Tricine-sodium Dodecyl Sulphate-polyacrylamide Gel Electrophoresis Research Articles

Hydrolyzing behaviors of endogenous proteases on proteins in sesame milk and application for producing low-phytate sesame protein hydrolysate

Endogenous proteases with high activity have been identified in sesame seeds. However, the hydrolyzing behaviors of endogenous proteases on proteins in sesame milk are not well understood. In this study, the endogenous proteases optimally hydrolyzed proteins at pH 4.5 and 50 °C for 6 h. Tricine–sodium dodecyl sulphate–polyacrylamide gel electrophoresis and liquid chromatography tandem mass spectrometry analyses revealed that endogenous proteases randomly cleaved the cleavable peptide bonds on 11S globulins, and amino acid analysis indicated that serine carboxypeptidases preferentially cleaved tryptophan, phenylalanine, methionine, tyrosine, and leucine. The hydrolyzed sesame milk was separated into cream, transparent skim, and precipitate fractions by centrifugation (3000g, 5 min). The major protein components in skim were 42% peptides (<1500 Da) and 35% free amino acids. The phytate in skim was greatly reduced by adjusting to neutral and alkaline pH. This study is meaningful at supplying a strategy for producing low-phyate sesame protein hydrolysate.

Endopeptidases, exopeptidases, and glutamate decarboxylase in soybean water extract and their in vitro activity

The proteolytic activity of some soybean endogenous proteases have been clarified in the previous studies, but the information concerning the roles of these proteases and some other unknown ones during soybean processing are scarce. Herein, 16 endopeptidases, 13 exopeptidases, 24 inhibitors (two serpin-ZX and one subtilisin inhibitor firstly identified), and one glutamate decarboxylase were identified in the soybean water extract by the liquid chromatography tandem mass spectrometry analysis. Amongst the identified endopeptidases, just the aspartic endopeptidases (optimal at pH 2.5–3 and 35–45 °C) showed the detectable proteolytic activity by the tricine–sodium dodecyl sulphate–polyacrylamide gel electrophoresis and protease inhibitor assay analyses, whereas serine, cysteine, and metallo- endopeptidases (except P34 probable thiol protease) did not. Free amino acid analysis showed that the exopeptidases and glutamate decarboxylase were optimal at pH 6 and 45 °C, and by 6 h incubation, the free amino acids and γ-aminobutyric acid almost doubled.

Partial Purification Characterization and Application of Bacteriocin from Bacteria Isolated Parkia biglobosa Seeds

Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains. Fermented Parkia biglobosa seeds (African locust bean) were screened for bacteriocin-producing lactic acid bacteria (LAB) with the characterization of putative bacteriocins. Bacteriocin-producing lactic acid bacteria (LAB) were identified by 16s rDNA sequencing. Molecular sizes of the bacteriocins were determined using the tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (tricine-SDS–PAGE) and effects of enzymes, pH, detergents and temperature on bacteriocin activity investigated, using standard procedures. Bacteriocins production and activities were measured by spectrophotometric analysis. Statistical analysis was carried out using student t-test and Analyses of Variance. Bacteriocigenic LAB isolated were Lactobacillus plantarum Z1116, Enterococcus faecium AU02 and Leuconostoc lactis PKT0003. They inhibited the growth of both Gram-positive and Gram-negative bacteria. The sizes of bacteriocins Z1116, AU02 and PKT0003 were 3.2 kDa, 10 kDa and 10 kDa, respectively. The synergistic effects of characterized bacteriocins and rifampicin tested on organisms showed significant differences (P &amp;lt; 0.05), as compared with the effects of only one of the two. The antimicrobial activity of the three bacteriocins was deactivated after treatment of the cell-free supernatants with proteinase K, papain, pepsin and trypsin. Parkia biglobosa seeds are, therefore, rich in LAB bacteriocins which could be explored. The biosynthetic mechanisms of LAB bacteriocins could be employed in food safety and security, preservation, peptide design, infection control and pharmacotherapy. This should help in the control of undesirable bacteria and in designing more potent and selective antimicrobial peptides.

Changes in physicochemical properties and gelation behaviour of caseinomacropeptide isolate by treatment with transglutaminase

The gelation behaviour of caseinomacropeptide isolate (CMPI) treated with transglutaminase at levels of 1 and 25 U g−1 protein was investigated at different pH and temperatures. Cross-linking of CMPI protein fractions by transglutaminase was confirmed using tricine-sodium dodecylsulphate-polyacrylamide gel electrophoresis. Cross-linking reduced the isoelectric point and hydrophobicity of CMPI. The gelation temperature of CMPI at pH 3 was reduced from 54 to 42 °C; a gel point (G’>1 Pa) was not observed at pH 4.5 after enzyme treatment during temperature sweep measurements. Cross-linked CMPI formed a gel with lower stiffness and fracture stress at 90 °C at pH 3.0 or 4.5 compared with gels of untreated CMPI. However, stiffness and fracture stress of CMPI gels formed at 70 °C at pH 3.0 increased by three- and four-fold, respectively, by cross-linking with 25 U g−1 protein of enzyme. Transglutaminase affected gelation of CMPI by cross-linking of both CMP and residual whey proteins.

Purification and antioxidant ability of peptide from egg in sea cucumber Apostichopus japonicus

ABSTRACTThe antioxidant peptide was prepared from hydrolyzed egg of Apostichopus japonicus. Ultra-filtration, high-speed counter-current chromatography and gel filtration were used in peptide purification. In each step of purification, meanwhile, peptide was tested with its antioxidant ability. The purity and molecular weight was tested by using Tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Results showed that the purified peptide obtained a sharply enhancing ability to scavenge hydroxyl radical from 37 to 89.82 U/mL. The purity of peptide was conformed and molecular weight was estimated about 30 KDa. It would be promising for purified peptide to be a good functional food resource.

Péptidos con Actividad Antimicrobiana obtenidos de Proteínas Lácteas con Extractos de Salpichroa origanifolia

In this work the hydrolytic activity of the extract from Salpichroa origanifolia ripe fruits on bovine, ovine and caprine caseins was studied. The enzymatic reaction was carried out at pH 6.2 and 37°C of temperature for twenty hours. Peptides obtained from the enzymatic hydrolysis of all caseins were quantified by spectrophotometry and analyzed by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) and densitometry. Casein hydrolysates were separated by ultrafiltration obtaining higher and lower fractions of 3 kDa molecular mass. Statistical analysis of the caseins hydrolysis was significant for the time of hydrolysis and the type of casein (p<0.05). A higher degree of hydrolysis of caprine casein was observed after 20 hours of incubation. Antimicrobial activity of the hydrolysates was assessed and it was found that less than 3 kDa molecular mass peptides from bovine and ovine caseins showed the highest inhibitory effect on the evaluated microorganisms.

Isolation and characterization of a novel lectin with antifungal and antiproliferative activities from &amp;lt;italic&amp;gt;Sophora alopecuroides&amp;lt;/italic&amp;gt; seeds

Sophora alopecuroides lectin (SAL), a novel lectin from the seeds of Sophora alopecuroides, was purified by ion-exchange chromatography on diethylaminoethyl (DEAE)- and carboxymethyl (CM)-Sepharose columns, followed by gel filtration on a Sephadex 75 10/300 GL column. SAL was found to be a monomer of 39916.3 Da, as determined by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC). The N-terminal 10-amino acid sequence of SAL, KPWALSFSFG, resembles those of other legume lectins. SAL exhibits hemagglutinating activity against rabbit erythrocytes at 11.9 μg/ml. Its hemagglutinating activity is stable in the pH range 7-11 and in the temperature range 30-90°C, and is stimulated by Mn(2+). The hemagglutinating activity of SAL is most potently inhibited by 50-mM d-galactose. SAL suppresses mycelial growth in Penicillium digitatum and Alternaria alternata; the IC(50) of the antifungal activity toward P. digitatum and A. alternata were found to be 3.125 and 3.338 μM, respectively. SAL suppresses the proliferation of human cervical cancer cells (HeLa) at an IC(50) of 6.25 μM (P< 0.05). But it has no inhibiting effect on bacteria. This is the first report of a lectin from seeds of S. alopecuroides.

Electrophoretic characterization of Amaranthus L. seed proteins and its systematic implications

The seed protein profiles of 11 Amaranthus taxa (Amaranthaceae) from Spain were studied. These profiles were evaluated as a chemical character to clarify the taxonomic complexity in the genus. Tricine-sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of the Amaranthus seed proteins studied showed a range of peptides varying from 64 to 12 kDa, with a larger number of protein bands observed between 25.1 and 12 kDa. For the taxonomic study, 14 bands, some of them subdivided into several isoforms, were considered. The similarity analysis based on the SDS-PAGE profile is a useful character for the discrimination of species in Amaranthus, except for A. cruentus and A. hypochondriacus, for which a hybrid population was found. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155, 57‐63. ADDITIONAL KEYWORDS: Amaranthaceae ‐ electrophoresis ‐ taxonomy.

Quantification of fresh meat peptides by SDS–PAGE in relation to ageing time and taste intensity

The extraction and quantification of fresh meat peptides in the molecular weight (MW) range between 3 and 17 kDalton (kDa) was examined, as well as their evolution during post-mortem ageing and their relation to taste intensity. Using a Tricine–sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) method, quantification of fresh meat peptides in this MW range was possible. Correlation coefficients higher than 0.98 were found between the absolute amounts of peptides loaded on the gel and the measured amounts expressed as cytochrome c equivalents. In contrast to the extraction in the presence of SDS, extraction with 0.1 N HCl, 3% HClO 4 and 0.9% NaCl only partially recovered peptides present in fresh meat samples in both the 3–10 and 10–17 kDa MW range. A substantial increase in peptide concentration in the 3–17 kDa MW range with time post-mortem in both pork and beef fresh muscle samples was found. For grilled beef longissimus lumborum and semimembranosus samples of different origin and ripening times, both the concentration of the peptides in the 3–10 kDa MW range and the intramuscular fat content were significantly related with taste panel intensity scores, while the correlation with the amount of peptides in the 10–17 kDa MW range was not significant.

Characteristics and identification of enterocins produced by Enterococcus faecium JCM 5804T.

To screen bacteriocin-producing lactic acid bacteria (LAB) in 52 type and reference strains, which have not previously been studied, with respect to bacteriocins, and to characterize the presence of bacteriocins. Only Enterococcus faecium JCM 5804T showed bacteriocin-like activity. It inhibited the growth of Lactobacillus spp., Enterococcus spp., Clostridium spp., Listeria monocytogenes, and vancomycin resistant Enterococcus (VRE). However, it was not effective against Gram-negative strains, Weisella spp., Leuconostoc spp., Lactococcus spp., or methicillin resistant Staphylococcus aureus (MRSA). The inhibitory activity of Ent. faecium JCM 5804T was inactivated by proteinase K, trypsin, alpha-chymotrypsin, and papain, but not by lysozyme, lipase, catalase, or beta-glucosidase. The inhibitory activity was stable at 100 degrees C for 30 min, and had a pH range from 2 to 10. The molecular weight of the partially purified bacteriocin(s) was approx. 4.5 kDa, according to tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Polymerase chain reaction and direct sequencing methods identified three different types of bacteriocins produced by Ent. faecium JCM 5804T, enterocin A, enterocin B, and enterocin P-like bacteriocin. Enterococcus faecium JCM 5804T produced three different types of bacteriocins, and they inhibited LAB and pathogens. This is the first report of enterocin A, enterocin B, and enterocin P-like bacteriocin, detected in Ent. faecium JCM 5804T among LAB type and reference strains.

Purification and characterization of a bacteriocin-like compound (Lichenin) produced anaerobically by Bacillus licheniformis isolated from water buffalo.

To characterize a bacteriocin-like factor from Bacillus licheniformis 26 L-10/3RA isolated from buffalo rumen. The culture supernatant exhibited the antibacterial activity against a number of indicator organisms in a cut-well agar assay under anaerobic conditions. The inhibitory component was purified by following ammonium sulphate precipitation, gel filtration and ion exchange chromatography and confirmed to be a single peptide. A single band on tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity and having an estimated molecular mass of approximately 1400 dalton. Complete amino acid sequence of the peptide yielded 12 amino acids from the N-terminal end (ISLEICXIFHDN). No homology with previously reported bacteriocins was observed and has been designated as Lichenin. Lichenin was found to be hydrophobic, sensitive to atmospheric oxygen, retained biological activity even after boiling for 10 min and was active over a pH range of 4.0-9.0. The Lichenin represents the first anaerobiosis specific expression of bacteriocin-like compound isolated from Bacillus licheniformis 26 L-10/3RA of buffalo rumen origin. Lichenin could be a potential candidate for manipulating the rumen function at molecular level intended for improving the productivity of the ruminant.

Phase variation of Haemophilus influenzae lipopolysaccharide: characterization of lipopolysaccharide from individual colonies.

The lipopolysaccharide (LPS) of Haemophilus influenzae expresses a number of core oligosaccharide epitopes on its outer surface. The expression of individual epitopes is subject to frequent (approximately 1% bacteria/generation) reversible phase variation, as determined by colony immunoblots. We have used a microtechnique for the extraction of LPS from individual colonies, whose LPS antigenic phenotype has been identified, so that the LPS can be studied by tricine sodium dodecylsulphate polyacrylamide gel electrophoresis (T-SDS-PAGE). This avoids the introduction of heterogeneous phase-varying LPS which is inevitable if bacteria from colonies are grown in broth culture prior to LPS extraction and analysis. Using these techniques we have investigated the repertoire of LPS phase variation exhibited by H. influenzae strain RM7004 (a serotype b meningitis isolate). This technique will facilitate the study of bacteria in which there is variable LPS expression.