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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 5 mg/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.

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 IC 50 value of 0.94 μg/μL whereas Neutrase® hydrolysate from Bacillus amyloliquefaciens (TRGH-Neutrase) showed an IC 50 value of 2.07 μg/μL. Regarding antioxidant activity, IC 50 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 IC 50 value of 170 μM, whereas GIPGAP showed the best ACE inhibitory activity in TRGH-Neutrase sample with an IC 50 value of 27.9 μM. The highest antioxidant activity was identified in peptide AVGAT, showing a 33% of activity at 3 mg/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.

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.

Potential utilization of skin by-product from thornback ray ( Raja clavata ) was investigated. Gelatin from alkali-pretreated skin was extracted without or with pepsin at pH 2.0 in 0.2 M acetic acid or 0.1 M glycine-HCl buffer. The addition of pepsin increased the yields of gelatin. Indeed, the yields of gelatin from thornbach ray obtained with pepsin treatment was about 30%, while those obtained in acetic acid or glycine-HCl buffer were 18.32% and 23.01%, respectively. The characteristics and functional properties of thornback ray gelatin (TRGP), obtained by treatment with 5 units of pepsin/g of skin in glycine-HCl buffer, were investigated and compared with halal bovine gelatin (HBG). TRGP had high protein content (90 g/100 g) similar to halal bovine gelatin (HBG). TRGP had a molecular weight (170 kDa) lower than HBG (226 kDa) and imino-acids content lower than HBG (10 and 22 residues per 100 residues, respectively). Its gel strength (140 g) was lower than that of HBG (260 g), possibly due to lower hydroxyproline content. Textural properties showed that the values hardness, elasticity, cohesiveness and chewiness of TRGP were lower than those of HBG. The functional properties of TRGP were examined in comparison to HBG. TRGP showed stronger ability to clarify apple juice than bovine gelatin. The nutritional component of clarified apple juice was practically not changed. As expected, the different origin and extraction process of HBG and TRGP influenced the properties of the corresponding gelatin solutions.

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.

This study was designed to test the hypolipidemic properties and antioxidative activities of sardinelle (Sardinella aurita) protein hydrolysates (SPHs) obtained by treatment with crude enzyme preparations from Bacillus pumilus A1(SPHA1), Bacillus mojavensis A21(SPHA21) and crude enzyme extract from sardinelle viscera (SPHEE). Wistar rats were fed during 7 weeks a standard laboratory diet, a cholesterol-enriched diet (1%) or a cholesterol SPH-enriched diet. The hypercholesterolemic diet induced the elevation of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). Supplementing cholesterol-enriched diet with SPHs or whole sardinelle protein (WSP) at a concentration of 5% (w/w) increased the serum level of high-density lipoprotein cholesterol (HDL-C) and HDL-C/TC ratio and decreased the serum levels of TC, TG, LDL-C and LDL-C/HDL-C ratio significantly. The thiobarbituric acid-reactive substances (TBARS) level, as an indicator of lipid peroxidation, and the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were examined. The hepatic antioxidant enzymes activities were significantly decreased and the malondialdehyde (MDA) level was increased in rats fed a cholesterol-enriched diet compared to those fed a standard diet. The treatment of hypercholesterolemic (HCD) diet rats with SPHs reduced the MDA concentration and increased the antioxidant enzyme activities. These results suggested that the hypolipidemic effect of SPHs might be due to their abilities to lower serum TC, TG, and LDL-C levels as well as to their antioxidant activities preventing the lipid peroxidation process.

Gelatin was extracted from the skin of grey triggerfish (Balistes capriscus) by the acid extraction process with a yield of 5.67 g/100 g skin sample on the basis of wet weight. The chemical composition and functional properties of gelatin were investigated. The gelatin had high protein (89.94 g/100 g) but low fat (0.28 g/100 g) contents. Differences in the amino acid composition between grey triggerfish skin gelatin (GSG) and halal bovine gelatin (HBG) were observed. GSG contained a lower number of imino acids (hydroxyproline and proline) (176 residues per 1000 residues) than HBG (219 residues per 1000 residues), whereas the content of serine was higher (40 versus 29 residues per 1000 residues, respectively). The gel strength of the GSG (168.3 g) was lower than that of HBG (259 g) (p < 0.05) possibly due to lower hydroxyproline content. Grey triggerfish skin gelatin exhibited a slightly lower emulsifying activity and water-holding capacity but greater emulsifying and foam stability, foam formation ability and fat-binding capacity than the halal bovine gelatin (p < 0.05). SDS-PAGE of GSG showed high band intensity for the major protein components, especially, α- and β-components and a similar molecular weight distribution to that of standard calf skin collagen type I.

In order to utilise sardinelle ( Sardinella aurita ) protein by-products, which is normally discarded as industrial waste in the process of fish manufacturing, heads and viscera proteins were hydrolysed by different proteases to obtain antioxidative peptides. All hydrolysates showed different degrees of hydrolysis and varying degrees of antioxidant activities. Hydrolysate generated with crude enzyme extract from sardine ( Sardina pilchardus ) displayed high antioxidant activity, and the higher DPPH radical-scavenging activity (87 ± 2.1% at 2 mg/ml) was obtained with a degree of hydrolysis of 6%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P 1 –P 8 ). Fraction P 4 , which exhibited the highest DPPH scavenging activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Seven antioxidant peptides were isolated. The molecular masses and amino acids sequences of the purified peptides were determined using ESI-MS and ESI-MS/MS, respectively. Their structures were identified as Leu-His-Tyr, Leu-Ala-Arg-Leu, Gly-Gly-Glu, Gly-Ala-His, Gly-Ala-Trp-Ala, Pro-His-Tyr-Leu and Gly-Ala-Leu-Ala-Ala-His. The first peptide displayed the highest DPPH radical-scavenging activity (63 ± 1.57%; at 150 μg/ml) among these peptides. The results of this study suggest that sardinelle by-products protein hydrolysates are good source of natural antioxidants.

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

Rabbit plasma low density lipoprotein (LDL) contains one major apolipoprotein of apparent molecular weight of 320 kDa, designated apolipoprotein (apo) Bh, while another component termed apoB1 of apparent molecular weight of 220 kDa is found in chylomicrons. The fragments generated by thrombin digestion of the protein moieties of rabbit and human LDL were separated by polyacrylamide gradient gel electrophoresis and compared. As in the human species, the enzyme produced limited cleavage patterns of rabbit LDL apoB. Within the first 2 h, two fragments (Tr1 and Tr2, with apparent molecular weights 280,000 and 44,000, respectively) appeared. Longer incubations led to the production of two additional peptides, Tr3 and Tr4 (apparent molecular weights 180,000 and 96,000, respectively). Ten monoclonal antibodies, developed against rabbit LDL and designated P01 to P10, were found to react with rabbit apoB. Some also cross-reacted with human apoB. Epitope mapping, performed with these antibodies, showed that Tr3 and Tr4 were derived from the further degradation of Tr1. The rabbit is one of the most frequently used animals in atherosclerosis research. Its LDL receptor has been characterized and there exists a strain of homozygous LDL receptor-deficient rabbits referred to as WHHL rabbits. Despite this, little has been done to characterize the structure of rabbit apoB; only a short region has been sequenced and shown to be the carboxyl-terminal region, the rabbit apoB1. The molecular weight of human apoB (550,000) is much larger than rabbit apoBh. In both species, a primary and secondary thrombin cleavage occur, but the size of the fragments produced is very different between the two species. Identification of the thrombolytic fragments of the rabbit apoB have afforded the opportunity to compare the structures of both apoB species.

Lipids, apolipoproteins, lipoproteins, as well as lipoproteins containing both apo A-I and apo A-II (Lp A-I:A-II) or apo A-I but no apo A-II (Lp A-I), proapolipoprotein (proapo) A-I and the activity of lecithin:cholesterol acyltransferase (LCAT), were investigated in umbilical cord sera of 67 term human neonates (30 females and 37 males). Lp A-I and Lp A-LA-II were present in umbilical cord sera with levels of 0.26 ± 0.1 and 0.33 ± 0.15 g/l, respectively. Furthermore, the absolute amount of proapo A-I was lower in cord blood than in adult plasma, but in view of the lower apo A-I levels in umbilical cord sera it comprised 10.48 ± 3.86% of total apo A-I and was thus significantly higher than in adult plasma (7.1 ± 0.9%). Proapo A-I was highly correlated with HDL cholesterol and apo A-I. Total serum LCAT activity was about 50% of adult plasma and was highly correlated with Lp A-I, but not with Lp A-I:A-II. We conclude that human umbilical cord serum contains both Lp A-I and Lp A-I:A-II particles and that the LCAT activity is predominantly related with the Lp A-I subfraction. The higher percentage in umbilical cord sera of proapo A-I may indicate a higher turnover of apo A-I or a lower activity of the proapo A-I cleaving enzyme which is still not identified.

Cholesterol efflux was studied in cultured Ob1771 adipose cells after preloading with LDL cholesterol. Exposure to particles containing apo A II (LpA I ) and particles containing apo A I and apo A II (LpA I : A II ) isolated from native human plasma, and from HDL 2 or HDL 3 , showed that only LpA I were able to promote cholesterol efflux, despite the fact that both kinds of particles were able to bind to receptor sites within the same range of concentrations (apparent K d values between 10 and 25 μg/ml). During this long-term exposure, LpA I : A II demonstrated a concentration-dependent inhibition (10–60 μg/ml) of LpA I -mediated cholesterol efflux from adipose cells under conditions where LpA I : A II did not deliver cholesterol to the cells and where no net change in the distribution of apo A I between LpA I and LpA I : A II was observed. The antagonizing and modulating role of LpA I : A II in preventing cholesterol efflux mediated by LpA I appears not to be related to the lipid composition and cholesterol content of the particles but, rather, appears dependent upon the presence of apo A I in LpA I particles and apo A I in LpA I : A II particles. The actual concentrations of these particles in the interstitial fluid bathing peripheral cells might be critical for the in vivo occurrence of cholesterol efflux.

The role of a high-affinity receptor site for high-density lipoproteins (HDL) has been investigated in parental Ob1771 adipose cells and their transformed counterparts after transfer of the complete early region of polyoma virus (Ob17PY cells). Binding of ApoAI, ApoAII and HDL3 occurs in Ob1771 cells and derived membranes, whereas no binding is observed in Ob17PY cells and derived membranes. After thymidine block, growth-arrested Ob17PY cells become able to bind ApoAI, ApoAII and HDL3; this recovery is prevented in actinomycin D- or cycloheximide-treated cells. In contrast to ApoAI, ApoAII or HDL3 binding, both growing and growth-arrested Ob17PY cells do show receptor activities for low density lipoproteins and transferrin, respectively, which are similar in affinity and maximal capacity. Following cholesterol accumulation which takes place in the presence of LDL cholesterol, subsequent exposure to HDL3 or ApoAI promotes cholesterol efflux from Ob1771 cells and growth-arrested Ob17PY cells but not from growing Ob17PY cells. These results show that the presence of a high-affinity receptor site for HDL in intact adipose cells is required for the promotion of reverse cholesterol transport.

Epidemiological studies have shown a relationship between low concentrations of high density lipoprotein (HDL) cholesterol and the incidence risk of cardiovascular diseases1,2. Recent pharmacological studies3 have clearl demonstrated the protective role of HDL in that respect. In the last decade much attention has been paid to the significance of apolipoprotein determination4,5. Thus, ApoAI, the major protein of HDL, appears to be a better predictor of coronary artery disease (CAD) than HDL cholesterol6,7. Two main types of lipoprotein particles are identified within HDL : those which contain ApoAI and ApoAII (LpAI:AII) and those which contain ApoAI but not ApoAII (LpAI). It has been shown quite recently that CAD subjects are characterized by a different distribution of ApoAI between LpAI and LpAI:AII, the data supporting the view that LpAI might represent the “antiatherogenic” fraction of HDL8.

The angiotensin I-converting enzyme (ACE) inhibitory activities and antioxidant properties of thornback ray gelatin hydrolysates (TRGHs) prepared by treatment with proteolytic proteases from Bacillus subtilis A26, Raja clavata crude alkaline protease extract, Alcalase and Neutrase were investigated. All gelatin hydrolysates showed different degrees of hydrolysis and hydrophobic/hydrophilic peptides ratio. Moreover, they possess high protein content (70.04 ± 0.55–74.14 ± 0.28%). The antioxidant activity was assayed using various in vitro tests. The highest antioxidant activity was observed with hydrolysate obtained by treatment with A26 proteases (TRGH-A26) which exhibited a 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity with a concentration that produces 50% of inhibition (IC50) of 1.98 ± 0.02 mg/ml of sample, reduced the ferric ions with an absorbance at 700 nm of 0.962 ± 0.07, prevents bleaching of β-carotene with 73.02 ± 1.90% inhibition and gave an antioxidative efficacy of 180 ± 0.08 µmol/ml α-tocopherol equivalents at 5 mg/ml in the phosphomolybdenum assay. However, gelatin hydrolysate treated with Alcalase (TRGH-Alcalase) was the most potent to prevent DNA oxidation. For the ACE inhibitory activity, all hydrolysates displayed ACE-inhibitory activity. TRGH-A26 and TRGH-Alcalase exhibited the highest activity with 85 ± 0.65 and 82 ± 0.49%, respectively, at 5 mg/ml. The results revealed that TRGHs could be used as ingredients to formulate functional foods.