Insecticidal components from field pea extracts: isolation and separation of peptide mixtures related to pea albumin 1b

Chromatographic fractionation of crude extracts (C8 extracts) from the protein-enriched flour of commercial field peas (Pisum sativum L.) has been shown here to yield peptide mixtures related to the pea albumin 1b (PA1b) family of cysteine-rich plant peptides. The mixtures were obtained initially by flash chromatography with silica gel. Following elution of soyasaponins and lysolecithins, the end fractions obtained with the use of two flash chromatographic solvent systems displayed activity in a flour disk antifeedant bioassay with the rice weevil [Sitophilus oryzae (L.)]. Chemical properties of these mixtures were compared by thin-layer chromatography, high-performance liquid chromatography (HPLC), IR, MS, and amino acid analyses. The major peptides of C8 extracts, with average masses of 3752, 3757, and 3805 Da, were isolated by anion exchange chromatography. Samples enriched in the peptide of mass 3752 were isolated by cation exchange chromatography. Reduction plus alkylation experiments in combination with electrospray ionization mass spectrometry showed that C8 extracts contained about 10 peptides and, like PA1b, each peptide possessed six cysteine residues (three disulfide bonds). Disulfide bond reduction with 2-mercaptoethanol destroyed the antifeedant activity. The native peptides of C8 extracts were found to be resolved into nine peaks with XTerra HPLC columns operating at alkaline pH. These columns were employed to assess the distribution of pea peptides in the isolated fractions, with photodiode array and electrospray detection.     

Fractionation of lentil seeds (Lens culinaris Medik.) for insecticidal and flavonol tetraglycoside components

Crude methanol extracts from four cultivated varieties of mature lentil seeds (Lens culinaris Medik.) were found to possess antifeedant and insecticidal properties in laboratory tests with the rice weevil (Sitophilus oryzae L.), an insect pest of stored products. Flash chromatography with silica gel on active Diaion HP-20 methanol extracts gave flavonol, lysolecithin, soyasaponin, and peptide fractions, as determined by HPLC and electrospray ionization LC/MS. The flavonol fraction was shown by high-resolution NMR experiments to contain a mixture of kaempferol 3-O-beta-glucopyranosyl(1-->2)-O-[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside and, tentatively, kaempferol 3-O-beta-glucopyranosyl(1-->2)-O-[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside-7-O-alpha-rhamnopyranoside. These inactive tetraglycosides, although inseparable under the reported HPLC conditions, were detected by NMR spectroscopy in nearly equal proportions. Three lysolecithins were identical to those previously identified in pea extracts. Soyasaponin I (soyasaponin Bb) and soyasaponin VI (soyasaponin betag) were found in Diaion HP-20 methanol extracts. An insecticidal lentil peptide with a mass of 3881 Da, isolated from an Eston variety in small quantities by anion exchange chromatography, was related to the cysteine-rich pea albumin 1b class of botanical insecticides. Binary mixtures of the insecticidal lentil peptide and soybean soyasaponin I were synergistic in tests with S. oryzae.     

Quantification of the group B soyasaponins by high-performance liquid chromatography

High-performance liquid chromatographic methods were developed for the isolation and quantitative determination of the group B soyasaponins, including 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins alphag, betag, and betaa, and their non-DDMP counterparts, soyasaponins V, I, and II, respectively, with formononetin used as the internal standard. The limits of quantification for soy products were 0.11-4.86 micromol/g. The within-day and between-days assay coefficients of variation were <9.8 and < 14.3%, respectively. The group B soyasaponin concentrations in 46 soybean varieties ranged from 2.50 to 5.85 micromol/g. Soy ingredients (soybean flour, toasted soy hypocotyls, soy protein isolates, textured vegetable protein, soy protein concentrates, and Novasoy) and soy foods (commercial soy milk, tofu, and tempeh) contained the group B soyasaponins from 0.20 to 114.02 micromol/g. There was no apparent correlation between isoflavone and soyasaponin concentrations in the soy products examined.     

Complete quantification of group A and group B soyasaponins in soybeans

A combination of high-pressure extraction and preparative chromatography was used to purify the group A and group B soyasaponins from soy germ for use as analytical standards and for use in biological assays. A standardized sample preparation and extraction method was developed for the analysis of phytochemicals found in soy and processed soy products, which is reproducible in other laboratories. The extracts can be analyzed with standard liquid chromatography-mass spectrometry and high-performance liquid chromatography methods to identify and quantitate the group A and group B forms of the soy saponins, as well as the soy isoflavones. Complete saponin analysis of the extracts prepared from soy germ (hypocots), hulls, and cotyledons shows that a significant portion of the saponins is concentrated in the germ. The germ contains nearly all of the group A soyasaponins, while the group B soyasaponins are nearly equally distributed between the germ and the cotyledons. The hulls contain little of either isoflavones or saponins. Whole (full fat) soybeans grown on a tract in central Illinois in 2003 contain approximately 4-6% saponins on a weight basis, of which about one-fifth or less of the total saponin content are group A soyasaponins; the balance is group B soyasaponins.     

Evaluation of soyasaponin, isoflavone, protein, lipid, and free sugar accumulation in developing soybean seeds

A combination of analytical techniques was used to examine and quantify seed compositional components such as protein, lipid, free sugars, isoflavones, and soyasaponins during soybean development and maturation in two Korean soybean cultivars. Protein accumulation was rapid during reproductive stages, while lipid content was only relatively moderately increased. The major carbohydrate saccarides sucrose and stachyose constantly increased during the reproductive stage. Previously published results suggest that the free sugar and lipid content reached their maximal concentrations at a relatively early stage of seed development and remain constant in comparison to other chemical components. The malonylglucosides were the predominant isoflavone form followed by the glucosides, acetyl glucosides, and aglycone forms. As soybean seed matures, total soyasaponin concentration was constantly decreased until the R8 stage. Soyasaponin beta(g) was the major soyasaponin in DDMP-conjugated group B soyasaponins, followed by the non-DDMP counterpart soyasaponin I and soyasaponin A1. The ratio of total isoflavone to total soyasaponin in the developing soybean increased from 0.06 to 1.31. Protein, lipid, and free sugar contents in the developing soybean seeds showed significant positive correlations with conjugated isoflavones and total isoflavone concentration, while the lipid contents showed a negative correlation with the isoflavone aglycone. Protein, lipid, and free sugar contents showed a negative correlation with total group A and B soyasaponins and total soyasaponins; however, only the soyasaponin A content was significantly negatively correlated with free sugar content. Total soyasaponin content was negatively correlated with isoflavone content (r = -0.828 at p < 0.01).     

Human fecal metabolism of soyasaponin I

The metabolism of soyasaponin I (3-O-[alpha-L-rhamnopyranosyl-beta-D-galactopyranosyl-beta-D-glucuronopyranosyl]olean-12-ene-3beta,22beta,24-triol) by human fecal microorganisms was investigated. Fresh feces were collected from 15 healthy women and incubated anaerobically with 10 mmol soyasaponin I/g feces at 37 degrees C for 48 h. The disappearance of soyasaponin I in this in vitro fermentation system displayed apparent first-order rate loss kinetics. Two distinct soyasaponin I degradation phenotypes were observed among the subjects: rapid soyasaponin degraders with a rate constant k = 0.24 +/- 0.04 h(-)(1) and slow degraders with a k = 0.07 +/- 0.02 h(-)(1). There were no significant differences in the body mass index, fecal moisture, gut transit time, and soy consumption frequency between the two soyasaponin degradation phenotypes. Two primary gut microbial metabolites of soyasaponin I were identified as soyasaponin III (3-O-[beta-D-galactopyranosyl-beta-D-glucuronopyranosyl]olean-12-ene-3beta,22beta,24-triol) and soyasapogenol B (olean-12-ene-3beta,22beta,24-triol) by NMR and electrospray ionized mass spectroscopy. Soyasaponin III appeared within the first 24 h and disappeared by 48 h. Soyasapogenol B seemed to be the final metabolic product during the 48 h anaerobic incubation. These results indicate that dietary soyasaponins can be metabolized by human gut microorganisms. The sugar moieties of soyasaponins seem to be hydrolyzed sequentially to yield smaller and more hydrophobic metabolites.     

Determination of saponins in legumes by direct densitometry.

Research has shown that dietary saponins may have health benefits. A simple, rapid method for the determination of saponins in legumes, using densitometry, is described. Saponin preparations, after pretreatment to remove nonsaponin components, are spotted in rows on a thin-layer chromatography plate, along with soyasaponin standards. The plate, without solvent development, is directly treated with sulfuric acid and heated. Violet spots develop which have a density proportional to the amount of saponin present. The standard curve has a correlation coefficient of 0.99 and is linear over the range of 1.25 to 10 microg of soyasaponins applied. The method has a coefficient of variation of less than 3% and compares favorably with quantitative thin-layer chromatography. Using this method the saponin contents of defatted soy flour (0.58%), dried navy beans (0.32%), and dried kidney beans (0.29%) were determined, and these results were found to be consistent with previous reports in the literature.     

Use of a simplified HPLC-UV analysis for soyasaponin B determination: study of saponin and isoflavone variability in soybean cultivars and soy-based health food products

Soyasaponins are phytochemicals of major interest for health. Their identification and quantification remain difficult owing to the large number of structural isomers in soybeans and the lack of stable standards. In this study, a rapid method using high performance liquid chromatography (HPLC) using a UV detector (205 nm) was developed to identify and quantify soyasaponins belonging to group B and compare them with isoflavones in different soy materials. 2,3-Dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins were determined using external calibration or a molecular mass ratio after alkaline hydrolysis to cleave their DDMP moieties. The detection limit of soyasaponin I, used as a reference molecule to simplify the analysis, was 0.065 micromol/g. Soyasaponin contents in seven soybean varieties ranged from 13.20 to 42.40 micromol/g in the germ and from 2.76 to 6.43 micromol/g in the cotyledons. The within-day and between-days variation coefficients did not exceed 7.9 and 9.0%, respectively, for the major soyasaponins. Soyasaponin B quantification in different soy-based health supplements was reported along with measurements of their isoflavone content to provide information on the variability of these bioactive compounds among different types of soy food materials.     

Effect of Germination and UV‐C Radiation on the Accumulation of Flavonoids and Saponins in Black Bean Seed Coats

In the present study, we evaluated UV‐C radiation and germination treatments as an approach to increase the concentration of bioactive molecules in black bean seed coats. Black beans were germinated for 20 h under UV‐C radiation. Germination rate was higher in seeds radiated with UV‐C light compared with the control (nonirradiated seeds). Flavonoid content was increased twofold in seed coats of beans germinated for 10 h under UV‐C compared with the control. Quercetin‐3O‐glucoside was the major flavonoid identified in stressed seed coats. Furthermore, the application of UV‐C radiation during germination increased the content of soyasaponin Af, Ba, and αg, and it induced the de novo biosynthesis of soyasaponins (phaseoside I, soyasaponin deacetyl Af, and soyasaponin deactyl Ah) not present in the control. Germination of black beans under UV‐C radiation was an effective and simple approach to increase the concentration of bioactive molecules in black bean seed coats.     

Insecticidal components from field pea extracts: sequences of some variants of pea albumin 1b

Methanol soluble insecticidal peptides with masses of 3752, 3757, and 3805 Da, isolated from crude extracts (C8 extracts) derived from the protein-enriched flour of commercial field peas [Pisum sativum (L.)], were purified by reversed phase chromatography and, after reduction and alkylation, were sequenced by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry with the aid of various peptidases. These major peptides were variants of pea albumin 1b (PA1b) with methionine sulfoxide rather than methionine at position 12. Peptide 3752 showed additional variations at positions 29 (valine for isoleucine) and 34 (histidine for asparagine). A minor, 37 amino acid peptide with a molecular mass of 3788 Da was also sequenced and differed from a known PA1b variant at positions 1, 25, and 31. Sequence variants of PA1b with their molecular masses were compiled, and variants that matched the accurate masses of the experimental peptides were used to narrow the search. MALDI postsource decay experiments on pronase fragments helped to confirm the sequences. Whole and dehulled field peas gave insecticidal C8 extracts in the laboratory that were enriched in peptides with masses of 3736, 3741, and 3789 Da, as determined by high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry. It was therefore concluded that oxidation of the methionine residues to methionine sulfoxide occurred primarily during the processing of dehulled peas in a mill.     

Insect antifeedant activity of flavones and chromones against Spodoptera litura

The antifeedant polymethylated flavones 5-hydroxy-3,6,7,8,4'-heptamethoxyflavone, 5-hydroxy-3,6,7,8-tetramethoxyflavone, and 5,6-dihydroxy-3,7-dimethoxyflavone have been isolated from the cudweed, Gnaphalium affine D. Don (Compositae). These flavonoids and authentic analogues showed insect antifeedant activity against the common cutworm (Spodoptera litura F.). In a previous paper, it was suggested that there was no substituent on the B-ring of the flavonoid for the beneficial antifeedant activity against the common cutworm. These flavonoids having a phenyl group as the B-ring and the chromone as elimination of the B-ring from the flavonoids were used to test the hypothesis of the previously described B-ring effect. The known fact is that Sculletaria baicarensis (Rutaceae) produced the 2-phenyl flavone. Test compounds and their methylated derivatives were prepared from this material for the structure-activity relationship (SAR) study of insect antifeedant activity. In spite of the 2-phenyl flavonoids, some tested compounds did not show any insect antifeedant activity against the common cutworm, although these inactive flavonoids were deficient in the 6-substituent group on the A-ring of the flavonoid. This 6-position-substituted derivative almost showed strong insect antifeedant activity against common cutworm. Moreover, the tested flavonoids having a hydroxyl group as a substituent on any of the positions tended to increase the activity. These results suggested the importance of the 6-position substitution on the flavonoid; however, hydrophilic substituents decreased the activity. Baicalein (5,6,7-trihydroxyflavone) derivatives did not show any activity despite having the 6-substituent derivative. Although the activity of some chromones increased the activity of the flavone, the bulky B-ring was a disadvantage for the antifeedant activity. It was suggested that the charge on C(3) and C(5) of the flavonoid was important for the biological activity. Additionally, an adequate hydrogen bonding property, which is different from lipophilicity, was an advantage for the activity on the basis of a QSAR analysis.     

Effect of soyasapogenol A and soyasapogenol B concentrated extracts on HEP-G2 cell proliferation and apoptosis

The growth inhibition and the induction of apoptosis brought about by soyasaponins extracted from soy flour ( Glycine max (L.)) and concentrated for soyasapogenols A and B formed by hydrolysis were tested for cytoactivity in the human hepatocellular carcinoma cell line Hep-G2. Concentrated soyasapogenol A (SG-A) and soyasapogenol B (SG-B) extracts contained approximately 69.3% and 46.2% of their respective aglycones (soyasapogenols) assessed by HPLC and ESI-MS, while the soyasaponin extract (TS), derived from crude methanol extraction, did not contain any detectable amounts of SG-A or SG-B. An MTT viability assay showed that all three extracts had an effect on Hep-G2 proliferation in a dose-response manner with 72 h LC50 values of 0.594+/-0.021 mg/mL for TS, 0.052+/-0.011 mg/mL for SG-A, and 0.128+/-0.005 mg/mL for SG-B. Apoptotic cells were determined by flow cytometry cell cycle analysis and confocal laser scanning microscopy (CLSM). Cell cycle analysis indicated a significant ( P< 0.05) greater sub-G1 buildup of apoptotic cells at 24 h (25.63+/-2.1%) and 72 h (47.1+/-3.5%) for the SG-A extract compared to SG-B, whereas the TS extract produced only a minor buildup of sub-G1 cells. CLSM confirmed a morphological change of all treatments after 24 h, at the respective LC50 concentrations. These results show that the samples that contained mainly soyasapogenols A and B showed a greater ability to inhibit proliferation of cultured Hep-G2 when compared to a total soyasaponin extract that did not contain any soyasapogenols.     

Simultaneous identification of soyasaponins and isoflavones and quantification of soyasaponin Bb in soy products,using liquid chromatography/electrospray ionization-mass spectrometry

A method has been developed for simultaneous identification of soyasaponins and soy isoflavones in soy products, based on liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Soy-based nutraceutical products were analyzed by LC/ESI-MS with detection of protonated and sodiated molecular ions, as well as characteristic fragment ions for these compounds. Soy isoflavones were characterized by a strong protonated molecular ion in addition to corresponding [aglycone + H](+) ions. Monitoring the soyasaponin-specific protonated aglycone and dehydrated aglycone ions throughout the chromatogram provided a unique fingerprint for soyasaponin content in the samples. This mass spectrometric fingerprint also allowed immediate classification of the soyasaponin analytes as group A or B soyasaponins, based on the unique masses of aglycone ions observed for each class. Quantification of soyasaponin B(b) in soy-derived materials, based on the use of a purified soyasaponin B(b) standard and a glycyrrhizin internal standard, has been accomplished.     

Diterpenes from Salvia broussonetii transformed roots and their insecticidal activity

The new diterpenes brussonol (1) and iguestol (6alpha,11-dihydroxy-12-methoxy-abieta-8,11,13-triene) (2) with an icetexane and a dehydroabietane skeleton, respectively, have been isolated from hairy root cultures of Salvia broussonetii. Other previously known diterpenes, 7-oxodehydroabietane, 11-hydroxy-12-methoxyabietatriene, taxodione, inuroyleanol, ferruginol, deoxocarnosol 12-methyl ether, cryptojaponol, pisiferal, sugiol, isomanool, 14-deoxycoleon U, 6alpha-hydroxydemethylcryptojaponol, demethylsalvicanol, and demethylcryptojaponol, were also obtained from these roots. The insect antifeedant and toxic effects of several of these compounds were investigated against the insect pests Spodoptera littoralis and Leptinotarsa decemlineata. Additionally, their comparative cytotoxic effects were tested on insect Sf9 and mammalian CHO cells. Demethylsalvicanol (4) was a moderate antifeedant to L. decemlineata, whereas brussonol (1) was inactive. 14-Deoxycoleon U (15) was the strongest antifeedant, whereas demethylcryptojaponol (11) was toxic to this insect. None of these compounds had antifeedant or negative effects on S. littoralis ingestion or weight gains after oral administration. Demethylcryptojaponol (11) was cytotoxic to mammalian CHO and insect Sf9 cell lines, followed by the icetexane derivative brussonol (1), with moderate cytotoxicity in both cases. The remainder of the test compounds showed a strong selective cytotoxicty to insect Sf9 cells, with demethylsalvicanol (4) being the most active.     

Group B saponins in soy products in the U.S. Department of Agriculture--Iowa State University isoflavone database and their comparison with isoflavone contents

Isoflavones in soy protein foods are thought to contribute to the cholesterol-lowering effect observed when these products are fed to humans. The group B saponins are another ethanol-soluble phytochemical fraction associated with soy proteins and isoflavones and have also been associated with cholesterol-lowering abilities. We measured the group B soyasaponin concentrations in a variety of soy foods and ingredients in the U.S. Department of AgricultureIowa State University Isoflavone Database. We compared the isoflavone and soy saponin concentrations and distributions in intact soybeans, soy ingredients, and retail soy foods. Group B saponins occur in six predominant forms. There appears to be no correlation between saponin and isoflavone concentrations in intact soybeans ranging from 5 to 11 mumol isoflavones/g soybean and from 2 to 6 mumol saponin/g soybean. Depending upon the type of processing, soy ingredients have quite different saponins/isoflavones as compared to mature soybeans. In soy foods, the saponin:isoflavone ration ranges from 1:1 to 2:5, whereas in soy protein isolates, the ratio is approximately 5:3. Ethanol-washed ingredients have very low saponins and isoflavones. These very different distributions of saponins and isoflavones in soy products may affect how we view the outcome of feeding trials examining a variety of protective effects associated with soy consumption.     

Insect repellent/antifeedant activity of 2,4-methanoproline and derivatives against a leaf- and seed-feeding pest insect

2,4-methanoproline is a natural product isolated from the seeds of Ateleia herbert smithii Pittier that was formerly suggested to have insect repellent/antifeedant activity; however, this was not tested quantitatively. In this study the insect repellent/antifeedant potency of methanoproline was measured against larvae of the cotton leafworm, Spodoptera littoralis (Boisd.), and adults of the cowpea weevil, Callosobruchus maculatus (F.). In addition, several N-alkyl, amino, and nitrile derivatives of methanoproline with varying stereodemanding substituents were synthesized and also tested. It was shown that in S. littoralis methanoproline itself did not show any significant activity but that derivatives 5, 7, 8, and 10 did show a reasonable repulsive/antifeedant activity that was comparable to the commercial repellent DEET. A significant repellent activity was scored for methanoproline in adults of C. maculatus that was similar to DEET.     

Determination of soyasaponins in soy with LC-MS following structural unification by partial alkaline degradation

High-performance liquid chromatography coupled with electrospray ionization mass spectrometer was used to study the soyasaponins in soy. It was found that each soyasaponin belonging to group A existed mainly in their genuine acetylated forms. The partially to fully deacetylated structures coexisted in various proportions. Likewise, the soyasaponins belonging to group B in soy were detected as both 2,3-dihydro-2,5-dihydroxy-6-methyl-4-pyrone (DDMP) conjugated forms and non-DDMP forms. The structural diversity of soyasaponins hinders the separation and determination of the individual compounds in soy. In the present studies, the soyasaponins extracted from soy were treated with sodium hydroxide under mild conditions to cleave the acetyl groups from soyasaponins in group A as well as the DDMP from soyasaponins in group B, while the glycoside structures remained unaffected. By doing so, all soyasaponins originating from the same initial structures were unified into well-defined structures and then quantified individually using the selective ion recording of their [M-H](-) ions. The pure deacetyl and non-DDMP soyasaponins were used as the external standards. The quantification limits of soyasaponins in group A and group B were 1.74 and 1.89 ng injected on column with recovery rates of 94.1% +/- 4.2% and 96.9% +/- 2.9%, respectively.     

Synthesis and insect antifeedant activity of aurones against Spodoptera litura larvae

A series of aurones were prepared from various phenols via phenoxy acetic acids and coumaranones and evaluated for insect antifeedant activity against the common cutworm (Spodoptera litura). The naturally occurring aurone was most active at an ED50 of 0.12 micromol/cm2. The synthetic precursor, coumaranones, showed that the introduction of methoxyl and methyl groups to the benzene ring increased insect antifeedant activity. Similarly, the tested aurones showed that the introduction of methoxyl group to the A and/or B rings increased the insect antifeedant activity, but 4,5,6- and 3',4',5'-trisubstituted compounds did not show this activity in this test. The hydroxylation of aurones in the B ring should be disadvantageous for insect antifeedant activity against S. litura. Although the melting points did not correlate well with the insect antifeedant activity, compounds that were nearly inactive had high melting points. A significant correlation was noted between biological activity (pED50) and a hydrogen-bonding parameter calculated from the Rf value obtained from SiOH thin-layer chromatography and a lipophilicity parameter (log k) calculated from the retention time in ODS high-performance liquid chromatography. The respective correlation coefficients (r) were -0.83 and -0.70. The introduction of alkoxy and alkyl groups along with adequate hydrogen bonding seems to contribute to the antifeedant activity of the compounds tested.     

Synthesis and insect antifeedant activity of precocene derivatives with lactone moiety

Starting from precocenes I and II, four of their derivatives with a lactone moiety were obtained. The compounds have been assessed as antifeedants against several diverse insect species including the storage pests the confused flour beetle (Tribolium confusum Duv., larvae and adults), the granary weevil beetle (Sitophilus granarius L., adults), and the khapra beetle (Trogoderma granarium Ev., larvae) and against the herbivorous pest insects Colorado potato beetle (Leptinotarsa decemlineata Say, adults and larvae) and aphids (Myzus persicae Sulz.). Precocenes, especially precocene II, showed a very strong antifeedant effect against all storage pests and aphids. The introduction of a lactone moiety caused a decrease in antifeedant activity against these species. Both precocenes were moderately active against L. decemlineata adults. The best antifeedants to this species were precocene derivatives, especially iodolactones. The introduction of iodine into a molecule had a great effect on the antifeedant activity of those compounds.     

Preparation of bean curds from protein fractions of six legumes.

Chickpeas, lentils, smooth peas, mung beans, and faba beans were milled into flours and fractionated to protein and starch fractions. Compositions of the seeds, cotyledons, and flours were compared for each legume and the weight and protein recovery of each fraction analyzed. Bean curds were prepared from the protein fractions through heat denaturation of protein milk, followed by coagulation with calcium sulfate or magnesium sulfate. The effect of chickpea protein concentration and coagulant dosage on the texture of bean curds was evaluated using a texture analyzer. Textural analysis indicated that curd prepared at 2.3-3.0% protein concentration and 1.5% CaSO(4) dosage had better yield and better texture than curds prepared under other conditions. Bean curds prepared from chickpeas and faba beans exhibited the second highest springiness and cohesiveness after those from soybeans. Curds of mung beans and smooth peas, on the other hand, had the highest yields and the highest moisture contents. The protein yield of the first and second soluble extracts used for curd preparation accounted for approximately 90% of the total protein of the seeds.