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Hydrolysis of bovine hemoglobin (bHb), the main constituent of bovine cruor by-product, releases a natural antimicrobial peptide (NKT) which could present a major interest for food safety. To enrich this, tangential ultrafiltration can be implemented, but ultrafiltration conditions are mainly empirically established. In this context, the application of a simulation method for predicting the NKT yield and enrichment was investigated. Ultrafiltration performances were studied for decolored bHb hydrolysates at different degrees of hydrolysis (DH, 3%, 5%, 10% and 18%) and colored hydrolysates (3% and 5% DH) with 1 and 3 kg&middot, mol&minus, 1 regenerated cellulose membranes. The simulation method helped to identify the most promising hydrolysate (in terms of NKT enrichment, yield and productivity) as the 3% DH colored hydrolysate, and UF conditions (volumetric reduction factor of 5 and 3 with 1 and 3 kg&middot, 1 membrane, respectively) for higher antimicrobial recovery. A maximal enrichment factor of about 29 and NKT purity of 70% in permeate were observed. The results showed that the antimicrobial activity was in relation with the process selectivity and NKT purity. Finally, this reliable method, applied for predicting the ultrafiltration performances to enrich peptides of interest, is part of a global approach to rationally valorize protein resources from various by-products.

Bovine cruor, a slaughterhouse waste, was mainly composed of hemoglobin, a protein rich in antibacterial and antioxidant peptides after its hydrolysis. In the current context of food safety, such bioactive peptides derived from enzymatic hydrolysis of hemoglobin represent potential promising preservatives for the food sector. In this work, the hemoglobin hydrolysis to produce bioactive peptides was performed in a regulated pH medium without the use of chemical solvents and by an eco-efficient process: electrodialysis with bipolar membrane (EDBM). Bipolar/monopolar (anionic or cationic) configuration using the H+ and OH&minus, generated by the bipolar membranes to regulate the pH was investigated. The aim of this study was to present and identify the bioactive peptides produced by EDBM in comparison with conventional hydrolysis and to identify their biological activity. The use of the EDBM for the enzymatic hydrolysis of hemoglobin has allowed for the production and identification of 17 bioactive peptides. Hydrolysates obtained by EDBM showed an excellent antimicrobial activity against six strains, antioxidant activity measured by four different tests and for the first time anti-fungal activities against five yeasts and mold strains. Consequently, this enzymatic hydrolysis carried out in regulated pH medium with bipolar membranes could provide bioactive peptides presenting antibacterial, antifungal and antioxidant interest.

International audience; Blood, from slaughterhouses, is an inevitable part of meat production, causing environmental problems due to the large volumes recovered and its low valorization. However, the α137-141 peptide, a natural antimicrobial peptide, can be obtained after hydrolysis of hemoglobin, the main constituent of blood red part. To recover it at a sufficient concentration for antimicrobial applications, a new sustainable technology, called electrodialysis with ultrafiltration membrane (EDUF), was investigated. The α137-141 concentration was increased about 4-fold at a feed peptide concentration of 8% with an enrichment factor above 24-fold. This feed peptide concentration also needed the lowest relative energy consumption. Moreover, this peptide fraction protected meat against microbial growth, as well as rancidity, during 14 days under refrigeration. This peptide fraction was validated as a natural preservative and substitute for synthetic additives against food spoilage. Finally, producing antimicrobial/antioxidant peptide from wastes by EDUF fits perfectly with the concept of circular economy.

In this work we evaluated the mode of action of six new synthesized peptides (Met-Asp-Asn; Glu-leu-Ala-Ala-Ala-Cys; Leu-Arg-Asp-Asp-Phe; Gly-Asn-Ala-Pro-Gly-Ala-Val-Ala; Ala-Leu-Arg-Met-Ser-Gly and Arg-Asp-Arg-Phe-Leu), previously identified, from the most active peptide fractions of RuBisCO peptic hydrolysate against Listeria innocua via a membrane damage mechanism. Antibacterial effect and the minimum inhibitory concentrations (MIC) of these peptides were evaluated against six strains and their hemolytic activities towards bovine erythrocytes were determined. Prediction of the secondary structure of peptides indicated that these new antibacterial peptides are characterized by a short peptide chains (3-8 amino acid) and a random coli structure. Moreover, it was observed that one key characteristic of antibacterial peptides is the presence of specific amino acids such as cysteine, glycine, arginine and aspartic acid. In addition the determination of the extracellular potassium concentration revealed that treatment with pure RuBisCO peptides could cause morphological changes of L. innocua and destruction of the cell integrity via irreversible membrane damage. The results could provide information for investigating the antibacterial model of antibacterial peptides derived from RuBisCO protein hydrolysates.

Production of bioactive peptides from slaughterhouse wastes with a final low salt content by electrodialysis with bipolar membranes (EDBM) has recently been demonstrated as an innovative and efficient technology. However, a limitation in the electro-acidification process was noticed due to membrane fouling. In this context, the aim of the present work was to identify process conditions that could reduce peptide fouling in EDBM. Hence, two different conductivity levels (0.9 mS/cm and 4.5 mS/cm) were investigated in bipolar/monopolar (anionic and cationic) configuration and compared to conventional hydrolysis in the same conductivity conditions as well as a conventional hydrolysis using a buffer solution. The impact of such conductivity levels was studied during EDBM especially on membrane fouling and hydrolysis parameters: 1) kinetics of the enzymatic mechanism, 2) degree of hydrolysis and 3) peptide population. The results showed the same enzymatic mechanism “one-by-one” for all the conditions but a slower enzymatic kinetics for EDBM-0.9 mS/cm. EDBM-0.9 mS/cm allowed the production of bioactive peptides but with fouling formation on MCP while EDBM-4.5 mS/cm allowed the production of bioactive peptides without any fouling. Finally, optimizing certain conditions for further studies would lead to a promising green process to substitute synthetic additives used to protect food by antimicrobial peptides from blood slaughterhouses.

The objective of this work was to examine the effectiveness of adsorption for the removal of polyphenols from an alfalfa white protein concentrate to produce a protein purified fraction. Six commercial macroporous resins were tested for their polyphenols adsorption characteristics. The screening results showed that the adsorbent HP20 and the anion-exchange resin AER1 had the best adsorption characteristics with adsorption capacities of 54.62 mg/g and 48.18 mg/g, respectively. The effect of pH, contact time and adsorption isotherms were successively investigated on the two preselected resins. The process kinetics was evaluated by the pseudo-first-order, the pseudo-second-order, Elovich, intra-particle diffusion and the modified pseudo-first-order models. Batch kinetic data have been well described by the pseudo second order model. Meantime, the adsorption process close equilibrium was governed by intra-particle diffusion. The adsorption data were fitted well with the Langmuir isotherm model. It was found that the process is favourable. These results will be used in future studies for the design of column adsorption systems for the purification of the alfalfa protein concentrate and the recovery of phenolic compounds contained in the latter.

European eel by-products have been hydrolysed in order to produce novel bioactive peptides with antibacterial and antioxidant activity. Protein hydrolysates from Anguilla by-products obtained by treatment with Savinase® (PHAB-s) have high protein content than that produced with Protamex® (PHAB-p). Furthemore, both protein hydrolysates had a high percentage of essential amino acids. In addition, results indicated that PHAB-s and PHAB-p exhibited different degrees of antioxidant activities, evaluated by several antioxidant assays. Thus, PHAB-s displayed high antioxidant activity, in terms of DPPH-radical scavenging effect (IC50 = 5.2 mg/ml), β-carotene bleaching inhibitory effect (CI50 = 4.4 mg/ml) and metal chelating activity. At 10 mg/ml, PHAB-p showed the highest reducing power. Antibacterial activities of PHAB-s and PHAB-p were also evaluated. PHAB-s showed the most important inhibitory effects against seven bacterial species. The application of PHAB-s and PHAB-p on minced meat as preservative reduced the lipid oxidation and inhibited the microbial growth during chilled storage for 11 days., This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia and the Spanish Ministry of Economy, Industry and Competitiveness under project AGL2014-52825-R.

RuBisCO is the main soluble protein of alfalfa juice. Despite its attractive applications in a wide range of food products this protein remains largely underutilized. Accordingly, the present study was undertaken to investigate the feasibility and efficiency of using ammonium hydroxide (NH4OH) and precipitation at pH 3 for purification and recovery of RuBisCO from industrial Alfalfa green juice. The performance of the proposed RuBisCO recovery method was evaluated through qualitative and quantitative analysis. The results obtained by the proposed method were compared to those achieved for industrial brown juice. The findings from gel electrophoresis (SDS-PAGE), mass spectrometry, and reverse phase high-performance liquid chromatography (RP-HPLC) revealed that RuBisCO was obtained at a high purity of about 90 %. RuBisCO was hydrolyzed using pepsin and the resulting hydrolysate was assayed for antioxidative activity using various antioxidant assays, including the inhibition of linoleic acid oxidation, reduction of ferric ion, and stabilization of ABTS*+ radicals. Overall, the results showed that RuBisCO could be considered as a promising alternative source for the production of novel industrial peptides for application in the pharmaceutical, nutraceutical and functional food industries.

Inhibition of DPP-IV may improve glycemic control in diabetics by preventing the rapid breakdown and there by prolonging the physiological action of incretin hormones. Barbel muscle protein hydrolysate (BMPH) was noted to exhibit DPP-IV inhibitory activity, with an IC value of 1.94mg/mL. It was fractionated into five major fractions (F-F) by size exclusion chromatography using a Superdex peptide. The F fraction was noted to display the highest inhibitory activity, with an IC value of 1.23mg/mL, and was, therefore, further fractionated by RP-HPLC. Four major peptide sub-fractions were selected. The results revealed that the SF sub-fraction showed the highest DPP-IV inhibitory activity, with an IC value of 0.21mg/mL. This sub-fraction was submitted to RP-HPLC, ESI-MS, and ESI-MS/MS analyses. The findings indicated that SF4 consisted of two peptides (IC=96μg/mL), namely PP1 and PP2, whose structures were identified as Trp-Ser-Gly (330Da) and Phe-Ser-Asp (349Da), respectively. This is the first report of these sequences from barbel proteins.The structural modelling through docking simulations results with DPP-IV showed that the Trp-Ser-Gly peptide bound to DPP-IV with high affinity. Overall, the results suggested that BMPH can be considered as a promising natural source of DPP-IV inhibitory peptides., This work was funded by the Ministry of Higher Education and Scientific Research, by the Spanish Ministry of Foreign

The goal of the present work was the extraction and structural characterization of novel sulfated glycosaminoglycans from European eel skin. The recovered glycosaminoglycans were physicochemically characterized and the uronic acid and sulfate contents were 35.12 ± 2.13% and 16.32 ± 0.4%, respectively. Cellulose acetate electrophoresis for extracted glycosaminoglycans was also investigated. Molecular weight of these sulfated glycosaminoglycans was determined (~37 kDa) by the gradient PAGE. Glycosaminoglycans obtained from the European eel were composed of non-sulfated, mono- and disulfated disaccharides. These sulfated glycosaminoglycans were evaluated for their in vitro anticoagulant activity using activated partial thromboplastin time, thrombin time and prothrombin time tests. The result showed that the recovered glycosaminoglycans exhibited interestingly anticoagulant activity. These glycosaminoglycans did not show haemolytic activity towards human erythrocytes. Furthermore, these bioactive substances can be explored as a functional food with antithrombotic function or used as source of anticoagulant drugs.

The present paper reports on the isolation and characterization of novel bioactive peptides by the hydrolysis of RuBisCO proteins. Antibacterial activity was evaluated against Gram-negative (Escherichia coli and Salmonella enterica) and Gram-positive (Listeria innocua, Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus and Bacillus subtilis) bacteria. A simple method, consisting in the precipitation of the active peptides under ionic strength and pH conditions, was used to separate antimicrobial peptides from complex peptic hydrolysates of RuBisCO obtained by treatment with Pepsin® (DH = 18.8%). The obtained peptide extract was fractionated using RP-HPLC, and the active fractions were analyzed by liquid chromatography, electrospray ionization, and tandem mass spectrometry (LC-ESI-MS). Among a total of twelve identified peptides, three new pure antibacterial peptides, namely (Met-Asp-Asn), (Glu-Leu-Ala-Ala-Ala-Cys) and (Leu-Arg-Asp-Asp-Phe), were obtained under hydrolytic conditions. The latter peptides were highly active against the tested strains. The three peptides did not show hemolytic activity towards bovine erythrocytes. Taken together, the results suggest that the new peptides isolated from RuBisCO could offer potential ingredients for the functional foods industry.

Bovine hemoglobin is the major component of the cruor (slaughterhouse by-product) and can be considered as an important source of active peptides that could be obtained by pepsic hydrolysis. The kinetics of appearance and disappearance of several antibacterial peptides from α 1-32 family during hydrolysis of synthesized α 1-32 peptide, of purified bovine hemoglobin and of cruor was studied, and reaction scheme for the hydrolysis of α 1-32 family peptides from these three sources was determined. On this basis, a mathematical model was proposed to predict the concentration of each peptide of interest of this family depending on hydrolysis time, and also on temperature (in the range 15-37 °C), pH (in the range 3.5-5.5) and enzyme to substrate ratio (in the range 1/50-1/200 for the synthesized peptide and 1/5-1/20 for purified bovine hemoglobin and cruor). Apparent rate constants of reactions were determined by applying the model on a set of experimental data and it was shown that they depended on the temperature according to Arrhenius's law, that their dependence on the pH was linear, and that enzyme to substrate ratio influence was limited (in the studied range).

Background Reactive oxygen species play a crucial role in the pathogenesis of diabetes and its complications. The present study was undertaken, in vivo , to examine the protective effect of astaxanthin extracted from the shell waste of deep-water pink shrimp ( Parapenaeus longirostris ) against oxidative stress of alloxanic adult male rats. Results Alloxan treatment revealed a significant elevation in plasma glycemia and lipid parameters such as total lipid, total cholesterol and triglycerides compared to the control group (C). In addition, liver malonaldialdehyde levels (MDA), an index of lipid peroxidation, significantly increased compared to control group. The activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) and reduced glutathione (GSH) levels decreased significantly compared to control group. Moreover, diabetic rats presented a significant increase in the activities of aspartate transaminase (AST) alanine transaminase (ALT) and alkaline phosphatase (ALP) in plasma, indicating considerable hepatocellular injury. Astaxanthin treatment restores these parameters near to control values. Histological studies on the liver tissue of alloxan and astaxanthin treated rats confirmed the protective effects of astaxanthin. Conclusions The results revealed that astaxanthin may be helpful in preventing diabetic complications in adult rats by reversing hepatotoxicity. It can be one of the ingredients in a number of healthy products.

Enzymatic hydrolysis of chickpea protein concentrate (CP) by Alcalase® and some physiochemical and antioxidant properties of the resulting hydrolysate (CPH) were characterised. CPH displayed higher antioxidant activity than CP. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into four major fractions (Fra.I, Fra.II, Fra.III, and Fra.IV). Fraction III, which exhibited the highest DPPH scavenging activity (54% at 1 mg/ml), was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Eleven antioxidant fractions were isolated and two peptide sub-fractions show antioxidant activity (P3 and P8). The P8 displayed the highest DPPH radical-scavenging activity (67%; at 200 µg/ml) among these peptides subfractions. The molecular masses and amino acids sequences of the purified peptides were determined using ESI-MS and ESIMS/MS, respectively. Their structures were identified as Asp-His-Gly and Val-Gly-Asp-Ile. These peptides did not show haemolytic activity towards bovine erythrocytes. The results suggest that CPH are good source of natural antioxidants.

There is great interest in the development of antimicrobial peptides as a potentially novel class of antimicrobial agents. In this study we evaluated the mode of action of new peptides (Gly-Val-His, Trp-His-Arg, Trp-His-Phe, Pro-Pro-Ser-Ser, Ala-Ala-Ala-Leu, Ala-Ala-Gly-Gly-Val, Ala-Ala-Val-Lys-Met, Ala-Ser-Ser-Ser), previously characterized, from barbel protein hydrolysates against Listeria monocytogenes via a membrane damage mechanism. The minimum inhibitory concentrations (MIC) of these peptides toward six strains and their hemolytic activity toward bovine erythrocytes were determined. Prediction of peptide secondary structure indicated that these peptides should have random coil structures and high content of hydrophobic amino acids. In addition, results of the determination of extracellular potassium, which is considered a good marker of membrane permeability, revealed that treatment with pure barbel peptides could cause morphological changes of L. monocytogenes and destruction of the cell integrity via irreversible membrane damage. The results could provide information for investigating the antibacterial model of antibacterial peptides derived from fish protein hydrolysates.

Peptides obtained by enzymatic hydrolysis of fish proteins exhibit not only nutritional but also biological properties of dietary uses, or even therapeutic potential. The objective of the present study was to isolate and characterize peptides from the protein hydrolysates of barbel muscle with antibacterial activity against Gram-positive ( Listeria monocytogenes , Staphylococcus aureus , Enterococcus faecalis , Micrococcus luteus and Bacillus cereus ) and Gram-negative ( Escherichia coli , Salmonella enterica , Pseudomonas aeruginosa , Klebsiella pneumoniae and Enterobacter sp.) bacteria. Barbel muscle protein hydrolysates (BMPHs), obtained by treatment with Alcalase ® (DH = 6.6%), was fractionated by size exclusion chromatography on a Sephadex G-25 and purified by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses and amino acid sequences of these peptides were determined using ESI–MS and ESI–MS/MS, respectively. Eleven peptides in F II-1 , F II-2 , F II-3 and F II-4 sub-fractions separated by RP-HPLC were identified. The most active peptide fraction (F II-3 ) contained three peptides: Ala–Ala–Ala–Leu; Ala–Ala–Gly–Gly–Val and Ala–Ala–Val–Lys–Met. These peptides don't show hemolytic activity towards bovine erythrocytes. These results suggest that some peptides from barbel could be a beneficial ingredient for nutraceuticals.

The incorporation of protein hydrolysates is of increasing commercial interest. The present study investigated the angiotensin-I converting enzyme (ACE) inhibitory activities and antioxidant properties of goby protein hydrolysates (GPHs) obtained by treatment with various gastrointestinal fish proteases. All hydrolysates displayed ACE-inhibitory activity. The hydrolysate generated by the grey triggerfish proteases (GPH-TF) displayed the highest ACE-inhibitory activity (99 ± 1.4%, at 1.5 mg/ml) followed by that obtained by smooth hound proteases (GPH-SH) (87.5 ± 0.7%). Additionally, results indicated that GPHs showed varying degrees of antioxidant activities, evaluated by using various antioxidant assays. Hydrolysate obtained by treatment with crude protease from golden mullet (GPH-GM) showed the most pronounced DPPH-radical scavenging effect, followed by bovine trypsin hydrolysate (GPH-T), while that obtained by triggerfish proteases (GPH-TF) displayed the highest β-carotene bleaching inhibitory effects. Moreover, all hydrolysates, except GPH-SH, showed high chelating effect and lipid peroxidation inhibition, even at low concentrations. Further, addition of GPH-TF in turkey meat sausage resulted in products with improved antioxidant activity. The results suggested that GPHs are good source of natural ACE-inhibitory and antioxidant peptides.

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.

The use of antimicrobial peptides (AMPs) is an alternative to traditional antibiotics. AMPs are obtained using different methods such as bacterial synthesis, chemical synthesis and controlled enzymatic hydrolysis. The later is an interesting approach that deserves our attention because of the yields gathered and peptides engineered. Usually, activities of AMPs obtained in such a way are tightly dependent on the hydrolysis mechanism used. This paper deals with the hydrolysis of hemoglobin mechanism as a potential source of AMPs. Production of AMPs from hemoglobin using enzymatic controlled system is linked to hemoglobin structure. Further, we show that bovine hemoglobin, which is sensitive to peptic hydrolysis, results upon enzymatic digestion as a great source of AMPs. The hemoglobin in native and denatured states was hydrolyzed by “one-by-one” and “zipper” mechanisms, respectively. Nevertheless, a new mechanism named “semi-zipper” mechanism is obtained when protein is in molten globule structural state, constituting an original strategy for AMPs production. Seventy seven percentage of the peptides obtained by this new strategy showed antibacterial activity against nine strains.

The anticoagulant activities of protein hydrolysates prepared from goby muscle by treatment with various bacterial alkaline proteases were investigated. All proteases exhibited varying degrees of hydrolysis (DH) and all goby protein hydrolysates (GPHs) caused a significant prolongation of both the thrombin time (TT) and the activated partial thromboplastin time (APTT). The hydrolysate generated by the crude protease from Bacillus licheniformis NH1 displayed the highest anticoagulant activity, and the higher TT (about 32 s) at a concentration of 5 mg/mL was obtained with hydrolysate having a DH of 8.86%. This hydrolysate was then fractionated by size exclusion chromatography on a Sephadex G-25 column into five major fractions (F1–F5). Fraction F2, which exhibited the highest anticoagulant activity, was then fractionated by reversed-phase high-performance liquid chromatography. The molecular masses and amino acid sequences of four peptides in peptide sub-fraction F2–6, which exhibited the highest anticoagulant activity, were determined using ESI-MS and ESI-MS/MS, respectively. The structures of these peptides were identified as Leu-Cys-Arg, His-Cys-Phe, Cys-Leu-Cys-Arg and Leu-Cys-Arg-Arg.

In this study we evaluated the short-term oral antihypertensive effect of several peptide sequences isolated from cuttlefish (Sepia officinalis) muscle proteins hydrolysates, previously characterized as in vitro angiotensin-I-converting enzyme-inhibitors, in spontaneously hypertensive rats (SHR). Systolic blood pressure (SBP) and the diastolic blood pressure (DBP) of the rats were measured by the tail cuff method before administration and also 2, 4, 6, 8 and 24 h post-administration. The peptide sequences Val-Tyr-Ala-Pro, Val-Ile-Ile-Phe and Ala-His-Ser-Tyr showed a clear decrease in SBP and DBP in SHR. Met-Ala-Trp caused a significant decrease of the SBP in the SHR, but this sequence caused a slight decrease in DBP in these animals. SBP and DBP returned to baseline values 24 h post-administration of all peptides. Thus, these peptides are bioactive ingredients with potential benefit in the prevention and treatment of hypertension.

Antioxidant activities of protein hydrolysates prepared from goby muscle by treatment with various bacterial alkaline proteases from Bacillus mojavensis A21, Bacillus subtilis A1, Bacillus pumilus A26, and Bacillus licheniformis NH1 and Alcalase were investigated. All hydrolysates showed different degrees of hydrolysis. The antioxidant activities of protein hydrolysates at different concentrations were evaluated using various in vitro antioxidant assays, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power assays, β-carotene bleaching, and DNA nicking assay. All protein hydrolysates showed varying degrees of antioxidant activity. The hydrolysate prepared using A26 proteases showed the strongest DPPH radical-scavenging activity, followed by NH1 and A21 hydrolysates. Moreover, NH1 hydrolysate exhibited notable effects against the discoloration of β-carotene. A1, A21, and alcalase protein hydrolysates exhibited high protection against hydroxyl radical induced DNA breakage.

Chymotrypsin from the hepatopancreas of cuttlefish (Sepia officinalis) was purified to homogeneity, with a 120-fold increase in specific activity and 23% recovery. The molecular weight of the purified chymotrypsin was estimated to be 28 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The optimum pH and temperature for the chymotrypsin activity were pH 8.5 and 55 °C, respectively, using succinyl- l -ala-ala-pro- l -phenylalanine-p-nitroanilide (SAAPFpNA) as a substrate. The enzyme was extremely stable in the pH range of 7.0–10.0 and highly stable up to 50 °C after 1 h incubation. This proteinase was strongly inhibited by chymostatin, soybean trypsin inhibitor, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but was not inhibited by tosyl- l -phenylalanine chloromethyl ketone, N-carbobenzoxy-phenylalanine chloromethyl ketone or Nα-tosyl- l -lysine chloromethyl ketone. The enzyme hydrolysed long chymotrypsin peptide substrates SAAPFpNA, SAAPLpNA and ZAALpNA and did not hydrolyse short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SAAPFpNA, with kcat 18 s−1 and Km 22 μM. However, the enzyme had a lower Km for SAAPLpNA, 54 μM. The N-terminal amino acid sequence of the first 20 amino acids of the purified chymotrypsin was IVGGQEATIGEYPWQAALQV.

Alpha-lactalbumin hydrolysate is of significant interest, due to its potential application as a source of bioactive peptides in nutraceutical and pharmaceutical domains. This study was focused on the cholecystokinin (CCK) family compounds which are small peptides involved in the satiety control. The action of chymotryptic hydrolysate of alpha-lactalbumin on cholecystokinin release from intestinal endocrine STC-1 cells was investigated. We demonstrated for the first time that a chymotryptic hydrolysate of alpha-lactalbumin was able to highly stimulate CCK-releasing activity from STC-1 cells. The peptidic hydrolysate was characterized by LC/MS and MS/MS, thus highlighting the presence of 11 fractions containing 21 peptides, each potentially having the desired activity.

Yeast extracts, by their nutritional characteristics, have a high potential as a source of biologically active molecules and functional food ingredients. In this work, in vitro and in vivo satietogenic effect of yeast extracts was examined. The in vitro results obtained for the first time showed that a yeast extract strongly stimulated the secretion of cholecystokinin (CCK) from endocrine STC-1 cells. The effects obtained with this yeast extract were compared to those obtained with other food products known for their potential involvement in satiety (milk proteins, soybean). This yeast extract showed a stronger ability to stimulate CCK secretion than all other products tested. Lack of toxicity of this yeast extract was demonstrated through cell proliferation assays. In vivo, the tests in rats confirmed the satietogenic potential of this yeast extract, particularly in terms of food intake and weight loss, but also for hormone levels.

The characteristics and functional properties of gelatin from skin cuttlefish ( Sepia officinalis ) were investigated and compared to those of halal bovine gelatin (HBG). The gelatin extraction efficiency was improved by an acid-swelling process in the presence of smooth hound crude acid protease extract (SHCAP). The yields of gelatins from cuttlefish skin after 48 h with acid and with crude acid protease (15 units/g alkaline-treated skin) were 2.21% and 7.84%, respectively. The gelatin from skin cuttlefish had high protein (91.35%) but low fat (0.28%) contents. Compared to HBG, the cuttlefish-skin gelatin (CSG) has different amino acids composition than halal bovine gelatin. CSG contained slightly low hydroxyproline and proline (180‰) than HBG (219‰), whereas the content of serine was higher (49‰ versus 29‰). The gel strength of the gelatin gel from CSG (181 g) was lower than that of HBG (259 g) (p

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 bovine hemoglobin hydrolysate obtained with pig pepsin in the presence of 30% ethanol. This hydrolysate was fractioned by reversed-phase high-performance liquid chromatography (RP-HPLC) into 12 major fractions (F1–F12). All fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize and identify the peptides in these fractions. This hydrolysis permitted to generate a new serial of bioactive peptides with both antimicrobial and ACE inhibitory activities. Identified peptides were TKAVEHLDDLPGALSELSDLHAHKLRVDPVNFKLLSHSLL, LDDLPGALSELSDLHAHKLRVDPVNFKLLSHSL, KLLSHSL, and LLSHSL corresponding respectively to the 67-106, 73-105, 99-105, and 100-105 fragments of the α chain of bovine hemoglobin. They were the first found from bovine hemoglobin. These purified peptides have an antibacterial activity against four bacteria strains: Kocuria luteus A270, Listeria innocua, Escherichia coli, and Staphylococcus aureus with a MIC between 187.1 and 35.2 μM. On the other hand, these peptides displayed at the same time ACE inhibitory activity with an IC50 range from 42.55 to 1,095 μM.

Cathepsin D from the hepatopancreas of cuttlefish ( Sepia officinalis ) was purified to homogeneity by precipitation with ammonium sulfate (30-60%, w/v), Sephadex G-100 gel filtration, Mono-S cation-exchange chromatography, Sephadex G-75 gel filtration, and Mono-S FPLC with a 54-fold increase in specific activity and 17% recovery. The molecular weight of the purified cathepsin D was estimated to be 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of the native-PAGE and hemoglobin zymography, the purified protease appeared as a single band. The optimum pH and temperature for the cathepsin D activity were pH 3.0 and 50 °C, respectively, using hemoglobin as a substrate. The purified enzyme was completely inhibited by pepstatin A; however, no inhibition was observed with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Moreover, the activity was strongly inhibited by SDS and molybdate and enhanced by ATP. The purified cathepsin D was activated by Mg(2+), Ni(2+), Zn(2+), Cu(2+), Cd(2+), Sr(2+), and Co(2+) ions, whereas it was not affected by Na(+), K(+), and Ca(2+) ions. The N-terminal amino acid sequence of the first 13 amino acids of the purified cathepsin D was APTPEPLSNYMDA. S. officinalis cathepsin D, which showed high homology with cathepsin D from marine vertebrates and invertebrates, had a Pro residue at position 6 and a Ser residue at position 8, where Thr and Lys are common in all marine vertebrates cathepsins D. S. officinalis cathepsin D showed high efficiency for the hydrolysis of myofibrillar proteins extracted from cuttlefish muscle.

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from muscle of cuttlefish ( Sepia officinalis ) by treatment with various digestive proteases were investigated. The most active hydrolysate was obtained with the crude protease extract from the hepatopancreas of cuttlefish (64.47 ± 1.0% at 2 mg of dry weight/ml) with a degree of hydrolysis of 8%. By gel filtration on Sephadex G-25 and RP-HPLC on C18 column, three novel peptides with high ACE-inhibitory activity were purified and their molecular masses and amino acid sequences were determined. The three peptides Val-Tyr-Ala-Pro, Val-Ile-Ile-Phe and Met-Ala-Trp with IC 50 values of 6.1, 8.7 and 16.32 μM, respectively, were novel ACE-inhibitory peptides. Lineweaver–Burk plots suggest that the three purified peptides act as non-competitive inhibitors against ACE. These results suggest that some peptides from cuttlefish could be a beneficial ingredient for nutraceuticals against hypertension.

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.

Two cuttlefish ( Sepia officinalis ) viscera protein hydrolysates were obtained with different enzymes extracted from cuttlefish and smooth hound ( Mustellus mustellus ). Their ability to stimulate the secretion of cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1), using the enteroendocrine STC-1 cell line, and to inhibit the DPP-IV activity during a simulated gastrointestinal digestion was assayed. The physico-chemical parameters of hydrolysates and their effects on intestinal cell viability were also determined. The hydrolysate obtained with cuttlefish enzymes (CVPH1) appeared to be the most promising for all assessed bioactivities. Thus CVPH1 was able to stimulate CCK and active GLP-1 releasing activities of enteroendocrine cells without any cytotoxicity and to inhibit DPP-IV activity. Moreover, these actions were enhanced after gastrointestinal digestion and CVPH1 was also able to inhibit the intestinal DPP-IV activity of Caco-2 cells. These very promising findings highlight, via two different mechanisms, the positive effect of CVPH1 on GLP-1 actions.

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the alpha-chain of bovine hemoglobin: alpha107-141, alpha137-141, and alpha133-141, and one peptide to the beta-chain: beta126-145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined.