Activity Of Peptide Fractions Research Articles

Isolation and structural elucidation of antioxidant peptides from oyster (Saccostrea cucullata) protein hydrolysate.

Protein derived from the oyster (Saccostrea cucullata) was hydrolyzed using protease from Bacillus cereus SU12 for isolation of antioxidant peptides. The oyster hydrolysate exhibited a strong antioxidant potential in DPPH (85.7±0.37%) followed by Hydrogen peroxide radical scavenging activity (81.6±0.3%), Hydroxyl radical-scavenging activity (79.32±0.6%), Reducing power assay (2.63±0.2 OD at 700nm). Due to the high antioxidant potential, hydrolysate was fractionated in Sephadex G-25 gel filtration chromatography. The active peptide fraction was further purified by UPLC-MS. Totally 7 antioxidant peptides were collected. Among 7 peptides (SCAP 1-7), 3 peptides (SCAP 1, 3 and 7) had highest scavenging ability on DPPH radicals. The amino acid sequence and molecular mass of purified antioxidant peptides (SCAP1, SCAP3 and SCAP7) were determined by Q-TOF ESI mass spectroscopy and structures of the peptides were Leu-Ala-Asn-Ala-Lys (MW=515.29Da), Pro-Ser-Leu-Val-Gly-Arg-Pro-Pro-Val-Gly-Lys-Leu-Thr-Leu (MW=1432.89Da) and Val-Lys-Val-Leu-Leu-Glu-His-Pro-Val-Leu (MW=1145.75Da), respectively. The unique amino acid composition and sequence in the peptides might play an important role in expression of their antioxidant activity. The results of this study suggest that oyster protein hydrolysate is good source of natural antioxidants.

Antioxidant Properties of Protein Hydrolysate Obtained from Oyster Saccostrea cucullata (Born, 1778)

The antioxidant activities of enzymatically hydrolyzed (protease from Bacillus cereus SU12) oyster (Saccostrea cucullata) protein were studied. The hydrolysate exhibited a strong antioxidant potential in 1, 1-diphenyl-2-picrylhydrazyl (DPPH, 85.7 ± 0.37%), followed by hydrogen peroxide radical scavenging activity (81.6 ± 0.3%), hydroxyl radical scavenging activity (79.32 ± 0.6%), and reducing power assay (2.63 ± 0.2 OD at 700 nm) at a concentration of 1 mg/mL. Due to the high antioxidant potential, the hydrolysate was purified in Sephadex G-25 gel filtration chromatography. The active peptide fraction was identified by DPPH and reducing power assay. The amino acid content of the purified active peptide fraction was analyzed by high performance liquid chromatography. The active fraction contained a good quantity of both essential and nonessential amino acids. The present study revealed that oyster (S. cucullata) protein hydrolysate is a potential source for natural antioxidants.

Purification and Characterization of Antioxidant Peptides from Oyster (Saccostrea cucullata) Hydrolysate and the Anticancer Activity of Hydrolysate on Human Colon Cancer Cell Lines

The focus of the study was to investigate antioxidant activity and characterize antioxidant peptides from oyster (Saccostrea cucullata) protein hydrolysate. The protease hydrolysate of oyster exhibited strong potential to donate hydrogen and was able to scavenge Hydrogen peroxide, Hydroxyl and DPPH radicals. Due to the high antioxidant potential, hydrolysate was purified in Sephadex G-25 gel filtration chromatography. The active peptide fraction was further purified by UPLC-MS. Totally seven antioxidant peptides were collected. Among seven peptides (SCAP 1–7), three peptides (SCAP 1, 3 and 7) had highest scavenging ability on DPPH radicals. The amino acid sequence and molecular mass of purified antioxidant peptides (SCAP1, SCAP3 and SCAP7) were determined by Q-TOF ESI mass spectroscopy and structures of the peptides were Leu-Ala-Asn-Ala-Lys (MW = 515.29 Da), Pro-Ser-Leu-Val-Gly-Arg-Pro–Pro-Val-Gly-Lys-Leu-Thr-Leu (MW = 1,432.89 Da) and Val-Lys-Val-Leu-Leu-Glu-His-Pro-Val-Leu (MW = 1,145.75 Da), respectively. The oyster hydrolysate was tested for cell cytotoxicity on Vero (kidney epithelial cells of the African Green Monkey) and HT-29 (human colon carcinoma) cell lines. It was found that the hydrolysate did not show any cytotoxic effect for Vero cell lines and exerted a significant cytotoxic effect on HT-29 cell lines. We thus conclude that the anticancer and antioxidative hydrolysate from oyster (S. cucullata) may be useful ingredients in food and nutraceutical applications.

Production and properties of health-promoting proteins and peptides from bovine colostrum and milk.

The high nutritive value and diverse functional properties of milk proteins are well known. Beyond these qualities, milk proteins have attracted growing scientific and commercial interest as a source of biologically active molecules. Such proteins are found in abundance in colostrum which is the initial milk secreted by mammalian species during late pregnancy and the first few days after birth of the offspring. The best characterized colostrum-based bioactive proteins include alpha-lactalbumin, beta-lactoglobulin, immunoglobulins, lactoferrin, lactoperoxidase and growth factors. All of them can nowadays be enriched and purified on an industrial scale from bovine colostral whey or cheese whey. These native proteins exhibit a wide range of biological activities that are known to affect the digestive function, metabolic responses to absorbed nutrients, growth and development of organs and disease resistance. Also, some of these proteins may prove beneficial in reduction of the risks of chronic human diseases reflected by the metabolic syndrome. It is speculated that such potentially beneficial effects are partially attributed to bioactive peptides derived from intact proteins. These peptides can be liberated during gastrointestinal digestion or fermentation of milk by starter cultures. The efficacy of a few peptides has been established in animal and human studies and the number of commercial products supplemented with specific milk peptides is envisaged to increase on global markets. Bovine colostrum appears as a highly potential source of biologically active native proteins and peptide fractions for inclusion as health-promoting ingredients in various food applications.

Antibacterial peptides from barbel muscle protein hydrolysates: Activity against some pathogenic bacteria

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 FII-1, FII-2, FII-3 and FII-4 sub-fractions separated by RP-HPLC were identified. The most active peptide fraction (FII-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.

Partial Characterization of Ultrafiltrated Soy Protein Hydrolysates with Antioxidant and Free Radical Scavenging Activities

Soy protein isolate (SPI) was hydrolyzed with Flavourzyme® (SHF) or chymotrypsin (SHC). Hydrolysates were sequencially fractionated by ultrafiltration using different membrane pore sizes (50, 10, and 3 kDa). The antioxidant ability of each hydrolysate protein fraction was tested in a liposome oxidizing system and their free radical scavenging activity (FRSA) was evaluated with the DPPH method (diphenylpicrilhydrazine radical). Molecular weight (MW) distribution, solubility, surface hydrophobicity, and amino acid composition of each SPI hydrolysate fraction were measured and their effect on antioxidant and scavenging activities was established by multivariate correlation. The most active ultrafiltrated peptide fractions (P < 0.05), from SHF and SHC, had of MW of <3 kDa (F3 and C3, respectively). These fractions decreased liposome oxidation by 83.2% and 84.5%, respectively, and also showed the highest FRSA (F3: 21.3% and C3: 24.4%). In addition to molecular size, the antioxidant activity and FRSA of soy protein fractions were related to their amino acid composition, especially to an increased content of Phe and a lowered content of Lys. Also, hydrophobicity of ultrafiltrated peptide fractions was an important characteristic (P < 0.001) associated with their ability to trap free radicals. Ultrafiltered peptide fractions with low MW have a high potential to be used as natural alternatives to prevent lipid oxidation in foods.

Free radical scavenging activity of peptide fractions from defatted soybean meal hydrolysates evaluated by electron spin resonance

Defatted soybean meal, a by-product of soybean oil extraction, was pretreated by ultrasonic and hydrolyzed with neutrase. The DSMH pretreated at 400 W of ultrasonic power, identified to possess the strongest antioxidant activity, was fractionated according to molecular weight into three fractions of DSMH-I (>10 kDa), DSMH-II (5-10 kDa), and DSMH-III (<5 kDa) using ultrafiltration. The fraction, DSMH-III (<5 kDa), exhibited the highest antioxidative activity and was further purified using ion-exchange chromatography. The DSMH-III was separated into five fractions (A, B, C, D, and E). Fraction C with molecular weight of 2434 Da exhibited the strongest free radical scavenging, which was evidenced by the electron spin resonance of 1,1-diphenyl-2-pycryl hydrazyl and hydroxyl radicals. Fraction C was subjected to reverse-phase high performance liquid chromatography and the sequences of the highest activity peptide were determined by liquid chromatography tandem mass spectrometry. The strongest antioxidant activity peptide had the amino acid sequence of Glu-Glu-Gln-Glu-Trp-Pro-Arg-Lys-Glu-Glu-Lys. In conclusion, ultrasonic treatment and ultrafiltration could enhanced antioxidant activity of DSMH.

Detection of antibacterial activity in an enzymatic hydrolysate fraction obtained from processing of Atlantic rock crab (Cancer irroratus) by-products

Peptide fractions generated via proteolytic processing of Atlantic rock crab by-products were tested for antibacterial activities against bacterial strains of interest in food and health. Of all fractions obtained the specific nano-filtered retentate exhibited an antibacterial activity. The peptide fraction demonstrated inhibitory activity against several Gram-negative bacilli and against some Gram-positive bacteria. The principal bioactive peptide fraction was of low molecular weight and was comprised mainly of proteins, 74%, and minerals, 16%. The amino acid content revealed that lysine, leucine, arginine, aspartic acid and glutamic acid residues were represented in the highest composition. The optimal inhibitory activity was observed at alkaline pH 9. The first step of purification, performed on a chromatography resin, led to the isolation of an anionic antibacterial peptidic fraction. Following gel permeation chromatography analysis, the active peptide fraction appears to be a complex of molecules ranging from 200Da to 750Da. Results indicating the production of antibacterial molecules from enzymatic hydrolysis of Atlantic rock crab by-products should be viewed in high-value bioproducts in several areas such as food and health sectors.

Isolation and Characterization of an Antibacterial Peptide Fraction from the Pepsin Hydrolysate of Half-Fin Anchovy (Setipinna taty)

Enzymatic proteolysis of food proteins is considered a promising method to generate antibacterial peptides. The objective of the present study was to isolate and characterize peptide fraction from the pepsin hydrolysate of half-fin anchovy (Setipinna taty) with antibacterial activity against Escherichia coli. The most active peptide fraction HAHp2-3-I was isolated by a series of chromatographic methods, including Sephadex G-25 chromatography, reverse high-performance liquid chromatography (RP-HPLC) and Source 5RPC ST. Peptides identification of HAHp2-3-I was carried out using UPLC-LTQ-Orbitrap mass spectrometer. HAHp2-3-I contained five cationic peptides (MLTTPPHAKYVLQW, SHAATKAPPKNGNY, PTAGVANALQHA, QLGTHSAQPVPF and VNVDERWRKL) and three anionic peptides (LATVSVGAVELCY, NPEFLASGDHLDNLQ and PEVVYECLHW). Prediction of peptide secondary structure indicated that these anionic peptides should have extended strand and random coil structures, whereas cationic peptides PTAGVANALQHA and VNVDERWRKL could form alpha helixes. In addition, results of scanning electron microscopy (SEM) revealed that treatment by HAHp2-3-I could cause the morphological changes of E. coli 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 from water buffalo cheese whey induced senescence cell death via ceramide secretion in human colon adenocarcinoma cell line

Milk proteins are a source of bioactive peptides. Recent studies have indicated that protein-derived peptides released in buffalo cheese acid whey exert a cytomodulatory effect in human epithelial colon cancer (CaCo2) cells. The aim of the present study was to explain the molecular mechanism involved in the response of CaCo2 cells to oxidative stress in the presence of peptide fractions of buffalo cheese whey, purified and characterized by mass spectrometry. We demonstrated that treatment of CaCo2 treated with H2O2 (H-CaCo2) cells with a partially purified peptide sub-fraction (f3) from buffalo cheese acid whey induced a reduction of mitochondrial superoxide anion with subsequent decrease in heat shock protein 70 and 90 expression. Moreover, we observed a 5-fold decrease in cyclin A expression and cell cycle arrest in G1/G0 phases. These responses were associated with increased activity of alkaline phosphatase and beta-galactosidase, markers of differentiation and senescence respectively. The structural characterization of the active peptide fraction and the elucidation of the effects induced by its treatment on H-CaCo2 cells in vitro demonstrated an activity of this peptide sub-fraction in the modulation of cell cycle, thus suggesting potential application for the development of nutraceuticals as well as health-promoting functional foods.

Evidence of Antibacterial Activities in Peptide Fractions Originating from Snow Crab (Chionoecetes opilio) By-Products.

Antibacterial peptide fractions generated via proteolytic processing of snow crab by-products exhibited activity against Gram-negative and Gram-positive bacteria. Among the bacterial strains tested, peptide fractions demonstrated inhibitory activity against the Gram-negative bacteria such as Aeromonas caviae, Aeromonas hydrophila, Campylobacter jejuni, Listonella anguillarum, Morganella morganii, Shewanella putrefasciens, Vibrio parahaemolyticus and Vibrio vulnificus and against a few Gram-positive bacteria such as Listeria monocytogenes, Staphylococcus epidermidis and Streptococcus agalactiae. The principal bioactive peptide fraction was comprised mainly of proteins and minerals (74.3 and 15.5%, respectively). Lipids were not detected. The amino acid content revealed that arginine (4.6%), glutamic acid (5.3%) and tyrosine (4.8%) residues were represented in the highest composition in the antibacterial peptide fraction. The optimal inhibitory activity was observed at alkaline pH. The V. vulnificus strain, most sensitive to the peptide fraction, was used to develop purification methods. The most promising chromatography resins selected for purification, in order to isolate peptides of interest and to carry out their detailed biochemical characterization, were the SP-Sepharose™ Fast Flow cation exchanger and the Phenyl Sepharose™ High Performance hydrophobic interaction media. The partially purified antibacterial peptide fraction was analyzed for minimum inhibitory concentration (MIC) determination, and the value obtained was 25μgml(-1). Following mass spectrometry analysis, the active peptide fraction seems to be a complex of molecules comprised of several amino acids and other organic compounds. In addition, copper was the main metal found in the active peptide fraction. Results indicate the production of antibacterial molecules from crustacean by-products that support further applications for high-value bioproducts in several areas such as food and health.

Enhancing the anti-adipogenic activity of soy protein by limited hydrolysis with Flavourzyme and ultrafiltration

Limited hydrolysis of soy protein isolate (SPI) with Flavourzyme for 2 h to obtain the hydrolysate (FH2h) revealed much higher suppression of glycerol-3-phosphate dehydrogenase (GPDH) activity and relative lipid accumulation (RLA) than intact SPI in 3T3-L1 preadipocytes during differentiation. Lower GPDH activity or RLA indicates higher anti-adipogenic activity. The GPDH significantly decreased from 673 to 477 U/mg protein ( p < 0.05). Sequentially fractionating FH2h with 30–1 kDa (kilo-daltons) molecular weight cut-off (MWCO) membranes to obtain the 1 kDa permeate resulted in further reduction of 59% GPDH activity. When comparing the high-performance size-exclusion chromatography (HPSEC) profiles, the most active peptide fraction for the anti-adipogenic activity was primarily composed of small peptides with molecular weight less than 1300 Da. According to the Western immunoblot analysis, 1 kDa permeate inhibits adipogenesis by affecting the expression of peroxisome proliferators-activated receptor γ (PPARγ) and the CCAAT/enhancer binding protein α (C/EBPα) during 3T3-L1 cells differentiation.

ACE-inhibitory activity of probiotic yoghurt

In this study, the in vitro angiotensin-converting enzyme (ACE)-inhibitory (ACE-I) activity of peptide fractions from different yoghurt batches was assessed. Inhibition of ACE activity resulted in an overall antihypertensive effect. Yoghurts were prepared either using a sole yoghurt culture including Lactobacillus delbrueckii ssp . bulgaricus Lb1466 and Streptococcus thermophilus St1342, or L. acidophilus L10, L. casei L26 and Bifidobacterium lactis B94 in addition to yoghurt culture. ACE-I activity was determined at weekly intervals during 28 days of cold storage. Peptide fractions showing high ACE-I activity were further purified using multiple-steps of RP-HPLC. All probiotic yoghurts showed appreciable ACE-I activity during initial stages of storage compared with the control yoghurt, with a significant ( p<0.05) decrease afterwards. The ACE-I activity ranged from IC 50 of 103.30–27.79 μg mL −1 with the greatest ACE inhibition achieved during first and third week of storage. The in vitro ACE-I activity could be related to the peptide liberation via degradation of caseins. In total, 8 ACE-I peptides were characterized originating from α s2-casein (1), κ-casein (2) and β-casein, of which two well-known ACE-inhibiting peptides, namely Val–Pro–Pro (VPP) and Ile–Pro–Pro (IPP), were identified. These peptides are already used in commercial products.

Antigenotoxicity of peptides produced from silk fibroin

This study was conducted to examine in vitro antigenotoxic effects of the peptides derived from the hydrolysates of silk fibroin in mouse embryo 3T3 cells. The hydrolysates were prepared by acid or enzymic hydrolysis of fibroin, and antigenotoxicities were determined by measuring the reduced levels of DNA damage using the Comet assay. The fibroin preparation isolated from cocoons was efficiently digested by hydrolytic reactions with acid or Alcalase, an industrial protease. The acid- and Alcalase hydrolysates showed higher antigenotoxic activities than peptic- and tryptic hydrolysates. Active peptide fractions were separated from acid- and Alcalase hydrolysate by gel filtration chromatography. It was deduced from the chromatograms and amino acid analyses that the size (3–7 amino acids) and the glycine plus alanine content of peptides might be important factors for their activities. In addition, it was found that the antigenotoxic effect of peptides was due both to the protective interactions between cells and peptide molecules and to the direct inactivation of the mutagen, MNNG by peptides. Considering the availability and safety of silk fibroin and the superior antigenotoxic effects of produced peptides, the results of this study showed the possibility of utilizing fibroin as a source for chemopreventive functional peptides.

Ovarian and breast cytotoxic T lymphocytes can recognize peptides from the amino enhancer of split protein of the Notch complex

In this study we investigated recognition by ovarian tumor associated lymphocyte (OVTAL), and breast tumor associated lymphocytes (BRTAL), of peptides corresponding to the sequence 125–135 of the Aminoenhancer of split (AES) protein. Three of these peptides designated as G75:AES1/2 (128–135), G60: AES1/2 (127–137) and G61: AES1/2 (125–133) correspond to the wild-type AES sequence, while the fourth G76: G PLTPLPV, AES1/2 (128–135) corresponds to a variant sequence of the peptide G75 with the N-terminal Leu substituted to glycine. These sequences were chosen for study because mass-spectrometric analysis (MS) of a CTL active HPLC peptide fraction eluted from immunoaffinity precipitated HLA-A2 molecule, revealed: (a) the presence of an ion with a mass-to-charge ratio ( m/z) of 793 which was more abundant than other ions of similar masses; (b) the tentatively reconstituted sequence of the ion 793 matched the sequence of peptide G76. We found that AES peptides G75 (128–135) and G76 (128–135) (L128G) reconstituted CTL recognition at concentrations ranging between 200–500 nM. These concentrations are lower than concentrations reported to activate effector function of CTL recognizing other epithelial tumor Ag. Furthermore, analysis with cloned CD8 + T cells indicated that G75 and G76 were not cross-reactive specificities, suggesting a key role for the N-terminal residues of the variant peptide in dictating specificities. Since the AES proteins are part of a set of transcriptional repressors encoded by the Enhancer of split [E(spl)] genes, and since these repressors are activated to suppress cell differentiation in response to Notch receptors signalling, the AES peptides may represent a novel class of self-antigens that deserve further consideration as tumor Ag in epithelial cancers.

Isolation of coeliac active peptide fractions from gliadin.

For the isolation of coeliac active peptide fractions the peptic tryptic digest of whole gliadin was successively separated by ultrafiltration, gel filtration, cation-exchange chromatography, anion exchange chromatography and high-performance liquid chromatography. After each separation step the peptide fractions obtained were characterized by amino acid analysis and examined for coeliac activity in an immunological test (LIF test) and in an organ-culture test. The most active fractions have molecular weights ranging from 7,000 to 14,000 daltons, high contents of Glx (greater than 40 mol-%), Pro (greater than 20 mol-%) and Phe (greater than 5 mol-%) and low contents of S-containing and basic amino acids (0 and less than 2.0 mol-%, respectively). The peptide fraction B3142 obtained after five separation steps seems to be a pure peptide, which shows activity in the immunological test for all coeliac patients examined in very low concentrations. This peptide consists of 53 amino acid residues and has the composition Glx24, Pro15, Val4, Phe3, Ser2, Leu2, Asx1, Gly1, Tyr1.

Small peptides controlling transcription in vitro are bound to chromatin DNA

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 μg/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.

Low molecular weight peptides controlling transcription are present in the calf thymus chromatin structure.

A calf thymus peptide fraction controlling DNA and chromatin template has been purified by DNA-cellulose and Dowex 50 WX2 chromatography and its amino acid composition determined. The active peptide fraction can be extracted in high pH buffer from calf thymus native chromatin previously deproteinized by chloroform-isamyl alcohol and phenol. These data demonstrate that the thymic peptide(s) is (are) a chromatin protein constituent strongly linked to DNA. The specificity in association of the peptide(s) to DNA has also been considered.

Pharmacologically active peptides in extracts from cortical and subcortical tissues of human and bovine brain.

1. The gut-contracting peptide components Fa, Fb and Fc were prepared from crude substance P preparations made from cortical and subcortical tissues of human and bovine brain. 2. Fc preparations of human cortex, amygdala, hypothalamus, and red nucleus were then chromatographed on columns of DEAE-cellulose. 3. These experiments and such using paper chromatography, thin-layer chromatography, and paper electrophoresis led to results which point to the possible existence in brain of numerous polypeptides with pharmacological, i.e. gut-contracting activity. 4. There exist species differences between active peptide fractions of corresponding areas of human and bovine brain.