Rim Nasri

Characterization and Potential Use of Cuttlefish Skin Gelatin Hydrolysates Prepared by Different Microbial Proteases

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

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.

Novel angiotensin I-converting enzyme inhibitory peptides from enzymatic hydrolysates of goby (Zosterisessor ophiocephalus) muscle proteins

UNLABELLED In recent years, food protein-derived bioactive peptides have received considerable attention because of their numerous health benefits. Amongst bioactive peptides, those with antihypertensive activity are receiving special attention due to their role in cardiovascular diseases. Goby protein hydrolysates (GPHs) prepared by treatment with five different crude bacterial proteases were found to exhibit varying degrees of angiotensin I-converting enzyme (ACE) inhibitory activity. The hydrolysate generated by the crude protease from Bacillus mojavensis A21, which displayed the highest ACE inhibitory activity, was further fractionated by size exclusion chromatography on a Sephadex G-25 and reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses and amino acid sequences of five peptides, in sub-fraction F5-2, which exhibited the highest ACE inhibitory activity, were determined using ESI-MS and ESI-MS/MS, respectively. The structures of these peptides were identified as Ala-Arg-Ser, Val-Val-Ala-Pro-Phe-Ala-His-Gly-Thr, Arg-Ser-Thr-Ala, Phe-Tyr-Pro-Pro, Arg-Cys-Ser-Ala-Gly-Val. Further, the sequences of fifteen peptides in the F5-4 sub-fraction, which exhibited high activity, were determined. Therefore, GPHs have a potential as hypotensive nutraceutical ingredients. BIOLOGICAL SIGNIFICANCE Peptides find many outlets of application in the biotechnological field, amongst which are pharmaceutical applications. Progression amongst new small molecules deposited like substance medicamentous blows itself. In this context, large pharmaceutical companies invest in peptide research to open therapeutic new prospects. Even if they are used as therapeutic agents for nearly one century in their natural form, the use of peptides remains parsimonious although we experienced a significant development since a few tens of years, in particular thanks to the clarification of the methods of production, chemical in solid or biological phase such as in phage display. Peptides present many advantages compared to traditional drugs that have small molecules, Generation of bioactive peptides by proteolysis of food proteins, using exogenous proteases, is a new and interesting approach for the production and identification of new and potent specific hypotensive agents. From another side, compared with natural peptides isolated from different sources, there is more diversity in structure and mode of action of the derived bioactive peptides. In fact, proteolysis of protein substrates, having different amino acid composition and sequences, by proteases having different specificities may generate numerous specific peptide inhibitors, with different lengths and amino acid sequences. These bioactive peptides have received considerable attention for their effectiveness in both the prevention and the treatment of hypertension.

Characterisation of trypsin purified from the viscera of Tunisian barbel (Barbus callensis) and its application for recovery of carotenoproteins from shrimp wastes

Trypsin was purified from the viscera of barbel by precipitation using ammonium sulphate (0-80%), Sephadex G-100, and Mono Q-Sepharose ion exchange chromatography. The trypsin was purified 27-fold, with 79U/mg specific activity and 31% recovery. The enzyme had a molecular weight of 24kDa; purified trypsin appeared as a single band on native-PAGE. The optimum pH and temperature for enzyme activity were pH 10.0 and 55°C with BAPNA used as a substrate. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECTPYSQ. The Michaelis-Menten constant (Km) and catalytic constant (kcat) values of the enzyme were 0.018mM and 1.21s-1, respectively. The study also investigated the effects of purified trypsin on the recovery of carotenoproteins from shrimp (Parapenaeus longirostris) shells through hydrolysis using 1.0U barbel trypsin/g shrimp shells for 1h at 30°C. The freeze-dried carotenoproteins recovered contained 71.09% protein, 16.47% lipid, 7.78% ash, and 1.79% chitin.

A laundry detergent-stable alkaline trypsin from striped seabream (Lithognathus mormyrus) viscera: purification and characterization.

An alkaline trypsin from the intestine of striped seabream (Lithognathus mormyrus) was purified and characterized. The enzyme was purified to homogeneity by precipitation with ammonium sulfate, Sephadex G-100 gel filtration and CM-Sephadex cation-exchange chromatography, with a 24.9-fold increase in specific activity and 13% recovery. The molecular weight of the purified alkaline trypsin was estimated to be 27.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. The purified trypsin appeared as a single band on native PAGE. Interestingly, the enzyme was highly active over a wide range of pH from 8.0 to 11.0, with an optimum at pH 10.0 using Nalpha-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The relative activities at pH 8.0, 11.0, and 12.0 were 73%, 67% and 50.4%, respectively. The enzyme was extremely stable over a broad pH range (5.0-12.0). The optimum temperature for enzyme activity was 50 degrees C. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI). In addition, the enzyme showed excellent stability toward various surfactants and bleache agents and compatibility with some commercial solid and liquid detergents. The trypsin kinetic constants, Km and kcat of the enzyme for BAPNA, were 0.29 mM and 1.36 s(-1), respectively, while the catalytic efficiency kcat/Km was 4.68 s(-1) mM(-1).

Biochemical properties of anionic trypsin acting at high concentration of NaCl purified from the intestine of a carnivorous fish: smooth hound (Mustelus mustelus).

Trypsin from the intestine of smooth hound (Mustelus mustelus) was purified by fractionation with ammonium sulfate, Sephadex G-75 gel filtration, and DEAE-cellulose ion exchange chromatography, with a 65-fold increase in specific activity and 15% recovery. The molecular weight of the purified trypsin was estimated to be 24 kDa using size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme showed esterase-specific activity on N(alpha)-p-tosyl-L-arginine methyl ester hydrochloride (TAME) that was four times greater than its amidase-specific activity on Nalpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the trypsin activity were pH 8.5 and 50 degrees C, respectively, using TAME as a substrate. The enzyme was extremely stable in the pH range of 7.0-9.0 and highly stable up to 40 degrees C after 1 h of incubation. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK), specific inhibitors for trypsin. In addition, smooth hound trypsin showed higher proteolytic activity at high NaCl concentration, demonstrating its potential for protein hydrolysis at high salt content. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECKPHSQ. This sequence showed high homology with trypsins from marine vertebrates and invertebrates. Purified trypsin had a Michaelis-Menten constant (K(m)) and catalytic constant (K(cat)) of 0.387 +/- 0.02 mM and 2.62 +/- 0.11 s(-1), respectively, when BAPNA was used as a substrate. For the hydrolysis of TAME, K(m) and K(cat) were 0.156 +/- 0.01 mM and 59.15 +/- 2.2 s(-1), respectively.

Ameliorating effects of goby fish protein hydrolysates on high-fat-high-fructose diet-induced hyperglycemia, oxidative stress and deterioration of kidney function in rats.

This study investigated the therapeutic potential of undigested goby fish (Zosterisessor ophiocephalus) muscle proteins (UGP) and their hydrolysates on high-fat-high-fructose diet (HFFD)-fed rats. HFFD induced hyperglycemia, manifested by a significant increase in the levels of glucose and glycogen as well as α-amylase activity when compared to normal rats. The administration of GPHs to HFFD-fed rats significantly decreased α-amylase activity and the contents of blood glucose and hepatic glycogen. By contrast, the UGP increased the glucose metabolic disorders in HFFD-fed rats. Furthermore, HFFD-fed rats showed oxidative stress, as evidenced by decreased antioxidant enzyme activities and glutathione (GSH) levels and increased concentration of the lipid peroxidation product malondialdehyde in liver and kidney. Interestingly, the daily gavage of UGP and GPHs improved the redox status in liver and kidney of HFFD-rats by ameliorating or reversing the above-mentioned changes. Moreover, GPHs exhibited a renal protective role by reversing the HFFD-induced decease of uric acid and increase of creatinine levels in serum and preventing some HFFD-induced changes in kidney architecture. The results demonstrate that GPHs contain bioactive peptides that possess significant hypoglycemic and antioxidant properties, and ameliorate renal damage in rats fed hypercaloric diet.

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

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

Sulfated polysaccharides from common smooth hound: Extraction and assessment of anti-ACE, antioxidant and antibacterial activities.

The present study investigates biological activities of sulfated polysaccharides (SPs) isolated from smooth hound by precipitation with cetylpyridinium chloride (SP1) or ethanol (SP2). SP1 showed the highest amounts of sulfated groups (10.2%) and proteins (7.84%) and high molecular weight sugars. Infrared spectroscopic analysis showed typical peaks of sulfated polysaccharides, particularly for the SP1 that was characterized by the presence of O=S=O groups and acetyl groups. Interestingly, SPs displayed important angiotensin I converting enzyme (ACE) inhibitory (IC50=1.04 and 0.75mg/ml for SP1 and SP2, respectively), antibacterial (Gram+ and Gram-) and antioxidant activities (reducing power, metal chelating activity, β-carotene bleaching inhibition and DNA nicking assay). Moreover, SPs fractionation by DEAE-cellulose column chromatography showed one peak during the buffer elution phase and three major fractions during the linear gradient of NaCl. The overall data suggested that SPs could be used as natural antioxidant, antimicrobial and anti-ACE ingredient to formulate functional foods.

Marine-derived bioactive peptides as new anticoagulant agents: a review.

The development of more effective antithrombotic agents with reduced side risk is necessary for the prevention of thromboembolic events. In recent years, bioactive peptides derived from food proteins have received considerable attention because of their numerous healths beneficial. Based on their amino acids sequences these peptides can exhibit various biological activities including, antihypertensive, antioxidative and antibacterial activities. Moreover, several naturally or food proteins-derived bioactive peptides that could prolong the activated partial thromboplastin time (APTT) and thrombin time (TT) have been isolated. Due to their therapeutic potential in the treatment or prevention of various diseases, bioactive peptides can be used as functional food ingredients, nutraceuticals or pharmaceuticals. Marine organisms represent a valuable source of new bioactive substances including bioactive peptides. This paper presents an overview of the bioactive anticoagulant peptides derived from fish protein sources, as well as the enzymatic proteolysis approach used for the development of these new biopeptides.