Electroseparation of Slaughterhouse By-Product: Antimicrobial Peptide Enrichment by pH Modification.

The fractionation of bioactive peptides from hydrolysate is a main challenge to produce efficient alternative for synthetic additives. In this work, electrodialysis with ultrafiltration membrane (EDUF) was proposed to increase the purity of one antimicrobial peptide from slaughterhouse by-product hydrolysate. This targeted-peptide, α137-141 (653 Da, TSKYR), inhibits a large spectrum of microbial growths and delays meat rancidity; therefore, if concentrated, it could be used as food antimicrobial. In this context, three pH values were investigated during EDUF treatment to increase the α137-141 purity: 4.7, 6.5, and 9. pH 9 showed the highest purity increase-75-fold compared to the initial hydrolysate. Although the whole hydrolysate contains more than 100 peptides, only six peptides were recovered at a significant concentration. In this fraction, the α137-141 peptide represented more than 50% of the recovered total peptide concentration. The EDUF α137-141-enriched fraction obtained in this optimized condition would be a promising natural preservative to substitute synthetic additives used to protect food.

Bovine hemoglobin: an attractive source of antibacterial peptides.

A peptic hemoglobin hydrolysate was fractioned by a semi-preparative reversed-phase HPLC and some fractions have an antibacterial activity against four bacteria strains: Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis. These fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize the peptides in these fractions. Each fraction contains at least three peptides and some fractions contain five peptides. All these fractions were purified several times by HPLC to obtain pure peptides. Thirty antibacterial peptides were identified. From the isolated antibacterial peptides, 24 peptides were derived from the alpha chains of hemoglobin and 6 peptides were derived from the beta chains of hemoglobin. The lowest concentration of these peptides (minimum inhibitory concentration (MIC)) necessary to completely inhibit the growth of four bacteria strain was determined. The cell population of all of the tested bacteria species decreased by at least 97% after a 24-h incubation with any of the peptides at the minimum inhibitory concentration.

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

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

Isolation and characterization of four antibacterial peptides from bovine hemoglobin.

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.

Purification, identification and structural modelling of DPP-IV inhibiting peptides from barbel protein hydrolysate.

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 IC50 value of 1.94mg/mL. It was fractionated into five major fractions (FI-FV) by size exclusion chromatography using a Superdex peptide. The FIII fraction was noted to display the highest inhibitory activity, with an IC50 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 SF4 sub-fraction showed the highest DPP-IV inhibitory activity, with an IC50 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 (IC50=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.

New antibacterial peptide derived from bovine hemoglobin.

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.

Nine novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttlefish (Sepia officinalis) muscle protein hydrolysates and antihypertensive effect of the potent active peptide in spontaneously hypertensive rats.

This study aimed to identify novel ACE inhibitory peptides from the muscle of cuttlefish. Proteins were hydrolyzed and the hydrolysates were then subjected to various types of chromatography to isolate the active peptides. Nine ACE inhibitory peptides were isolated and their molecular masses and amino acid sequences were determined using ESI-MS and ESI-MS/MS, respectively. The structures of the most potent peptides were identified as Val-Glu-Leu-Tyr-Pro, Ala-Phe-Val-Gly-Tyr-Val-Leu-Pro and Glu-Lys-Ser-Tyr-Glu-Leu-Pro. The first peptide displayed the highest ACE inhibitory activity with an IC50 of 5.22µM. Lineweaver-Burk plots suggest that Val-Glu-Leu-Tyr-Pro acts as a non-competitive inhibitor against ACE. Furthermore, antihypertensive effects in spontaneously hypertensive rats (SHR) also revealed that oral administration of Val-Glu-Leu-Tyr-Pro can decrease systolic blood pressure significantly (p<0.01). These results suggest that the Val-Glu-Leu-Tyr-Pro would be a beneficial ingredient for nutraceuticals and pharmaceuticals acting against hypertension and its related diseases.

Ability of natural astaxanthin from shrimp by-products to attenuate liver oxidative stress in diabetic rats.

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.

Biochemical and antioxidant properties of peptidic fraction of carotenoproteins generated from shrimp by-products by enzymatic hydrolysis.

The composition, functional properties and in vitro antioxidative activity of the peptidic fraction of carotenoproteins from shrimp (Parapenaeus longirostris) by-products generated by enzymatic treatment with Alcalase was evaluated. The peptidic fraction of carotenoproteins (PFCP) contained 80.8 ± 0.21% protein, 2.74 ± 0.3% lipid, 14.4 ± 0.14% ash, 1.13 ± 0.08% chitin and 1.08 ± 0.02 µg total carotenoid/g of sample. The amino acid profile of PFCP showed a high percentage of essential amino acids, such as arginine, lysine, histidine and leucine. Therefore, PFCP had a high nutritional value and could be used as a supplement to poorly balanced dietary proteins. PFCP showed an excellent solubility and possessed interfacial properties, which were governed by their concentrations. The antioxidant activities of PFCP at different concentrations were evaluated using various in vitro antioxidant assays, including the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power, chelating effects assay and ß-carotene bleaching. The antioxidant activity of PFCP, based on their protection of supercoiled DNA strand from scission by peroxyl and hydroxyl radicals into the nicked circular form was also investigated. Results from this study suggest that the peptidic fraction of carotenoproteins is a good source of natural antioxidants and peptides with interesting functionalities.

Controlled Enzymatic Hydrolysis: A New Strategy for the Discovery of Antimicrobial Peptides.

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.

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

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.

Characterization and identification of a chymotryptic hydrolysate of alpha-lactalbumin stimulating cholecystokinin release in STC-1 cells.

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.

RYH: a minimal peptidic sequence obtained from beta-chain hemoglobin exhibiting an antimicrobial activity.

Bovine hemoglobin is an animal protein described as source of bioactive peptides. Enzymatic hydrolysis of this protein results into some peptides exhibiting antimicrobial activity against Gram-positive and Gram-negative bacteria. In this study, a family of peptides from the beta chain (beta-114-145 derived peptides) obtained by peptic hydrolysis of bovine hemoglobin, was purified by reverse-phase HPLC and characterized by different analytical techniques (mass spectrometry, circular dichroism). The minimum inhibitory concentration was determined to show the antimicrobial activity of these peptides. Four bacterial strains were used: two Gram-negative (Escherichia coli and Salmonella Enteritidis) and two Gram-positive strains (Listeria innocua and Micrococcus luteus). The effect of these peptides on artificial membrane was also measured. Our findings showed that the peptide ß114-145 and its peptic derivatives contain the RYH sequence. The most antimicrobial peptide is the RYH peptide which was the shortest one.

Minimal antimicrobial peptidic sequence from hemoglobin alpha-chain: KYR.

Hemoglobin is an animal protein described as a source of biologically active peptides. Peptic digestion of bovine hemoglobin alpha-chain allowed obtaining peptide fractions with antimicrobial activity. These peptides were purified by reverse-phase High-Performance Liquid Chromatography (HPLC) and characterized by mass spectrometry. The minimal inhibitory concentration and mode of action of these peptides were studied against five bacterial strains including Escherichia coli and Salmonella enteritidis as Gram-negative bacteria and Listeria innocua, Micrococcus luteus and Staphylococcus aureus as Gram-positive bacteria. The action aforementioned peptides were studied on artificial membranes as well. The most active peptides resulted to be the short ones. Consequently, the minimal peptidic sequence necessary for the antibacterial activity was clearly determined: KYR.

Obtaining antimicrobial peptides by controlled peptic hydrolysis of bovine hemoglobin.

Under standard conditions, the peptides and specially the active peptides were obtained from either the denatured hemoglobin that all structures are completely modified or either the native hemoglobin where all structures are intact. In these conditions, antibacterial peptides were isolated from a very complex peptidic hydrolysate which contains more than one hundred peptides having various sizes and characteristics, involving a complex purification process. The new hydrolysis conditions were obtained by using 40% methanol, 30% ethanol, 20% propanol or 10% butanol. These conditions, where only the secondary structure of hemoglobin retains intact, were followed in order to enrich the hydrolyzed hemoglobin by active peptides or obtain new antibacterial peptides. In these controlled peptic hydrolysis of hemoglobin, a selective and restrictive hydrolysate contained only 29 peptides was obtained. 26 peptides have an antibacterial activity against Micrococcus luteus, Listeria innocua, and Escherichia coli with MIC from 187.1 to 1 µM. Among these peptides, 13 new antibacterial peptides are obtained only in these new hydrolysis conditions.

Comparative Study on Biochemical Properties and Antioxidative Activity of Cuttlefish (Sepia officinalis) Protein Hydrolysates Produced by Alcalase and Bacillus licheniformis NH1 Proteases.

Antioxidative activities and biochemical properties of protein hydrolysates prepared from cuttlefish (Sepia officinalis) using Alcalase 2.4 L and Bacillus licheniformis NH1 proteases with different degrees of hydrolysis (DH) were determined. For the biochemical properties, hydrolysis by both enzymes increased protein solubility to above 75% over a wide pH range. The antioxidant activities of cuttlefish protein hydrolysates (CPHs) increase with increasing DH. In addition, all CPHs exhibited antioxidative activity in a concentration-dependent manner. NH1-CPHs generally showed greater antioxidative activity than Alcalase protein hydrolysates (P < 0.05) as indicated by the higher 1,1-diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity and ferrous chelating activity. Both Alcalase and NH1 protein hydrolysates were able to retard lipid peroxidation and ß-carotene-linoleic acid oxidation. Alcalase-CPH (DH = 12.5%) and NH1-CPH (DH = 15%) contained 75.36% and 80.11% protein, respectively, with histidine and arginine as the major amino acids, followed by glutamic acid/glutamine, serine, lysine, and leucine. In addition, CPHs have a high percentage of essential amino acids made up 48.85% and 50.04%. Cuttlefish muscle protein hydrolysates had a high nutritional value and could be used as supplement to poorly balanced dietary proteins.

Analysis of novel angiotensin I-converting enzyme inhibitory peptides from enzymatic hydrolysates of cuttlefish (Sepia officinalis) muscle proteins.

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from cuttlefish (Sepia officinalis) proteins by treatment with various bacterial proteases were investigated. The hydrolysate generated by the crude enzyme from Bacillus mojavensis A21 displayed the highest ACE inhibitory activity, and the higher inhibition activity (87.11 +/- 0.92% at 2 mg/mL) was obtained with hydrolysis degree of 16%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P(1)-P(8)). Fraction P(6), which exhibited the highest ACE inhibitory activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Eleven ACE inhibitory peptides were isolated, and their molecular masses and amino acids sequences were determined using ESI-MS and ESI-MS/MS, respectively. The structures of the most potent peptides were identified as Ala-His-Ser-Tyr, Gly-Asp-Ala-Pro, Ala-Gly-Ser-Pro and Asp-Phe-Gly. The first peptide displayed the highest ACE inhibitory activity with an IC(50) of 11.6 microM. The results of this study suggest that cuttlefish protein hydrolysates are a good source of ACE inhibitory peptides.

Cathepsin D from the hepatopancreas of the cuttlefish (Sepia officinalis): purification and characterization.

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.

Antibacterial activity of some lactic acid bacteria isolated from an Algerian dairy product.

In the present study, the antibacterial effect of 20 lactic acid bacteria isolates from a traditional cheese was investigated. 6 isolates showed antibacterial effect against Gram positive bacteria. Streptococcus thermophilus T2 strain showed the wide inhibitory spectrum against the Gram positive bacteria. Growth and bacteriocin production profiles showed that the maximal bacteriocin production, by S. thermophilus T2 cells, was measured by the end of the late-log phase (90 AU ml(-1)) with a bacteriocine production rate of 9.3 (AU ml(-1)) h(-1). In addition, our findings showed that the bacteriocin, produced by S. thermophilus T2, was stable over a wide pH range (4-8); this indicates that such bacteriocin may be useful in acidic as well as nonacidic food. This preliminarily work shows the potential application of autochthonous lactic acid bacteria to improve safety of traditional fermented food.

Continuous production of a peptidic fraction containing the intermediate opioid peptide LVV-haemorphin-7 (LVVh-7) by peptic hydrolysis of bovine haemoglobin in a continuous membrane reactor.

Peptic hydrolysis of native bovine haemoglobin at pH 3 yields the LVV-haemorphin-7 (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe; LVVh-7) opioid peptide corresponding to the residues-31-40 fragment of the beta-chain of haemoglobin. This peptide is intermediate in the course of batch hydrolysis and is rapidly degraded. Indeed, it shows an optimum at 3% degree of hydrolysis (i.e. 2 min of reaction time). The hydrolysis was carried out in a continuous membrane reactor with a space time (ratio of the flux to the reactor volume) set to 2 min (corresponding to optimum LVVh-7 production). This process allows the continuous production of a constant fraction of intermediate peptides containing LVVh-7 for 48 min.