Composition, functional properties and in vitro antioxidant activity of protein hydrolysates prepared from sardinelle (Sardinella aurita) muscle.

Composition, functional properties and in vitro antioxidative activities of protein hydrolysates prepared from muscle of sardinelle (Sardinella aurita) were investigated. Sardinelle protein hydrolysates (SPH) were obtained by treatment with crude enzyme preparations from Bacillus pumilus A1 (SPHA1), Bacillus mojavensis A21 (SPHA21) and crude enzyme extract from sardinelle (Sardinella aurita) viscera (SPHEE). The protein hydrolysates SPHA1, SPHA21 and SPHEE contained high protein content 79.1%, 78.25% and 74.37%, respectively. The protein hydrolysates had an excellent solubility and possessed interfacial properties, which were governed by their concentrations. The antioxidant activities of protein hydrolysates at different concentrations were evaluated using various in vitro antioxidant assays, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power assay, chelating activity, ß-carotene bleaching and DNA nicking assay. All protein hydrolysates showed varying degrees of antioxidant activity. SPHA21 had the highest DPPH radical scavenging activity (89% at 6 mg/ml) and higher ability to prevent bleaching of ß-carotene than SPHA1 and SPHEE (p < 0.05). However, SPHEE exhibited the highest metal chelating activity (89% at 1 mg/ml) and the strongest protection against hydroxyl radical induced DNA breakage (p < 0.05).

Protective effects of thornback ray muscle protein hydrolysate against dyslipidemia, oxidative stress and reduced fertility induced by high cholesterol diet in adult male rats.

Enzymatic thornback ray (Raja clavata) muscle hydrolysates have been shown to have antioxidant and antihypertensive activities in vitro. The Neutrase hydrolysate exhibited the highest activities, so it was investigated along with the undigested muscle to test their hypolipidemic, antioxidative and fertility effects in rats fed with a high-cholesterol diet (HCD). Animals were allocated into four groups of 5 rats each: a normal diet group (control), a HCD group, and two groups of HCD with a daily dose of undigested muscle (Und) or the hydrolysate (MH) at 0.7 g kg-1 of body weight. All animals received their respective treatments daily for 1 month. After the treatment period, serum lipid profiles, the activities of alanine aminotransferase and aspartate aminotransferase, the level of malonaldehyde, the activities of antioxidant enzymes (catalase and glutathione peroxidase) in the liver and sperm fertility parameters (in the epididymis and testis) were determined. Compared with those fed a standard diet, HCD induced dyslipidemia and oxidative stress, and decreased numerous reproductive parameters (mobility, count and viability). Interestingly, supplementing the HCD with thornback ray proteins attenuated all these anomalies, especially in the case where they were hydrolysed. These observations suggested that these proteins might contain bioactive peptides that possess hypocholesterolemic and antioxidant activities that ameliorate sperm damage.

Digestive alkaline proteases from thornback ray (Raja clavata): Characteristics and applications.

This study describes the characterization of a crude protease extract from thornback ray (Raja clavata) and its evaluation in liquid detergent and in deproteinizattion of shrimp waste. At least five clear caseinolytic proteases bands were observed in a zymogram. The crude protease showed optimum activity at pH 8.0 and 50 °C, and it was highly stable over pH range from 8.0 to 11.0. Proteolytic enzymes were very stable in non-ionic surfactants and in the presence of oxidizing agents, maintaining 70% of their activity after incubation for 1 h at 30 °C in the presence of 1% sodium perborate. In addition, they showed high stability and compatibility with various liquid laundry-detergents available in the Tunisian market. The crude extract retained 100% of its activity after preincubation for 60 min at 30 °C in the presence of Nadhif Perfect, Textil and Carrefour laundry detergents. Further, proteases from R. clavata viscera were used for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3 h hydrolysis at 45 °C with an enzyme/substrate ratio of 30 U/mg of proteins was 74%. These results suggest that enzymatic deproteinization of shrimp wastes by fish endogenous alkaline proteases could be applicable to the chitin production process.

Bioactive peptides identified in thornback ray skin's gelatin hydrolysates by proteases from Bacillus subtilis and Bacillus amyloliquefaciens.

Thornback ray skin gelatin has been hydrolyzed with two different proteases in order to obtain peptides with ACE inhibitory and antioxidant activity. Hydrolysates with protease from Bacillus subtilis A26 (TRGH-A26) displayed ACE inhibitory activity with an IC50 value of 0.94 µg/µL whereas Neutrase® hydrolysate from Bacillus amyloliquefaciens (TRGH-Neutrase) showed an IC50 value of 2.07 µg/µL. Regarding antioxidant activity, IC50 values of 1.98 and 21.2 µg/µL in TRGH-A26 and TRGH-Neutrase, respectively, were obtained using the DPPH radical-scavenging assay. The most active fractions identified by size-exclusion chromatography were further purified by RP-HPLC and analysed using nanoESI-LC-MS/MS to identify the sequence of the peptides. APGAP was the most active peptide inTRGH-A26 for ACE inhibitory activity with an IC50 value of 170 µM, whereas GIPGAP showed the best ACE inhibitory activity in TRGH-Neutrase sample with an IC50 value of 27.9 µM. The highest antioxidant activity was identified in peptide AVGAT, showing a 33% of activity at 3mg/mL using the DPPH radical-scavenging assay. The obtained results proved the potential of thornback ray skin gelatin hydrolysates as a source of bioactive peptides. STATEMENT OF SIGNIFICANCE: This study describes a peptidomic approach for the identification of ACE-inhibitory and antioxidant peptides generated from thornback ray gelatin (Raja clavata) hydrolysates from Bacillus subtilis A26 and Bacillus amyloliquefaciens Neutrase® enzymes and expose the potential of thornback ray gelatin hydrolysate as a source of bioactive peptides. In this sense, the decrease of systolic blood pressure is one of the main measurements considered in public health for the treatment of cardiovascular diseases, stroke and even end-stage renal disease. Traditionally, synthetic drugs such as captopril and enalapril have been used as ACE inhibitors despite their secondary effects, but the finding of new sources for the generation of natural bioactive peptides such as thornback ray muscle results is very important in the knowledge of less hostile but highly effective antihypertensive peptides as well as the development of new uses for waste and by-products generated from marine products, helping to solve the already existing environmental problem affecting this industry.

Characterization, antioxidative and ACE inhibitory properties of hydrolysates obtained from thornback ray (Raja clavata) muscle.

Thornback ray muscle hydrolysates (TRMHs) prepared by treatment with proteases from Bacillus subtilis A26 (TRMH-A26), Raja clavata crude alkaline protease extract (TRMH-Crude), Alcalase (TRMH-Alcalase) and Neutrase (TRMH-Neutrase) were elaborated and their antioxidant properties and angiotensin I-converting enzyme (ACE) inhibitory activities were tested. TRMHs showed different degrees of hydrolysis (DH from 11 to 22%) and hydrophobic/hydrophilic peptide ratio. Protein content varied from 71 to 74%. Gly, Pro, Asp and Asn were the most prominent amino acids, while hypoxanthine was the major nucleotide related compound present. The antioxidant activity was assayed using various tests. TRMH-Neutrase exhibited the highest antioxidant activity in DPPH scavenging, reducing power and inhibition of ß-carotene bleaching tests. However in the total antioxidative efficacy, TRMH-Crude exhibited the highest activity. TRMH-Crude and TRMH-Neutrase were the most potent to prevent DNA oxidation by Fenton reagent. Concerning anti-ACE activity, TRMH-A26 and TRMH-Neutrase exhibited the highest activity with 87% at 5mg/ml. The results revealed that TRMHs could be employed as a protein source in food additive processing or diets for aquatic organisms and other farmed animals. BIOLOGICAL SIGNIFICANCE: The present study explores for the first time the elaboration of enzymatic hydrolysates from thornback ray R. clavata. The hydrolysates are well characterized and showed an interesting protein content as well as the presence of nucleotide related compounds, essential amino acids and taurine, which make them an interesting source of fish meal in aquaculture feeds. The hydrolysates were found to exhibit ACE inhibitory activity and antioxidant activity. The hydrolysates could serve also as a potential protein source for functional foods.

Characterization and potential use of cuttlefish skin gelatin hydrolysates prepared by different microbial proteases.

Composition, functional properties, and in vitro antioxidant activities of gelatin hydrolysates prepared from cuttlefish skin were investigated. Cuttlefish skin gelatin hydrolysates (CSGHs) were obtained by treatment with crude enzyme preparations from Bacillus licheniformis NH1, Bacillus mojavensis A21, Bacillus subtilis A26, and commercial alcalase. All CSGHs had high protein contents, 74.3-78.3%, and showed excellent solubility (over 90%). CSGH obtained by alcalase demonstrated high antioxidant activities monitored by ß-carotene bleaching, DPPH radical scavenging, lipid peroxidation inhibition, and reducing power activity. Its antioxidant activity remained stable or increased in a wide range of pH (1-9), during heating treatment (100°C for 240 min) and after gastrointestinal digestion simulation. In addition, alcalase-CSGH was incorporated into turkey meat sausage to determine its effect on lipid oxidation during 35 days of storage period. At 0.5 mg/g, alcalase-CSGH delayed lipid oxidation monitored by TBARS and conjugated diene up to 10 days compared to vitamin C. The results reveal that CSGHs could be used as food additives possessing both antioxidant activity and functional properties.

The influence of the extent of enzymatic hydrolysis on antioxidative properties and ACE-inhibitory activities of protein hydrolysates from goby (Zosterisessor ophiocephalus) muscle.

Antioxidant properties and angiotensin-converting enzyme (ACE) inhibitory activities of protein hydrolysates from goby (Zosterisessor ophiocephalus) muscle, with different degrees of hydrolysis (DH) from 5 to 25%, prepared by treatment with crude proteases extract from smooth hound intestines, were investigated. Goby protein hydrolysates (GPHs) are rich in Gly and Thr, which accounted for 14.1-15% and 11.6-13.2% of the total amino acids, respectively. The antioxidant activities of GPHs were investigated by using several in vitro assay systems. All GPHs exhibited significant metal chelating activity and DPPH free radical-scavenging activity, and inhibited linoleic acid peroxidation. For the ACE-inhibitory activity, as the DH increased, the activity of GPHs increased. The obtained results revealed that antioxidant and ACE-inhibitory activities of GPHs were influenced by the degree of hydrolysis. A medium degree of enzymatic hydrolysis was appropriate to obtain GPHs with good antioxidant activity, while small peptides were essential to obtain high ACE inhibitory activity.

Physical, structural, antioxidant and antimicrobial properties of gelatin-chitosan composite edible films.

Physico-chemical and mechanical properties of cuttlefish skin gelatin (G), chitosan (C) from shrimp (Penaeus kerathurus) and composite films (G75/C25, G50/C50, G25/C75) plasticized with glycerol were investigated. The results indicated that chitosan film had higher tensile strength and lower elongation at break when compared with the other films. Composite films show no significant difference in tensile strength (TS), thickness and transparency. The structural properties evaluated by FTIR and DSC showed total miscibility between both polymers. DSC scans showed that the increase of chitosan content in the composite films increases the transition temperature (Tg) and enthalpy (ΔHg) of films. The morphology study of gelatin, chitosan and composite films showed a compact and homogenous structure. In addition, gelatin and G75/C25 films demonstrated a high antioxidant activities monitored by ß-carotene bleaching, DPPH radical-scavenging and reducing power activities, while films contained chitosan exhibited higher antimicrobial activity against Gram-positive than Gram-negative bacteria.

Evaluation of hypocholesterolemic effect and antioxidant activity of Boops boops proteins in cholesterol-fed rats.

Dietary proteins affect blood cholesterol concentrations and antioxidant status, which are related to several diseases, including cardiovascular disease. The present study attempts to investigate the potential of Boops boops proteins (Bb-NHP) and its hydrolysate (Bb-HP) in the prevention of hypercholesterolemia and oxidative stress in rats fed a high cholesterol diet (HCD). After four weeks' treatment, serum lipid profiles (total cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol), the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the level of malonaldehyde (MDA) and the activities of antioxidant enzymes [catalase (CAT) and glutathione peroxidase (GPx)] in liver were determined. Compared with those fed a standard diet, high cholesterol diet induced dyslipidemia, oxidative stress, and aortic structure alterations. Interestingly, supplementing the HCD with Boops boops proteins attenuated these anomalies in a dose-dependent manner. These observations suggested that B. boops proteins might provide health benefits by helping to reduce the deleterious effects of increased intake of cholesterol that characterize modern diets.

Digestive Alkaline Proteases from Zosterisessor ophiocephalus, Raja clavata, and Scorpaena scrofa: Characteristics and Application in Chitin Extraction.

The aim of this work was to study some biochemical characteristics of crude alkaline protease extracts from the viscera of goby (Zosterisessor ophiocephalus), thornback ray (Raja clavata), and scorpionfish (Scorpaena scrofa), and to investigate their applications in the deproteinization of shrimp wastes. At least four caseinolytic proteases bands were observed in zymogram of each enzyme preparation. The optimum pH for enzymatic extracts activities of Z. ophiocephalus, R. clavata, and S. scrofa were 8.0-9.0, 8.0, and 10.0, respectively. Interestingly, all the enzyme preparations were highly stable over a wide range of pH from 6.0 to 11.0. The optimum temperatures for enzyme activity were 50°C for Z. ophiocephalus and R. clavata and 55°C for S. scrofa crude alkaline proteases. Proteolytic enzymes showed high stability towards non-ionic surfactants (5% Tween 20, Tween 80, and Triton X-100). In addition, crude proteases of S. scrofa, R. clavata, and Z. ophiocephalus were found to be highly stable towards oxidizing agents, retaining 100%, 70%, and 66%, respectively, of their initial activity after incubation for 1 h in the presence of 1% sodium perborate. They were, however, highly affected by the anionic surfactant SDS. The crude alkaline proteases were tested for the deproteinization of shrimp waste in the preparation of chitin. All proteases were found to be effective in the deproteinization of shrimp waste. The protein removals after 3 h of hydrolysis at 45°C with an enzyme/substrate ratio (E/S) of 10 were about 76%, 76%, and 80%, for Z. ophiocephalus, R. clavata, and S. scrofa crude proteases, respectively. These results suggest that enzymatic deproteinization of shrimp wastes by fish endogenous alkaline proteases could be applicable to the chitin production process.