Composition of Acid-sensitive Fraction in Soybean Proteins

An acid-sensitive fraction (ASF) was prepared from defatted soybean meals by two procedures. ASF1 was prepared by precipitation at pH 4.5 followed by removal of 1 m NaCl-soluble materials from the precipitate. ASF2 was prepared by precipitation in solution containing 1 m NaCl at pH 4.5. The protein components of the two fractions were analyzed by gel electrophoresis in a dissociating-buffer system and found to contain β-conglycinin, glycinin and whey proteins. In addition to these, several other bands appeared.

Appreciable amounts of lipid (8.2% in ASF1 and 8.8% in ASF2) were also found in the fractions. They were separated by column chromatography and thin-layer chromatography. Glycolipids were the major components of the lipids. Both glycolipid and phospholipid fractions contained slower-moving materials on thin-layer chromatography.     

Disappearance of immunoreactive glycinin and 𝛃-conglycinin in the digestive tract of piglets

Soybean allergy represents a health threat to human and animals. Glycinin and β-conglycinin, the main storage proteins in soybean, have been identified as major food/feed allergens. The present study was conducted to investigate the disappearance of immunoreactive glycinin and β-conglycinin in the digestive processes of piglets. Twelve crossbred piglets, weaned at 21 days of age, were allocated to three dietary treatments in a complete block design, each treatment with four replicates (female/male = 1:1). From day 22–28, the control group was fed diets without leguminous products, while the two treatment groups received diets containing 2.2% purified glycinin or β-conglycinin. All piglets were slaughtered at 29 days of age and digesta was sampled from stomach, middle jejunum, caecum and colon. Results indicated that immunoreactive glycinin and β-conglycinin decreased as the digesta descended down the digestive tract to 0.12% and 0.47%, respectively. Little immunoreactive glycinin was found in the digesta of caecum and colon, while immunoreactive β-conglycinin was detected in the colon. Along the whole digestive tract the disappearance of immunoreactive glycinin was significantly higher than β-conglycinin (p < 0.05).     

Opposite Contributions of Glycinin- and β-Conglycinin-Derived Peptides to the Aggregation Behavior of Soy Protein Isolate Hydrolysates

The aggregation behavior as a function of pH was studied for hydrolysates obtained by hydrolysis of soy protein isolate (SPI) and glycinin- and β-conglycinin-rich protein fractions with subtilisin Carlsberg. The substrates were hydrolyzed up to degrees of hydrolysis (DH) of 2.2% and 6.5%. Compared with nonhydrolyzed SPI, a decrease in solubility was observed for the hydrolysates of SPI [0.8% (w/v) protein, I = 0.03 M] around neutral pH. At pH 8.0, glycinin hydrolysates had a much lower solubility (∼43% and 60%, respectively, for DH 2.2% and 6.5%) than SPI and β-conglycinin-derived hydrolysates, which were almost completely soluble. Peptides that aggregated were all larger than 5 kDa, and as estimated by size-exclusion chromatography their composition was almost independent of the aggregation pH. The solubility of hydrolysates of SPIs with a varying glycinin and β-conglycinin composition showed that glycinin-derived peptides are the driving force for the lower solubility of SPI hydrolysates. The solubility of SPI hydrolysates at pH 8.0 was shown not to be the sum of that of glycinin and β-conglycinin hydrolysates. Assuming that the separate hydrolysis of glycinin and β-conglycinin did not differ from that in the mixture (SPI), this indicates that β-conglycinin-derived peptides have the ability to inhibit glycinin-derived peptide aggregation.     

大豆中β-伴大豆球蛋白提纯方法的优化

β-伴大豆球蛋白（7S）是大豆种子储藏蛋白质的主要成分之一,对大豆蛋白质的营养品质有着重要影响。然而,如何提高该蛋白的纯度一直是影响其表征鉴定的一个主要问题。在本研究中,我们在Liu等报道方法的基础上,通过两个关键提取环节的条件优化,使β-伴大豆球蛋白的纯度得到了明显提高。一是在11S球蛋白去除之前提高所加还原剂SBS的浓度,分析SBS浓度提高对β-伴大豆球蛋白提取纯度的影响,将SBS的最适浓度确定为0.03 mmol·L^-1;二是在β-伴大豆球蛋白析出之前,通过加水析出/离心法去除一次混杂的11S球蛋白。这两个环节的条件优化最终使得β-伴大豆球蛋白的提取纯度达到98%。本研究结果对大豆蛋白质遗传育种研究中β 伴大豆球蛋白的表征鉴定具有重要意义。     

Enzymatic Production of Ribavirin from Pyrimidine Nucleosides by Enterobacter aerogenes AJ 11125

Microorganisms that produce ribavirin(1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide; virazole^®) directly from pyrimidine nucleosides and TCA (1,2,4-triazole-3-carboxamide) were screened from our stock cultures. Of the 400 strains tested, 16 were isolated as ribavirin-producers from uridine or cytidine. In particular, Enterobacter aerogenes AJ 11125, Bacillus brevis AJ 1282 and Sarcina lutea AJ 1212 were found to possess potent activities of ribavirin production from them. In the presence of intact cells of Enterobacter aerogenes AJ 11125, which was selected as the best strain, 110.2mm and 67.6 mm ribavirin were produced from uridine and cytidine, respectively, on 96 hr reaction at 60°C. In addition, this strain could also produce ribavirin from guanosine, but could not produce it from orotidine, which is also a pyrimidine nucleoside.     

Thermal Stability of Individual Molecular Species of Soybean β-Conglycinin

The thermal stability of three individual molecular species of soybean β-conglycinin, α₃, β₃, and modified α₃ (amino terminal Vall-Argl26 amino acid groups deleted), was studied by the change in far- and near-ultraviolet CD spectra and in surface hydrophobicity. The α₃ molecule was less thermostable than β₃, its lower stability being derived from the core of the molecule rather than the extra polypeptide chain on the amino terminal side.     

Effect of Soy Protein Subunit Composition on the Rheological Properties of Soymilk during Acidification

The effect of soy protein subunit composition on the acid-induced aggregation of soymilk was investigated by preparing soymilk from different soybean lines lacking specific glycinin and β-conglycinin subunits. Acid gelation was induced by glucono-δ-lactone (GDL) and analysis was done using diffusing wave spectroscopy and rheology. Aggregation occurred near pH 5.8 and the increase in radius corresponded to an increase in the elastic modulus measured by small deformation rheology. Diffusing wave spectroscopy was also employed to follow acid gelation, and data indicated that particle interactions start to occur at a higher pH than the pH of onset of gelation (corresponding to the start of the rapid increase in elastic modulus). The protein subunit composition significantly affected the development of structure during acidification. The onset of aggregation occurred at a higher pH for soymilk samples containing group IIb (the acidic subunit A₃) of glycinin, than for samples prepared from Harovinton (a commercial variety containing all subunits) or from genotypes null in glycinin. The gels made from lines containing group I (A₁, A₂) and group IIb (A₃) of glycinin resulted in stiffer acid gels compared to the lines containing only β-conglycinin. These results confirmed that the ratio of glycinin/β-conglycinin has a significant effect on gel structure, with an increase in glycinin causing an increase in gel stiffness. The type of glycinin subunits also affected the aggregation behavior of soymilk.     

Dynamic viscoelastic properties of glycinin and β-conglycinin gels from soybeans

The effects of heating temperature on gel properties and conformational changes were investigated in glycinin and β-conglycinin gels using Theological and Fourier transform ir (FTIR) methods. Solutions of 15 wt % glycinin or β-conglycinin in 35 mM phosphate buffer at pH 7.6 were heated at various temperatures for 30 min and rheological properties were measured at 20°C. The storage modulus G′ as a function of frequency changed from a monotonical decrease with decreasing frequency to a plateau in the range from 0.0018 to 40 Hz by heating at temperatures higher than 80°C for glycinin and 65°C for β-conglycinin. A band at 1618 cm^?1 (associated with the β-sheet structure) on ir spectra increased with the formation of heat-induced gels. The value of the storage modulus G′ correlated well with the increase in absorbance at 1618 cm^?1. These results suggest that the formation of a β-sheet structure may be closely related to the value of the storage modulus G′ for heat-induced gels in soybean proteins and that heat-induced gels of glycinin and β-conglycinin are formed by cross-links with intermolecular β-sheet structures. © 1994 John Wiley & Sons, Inc.     

Expression of a Soluble Functional Form of the Integrin α4β1 in Mammalian Cells

The integrin α₄β₁(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human α₄ and β₁ subunits were fused to the genomic DNA encoding the human γ1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote α₄β₁ heterodimer formation. The soluble α₄β₁ Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent K_d for VCAM-1 binding were similar for both the soluble and native forms of α₄β₁. In addition, the integrin–Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble α₄β₁ should be generally applicable to a range of integrins.     

Solubility of soy lipophilic proteins: comparison with other soy protein fractions

Solubility of soy lipophilic proteins (LP) was studied as compared with that of other soy protein fractions. LP, β-conglycinin, glycinin, and soy protein isolate (N-SPI) were prepared under the condition to avoid heat denaturation. Solubility of LP was lower than that of other soy protein fractions under all the tested conditions varying in pH values and ionic strength. The solubility of LP was increased constantly by elevating temperature until 90 °C, whereas that of β-conglycinin and glycinin dropped at high temperature. Temperature-dependent change in solubility of N-SPI might reflect the balance among that of glycinin, β-conglycinin and LP. Based on the results of SDS-PAGE, determination of phospholipid content and Fourier Transform Infrared spectroscopy, we discussed the solubilization behavior of LP relating to its origin and composition.     

Salt-induced Reconstitution of β-Conglycinin from Its Thermal Dissociates

The salt-free heat denaturated β-conglycinin, heated to 99°C for 5 min, exhibited dissociation of the protein into subunits (α, α′ and β). Heat-induced dissociates could be converted to β-conglycinin with an increase in ionic strength of the protein solution. The reassociation of these dissociates depended on the salt concentration and on the species of the constituent subunits. Adding salt above 0.1 m NaCl favored reassociation of the thermal dissociates. The β subunit has a tendency to form an aggregate of higher molecular size, while the α and α′ subunits have an ability to form the 7S aggregate. Reconstituted β-conglycinin possessed the characteristic of 7S ? 9S interconversion with a change of ionic strength, which has been considered as a feature of native conglycinin. The restoration of electrophoretical mobility, ultracentrifugal characteristics and the secondary structure of CD properties was distinct evidence supporting the reconstitution of β-conglycinin from its thermally denatured state. However, the reconstituted conformation differed from the native β-conglycinin in its quantitative precipitin curve and ultraviolet difference spectrum.     

Subunit structure of γ-conglycinin in soybean seeds

Dissociated subunits of purified γ-conglycinin were isolated on a DEAE-Sephadex A-50 column. A single band was seen on two kinds of gel electrophoresis and isoleucine was shown as the only N-terminal amino acid. The isolated subunit reacted with antisera to the native γ-conglycinin. The M_r of the subunit was 51 000–51 500 estimated by urea-acetic acid and SDS-urea gel electrophoresis. A value of 50 000 was obtained by gel filtration with guanidine-hydrochloric acid on Sepharose CL-6B. The γ-conglycinin molecule was found to be made up of three subunits. This was determined by cross-linking the subunits and then submitting them to gel electrophoresis. Differences and similarities of subunit structure among γ-conglycinin, β-conglycinin and glycinin are discussed.     

Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth

The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and β-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and β-conglycinin specific antibodies. The three subunits of β-conglycinin were preferentially metabolized. Of the three subunits of β-conglycinin, the larger α and α′ subunits are rapidly degraded, generating new β-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the β-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of β-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.     

Determination of Seed Storage Proteins and Total Isoflavones in Wild and Cultivated Soybeans

Two soybean components namely, storage proteins and isoflavone content in a wild and three cultivated soybean genotypes were characterized and compared. The storage proteins, β-conglycinin and glycinin were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and two major storage proteins and their subunits were characterized using mass spectrometry. The three isoflavones, aglycon and the nine conjugated forms were separated by HPLC (high performance liquid chromatography) and identified by comparison of retention time, ultraviolet and mass spectral analyses. Comparison between the number of 2D-PAGE protein spots of the storage protein subunits and HPLC area of twelve isoflavones was also evaluated. The analysis of proteins and isoflavones from the wild genotype and the three cultivated genotypes suggested possible interactions between proteins and isoflavones. The same wild genotype, which showed significant statistical differences in β-conglycinin and glycinin protein profiles also revealed considerable reduction in total isoflavones (> 55%) content.     

Occurrence and biosynthesis of catalase at different stages of seed maturation

It was to be shown whether during the biogenesis of microbodies some of their components were already present in the cell prior to the organelle's assembly. To this end, the occurrence and properties of catalase in soluble and particular fractions of ripening cucumber seeds were examined. Homogenates of seeds from ripening fruits were fractionated by isopycnic density gradient centrifugation, and thus catalase was found in three different fractions: as a soluble enzyme in the gradient supernatant, as a membrane fraction at density d=1.18 kg l^-1, and in association with microbodies. In the early steps of seed formation, catalase was detected at density d=1.18 kg l^-1 and in the gradient supernatant. At a later stage of seed maturation, however, catalase was primarily associated with microbodies which exhibited an equilibrium density of d=1.23 kg l^-1. M _r as well as subunit M _r of catalase were determined, and their close immunological relationship to leaf peroxisomal catalase and glyoxysomal catalase was demonstrated. Biosynthesis of catalase at different stages of seed maturation was investigated by in vivo labeling with l-[³⁵S]methionine, l-[¹⁴C]leucine and -[³H]aminolaevulinic acid. Electrophoretic analysis of de novo synthesized catalase subunits revealed the occurrence of a heavy form (M _r 57,500) in the soluble fraction; this form was preferentially labeled. A light form, M _r 53,500, was detected in microbodies and also in the soluble fraction. The findings lend support to the hypothesis that the rate of catalase synthesis is highest in an early stage of seed formation, when globulins have already been formed, but before de novo synthesis of malate synthase has commenced. Prior to microbody assembling, a cytoplasmic pool of catalase was labeled.Abbreviations EDTA Na₂-ethylenediaminotetraacetate - Hepes 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - M _r molecular weight     

Accumulation of β-conglycinin in soybean cotyledon through the formation of disulfide bonds between α'- and α-subunits

β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α'- and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α'/α-subunits were complexes of more than 720 kD. The α'- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α'/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α'- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions.     

Bioassay‐Guided Fractionation,Chemical Compositions and Antibacterial Activity of Extracts from Rhizomes of Globba schomburgkii Hook.f.

Bioassay‐guided fractionation was conducted on dichloromethane extract from the rhizomes of Globba schomburgkii Hook.f., which have previously been reported as the part with the highest antibacterial activity. 10 fractions and 20 sub‐fractions were obtained and evaluated for their potency against various strains of bacteria. The most active sub‐fractions were 8 times more effective against Staphylococcus aureus and Micrococcus luteus than the original crude extract. Moreover, two pure compounds, namely petasol and (E)‐15,16‐dinorlabda‐8(17),11‐dien‐13‐one, were successfully isolated and characterized for the first time from this plant species. Untargeted compound analysis of all fractions and sub‐fractions was performed by gas chromatography hyphenated with mass spectrometry, leading to positive identification of 167 compounds according to comparison with the mass spectrum and retention index database, 137 of which have never been reported for G. schomburgkii. The correlation between antibacterial activity and composition of each fraction suggests that the bioactive compounds could be 4,8‐β‐epoxycaryophyllene, methyl isocostate, (E)‐labda‐8(17),12‐diene‐15,16‐dial, α‐kessyl acetate, zederone, clovanediol, ledene oxide‐(I), alantolactone, or 8α,11‐elemadiol.     

Thermal Versus High Hydrostatic Pressure Treatments on Calcium-added Soybean Proteins. Protein Solubility,Colloidal Stability and Cold-set Gelation

The effects of thermal treatment (TT) and high hydrostatic pressure treatment (HHPT) on calcium-added soybean protein 1% (w/w) aqueous dispersions at pH 7.0 were compared. High hydrostatic pressure, but not thermal treatment, improved protein solubility and colloidal stability. Despite the fact that the glycinin solubility is more affected by calcium than that of β-conglycinin, glycinin could remain in dispersion in the presence of calcium when denatured by HHPT (calcium added before or after treatment), but not when denatured by TT or without denaturing treatment. Thus, polypeptide composition of soluble aggregates depended on type of treatment. Colloidal stability and molecular weight of soluble aggregates depended on the order of application of calcium and denaturing treatment: when calcium was present during either HHPT or TT, the dispersions had higher stability and higher proportion of soluble aggregates with high molecular weight than when calcium was added after treatments. After freeze drying and re-dispersing at higher protein content (10% w/w) calcium-added dispersions subjected to HHPT formed cold-set gels that were transparent and exhibited excellent water holding capacity. Our results provide the basis for the development of ready-to-use functional ingredients.

     

Trace elements distribution and species fractionation in the wheat (Triticum aestivum) plant

Abstract

The status of 13 trace elements’ (both essential and toxic) was investigated in individual parts of the winter wheat plant(Triticum aestivum) taken during its whole cultivation period. The study includes the determination of total concentrations, portions soluble in 0.02 mol L^?1 Tris-HCI buffer solution (pH = 7.5), and the fractionation of soluble species of elements by SEC and ICP/MS. Ligands of trace elements from a low-molecular weight SEC fraction were isolated by affinity chromatography and characterised by MALDI/MS analyses and by amino acids composition. Inhomogeneous accumulation of trace elements was found in the analysed plant tissues. The concentrations of elements are also affected by the maturity of the plants. The distribution of the soluble species of the elements between chromatographic fractions exhibited some regularity in all the samples. Substantial amounts of trace elements are located in a low-molecular weight fraction (< 2 kDa). Only chromatograms of Zn (grain) and Cu (all samples) contain significant medium-molecular and high-molecular weight fractions. Compounds isolated from the low-molecular weight fractions are rich in cystein and dicarboxylic amino acids or their amides. MALDI/MS spectra of these compounds isolated from shoots, straw and grain confirmed the presence of the phytochelatin PC₅.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.