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.     

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.     

Aminopeptidase from jumbo squid (Dosidicus gigas) hepatopancreas: purification,characterisation, and casein hydrolysis

An aminopeptidase (AP) was partially purified from jumbo squid (Dosidicus gigas) hepatopancreas with 154.24‐fold and yield of 6.15%. The purification procedure consisted of ammonium sulphate fractionation and DEAE‐Sephacel chromatography. The enzyme was approximately 48–53 kDa as estimated by SDS‐PAGE. With l ‐leu‐p‐NA, it had optimum activity at pH 8.0 and 30 °C. The K_m and V_max/K_m values of the enzymes for l ‐leu‐p‐NA were 0.326 mm and 2787 at 37 °C, respectively. Activation energy (E_a) of the enzyme was 53.50 kJ M^?1.The AP showed activity against seven synthetic substrates: l ‐proline>l ‐methionine>Ac. l ‐γ‐glutamic>l ‐glycine>l ‐leucine>l ‐alanine>l ‐lysine‐p‐NA. The enzyme was strongly inhibited by Bestatin, partially inhibited by a metal‐chelating agent and by PCMB, a cystein protease inhibitor. Zn²⁺ and (or) Ca²⁺ seemed to be its metal cofactor(s). Incubation of casein with the partially purified AP resulted in a degree of hydrolysis of 6%.     

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.     

Chymotrypsin isolation from jumbo squid (<Emphasis Type="Italic">Dosidicus gigas</Emphasis>) hepatopancreas: Partial characterization and effect on muscle collagen

Chymotrypsin was purified from jumbo squid hepatopancreas (HP) with 2.4-fold and yield 1.9%, and characterized with a molecular weight of 31 kDa, as estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Chymotrypsin effect over collagen extracted from the mantle, fins and arms of the jumbo squid was evaluated. The enzyme exhibited the maximum activity at pH 7 and 65°C using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as a substrate and it was identified using the specific inhibitors N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and phenyl methyl sulfonyl fluoride (PMSF), showing residual activities of 6% and 0%, respectively. Furthermore, high activity was observed in the pH range of 4.0 to 8.0. Purified enzyme showed a moderate in vitro activity using muscle collagen as a substrate. Although further research is needed, the results suggest that the enzyme has a potential application where acidic or slightly alkaline conditions are needed.     

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.     

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.     

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     

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.     

Changes in firmness and thermal behavior of ice-stored muscle of jumbo squid (<Emphasis Type="Italic">Dosidicus gigas</Emphasis>)

Changes in firmness, muscle total protease activity, and the thermal behavior of jumbo squid (Dosidicus gigas) were measured throughout 15 days of ice-storage. A significant decrease (p<0.05) in the shear force of raw mantle muscle was observed after 7-days ice-storage. The highest total protease activity detected was 1.24 U/g mantle. The thermograms obtained at day zero showed four transition states. The first three transition states were endothermic and correspond to myosin (50 °C), sarcoplasmic proteins (69 °C), and actin (79 °C). The fourth transition state was exothermic at 107 °C, and was probably associated with protein aggregation. The thermal behavior of the muscle showed a decreasing trend in temperature and enthalpy of transition for myosin, sarcoplasmic proteins, and actin with storage time. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a change in the myofibrillar protein pattern, which with the shear force, and differential scanning calorimetry data, suggests a partial denaturation of that protein fraction during ice-storage.     

Preparation of alcalase hydrolysed rice bran protein concentrate and its inhibitory effect on soybean lipoxygenase activity

Rice bran protein concentrate (RBPC) was prepared from defatted rice bran and hydrolysed by alcalase at different hydrolysis times. As the hydrolysis times increased, the degree of hydrolysis (DHs) increased. RBPC hydrolysate obtained at 50 min (RBPCH‐50) had the highest inhibitory efficiency on soybean lipoxygenase (LOX) activity (66%). The inhibition kinetics of the reaction analysed by Lineweaver–Burk plots indicates that RBPCH‐50 is a competitive inhibitor. RBPCH‐50 inhibited soybean LOX with an IC₅₀ of 11.73 μg μL^?1 RBPCH‐50, and the obtained K_I was 4.59 μg μL^?1 RBPCH‐50. LOX inhibitory activity of RBPCH‐50 was significantly higher than that of 50 and 100 ppm butylated hydroxyanisole (BHA) and 50, 100, and 200 ppm butylated hydroxytoluene (BHT) (P ≤ 0.05); however, LOX inhibitory activity of RBPCH‐50 was similar to that of 200 ppm BHA (P > 0.05). Therefore, RBPCH might potentially be used as a natural LOX inhibitor.     

Mass Transfer Modelling During Osmotic Dehydration of Jumbo Squid (Dosidicus gigas): Influence of Temperature on Diffusion Coefficients and Kinetic Parameters

Mathematical modelling was used to study the effect of process temperature on moisture and salt mass transfer during osmotic dehydration (OD) of jumbo squid with 6% (w v ⁻¹) NaCl at 75, 85 and 95 °C. The diffusion coefficients for moisture and salt increased with temperature. Based on an Arrhenius-type equation, activation energy values of 62.45 kJ mol⁻¹ and 52.14 kJ mol⁻¹ for moisture and salt, respectively, were estimated. Simulations of mass transfer for both components were performed according to Newton, Henderson and Pabis, Page, Weibull and logarithmic mathematical expressions. The influence of drying temperature on the kinetic parameters was also studied. Based on statistical tests, the Weibull and logarithmic models were the most suitable to describe the mass transfer phenomena during OD of jumbo squid.     

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.     

Lipid Oxidation in Microsomal Fraction of Squid Muscle (Loligo peali)

Frozen squid is susceptible to both lipid oxidation and yellow/brown discoloration during frozen storage. The involvement of lipid oxidation in the microsomal fraction of squid muscle on oxidative rancidity and discoloration was investigated using iron and either enzymatic or non‐enzymatic redox cycling pathways. Lipid oxidation was measured by thiobarbituric acid‐reactive substances (TBARS), and color changes were measured spectrophotometrically using an integrating sphere. The lipid oxidation was not observed in the squid microsomes in the presence of Fe³⁺ and β‐nicotinamide adenine dinucleotide disodium salt (NADH) or β‐nicotinamide adenine dinucleotide phosphate, reduced (NADPH), suggesting that the enzymatic redox cycling pathway was not active. Iron‐promoted TBARS formation was observed in the non‐enzymatic pathway when ascorbic acid was used as a reducing compound. Non‐enzymatic lipid oxidation increased with increasing temperature (4 °C to 37 °C), iron (0 to 100 μM), and ascorbic acid (0 to 200 μM) concentrations. As lipid oxidation in the microsomes or isolated microsomal lipids increased, color changes were observed as could be seen by an increase in b* values (yellowness) and a decrease in a* (redness) values. The ability of iron and ascorbate to promote both lipid oxidation and pigment formation in the microsomal fraction suggests that this pathway could be responsible for quality deterioration of squid muscle during storage.     

Effects of hydroxyl radical induced oxidation on water holding capacity and protein structure of jumbo squid (Dosidicus gigas) mantle

Protein oxidation is considered as an important issue in food preservation process. In the present study, the potential influence of protein oxidation on water holding capacity and protein structure of jumbo squid (Dosidicus gigas) mantle was investigated. After the hydroxyl radical oxidation, it was found that the carbonyl, surface hydrophobicity and dityrosine content of myofibrillar protein significantly increased (P < 0.05), while the content of total sulphydryl decreased significantly (P < 0.01). Meanwhile, the fluorescence intensity of squid was weakened, and the maximum absorption peak of fluorescence red shift as the H₂O₂ concentration increased. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that not only the protein cross-linking but also degradation could have occurred. The content of α-helix decreased, the content of β-sheet, β-turn and the unordered structures increased after oxidation. In addition, oxidation resulted in a decrease in water holding capacity. Taken together, oxidation resulted in the damage of the myofibrillar structure, the increase in muscle loss rate and the decrease in water holding capacity.     

Isolation and properties of 5′-nucleotidase isolated from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California,Mexico

The enzyme 5′-nucleotidase of jumbo squid (Dosidicus gigas) mantle was purified and its SDS–PAGE showed a single band of 33 kDa, whereas a protein with a molecular mass of 107 kDa was detected by gel filtration suggesting a homotrimeric nature of this enzyme. Subunits of the named enzyme were not linked by covalent bonds. Isoelectric focusing of this enzyme showed a pI of 3.6–3.8 and presented a hyperbolic kinetics with V_max of 1.16 μM/min/mg of protein, K_m of 1.49 mM, K_cat of 3.48 μM of P_ι s⁻¹ and K_cat/K_m relation of 356.52 ((mol/L)⁻¹ s⁻¹). Purified enzyme preferred AMP as substrate (by 6.7-folds) than IMP, showing a K_m of 6.34 mM, V_max of 0.19 μM/min/mg of protein a K_cat of 0.3388 mol of P_ι s⁻¹ and K_cat/K_m relation of 53.44 ((mol/L)⁻¹ s⁻¹). The low K_m in relation to purified AMP deaminase of the same organism suggested a high contribution of 5′-nucleotidase in AMP degradation in jumbo squid mantle.     

The Interaction Between Lipoxygenase‐Catalyzed Oxidation and Autoxidation in Dry‐Cured Bacon and a Model System

A model system was conducted to characterize the interaction between lipid autoxidation and enzyme‐catalyzed oxidation in dry‐cured bacon. This involved the use of a hydroxyl radical (HO?) generating system and the extraction and purification of lipoxygenases (LOX) from pork belly. The results showed that LOX activity rapidly (P < 0.05) increased during the curing of dry‐cured bacon. This may be because of the hydroxyl‐radical‐mediated oxidation from LOX‐Fe²⁺ to LOX‐Fe³⁺, which activates LOX. In addition, experiments of the model system also showed that LOX activity could be inhibited by increasing the substrate concentration, although substrate type and concentration had no effect on autoxidation. Moreover, LOX enzyme‐catalyzed oxidation and autoxidation could act synergistically to promote lipid oxidation irrespective of the substrate (linoleic or arachidonic acid). These results provide useful information for regulating lipid oxidation during the production of dry‐cured pork products.     

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.     

Nonthermal Inactivation of Soy (Glycine Max Sp.) Lipoxygenase by Pulsed Ultraviolet Light

This study investigated pulsed ultraviolet (PUV) illumination at different distances from the PUV source on soybean lipoxygenase (LOX) (0.4 mg/mL in 0.01 M Tris‐HCl buffer, pH 9) activity. Samples (5 mL) were illuminated for 1, 2, 4, 8, and 16 s at 3 distances 6, 8.5, and 11 cm from the PUV lamp's quartz window. The temperature of 33.5 ± 1.8°C was observed for the highest treatment time of 16 s at the shortest distance of 6 cm, and resulted in a 3.5 log reduction (99.95%) in initial LOX activity. Illumination time and distance from the lamp significantly (P ≤ 0.05) affected LOX inactivation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) was performed on treated LOX samples and further protein profile for treated LOX filtrate (≤10 kDa), was analyzed by reverse phase high‐performance liquid chromatography (RP‐HPLC). The protein profile analysis revealed that LOX protein degradation was influenced significantly (P ≤ 0.05) by PUV illumination time.     

Determination of the veterinary drug maduramicin in food by fluorescence polarisation immunoassay

A fluorescence polarisation immunoassay (FPIA) using a specific polyclonal antiserum for the detection of maduramicin (MD) was developed and optimised. The polyclonal antiserum was produced against MD linked to bovine serum albumin. Fluorescein‐labelled MD (tracer) was synthesised by N‐hydroxysuccinimide active ester method and purified using thin layer chromatography. The developed FPIA for MD had a dynamic range from 0.01 to 5.6 μg mL^?1 with an IC₅₀ value of 0.16 μg mL^?1 and a limit of detection of 0.002 μg mL^?1. Recoveries from chicken muscle, fat and egg samples spiked at 0.25, 5 and 10 μg g^?1 levels were 82–130%. The FPIA results from analysis of incurred residues in chicken muscle samples showed that the simple procedure is viable.