Lunasin concentration in different soybean genotypes, commercial soy protein, and isoflavone products

Lunasin is a unique and novel cancer preventive peptide originally isolated from soy. Information on lunasin concentration of soybean cultivars and commercial soy proteins would be useful in developing lunasin-enriched cultivars and soy products. We report the development of an enzyme-linked immunosorbent assay (ELISA) method to identify lunasin and quantify the variations in concentration in 144 selected, diverse soybean accessions from the U.S. Department of Agriculture Soybean Germplasm Collection, several commercially available soy protein fractions and isoflavone-enriched products. With synthetic lunasin and monoclonal antibody, ELISA shows a linear concentration range of 24-72 ng/mL, good reproducibility, a detection limit of 8 ng/mL, and a recovery of 90% on spiked soy samples. Lunasin concentrations in the tested materials range from 0.10 to 1.33 g/100 g flour. Differences that exceeded 100% have been observed among accessions of similar maturity that were grown in the same environment, indicating that genetic differences in soybeans exist for lunasin. The mean of 23 major ancestral lines of U.S. cultivars is similar to the mean of 16 modern cultivars selected to represent the current diversity of the crop, but the highest values were found within the ancestral and exotic accessions. Soy protein concentrate, isolate, and hydrolyzate contain 2.81 +/- 0.30, 3.75 +/- 0.43, and 4.43 +/- 0.59 g lunasin/100 g flour, respectively, while soy flour and soy flakes contain 1.24 +/- 0.22 g lunasin/100 g flour. Isoflavone-enriched products contain very little or no lunasin. The relative mass (M(r)) of lunasin in the samples is 5.45 +/- 0.25 kDa. The wide range of lunasin concentrations within the Glycine max species indicates that the levels of this important bioactive peptide can be genetically manipulated. Furthermore, soy isolates and hydrolyzed soy proteins contain the highest concentrations of lunasin.     

Barley lunasin suppresses ras-induced colony formation and inhibits core histone acetylation in mammalian cells

Lunasin is a novel peptide originally identified in soybean that suppresses chemical carcinogen-induced transformation in mammalian cells and skin carcinogenesis in mice. Since the lunasin gene was cloned from soybean and the chemically synthesized form of the lunasin peptide has been used in experiments conducted so far, the isolation of lunasin from other natural sources and testing of its biological properties have not been carried out. We report here the isolation, purification, and biological assay of lunasin from barley, a newly found rich source of the peptide. The identity of lunasin was established by Western blot analysis and mass spectrometric peptide mapping of the in-gel tryptic digest of the putative protein band. Lunasin was partially purified with anion exchange and immunoaffinity chromatography. The crude and partially purified lunasin from barley suppressed colony formation in stably ras-transfected mouse fibroblast cells induced with IPTG. These fractions also inhibited histone acetylation in mouse fibroblast NIH 3T3 and human breast MCF-7 cells in the presence of the histone deacetylase inhibitor sodium butyrate.     

In vitro digestibility of the cancer-preventive soy peptides lunasin and BBI

Lunasin and BBI (Bowman Birk protease inhibitor) are bioactive soy peptides that have been shown to be effective suppressors of carcinogenesis in in vitro and in vivo model systems. Since they are subject to digestion in the gastrointestinal tract, we investigated here the stabilities of lunasin and BBI to digestion in vitro by simulated intestinal fluid (SIF) and simulated gastric fluid (SGF). Samples containing lunasin and BBI of varying purities were subjected to in vitro digestion by SIF and SGF at different times and analyzed by Western blot. While the pure BBI reaction is stable after SIF and SGF digestions, the purified lunasin from soybean and synthetic lunasin are easily digested after 2 min in both in vitro digestions. In contrast, lunasin from soy protein containing BBI is comparatively stable after SIF and SGF digestions. Both lunasin and BBI are able to internalize into the cell and localize in the nucleus even after digestion, suggesting that some of the peptides are intact and bioactive. These data suggest that BBI plays a role in protecting lunasin from digestion when soy protein is consumed orally. The role of other soy protease inhibitors such as Kunitz Trypsin Inhibitor (KTI) cannot be excluded from these experiments.     

Cancer-preventive peptide lunasin from Solanum nigrum L. inhibits acetylation of core histones H3 and H4 and phosphorylation of retinoblastoma protein (Rb)

Lunasin, a unique 43 amino acid, 4.8 kDa cancer-chemopreventive peptide initially reported in soybean and now found in barley and wheat, has been shown to be cancer-chemopreventive in mammalian cells and in a skin cancer mouse model against oncogenes and chemical carcinogens. To identify bioactive components in traditional herbal medicines and in search for new sources of lunasin, we report here the properties of lunasin from Solanum nigrum L. (SNL), a plant indigenous to northeast Asia. Lunasin was screened in the crude extracts of five varieties of the medicinal plants of Solanaceae origin and seven other major herbal plants. An in vitro digestion stability assay for measuring bioavailability was carried out on SNL crude protein and autoclaved SNL using pepsin and pancreatin. A nonradioactive histone acetyltransferase (HAT) assay and HAT activity colorimetric assay were used to measure the inhibition of core histone acetylation. The inhibitory effect of lunasin on the phosphorylation of retinoblastoma protein (Rb) was determined by immunoblotting against phospho-Rb. Lunasin isolated from autoclaved SNL inhibited core histone H3 and H4 acetylation, the activities of the HATs, and the phosphorylation of the Rb protein. Lunasin in the crude protein and in the autoclaved crude protein was very stable to pepsin and pancreatin in vitro digestion, while the synthetic pure lunasin was digested at 2 min after the reaction. We conclude that lunasin is a bioactive and bioavailable component in SNL and that consumption of SNL may play an important role in cancer prevention.     

Secoisolariciresinol and matairesinol of sea buckthorn (Hippophaë rhamnoides L.) berries of different subspecies and harvesting times

Sea buckthorn (Hippopha? rhamnoides) seeds, berries, and berry fractions are often used as sources of bioactive ingredients for health products. The aim of the present study was to analyze lignans in these fractions of sea buckthorn. Secoisolariciresinol and matairesinol in seeds, fruit pulp/peel, and whole berries of sea buckthorn of three subspecies were analyzed by isotope dilution gas chromatography-mass spectrometry. The total content of the two lignans secoisolariciresinol and matairesinol varied widely from 8 to 139 microg/100 g in fresh berries and from 51 to 319 microg/100 g in dry berries. The content of secoisolariciresinol varied in the range of 34-313 microg/100 g of dry mass in the fruit pulp/peel and 93-355 microg/100 g in dry seeds. The content of matairesinol fell within the range of 3-25 microg/100 g in dry pulp/peel and 1-13 microg/kg in dry seeds. Wild H. rhamnoides ssp. sinensis contained a significantly higher total level of secoisolariciresinol and matairesinol in dry seeds, dry berries, and fresh berries compared with wild ssp. rhamnoides (253 vs 135 microg/100 g, P < 0.01, in seeds; 224 vs 153 microg/100 g, P < 0.05, in dry berries; 71 vs 29 g/100 g, P < 0.01, in fresh berries) and the cultivar of ssp. mongolica (253 vs 112 microg/100 g in seeds, 71 vs 9 microg/100 g in fresh berries). Harvesting dates had a significant influence on the content of the two lignans in seeds, fruit pulp/peel, and whole berries. This is the first report of lignans in sea buckthorn.     

Oxalate in grain amaranth

Grain amaranth (Amaranthus spp.) is a widely adaptable C4 pseudo-cereal crop that has interesting nutritional characteristics including high protein and calcium concentrations and a lack of gluten. To date, no antinutrient has been found at problematic levels in grain amaranth; however, oxalate has not been thoroughly studied. Dietary oxalate is a potential risk factor for kidney stone development, and its presence in food lowers calcium and magnesium availability. Oxalate concentration and forms and calcium and magnesium concentrations were determined in 30 field-grown grain amaranth genotypes from the species A. cruentus, A. hybrid, and A. hypochondriacus. The effects of seeding date and fertilization with calcium ammonium nitrate were evaluated in field experiments conducted in multiple environments; the effects of cooking were also evaluated. Mean total oxalate concentration in the 30 genotypes analyzed was 229 mg/100 g, with values ranging between 178 and 278 mg/100 g, the greatest proportion being insoluble (average of 80%). Calcium concentration averaged 186 mg/100 g and ranged between 134 and 370 mg/100 g, whereas magnesium averaged 280 mg/100 g and ranged between 230 and 387 mg/100 g. Fertilization only marginally increased total oxalate concentration and had no effects on other variables. Seeding date had no effects on any of the variables studied. Boiling increased the proportion of soluble oxalate but did not affect total oxalate concentration. Grain amaranth can be considered a high oxalate source, however, as most is in insoluble form, and due to its high calcium and magnesium concentrations, oxalate absorbability could be low. This should be confirmed by bioavailability studies.     

Catalytic inhibition of human DNA topoisomerase II by interactions of grape cell culture polyphenols

Previously, we isolated mixed polyphenolic fractions on a toyopearl matrix (TP-2 to TP-6) from grape cell cultures that were highly potent catalytic inhibitors in a human DNA topoisomerase II assay for cancer chemoprevention. The objectives of this study were to evaluate the potency of, and potential interactions between, individual fractions and some of the purified bioactive polyphenols that comprise these fractions on human DNA topoisomerase II catalytic activity. Treatments that combined anthocyanin-rich fractions (TP-2; 0.5 or 2.0 microg of dried material/mL), fractions containing catechins, procyanidin dimers, and flavanones (TP-4; 0.25 microg of dried material/mL), and/or fractions enriched with procyanidin oligomers and polymers (TP-6; 0.15 or 0.5 microg of dried material/mL) showed additive effects toward catalytic inhibition of the enzyme. Epicatechin gallate (IC50 = 0.029 microM), myricetin (0.39 microM), procyanidin B2 (PB2, 4.5 microM), and resveratrol (65.7 microM), constituents of the most bioactive mixed fraction from grape cell culture (TP-4), each individually provided potent catalytic inhibition of topoisomerase II. In addition, potentiating interactions between the PB2 and the other polyphenolic constituents mentioned above and between myricetin and resveratrol were clearly demonstrated. A synergistic interaction between myricetin and resveratrol was also confirmed with isobolographic analysis at a molar ratio of 1:70.     

Digested Ara h 1 loses sensitizing capacity when separated into fractions

The major peanut allergen Ara h 1 is an easily digestible protein under physiological conditions. The present study revealed that pepsin digestion products of Ara h 1 retained the sensitizing potential in a Brown Norway rat model, while this sensitizing capacity was lost by separating the digest into fractions by gel permeation chromatography. Protein chemical analysis showed that the peptide composition as well as the aggregation profiles of the fractions of Ara h 1 digest differed from that of the whole pool. These results indicate that the sensitizing capacity of digested Ara h 1 is a consequence of the peptides being in an aggregated state resembling the intact molecule or that most peptides of the digests need to be present in the same solution, having a synergistic or adjuvant effect and thereby augmenting the immune response against other peptides.     

Effect of physicochemical conditions on peptide-peptide interactions in a tryptic hydrolysate of beta-lactoglobulin and identification of aggregating peptides

The objective of this study was to characterize the changes in peptide solubility resulting from changing some physicochemical conditions in a tryptic hydrolysate of beta-lactoglobulin (beta-LG). The turbidity (500 nm) of a 1% solution of tryptic peptides was measured at pH 3-10, at 5, 25, and 50 degrees C, in the presence of different salt concentrations (0, 0.5, and 1 M NaCl), in the presence of denaturing and reducing agents (6 M urea, 5% SDS, or 5% beta-mercaptoethanol), and under an electric field (isoelectric focusing). The results reveal an increase in turbidity of the peptide solution at pH 4, but a slight increase in turbidity was also observed at pH 8, which is attributable to peptides linked by disulfide bridges. The effect of temperature and ionic strength on the turbidity occurring at pH 4 indicates that mainly hydrophobic interactions are involved in the aggregation process. The material in the precipitate at pH 4 was identified as the peptides beta-LG 1-8, 15-20, and 41-60 and non-hydrolyzed alpha-lactalbumin. These results suggest that a limited number of peptides are involved in the aggregation process observed at pH 4, some of which having bioactive (beta-LG 15-20, ACE inhibitor, and opioid) or emulsifying properties (beta-LG 41-60). Aggregation of these peptides at acidic pH indicates that a simple acidification step could represent an easy process for isolating peptidic fractions enriched in bioactive or functional peptides.     

Bowman-Birk and Kunitz Protease Inhibitors among Antinutrients and Bioactives Modified by Germination and Hydrolysis in Brazilian Soybean Cultivar BRS 133

Soybean contains constituents that have antinutritional and bioactive properties. Enzymatic hydrolysis and germination can enhance the biological activity of these compounds in soybean. The objective of this study was to investigate the effect of germination, Alcalase (protease) hydrolysis, and their combination on the concentrations of antinutritional and bioactive compounds in Brazilian soybean cultivar BRS 133. A combination of germination and Alcalase hydrolysis resulted in the degradation of Bowman-Birk inhibitor (BBI), Kunitz trypsin inhibitor (KTI), and lunasin by 96.9, 97.8, and 38.4%. Lectin was not affected by any of the processing treatments when compared to nongerminated and nonhydrolyzed soy protein extract. Total isoflavones (ISF) and total saponins (SAP) increased by 16.2 and 28.7%, respectively, after 18 h of germination, while Alcalase hydrolysis led to the reduction of these compounds. A significant correlation was found between concentrations of BBI and KTI, BBI and lunasin, BBI and ISF, KTI and lunasin, KTI and ISF, KTI and SAP, lunasin and ISF, and ISF and SAP. Germination and Alcalase hydrolysis interacted in reducing BBI, ISF, and SAP. This study presents a process of preparing soy flour ingredients with lower concentrations of antinutritional factors and with biologically active constituents, important for the promotion of health associated with soybean consumption. In conclusion, 18 h of germination and 3 h of Alcalase hydrolysis is recommended for elimination of protease inhibitors, while bioactives are maintained by at least 50% of their original concentrations.     

Determination and toxicity of saponins from Amaranthus cruentus seeds.

The concentrations of four triterpene saponins present in amaranth seeds were determined with high-performance liquid chromatography. It was shown that the total concentration of saponins in seeds was 0. 09-0.1% of dry matter. In germinating seeds an increase in concentration to 0.18% was observed after 4 days of germination, which remained stable for the next 3 days and later dropped to 0.09%. Highly purified extracts from the seeds were tested for their toxicity against hamsters. The hydrophobic fraction obtained by the extraction of seeds with methylene chloride showed no toxicity; the behavior of tested animals was similar to that of the group given an equivalent dose of rapeseed oil. A crude saponin fraction, containing approximately 70% of pure saponins in the matrix, showed some toxicity; the approximate lethal dose was calculated as 1100 mg/kg of body weight. It is concluded that low contents of saponins in amaranth seeds and their relatively low toxicity guarantee that amaranth-derived products create no significant hazard for the consumer.     

Production of lactoferricin and other cationic peptides from food grade bovine lactoferrin with various iron saturation levels

Purification of lactoferricin (Lfcin), a cationic antimicrobial peptide, was achieved by peptic digestion of food grade bovine lactoferrin (LF) followed by fractionation on an industrial grade cation exchange resin with stepwise salt gradient elution. The digest and eluted fractions were partially characterized by MALDI-ToF MS and N-terminal sequencing. A fraction eluted using phosphate buffer with 2.0 M NaCl contained predominantly two peptides with masses of 3196 and 3124 Da, which corresponded to the 26- and 25-amino acid peptides FKCRR WQWRM KKLGA PSITC VRRAF (A), containing the Lfcin sequence. Putative sequences of cationic peptides in other eluted fractions included FKNKS RSFQ, WRMKK LGAPS ITCVR RA, and GAPSI TCVRR AFALE CIRAI AEKKA. The iron saturation level of LF had no effect on the production of Lfcin. Nevertheless, the digestion of LF containing lower iron content led to the production of a higher quantity of low molecular weight cationic peptides. A two-step process using industrial grade cation exchange resin led to 35% recovery of Lfcin and also produced other cationic peptides with potential bioactive properties.     

Identification and differences of total proteins and their soluble fractions in some pseudocereals based on electrophoretic patterns

Genetic diversity and relationships of 11 species and cultivars belonging to different Angiosperms families were examined using sodium dodecyl sulfate seed protein markers. The protein was resolved into 36 bands (for soybean), 41 (for quinoa), 35 (for buckwheat), and 28 to 39 bands of Amaranth species, respectively. All species and cultivars can be distinguished from each other. Soybean, quinoa, and buckwheat species had a characteristic protein pattern showing a high degree of polymorphism. The protein patterns of soybean were considerably different from other species. Amaranth species had similar seed protein electrophoretic profile. The similarity coefficients calculated on the basis of presence and absence of bands ranged from 0.08 to 0.97. Following the UPGMA algorithm of similarity coefficients, the examined species and varieties could be clustered into two similarity groups. Our results did not confirm the Tachtadzjan hypothesis that Polygonales (e.g., buckwheat) and Caryophyllales (e.g., quinoa and amaranth) are closely related. Our data rather indicate occurrence of significant genetic distance (similarity coefficients 0.05-0.10). Also, it is doubtful that amaranth and quinoa species are also closely related (similarity coefficients varied from 0.16 to 0.25). It seems that soybean, quinoa, buckwheat, and amaranth (as a genus) can be considered as phylogenetic distant taxa. Differences and similarities in the secondary structure were observed by circular dichroism spectra. Some similarity was found between these plants in their soluble protein fractions and amino acid composition. These plants can be a substitution of each other as well as for cereals.     

Contents and bioactivities of lunasin, bowman-birk inhibitor, and isoflavones in soybean seed

It has been previously demonstrated that lunasin is a novel and promising cancer preventive peptide from soybean. The Bowman-Birk protease inhibitor (BBI) and isoflavones are well-studied substances from soy. This study evaluated the levels and bioactivities of these three compounds as affected by stages of seed development and sprouting under light and dark conditions. BBI and lunasin appear at 7 and 6 weeks, respectively, after flowering and increase as the seed matures. Daidzein and genistein both decrease during seed maturation. During sprouting under light, BBI increases up to the 6th day and decreases thereafter, disappearing at the 9th day after soaking. Under dark conditions, BBI increases up to the 7th day after soaking and decreases thereafter, disappearing at the 10th day. Lunasin starts to decrease at 2 days after soaking and disappears completely at 7 days under light and dark conditions. Daidzein and genistein increase continuously during the 10 days of soaking, and both increase more in the dark than under light conditions. Protein extracts from early seed development (2-5 weeks after flowering) suppress cell viability to a greater degree than those from later stages (6-9 weeks). Inhibition of foci formation by protein extracts from later stages is greater than those from earlier stages. Lunasin and BBI suppress foci formation more than the isoflavones. Sprouting decreases lunasin and BBI contents but increases isoflavones. Protein extracts from early soaking times inhibit foci formation more and suppress cell viability less than those from later soaking times. Light and dark conditions have no influence on the bioactivities of protein extracts. These data are useful in the preparation of soy fractions enriched in lunasin, BBI, and isoflavones and in making dietary recommendations.     

Hydrophobicity of bitter peptides from soy protein hydrolysates

Soy peptides were characterized for flavor, chemical properties, and hydrophobicity to investigate their relationships with bitterness. Five peptide fractions ranging in average molecular mass from 580 to 11300 Da were fractionated by ultrafiltration from two commercial soy protein hydrolysates. The bitterness of fractionated peptides was related to molecular mass, with maximum bitterness observed at approximately 4000 Da for one hydrolysate and 2000 Da for the other. The bitterness increased as the peptide M(w) decreased to 3000 Da for the first hydrolysate and to 2000 Da for the second one and then decreased as the peptide M(w) decreased below 1000 Da. The peptide fraction with molecular mass of <1000 Da showed the lowest bitterness for both. The hydrophobicity data based on Q values do not support Ney's Q rule as a predictor of bitterness for soy peptides.     

Hydrolysis of whey protein isolate with Bacillus licheniformis protease: aggregating capacities of peptide fractions

In a previous study, peptides aggregating at pH 7.0 derived from a whey protein hydrolysate made with Bacillus licheniformis protease were fractionated and identified. The objective of the present work was to investigate the solubility of the fractionated aggregating peptides, as a function of concentration, and their aggregating capacities toward added intact proteins. The amount of aggregated material and the composition of the aggregates obtained were measured by nitrogen concentration and size exclusion chromatography, respectively. The results showed that of the four fractions obtained from the aggregating peptides, two were insoluble, while the other two consisted of 1:1 mixture of low and high solubility peptides. Therefore, insoluble peptides coaggregated, assumedly via hydrophobic interactions, other relatively more soluble peptides. It was also shown that aggregating peptides could aggregate intact protein nonspecifically since the same peptides were involved in the aggregation of whey proteins, beta-casein, and bovine serum albumin. Both insoluble and partly insoluble peptides were required for the aggregation of intact protein. These results are of interest for the applications of protein hydrolysates, as mixtures of intact protein and peptides are often present in these applications.     

Use of a commercial protease and yeasts to obtain CGRP-like molecules from saithe protein

Different bioactive molecules, such as CGRP-like peptides, can be found in fish protein hydrolysates. Calcitonin gene-related peptide (CGRP) is a neuropeptide known to act as a potent arterial and venous vasodilator in humans. This study focuses on the industrial obtaining of CGRP-like molecules from saithe (Pollachius virens) byproduct, derived from the filleting process. Protein from P. virens was primarily hydrolyzed with Alcalase and later treated with Saccharomyces cerevisiae live cells. Treatment with Saccharomyces doubled the quantity of bioactive molecules obtained. The CGRP-like molecules were partially purified by chromatography, and the immunoreactive material was further analyzed for its CGRP-like bioactivity, using a specific radioreceptor assay. The concentration of CGRP-like molecules increased over 100-fold after purification. The bioactive molecules were able to induce cyclic AMP stimulation in rat liver membranes. Finally, partial sequencing of the bioactive peptide was performed, showing some homology with alpha-actin and myosin of several fish species.     

Impact of ultrafiltration membrane material on Peptide separation from a snow crab byproduct hydrolysate by electrodialysis with ultrafiltration membranes

Electrodialysis with ultrafiltration membrane (EDUF) is a technology based on the separation of molecules according to their charge and molecular mass. Some works have already successfully demonstrated the recovery of bioactive peptide fractions. However, the impact of ultrafiltration membrane (UFM) material, used in the EDUF system, on the peptide migration has never been studied. Consequently, the objectives of this work were (1) to evaluate the effect of two different UFM materials on the selective separation of peptides from a snow crab byproduct hydrolysate by electrodialysis with ultrafiltration membranes and (2) to determine the effect of UFM material on their potential fouling by peptides. It appeared that, after 6 h of EDUF separation using polyether sulfone (PES) and cellulose acetate (CA) UFM, peptides with low molecular weights ranging from 300 to 700 Da represented the most abundant population in the KCl1 (compartment located near the anode for the recovery of anionic/acid peptide fractions) and KCl2 (compartment located near the cathode for the recovery of cationic/basic peptide fractions) permeates. Peptides with molecular weights ranging from 700 to 900 Da did not migrate during the EDUF treatment. Moreover, only CA UFM allowed the recovery of high molecular weight molecules (900-20000 Da) in both KCl compartments. Peptides desorbed from PES and CA UFM after 6 h of EDUF separation had low molecular weights and belonged mainly to the 600-700 Da molecular weight range. These peptides represented a low proportion of the peptides initially present in the snow crab byproduct hydrolysate with individual molecular weight range proportions from 1.52 ± 0.31 to 10.2 ± 2.32%.     

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.