Preparation and Characterization of Protein Hydrolysates From Little Loligo Squid (Uroteuthis chinensis)

Research on the little loligo squid (Uroteuthis chinensis) protein hydrolyzed by papain and Flavourzyme and characterization of hydrolysates was carried out in this article. The optimum hydrolysis conditions of papain were obtained by orthogonal experiments, and the influence factors on the degree of hydrolysis such as enzyme/substrate ratio, hydrolysis time, pH, and temperature were investigated. The effect of Flavourzyme on the degree of hydrolysis was also studied. The characteristics of freeze-dried hydrolysates such as yield, moisture, nitrogen recovery, molecular mass distribution, and amino acid composition were determined. The results showed that papain and Flavourzyme could hydrolyze the little loligo squid protein efficiently, and the degree of hydrolysis was up to 25.82% under the optimum hydrolysis conditions. The average molecular mass of hydrolysates obtained with papain and Flavourzyme was 1,246 Da, and the low molecular mass peptides were the main fraction of hydrolysates. The yield, moisture, and nitrogen recovery of freeze-dried hydrolysates was 19.8, 4.4, and 74.2%, respectively. Compared with suggested patterns of amino acid requirements by the Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO), the hydrolysates of the little loligo squid had high nutritional value, and was a potential nutritious supplement used in various food products.     

Effect of Enzyme Type on the Antioxidant Activities and Functional Properties of Enzymatic Hydrolysates from Sea Cucumber (Cucumaria frondosa) Viscera

ABSTRACT

Effects of various proteases were obvious on the amino acid composition, antioxidant activities, and functional properties of enzymatic hydrolysates prepared from sea cucumber viscera (SCV). Alcalase, trypsin, and flavourzyme conferred better antioxidant activities on hydrolysates and endowed them with a higher degree of hydrolysis, coming to 19.08, 32.38, and 15.94%, respectively. For functional properties, neutrase- and flavourzyme-derived hydrolysates showed better foaming properties. Amino acid composition suggested that papain, neutrase, alcalase, and bromelain granted the hydrolysate’s high nutritional quality. The results indicated that SCV hydrolysates could be used in food systems to impart desirable characteristics to products.     

Enzymatic Hydrolysis of Catfish (Pangasius hypophthalmus) By-Product: Kinetic Analysis of Key Process Parameters and Characteristics of the Hydrolysates Obtained

In this study, kinetic evaluation of enzymatic hydrolysis of catfish (Pangasius hypophthalmus) by-product meat was conducted. The effects of key factors such as type of proteases (neutrase, papain, and bromelain), enzyme to substrate ratio (E/S), substrate concentration, and extent of hydrolysis on process efficiency, properties and functionalities of hydrolysates were evaluated. The results demonstrated that, for catfish shredded meat, bromelain and papain had higher affinity than neutrase. Kinetics parameters V_m and K_m of neutrase, papain, and bromelain were 4.486, 9.060, and 18.249 μM/min and 35.433, 6.582, and 7.034 g/l, respectively. Optimal E/S ratios of neutrase, papain, and bromelain were estimated to be 0.92, 2.76, and 3.31 mg/g. At optimal E/S, the highest possible substrate concentrations were found to be 108.4 g/l for neutrase, 36.2 g/l for papain, and 135.8 g/l for bromelain. Both kinetics study and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that bromelain and papain had higher proteolytic activity toward catfish protein than neutrase. A significant increase in antioxidant activity of hydrolysates was observed with neutrase and papain hydrolysate, but not with bromelain. The results could be helpful for industrial processors to develop an efficient process for catfish by-product valorization.     

紫菜活性肽复合酶法制备与清除羟自由基作用

以条斑紫菜为原料,利用胰蛋白酶和木瓜蛋白酶组成的复合酶水解紫菜蛋白制备生物活性肽并研究清除羟自由基作用。采用单因素试验考察胰蛋白酶与木瓜蛋白酶的比例、底物质量浓度、酶用量、水解温度、水解时间、pH对羟自由基清除率及水解度的影响,响应面法优化酶解条件,SephadexG-15凝胶层析法测定活性肽的分子质量分布。试验结果表明,复合酶最佳酶解工艺条件为胰蛋白酶与木瓜蛋白酶复合酶比例1.67,底物质量浓度20mg/mL,酶用量1.67%,pH 7.0,温度55℃,酶解4h,紫菜活性肽对羟自由基的清除率为80.6%,清除率为50%时的质量浓度为0.744mg/mL,分子质量大多分布于500～1500ku。     

扇贝边酶解技术研究

研究了混合酶水解扇贝边蛋白质的技术条件，并和单酶水解情况进行了比较。结果表明，混合酶水解可提高水解液中氨基酸态氮（AAN）含量，同时改善了水解液风味。本文引进二次正交旋转组合试验设计方案，系统地研究了影响混合酶水解效果的相关因素，得出了扇贝边水解液中氨基酸态氮生成量的变化规律一回归模型。应用该模型可对水解液中AAN生成量进行预测，也可进一步优化选择扇贝边的酶解条件。     

Optimization of the Enzymatic Hydrolysis Assisted by Ultra-high Pressure Processing of Alaska Pollock Frame for Improving Flavour

ABSTRACT

In order to improve the flavor of aquatic enzymatic hydrolysate, this study determined the effect of ultra-high pressure processing (UHP) coupled with enzymatic hydrolysis on the flavor of Alaska pollock frame (APF) hydrolysates. The optimal conditions for UHP enzymatic hydrolysis were as follows: pressure time was 60 min, pH value was 7.5, and pressure was 150 MPa. The content of soluble peptide, total-free amino acid, and delicious amino acid (DAA) content of atmospheric pressure and UHP hydrolysates were 11.37 vs 16.70 mg/mL, 5 528 vs 6 234 mg/100 g, and 2145 vs 2411 mg/100 g, respectively. The major compounds in the enzymatic hydrolysates of UHP included seven types of ketones (18.31%), 8 types of aldehydes (6.35%), 3 types of alcohols (2.61%), one type of ester (0.38%), and one type of furan (7.89%). The results of electronic tongue (e-tongue) demonstrated good agreement with gas chromatography-mass spectrometry (GC-MS) analysis. In conclusion, UHP enzymatic hydrolysis can be a potential tool to improve the flavor compounds of hydrolysates.     

Structural Changes,Volatile Compounds and Antioxidant Activities of Maillard Reaction Products Derived from Scallop (Patinopecten yessoensis) Female Gonad Hydrolysates

In order to improve the utilization of scallop byproducts, scallop female gonad hydrolysates (SFGHs) were prepared using alcalase and ribose at a mass ratio of 1:2 and pH 7.0 (95°C, up to 12 h) to develop heterogeneous Maillard-type antioxidant and flavor compounds. The formation of Schiff’s base as well as modification of the amide bands in Maillard reaction products (MRPs) were investigated using ultraviolet–visible, fluorescence, and Fourier transform infrared spectroscopy. The significant decrease of amino acid residues explained the strong Maillard reactivity of SFGHs. MRPs with enhanced ABTS⁺ radical scavenging capacity over SFGHs promoted the survival of HepG2 cells treated with hydrogen peroxide. Moreover, 19 new volatile compounds were produced by the reaction of SFGHs with ribose. These results suggest that SFGHs–ribose MRPs can be potentially used as antioxidants with flavor properties.

Abbreviations: SFGs: scallop female gonads; SFGHs: scallop female gonad hydrolysates; ABTS: 2?2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); BHT: butylated hydroxytoluene; Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; MRPs: Maillard reaction product; H₂O₂: hydrogen peroxide; HS-SPME/GC/MS: Headspace solid phase micro-extraction/gas chromatography/mass spectrometry.     

Optimization of Hydrolysis Conditions for Production of Angiotensin-Converting Enzyme Inhibitory Peptides from Basa Fish Skin Using Response Surface Methodology

ABSTRACT

Basa fish skin free from fat and noncollagenous proteins was hydrolyzed by different proteinases to produce angiotensin-converting enzyme (ACE) inhibitory peptides. Protamex hydrolysates were found to have the highest ACE inhibitory activity. Response surface methodology (RSM) was applied to optimize the hydrolysis conditions of Protamex—including enzyme-to-substrate ratio, hydrolysis time, and solid (fish skin)-to-liquid (water for hydrolysis)—at a temperature of 50°C and a pH of 7.0. The mathematical model demonstrated a good fit with the experimental data. An enzyme-to-substrate ratio of 6,108 U/g, a hydrolysis time of 1.24 h, and a solid-to-liquid ratio of 1:8.05 g/mL were found to be the optimal conditions to achieve the highest value of ACE inhibitory activity. The IC₅₀ of the hydrolysates for ACE inhibitory activity was 1.417 mg/mL under these conditions.     

酶解扇贝裙边制备复合氨基酸螯合钙的研究

扇贝裙边富含蛋白质、脂质等营养成分。为了高值化利用扇贝裙边,本研究选用中性蛋白酶、动物蛋白酶、风味蛋白酶、木瓜蛋白酶和酸性蛋白酶5种酶,以酶解液中游离氨基酸态氮为考察指标,对扇贝裙边进行酶解工艺条件探讨。首先,将5种酶制成复合蛋白酶进行正交实验,确定最佳酶解时间、温度、pH及加酶量,经检验,氨基酸转化率为77%;然后,通过实验确定CaCl2为最适钙源,以贝壳为原料,通过水飞法和酸法转化可制得贝壳源CaCl2。将扇贝裙边酶解液中复合氨基酸与来源于贝壳的钙螯合制备复合氨基酸螯合钙,以正交实验筛选出最佳螯合条件。经检验,该螯合反应螯合率达92%。     

Antioxidant Properties of Scomber japonicus Hydrolysates Prepared by Enzymatic Hydrolysis

The antioxidant properties of the Pacific chub mackerel (Scomber japonicus) muscle protein hydrolysates prepared by enzymatic hydrolysis were investigated. After enzyme hydrolysis at 50°C for 60 min, more than 80% of the S. japonicus muscle protein was hydrolyzed. The highest 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity (71.69%) occurred in whole muscle protein hydrolysates treated at 50°C for 30 min with Protamex, and the highest 2,2?-azino-bis(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS) radical-scavenging activity (95.39%) was observed in white muscle protein hydrolysates treated at 50°C for 30 min with Neutrase. The highest superoxide dismutase (SOD)-like activity (32.84%) was recorded in white muscle protein hydrolysates treated at 50°C for 120 min with Protamex. Changes in the molecular weight distribution of S. japonicus muscle proteins after enzymatic hydrolysis were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A robust and a convenient enzyme hydrolysis technique for obtaining S. japonicus muscle protein hydrolysates with useful biological activities, within a short time (<2 h) is proposed.     

Utilization of Chum Salmon (Oncorhynchus keta) Skin Gelatin Hydrolysates to Attenuate Hydrogen Peroxide-Induced Oxidative Injury in Rat Hepatocyte BRL Cell Model

Six chum salmon skin gelatin hydrolysates with degree of hydrolysis of 4.7–13.5% were generated by Alcalase and papain and showed scavenging activities to 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and superoxide anion radicals. Potential protection of these hydrolysates against H₂O₂-induced oxidative injury in rat hepatocyte line (BRL cells) was assessed in vitro, based on cell viability, leakage of lactate dehydrogenase, thiobarbituric acid reactive substances (TBARS), glutathione content, glutathione reductase, and catalase of the cells. The hydrolysates exhibited protection on the hepatocytes, reflected by an enhanced cell viability (about 6.2–87.9%), glutathione reductase (about 28.2–85.9%) or catalase (about 43.3–116.9%) activity, and decreased lactate dehydrogenase leakage (about 2.7–34.7%) or TBARS content (about 4.1–39.3%). On the contrary, cellular glutathione content gave an unnoticeable difference among the investigated groups. These hydrolysates also showed protection against DNA damage in the cells. Cytoprotection of these hydrolysates possessed a dosage-dependence manner. More importantly, there was a high correlation (0.7 < r < 0.9) between DPPH or superoxide anion radical scavenging activity of these hydrolysates and three cellular antioxidant indices (glutathione content, glutathione reductase, and catalase activity).     

Influence of Different Hydrolysis Processes by Trypsin on the Physicochemical,Antioxidant, and Functional Properties of Collagen Hydrolysates from Sphyrna lewini,Dasyatis akjei,and Raja porosa

ABSTRACT

Acid soluble collagen hydrolysates from the cartilages of Sphyrna lewini, Dasyatis akjei, and Raja porosa on different hydrolysis conditions by trypsin were prepared and named as S, D, and R, respectively. The hydrolysates (S1, D1, and R1) obtained from hydrolysis in pH 2.5, 3 h, 37°C presented wonderful foaming and emulsifying capacities, caused by their high average molecular weights (AMWs), high degree of Pro and Lys hydroxylation, and imino acid contents (17.1, 15.3, and 14.1%). At the concentration of 0.1% (w/v), the foaming capacities of S1, D1, and R1 were 104.75 ± 2.57, 63.87 ± 2.73, and 76.87 ± 2.02%; and the emulsifying activity indexes of them were 116.07 ± 1.89, 91.04 ± 1.79, and 123.85 ± 2.14 m²/g, respectively. Conversely, hydrolysates (S2, D2, and R2) obtained from hydrolysis in pH 7.8, 3 min, 37°C exhibited strong scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (EC₅₀ 0.568, 0.680, and 1.634 mg/mL), hydroxyl radical (EC₅₀ 0.253, 0.376, and 0.438 mg/mL), and moderate reducing power (0.0465–0.4702 at the concentration of 5 mg/mL), due to their low AMWs. The results indicated that S1, D1, and R1 could be used as emulsifiers and foaming agents, and S2, D2, and R2 could be used as natural antioxidants in food systems.     

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 and Angiotensin-Converting Enzyme Inhibitory Activities of Protein Hydrolysates Prepared from Threadfin Bream (Nemipterus spp.) Surimi By-products

Solid wastes from threadfin bream (Nemipterus spp.) surimi production composed of head and frame were hydrolyzed by various commercial proteases (Alcalase, Flavourzyme, Neutrase, Protamex, papain, and pepsin) to produce protein hydrolysates with bioactive properties. An Alcalase-hydrolyzed sample at 24.4% degree of hydrolysis (DH) displayed the highest antioxidant activity based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP) assay, and potassium ferricyanide method. In addition, it showed an inhibitory activity toward angiotensin-converting enzyme (ACE) of 25.5%. Antioxidant activity of threadfin bream by-product hydrolysates increased with hydrolysis time and reached the highest DPPH activity after 6 h, while that hydrolyzed for 3 h showed the highest reducing power based on FRAP and potassium ferricyanide assays. In addition, ACE inhibitory activity was found to be at an optimum after 3 h of hydrolysis. The hydrolysates (1 mg/mL) also retarded oxidation of a linoleic acid emulsion system to a similar extent as 0.1 mg/mL 3-tert-butyl-4-hydroxy anisole (BHA), indicating a potential use in the food system. Protein hydrolysates from threadfin bream surimi by-products could be tailor-made to possess both antioxidant and ACE inhibitory activity through controlling DH of Alcalase-catalyzed reactions.     

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)。     

The Feasibility of Increasing Lipid Extraction in Tilapia (Oreochromis niloticus) Waste by Proteolysis

ABSTRACT

The aim of the present study was to evaluate the feasibility of increasing lipid extraction in tilapia (Oreochromis niloticus) waste using different enzymes and to minimize the periods of hydrolysis to evaluate the performance of the lipid fractions. When processing waste tilapia, commercial enzymes were added that were composed of neutrase and alcalase acting at different hydrolysis times to produce the best yields. The treatment proved feasible for obtaining the protein hydrolysates. The type of enzyme and the hydrolysis time determined the degree of fractionation. The enzymes performed well, and the highest efficiency occurred within 2 h of hydrolysis. By fractionating, the obtained by-products can be applied in the preparation of feed, as the lipid fraction treatments yielded significant amounts of polyunsaturated fats and suitable n-6/n-3 ratios.     

Effect of Degree of Hydrolysis and Protease Type on the Antioxidant Activity of Protein Hydrolysates From Cuttlefish (Sepia officinalis) By-Products

We have investigated the antioxidant activities of eight hydrolysates from cuttlefish by-products obtained by treatment with various gastrointestinal proteases (chymotrypsin, trypsin, and crude alkaline enzyme extracts from cuttlefish and sardinelle) and bacterial proteases (Alcalase and crude enzymes from Bacillus pumilus A1, Bacillus mojavensis A21, and Bacillus cereus BG1). The antioxidant activities of the cuttlefish by-products protein hydrolysates (CPHs) were evaluated using various in vitro antioxidant assays, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducing power, and total antioxidant capacity. All hydrolysates showed different degrees of hydrolysis (DH) and varying degrees of antioxidant activity. Among the different hydrolysates, cuttlefish crude enzyme hydrolysate exhibited the highest antioxidant activity, followed by sardinelle crude enzyme and Alcalase hydrolysates. Further, CPHs with different degrees of hydrolysis were prepared by treatment with proteolytic enzymes from cuttlefish, sardinelle, and B. mojavensis A21. All hydrolysates showed a greater antioxidative activity as indicated by all the methods considered. In addition, antioxidant activity in hydrolysates was positively correlated with the increase of DH. The results of this study indicated that CPHs might be a good candidate for further investigation in developing new antioxidants.     

Preparation of Water-Soluble Chitosan and its Suppressive Effect on the Denaturation of Scallop Adductor Muscle Myofibrillar Protein During Frozen Storage

ABSTRACT

In the present study, investigated were the preparation of water-soluble chitosan (WSC) by hydrolysis of original chitosan with commercial α-amylase and the suppressive effect of WSC on denaturation of bay scallop adductor muscle myofibrillar protein (Mf) during frozen storage at ?18°C for 12 months. The WSC (2.5- to 10-g dry weight) was added to 100 g of the Mf, and the changes in the Ca-ATPase activity, solubility, and sulfhydryl content were examined during frozen storage. The addition of WSC significantly reduced the denaturation of the Mf Ca-ATPase. Moreover, a significant increase in Mf solubility and sulfhydryl content was observed in the WSC-treated bay scallop adductor muscles compared with the control (p < 0.05) during frozen storage at ?18°C.     

响应面法优化中性蛋白酶酶解海湾扇贝副产物制备抗氧化酶解物研究

采用响应面法优化中性蛋白酶酶解海湾扇贝(Argopecten irradias)副产物蛋白制备抗氧化酶解物工艺。以DPPH自由基清除率和还原能力为响应值,在底物浓度、温度、时间、酶与底物比4个单因素实验基础上,优化确定3个显著性因素。获得实验条件下最佳制备工艺为:底物浓度25 mg·mL^-1、温度50℃、时间3.8 h、酶与底物比1.5%。在此条件下,实验验证酶解海湾扇贝副产物蛋白制备抗氧化酶解物的DPPH清除率为81.93%(预测值为83.19%),还原能力为58.12%(预测值为58.92%),DPPH自由基清除率和还原能力的IC 50值分别为0.264 mg·mL^-1和3.136 mg·mL^-1,且表现出较强的抗氧化活性。研究表明,优化后的回归模型用于预测中性蛋白酶酶解海湾扇贝副产物蛋白制备抗氧化酶解物是可行的。     

Common Kilka Hydrolysis: Investigation of Functional and In Vitro Antioxidant Properties of Hydrolysates Obtained by Kiwifruit and Ginger Proteases

The functional and in vitro antioxidant properties of common kilka fish protein hydrolysates with different degree of hydrolysis (DH) obtained by kiwifruit protease (KP) and ginger protease (GP) were evaluated. The electrophoretic patterns of hydrolysates showed the presence of one major band in different DHs with a molecular weight of less than 25 kDa. Protein solubility was positively correlated with DH, and the solubility of KP and GP hydrolysates (HKP and HGP, respectively) at 15% DH was higher than other DHs (p ≤ 0.05). Higher emulsifying and foaming properties were observed in HKP over a pH range of 2–10 (p ≤ 0.05), and in vitro antioxidant activity was higher in HKP at 15% DH as seen from 2,2-diphenyl-1-picrylhydrazyl radical scavenging and ferric-reducing power. Thus, the results reveal that DH and enzyme type affects functional properties and antioxidant activity of the hydrolysates.