ABSTRACT
A new colorimetric method based on the bleaching of the iodoplatinate ion has been developed for fast and easy determination of γ-glutamyl-S-ethenyl-cysteine (GEC) in narbon vetch (Vicia narbonensis L.) seeds. The calibration curve showed a good correlation (r(2)=0.9959) between absorbance and GEC amounts from 5.5 to 33 µg (10-59.78 µmol/L). The limits of detection and quantification were 1.16 and 3.55 µmol/L, respectively, and no significant interferences from other sulfur-containing compounds were observed. The method showed excellent repeatability (relative standard deviation [RSD]=0.28%), reproducibility (RSD=4.4%), and accuracy (94%). Determination of GEC in 20 narbon vetch accessions yielded values that were in agreement with those reported previously using capillary electrophoresis and high-performance liquid chromatography methods. The method could be especially valuable for determination of GEC during the process of production of new low-GEC narbon vetch varieties.
Subject(s)
Colorimetry/methods , Dipeptides/analysis , Seeds/chemistry , Vicia/chemistry , Chromatography, High Pressure Liquid/methods , Electrophoresis, Capillary/methods , Iodides/chemistry , Limit of Detection , Platinum Compounds/chemistry , Reproducibility of ResultsABSTRACT
Two hydrolysis methods used to obtain rapeseed isolate derivates were compared: chemical hydrolysis performed under alkaline conditions and pepsic proteolysis performed under acidic conditions. The mean molecular weights obtained for the hydrolysates varied from 26 to 2.5 kDa, depending on the level of hydrolysis. Further characterisation showed that, at the same level of hydrolysis, the chemical hydrolysates differed by their charges and hydrophobicity from those derived from enzymatic digestion. Analysis of the foaming properties showed, for both cases, that a limited degree of hydrolysis, around 3%, was sufficient to optimise the foaming properties of the isolate despite the different physicochemical properties of the peptides generated. The study of foaming properties at basic, neutral and acidic pHs showed that the hydrolysate solutions yielded dense foams which drained slowly and which maintained a very stable volume under the three pH conditions tested.
Subject(s)
Brassica rapa/chemistry , Protein Hydrolysates/chemistry , Chemical Phenomena , Chemistry, Physical , Hydrogen-Ion Concentration , Hydrolysis , Hydrophobic and Hydrophilic Interactions , Molecular Weight , Peptides/chemistry , Solubility , Solutions/chemistry , Time FactorsABSTRACT
Extensive rapeseed protein hydrolysate obtained sequentially with Alcalase and Flavourzyme showed inhibitory activity towards Alcalase. Inhibitory activity decreased as the hydrolytic process progressed probably by heat denaturation and/or partial protease degradation. Alcalase rapeseed inhibitors were purified by gel filtration and subsequent ion exchange chromatography. They are composed of peptides of 8.4 and 6.1 kDa linked by interchain disulphide bonds, as observed by reducing SDS-PAGE, with a native molecular weight of 18 kDa. Aminoacid composition of the inhibitors was characterized by the high proportion of methionine (4.2%) and cysteine (4.6%). Alcalase inhibitors were partially resistant to heat treatment; after heating at 70 degrees C for 45 minutes more than 50% of the original inhibitory activity remained in the purified protein but after heating at 90 degrees C for 5 minutes, inhibitory activity decreased very fast to a basal level. The possible relation of these protease inhibitors with the 2S albumin storage proteins is discussed.
Subject(s)
Brassica rapa/chemistry , Plant Proteins/pharmacology , Protease Inhibitors/isolation & purification , Protease Inhibitors/pharmacology , Subtilisins/antagonists & inhibitors , Amino Acids/analysis , Hot Temperature , Hydrolysis , Plant Proteins/isolation & purification , Protease Inhibitors/chemistry , Protein DenaturationABSTRACT
Chickpea legumin has been purified and incubated under oxidizing conditions with linoleic acid to investigate the influence of this acid on the structure and nutritional quality of the protein. At the end of the incubation time, >30% of the linoleic acid was oxidized. The oxidized linoleic acid was highly detrimental to legumin, and the electrophoretic pattern of the protein was completely changed after the incubation period. Nevertheless, neither polymerization nor cleavage of the protein was observed as deduced from gel filtration chromatography, suggesting that the changes observed in native electrophoresis were probably due to oxidation of legumin. The incubation of legumin with linoleic acid also produced a diminution of the contents of methionine and histidine, by 81.3 and 24.3%, respectively. Finally, in vitro protein digestibility of chickpea legumin was also seriously affected by the incubation with linoleic acid, decreasing from 84.1 to 69.2%.
Subject(s)
Fabaceae , Linoleic Acid/chemistry , Plant Proteins/chemistry , Plants, Medicinal , Amino Acids/analysis , Chromatography, Gel , Oxidation-Reduction , Plant Proteins/isolation & purification , Seeds/chemistry , LeguminsABSTRACT
Chickpea protein isolate was used as starting material for the production of hypoallergenic protein hydrolysates. Western blotting of the protein isolate showed that IgE in sensitized patient sera strongly bound to the basic polypeptidic chains and recognized the acidic ones of 11S globulin. During the hydrolysis process by the individual and/or sequential action of endo- and exoproteases, a high reduction of antigenic activity was observed. Results suggest that the presence of intact or partially hydrolyzed basic polypeptide chains of 11S globulin are responsible for the formation of IgE complexes in protein hydrolysates obtained by exoprotease treatment; however, the digestion of these polypeptide chains by individual action of endoprotease caused a high loss of antigenic activity. The most effective reduction of antigenicity, >90%, was observed in extensive hydrolyzed chickpea proteins obtained by sequential treatment with endo- and exopeptidases. This chickpea protein hydrolysate could be useful for the elaboration of specialized hypoallergenic food products.
Subject(s)
Fabaceae , Food Hypersensitivity/immunology , Plant Proteins/immunology , Plants, Medicinal , Protein Hydrolysates/immunology , Blotting, Western , Endopeptidases , Food Hypersensitivity/blood , Humans , Immunoglobulin EABSTRACT
A chickpea 2S albumin has been purified by solubilization in 60% methanol and ion-exchange chromatography. Under denaturing conditions it is composed of two peptides of 10 and 12 kDa. Native molecular mass determined by gel filtration chromatography is 20 kDa. Amino acid composition shows that it is rich in sulfur amino acids, mainly cysteine with 4.6% of the total. On the other hand, it has antinutritional characteristics of being allergenic for chickpea-sensitive individuals and inhibitory against porcine chymotrypsin with a lesser degree toward trypsin. The results of interest from a nutritional point of view are discussed.
