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.

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.

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.

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.

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.

Change of diet, plasma lipids, lipoproteins, and fatty acids during Ramadan: a controversial association of the considered Ramadan model with atherosclerosis risk.

Different Islamic populations have different alimentary habits, notably during Ramadan. The paper reports the change of diet, lipids, and lipoproteins produced during Ramadan in one Tunisian population. During Ramadan, the study subjects consumed more proteins, cholesterol, vitamin E (p<0.01), and polyunsaturated fatty acids (p<0.05). At the same time, they exhibited an increase in total cholesterol, low-density lipoprotein-cholesterol (p<0.01) and apoprotein B (p<0.05) and a decrease in the ratio of apoprotein AI to apoprotein B (p<0.01). All assayed saturated fatty acids were unaffected by Ramadan fasting while three unsaturated fatty acids (C18:1cis9, C18:2n-6, and C30:4n-6) increased significantly. A return to the habitual diet for a four-week period was not sufficient to restore the pre-fasting patterns. For the study subjects, Ramadan was clearly associated with a change of diet and biochemical profile but its effective impact on atherosclerosis risk was unclear, perhaps, because other non-alimentary changes ought to be considered too. Future studies considering the no-alimentary factors, such as sleep and physical activity, would be useful to clarify the contribution of dietary change in the observed modification of biological profile.

Purification and characterization of three trypsin isoforms from viscera of sardinelle (Sardinella aurita).

Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20-70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55°C, while trypsin B at 50°C. All isoforms showed the same optimal pH (pH 9.0) using Nα-benzoyl-DL: -arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40°C and over a broad pH range (7.0-11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25°C and pH 9.0. The values of K (m) and k (cat) were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 s(-1), respectively. The N-terminal sequences of the first 10 amino acids were "I V G G Y E C Q K Y" for trypsin A and "I V G G Y E A Q S Y" for trypsins B and C. These sequences showed highly homology to other fish trypsins.

Influence of degree of hydrolysis on functional properties and angiotensin I-converting enzyme-inhibitory activity of protein hydrolysates from cuttlefish (Sepia officinalis) by-products.

BACKGROUND: In Tunisia the cuttlefish-processing industry generates large amounts of solid wastes. These wastes, which may represent 35% of the original material and constitute an important source of proteins, are discarded without any attempt at recovery. This paper describes some functional properties and the angiotensin I-converting enzyme (ACE)-inhibitory activity of protein hydrolysates prepared by hydrolysis of cuttlefish (Sepia officinalis) by-products with crude enzyme extract from Bacillus licheniformis NH1. RESULTS: Cuttlefish by-product protein hydrolysates (CPHs) with different degrees of hydrolysis (DH 5, 10 and 13.5%) were prepared. All CPHs contained 750-790 g kg(-1) proteins. Solubility, emulsifying capacity and water-holding capacity increased while fat absorption and foaming capacity decreased with increasing DH. All hydrolysates showed greater fat absorption than the water-soluble fraction from undigested cuttlefish by-product proteins and casein. CPHs were also analysed for their ACE-inhibitory activity. CPH3 (DH 13.5%) displayed the highest ACE inhibition (79%), with an IC(50) value of 1 mg mL(-1). CONCLUSION: Hydrolysis of cuttlefish by-product proteins with alkaline proteases from B. licheniformis resulted in a product with excellent solubility over a wide pH range and high ACE-inhibitory activity. This study suggests that CPHs could be utilised to develop functional foods for prevention of hypertension.

Trypsin from the viscera of Bogue (Boops boops): isolation and characterisation.

Trypsin from the viscera of Bogue (Boops boops) was purified to homogeneity by precipitation with ammonium sulphate, Sephadex G-100 gel filtration and Mono Q-Sepharose anion exchange chromatography, with an 8.5-fold increase in specific activity and 36% recovery. The molecular weight of the purified enzyme was estimated to be 23 kDa by SDS-PAGE and size exclusion chromatography. The purified trypsin appeared as a single band on native-PAGE and zymography staining. The purified enzyme showed esterase-specific activity on N-α-benzoyl-L-arginine ethyl ester (BAEE) and amidase activity on N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity, after 10 min incubation, were pH 9.0 and 55°C, respectively, using BAPNA as a substrate. The trypsin kinetic constants Km and kcat on BAPNA were 0.13 mM and 1.56 s(-1), respectively, while the catalytic efficiency kcat/Km was 12 s(-1) mM(-1). Biochemical characterisation of B. boops trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish processing and food industries.

[Biologic profile in Tunisian infants (0-2 years), malnourished]. / Profil biologique d'une population de nourrissons Tunisiens (0-2 ans), malnutris.

BACKGROUND: The malnutrition of the infants could be explained by a delay of the growth and the perturbation of biological parameters. AIM: To establish the nutritional profile of the Tunisian infant of less than two years. To specify the principal deficiencies and the possible origins of these deficiencies. METHODS: In our transverse exploratory study, carried out in period of 9 month. This study was conducted in two groups of Tunisian young children less than two years old: a control group and a malnourished group (Z score < or = 2SD). RESULTS: Our data consolidate the important impact of pregnant women nutritional state and of breastfeeding on the foetus ant infant growth.Compared to control infants, the malnourished young showed a significant alteration of different biologic parameters. This alteration appeared positively linked to the gravity of malnutrition as indicated by the positive relation obtained between the weight/height ratio and many studied parameters. The malnourished infants showed, notably, a significant reduction of the average values of Chol-HDL, apo AI, Vit E, TSH and TF4 levels and Chol-HDL/Chol LDL ratio. Chol-HDL, apo AI and HDL/Chol-LDL are found positively and significantly correlated with TF4. So, their reduction in ill children would be, at least in part, a side effect of the thyroid function reduction. CONCLUSION: Our results confirm the existence of an important change of biological profile in malnourished young children. Besides, they emphasize that studies about young children could be helpful, notably, in the prevention and the fight against atherosclerosis.

Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases.

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from heads and viscera of sardinelle (Sardinella aurita) by treatment with various proteases were investigated. Protein hydrolysates were obtained by treatment with Alcalase(®), chymotrypsin, crude enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina pilchardus) viscera. All hydrolysates exhibited inhibitory activity towards ACE. The alkaline protease extract from the viscera of sardine produced hydrolysate with the highest ACE inhibitory activity (63.2±1.5% at 2mg/ml). Further, the degrees of hydrolysis and the inhibitory activities of ACE increased with increasing proteolysis time. The protein hydrolysate generated with alkaline proteases from the viscera of sardine was then fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P1-P8). Biological functions of all fractions were assayed, and P4 was found to display a high ACE inhibitory activity. The IC50 values for ACE inhibitory activities of sardinelle by-products protein hydrolysates and fraction P4 were 1.2±0.09 and 0.81±0.013mg/ml, respectively. Further, P4 showed resistance to in vitro digestion by gastrointestinal proteases. The amino acid analysis by GC/MS showed that P4 was rich in phenylalanine, arginine, glycine, leucine, methionine, histidine and tyrosine. The added-value of sardinelle by-products may be improved by enzymatic treatment with visceral serine proteases from sardine.