Characterization of yam bean (Pachyrhizus erosus) proteins

Seed proteins from Mexican yam bean seeds (Pachyrhizus erosus L.) were sequentially extracted according to the Osborne classification. Albumins were the major fraction (52.1-31.0%), followed by globulins (30.7-27.5%). The minor protein fraction was prolamins (0.8%). Defatting with chloroform/methanol remarkably affected the distribution of protein solubility classes; albumins were the most affected fraction (4.3-17.5%). Electrophoretic patterns of albumins showed bands at 55, 40, 35, and 31 kDa. After reduction of the globulin fraction exhibited two triplets, one from 35 to 31 kDa and the second from 19 to 21 kDa, these could be compared to the acid and basic polypeptides of 11S-like proteins. Prolamins showed one band at 31 kDa, and glutelins after reduction showed three main bands at 52, 27, and 14 kDa. Trypsin inhibitors were assayed in saline extracts; the values found (1232-2608 IU/g of meal) were lower than those of other legumes. In general, yam bean seed proteins showed an excellent balance of all essential amino acids; albumins contain the highest amount of essential amino acids.     

One- and two-dimensional electrophoretic identification of IgE-binding polypeptides of Lupinus albus and other legume seeds

The prevalence of food allergies in the world population requires integrated approaches to identify new potential allergens, especially those of plant origin. The aim of this work was the allergen in vitro analysis of Lupinus albus seed proteome, a promising food protein source, and the assessment of IgE cross-reactivities with other more diffused legume species. A combination of one- and two-dimensional gel electrophoresis and immunoblotting analyses with specific IgGs for band identification and lupin-sensitized patients' circulating IgEs for allergenicity studies has been used. Two lupin proteins, namely, conglutin gamma and 11S globulin basic subunits, strongly reacted with all patients' sera. Also, cross-reactivities with the homologous polypeptides of other legume species were observed. Otherwise, no reaction at all was detected with a 2S-type lupin protein. This global electrophoretic approach has allowed the identification of a new potential lupin allergen and confirmed the cross-reactivity among the legume 11S globulin basic subunits.     

Characterization of the major proteins of tubers of yam bean (Pachyrhizus ahipa)

Tubers of six accessions of ahipa (Pachyrhizus ahipa) contained between 0.77 and 1.34% nitrogen on a dry weight basis. This corresponds to 4.8 to 8.4% crude protein based on a nitrogen to protein conversion factor of 6.25; but detailed analysis of AC230 showed that although 93% of the total N was extracted with buffer containing 1.0 M NaCl, about a third of this was lost on dialysis. It was calculated, therefore, that salt-soluble proteins comprise about 60% of the total tuber nitrogen, with low-molecular-mass nitrogenous components comprising a further 30%. Electophoretic analysis of the salt-soluble proteins showed similar patterns of components in the six accessions, with none being present in amounts sufficiently high to suggest a role as storage proteins. Furthermore, light microscopy failed to show significant deposits of protein within the tuber cells. Five "major" protein bands, which together accounted for about 19% of the total salt-soluble protein fraction were purified and subjected to N-terminal amino acid sequencing. Comparison of these with sequences in protein databases revealed similarities to alpha-amylases, chitinases and chitin binding proteins, cysteine proteinases (including major components from P. erosus tubers), a tuberization-specific protein from potato, and proteins induced in soybean and pea by stress or the plant hormone abscisic acid, respectively. It was concluded that the primary roles of these proteins are probably in aspects of tuber metabolism and development and/or conferring protection to pests and pathogens, and that true storage proteins are not present. The absence of storage proteins is consistent with the biological role of the tubers as storage organs for carbohydrates (cf cassava tuberous roots) rather than as propagules (cf yam and potato tubers).     

Nodulation of African yam bean (Sphenostylis stenocarpa) by Bradyrhizobium sp. isolated from Erythrina brucei

African yam bean (Sphenostylis stenocarpa), which is widely cultivated in Africa because of its growth capability on marginal soils, was nodulated by an endosymbiont (characterized and designed Bradyrhizobium sp. AUEB20) isolated from the Ethiopian tree Erythrina brucei with the formation of a small number of large, indeterminate N₂-fixing nodules. In contrast, 24 other isolates from Ethiopian woody legumes were ineffective. Strain AUEB20 promiscuously nodulated a number of tropical legumes, but none out of five European crop plants tested. Received: 17 September 1996     

Characterization of starch from tubers of yam bean (Pachyrhizus ahipa).

Detailed studies of the starch present in tubers of six accessions of Pachyrhizus ahipa (ahipa) have been carried out using starches from tubers of P. erosus (Mexican yam bean) and seeds of ahipa and wheat for comparison. Starch accounted for 56-58% of the tuber dry weight with granules occurring in a range of geometric forms and in sizes from below 5 microm to about 35 microm (mean about 10 microm in all accessions except two). The amylose content ranged from 11.6 to 16.8% compared with 16.9% in P. erosus tubers and over 23% in the seed starches. X- ray diffraction analysis showed A-type or C(A)-type diffraction patterns. The chain-length distribution of the amylopectin after enzyme debranching showed a peak at DP11 similar to that of wheat starch, but had a less marked shoulder at DP 21-22 and contained a higher proportion of longer chains. Differential scanning calorimitry showed an endothermic peak corresponding to gelatinization with T(max) ranging from 59 to 63 degrees C, which was similar to the T(max) of wheat (about 64 degrees C). The composition of the ahipa starch may mean that it is suitable for food applications that require low amylose content and low retrogradation after processing.     

One- and two-dimensional direct chiral liquid chromatographic determination of mixtures of diclofop-Acid and diclofop-methyl herbicides

Simple one- and two-dimensional high-performance liquid chromatography (HPLC) methods for the simultaneous enantiomeric determination of alkyloxyphenoxypropionic acid herbicides is presented. Compounds studied were ( R, S)-2-[4-(2,4-dichlorophenoxy)phenoxy]propionic acid (diclofop-acid) and ( R, S)-2-[4-(2,4-dichlorophenoxy)]methyl propionate (diclofop-methyl). Mobile phases necessary to separate their enantiomers on an alpha1-acid glycoprotein chiral stationary phase are different; therefore, the simultaneous separation by an isocratic mode is not possible. The chiral separation method proposed involves a one-step gradient allowing for the simultaneous determination of both racemic enantiomers. Detection limits of the method were 0.03 mg/L for both diclofop-acid enantiomers and 0.14 and 0.15 mg/L for diclofop-methyl enantiomers, respectively. The two-dimensional method involves the use of two chromatographs in one achiral-chiral coupling. The LC-LC method is more suitable for complex samples because it involves an online cleanup effect. Detection limits were 1.25 and 1.87 mg/L for diclofop-acid and 2.70 and 3.02 mg/L for diclofop-methyl enantiomers, respectively. Accuracy, repeatability, and reproducibility have been studied in standard samples and a technical product.     

Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis

In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.     

Nutritional evaluation of tepary bean: Elemental analysis of seed

Tepary bean (Phaseolus acutifolius var. latifolius), a potential arid‐land legume crop, was analyzed for total protein, oil, fiber and mineral content. Results indicate the bean would be adequate in these nutrients for a human diet if deficient amino acids in the seed‐protein were complimented.     

Analysis of dry matter and protein contents in fresh yam bean tubers by near‐infrared reflectance spectroscopy

Abstract

Fast screening methods are needed for plant breeding. The objective of this research was to evaluate the potential of near‐infrared reflectance spectroscopy (NIRS) for the simultaneous analysis of dry matter and protein contents in intact discs of fresh yam bean (Pachyrhizus spp.) tubers. Discs from 210 tubers were extracted with a punch few hours after harvesting and scanned by NIRS using a specially designed adapter. External validation revealed a close relationship between NIRS and reference methods for dry matter content (r²=0.94; standard error of performance, SEP=1.2%) and protein content (r²=0.87; SEP=1.94%). The calibration for protein content was compared with another one developed using dried‐ground tuber samples (r²=0.97; SEP=0.97%). These results suggested that NIRS can be used to determine dry matter and protein contents in fresh tuber samples of yam beans with acceptable accuracy. Further research will have to determine if additional traits can be incorporated into this scheme.     

The characterization of Nigerian varieties of pepper, Capsicum annuum and Capsicum frutescens by SDS- polyacrylamide gel electrophoresis of seed proteins

The possibility of using electrophoresis to characterize varieties of pepper, Capsicum annuum and Capsicum frutescens cultivated in Nigeria was investigated. The SDS- polyacrylamide gel electropherogram of extracted total seed proteins of 10 breeding lines in each of the 6 varieties investigated, revealed a pattern in which 12 polypeptide bands with apparent molecular weight range of 22 to 98 kilodaltons could be distinguished. The result showed that the six varieties could be characterized on the basis of presence/absence and staining intensities of 7 polypeptide bands. It is suggested that SDS- polyacrylamide gel electrophoresis of seed proteins provides a useful analytical technique for the characterization of varieties of pepper and there may be genotype duplicates in the collection of Nigerian Capsicum germplasm.     

Accumulation of zinc,phosphorus, and magnesium by navy bean seed

Considerable variation is found in zinc (Zn Concentration in navy bean (Phaseolus vulgaris L.) seed, an important food source of Zn and magnesium (Mg). The influence that phosphorus (P) and Zn fertilizers, and source of nitrogen (N) (inoculation with Rhizobium phaseoli versus 150 mg/kg NH₄NO₃‐N) had on growth and nutrient uptake of ‘Upland’ navy bean was studied under greenhouse conditions on a Glyndon loamy fine sand, a Calciaquoll low in available N, P, and Zn. Yields of stems (+ pod walls), blades, and seed were increased by N, P, and Zn fertilizers. Zinc concentration at maturity varied between 13 and 37 mg/kg for seed, 15 and 39 mg/kg for blades, and 5 and 30 mg/kg for stems. Zinc fertilizer was the chief factor responsible for this variability. Phosphorus concentration at maturity varied between 4.0 and 6.2 g/kg for seed, 1.9 and 11.8 g/kg for blades, and 1.0 to 2.7 g/kg for stems. Added P fertilizer increased P concentration in the three tissues, but the effect was most pronounced for blades of plants without added Zn. Magnesium concentration at maturity was mainly affected by N fertilizer.and ranged from 1.6 to 1.9 g/kg for seed, 4.0 and 9.7 g/kg for blades, and 4.0 to 7.1 g/kg for stems. Soil management can greatly affect Zn concentration in navy bean seed.     

Compositional and conformational analysis of yam proteins by near infrared fourier transform Raman spectroscopy

Fourier transform (FT)-Raman spectroscopy was employed to study the molecular structure of yam proteins isolated from three commonly consumed yam species including Dioscorea alata L., D. alata L. var. purpurea, and Dioscorea japonica. Although D. alata L. and D. alata L. var. purpurea consisted of similar amino acid residues, they still exhibited significant differences in conformational arrangement. The secondary structure of D. alata L. was mainly an alpha-helix, while D. alata L. var. purpurea was mostly in antiparallel beta-sheets. In contrast, D. japonica, which belongs to a different species, exhibited explicit differences in amino acid compositions and molecular structures of which the conformation was a mixed form of alpha-helices and antiparallel beta-sheets. FT-Raman directly proved the existence of S-S in yam proteins, implying that oligomer formation in yam proteins might be due to disulfide linking of dioscorin (32 kDa). The microenvironment of aromatic amino acids and the state of S-S in yam proteins were also discussed.     

Isolation and identification of steroidal saponins in Taiwanese yam cultivar (Dioscorea pseudojaponica Yamamoto)

A new furostanol pentaoligoside and spirostanol tetraoligoside were isolated for the first time from yam tubers (Dioscorea pseudojaponica Yamamoto) from Taiwan, together with four known yam saponins, methyl protodioscin, methyl protogracillin, dioscin, and gracillin. Their structures were characterized as 26-O-beta-D-glucopyranosyl-22alpha-methoxyl-(25R)-furost-5-en-3beta,26-diol, 3-O-alpha-L-rhamnopyranosyl-(1-->2)-O-([alpha-L-rhamnopyranosyl-(1-->4)]-O-[alpha-L-rhamnopyranosyl-(1-->4)])-beta-D-glucopyranoside, and (25R)-spirost-5-en-3beta-ol 3-O-alpha-L-rhamnopyranosyl-(1-->2)-O-([alpha-L-rhamnopyranosyl-(1-->4)]-O-[alpha-L-rhamnopyranosyl-(1-->4)])-beta-D-glucopyranoside. The structural identification was performed using LC-MS and 1H and 13C NMR. The methanol extract of yam tubers was fractionated by XAD-2 column chromatography using a methanol/water gradient elution system to yield furostanol and spirostanol glycoside fractions. Preparative high-performance liquid chromatography, employing a C18 column and a mobile phase of methanol/water (69:31, v/v), was used to separate each furostanol glycoside, whereas a mobile phase of methanol/water (79:21, v/v) was used to resolve the individual spirostanol glycosides. The conversions from steroid saponins to diosgenin after acid hydrolysis were around 68 and 90% for furostanol and spirostanol glycosides, respectively.     

Effect of seed phosphorus concentration on nodulation and growth of three common bean cultivars

Abstract

An experiment was conducted in the greenhouse to evaluate the effects of seed phosphorus (P) concentration on growth, nodulation, and nitrogen (N) and P accumulation of three common bean (Phaseolus vulgaris L.) cultivars. Seeds were produced under low or high soil P levels, and soaked, or not, in 200 mM KH₂PO₄ solution. The experiment had a 3×3×2×2 factorial block design: three cultivars (ICA Col 10103, Carioca and Honduras 35), three levels of applied P (15, 30 and 45 mg P kg^?1 soil), two native seed P concentrations, and two seed soaking treatments. Plants were harvested at flowering. Soaked seeds increased the number, dry mass and P content of nodules, but did not affect plant growth. Plants originated from seeds with high native P concentration presented higher shoot dry mass and nodule number and mass at every soil P level, and were less responsive to increased soil P supply, than plants from low seed P. In plants from seeds with high P, soil P levels did not alter significantly root dry mass, while in plants from seeds with low P bean cultivars expressed wider differences in root dry mass. The genotypic variability of nodulation was influenced by soil P levels and seed P concentration. Both higher soil or seed P supply enhanced N and P accumulation in shoots. These results indicate that a high seed P concentration produces plants less dependent on soil P supply, and can enhance nodulation and N₂ fixation of common bean. Seed P supply affected the cultivar performance, and should be considered in evaluation of bean genotypes.     

Stepwise extraction of proteins and carbohydrates from soybean seed

The stepwise hot water extraction of soybeans, which were extractions in a series of procedures of whole soybean seeds, dehulled and sliced ones, and pressed ones carried out by autoclaving, was investigated to study the localization in the seed and their characteristics. The characteristics of each extraction were studied by HPLC, SDS-PAGE, components analysis, microscopic observation, and effect for some enzymes. Carbohydrates were easier to extract than protein. In the extractions, the ratio of uronic acid per total sugar was constantly about 0.3. A comparison of these extracts, soybean milk, extraction from defatted soybean meal, and soybean milk residues was also carried out, and the characteristics and the localization were investigated. Mid-sized proteins in soybean milk were easy to extract. However, hardly any high molecular weight proteins or high molecular weight carbohydrates were extracted. The proteins and carbohydrates were considered to be localized in the middle lamella and in the protein and/or oil bodies of the cell, and the proteins and carbohydrates were gradually extracted through seed and cell breaking. Gelation was observed only in the boiled extracts from whole seeds. Pepsin and trypsin digests of the high molecular weight protein had inhibitory activity against the angiotensin I converting enzyme.     

Nutritive evaluation of the seed,germinated seed,and string bean of Erythrina americana and the detoxification of the material by boiling

The nutritive and toxicological values of the dry seeds, germinated seeds, and string beans of Erythrina americana were studied using raw and boiled samples. Raw germinated seeds had a higher protein content and lower fiber content than dry seeds. The whole string bean had lower protein content and higher fiber content. However, the seeds of the green pod showed the same protein concentration as the dry seeds (dry basis). Boiling and elimination of broth was beneficial in diminishing the alkaloid concentration in all the samples. The trypsin inhibitors, lectins, and tannins were also diminished as was expected. The raw string bean showed the lowest LD(50). Although the total essential amino acids content of the boiled germinated seeds was increased, the quality of protein, protein efficiency ratio (PER), was lower than in boiled dry seeds, and in these, the PER was similar to the control (casein). The present results suggest that for the protein quality and low alkaloid content, the boiled dry seeds and string beans could be used for animal feeding. It could be interesting to test the raw string beans in ruminants since in this stage E. americana showed the lowest toxicity.     

Calcium- and magnesium-dependent aggregation of legume seed storage proteins.

The solubility characteristics and sedimentation behavior of total or individual globulins from legume seeds [Lupinus albus L., Pisum sativum L., and Glycine max (L.) Merr.] were investigated. The typical insolubility of globulins detected during their extraction seems to be due to the presence of a low molecular weight factor(s) in the seed extract. The solubility of the purified globulins decreases with increasing concentrations of calcium and/or magnesium, but not of other cations, showing minimum values at concentrations that vary with the particular globulin considered. Ultracentrifugation analyses revealed that the Ca(2+)- and/or Mg(2+)-induced insolubilization of the globulins involves the formation of high-order aggregates of molecules of the same or of different globulins. These macromolecular structures are dissociated under conditions of high ionic strength, suggesting the involvement of electrostatic interactions in the aggregation process. The degree of association relies heavily on the amount of Ca(2+) and/or Mg(2+) available, on the presence of chelating agents for these divalent cations, and on the ionic strength of the surrounding medium. The possible physiological significance of the findings is discussed.     

Interactions of grape seed tannins with salivary proteins

To evaluate the amount and type of condensed tannins binding salivary proteins, which are supposed to be involved in astringent sensation, model systems allowing further analyses of proteins and condensed tannins were developed. The precipitates formed after addition of grape seed tannins to salivary proteins indicate that a binding interaction occurs. Dissociation of insoluble complexes was achieved by sodium dodecyl sulfate treatment. Thiolysis reaction allowed the quantification and characterization of proanthocyanidins on both the resulting pellet and the supernatant. Binding proteins were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The higher polymerized tannins predominantly precipitated together with the salivary proteins. The condensed tannins remaining in solution were low molecular weight polymers.