g protein) compared with other six fractions. The molecular weight distribution of F2 was found to vary from 170 to 1500 Da.     

响应面法优化秋刀鱼酶解制备抗氧化活性肽的工艺

采用酶解法从秋刀鱼肌肉中提取抗氧化活性肽,以水解度(DH)、TCA-可溶性肽含量、DPPH自由基清除率、Fe3+还原力、·OH清除率以及O2?·清除率为指标,从六种商业用酶中(中性蛋白酶、碱性蛋白酶、风味蛋白酶、木瓜蛋白酶、菠萝蛋白酶、胰蛋白酶)筛选出最适蛋白酶.以料液比、酶添加量、酶解时间、温度、pH五个因素进行单因...     

海蜇生殖腺酶解肽的抗氧化活性和ACE抑制活性研究

为实现海蜇加工副产物生殖腺的高值化利用,以海蜇性腺脱脂粉为原料,选用中性蛋白酶、木瓜蛋白酶、胰蛋白酶和碱性蛋白酶分别对海蜇生殖腺进行水解,以酶解产物（JGHP）的水解度、DPPH清除活性和ACE抑制活性为指标,选择制备海蜇生殖腺活性肽的工具酶。经超滤膜将酶解产物分成JGPH-P1（大于3000 u）、P2（1000³000 u）、P3（小于1000 u）三个部分,分别测定三部分的DPPH清除活性和ACE抑制活性。结果表明,中性蛋白酶水解所得产物的水解度、DPPH清除率和ACE抑制活性均优于其他三种蛋白酶;JGHP超滤后,其中组分JGHP-P3的活性较高。利用电子自旋共振（ESR）技术检测JGHP-P3对DPPH·、·OH、O-2·的清除活性。其ACE抑制活性IC50为1.06 mg/m L,DPPH·、·OH、O-2·的清除活性IC50分别为0.38、0.63、0.59 mg/m L。由此可见,经中性蛋白酶水解得到的JGPH中小于1000 u的组分具有较高的抗氧化和ACE抑制活性。      

Use of pyloric caeca extract from bigeye snapper (Priacanthus macracanthus) for the production of gelatin hydrolysate with antioxidative activity

Proteolytic activity of pyloric caeca extract (PCE) from bigeye snapper (Priacanthus macracanthus) was studied. The highest activity was observed at 55 °C and pH 8.0 when casein, N^α-Benzoyl-dl-arginine-p-nitroanilide (BAPNA) and N^α-p-Tosyl-l-arginine methyl ester hydrochloride (TAME) were used as the substrates. The activity was inhibited markedly by 1 mg/ml soybean trypsin inhibitor, whereas E-64, pepstatin A and EDTA exhibited a negligible effect on activity. The results suggested that a trypsin-like enzyme was most likely the major proteinase in PCE. As determined by activity staining, two proteolytic activity bands with apparent molecular weights of 55 and 24 kDa were found. Gelatin hydrolysate from bigeye snapper skin prepared using PCE exhibited the increases in 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and ferric reducing antioxidative power (FRAP) as degrees of hydrolysis (DHs) increased (P < 0.05). Hydrolysates derived from gelatin using Alcalase combined with PCE showed the highest ABTS radical scavenging activity (P < 0.05). Gelatin hydrolysate prepared using Alcalase in combination with Neutrase or PCE at 500 and 1000 ppm, respectively, retarded the oxidation in both linoleic acid oxidation and lecithin liposome oxidation systems. The antioxidative peptide of gelatin hydrolysate had a molecular weight of 1.7 kDa.     

Influence of degree of hydrolysis on functional properties, antioxidant and ACE inhibitory activities of egg white protein hydrolysate

Functional properties, antioxidant, and angiotensin-I converting enzyme (ACE) inhibitory activities of egg white protein hydrolysate (EWPH) prepared with trypsin at different degree of hydrolysis (DH) were investigated. The DPPH radical scavenging activity, reducing power, lipid peroxidation inhibitory activity, and ACE inhibitory activity increased with DH at first and then decreased gradually. Hydrolysates with 12.4% DH had the highest antioxidant and ACE inhibitory activities. As DH increased, the solubility of EWPH increased while the emulsifying and foaming properties decreased. The functional properties of EWPH were also controlled by pH. Ultrafiltration of the hydrolysate with 12.4% DH revealed that the fractions of molecular weight lower than 3 kDa exhibited the highest antioxidant and ACE inhibitory activities. The results indicated that EWPH with different DH have different bioactive and functional properties and EWPH by controlled hydrolysis may be useful ingredients in food and nutraceutical applications with potential bioactive properties.     

琥珀酸脱酰胺对小麦面筋蛋白酶解产物抗氧化性的影响

研究琥珀酸不同脱酰胺程度对小麦面筋蛋白胰酶的酶解产物抗氧化性的影响。在相同水解度下,以DPPH.清除率、.OH清除率、ORAC值为抗氧化指标,结果表明低脱酰胺程度(22.4%)的小麦面筋蛋白酶解产物的抗氧化性有较大幅度的提高,高脱酰胺程度(60.4%)的小麦面筋蛋白酶解产物的抗氧化性降低。水解度15%的酶解产物的肽分子量分布表明,低脱酰胺的小麦面筋蛋白酶解产物中分子量小于3000u的肽段增加,高脱酰胺的小麦面筋蛋白酶解产物中分子量小于3000u的肽段减少;在相同水解度下的氨基酸分析结果表明,脱酰胺后的小麦面筋蛋白酶解生成的肽的疏水性和抗氧化性的氨基酸含量提高。因此,低脱酰胺的小麦面筋蛋白酶解产物抗氧化性提高的主要原因是产物中含疏水性和抗氧化性氨基酸的小分子肽含量提高。      

谷朊粉酶解条件的优化及其抗氧化活性的研究

以溶解性为指标,采用单酶(Alcalase 2.4 L)和双酶(Alcalase 2.4 L+Flavourzyme 500 MG)2种方法水解谷朊粉,在单因素试验的基础上通过正交试验,优化了单酶和双酶水解的最佳条件,并分析了最优酶解条件下得到的2种产物的抗氧化活性、相对分子质量分布及其氨基酸组成。结果表明,单酶和双酶水解物的最大溶解性分别达到了61.38%和82.21%。水解物质量浓度为3.0 mg/m L时,单酶水解物的还原力、DPPH自由基、超氧阴离子、羟自由基的清除率分别达到0.81、71.82%、56.83%和70.82%,而双酶水解物相应的指标分别达到1.01、85.33%、74.84%和84.33%。结果表明,双酶水解不仅能提高谷朊粉的溶解性,而且也能明显提高其抗氧化活性。双酶水解物中小分子肽(分子质量<1 500 u)和疏水性氨基酸的质量分数分别达到61.63%和33.74%,均高于单酶水解物,表明谷朊粉酶解物的抗氧化活性与小分子肽含量、疏水性氨基酸呈现正相关。     

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.     

Physicochemical and bitterness properties of enzymatic pea protein hydrolysates

ABSTRACT:  The effects of different proteolytic treatments on the physiochemical and bitterness properties of pea protein hydrolysates were investigated. A commercial pea protein isolate was digested using each of 5 different proteases to produce protein hydrolysates with varying properties. After 4 h of enzyme digestion, samples were clarified by centrifugation followed by desalting of the supernatant with a 1000 Da membrane; the retentates were then freeze-dried. Alcalase and Flavourzyme™ produced protein hydrolysates with significantly higher ( P < 0.05) degree of hydrolysis when compared to the other proteases. Flavourzyme, papain, and alcalase produced hydrolysates that contained the highest levels of aromatic amino acids, while trypsin hydrolysate had the highest levels of lysine and arginine. Papain hydrolysate contained high molecular weight peptides (10 to 178 kDa) while hydrolysates from the other 4 proteases contained predominantly low molecular weight peptides (≤ 23 kDa). DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity of the Flavourzyme hydrolysate was significantly ( P < 0.05) the highest while alcalase and trypsin hydrolysates were the lowest. Inhibition of angiotensin converting enzyme (ACE) activity was significantly higher ( P < 0.05) for papain hydrolysate while Flavourzyme hydrolysate had the least inhibitory activity. Sensory analysis showed that the alcalase hydrolysate was the most bitter while papain and α-chymotrypsin hydrolysates were the least. Among the 5 enzymes used in this study, papain and α-chymotrypsin appear to be the most desirable for producing high quality pea protein hydrolysates because of the low bitterness scores combined with a high level of angiotensin converting enzyme inhibition and moderate free radical scavenging activity.     

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.     

鸭肉蛋白酶解产物的抗氧化特性

为了解酶解时间、蛋白酶种类对鸭肉蛋白酶解产物抗氧化特性的影响,分别用复合蛋白酶、风味蛋白酶和胰酶对鸭肉进行单酶酶解和双酶分步酶解（胰酶＋复合蛋白酶、胰酶＋风味蛋白酶）,制备不同时间段的酶解产物,并对其自由基清除能力（1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基、羟自由基（hydroxyl radical,·OH）和超氧阴离子自由基（superoxide radical,O2－·））和总还原力进行分析。结果表明：各鸭肉蛋白酶解产物的DPPH自由基清除率随着酶解时间的延长而增加,但·OH和O2－·清除率及总还原力随着酶解时间的延长先增加后降低（P＜0.05）。在5 种鸭肉蛋白酶解产物中,复合蛋白酶酶解物表现出最强的DPPH自由基清除能力（75.70±1.54）%、·OH清除能力（59.41±1.24）%和O2－·清除能力（98.50±4.51）%,但用双酶分步酶解得到的酶解产物表现出最强的总还原力（0.330±0.017）。因此鸭肉蛋白酶解产物的抗氧化特性受酶解时间和蛋白酶种类的影响,复合蛋白酶是制备鸭肉蛋白源抗氧化肽的最适蛋白酶。     

岩藻低聚糖的酶法制备及活性分析

利用岩藻聚糖硫酸酯酶对岩藻聚糖硫酸酯进行降解,获得岩藻低聚糖。利用均匀设计和正交设计方法,设计122组试验,通过Matlab软件建立3层BP网络模型模拟酶解过程,运用遗传算法对上述网络进行寻优。并利用D-半乳糖致衰老小鼠模型,对岩藻低聚糖和岩藻聚糖硫酸酯的抗氧化活性进行对比研究。结果表明,在加酶量0～24U/mg、酶解温度20～40℃、酶解时间0～4h的酶解条件下,产物分子质量会发生显著性变化,选取上述范围作为寻优范围。遗传算法寻优结果表明,当加酶量20.7U/mg、酶解温度26℃、酶解时间2.3h时,能够获得最大抗氧化活性的岩藻低聚糖,该产物的1,1-二苯基-2-三硝基苯肼自由基清除率达到85.1%,分子质量为18.2kDa。体内动物实验结果表明,岩藻低聚糖较岩藻聚糖硫酸酯在羟自由基清除率、丙二醛活力及减少过氧化氢酶含量等抗氧化指标方面都有显著提高（P＜0.05）。本实验为工业化制备高抗氧化活性岩藻聚糖硫酸酯水解产物提供了一定实验支撑。     

Production and characterisation of duck albumen hydrolysate using enzymatic process

Influences of different ultrasound treatments combined with heat pretreatment on enzymatic hydrolysis, emulsifying properties and antioxidant activities of hydrolysates from duck egg albumen were studied. Heat pretreatment at 95 °C for 30 min inhibited both serine and cysteine protease inhibitors effectively. Ultrasonication of heated duck albumen at 60% amplitude for 10 min yielded the highest surface hydrophobicity. Coincidentally, aforementioned pretreatment rendered the hydrolysate with highest degree of hydrolysis (DH) than other pretreatments when Alcalase was used. The resulting hydrolysate showed the highest antioxidant activities including DPPH radical and ABTS radical cation scavenging activities and ferric reducing antioxidant power as well as emulsifying properties when hydrolysis time of 90 min was used. The hydrolysate possessed the peptides with molecular weight of 219–255 Da with the highest ABTS radical scavenging activity. Thus, heat pretreatment, followed by ultrasonication of duck albumen under appropriate condition could increase DH, antioxidant activities and emulsifying properties of duck albumen hydrolysate.     

Effect of defatting and enzyme type on antioxidative activity of shrimp processing byproducts hydrolysate

Shrimp processing byproducts (SPB) was digested by 6 proteases (trypsin, pepsin, neutrase, Protamex, Flavourzyme, and Alcalase) to produce antioxidative peptides. Both degree of hydrolysis (DH) and DPPH radical scavenging activity (DSA) of the Alcalase hydrolysate were the highest of all. The effect of defatting on DH and DSA of the Alcalase hydrolysate was significant. The DH decreased while the DSA increased after defatting of the byproducts. The antioxidative activity of Alcalase hydrolysate was also investigated using several in vitro assays, including DPPH, ABTS radical scavenging assays (ASA), reducing power assay, and chelating activity. The antioxidative activity of the hydrolysate was obviously concentration dependent. The SPB Alcalase hydrolysate exhibited notable DSA and ASA with the IC50 values of 500 and 7.4 μg/mL, respectively. And the hydrolysate showed 38.9% chelating activity at 120 μg/mL level. The SPB Alcalase hydrolysate was a potential source of natural antioxidants.     

酶解鸡血球制备抗氧化肽的工艺优化和分析鉴定

为挖掘家禽血液潜在的抗氧化特性,以鸡血球为原料,筛选最佳蛋白酶酶解制备抗氧化肽,并对酶解工艺进行响应面优化。采用超滤、阳离子交换色谱、凝胶色谱及高效液相色谱对酶解物进行连续分离纯化,并用质谱进行结构鉴定。结果表明,酸性蛋白酶水解血球蛋白制备的酶解物具有最高的1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基清除能力（＞80%）。其最佳酶解条件为酶用量2%、酶解温度45?℃、酶解时间3.0?h,该条件下的DPPH自由基清除能力、超氧阴离子自由基清除能力和还原力分别为（98.31±0.66）%、（28.89±0.31）%和1.94±0.03。经系列分离纯化获得抗氧化活性最强的组分,其在质量浓度为0.1?mg/mL时,DPPH自由基清除能力和还原力分别为（87.16±1.59）%和0.21±0.01。经高效液相色谱-质谱联用技术鉴定,目标肽的氨基酸序列为MGQKDSYVGDEAQSKRGILT,分子质量为2?182.1?Da。