Differential effects of heating modes on the immunogenic potential of soy-derived peptides released after in vitro infant digestion.

During production of soy-based infant formula, soy protein undergoes heating processes. This study investigated the differential impact of heating modes on the immunogenic potential of peptides in soy protein digests. Wet or dry heating was applied, followed by in vitro gastrointestinal infant digestion. The released peptides were analyzed by LC-MS/MS. Bioinformatics tools were utilized to predict and identify potential linear B-cell and T-cell epitopes, as well as to explore cross-reactivity with other legumes. Subsequently, the peptide intensities of the same potential epitope across different experimental conditions were compared. As a result, we confirmed the previously observed enhancing effect of wet heating on infant digestion and inhibitory effect of dry heating. A total of 8,546 peptides were detected in the digests, and 6,684 peptides were with a score over 80. Among them, 29 potential T-cell epitopes and 27 potential B-cell epitopes were predicted. Cross-reactivity between soy and other legumes, including peanut, pea, chickpea, lentil, kidney bean, and lupine, was also detected. Overall, heating and digestion time could modulate the potential to trigger peptide-induced immune responses.

Lunasin Peptide is a Modulator of the Immune Response in the Human Gastrointestinal Tract.

INTRODUCTION: Lunasin is a soybean bioactive peptide with a variety of beneficial properties against chronic disorders. However, its effect in human primary intestinal cells remains unknown. Hence, this study aims to characterize its ex vivo biological activity in the human intestinal mucosa. METHODS AND RESULTS: Human intestinal biopsies, obtained from healthy controls, are ex vivo conditioned with lunasin both in the presence/absence of lipopolysaccharide (LPS). Peptide maintains its stability during biopsy culture by HPLC-MS/MS analysis. Lunasin is bioactive in the human mucosa, as it induces IL-1ß, TNF-α, IL-17A, CCL2, and PGE2/COX-2 gene expression together with an increased expression of tolerogenic IL-10 and TGFß, while it also downregulates the expression of iNOS and subunit p65 from NF-κB. Indeed, lunasin also abrogates the LPS-induced pro-inflammatory response, downregulating IL-17A, IFNγ, and IL-8 expression, and inducing IL-10 and TGFß expression. These results are also mirrored in the cell-free culture supernatants at the protein level by Multiplex. Moreover, lunasin further induces a regulatory phenotype and function on human intestinal conventional dendritic cell and macrophage subsets as assessed by flow cytometry. CONCLUSIONS: We hereby have characterized lunasin as an immunomodulatory peptide with potential capacity to prevent immune and inflammatory-mediated disorders in the human gastrointestinal tract.

Identification of the linear immunodominant epitopes in the ß subunit of ß-conglycinin and preparation of epitope antibodies.

ß-conglycinin is one of the major allergens in soybean protein. The purpose of this study was to predict and to identify the major linear epitopes of the ß subunit of ß-conglycinin. Potential linear epitopes were predicted and confirmed by three immunoinformatics tools combined with the Immune Epitope Database (IEDB). Ten potential epitope peptides were synthesized by Fmoc (9-fluorenylmethoxycarbonyl) solid phase peptide synthesis and were validated by the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) using sera from soybean allergic patients. Polyclonal antibodies, which were prepared by immunizing rabbits with synthesized peptides, were used to confirm their binding ability with ß-conglycinin through western blot and dot blot assays. The results showed that 10 peptides were screened as the main epitopes for the ß subunit of ß-conglycinin. All 10 peptides (P1-P10) presented IgG binding activity, and P2 and P6 were also validated as IgE binding peptides. Moreover, the results of dot blot showed that P5 and P8 might be located inside the protein molecule. Western blot indicated that most of polyclonal antibodies were bound effectively to the ß subunit of ß-conglycinin. In addition, few polyclonal antibodies exhibited an immune cross-reaction with the α and α' subunits.

MHC II-PI3K/Akt/mTOR Signaling Pathway Regulates Intestinal Immune Response Induced by Soy Glycinin in Hybrid Grouper: Protective Effects of Sodium Butyrate.

Soy glycinin (11S) is involved in immune regulation. As an additive, sodium butyrate (SB) can relieve inflammation caused by 11S. To further delve into the mechanisms. A diet containing 50% fishmeal was the control group (FM group), and the experimental groups consisted of the FM group baseline plus 2% glycinin (GL group), 8% glycinin (GH group), and 8% glycinin + 0.13% sodium butyrate (GH-SB group). The specific growth ratio (SGR), feed utilization, and density of distal intestinal (DI) type II mucous cells were increased in the GL group. In the serum, IFN-γ was significantly upregulated in the GL group, and IgG and IL-1ß were upregulated in the GH group. IgG, IL-1ß, and TNF-α in the GH-SB group were significantly downregulated compared to those in the GH group. The mRNA levels of mTOR C1, mTOR C2, and Deptor were upregulated in the GL, GH, and GH-SB groups in the DI compared with those in the FM group, while the mRNA levels of mTOR C1 and Deptor in the GH group were higher than those in the GL and GH-SB groups. 4E-BP1, RICTOR, PRR5, MHC II, and CD4 were upregulated in the GH group. TSC1, mLST8, and NFY mRNA levels in the GL and GH-SB groups were upregulated compared with those in the FM and GH groups. Western blotting showed P-PI3KSer294/T-PI3K, P-AktSer473/T-Akt, and P-mTORSer2448/T-mTOR were upregulated in the GH group. Collectively, our results demonstrate that low-dose 11S could improve serum immune by secreting IFN-γ. The overexpression of IgG and IL-1ß is the reason that high-dose 11S reduces serum immune function, and supplementing SB can suppress this overexpression. Low-dose 11S can block the relationship between PI3K and mTOR C2. It can also inhibit the expression of 4E-BP1 through mTOR C1. High-dose 11S upregulates 4E-BP2 through mTOR C1, aggravating intestinal inflammation. SB could relieve inflammation by blocking PI3K/mTOR C2 and inhibiting 4E-BP2. Generally speaking, the hybrid grouper obtained different serum and DI immune responses under different doses of 11S, and these responses were ultimately manifested in growth performance. SB can effectively enhance serum immunity and relieve intestinal inflammation caused by high dose 11S.

Effect of microbial transglutaminase cross-linking on the quality characteristics and potential allergenicity of tofu.

Microbial transglutaminase (MTGase) has been developed as a new tofu coagulant in recent years due to its good hydrophilicity, high catalytic activity, and strong thermal stability. This study aimed to investigate the effect of MTGase on the physicochemical properties and immunoreactivity of tofu relative to conventional coagulants [brine and glucono-δ-lactone (GDL)]. Structural changes of the MTGase cross-linked soymilk protein were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD) spectroscopy, ultraviolet (UV) absorption spectroscopy, and fluorescence spectroscopy. The IgE-binding capacity of MTGase cross-linked proteins was tested by enzyme-linked immunosorbent assay (ELISA). The physicochemical properties, quality characteristics, and surface microstructures of five different types of tofu were determined by the Kjeldahl nitrogen method, texture analysis, and scanning electron microscopy (SEM). The digestibility of tofu was evaluated in vitro by simulated gastrointestinal (GIS) digestion. A cell sensitization experiment was performed in vitro to evaluate the capability of tofu digestion products to induce the release of bioactive mediators from human basophil leukemia (KU812) cells. Results indicated that MTGase significantly changed the advanced structure of the soymilk protein. Compared with tofu without MTGase, the composite coagulant tofu containing MTGase exhibited better quality. MTGase improved the water-holding capacity (WHC) of the internal mesh structure and increased the yield of tofu. The digestion products of the composite coagulant tofu, especially the GDL plus MTGase tofu, induced KU812 cells to release fewer bioactive mediators compared with those of MTGase-free tofu. MTGase can not only improve the quality of conventional coagulant tofu but also reduce the potential allergenicity of tofu to a certain extent.

ß-Conglycinin-Induced Intestinal Porcine Epithelial Cell Damage via the Nuclear Factor κB/Mitogen-Activated Protein Kinase Signaling Pathway.

Soybean allergy is a serious health risk to humans and animals; ß-conglycinin is the primary antigenic protein in soybean. Intestinal porcine epithelial (IPEC-J2) cells were used as an in vitro physiological model of the intestinal epithelium to study the effects of different concentrations of soybean antigen protein ß-conglycinin to identify the involved signaling pathways. The cells were divided into eight groups and either untreated or treated with different concentrations of ß-conglycinin, pyrrolidine dithiocarbamate (PDTC), Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), SP600125, and SB202190 either alone or in combination. The cells were incubated with 1, 5, and 10 mg·mL-1 ß-conglycinin or 5 mg·mL-1 ß-conglycinin and 1 µmol·L-1 nuclear factor κB (NF-κB) inhibitor (PDTC), inducible nitric oxide synthase inhibitor (l-NAME), c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and p38 inhibitor (SB202190) for 24 h, separately; controls were left untreated. The mRNA, protein, and phosphorylation levels of NF-κB, p38, and JNK were higher in the treated groups than in the control group. ß-Conglycinin decreased tight junction distribution, destroyed the cytoskeleton of IPEC-J2 cells, and caused cell death. After the addition of the inhibitors, ß-conglycinin-induced IPEC-J2 cell damage was significantly reduced. ß-Conglycinin caused damage to IPEC-J2 cells via the mitogen-activated protein kinase/NF-κB signaling pathway. The results of this study are crucial for exploring the mechanisms underlying allergic reactions caused by soybean antigen proteins.

Acidic polypeptides A1a, A3 and A4 of Gly m 6 (glycinin) are allergenic for piglets.

Gly m 6 (glycinin) is one of the major antigenic proteins in soybeans responsible for transient hypersensitivity to soybean meal in weaned piglets. The globulin is a hexamer consisting of subunits containing basic and acidic polypeptides. Multiple acidic polypeptides have long been demonstrated to be allergens for humans and play a key role in the overall allergenicity of Gly m 6. To date, knowledge on the allergenicity of the acidic polypeptides for piglets is very limited. The purpose of this study was to identify the acidic polypeptides that were allergenic for piglets and to characterize these acidic polypeptides by ELISA, western blot, skin prick and basophile histamine release test. The IgG and IgE antibody binding capacities of the acidic polypeptides of Gly m 6 were determined using ELISA and western blot analysis with sera from Gly m 6 sensitized piglets. Skin prick test and basophile histamine release test were conducted to measure the effector cell response to the polypeptides. Specific IgG and IgE antibodies against A1a, A3 and A4 of Gly m 6 were identified in the sera of Gly m 6 sensitized piglets. Meanwhile, positive skin prick test and specific histamine release responses were also induced by the acidic polypeptide A1a, A3 and A4 of Gly m 6 from the basophiles of Gly m 6 sensitized piglets. The results demonstrate that the acidic polypeptide A1a, A3 and A4 of Gly m 6 are allergenic for piglets.

Soybean Glycinin- and ß-Conglycinin-Induced Intestinal Damage in Piglets via the p38/JNK/NF-κB Signaling Pathway.

ß-Conglycinin (7S) and glycinin (11S) are known to induce a variety of hypersensitivity reactions involving the skin, intestinal tract, and respiratory tract. The present study aimed to identify the mechanism underlying the development of allergy to soybean antigen proteins, using piglets as an animal model. Weaned "Duroc × Landrace × Yorkshire" piglets were fed a diet supplemented with 7S or 11S to investigate the signaling pathway involved in intestinal damage in piglets. Results showed that serum nitric oxide (NO), tumor necrosis factor-α (TNF-α), and caspase-3 levels were significantly higher in 7S- and 11S-fed piglets compared to those in suckling or weaned ones. mRNA, protein, and phosphorylation levels of nuclear factor-kappa B (NF-κB), p38, and Jun N-terminal kinase (JNK) were higher in 7S- and 11S-fed piglets than in suckling and weaned ones. Overall, our results indicate that 7S and 11S damaged the intestinal function in piglets through their impact on NF-κB, JNK, and p38 expression.

Degradation of major allergens and allergenicity reduction of soybean meal through solid-state fermentation with microorganisms.

In this study, we determined whether solid-state fermentation could degrade major allergens and reduce potential allergenicity of soybean meal (SBM). Solid-state fermentation was realized through a mixture of Lactobacillus casei, yeast, and Bacillus subtilis. High-performance liquid chromatography, size exclusion-high-performance liquid chromatography, and capillary liquid chromatography/tandem mass spectrometry coupled with electrospray ionization were used to examine the total amino acids and molecular weight distribution of the fermented soybean meal (FSBM). In addition, the potential allergenicity of FSBM was assessed by conducting in vitro competitive inhibition ELISA and oral sensitization and challenge of a BALB/c mice model. The results indicated that the total amino acid content increased and soy protein was degraded into polypeptides with low molecular weights that were derived from the hydrolysis of the allergen sequences N232-D383, G253-I265, E169-S215, G68-G98, A365-I375, and V153-A167. Moreover, the FSBM group exhibited a lower in vitro immunoglobulin E (IgE)-binding capacity than the SBM group. The BALB/c model indicated that the FSBM group manifested milder damage to the intestine, lower mMCP-1 and IgE levels, and higher IFN-γ levels as compared to the SBM group. These findings suggested that the potential allergenicity of SBM was reduced by the solid-state fermentation induced by the mixture of Lactobacillus casei, yeast, and Bacillus subtilis.

Soymilk fermentation by Enterococcus faecalis VB43 leads to reduction in the immunoreactivity of allergenic proteins ß-conglycinin (7S) and glycinin (11S).

Food allergies represent a serious problem affecting human health and soy proteins rank among the most allergenic proteins from food origin. The proteolytic enzymes produced by lactic acid bacteria (LAB) can hydrolyse the major allergens present in soybean, reducing their immunoreactivity. Many studies have reported the ability of LAB to ferment soy-based products; while the majority of them focus on the improvement of the sensory characteristics and functionality of soy proteins, a lack of information about the role of lactic fermentation in the reduction of immunoreactivity of these proteins exists. The aim of the present study was to evaluate the capability of the proteolytic strain Enterococcus faecalis VB43 to hydrolyse the main allergenic proteins present in soymilk and to determine the immunoreactivity of the obtained hydrolysates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results of fermented soymilk demonstrated complete hydrolysis of the ß-subunit from ß-conglycinin and the acidic polypeptide from glycinin. Reversed phase high performance liquid chromatography (RP-HPLC) analysis of the peptides released after hydrolysis revealed the appearance of new peptides and the disappearance of non-hydrolysed proteins, indicating extensive hydrolysis of the substrate. Results from competitive enzyme-linked immunosorbent assay (ELISA) tests clearly indicated a reduction in the immunoreactivity (more than one logarithmic unit) in the fermented sample as compared to the non-fermented control. Our results suggest that the soymilk fermented by E. faecalis VB43 may induce lower allergic responses in sensitive individuals. The strain E. faecalis VB43 may be considered as an excellent candidate to efficiently reduce the immunoreactivity of soymilk proteins.

Insight into the gastro-duodenal digestion resistance of soybean proteins and potential implications for residual immunogenicity.

Soy is an important component of the human diet thanks to its nutritional value and high protein content; however, it also represents a risk for allergenic consumers due to its potential to trigger adverse reactions in sensitized individuals. The putative correlation between immunoreactivity and resistance to the human gastrointestinal (GI) digestion has drawn attention to investigating soybean proteins digestibility. In this work, we provided further insights into this field by performing in vitro simulated GI digestion experiments directly on ground soybean seeds, to provide more realistic results obtained from the digestion of the whole food matrix. Soybean digestion products were analyzed by SDS-PAGE followed by untargeted HPLC-MS/MS analysis and the final data were software treated to enable protein/peptide identification. The latter allowed monitoring the proteolytic degradation of the main soybean proteins during the gastric and duodenal phases. In particular, ß-conglycinin and trypsin inhibitors showed the highest resistance to the combined activity of GI enzymes, showing a partial degradation at the end of the duodenal phase as ascertained by the strong electrophoretic bands displayed at 50 kDa and 20 kDa, respectively. Glycinin subunits also presented, even if to a lower extent, resistance to the complete proteolytic degradation, as demonstrated by polypeptide fragments with molecular weight lower than 20 kDa displayed in the gel at the end of duodenal digestion. In addition, by bioinformatics analysis it was demonstrated that the GI resistant fragments of the allergenic proteins, ß-conglycinin and glycinin, retained in their primary structure linear epitopes potentially able to trigger an immunoreaction when exposed to the intestinal mucosa. Moreover, such resistant peptides also presented a structural homology with epitope sequences recognized in other legume species, presenting a potential risk of adverse cross-reaction for a larger category of allergic consumers.

In vitro digestibility and IgE reactivity of enzymatically cross-linked heterologous protein polymers.

Homologous and heterologous cross-linked polymers of whey protein isolate (WPI), soy protein isolate (SPI) and casein (CN) and their binary mixtures, viz., WPI+SPI, WPI+CN and SPI+CN, were produced using transglutaminase, and their in vitro IgE reactivity and digestibility under simulated gastro-intestinal conditions were studied. The results showed that the IgE reactivity of protein components in heterologous polymers was significantly lower than that in homologous polymers, suggesting that each protein component masked the IgE-reactive epitopes in the other protein component more effectively in heterologous polymers than in homologous polymers. In vitro digestion under simulated gastro-intestinal conditions revealed that both homologous and heterologous polymers were less digestible than untreated proteins, but the peptides released during the time course of digestion were less IgE-reactive. The results of this study indicate that hypoallergenic protein products could be produced by transglutaminase-mediated heterologous polymerization of protein mixtures.

Epitope mapping and identification of amino acids critical for mouse IgG-binding to linear epitopes on Gly m Bd 28K.

Gly m Bd 28K is one of the major allergens in soybeans, but there is limited information on its IgG-binding epitopes. Thirty-four overlapping peptides that covered the entire sequence of Gly m Bd 28K were synthesized, and 3 monoclonal antibodies against Gly m Bd 28K were utilized to identify the IgG-binding regions of Gly m Bd 28K. Three dominant peptides corresponding to (28)GDKKSPKSLFLMSNS(42)(G28-S42), (56)LKSHGGRIFYRHMHI(70)(L56-I70), and (154)ETFQSFYIGGGANSH(168)(E154-H168) were recognized. L56-I70 is the most important epitope, and a competitive ELISA indicated that it could inhibit the binding of monoclonal antibody to Gly m Bd 28K protein. Alanine scanning of L56-I70 documented that F64, Y65, and R66 were the critical amino acids of this epitope. Two bioinformatics tools, ABCpred and BepiPred, were used to predict the epitopes of Gly m Bd 28K, and the predictions were compared with the epitopes that we had located by monoclonal antibodies.

Enzymatic Hydrolysis Does Not Reduce the Biological Reactivity of Soybean Proteins for All Allergic Subjects.

Many soybean protein products are processed by enzymatic hydrolysis to attain desirable functional food properties or in some cases to reduce allergenicity. However, few studies have investigated the effects of enzymatic hydrolysis on the allergenicity of soybean products. In this study the allergenicity of soybean protein isolates (SPI) hydrolyzed by Alcalase, trypsin, chymotrypsin, bromelain, or papain was evaluated by IgE immunoblots using eight soybean-allergic patient sera. The biological relevance of IgE binding was evaluated by a functional assay using a humanized rat basophilic leukemia (hRBL) cell line and serum from one subject. Results indicated that hydrolysis of SPI by the enzymes did not reduce the allergenicity, and hydrolysis by chymotrypsin or bromelain has the potential to increase the allergenicity of SPI. Two-dimensional (2D) immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the chymotrypsin-hydrolyzed samples indicated fragments of ß-conglycinin protein are responsible for the apparent higher allergenic potential of digested SPI.

Serine versus threonine glycosylation with α-O-GalNAc: unexpected selectivity in their molecular recognition with lectins.

The molecular recognition of several glycopeptides bearing Tn antigen (α-O-GalNAc-Ser or α-O-GalNAc-Thr) in their structure by three lectins with affinity for this determinant has been analysed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the molecular recognition. In fact, while Soybean agglutinin and Vicia villosa agglutinin lectins prefer Tn-threonine, Helix pomatia agglutinin shows a higher affinity for the glycopeptides carrying Tn-serine. The different conformational behaviour of the two Tn biological entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topology of the binding site of the lectins are at the origin of these differences.

A rapid test for soy aeroallergens exposure assessment.

BACKGROUND: Determining soy aeroallergens levels is extremely important in the assessment of health risks due to these airborne substances. Currently, soy aeroallergens exposure in the environment is monitored using enzyme immunoassays (EIA) which must be evaluated in a specialized laboratory by skilled personnel. OBJECTIVE: To describe the development and performance of a rapid immunochromatography assay for the detection of soy aeroallergens in environmental samples. METHODS: A test strip using gold labeled anti-soy hull low molecular weight extract (SHLMWE) antibody for the rapid detection of soy aeroallergens in environmental samples was developed. One hundred nineteen airborne samples were analysed in parallel by the strip assay and the anti-SHLMWE sandwich EIA. The assay results were visually analysed by three independent observers who ranked samples as: -, + or ++. Strips were also scanned and analysed by densitometry. RESULTS: The rapid test detected a range of concentrations from 6.25 to 25 ng/mL. Agreement in strip assay interpretations between evaluators was substantial (Kappa = 0.63; CI 0.544-0.715). Visual interpretation also gave a good concordance with EIA results, with sensitivity ranging from 77.3 to 100 and specificity from 65 to 83.5 depending on the observer. Furthermore, a strong correlation was observed between densitometry results of strip assay and EIA determinations. CONCLUSIONS: The strip assay developed is rapid, simple, and sensitive and does not require expensive equipment or specific skills. It has considerable potential in the environmental monitoring field for screening soy aeroallergens levels in port cities where allergen measurements are not currently performed. Due to its simplicity, the test will improve the management of soy allergic patients by controlling environmental allergen exposure without the need for apparatus or skilled personnel.

Targeting a cross-reactive Gly m 5 soy peptide as responsible for hypersensitivity reactions in a milk allergy mouse model.

BACKGROUND: Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity. METHODS: Cow's milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy. RESULTS: Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice. CONCLUSIONS: Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide.

The integral and extrinsic bioactive proteins in the aqueous extracted soybean oil bodies.

Soybean oil bodies (OBs), naturally pre-emulsified soybean oil, have been examined by many researchers owing to their great potential utilizations in food, cosmetics, pharmaceutical, and other applications requiring stable oil-in-water emulsions. This study was the first time to confirm that lectin, Gly m Bd 28K (Bd 28K, one soybean allergenic protein), Kunitz trypsin inhibitor (KTI), and Bowman-Birk inhibitor (BBI) were not contained in the extracted soybean OBs even by neutral pH aqueous extraction. It was clarified that the well-known Gly m Bd 30K (Bd 30K), another soybean allergenic protein, was strongly bound to soybean OBs through a disulfide bond with 24 kDa oleosin. One steroleosin isoform (41 kDa) and two caleosin isoforms (27 kDa, 29 kDa), the integral bioactive proteins, were confirmed for the first time in soybean OBs, and a considerable amount of calcium, necessary for the biological activities of caleosin, was strongly bound to OBs. Unexpectedly, it was found that 24 kDa and 18 kDa oleosins could be hydrolyzed by an unknown soybean endoprotease in the extracted soybean OBs, which might give some hints for improving the enzyme-assisted aqueous extraction processing of soybean free oil.

The origin and functional transition of P34.

P34, a storage protein and major soybean allergen, has undergone a functional transition from a cysteine peptidase to a syringolide receptor. An exploration of the evolutionary mechanism of this functional transition is made. To identify homologous genes of P34, syntenic network was constructed using syntenic relationships from the Plant Genome Duplication Database. The collected homologous genes, along with SPE31, a highly homologous protein to P34 from the seeds of Pachyrhizus erosus, were used to construct a phylogenetic tree. The results show that multiple gene duplications, exon shuffling and following granulin domain loss and some critical point mutations are associated with the functional transition. Although some tests suggested the existence of positive selection, the possibility that random fixation under relaxation of purifying selection results in the functional transition is also supported. In addition, the genes Glyma08g12340 and Medtr8g086470 may belong to a new group within the papain family.