Ahmed Barkia

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

UNLABELLED 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.

UNLABELLED 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 β-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.

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.

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.

Unfavorable apoAI-containing lipoproteins profile in Tunisian obese women group

Medicinal plants are important elements of indigenous medical system that have persisted in developing countries. Many of the pharmacological principles currently used as anticancer agents were first isolated from plants. However, some important anticancer agents are still extracted from plants because they cannot be synthesized chemically on a commercial scale due to their complex structures that often contain several chiral centers. The aim of this study was to test different extracts from the leaves of Moringa or drumstick tree ( Moringa oleifera ) for activity against leukemia and hepatocarcinoma cells in vitro . The extracts could kill majority (70 - 86%) of the abnormal cells among primary cells harvested from 10 patients with acute lymphoblastic leukemia (ALL) and 15 with acute myeloid leukemia (AML) as well as a culture of hepatocarcinoma cells (75% death), but most significantly by the hot water and ethanol extracts. In conclusion, M. oleifera may have potential for use as source of natural treatment for diseases such as cancer.