Bioactive peptides from collagen hydrolysates from squid (Dosidicus gigas) by-products fractionated by ultrafiltration

The bioactive properties of peptide fractions obtained from the hydrolysis of squid (Dosidicus gigas) by-products collagen, using Protease type XIV and ultrafiltration (UFI), were studied. The basic objective was to improve the bioactivity of squid hydrolysates via the application of UFI. Peptide fractions obtained after UFI had higher antioxidant and antimutagenic activities, but the antiproliferative activity did not improve after UFI. Peptides <5 kDa (Fraction F3) showed higher antioxidant and antimutagenic activities, as well as lower antioxidant and antiproliferative activities than both, peptides >10 kDa (F1) and those within the range of >5 to <10 kDa (F2). Band at lower field observed in FT-IR spectra and proton-peaks observed at higher ¹H-NMR fields, both associated to aromatic amino acids, as well as to other antioxidant amino acids such as hydroxyproline, glycine, arginine and lysine, may explain F3 bioactivity. Ultrafiltration can, therefore, be used to improve some bioactivities of squid collagen hydrolysates.     

Bioactive properties of enzymatic hydrolysates from abdominal skin gelatin of yellowfin tuna (Thunnus albacares)

Gelatin (90.6 ± 0.1%) was optimally prepared by response surface methodology from yellowfin tuna (Thunnus albacares, YT) abdominal skin. To investigate bioactive properties of enzymatic hydrolysates from the abdominal skin gelatin (ASG), ASG was hydrolysed with alcalase, protamex, neutrase and flavourzyme as affected by hydrolysis time. Antioxidant, nitrite scavenging and angiotensin‐I converting enzyme (ACE) inhibitory activities of the hydrolysates were determined. Antioxidant activities of the hydrolysates were found through linoleic acid peroxidation inhibitory effects. Alcalase‐derived hydrolysates (AHs) were more effective than others in metal ions chelating, superoxide anion scavenging and hydroxyl radical scavenging activities (P < 0.05). AHs showed significantly stronger nitrite scavenging activities (44.4–60.7%) than others (P < 0.05). Fraction A from AH showed strong ACE inhibitory activity (IC₅₀ of 0.75 mg mL^?1). These results suggest that YT ASG and its enzymatic hydrolysates could be functional food and/or pharmaceutical ingredients with potent antioxidant, anticarcinogenic and antihypertensive benefits.     

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。      

Distinctive characteristics of collagen and gelatin extracted from Dosidicus gigas skin

Squid skin, often discarded as processing by-product, is a good resource of collagen/gelatin. In this study, acid soluble collagen (ASC), pepsin soluble collagen (PSC) and water soluble gelatin (WSG) were extracted from squid (Dosidicus gigas) skin and physicochemically examined. The lowest yield of 33.5% was obtained for ASC extracted at 4 °C, and the addition of pepsin increased the collagen yield by around 35.0% (PSC). The highest yield of 81.9% (WSG) was achieved by thermal extraction at 60 °C. A low temperature can largely retain the native helix structures of ASC and PSC, contrariwise, thermal treatment converted collagen into gelatin with unordered and renatured structures. The proline and hydroxyproline contents of ASC, PSC and WSG were 183/1000 residues, 194/1000 residues and 175/1000 residues, respectively. In addition, WSG showed a denaturation temperature at 80.7 °C which was much higher than that of ASC (24.2 °C) and PSC (26.2 °C), while a significant lower resistance towards enzymatic digestion.     

Jumbo squid (Dosidicus gigas) mantle muscle gelled-emulsified type product: formulation, processing and physicochemical characteristics

Jumbo squid ( Dosidicus gigas ), an abundant species in the Gulf of California, can have a great potential for production of gelled-emulsified type products. Thus, formulation, processing and physicochemical characteristics of frankfurter-type product from jumbo squid mantle muscle (JSF) was achieved. JSF were vacuum-packed and stored at 2–4 °C. Samples were analysed for physicochemical (colour, texture, TBARS, peroxide value, folding test, pH, and water content and holding capacity) and microbial changes at regular intervals during storage for up to 27 days. The sensory quality of the product was also evaluated. Shear force, cohesiveness and colour (hue angle and total colour difference) were the most affected ( P  <   0.05) parameters at day 27, changes most probably because of microbial growth as total aerobic counts increased to >2.7 × 10⁵ CFU g⁻¹ (day 21). Product showed acceptability. Results suggest a stable gelled-emulsified type product can be developed from jumbo squid mantle muscle opening a range of possibilities for product development.     

Molecular and physical characteristics of squid (Todarodes pacificus) skin collagens and biological properties of their enzymatic hydrolysates

ABSTRACT:  The physicochemical properties of squid skin collagens and biological activity of their enzymatic hydrolysates were determined to produce more value-added materials. The amino acid compositions of the inner and outer squid skin collagens were similar, but distinct from that of bovine tendon collagen in respect to the higher levels of aspartic acid, arginine, threonine, and serine, and of the lower levels of alanine, proline, and hydroxyproline. SDS-PAGE patterns suggested that squid skin collagen consisted of at least 2 different polypeptides (α1 and α2 chains) and their cross-linked chains. The molecular weights of α1 and α2 chains of bovine tendon collagens were higher than those of the corresponding α1 and α2 chains of squid skin collagens. The denaturation temperatures of inner and outer skin collagens were 27.1 and 27.3 °C, respectively, which were about 9 °C lower than that of bovine tendon collagen. Water holding capacities of inner and outer squid skin collagens were relatively similar, but were significantly greater than that of bovine tendon collagen. The maximum hydrolysis of squid skin collagens was obtained as follows: for outer skin collagen, enzyme concentration, 3.5%; hydrolysis time, 83 min; pH 7.0; hydrolysis temperature, 60 °C, whereas for inner skin collagen, enzyme concentration, 3.2%; hydrolysis time, 83 min; pH 7.0; hydrolysis temperature, 60 °C. The enzymatic hydrolysates of outer and inner skin collagens were separated by Sephacryl S-100 column, resulting in the production of 3 fractions (F1, F2, and F3). F3 fraction exhibited higher antioxidant, tyrosinase inhibitory, and antielastase activities than the other fractions.     

Lysyl oxidase from jumbo squid (Dosidicus gigas) muscle: detection and partial purification

Lysyl oxidase (LOX) was detected and partially purified from jumbo squid (Dosidicus gigas) muscle, for the first time. A procedure for the purification of LOX from jumbo squid muscle which consisted of urea extraction, Sephadex G‐75 and anion exchange chromatography was developed. Activity of partially purified LOX was 390‐fold higher than the original extract. Two protein fractions with 32 and 24 kDa were detected by SDS‐PAGE. The enzyme was strongly inhibited by β‐aminopropionitrile fumarate, a specific LOX inhibitor. LOX was purified with 3.8% yield, showing a specific activity of 0.078 IU mg^?1 protein. This knowledge will help understand the behaviour of jumbo squid protein during cool storage or manufacture.     

Identification of a cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas as cathepsin L

A cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas was partially purified by a two step procedure involving ammonium sulfate precipitation and gel filtration chromatography and further by SDS–PAGE. The molecular weight of the proteinase was 24 kDa determined by SDS–PAGE and 23.7 kDa with mass spectrometry. The activity had an optimum pH of 4.5 and optimum temperature of 55 °C under the assay for cathepsin L specific synthetic substrate Z-PAAFC. The cathepsin B and H specific synthetic substrates Z-AAAFC and H-AMC did not show any hydrolysis with the partially purified enzyme. Peptide mapping of trypsin digests of the 24 kDa band from SDS–PAGE showed the squid cysteine proteinase was homologous to cathepsin L from different animal sources. The activity of the partially purified fraction with the cathepsin L specific substrate Z-PAAFC was inhibited 75–89% by enzyme inhibitors specific for cysteine proteinases but was also significantly inhibited by serine and aspartate proteinase inhibitors.     

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.     

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.     

Effects of pH and Sodium Chloride on the Gelling Properties of a Protein Concentrate Obtained from Jumbo Squid Mantle (Dosidicus Gigas)

Jumbo squid (Dosidicus gigas) is an underutilized fishery resource in the Gulf of California. Jumbo squid muscle could potentially be utilized to manufacture protein concentrates to be used as a base for surimi or as a food ingredient. However, the main problem is the low gelling ability of these protein concentrates compared those of with fish species. Consequently, the present study evaluated the effects of pH and the NaCl concentration on the gelling properties of protein concentrates from squid mantle. According to texture profile analysis, improved gelling properties were obtained at pH 6.0 and 1% of NaCl. During the sol-gel transition, decreased surface hydrophobicity that corresponded to an increased intensity of α-helix structures was detected. Moreover, the total sulfhydryl content decreased during gel formation. Viscoelasticity and water holding capacity analyses showed the formation of a more structured gel at pH 7.0. The higher enthalpy and denaturation temperature detected by differential scanning calorimetry at pH 7.0 and 1% NaCl showed that both variables (pH and NaCl concentration) affected the protein structure and conformation. The results demonstrated that varying pHs and NaCl concentrations improved the gelling properties of protein concentrates from squid.     

具有自由基清除活性的鱿鱼皮胶原蛋白肽酶解工艺研究

以蛋白水解度和羟自由基清除率为指标,采用胰蛋白酶法提取秘鲁鱿鱼皮中的胶原蛋白肽,并通过单因素试验和正交试验优化了鱼皮胶原蛋白肽的制备工艺条件,同时测定了鱼皮的基本组成成分.结果表明,加酶量5％,酶解时间7h,酶解温度50℃,pH值8.0,酶解效果最好.此条件下,鱿鱼皮胶原蛋白肽的水解度为26.1％,羟自由基清除率为83.3％,在医用保健食品和抗衰老美容护肤产品上具有很好的实用价值.     

响应面法优化酶解鸢乌贼制备抗氧化肽的工艺研究

利用响应面分析法对酶法水解鸢乌贼蛋白制备抗氧化肽的工艺条件进行优化。首先通过单因素实验最终确定木瓜蛋白酶为水解鸢乌贼蛋白制备抗氧化肽的最佳用酶,然后在单因素的基础上以加酶量、液料比、酶解时间为自变量,DPPH自由基清除率为响应值,建立二次回归设计模型,经修正后得到最佳酶解条件为:加酶量为0.66%(w/w),液料比为10.2(v/w),酶解时间为380 min,在此条件下DPPH自由基清除率为89.80%,与模型预测值相吻合。另外,氨基酸分析表明与鸢乌贼蛋白相比,优化后的酶解产物中必需氨基酸含量提高至原蛋白的2.2倍,蛋氨酸、色氨酸等抗氧化性氨基酸分别提高至7.2、25.6倍。      

Angiotensin-I-converting enzyme (ACE)-inhibitory and antihypertensive properties of squid skin gelatin hydrolysates

Gelatin extracted from squid (Dosidicus eschrichitii Steenstrup) skin was hydrolysed with pepsin to prepare Angiotensin-I-Converting Enzyme (ACE) inhibitory peptide. The ACE-inhibitory activity was measured by spectrophotometric assay. The hydrolysate was fractionated into three ranges of molecular weight (6 kDa < HSSG-I < 10 kDa, 2 kDa < HSSG-II < 6 kDa, HSSG-III < 2 kDa) using an ultrafiltration unit. The HSSG-III showed the most potent ACE inhibitory activity in vitro with IC₅₀ of 0.33 mg/ml. Renovascular hypertensive rats (RHR) model was made with two-kidney one clip assay, and antihypertensive effects were studied in RHR treated with HSSG-III for 30 days by oral administration. Arterial blood pressure were measured respectively. The HSSG-III remarkably reduced the arterial blood pressure of RHR. These results suggested that hydrolysate of squid skin gelatin obtained by treatment with pepsin was a good source of peptides with ACE-inhibitory activity and had an antihypertensive effect by oral administration.     

风味酶辅助下蛤蛋白酶解物自由基清除能力研究

采用5种酶解方式酶解美洲帘蛤,并对5种酶解液的自由基清除能力及风味进行比较分析,结果表明复合蛋白酶和风味蛋白酶双酶酶解组合的自由基清除能力最高,风味最好,其DPPH自由基和羟自由基清除率分别为(84.53±2.81)%和(78.19±3.11)%。在此基础上,将其冻干,进一步研究其浓度与自由基清除能力的关系,结果表明,自由基清除能力随着浓度的增加而升高,其DPPH自由基和羟自由基EC50分别为24mg/mL和12mg/mL;最后,对其酶解工艺条件进一步优化,最优酶解条件为pH6.5、酶解温度50℃、酶解时间4h、底物浓度1:2、加酶量0.8%,在此酶解条件下,蛤酶解液的DPPH自由基清除率为(86.6±3.72)%。     

Physicochemical changes of pepsin-solubilized and insoluble collagen in jumbo squid (Dosidicus gigas) muscle after cooking process

Collagen is the major connective tissue (CT) protein and one of the main constituents of the jumbo squid (Dosidicus gigas). Therefore, physicochemical changes of pepsin-solubilized collagen (PSC) and insoluble collagen (IC) were studied after cooking (100°C/30 min) of muscle (mantle, fins, and arms). Different pyridinoline (Pyr) contents (the major cross-linking molecule in collagen fibers) were found in the fresh muscle of the three anatomical regions. After the cooking process, a decrease from 10 to 30% in the thermal resistance of collagen was observed, depending on the anatomical region and fraction evaluated. Furthermore, the electrophoretic profile, Fourier transform infrared (FTIR) spectroscopy, and the amino-acid profile revealed that structural changes occurred in the two different collagen fractions caused by the thermal process, and the changes were greater in the mantle. Under the conditions applied in this study, collagen fractions from the squid arms showed more stability during the cooking process due to the high cross-linking degree of their fibers.     

牡蛎酶解产物的ACE抑制活性和清除自由基活性研究

研究了牡蛎蛋白质分别经537酸性蛋白酶和Alcalase碱性蛋白酶作用所得酶解产物的ACE抑制活性以及对DPPH·、·OH、O2·三种自由基的清除作用。结果表明,两种酶的作用产物都具有一定的ACE抑制活性和清除自由基活性。综合评价工艺条件以及功能实验结果得出,采用537酸性蛋白酶可以简化工艺,对于水解度为16％的酸性蛋白酶酶解产物,蛋白浓度为1mg/mL时,对ACE的抑制率为71.71％;20mg/mL时,对DPPH·、O2·自由基的清除率分别为76.23％、24.36％;8mg/mL时,对·OH自由基的清除率为8947％。相对分子质量分布结果表明,体系主要存在2肽到5肽的多肽类物质,HPLC图谱中对应峰面积约占总面积的76.99％。     

鲑鱼皮明胶水解物的抗氧化活性及其对细胞氧化损伤的保护作用

采用碱性蛋白酶、木瓜蛋白酶和中性蛋白酶水解鲑鱼皮明胶,得到水解度为4.7%到13.5%的7个明胶水解物,分别评价明胶水解物对自由基的清除能力。明胶水解物(0.5、1、2mg/mL)预先作用BRL大鼠肝细胞2h后,通过H2O2(5mmol/L)诱导氧化损伤,分别测定细胞存活率、乳酸脱氢酶(LDH)、丙二醛(MDA)和谷胱甘肽(GSH)等细胞内抗氧化产物的含量变化。结果表明,明胶水解物的抗氧化活性随着水解度的增大呈增强趋势。明胶水解物对细胞具有保护作用,可显著提高细胞存活率,降低LDH渗出量和MDA生成量,且存在一定的剂量关系,但细胞中GSH含量变化不显著;7个明胶水解物对H2O2诱导肝细胞损伤保护时,细胞存活率与水解物的体外抗氧化活性大小显著正相关,而LDH渗出量和MDA生成量与水解物的体外抗氧化活性大小负相关。      

Isolation and properties of AMP deaminase from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California,Mexico

Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5′-AMP sepharose. Specific activity of 2.5 U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS–PAGE showed a single band with 87 kDa molecular mass, native PAGE proved a band of 178 kDa, whereas gel filtration detected a 180 kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with V_max of 1.16 μM/min/mg, K_m of 13 mM, K_cat of 3.48 μM.s⁻¹ and a K_cat/K_m of 267 (mol/L)⁻¹.s⁻¹. The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms.     

Preparation and antioxidant activity of wheat gluten hydrolysates (WGHs) using ultrafiltration membranes

BACKGROUD: Many hydrolysates from animal and plant proteins have been found to possess physiological activities. Wheat gluten, an important by‐product of the wheat starch industry, is produced worldwide in enormous quantities. In this study, wheat gluten hydrolysates (WGHs) were obtained by enzymatic hydrolysis and fractionated using ultrafiltration membranes. The antioxidant activities of the hydrolysates were investigated by various antioxidant assays, including the ability to inhibit the autoxidation of linoleic acid and the scavenging effect on free radicals. Amino acid composition and molecular weight distribution were also evaluated to determine their relationship with antioxidant activity. RESULTS: The pepsin hydrolysate (PeWGH) had the highest activity and was ultrafiltrated into three major types, PeWGH I (5–10 kDa), PeWGH II (3–5 kDa) and PeWGH III (<3 kDa). PeWGH III showed stronger inhibition of the autoxidation of linoleic acid and higher scavenging activity against 2,2‐diphenyl‐1‐picrylhydrazyl, superoxide and hydroxyl free radicals. Furthermore, PeWGH III had the highest total hydrophobic amino acid content (45.11 g per 100 g protein), and its molecular weight distribution ranged from 1700 to 100 Da. CONCLUSION: The low molecular weight and amino acid composition of PeWGHs were found to be strongly correlated with their antioxidant activity. PeWGH could be used as a natural antioxidant in the pharmaceutical and food industries in the future. Copyright © 2008 Society of Chemical Industry