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

Lysyl oxidase (LOX; E.C.1.4.3.13) was purified from jumbo squid muscle (Dosidicus gigas) with 1900‐fold and yield 1.9%, and characterized for the first time. The purification procedure consisted of fractionation with urea and a combination of size‐exclusion and anion‐exchange chromatography. The enzyme had a molecular weight of 32 kDa, as estimated by SDS‐PAGE. Using a specific LOX substrate (1,5‐diaminopentane), its optimum activity was determined at pH 8.2 and 65 °C. Activation energy (E_a) of the enzyme was 69.94 kJ K^?1 mol^?1. The enzyme was strongly inhibited by β‐aminopropionitrile fumarate (BAPN), a specific LOX inhibitor. Moreover, purified LOX was able to work at different temperatures (20–90 °C) at pH 8.2. Although further research is needed, the results from this work suggest that based on LOX activity, this enzyme may be of practical use in preventing textural changes in jumbo squid during storage or processing.     

CYSTEINE PROTEINASE ACTIVITY IN JUMBO SQUID (DOSIDICUS GIGAS) HEPATOPANCREAS EXTRACTS

Jumbo squid specimens were captured, dissected, and the hepatopancreas was freeze dried for the extraction of proteolytic enzymes. An autolysis experiment conducted at 25C showed two peaks with maximum proteolysis at pH 3 and 5. The proteinase activity of the extract was measured using azocasein, BANA, Z‐PAAFC and Z‐AAAFC substrates. Activity of the extract with azocasein (pH 5) had a maximum at 55C and increased threefold with the inclusion of cysteine or DTT. The proteinase activity remained at least at 60% of the original after 45 h at 4C in the pH range of 3–8. Activity was inhibited 70–85% when extracts were treated with cysteine proteinase specific inhibitors. The proteinases extracted from jumbo squid hepatopancreas are mainly of the cysteine type and have significant activity towards a cathepsin L specific substrate. The understanding of proteinases from this tissue could have implications for quality control of jumbo squid food products.     

EFFECT OF pH AND TEMPERATURE ON JUMBO SQUID PROTEINS

Evaluation of the effect of pH (2 to 13) and temperature (0 to 50C) on functional properties of jumbo squid proteins was performed, followed by a 2  ×  3 factorial design for producing squid protein hydrolysates bearing useful functional properties. In particular, the effects of pH (8, 9 and 10) and temperature (30, 35, 40C) were evaluated. Alcalase and papain were tested on each treatment. The protein recovery, whippability and emulsifying capacity of the hydrolysates were evaluated. Almost 80% of the proteins were recovered in water-soluble form after hydrolysis with papain at pH 10 at the three temperatures. The highest values of whippability (245  ±  17.7%), foam stability (100%), emulsion-forming capacity (27  ±  0.97%) and stability (99.99  ±  8.8%) occurred with papain-produced hydrolysates. When squid protein was treated at 50C and pH 8, the highest whippability value (390.0  ±  0.1%) and foam stability (100%) were obtained when no enzyme was added.

PRACTICAL APPLICATIONS

This paper assesses how process variables, particularly temperature and pH, affect the functional properties of squid proteins. Making use of such process variables will produce more useful and efficient processes, as the application of a hydrolysis system. The processing of jumbo squid protein to obtain proteins bearing adequate functional properties would be an inexpensive way to provide added value to this marine resource and the production of a high-quality protein ingredient. These results hold promise for jumbo squid proteins as useful food ingredient because of their functional properties.     

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.     

PROTEIN SOLUBILITY AND PRODUCTION OF GELS FROM JUMBO SQUID

Solubility at several ionic strengths (0 to 1.0 M), pH (2 to 13) and gelling capacity of jumbo squid Dosidicus gigas muscle proteins were evaluated. Protein recovery was > 90% at pH 9 – 12. Autohydrolysis was evaluated and affected only sarcoplasmic proteins. Folding score was 5 on all gels. Strength was higher for thermal gels prepared from squid fin (50.2  ±  1.2 N) than that prepared from the mantle (23.4  ±  2.5 N). There was no significant difference in gel strengths from previously frozen (46.4  ±  7.5 N) and never-frozen samples (43  ±  5.5 N). Moisture, water drip and water-holding capacity were evaluated on thermal gels. There were significant differences between frozen and never-frozen samples. Results on solubility and gel forming capacity of the proteins from mantle and fin of jumbo squid suggest that these properties can provide additional value to this resource.

PRACTICAL APPLICATIONS

Jumbo squid is a rich source of high-quality protein. Obtaining sophisticated products would be an easy way to take advantage of the high-quality proteins in jumbo squid muscle. Knowledge of the functional properties of those proteins is needed to achieve this. The use of such properties, such as gel-forming capacity, which are similar to other proteins used in the food industry, will give jumbo squid added value. The results described here indicate that jumbo squid proteins may be useful as a food ingredient because of their solubility and gel-forming capacity.     

Relationship between lysyl oxidase activity,pyridinoline content and muscle texture during ice storage of jumbo squid (Dosidicus gigas)

Collagen fibres, stabilised by lysyl oxidase (LOX), play an important role in jumbo squid because they are responsible for the union between various cells; therefore, a close interdependence between their functions and muscle firmness during ice storage has been suggested. In this study, the relationship between LOX activity, pyridinoline (Pyr) content and muscle texture (SF) during ice storage of jumbo squid mantle was evaluated. LOX activity was confirmed within the range of 4.1–7.1 × 10^?3 U g^?1 of protein, leading to an increase in Pyr content, detected in the range of 0.85–1.32 mmol mol^?1 of collagen after 5–20 days. The SF of the muscle became harder during the ice storage time, increasing from 21.08 to 37.95 N. It was therefore possible to establish the relationship between LOX activity, collagen cross‐links (Pyr content) and texture patterns during ice storage of jumbo squid muscle, which increased after 20 days.     

Proteomic identification and physicochemical characterisation of paramyosin and collagen from octopus (Octopus vulgaris) and jumbo squid (Dosidicus gigas)

To perform improvements in food science, it is fundamental to understand the physicochemical properties of proteins since their interaction with other macromolecules plays an essential role in food systems. Collagen and paramyosin help in the maintenance of the matrix structure cells, the textural behaviour and the technical functionality of the protein concentrates; because of this, their identification and characterisation are necessary. Cephalopods species have shown differences in the distribution of its muscle fibres. The amino acid profile of jumbo squid showed a high content of glycine and hydroxyproline, while octopus showed a high content of acidic amino acids. The thermal profile of jumbo squid showed an endothermic transition at 117 °C, which octopus did not present. Moreover, the proteomic identification confirms the identity of paramyosin with 33% coverage to paramyosin from Dosidicus gigas and a 4% coverage to collagen type II from Sepia pharaonis on octopus and jumbo squid, respectively.     

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.     

Effects of thermal processing and various chemical substances on formaldehyde and dimethylamine formation in squid Dosidicus gigas

BACKGROUND: Trimethylamine oxide (TMAO) in squid is demethylated to dimethylamine (DMA) and formaldehyde (FA) during storage and processing. This study examined the effects of thermal processing and various chemical substances on FA and DMA formation in squid. RESULTS: The thermal conversion of TMAO was assessed by analysing four squid and four gadoid fish species, which revealed that FA, DMA and trimethylamine (TMA) were gradually produced in squid, whereas TMA increased and FA decreased in gadoid fish. A significant increase in both FA and DMA levels was observed in the supernatant of jumbo squid with increased heating temperature and extended heating time at pH 6–7. Ferrous chloride combined with cysteine and/or ascorbate had a significantly positive effect on FA formation in the heated supernatant of jumbo squid. No significant difference was observed in the levels of Cu and Fe in squid and gadoid fish. The capability of Fe²⁺ to promote the formation of FA and DMA was not completely attributable to its reducing power in squid. CONCLUSION: Non‐enzymatic decomposition of TMAO was a key pathway during the thermal processing of jumbo squid, and Fe²⁺ was a crucial activator in the formation of FA and DMA. Copyright © 2012 Society of Chemical Industry     

鱿鱼肝脏自水解过程中内源蛋白酶的作用研究

对鱿鱼肝脏蛋白酶的酶学性质及其在鱿鱼肝脏自水解过程中的作用进行研究。结果表明：以偶氮酪蛋白为底物,TCA可溶性肽为评价指标,测得鱿鱼肝脏蛋白酶粗酶的最适反应温度为40℃,最适反应pH值为5.0;该粗酶在pH3.0～8.0较为稳定。在鱿鱼肝脏自水解过程中,内源性半胱氨酸蛋白酶发挥主要作用。此外,丝氨酸蛋白酶和金属蛋白酶也有一定作用。     

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.     

Effect of storage at 0 °C on mantle proteins and functional properties of jumbo squid

Effect of iced storage of jumbo squid mantle with fin on gelling capacity and changes in protein fractions and functional properties of jumbo squid mantle protein during storage at 0 °C were assessed. Most values of texture variables in gels did not significantly change during storage. On average, they were: strength 65.07 ± 4.71 N; elasticity 68.14 ± 5.3%, fracturability 52.97 ± 1.28 N and cohesiveness 36.6 ± 0.1%. Protein solubility increased more than 40%. Whippability increased during storage for 16 days at 0 °C (81–162%), as did foam stability (73–94%). Results suggest that iced squid mantle protein is a suitable ingredient for food products where these functional properties are desirable. Muscle fibres of squid mantle undergo various changes during storage. At 0 °C, they are disrupted, whereas at −20 °C, they aggregate and develop empty spaces in the tissue.     

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.     

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.     

Effect of dietary protein on muscle collagen,collagenase and shear force of farmed white shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>)

The effect of three protein sources (sardine-based diet, squid-based diet and commercial diet) in feed on white shrimp (Litopenaeus vannamei) growth, muscle collagen, collagenase activity and shear force was determined. Shrimp fed on diets with squid and sardine protein exhibited greater growth (p<0.05) than those fed with commercial feed. Shrimp muscle collagen obtained from each treatment group showed similar molecular weight to that of bovine collagen type I as determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Denaturation thermograms by differential scanning calorimetry (DSC) for shrimp collagen showed a transition peak at 47 °C, whereas that for bovine collagen type I was 65 °C. The lowest enthalpy of transition was detected in collagen from shrimp fed on squid. The highest muscle collagenase activity was detected in shrimp fed with commercial feed. After 10-day ice storage, muscle from shrimp fed commercial feed required somewhat less shear force than those fed with sardine-based and squid-based meal. The results suggest that the source of protein influences the enthalpy of transition of collagen from shrimp muscle, collagenase activity, and texture in shrimp tail meat as well as growth rate.     

Radical scavenging properties of protein hydrolysates from Jumbo flying squid (Dosidicus eschrichitii Steenstrup) skin gelatin

Gelatin extracted from Jumbo flying squid skin was hydrolyzed with serials of protease. The scavenging effects of gelatin hydrolysates on superoxide and hydroxyl radical were assessed by chemiluminescence analysis. Properase E and pepsin have shown efficient hydrolysis of squid skin gelatin to obtain high radical scavenging activity peptides. The conditions chosen for enzymic hydrolysis of squid skin gelatin with properase E were pH 9.0, 45 °C, 3 h reaction time, and enzyme‐to‐substrate ratio of 1:50. Hydrolysates combining properase E and pepsin showed the best radical scavenging activity. For fragmented hydrolysates by ultrafiltration, fractions UF‐2 (M_w < 2000 Da) had high yield and radical scavenging activity. Copyright © 2006 Society of Chemical Industry     

Parameters affecting the isolation of collagen from squid (Illex argentinus) skins

The solubility of collagen from squid (Illex argentinus) skin in salt solutions and the efficiency of removal of skin chromatophores were determined. Homogenization of minced squid skin in 5–15% NaCl solution at 0°C solubilized 35–24% of total amount of crude protein and caused 2–5% loss of collagen but was not effective in removing pigments from the skin. The treatment of whole squid skins in 5–15% NaCl solutions at room temperature led to separation of the chromatophores, but the loss of soluble collagen was 57–16%. Collagen, soluble in dilute acid solutions was isolated from squid skins by 24 h soaking in 10% NaCl solution at room temperature, washing with water and bleaching for 48 h in 1% H₂O₂ in 0.01 M NaOH. The yield of collagen was 53%. It could be increased to 90% by using NaOH solution at pH 11.5 instead of 10% NaCl but the isolate was less soluble in dilute acid and the viscosity of 0.5% dispersion of collagen was four times lower. The rancid off-odour could be prevented by adding 0.5% of a non-ionic detergent to all solution used in the procedure. ©     

鱿鱼肝脏蛋白水解液及ACE 抑制肽的制备

为高效利用鱿鱼及其下脚料肝脏蛋白水解物,采用酶解技术和凝胶过滤分离等技术对鱿鱼肝脏蛋白水解液中抑制肽进行研究。结果表明：胃蛋白酶为鱿鱼肝脏蛋白水解的最佳酶类,同时以水解度和ACE 抑制活性为指标,得出胃蛋白酶水解的最佳条件：在36℃条件下酶解22h,酶与底物的质量比2%,底物质量分数2.5%。经过上述条件处理的水解液再经超滤处理(截留分子质量为20kD)后,用Sephadex G-50 进行分离,洗脱得到5 个峰,其中组分B 的ACE 抑制活性最高,其半抑制浓度(IC50)达到1.80mg/mL。     

秘鲁鱿鱼皮酸溶性胶原蛋白提取工艺优化及其特性

以秘鲁鱿鱼皮为原料,通过单因素试验及响应面优化法得到秘鲁鱿鱼皮酸溶性胶原蛋白（acid soluble collagen,ASC）最佳提取工艺,并对ASC的理化特性进行研究。结果表明：最佳提取工艺为在4 ℃条件下,冰醋 酸浓度0.47 mol/L、液料比24.5 mL/g,浸泡43 h后,ASC提取率为31.76%;紫外全波长扫描结果表明,所得ASC在 231 nm波长处有最大吸收峰,符合Ⅰ型胶原蛋白特征;凝胶电泳分析表明,提取的ASC含有α1、α2和β链;拉曼光 谱分析表明,所得ASC结构完整,三螺旋结构未受到破坏;ASC的相变温度为50.07 ℃。     

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.