Utilisation of collagenolytic enzymes from sierra fish (Scomberomorus sierra) and jumbo squid (Dosidicus gigas) viscera to generate bioactive collagen hydrolysates from jumbo squid muscle.

Crude extracts of collagenases from jumbo squid (Dosidicus gigas) hepatopancreas and sierra fish (Scomberomorus sierra) viscera were used to hydrolyse squid muscle collagen into peptides with inhibitory capacity over angiotensin I-converting enzyme (ACE) and ABTS free radicals [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)], as a measure of their antihypertensive potential and antioxidant activity, respectively. Proteins from 20 to 200 kDa were found in both enzyme extracts; however, in comparison to the jumbo squid extract (JSE), the extraction yield and specific activity of the enzymatic sierra fish extract (SFE) were ≈ 40% greater, suggesting the presence of enzymes with different collagenolytic activity. Moreover, the utilised collagen was obtained with a yield of 0.98 ± 0.09 g/100 g muscle from jumbo squid arms, which after an incubation with JSE and SFE generated peptides with different biological activity. However, the collagen hydrolysates from the enzymatic SFE contained a higher proportion of low-molecular-weight peptides than that obtained from JSE (15.2 and 7.9% of < 3 kDa peptides, respectively). Finally, the antioxidant potential and ACE-inhibitory activity were increased after hydrolysis, being the SFE the one that showed a greater increase of both biological activities (82.28% of ACE inhibition and 64% of ABTS inhibition).

Obtaining glycoconjugates of marine origin via Maillard reaction and their cytotoxic effect: an alternative for the use of animal byproducts.

BACKGROUND: Protein glycation by Maillard reaction is commonly used to improve the functional and bioactive properties of food proteins. It is also known that this glycation method can be accelerated by heat without the need for chemical reagents that could be harmful to health. In this study, glycoconjugates were obtained from a mixture of connective tissue proteins (CTP) from jumbo squid (Dosidicus gigas) and two different sugars, dextran (DEX; 10 kDa) and glucose (GLU), using protein-to-carbohydrate ratios of 1:2 and 1:3, in solution at 50 °C for 6 h. The glycation products were characterized by means of their physicochemical properties and cytotoxic effect. RESULTS: The intensity of the browning measured at A420nm and A294nm in glycoconjugates showed no significant difference (P < 0.05). CTP-DEX (1:2) and CTP-DEX (1:3) were those products with the greatest fluorescence related to the intermediate stage in the Maillard reaction, and also with the highest degree of glycation, which was confirmed using o-phthaldialdehyde assay and Fourier transform infrared analysis. The values of cellular viability for CTP-GLU (1:3), CTP-DEX (1:2, 1:3) as well as CTP (0, 6 h) were around 92-103%. CONCLUSIONS: The operational parameters used in the glycation process achieved the formation of glycoconjugates from proteins of D. gigas, showing no cytotoxic effect on the HaCaT cell line. This research proposes an alternative for the modification of proteins and opens the way to future investigations regarding the bioactivity of these macromolecules to have applications for the use of byproducts in food science and technology. © 2020 Society of Chemical Industry.

Effect of temperature on the actomyosin-paramyosin structure from giant squid mantle (Dosidicus gigas).

BACKGROUND: The secondary structure of a protein determines its functional properties, such as its gelling capacity. The α-helix and ß-sheet comprise its main structures. Myofibrillar proteins from jumbo squid are composed mainly of the actomyosin-paramyosin complex; this complex contains a high percentage of α-helix, because actin, paramyosin, and myosin constitute 30%, 100%, and 55% of the α-helix, respectively. It is important to elucidate the role of the secondary structures in the gelation of giant squid proteins as they form gel. The role of the secondary structures in the gelation of giant squid proteins is therefore very important. For this reason, the objective of this work was to evaluate the effect of temperature on the structural behavior of actomyosin-paramyosin isolate (API) from Dosidicus gigas. RESULTS: The unfolding of the API system, which is composed of the actomyosin-paramyosin complex, was clarified by studying surface hydrophobicity and viscosity. Three characteristic peaks were found, associated with myosin, paramyosin, and actin. Infrared and circular dichroism corroborated the view that API undergoes major structural changes, because it proceeds from mostly an α-helix structure to 100% ß-sheet. CONCLUSION: The structural rearrangement favors gelation by cross-linking, generating new protein-protein and water-protein interactions, which create a more stable structure compared to mantle proteins (MP). Likewise, the presence of sarcoplasmic and stromal proteins in D. gigas muscle prevents the unfolding of myofibrillar proteins, favoring gelation by agglomeration, decreasing the ability to trap water and thus its gelling capacity. © 2019 Society of Chemical Industry.

Muscle lysyl oxidase activity and structural/thermal properties of highly cross-linked collagen in jumbo squid (Dosidicus gigas) mantle, fins and arms.

Muscle from mantle, fins and arms of squid (Dosidicus gigas) were compared based on lysyl oxidase activity (LOX), chemical/structural and thermodynamic properties of highly cross-linked collagen. The arms collagen presented the highest temperature (Tp) and enthalpy of transition. The arms collagen thermic properties may be explained by the higher imino amino acid content, proline and lysine hydroxylation degrees. Moreover, among the regions, the collagen from the arms had a more intense ß band chain, hydroxymerodesmosine peak in the resonance magnetic nuclear spectra and pyridinoline peak in the Raman spectra. Fins showed the highest LOX activity. The LOX activity was associated with the Tp, proline and lysine hydroxylation degrees. These results implied that the collagen in the arms was more intermolecularly ordered than the mantle and fins, and may provide a theoretical basis for a better understanding of the thermal behaviour of squid tissues during management and processing.

Activity of chitosan-lysozyme nanoparticles on the growth, membrane integrity, and ß-1,3-glucanase production by Aspergillus parasiticus.

Synthesis of nanocomposites from antimicrobial biopolymers such as chitosan (CS) and lysozyme (LZ) is an important and promising area in bionanotechnology. Chitosan-lysozyme (CS-LZ) nanoparticles (NPs) were prepared by the nanoprecipitation method, using commercial chitosan of 153 kDa. TEM and dynamic light scattering (DLS) analysis were carried out to evaluate the morphology, size, dispersion, and Z potential. Association efficiency of lysozyme was determined using Coomassie blue assay. The antifungal activity of NPs against Aspergillus parasiticus was evaluated through cell viability (XTT), germination and morphometry of spores, and reducing sugars production; the effects on membrane integrity and cell wall were also analyzed. NPs' size were found in the range of 13.4 and 11.8 nm for CS-LZ and CS NPs, respectively, and high Z potential value was observed in both NPs. Also, high association of lysozyme was presented in the CS matrix. With respect to the biological responses, CS-LZ NPs reduced the viability of A. parasiticus and a strong inhibitory effect on the germination of spores (100% of inhibition) was observed at 24 h in in vitro assays. CS-LZ and CS NPs affected the membrane integrity and the cell wall of spores of fungi with respect to control, which is consistent with the low amount of reducing sugars detected. CS-LZ NPs prepared by nanoprecipitation promise to be a viable and safe alternative for use in biological systems, with a possible low or null impact to humans and biota. However, the potential benefits and the environmental and health implications of NPs need to be globally discussed due to its possible negative effects.

Role of Endogenous and Exogenous Tocopherols in the Lipid Stability of Marine Oil Systems: A Review.

In marine organisms primarily intended for human consumption, the quality of the muscle and the extracted oils may be affected by lipid oxidation during storage, even at low temperatures. This has led to a search for alternatives to maintain quality. In this sense, antioxidant compounds have been used to prevent such lipid deterioration. Among the most used compounds are tocopherols, which, due to their natural origin, have become an excellent alternative to prevent or retard lipid oxidation and maintain the quality of marine products. Tocopherols as antioxidants have been studied both exogenously and endogenously. Exogenous tocopherols are often used by incorporating them into plastic packaging films or adding them directly to fish oil. It has been observed that exogenous tocopherols incorporated in low concentrations maintain the quality of both muscle and the extracted oils during food storage. However, it has been reported that tocopherols applied at higher concentrations act as a prooxidant molecule, probably because their reactions with singlet oxygen may generate free radicals and cause the oxidation of polyunsaturated fatty acids in fish oils. However, when tocopherols are included in a fish diet (endogenous tocopherols), the antioxidant effect on the muscle lipids is more effective due to their incorporation into the membrane lipids, which can help extend the shelf life of seafood by reducing the lipid deterioration that occurs due to antioxidant synergy with other phenolic compounds used supplements in fish muscle. This review focuses on the most important studies in this field and highlights the potential of using tocopherols as antioxidants in marine oils.

An in vitro study of alkaline phosphatase sensitivity to mixture of aflatoxin B1 and fumonisin B1 in the hepatopancreas of coastal lagoon wild and farmed shrimp Litopenaeus vannamei.

This study aimed to establish the combined effect of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) on wild Litopenaeus vannamei hepatopancreas alkaline phosphatase (AP) activity compared with that of farmed shrimp. AP activity in hepatopancreas extract was confirmed by several specific inhibitor assays. AP activity of wild shrimp was higher than that of farmed shrimp (p < 0.05). However, AP activity from both wild and farmed shrimp was inhibited when incubated with AFB1 and FB1. The greatest inhibition occurred when AP was incubated with a mixture of AFB1 and FB1. The IC50 for AFB1 on AP activity of wild and farmed shrimp hepatopancreases was 0.790 and 0.398 µg/mL, respectively. The IC50 of FB1 was 0.87 µg/mL for wild shrimp and 0.69 µg/mL for farmed shrimp. These results suggest that, at the mycotoxins concentrations used in the study, AP from farmed L. vannamei was sensitive to the presence of both mycotoxins; however, AP is more sensitive to the combination of AFB1 + FB1 suggesting a possible synergistic or potentiating inhibitory effect.

Chymotrypsin isolation from jumbo squid (Dosidicus gigas) 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.

Effect of a low-density polyethylene film containing butylated hydroxytoluene on lipid oxidation and protein quality of Sierra fish (Scomberomorus sierra) muscle during frozen storage.

Fresh sierra fish (Scomberomorus sierra) fillets were packed in low-density polyethylene films with butylated hydroxytoluene (BHT-LDPE) added. Fillets packed in LDPE with no BHT were used as controls (LDPE). The packed fillets were stored at -25 degrees C for 120 days in which the film released 66.5% of the antioxidant. The influence of the antioxidant on lipid and protein quality, lipid oxidation, muscle structure changes, and shear-force resistance was recorded. As compared to LDPE films, fillets packed in BHT-LDPE films showed lower lipid oxidation, thiobarbituric acid values (4.20 +/- 0.52 vs 11.95 +/- 1.06 mg malonaldehyde/kg), peroxide values (7.20 +/- 1.38 vs 15.15 +/- 1.48 meq/kg), and free fatty acids (7.98 +/- 0.43 vs 11.83 +/- 1.26% of oleic acid). Fillets packed in BHT-LDPE films showed less tissue damage and lost less firmness than fillets packed in LDPE. A significant relationship between lipid oxidation and texture was detected (R2 adjusted, 0.70-0.73). BHT-LDPE films may be used not only to prevent lipid oxidation but also to minimize protein damage to prolong the shelf life of sierra fish.