Characterization of Protein Hydrolysates from Fish Discards and By-Products from the North-West Spain Fishing Fleet as Potential Sources of Bioactive Peptides

Fish discards and by-products can be transformed into high value-added products such as fish protein hydrolysates (FPH) containing bioactive peptides. Protein hydrolysates were prepared from different parts (whole fish, skin and head) of several discarded species of the North-West Spain fishing fleet using Alcalase. All hydrolysates had moisture and ash contents lower than 10% and 15%, respectively. The fat content of FPH varied between 1.5% and 9.4% and had high protein content (69.8–76.6%). The amino acids profiles of FPH are quite similar and the most abundant amino acids were glutamic and aspartic acids. All FPH exhibited antioxidant activity and those obtained from Atlantic horse mackerel heads presented the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power and Cu²⁺ chelating activity. On the other hand, hydrolysates from gurnard heads showed the highest ABTS radical scavenging activity and Fe²⁺ chelating activity. In what concerns the α-amylase inhibitory activity, the IC₅₀ values recorded for FPH ranged between 5.70 and 84.37 mg/mL for blue whiting heads and whole Atlantic horse mackerel, respectively. α-Glucosidase inhibitory activity of FPH was relatively low but all FPH had high Angiotensin Converting Enzyme (ACE) inhibitory activity. Considering the biological activities, these FPH are potential natural additives for functional foods or nutraceuticals.     

花生蛋白碱性蛋白酶水解物对血管紧张素转化酶抑制作用的初步研究

分别以碱性蛋白酶Alcalase和中性蛋白酶Neutrase对花生分离蛋白进行水解．制备花生分离蛋白水解物．并测定不同水解时间所得产物对血管紧张素转化酶(ACE)的抑制活性。未水解的花生分离蛋白没有ACE抑制活性．用中性蛋白酶Neutrase水解所得的水解物显示弱ACE抑制活性。然而，碱性蛋白酶Alcalasc水解物具有很强的ACE抑制活性．水解0．5h时水解物活性最高，其半抑制浓度为(IC50)0．56mg／mL。本研究表明，当用碱性蛋白酶Alcalase水解时，花生分离蛋白是生产ACE抑制肽的良好蛋白质来源，花生分离蛋白碱性蛋白酶Alcalase水解物可作为具有降压功能的功能食品添料。     

Effects of enzymatic hydrolysis on molecular structure and antioxidant activity of barley hordein

Hordein, the major storage protein of barley (Hordeum vulgare L.), was hydrolysed by three selected proteases, including alcalase, flavourzyme and pepsin. The effects of protease type and hydrolysis time on hordein molecular weight, surface hydrophobicity, secondary structure and antioxidant activity were investigated. Flavourzyme hydrolysis of hordein was relatively more extensive and rapid, resulting in the formation of medium- and small-sized peptides with a broad distribution within 30 min. Alcalase and pepsin more gradually and less extensively hydrolysed hordein into medium- and larger-sized peptides, respectively. Protein surface hydrophobicity decreased with an increasing degree of hydrolysis. The flavourzyme and alcalase hydrolysates had superior DPPH (1,1-diphenyl-2-picryl hydrazyl) free radical scavenging activity (44-70 and 48-58%, respectively, at 0.5 mg/mL), Fe²⁺-chelating ability (21-64% and 39-73%, respectively, at 1 mg/mL), and superoxide radical scavenging capacity. It is proposed that the large- and medium-size hydrolysate fractions were most likely responsible for the antioxidant activities of hordein hydrolysates, and could be used as antioxidant peptides in food and nutraceutical applications.     

不同干燥方式对橄榄多酚抗氧化活性的影响

研究不同干燥方式对橄榄多酚抗氧化活性的影响。采用乙醇浸提法来提取橄榄多酚,AB-8 大孔树脂纯化后干燥,考察热风干燥温度、真空冷冻干燥及远红外干燥方式对橄榄多酚清除 1,1-二苯基-2-三硝基苯肼自由基（DPPH?）、羟自由基（?OH）和 2,2′-二氮-双铵盐自由基（ABTS+?）能力的影响。结果表明：热风干燥处理对橄榄多酚清除 3 种自由基的能力差异显著,以 80 ℃干燥效果最好;3 种处理方式对橄榄多酚清除 DPPH?的影响无显著差异,对?OH 和 ABTS+?清除能力影响差异显著。因此,在选择干燥方式时需要考虑橄榄多酚的用途。     

椰子种皮油提取物的抗氧化活性研究

测定椰子种皮油提取物中的总酚含量,对羟基自由基、DPPH（1,1-二苯基-2-三硝基苯肼）自由基、ABTS（2,2连氮-双-（3-乙基苯并噻咪唑-6-磺酸）自由基清除能力和总抗氧化能力等抗氧化活性指标。结果表明,椰子种皮油提取物中含有较高的总酚含量（68 mg/g）,提取物浓度为0.1 mg/mL时对羟基自由基的清除率为56.89％,对DPPH自由基的清除率为71.0％,对ABTS自由基的清除率为96.4％,表明椰子种皮油提取物具有很好的抗氧化活性。     

刺五加叶不同部位抗氧化活性的研究

目的研究刺五加叶不同部位在不同浓度下的体外抗氧化活性。方法采用清除ABTS+自由基、DPPH自由基评价法和测定还原性法,利用紫外分光光度计法测定刺五加叶的总提液、10%、30%和50%大孔吸附树脂醇洗脱部位在不同浓度下的抗氧化能力。结果刺五加叶不同部位抗氧化活性的强弱顺序为:30%部位>10%部位>总提液>50%部位,随着各部位浓度的增加,抗氧化能力增强。ABTS+自由基和DPPH自由基模型中,在浓度为0.64mg/mL时,30%的部位对ABTS+DPPH自由基模最大清除率分别达到99.541%、95.331%,与同浓度VC的清除率接近,其次浓度0.64mg/mL的10%部位的最大清除率分别达到96.324%、88.033%。在还原性法测定中30%部位在浓度为0.64mg/mL时,吸光度为2.269,与VC较接近,同浓度时10%部位的吸光度为2.155,次于30%部位。结论刺五加叶抗氧化能力较好,各种评定方法结合分析,其30%部位最好,可以开发为抗氧化剂,为进一步研究和开发刺五加叶提供了科学依据。     

Antioxidant Activities of Hydrolysates of Arca Subcrenata Prepared with Three Proteases

In order to get products with antioxidant activity from Arca subcrenata Lischke, the optimal hydrolase and hydrolysis conditions were investigated in the paper. Three proteases (neutrase, alcalase and papain) were applied to hydrolyze the homogenate of A. subcrenata. An orthogonal design was used to optimize hydrolysis conditions, and the pH-stat methods was used to determine the degree of hydrolysis. Viewed from the angle of reducing power, such as scavenging activities against α,α-diphenyl-β-picrylhydrazyl (DPPH) radical and hydrogen peroxide, the antioxidant activities of the alcalase hydrolysate (AH) were superior to neutrase hydrolysate (NH) and papain hydrolysate (PH), and its EC₅₀ values in DPPH radical and hydrogen peroxide scavenging effect were 6.23 mg/ml and 19.09 mg/ml, respectively. Moreover, compared with products hydrolyzed by neutrase and papain, the molecular mass of AH was lower and its content of amino acid of peptides was higher. Therefore, alcalase was selected as the optimal enzyme to produce active ingredients since its hydrolysate exhibited the best antioxidant activity among them and possessed large amount of potential active peptides.     

复合酶法制备澳洲坚果蛋白肽及其抗氧化活性研究

本研究以前期制备的澳洲坚果蛋白为原料,经复合酶（木瓜蛋白酶和中性蛋白酶）酶解制备澳洲坚果蛋白肽,采用水解度为指标,利用单因素试验与正交试验考察各酶解因素对澳洲坚果蛋白水解度的影响,同时通过不同分子量（3、10、30 kDa）的超滤离心管对制备的蛋白肽进行初步的分离,并基于DPPH自由基清除能力对不同分子量的澳洲坚果蛋白肽的抗氧化活性进行评价。结果表明：各酶解因素对复合酶酶解制备澳洲坚果蛋白水解度的影响依次为酶解初始pH>复合酶配比>酶添加量>酶解时间;最佳复合酶酶解条件为复合酶配比1∶5、酶添加量12 000 U/g、酶解液初始pH 9.0、酶解时间360 min,在此条件下澳洲坚果蛋白的水解度为21.88%;同时,通过分析不同分子量的澳洲坚果蛋白肽组分发现,不同分子量的澳洲坚果蛋白肽均具有抗氧化活性,分子量在3~10 kDa的肽段组分具有较强的抗氧化能力,其DPPH自由基清除能力达到80.97%,且随着蛋白肽组分浓度的增加,其抗氧化能力也逐渐增强。     

Influence of Amino Acid Compositions and Peptide Profiles on Antioxidant Capacities of Two Protein Hydrolysates from Skipjack Tuna (Katsuwonus pelamis) Dark Muscle

Influence of amino acid compositions and peptide profiles on antioxidant capacities of two protein hydrolysates from skipjack tuna (Katsuwonus pelamis) dark muscle was investigated. Dark muscles from skipjack tuna were hydrolyzed using five separate proteases, including pepsin, trypsin, Neutrase, papain and Alcalase. Two hydrolysates, ATH and NTH, prepared using Alcalase and Neutrase, respectively, showed the strongest antioxidant capacities and were further fractionated using ultrafiltration and gel filtration chromatography. Two fractions, Fr.A3 and Fr.B2, isolated from ATH and NTH, respectively, showed strong radical scavenging activities toward 2,2-diphenyl-1-picrylhydrazyl radicals (EC₅₀ 1.08% ± 0.08% and 0.98% ± 0.07%), hydroxyl radicals (EC₅₀ 0.22% ± 0.03% and 0.48% ± 0.05%), and superoxide anion radicals (EC₅₀ 1.31% ± 0.11% and 1.56% ± 1.03%) and effectively inhibited lipid peroxidation. Eighteen peptides from Fr.A3 and 13 peptides from Fr.B2 were isolated by reversed-phase high performance liquid chromatography, and their amino acid sequences were determined. The elevated antioxidant activity of Fr.A3 might be due to its high content of hydrophobic and aromatic amino acid residues (181.1 and 469.9 residues/1000 residues, respectively), small molecular sizes (3–6 peptides), low molecular weights (524.78 kDa), and amino acid sequences (antioxidant score 6.11). This study confirmed that a smaller molecular size, the presence of hydrophobic and aromatic amino acid residues, and the amino acid sequences were the key factors that determined the antioxidant activities of the proteins, hydrolysates and peptides. The results also demonstrated that the derived hydrolysates and fractions from skipjack tuna (K. pelamis) dark muscles could prevent oxidative reactions and might be useful for food preservation and medicinal purposes.     

Potential of Thermolysin-like Protease A69 in Preparation of Bovine Collagen Peptides with Moisture-Retention Ability and Antioxidative Activity

Bovine bone is rich in collagen and is a good material for collagen peptide preparation. Although thermolysin-like proteases (TLPs) have been applied in different fields, the potential of TLPs in preparing bioactive collagen peptides has rarely been evaluated. Here, we characterized a thermophilic TLP, A69, from a hydrothermal bacterium Anoxybacillus caldiproteolyticus 1A02591, and evaluated its potential in preparing bioactive collagen peptides. A69 showed the highest activity at 60 °C and pH 7.0. We optimized the conditions for bovine bone collagen hydrolysis and set up a process with high hydrolysis efficiency (99.4%) to prepare bovine bone collagen peptides, in which bovine bone collagen was hydrolyzed at 60 °C for 2 h with an enzyme–substrate ratio of 25 U/g. The hydrolysate contained 96.5% peptides that have a broad molecular weight distribution below 10000 Da. The hydrolysate showed good moisture-retention ability and a high hydroxyl radical (•OH) scavenging ratio of 73.2%, suggesting that the prepared collagen peptides have good antioxidative activity. Altogether, these results indicate that the thermophilic TLP A69 has promising potential in the preparation of bioactive collagen peptides, which may have potentials in cosmetics, food and pharmaceutical industries. This study lays a foundation for the high-valued utilization of bovine bone collagen.     

Antioxidant and Antihypertensive Potential of Protein Fractions from Flour and Milk Substitutes from Canary Seeds (<Emphasis Type="Italic">Phalaris canariensis</Emphasis> L.)

Canary seed (Phalaris canariensis) is used to feed birds but it has been recently considered a promising cereal with nutraceutical potential for humans. The aim of this work was to analyze the protein fractions from canary seed flour and from milk substitutes (prepared by soaking the seeds in water 12 and 24 h), and to evaluate antioxidant and antihypertensive capacity of peptides obtained after in vitro digestion. Prolamins were the major protein fraction, followed by glutelins. After digestion, albumins and prolamins fractions from milks presented higher levels of peptides than flour, globulins showed more peptides in flour and glutelins were found in similar concentrations in all samples; 24 h milk prolamins had the highest concentration of peptides. Purification by high performance liquid chromatography (HPLC), sequencing of peptides, in vitro antioxidant ABTS (2,2′-azino-bis, 3-ethylbenzothiazoline-6-sulphonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays, and antihypertensive capacity (angiotensin converting enzyme (ACE) assay), indicated that peptides from canary seed prolamins were the most efficient compounds with antioxidant and antihypertensive activity. Canary seeds may be considered an accessible and cheap source to prepare milk substitutes with high contents of bioactive peptides with remarkable functional properties to promote better human health and healthy ageing.     

Optimization of hydrolysis conditions for the production of angiotensin-I converting enzyme-inhibitory peptides and isolation of a novel peptide from lizard fish (Saurida elongata) muscle protein hydrolysate

Lizard fish (Saurida elongata) muscle protein was hydrolyzed using neutral protease to produce protein hydrolysate (LFPH), and the hydrolysis conditions were investigated using response-surface methodology. The optimum conditions for producing peptides with the highest angiotensin-I converting enzyme (ACE)-inhibitory activity were the following: enzyme-to-substrate ratio of 10,000 U/g, temperature of 48 °C, pH 7.0, and hydrolysis time of 2 h. Under these conditions, the ACE-inhibitory activity of LFPH and the degree of hydrolysis were 84% and 24%, respectively. A novel ACE-inhibitory peptide was isolated from LFPH using ultrafiltration, Sephadex G-15, and high-performance liquid chromatography. The amino acid sequence of the ACE-inhibitory peptide was identified as Ser-Pro-Arg-Cys-Arg (SPRCR), and its IC₅₀ was 41 ± 1 µM.     

厌氧处理对不同类型茶叶的氨基酸组成及生物活性的影响

采用经厌氧处理7 h后的同一批茶鲜叶原料,根据不同加工工艺分别制备成绿茶、白茶、乌龙茶、红茶和黑毛茶,分析其氨基酸组成和含量变化、以及其对血管紧张素转化酶(Angiotensin converting enzyme,ACE)抑制活性和抗氧化活性(包括细胞抗氧化能力以及1,1-二苯基-2-三硝基苯肼自由基清除能力、2,2...     

红树林植物海莲不同部位提取物抗氧化活性研究

植物抗氧化研究对寻找高效率、低毒性天然抗氧化剂具有重要的指导意义。以在海南分布较广的红树林植物海莲进行抗氧化活性研究,以期对海莲开发利用的可能性提供理论依据。采用清除DPPH自由基和清除ABTS自由基对海莲不同部位———根、茎、叶、花的乙酸乙酯提取物进行抗氧化能力评价,采用比色法和福林酚法测定总黄酮和总酚含量。结果显示,海莲茎的提取物在4个部位中抗氧化活性最好,清除二者的IC50分别为(0.234±0.053)mg/m L和(0.196±0.080)mg/m L,海莲中酚类物质含量较大,4个部位中茎的总酚含量最多,含量为13.2 mg。说明海莲各部位均显示出了较好的抗氧化能力,具有较好的开发潜力。     

Preparation and Hepatoprotective Activities of Peptides Derived from Mussels (Mytilus edulis) and Clams (Ruditapes philippinarum)

Low molecular weight (<5 kDa) peptides from mussels (Mytilus edulis) (MPs) and the peptides from clams (Ruditapes philippinarum) (CPs) were prepared through enzymatic hydrolysis by proteases (dispase, pepsin, trypsin, alcalase and papain). Both the MPs and the CPs showed excellent in vitro scavenging ability of free radicals including OH, DPPH and ABTS in the concentration range of 0.625–10.000 mg/mL. By contrast, the MPs hydrolyzed by alcalase (MPs-A) and the CPs hydrolyzed by dispase (CPs-D) had the highest antioxidant activities. Furthermore, MPs-A and CPs-D exhibited protective capabilities against oxidative damage induced by H₂O₂ in HepG2 cells in the concentration range of 25–800 μg/mL. Meanwhile, compared with the corresponding indicators of the negative control (alcohol-fed) mice, lower contents of hepatic MDA and serums ALT and AST, as well as higher activities of hepatic SOD and GSH-PX were observed in experiment mice treated with MPs-A and CPs-D. The present results clearly indicated that Mytilus edulis and Ruditapes philippinarum are good sources of hepatoprotective peptides.     

Antioxidant potential of sesame (Sesamum indicum) cake extract in stabilization of sunflower and soybean oils

Recently natural antioxidants have gained increased interest because natural food ingredients are safer than synthetic ones. Antioxidant activities and protective effects of sesame cake extract (SCE) in stabilizing sunflower oil (SFO) and soybean oil (SBO) were tested. Since different antioxidant compounds have different mechanisms of action, 2,2-azinobis (3-ethylbenzthiazoline sulfonate) (ABTS) radical scavenging activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity, and β-carotene/linoleic acid test system were used to assess the antioxidant efficacy of SCE. Total phenolic, flavonoid and flavonol contents in SCE were 1.94 (mg gallic acid equivalent (GAE) g⁻¹ dry weight (DW)), 0.88 (mg quercetain equivalent (QE) g⁻¹ DW), and 0.40 (mg QE g⁻¹ DW), respectively. Protective effects of SCE in stabilizing SFO and SBO were tested, compared to synthetic antioxidants, by measuring their peroxide values (PV), conjugated dienes (CD), conjugated trienes (CT) and p-anisidine value during accelerated storage. Results indicated that SCE exhibited stronger antioxidant activity in SFO and SBO than butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), while its antioxidant activity was less than that of tert-butyl hydroquinone (TBHQ).     

花生非淀粉多糖和抗氧化肽同步提取技术研究

研究米曲霉和酿酒酵母混合固态发酵热榨花生粕同步提取花生非淀粉多糖和抗氧化肽,为花生粕的高值化利用提供理论基础。以发酵产物的可溶性氮浓度、1,1-二苯基苦基苯肼(DPPH)自由基清除率、羟自由基清除率和非淀粉多糖得率为考察指标,研究了菌种比例、接种量、营养盐溶液量、发酵温度、发酵时间、水浴温度和水浴时间对发酵效果的影响,确定最佳工艺条件为:菌种比例1∶2,接种量3mL,营养盐溶液添加量30mL,发酵温度33℃,发酵时间42h,水浴温度40℃,水浴时间6h。此工艺下发酵产物的可溶性氮浓度为46.32mg/mL、DPPH自由基清除率为71.90%、羟自由基清除率为96.96%、非淀粉多糖得率为4.36%。发酵产物经乙醇沉淀和超滤分离得到的花生非淀粉多糖和抗氧化肽产品具有清除自由基、抑制脂质过氧化、金属离子螯合力和还原力等4大类抗氧化活性。     

超滤法分离花生肽及其抗氧化活性的研究

用碱性蛋白酶解法制备花生多肽,将得到的花生肽酶解液通过超滤的方法进行分离得到不同分子量段的多肽,对其进行抗氧化活性的研究得到抗氧化活性最强的分子量段的多肽。将花生短肽酶解液稀释并分别通过5KD、3KD的卷式纤维膜进行超滤得到三个分子量段的花生短肽。比较各分子量段的多肽对二苯代苦味酰基（DPPH）自由基的清除率、抗亚油酸氧化能力、羟自由基的清除率及还原力。结果表明：花生肽具有一定的抗氧化能力,且3KD以下的多肽抗氧化能力最强,3-5KD分子量段的多肽次之,大于5KD分子量的多肽表现出较弱的抗氧化能力。     

Reverse-phase HPLC Separation of Hemp Seed (Cannabis sativa L.) Protein Hydrolysate Produced Peptide Fractions with Enhanced Antioxidant Capacity

Hemp seed protein hydrolysate (HPH) was produced through simulated gastrointestinal tract (GIT) digestion of hemp seed protein isolate followed by partial purification and separation into eight peptide fractions by reverse-phase (RP)-HPLC. The peptide fractions exhibited higher oxygen radical absorbance capacity as well as scavenging of 2,2-diphenyl-1-picrylhydrazyl, superoxide and hydroxyl radicals when compared to HPH. Radical scavenging activities of the fractionated peptides increased as content of hydrophobic amino acids or elution time was increased, with the exception of hydroxyl radical scavenging that showed decreased trend. Glutathione (GSH), HPH and the RP-HPLC peptide fractions possessed low ferric ion reducing ability but all had strong (>60 %) metal chelating activities. Inhibition of linoleic acid oxidation by some of the HPH peptide fractions was higher at 1 mg/ml when compared to that observed at 0.1 mg/ml peptide concentration. Peptide separation resulted in higher concentration of some hydrophobic amino acids (especially proline, leucine and isoleucine) in the fractions (mainly F5 and F8) when compared to HPH. The elution time-dependent increased concentrations of the hydrophobic amino acids coupled with decreased levels of positively charged amino acids may have been responsible for the significantly higher (p?<?0.05) antioxidant properties observed for some of the peptide fractions when compared to the unfractionated HPH. In conclusion, the antioxidant activity of HPH after simulated GIT digestion is mainly influenced by the amino acid composition of some of its peptides.     

鹅掌柴提取物的抗氧化活性

从清除超氧阴离子自由基(O-2)、还原力、清除DPPH自由基、清除羟基自由基(·OH)等方面,研究鹅掌柴提取物的抗氧化活性.结果表明:鹅掌柴提取物对DPPH自由基和羟基自由基的清除能力较强,清除率分别为78.57%和74.99%,对脂质体过氧化的的抑制率为21.41%;与VE、VC、BHT相比,鹅掌柴提取物的还原能力和对Fe2+的络合能力也较强;但对超氧阴离子自由基的清除能力较弱.因此.鹅掌柴提取物是一种抗氧化功效较强的活性物质,具有很好的保健功能.