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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.

Angiotensin I-converting enzyme (ACE) inhibitory peptides have been searched in thornback ray (Raja clavata) muscle hydrolysed with Bacillus subtilis A26 proteases until a hydrolysis degree of 18.35%. The hydrolysate showed an IC50 of 0.83 mg mL−1. To identify peptides responsible for this activity, the extract was eluted through size-exclusion chromatography and fractions collected. The highest ACE inhibitory activity was found for fractions F2 and F3 which had IC50 of 0.42 and 0.51 mg mL−1, respectively. These fractions were analysed by nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS). A total of 131 and 108 peptide sequences mainly derived from actin, myosin heavy chain and procollagen alpha 1 chain proteins were identified in fractions F2 and F3, respectively. FQPSF and LKYPI showed the best results with an IC50 of 12.56 and 27.07 μM, respectively. These results prove the potential of thornback ray muscle hydrolysate as a source of ACE inhibitory peptides., This work was funded by a grant from the Ministry of Higher Education and Scientific Research of Tunisia and grant Prometeo/2012/001 from Conselleria d´Educació Cultura i Sport of Generalitat Valenciana, both are acknowledged. JAEDOC- CSIC postdoctoral contract cofounded by ESF to L.M. is also acknowledged

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.

Gelatins from alkali-pre-treated skin of Octopus (Octopus vulgaris) were extracted with different concentrations of pepsin at pH 2.0. The resulting octopus skin gelatins OSG0, OSG5, OSG10 and OSG15, extracted, respectively, without enzyme treatment or with 5, 10 and 15 U of pepsin/g alkaline treated skin were evaluated for gel strength, textural parameters, thermal and gelling properties. The yield of gelatin extracted without enzymatic pretreatment was only 3.21% and the addition of pepsin (15 U/g) increased the yield of gelatin extraction to 7.78%. Molecular weight distribution of gelatins indicates that OSG10 and OSG15 contain peptides with molecular weights less than 10 kDa (>40%). In addition, Fourier transform infrared (FTIR) spectra of extracted gelatins were slightly different, indicating that the triple helical structure of gelatins was affected by pepsin treatment. Compared to OSG0, pepsin gelatins exhibited lower gel strength, hardness, adhesiveness, transition, gelling and melting temperatures and all of the values decreased with increasing enzyme concentration. In addition foam expansion (FE) was affected by enzyme treatment, and the values decreased as pepsin increased. However, the emulsifying activity index (EAI) values of all gelatins were the same. Further, FE and EAI increased with increasing concentrations (1–3%, w/v). The results showed that octopus skin can be a good source for gelatin.

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.

Gelatin was extracted from alkali-pretreated skin of zebra blenny (Salaria basilisca) using commercial pepsin with a yield of 18 g/100 g of skin sample. The polypeptides pattern, gel strength, viscosity, textural parameters and functional properties of the zebra blenny skin gelatin (ZBSG) were investigated. Amino acid analysis revealed that ZBSG contained almost all essential amino acids, with glycine being the most predominant one. ZBSG was identified as a type I gelatin, containing α1 and α2-chains as the major constituents. Its gel strength and viscosity were 170.2 g and 5.95 cP, respectively. Fourier transformed infrared spectroscopy (FT-IR) spectra showed helical arrangements in its structure. Its solubility and functional properties were concentration-dependent. While foam expansion (FE) and foam stability (FS) increased with the increase of concentration, emulsifying activity index (EAI) and emulsion stability index (ESI) were noted to decrease. ZBSG also showed strong clarification ability particularly for apple juice, without affecting nutritional value.

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.

Resume But de l’etude Etudier l’effet du jeune intermittent subit par les rates adultes depuis la periode de gestation jusqu’a celle de lactation, sur le developpement et la maturation des ratons. Materiels et methodes Deux groupes comprenant chacun quatre rates Wistar gestantes sont constitues. Les rates du premier groupe sont soumises a un jeune intermittent qui commence des le 14e jour de gestation et se poursuit 21 jours apres la mise-bas. Celles du second groupe sont normalement nourries. Les jeunes des deux groupes de rates sont sacrifies a l’âge de 21 jours. Resultats La restriction alimentaire appliquee aux meres produit, chez les ratons, une reduction du poids corporel (–36 %), du contenu des thyroides en iode (p Conclusion La restriction alimentaire imposee aux rates adultes, depuis la gestation, conduit a l’installation d’un etat de denutrition chez leurs petits et a une perturbation histologique de leur thyroide. Celle-ci est associee a un hypothyroidisme qui serait responsable, au moins en partie, de l’effondrement de la capacite a reguler le remodelage osseux.

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.

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.

The antioxidant activities of protein hydrolysates prepared from heads and/or viscera of sardinelle (Sardinella aurita) by treatment with different proteases were evaluated using various in vitro antioxidant assays. All hydrolysates showed different degrees of hydrolysis and varying degrees of antioxidant activity. The hydrolysates obtained by treatment with crude enzyme from Mustelus mustelus intestines exhibited the highest radical-scavenging activity. However, Alcalase hydrolysates displayed the greater reducing power activities. Further, sardinelle heads protein hydrolysates were found to strongly suppress the discoloration of β-carotene compared with control. Both Alcalase protein hydrolysates obtained from heads or viscera were then fractionated by size exclusion chromatography on a Sephadex G-25, into two and four major peptide fractions, respectively. All fractions exhibited antioxidant activity, and fraction P4 with molecular mass around 3.5 kDa from sardinelle viscera protein hydrolysates was found to exhibit the highest radical-scavenging activity. PRACTICAL APPLICATIONS In Tunisia, sardinelle (Sardinella aurita) catches were about 13,300 tons in 2002. During processing, solid wastes including heads and viscera are generated and can be seen as 30% of the original raw material. These by-products constitute an important source of proteins. An interesting alternative to valorize these by-products is to transform sardinelle proteins by-products into biologically active peptides by protease treatments. The results clearly demonstrated that sardinelle by-products protein hydrolysates have excellent antioxidant activities, and thus, they have great potential as a source for natural antioxidants.

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.

Protein hydrolysates are of a significant interest, due to their potential application as a source of bioactive peptides in nutraceutical and pharmaceutical domains. The present study was focused on the effect of protein hydrolysate from smooth hound (Mustelus mustelus) (SHPH) in the regulation of components of the food intake control such as satiety. SHPH was produced by intestinal digestive proteases from the same species. The amino acid analysis by GC/MS showed that the hydrolysate was rich in leucine, alanine, glycine, threonine, serine, lysine and glutamate. The molecular weights of peptides in SHPH were estimated by ESI-MS to be between 200 and 2500 Da. Biological in vivo capacities of SHPH in rats were evaluated by determination of the CCK-like peptides and insulin content using a clinical human radioimmunoassay. The food intake and the body weight of rats were measured during the period of treatment. Rats treated with SHPH showed a significant decrease in body weight at the end of treatment, as well as a decrease of food intake. Our findings revealed a possible mechanism of the beneficial effects of SHPH in appetite regulation, and this might be important to prevent the risk of a number of medical conditions including type II diabetes.

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.

A highly alkaline trypsin from the intestine of Grey triggerfish ( Balistes capriscus ), with high activity at low temperature, was purified and characterised. The enzyme was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography, with a 13.9-fold increase in specific activity and 41.3% recovery. The molecular weight of the purified alkaline trypsin was estimated to be 23.2 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and size exclusion chromatography. Purified trypsin appeared as a single band on native–PAGE. Interestingly, the enzyme was highly active over a wide range of pH, from 9.0 to 11.5, with an optimum at pH 10.5, using Nα -benzoyl-DL-arginine- p -nitroanilide (BAPNA) as a substrate. The relative activities at pH 9.0, 11.5 and 12.0 were 86.5%, 92.6% and 52.4%, respectively. The enzyme was extremely stable in the pH range 7.0–12.0. In addition, the enzyme had high activity at low and moderate temperatures with an optimum at around 40 °C and had more than 80% of its maximum activity at 20 °C. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. The enzyme showed extreme stability towards oxidising agents, retaining about 87% and 80% of its initial activity after 1 h incubation at 40 °C in the presence of 1% sodium perborate and 1% H 2 O 2 , respectively. In addition, the enzyme showed excellent stability and compatibility with some commercial solid detergents. The N -terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECTPNST . B. capriscus trypsin, which showed high homology with trypsins from marine vertebrates, had a basic residue, Asn, at position 10, where His and Tyr are common in all marine vertebrates trypsins. The trypsin kinetic constants, K m and k cat for BAPNA, were 0.068 mM and 2.76 s −1 , respectively, while the catalytic efficiency, k cat / K m , was 40.6 s −1 mM −1 .

Thermostable trypsin from the hepatopancreas of Sepia officinalis was purified by fractionation with ammonium sulphate, Sephadex G-100 gel filtration, DEAE-cellulose an ion-exchange chromatography, Sephadex G-75 gel filtration and Q-Sepharose anion-exchange chromatography, with a 26.7-fold increase in specific activity and 21.8% recovery. The molecular weight of the purified enzyme was estimated to be 24,000 Da by SDS-PAGE and size exclusion chromatography. The purified enzyme showed esterase specific activity on Nα -benzoyl-L-arginine ethyl ester (BAEE) and amidase activity on Nα -benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity were pH 8.0 and 70 °C, respectively, using BAPNA as a substrate. The enzyme was extremely stable in the pH range 6.0–10.0 and highly stable up to 50 °C after 1 h of incubation. The purified enzyme was inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine-protease inhibitor. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGKESSPYNQ. S. officinalis trypsin, which showed high homology with trypsins from marine vertebrates and invertebrates, had a charged Lys residue at position 5 and a Ser residue at position 7, where Tyr and Cys are common in all marine vertebrates and mammalian trypsins. Further, the enzyme had an Asn at position 11, not found in any other trypsins. The trypsin kinetic constants, Km and kcat for BAPNA, were 0.064 mM and 2.32 s−1, respectively, while the catalytic efficiency kcat/Km was 36.3 s−1 mM−1.

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

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 IC50 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. Copyright © 2010 Society of Chemical Industry

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.

The malnutrition of the infants could be explained by a delay of the growth and the perturbation of biological parameters.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.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 scoreor = 2SD).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.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.

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.

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.

Introduction Parmi les lipoproteines contenant l’apoAI, seules les LpAI (sans apoAII) auraient des proprietes anti-atherogenes. Leur concentration et/ou leur proportion auraient la valeur d’un marqueur de risque d’atherosclerose. Notre etude examine le profil serique des lipoproteines contenant l’apoAI en cas d’obesite dont la nette progression et son association au developpement de l’atherosclerose en font un terrain de choix. Patients et Methodes 33 femmes obeses et 36 temoins, d’age comparable, ont ete selectionnees sur leur exces ponderal par rapport au poids ideal : > 125 % et Resultats En meme temps que l’exces ponderal (38,2 ± 7,8 contre 5,8 ± 4,1), les sujets obeses une alteration (p Discussion L’augmentation conjointe des TG et du rapport LpAI : AII/LpAI est en accord avec la correlation negative entre les TG et le rapport HDL2/HDL3. Tenant compte des donnees de la litterature indiquant l’association des HDL de grosse taille a une diminution du risque d’atherosclerose et des effets antagonistes des deux types de lipoproteines contenant l’apoAI sur le transport reverse du cholesterol, la reduction de la proportion des LpAI (plus grosses que LpAI : AII) par rapport aux LpAI : AII pourrait signifier un accroissement du risque de maladies cardiovasculaires chez nos obeses. Conclusion Par rapport a sa concentration, la repartition de l’apoAI apparait plus sensible aux variations et serait un marqueur de risque plus efficace.

Introduction Ramadan est associe a des changements alimentaires et biologiques variables selon la population. L’identification et la comparaison des variations alimentaires et des profils biologiques dans divers groupes de pratiquants serait utile pour evaluer l’impact des changements alimentaires sur le risque d’atherosclerose, et dans la recherche du regime adequat. Patients et Methodes 25 sujets, de 22 a 55 ans, etaient suivis. Leur regime habituel etait determine par questionnaire et celui de Ramadan par inventaire consigne sur carnet. Le BMI et divers parametres lipidiques et lipoproteiniques incluant les acides gras plasmatiques etaient determines avant (T0), a deux (T1) et quatre (T2) semaines de Ramadan et enfin un mois apres (T4). Resultats Durant le mois de Ramadan, nos sujets consomment significativement moins de glucides, et plus de protides, de cholesterol, de vitamine E (P Discussion Chez nos sujets, le mois de Ramadan etait associe a un changement du profil biologique qui etait de nature antiatherogene par certains de ses cotes (augmentation du rapport AGI/AGS) mais atherogene par d’autres. Des changements non alimentaires (tabagisme, alcoolisme, nombre d’heure de sommeil, …), eventuellement capables d’affecter le profil biologique, devraient etre consideres en meme temps que ceux alimentaires. Conclusion La relation Ramadan-atherosclerose est complexe et son exploration necessite l’investigation des changements aussi bien alimentaires que non alimentaires.

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.

Apolipoprotein A-I (apoA-I), the major protein component of human high density lipoprotein, appears intracellularly as an intermediate precursor (proapoA-I) with a hexapeptide extension (Arg-His-Phe-Trp-Gln-Gln) at its amino terminus. To investigate the regulation of processes that regulate plasma apoA-I levels, a sensitive and simple assay for proapoA-I is required. We describe a specific enzyme-linked immunosorbent assay (ELISA) for quantification of proapoA-I using monospecific rabbit antibodies raised against the peptide: Arg-His-Phe-Trp-Gln-Gln-Asp-Glu-Pro. The monospecificity of antibodies to propeptide has been checked and no cross-reaction with mature apoA-I has been found although three first mature apoA-I amino acids (Asp-Glu-Pro) were included in the immunizing peptide. The assay is a non-competitive sandwich ELISA in which polystyrene microtiter plates were used with antibodies to propeptide adsorbed on the wells. After incubation with plasma samples, the bound proapoA-I was revealed by labeled rabbit polyclonal antibodies directed against mature apoA-I. The working range was 10 to 100 ng/ml, recovery of proapoA-I added to plasma was 94.6 to 106.5%, and the intra- and interassay coefficients of variation were 3.8% and 7.9%, respectively. A delipidation step using diisopropylether-n-butanol was necessary to expose antigen sites of proapoA-I in native lipoproteins. Mean level of proapoA-I in normal subjects was 87 +/- 15 micrograms/ml. It represented 7.1% of total apoA-I while in Tangier serum it represented 29%.

Apolipoproteins were determined in 50 human amniotic fluids obtained by amniocentesis during weeks 16 and 22 (n = 26) or 33 and 41 (n = 24) of gestation. Whereas apo A-I, A-II, A-IV, and E were identified at levels of 1, 0.7, 0.8 and 1% of normal human adult plasma, respectively, apo B levels were only 0.04% of plasma concentration and apo C-III levels were below the detection limits of the assay. Amniotic fluid levels of apo A-II, A-IV and E did not differ between early and late stages of pregnancy, but levels of apo B decreased and apo A-I increased significantly in late pregnancy. Isofocusing showed apo A-I, A-II and A-IV in identical positions as compared to human adult plasma. Furthermore the known genetic polymorphism of apo A-IV was detectable. Individuals heterozygous, for the variant form apo A-IV-2, showing the phenotype apo A-IV (2-1), had significantly higher levels of apo A-I and A-II as compared to the common phenotype apo A-IV (1-1). We conclude that human amniotic fluid contains the major plasma apolipoproteins at about 1% of plasma levels with the exception of apo B which shows a level at an order of magnitude less than high-density lipoprotein apoproteins in comparison to their plasma counterparts.

This paper describes a procedure for the rapid isolation of urea-soluble apolipoproteins (apo) from delipidated human very-low- and high-density lipoproteins using anion-exchange fast protein liquid chromatography. The separation was complete within 30 min and peaks corresponding to apolipoproteins A-I, A-II, C-I, C-II, C-III0, C-III1, C-III2 and E were identified by comparing their chromatographic, electrophoretic and immunological behaviour with that of purified standards of each protein. A second purification step is necessary to obtain pure apolipoproteins. Apo E, which is difficult to purify by conventional chromatography, has been obtained in a good yield. The apo C-II that was obtained produced a symmetrical peak on chromatography but three bands in isoelectric focusing. The method can be upgraded to a preparative scale and offers the possibility of direct purification of apolipoproteins both from high-density lipoproteins and (following preliminary gel chromatography) from very-low-density lipoproteins.

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.