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.     

A supernodulating mutant isolated from soybean cultivar enrei

Abstract

Symbiotic nitrogen fixation in nodules of legumes depends on the complex interaction between the legume plant and (Brady)Rhizobium bacteria. Nodule formation and nitrogen fixation are closely regulated by both the host plant and the microsymbiont. Plant mutants with altered symbiotic performance are considered to be useful to gain a better understanding of the plant—microbe interactions in the legume—(Brady)Rhizobium symbiosis (Jacobsen 1984; Carroll et al 1985a, b; Park and Buttery 1988; Duc and Messager 1989; Gremaud and Harper 1989). Recently, Carroll et al. (1985a, b) have isolated the supernodulating mutants of the soybean cv. “Bragg,” which display a very large number of nodules and “nitrate-tolerant-symbiotic” (nts) characteristics. More recently, Gremaud and Harper (1989) have also isolated similar mutants from the soybean cv. “Williams.” These mutants not only provide materials that are useful for investigatings on the interaction in the nodule formation processes but also for agricultural practice. In particular, the nitrate-tolerance of these supernodulating mutants (Carroll et al. 1985b; Gremaud and Harper 1989) is useful for their cultivation in Japan where the level of soil nitrogen in fields is generally high. However, the cultivars previously used for the isolation of these mutants cannot adapt easily in Japanese climate due to different Maturity Group. Therefore, we attempted to isolate mutants with altered symbiotic phenotypes from the soybean cultivar “Enrei,” one of the most common cultivars in Japan.     

Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean fermentation starter

Bacillus amyloliquefaciens FSE-68 isolated from meju, a Korean soybean fermentation starter, was identified on the basis of biophysical tests and 16S rRNA gene sequence. A neutral metalloprotease (NPR68) and an alkaline serine-protease (APR68) were purified by ammonium sulfate precipitation and cation exchange chromatography and identified on the basis of their activities at different pH values and the selective protease inhibitors. The molecular weights of NPR68 and APR68 measured with ESI-MS were 32743 (+/- 0.8) and 27443 (+/- 0.5) Da, respectively. Against oxidized insulin chains, the NPR68 has a cleavage preference at the site where leucine is located as a P1' residue followed by phenylalanine, and the APR68 has broad specificity and favors leucine at the P1 site. These results indicate that the proteases are natural variants of subtilisin and bacillolysin.     

Antifungal activity of borrelidin produced by a Streptomyces strain isolated from soybean

In this study, an endophytic Streptomyces sp. neau-D50 with strong antifungal activity against Phytophthora sojae was isolated from healthy soybean root, using an in vitro screening technique. A bioactivity-guided approach was then employed to isolate and determine the chemical identity of bioactive constituents with antifungal activity from strain neau-D50. The structure of the antifungal metabolite was elucidated as borrelidin on the basis of spectral analysis. To our knowledge, this is the first report that borrelidin has strong antifungal activity against dominant race 1 of P. sojae with EC(50) and EC(95) of 0.0056 and 0.026 mg/L, respectively. The values were respectively 62.5- and 262.3-fold lower than those of the commercial fungicide metalaxyl, which has been used to treat soybean seed for the control of P. sojae . The in situ bioassays demonstrated that borrelidin at 10 mg/L reduced P. sojae race 1 lesions on soybean seedlings by 94.72% without affecting root growth. Thus, borrelidin might be a promising candidate for new antifungal agents against P. sojae.     

Serological properties and intrinsic antibiotic resistance of soybean bradyrhizobia isolated from Thailand

A group of Bradyrhizobium strains isolated from soybean plants in Thailand did not correspond to any known DNA homology groups of Bradyrhizobium japonicum and Bradyrhizobium elkanii reported by Hollis et al. (J. Gen. Microbiol., 123, 215–222, 1981). To clarify the phenotypic characteristics of the group, serological properties and intrinsic antibiotic resistance (IAR) profile of 94 Thai strains were compared with those of USDA and Japanese strains. Indirect ELISA tests for each Thai strain were performed agaiIl.st polyclonal antisera prepared against 15 USDA standard serotype strains of B. japonicum and B. elkanii. Among the 94 Thai strains tested, 36 which were previously identified as B. elkanii, with the exception of one strain, were strongly responsive to an antiserum prepared against USDA 31. The remaining 58 strains, with the exception of two strains, showed multiple cross reactions which were peculiar to the Thai strains. These serological reaction patterns did not correspond to any known serogroups labeled as B. japonicum and B. elkanii. In the IAR test, the taxonomically unknown Thai soybean bradyrhizobia exhibited a high level of resistance to neomycin (50 µg/mL), polymyxin (50 µg/mL), nalidixic acid (15 µg/mL), and kanamycin (15 µg/mL). Kanamycin could thus be useful in combination with neomycine and nalidixic acid for distinguishing between the unknown Thai strains and strains of B. japonicum and B. elkanii. Our results demonstrated that the unknown Thai strains were serologically and IAR-phenotypically remote from both B. japonicum and B. elkanii.     

Characterization of Rhizobium trifolii isolated from soils of different pH

Rhizobium trifolii were isolated from soils along a transect covering a range of soil pH (3.6–5.6) using two varieties of white clover by either growing seedlings directly in soil or in nutrient solution in tubes inoculated with soil. Rhizobia were present at pH 4.5 but absent at pH 3.9. Neither nodule number nor effectiveness were influenced by the method of isolation and the clover variety on which the strain was isolated. There was no relationship between the pH of the soils and either the number of nodules or the effectiveness of the isolates from those soils. Screening the isolates for tolerance of acidity and Al showed that multiplication was unaffected at pH 5.0 but was slowed for all strains at pH 4.5. Multiplication at pH 5.5 was unaffected by 10 μM Al but was inhibited by 50 μM Al. At pH 4.5 all but 16% of the isolates were inhibited by 10 μM Al; none multiplied with 50 μM Al. The strains which multiplied at pH 4.5 with and without Al were isolated equally from soils in the range pH 4.5–5.6. They were also isolated in almost equal proportions from the two varieties of clover and by the two isolation methods. Overall there was little variation in the effectiveness and acid- and Al-tolerance of isolates from these soils of different pH.     

Diversity and symbiotic effectiveness of rhizobia isolated from field-grown soybean nodules in Paraguay

Soybean was introduced in Paraguay in the 1920s and commercial crops have been grown since the 1970s. Root nodulation occurs at the majority of the producing sites, although inoculation has been practiced in only 15-20% of the cropping areas. The diversity and symbiotic effectiveness of soybean rhizobia was studied using 78 isolates obtained from root nodules of field-grown plants at 16 sites located in the two main producing states. The rhizobial isolates were characterized in relation to several parameters in vitro (colony morphology, tolerance to high temperature and salinity, intrinsic resistance to antibiotics, synthesis of indole acetic acid, profiles of proteins and lipopolysaccharides) and in vivo (nodulation, plant growth and total N accumulated in shoots). Fifty-eight isolates had slow growth rates and alkaline reaction in medium containing mannitol as the carbon source, whereas 20 had fast growth rates and an acid reaction. Most isolates did not tolerate acidity (pH 4.5) or high temperature (40°C). Very few isolates shared similar protein and lipopolysaccharide profiles; therefore a high level of diversity was detected, with most of the isolates representing unique strains. Some of the isolates with an outstanding symbiotic performance were identified, and will now be tested under field conditions in a search for efficient and competitive strains for use in commercial inoculants in Paraguay.     

Characterization of a chitosanase isolated from a commercial ficin preparation

A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).     

Characterization of beta-conglycinin and glycinin soy protein fractions from four selected soybean genotypes

The beta-conglycinin and glycinin fractions of soy protein were isolated from Macon, Ohio FG1, Enrei, and IL2 genotypes that were grown under the same environmental conditions. The soy protein fractions were evaluated to determine whether chemical composition and gel-forming properties were related. Amino acid analyses suggested that the hydrophobic residues may be the primary cause of differences in soy protein gel characteristics as the storage moduli increased with higher percentages of hydrophobic residues. Reversed-phase high-performance liquid chromatography profiles revealed variations in the composition of each fraction that corresponded to differences observed among the storage moduli. The gel-forming properties may be related to more than just protein content, such as the amount and type of amino acid in the fraction.     

Evaluation of bradyrhizobia strains isolated from field-grown soybean plants in Argentina as improved inoculants

Bradyrhizobium strains were isolated from nodules obtained from field-grown soybean plants sampled in 12 soybean production locations in Argentina. These fields had been annually cropped with soybean and did not show decreases in yields even though they had been neither N-fertilized nor inoculated for at least the last 5 years. We hypothesized that the isolated strains maintained high competitiveness and efficiency in fixing adequate N₂ levels. A set of strains that showed the highest nodular occupancy in each sampling location were assayed for symbiotic performance under greenhouse and field conditions and comparatively evaluated with Bradyrhizobium japonicum E109, the strain officially recommended for inoculant formulation in Argentina. An inoculant pool, formed by four strains obtained from nodules collected from Cañada Rica, developed higher nodular biomass than B. japonicum E 109 in assays carried out in greenhouses under well irrigated conditions. Additionally, neither nodule production nor specific nitrogenase activity decreased with respect to B. japonicum E 109 when plants were drought stressed during 7 days from sowing. The mean yields obtained under field conditions and plotted against the principal component one (CP1) obtained with an additive main effect and multiplicative interaction (AMMI) model showed that the inoculant pool from Cañada Rica had higher contribution to yield than strain E 109, although with lower environmental stability. The inoculant pool from Cañada Rica could be considered an improved inoculant and be used for preliminary assays, to formulate inoculants in Argentina.     

高压脉冲电场对大豆分离蛋白功能性质的影响

该文研究了高压脉冲电场对大豆分离蛋白功能性质的影响。结果表明：随着脉冲强度和脉冲处理时间的延长，大豆分离蛋白的溶解度、乳化性、起泡性及疏水性都增加。当脉冲强度或处理时间大于35 kV/cm或432 μs(溶解度)、30 kV/cm或144 μs(乳化性)、35 kV/cm或432 μs(起泡性)及30 kV/cm或288 μs(疏水性)时，由于蛋白质分子变性程度增加，分子发生聚集，功能性质反而下降。     

Inhibition of core histone acetylation by the cancer preventive peptide lunasin

Lunasin is a unique 43 amino acid soy peptide that has been shown to be chemopreventive in mammalian cells and in a skin cancer mouse model in this work against oncogenes and chemical carcinogens. The observation that lunasin inhibits core histone acetylation led to the proposal of an epigenetic mechanism by which lunasin selectively kills cells that are being transformed by disrupting the dynamics of cellular histone acetylation-deacetylation when the transformation event is triggered by the inactivation of tumor suppressors that function via histone deacetylation. Here is reported for the first time the core histone H3- and H4-acetylation inhibitory properties of lunasin from different Korean soybean varieties used for various food purposes and from tissues of rats fed with lunasin-enriched soy (LES) to measure bioavailability. Lunasin was analyzed by immunostaining and inhibition of core histone acetylation by a non-radioactive histone acetyl transferase assay. Various amounts of lunasin are found in the soybean varieties, which correlated with the extent of inhibition of core histone acetylation. Both soy lunasin and synthetic lunasin inhibit core histone acetylation in a dose-dependent manner. Lunasin in LES is protected from in vitro digestion by pepsin. Lunasin extracted from blood and liver of rats fed with LES is intact and inhibits core histone acetylation.     

Characterization of three chitosanase isozymes isolated from a commercial crude porcine pepsin preparation

Three chitosanases designated PSC-I, PSC-II, and PSC-III were purified from commercial pepsin preparation by sequentially applying pepstatin A-agarose affinity chromotography, DEAE-Sephacel ion-exchange chromatography, Mono Q column chromatography, and Mono P chromatofocusing. With respect to chitosan hydrolysis, the optimal pHs were 5.0, 5.0, and 4.0 for PSC-I, PSC-II, and PSC-III, respectively; optimal temperatures were 40, 40, and 30 degrees C; and the Km's were 5.2, 4.0, and 5.6 mg/mL. The molecular masses of the three isozymes were approximately 40 kDa, as estimated by both gel filtration and SDS-PAGE, and the isoelectric points were 4.9, 4.6, and 4.4, respectively, as estimated by isoelectrofocusing electrophoresis. All three chitosanase isozymes showed activity toward chitosan polymer and N,N",N' "-triacetylchitotriose oligomer. Most effectively hydrolyzed were chitosan polymers that were 68-88% deacetylated.     

Characterization of functional traits of two fluorescent pseudomonads isolated from basidiomes of ectomycorrhizal fungi

Some functional traits of Pseudomonas fluorescens 92 and BBc6, two strains isolated, respectively, from the basidiome of the ectomycorrhizal fungi Suillus grevillei and Laccaria laccata, were evaluated. A rifampicin-resistant mutant of P. fluorescens 92 (P. fluorescens 92R1) showed a significant in vivo plant growth promotion effect on cucumber plants. Quantitative analysis of enzymatic and physiological activities on different substrates showed that P. fluorescens 92 produced about a three times higher level of avicelase than BBc6, while equivalent amounts of β-glucosidase were produced by both strains. Satisfactory levels of neutral phosphomonoesterase and medium levels of acid phosphomonoesterase were produced by both. Only P. fluorescens BBc6 produced a very low amount of phosphodiesterase. Both strains produced high amounts of IAA and siderophores. Both strains showed on an iron deficient medium a very high antagonistic activity against the phytopathogenic fungus Heterobasidion annosum. Purification of fluorescent siderophores by copper-chelate chromatography showed that P. fluorescens 92 produced one pyoverdin (Pf92) and BBc6 two pyoverdins (PfBI and PfBII). A good inhibitory activity against mycelial growth of H. annosum was also observed when using the pyoverdines purified by affinity chromatography. Further purification by reverse phase high pressure liquid chromatography produced multiple fractionation of the three pyoverdins. Analysis of the reverse-phase purified pyoverdines by electronspray ionization mass spectrometry gave for pyoverdin Pf92 the mass value of 1213.8 and for both PfBI and PfBII the mass value of 1305.7. The presence of iron-chelating forms and sodium adducts were also evidenced.     

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.     

Characterization of Desulfovibrio sp. isolated from some lowland paddy field soils of Burkina Faso

In a wetland ecosystem such as lowland ricefields, the anaerobic mineralization of organic matter is a key mechanism for nutrient recycling (Jørgensen 1982; Freney et al. 1982). In the process which involves several bacterial groups, sulfate reducers (Watanabe and Furusaka 1980; Widdel 1988; Ouattara and Jacq 1992; Nozoe 1997), methanogens (Asakawa and Hayano 1995; Dianou et al. 1997), sulfur and ferric ion reducers (Jacq et al. 1991) become active when the soil becomes anoxic (Amstrong 1969). Sulfate reducers are common in flooded soils (Watanabe and Furusaka 1980; Widdel 1988; Furusaka et al. 1991). Reported densities in rice soils ranged from 10³ to 10⁵ g^-1 dry soils in Asia (Watanabe and Furusaka 1980) and from 10² to 10⁹ g^-1 dry soil in Sénégal (Ouattara and Jacq 1992). Among the sulfate reducers, the “classical” Desulfovibrio species are known to oxidize typical fermentation products such as hydrogen, lactate, pyruvate, and dicarboxylic acids (Widdel 1988). So far, data on the isolation and characterization of sulfate-reducing bacterial (SRB) strains from African rice paddy field soils study have been very limited.     

Characterization of allergens isolated from the freshwater fish blunt snout bream (Megalobrama amblycephala)

Fish are an important source of dietary protein for humans throughout the world. However, they are recognized as one of the most common food allergens and pose a serious health problem in countries where fish consumption is high. Many marine fish allergens have been extensively studied, but relatively little is known about freshwater fish allergens. This study identified two main allergens from blunt snout bream (Megalobrama amblycephala), a freshwater fish widely consumed in China. Sera from 11 patients with convincing clinical history of blunt snout bream allergy were utilized in IgE immunoblot analysis to identify prominent allergens. Several blunt snout bream proteins revealed specific binding to serum IgE, with the 47 and 41 kDa proteins being the most immunodominant among them. Two-dimensional gel electrophoresis (2D SDS-PAGE) enabled resolution of the 47 and 41 kDa proteins into several protein spots with distinct isoelectric points. 2D SDS-PAGE along with IgE immunoblot analysis further confirmed the strong reactivity of these protein spots with the pooled sera from blunt snout bream-sensitive patients. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of the peptides generated by trypsin digestion of the different spots corresponding to the 47 and 41 kDa proteins indicated that these spots were isoforms of enolase and muscle creatine kinase, respectively. The potential allergenicity of these proteins was further verified by an bioinformatics approach using the full-length and 80 amino acid sliding window FASTA searches, which revealed a significant amino acid sequence homology between blunt snout bream allergens and several known inhaled and crustacean allergens.     

Extraction of soybean oil from single cells

Single cells prepared from autoclaved soybeans and cellulase treatment of the cells were effective in digesting the cell walls of and extracting the oil from soybeans. The first cell wall of the soybean single cell was completely removed using cellulases; the thin and transparent second cell wall of the cell was swollen. Oil in the cell formed spherical or hemispherical oil drops, and oil leaking from the oil bodies was observed. The oil was almost retained within the second cell wall. Water-extractable substances were obtained at approximately >60% of the weight. Flotation of oil drops by centrifugation was easily done. Ambient n-hexane extraction was also possible; however, residual oil remained in the oil bodies. Protease or peptidase digested the structure of the oil bodies; however, separation of the oil and the hydrolysates was impossible. The oil from the oil bodies was obtained effectively (>85%) by pressing the single cells and/or cellulase-treated single cells.     

Nitrous oxide emission from herbicide-treated soybean

 The emission of N₂O from soybean plants treated with the herbicides dichlorophenoxyacetic acid (2,4-D) and bromoxynil was studied. The N₂O flux from 2,4-D- and bromoxynil-treated soybean was 14.1 ng N₂O-N g^–1 fresh weight h^–1 and 19.7 ng N₂O-N g^–1 fresh weight h^–1, respectively, i.e. approximately twice that of the controls. The NO₂ ^–-N concentration in 2,4-D- and in bromoxynil-treated soybean was about 8 μg N g^–1 fresh weight, i.e. fivefold the concentration found in control plants. The NO₃ ^– content in herbicide-treated soybean did not differ significantly from that of the control plants. Consequently, the accumulation of NO₂ ^–-N during the assimilation of NO₃ ^–-N was thought to cause the observed N₂O release. Probably, N₂O is a by-product produced during either the reaction of NO₂ ^–-N with plant metabolites or NO₂ ^–-N decomposition. Final conclusions must await further experiments. Received: 5 November 1999