Soybean Allergen Research Articles

Colorimetric LAMP microfluidic chip for detecting three allergens: peanut, sesame and soybean

Food allergies can greatly harm people’s health, and therefore detecting allergens in foods is extremely important. By integrating loop-mediated isothermal amplification (LAMP) with a microfluidic chip, we have developed a method for detecting the allergen genes of peanut (Arachis hypogaea), sesame (Sesamum indicum), and soybean (Glycine max) using a colorimetric method suitable for the naked eye, known as the colorimetric LAMP microfluidic chip. In the presence of peanut, sesame, or soybean in the samples, the corresponding reaction well of the microfluidic chip will appear pink, or otherwise remain light brown. This method of detection is specific and can easily distinguish these three allergens from others in foods. The detection limit for peanut, sesame and soybean allergens was 0.4 ng/μL using the LAMP-microfluidic chip. The accuracy of this novel and rapid method was validated using allergenic foods obtained commercially and was comparable with that of the typical TaqMan real-time PCR method.

Digital PCR for analysis of peanut and soybean allergens in foods

Digital PCR for analysis of peanut and soybean allergens in foods

The influence of composite enzymatic hydrolysis on the antigenicity of β-conglycinin in soy protein hydrolysates

The β-conglycinin is considered one of the major soybean allergens. The effects of hydrolysis with Neutrase and Flavourzyme on the antigenicity of β-conglycinin were investigated by indirect competitive, enzyme-linked immunosorbent assay (ELISA) and western blotting analysis. The molecular weight distribution was performed by SDS-PAGE. The hydrolysate with the lowest antigenicity was further separated by ultrafiltration. The results showed that enzymatic hydrolysis significantly reduced the antigenicity of β-conglycinin in soy protein hydrolysates. Flavourzyme hydrolysis for 15 min, inactivating the enzyme and then Neutrase hydrolysis for 15 min was the optimal combination of enzymatic hydrolysis, and the antigenicity of β-conglycinin in the hydrolysates was reduced by 64.46% compared with unhydrolyzed soy protein. The molecular weight distributions of hydrolysates were 31, 26, 20, and 15 kDa or lower. The low molecular weight (<10 kDa) ultrafiltration fractions exhibited the lowest antigenicity. Practical applications Enzymatic hydrolysis could be an efficient method in the reduction of soybean protein antigenicity. Flavourzyme hydrolysis for 15 min, inactivating the enzyme and then Neutrase hydrolysis for 15 min was the optimal hydrolysis mode for the reduction of β-conglycinin antigenicity. The results suggested that less than 10 kDa ultrafiltration fractions of hydrolysates showed the lowest antigenicity, which could be useful in the development of hypoallergenic soybean peptide powder.

Combining 2-DE immunoblots and mass spectrometry to identify putative soybean (Glycine max) allergens

Soybean is recognized as a commonly allergenic food, but the identity of important allergens is not well studied. Recently, some global regulatory agencies started requiring quantitative analysis of individual allergens, including unproven allergens, as part of the risk assessment for genetically engineered (GE) soybeans. We sought to identify soybean proteins that bind IgE from any of 10 individual soybean-sensitized subjects. Soybean IgE binding proteins were identified by 2-DE immunoblots using sera from four soy-allergic and plasma from six soy-sensitized human subjects. Corresponding spots were excised from stained gels, digested, and analyzed using a quadrupole TOF Synapt G2-S tandem mass spectrometer. Results showed the major IgE binding proteins were subunits of either β-conglycinin (Gly m 5) or glycinin (Gly m 6). Soybean Kunitz trypsin inhibitor (SKTI) was a significant IgE binding protein for four subjects. Soybean agglutinin, seed biotinylated protein (SBP) of 65 kDa, late embryogenesis protein (LEP), and sucrose-binding protein were identified as IgE binding only for soy-sensitized subjects. We conclude that the major soybean allergens are isoforms of Gly m 5, Gly m 6, and possibly SKTI and that requirements for quantitative measurement of proteins that are not clear allergens is not relevant to safety.

Boiling and Pressure Cooking Impact on IgE Reactivity of Soybean Allergens

Background: Soybean is one of the 8 foods that causes the most significant rate of food allergies in the USA and Europe. Thermal processing may impact on the allergenic potential of certain foods. We aimed to investigate modifications of the IgE-binding properties of soybean proteins due to processing methods that have been previously found to impact on the allergenicity of legumes such as peanut. Methods: Soybean seeds were subjected to different thermal processing treatments. To evaluate their impact on the IgE-binding capacity of soybean proteins, individual sera from 25 patients sensitized to soybean were used in in vitro immunoassays. Detection of specific soybean allergens in untreated and treated samples was carried out with specific monoclonal and polyclonal antibodies. In vivo studies of skin prick testing (SPT) were also performed. Results: The IgE reactivity of soybean was resistant to boiling up to 30 min, and this treatment had a higher impact when applied for 60 min. Treatment that combined heat and pressure produced a fragmentation of proteins in both soluble and insoluble fractions that went along with a decreased capacity to bind IgE and reduced the SPT wheal size. However, allergens such as 7S globulins survived this treatment. Conclusions: Thermal-processing methods able to attenuate the capacity of soybean proteins to bind IgE may contribute to the improvement of food safety and could constitute a potential strategy for the induction of tolerance to soybean.

Degradation and removal of wheat and soybean allergens in the brewing of soy sauce

Degradation and removal of wheat and soybean allergens in the brewing of soy sauce

Safety evaluation of the food enzyme peroxidase obtained from soybean (Glycine max) hulls.

The food enzyme considered in this opinion is a peroxidase (hydrogen‐peroxide oxidoreductase; EC 1.11.1.7) obtained from hulls of soybeans (Glycine max) by the company Kerry Ingredients & Flavours. The compositional data provided were considered sufficient. The manufacturing process did not raise safety concerns. The enzyme is intended to be used in baking processes. Based on the maximum recommended use level, dietary exposure to the food enzyme total organic solids (TOS) was estimated on the basis of individual data from the EFSA Comprehensive European Food Consumption Database. This exposure estimate is lower than the exposure to the fraction of soybean comparable to the food enzyme TOS resulting from the consumption of whole soybean‐derived foods by roughly an order of magnitude. As the food enzyme is derived from edible parts of soybean, in line with the requirements of the guidance document on food enzyme assessment, the Panel concluded that the provision of toxicological data was unnecessary. The potential allergenicity was evaluated by searching for similarity between the amino acid sequence of soybean peroxidase retrieved from the database Uniprot and the sequences of known food allergens; no match was found. Peroxidase from soybean hulls is not listed as an allergen in allergen databases. However, several soybean‐ and soybean hull proteins are known to be respiratory or food allergens. Based on the origin of the food enzyme from edible parts of soybean, the enzyme manufacturing process, the compositional and biochemical data provided, and the dietary exposure assessment, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. However, the Panel noted that this food enzyme may contain allergenic soybean proteins, thus, adverse reactions in susceptible soybean‐allergic individuals cannot be ruled out.

Different Damage to the Mechanical Barrier Function of IPEC-J2 Induced by Soybean Allergen β-conglycinin Hydrolyzed Peptides

Abstract Three major enzyme-hydrolyzed peptides have been produced after simulative digestion in vitro of soybean β-conglycinin. The intestinal barrier of IPEC-J2 induced by β-conglycinin enzyme-hydrolyzed peptides was evaluated in this study. The increased alkaline phosphatase (AP) activity was actually linearly correlated with the incubation time by the hydrolysate, the purified 52 kD peptide, or the mixture of 25 and 30 kD peptides. The MTT and TEER values declined in dose-dependence (0–2 mg/mL, p \lt 0.05) or in time-dependence (2–24 h, p \lt 0.05). After treatment with different hydrolyzed peptides, the tight junction expression of claudin-3, claudin-4, occludin, and ZO-1 were reduced (p \lt 0.05). Finally, it is found out that the maximum damage to the epithelial barrier function was induced by the mixture of 25 and 30 kD peptide, whereas the minimum damage was caused by the 52 kD peptide.

Transgenesis affects endogenous soybean allergen levels less than traditional breeding

The regulatory body that oversees the safety assessment of genetically modified (GM) crops in the European Union, the European Food Safety Authority (EFSA), uniquely requires that endogenous allergen levels be quantified as part of the compositional characterization of GM versions of crops, such as soybean, that are considered to be major allergenic foods. The value of this requirement for assessing food safety has been challenged for multiple reasons including negligible risk of altering allergen levels compared with traditional non-GM breeding. Scatter plots comparing the mean endogenous allergen levels in non-GM soybean isoline grain with the respective levels in GM grain or concurrently grown non-GM commercial reference varieties clearly show that transgenesis causes less change compared with traditional breeding. This visual assessment is confirmed by the quantitative fit of the line of identity (y = x) to the datasets. The current science on allergy does not support the requirement for quantifying allergen levels in GM crops to support safety assessment.

Effects of diesel exhaust particle exposure on a murine model of asthma due to soybean.

BackgroundExposure to soybean allergens has been linked to asthma outbreaks. Exposure to diesel exhaust particles (DEP) has been associated with an increase in the risk of asthma and asthma exacerbation; however, in both cases the underlying mechanisms remain poorly understood, as does the possible interaction between the two entities.ObjectiveTo investigate how the combination of soybean allergens and DEP can affect the induction or exacerbation of asthma in a murine model.MethodsBALB/c mice received intranasal instillations of saline, 3 or 5 mg protein/ml soybean hull extract (SHE), or a combination of one of these three solutions with DEP. Airway hyperresponsiveness (AHR), pulmonary inflammation in bronchoalveolar lavage, total serum immunoglobulin E and histological studies were assessed.ResultsA 5 mg protein/ml SHE solution was able by itself to enhance AHR (p = 0.0033), increase eosinophilic inflammation (p = 0.0003), increase levels of IL-4, IL-5, IL-13, IL-17A, IL-17F and CCL20, and reduce levels of IFN-γ. The combination of 5 mg protein/ml SHE with DEP also produced an increase in AHR and eosinophilic inflammation, but presented a slightly different cytokine profile with higher levels of Th17-related cytokines. However, while the 3 mg protein/ml SHE solution did not induce asthma, co-exposure with DEP resulted in a markedly enhanced AHR (p = 0.002) and eosinophilic inflammation (p = 0.004), with increased levels of IL-5, IL-17F and CCL20 and decreased levels of IFN-γ.Conclusions & clinical relevanceThe combination of soybean allergens and DEP is capable of triggering an asthmatic response through a Th17-related mechanism when the soybean allergen concentration is too low to promote a response by itself. DEP monitoring may be a useful addition to allergen monitoring in order to prevent new asthma outbreaks.

Three-dimensional structure of Gly m 5 (β-conglycinin) plays an important role in its stability and overall allergenicity

To date, there is insufficient knowledge regarding the relationship of the allegenicity and the three-dimensional structure of Gly m 5 (β-conglycinin), a major allergen in soybean. In the present study, allergen Gly m 5 was demonstrated to generate three major digestion-resistant fragments when it was subjected to in vitro digestion. The largest fragment corresponded to the main body of the monomer and two smaller fragments corresponded to the main bodies of two modules of the monomer. Two major protease cleavage sites were located in the regions near to the connection between two modules. Coincidentally, the major digestion-resistant fragments were demonstrated to contain intact IgE epitopes and be capable to induce basophil histamine release in Gly m 5 sensitised piglets, indicating that the three-dimensional structure of Gly m 5 afforded the molecule some protection from complete degradation into small peptides and amino acids, and contributed to its overall allergenicity.

Label-free biochips for rapid detection of soybean allergen &lt;i&gt;Glym&lt;/i&gt;Bd 30K (P34) in foods

Purpose: To develop an innovative method for detection of soybean allergen, Gly m Bd 30K (P34) in foods using a biosensor based on high spatial imaging ellipsometer. Methods: Two monoclonal antibodies, 2D1 and 5F9, each known to have specific bioactivity against P34 allergen, were selected and separately immobilized as ligands on silicon wafer surface to allow capture of the P34 allergen. The resultant changes on the wafer surface were viewed directly as images in gray scale. Results: Images indicated that these two antibodies detected the presence of P34 allergen in soybean extract with sensitivity of 1 mg/L and a detection time of about 15 min. For the detection of P34 allergen in foods, results from biochip detection were consistent with those obtained using ELISA detection. Conclusion: These results show that the biochip may be an effective analytical tool for food allergen detection. Keywords: Soybean Allergen, Gly m Bd 30K, Biochip, Detection, Foods

Safety evaluation of the food enzyme β-amylase obtained from soybean (Glycinemax).

The food enzyme considered in this opinion is a β‐amylase (EC 3.2.1.2) from soybean submitted by Nagase (Europa) GmbH. This β‐amylase is intended to be used in the starch processing for maltose syrup production and the manufacture of a Japanese rice cake type. Based on the maximum use levels recommended for the respective food processes, dietary exposure to the food enzyme–total organic solids (TOS) was estimated on the basis of Japanese consumption data. Conservative average infant formula consumption, as reported in the EFSA Draft Guidance on risk assessment of substances present in food intended for infants below 16 weeks of age, was used to estimate the exposure to a fraction of soybean comparable to the food enzyme–TOS, resulting from the consumption of soybean‐derived foods. The exposure estimate to the food enzyme–TOS was found to be lower than the comparable fraction from the source material. Potential allergenicity of the β‐amylase was evaluated by searching for similarity of the amino acid sequence to those of known allergens, and no match was found. The β‐amylase is produced from soybean, which is a known allergenic food. Japanese rice cake, consequently, may contain traces of soybean allergens, which may give rise to safety concerns in soybean‐allergic consumers. Based on the origin of the food enzyme from edible parts of soybean, the manufacturing process, the compositional and biochemical data provided and the dietary intake estimates, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use, except that Japanese rice cake produced with this food enzyme may contain traces of soybean allergens.

A comparative secretome analysis of industrial Aspergillus oryzae and its spontaneous mutant ZJGS-LZ-21

Aspergillus oryzae koji plays a crucial role in fermented food products due to the hydrolytic activities of secreted enzymes. In the present study, we performed a comparative secretome analysis of the industrial strain of Aspergillus oryzae 3.042 and its spontaneous mutantZJGS-LZ-21. One hundred and fifty two (152) differential protein spots were excised (p<0.05), and 25 proteins were identified. Of the identified proteins, 91.3% belonged to hydrolytic enzymes acting on carbohydrates or proteins. Consistent with their enzyme activities, the expression of 14 proteins involved in the degradation of cellulose, hemicellulose, starch and proteins, increased in the ZJGS-LZ-21isolate. In particular, increased levels of acid protease (Pep) may favor the degradation of soy proteins in acidic environments and promote the cleavage of allergenic soybean proteins in fermentation, resulting in improvements of product safety and quality. The ZJGS-LZ-21 isolate showed higher protein secretion and increased hydrolytic activities than did strain 3.042, indicating its promising application in soybean paste fermentation.

Degradation and removal of soybean allergen in Japanese soy sauce.

Soy sauce is a traditional fermented seasoning of Japan and is available throughout the world. The two main raw ingredients of soy sauce are soybean and wheat, both of which are established food allergens. The present study examined the degradation and removal of soybean allergens in soy sauce by immunoblotting with anti‑soybean protein antibody from rabbit and sera from two children with soybean allergy. It was demonstrated that soybean allergens were gradually degraded during the fermentation process, but were not completely degraded in raw soy sauce. During the processes of heat‑treatment and filtration, the soluble soybean allergens in raw soy sauce were denatured to insoluble allergens by heat‑treatment and subsequently completely removed from soy sauce by filtration. Therefore, to reduce the allergenicity of soy sauce, heat‑treatment and filtration are very important processes in addition to the enzymatic degradation during the fermentation of soy sauce.

Combination of two epitope identification techniques enables the rational design of soy allergen Gly m 4 mutants.

Detailed IgE-binding epitope analysis is a key requirement for the understanding and development of diagnostic and therapeutic agents to address food allergies. An IgE-specific linear peptide microarray with random phage peptide display for the high-resolution mapping of IgE-binding epitopes of the major soybean allergen Gly m 4, which is a homologue to the birch pollen allergen Bet v 1 is combined. Three epitopes are identified and mapped to a resolution of four key amino acids, allowing the rational design and the production of three Gly m 4 mutants with the aim to abolish or reduce the binding of epitope-specific IgE. In ELISA, the binding of the mutant allergens to polyclonal rabbit-anti Gly m 4 serum as well as IgE purified from Gly m 4-reactive soybean allergy patient sera is reduced by up to 63% compared to the wild-type allergen. Basophil stimulation experiments using RBL-SX38 cells loaded with patient IgE are showed a decreased stimulation from 25% for the wild-type Gly m 4 to 13% for one mutant. The presented approach demonstrates the feasibility of precise mapping of allergy-related IgE-binding epitopes, allowing the rational design of less allergenic mutants as potential therapeutic agents.

Establishment of a Lateral Flow Colloidal Gold Immunoassay Strip for the Rapid Detection of Soybean Allergen β-Conglycinin

A rapid lateral flow colloidal gold immunoassay strip was established in a sandwich format with the colloidal gold-labelled mouse anti-β-conglycinin monoclonal antibody (mAb) and the rabbit anti-β-conglycinin polyclonal antibody (pAb) in order to specifically identify a soybean allergen, β-conglycinin. Soybean and soy derivatives have become ubiquitous in many vegetarian and meat-based food products, and as a result, dietary avoidance has become difficult. Therefore, soybean avoidance is challenging for individuals with soybean allergy. In our study, the selected mAb showed high affinity to the target and no cross-reactivity with other soybean allergens. Based on the sandwich immunoassay principle, the rabbit pAb was applied in the test line with high titre. The assay could be accomplished within 10 min without sophisticated procedures. The limit of detection of the test strip was calculated at 1.66 mg/kg in powdered milk using an optical density scanner that measures relative optical density. The assay showed high specificity for β-conglycinin, with no cross-reactions with other food allergens. The efficacy of the lateral flow test strip to detect β-conglycinin in powdered milk samples ranged between 80.8 and 89.2% with relative standard deviations of less than 7.2%. Therefore, the test strip is useful as a rapid and reliable detection method for β-conglycinin in powdered milk. In addition, this assay is suitable for monitoring β-conglycinin in other processed foods when samples are appropriately extracted.

Natural Variability of Allergen Levels in Conventional Soybeans: Assessing Variation across North and South America from Five Production Years.

Soybean (Glycine max L. Merrill) is one of eight major allergenic foods with endogenous proteins identified as allergens. To better understand the natural variability of five soybean allergens (Gly m 4, Gly m 5, Gly m 6, Gly m Bd 28k, and Gly m Bd 30k), validated enzyme-linked immunosorbent assays (ELISAs) were developed. These ELISAs measured allergens in 604 soybean samples collected from locations in North and South America over five growing seasons (2009-2013/2014) and including 37 conventional varieties. Levels of these five allergens varied 5-19-fold. Multivariate statistical analyses and pairwise comparisons show that environmental factors have a larger effect on allergen levels than genetic factors. Therefore, from year to year, consumers are exposed to highly variable levels of allergens in soy-based foods, bringing into question whether quantitative comparison of endogenous allergen levels of new genetically modified soybean adds meaningful information to their overall safety risk assessment.

Analysis of antigenic epitopes and cloning of soybean major allergen β-conglycinin beta-subunit

The model of tertiary of β-conglycinin beta-subunit was constructed by the bioinformation tool SWISS-MODEL, and the conformational epitopes were predicted by Disco Tope 2.0 server.The overlapping fragments of β-conglycinin beta-subunit genes were amplified by PCR and inserted into p MD-18 T vector, then preserved into competent cell of Escherichia coli JM109.The recombinant plasmids were evaluated by blue-white selection, PCR and double enzyme digestion, and DNA sequence analysis showed that the cloned sequence was the very fragment designed. The successful cloning of the genes can provide a reference for the study of the expresstion of β-conglycinin beta-subunit and screening of antigenic epitopes.

Mass Spectrometric Analysis of N-Glycoforms of Soybean Allergenic Glycoproteins Separated by SDS-PAGE.

Glycosylation of many proteins has been revealed to be closely related with food allergies, and screening and structural analysis of related glycoproteins and glycoallergens are essential for studies in this field. Herein, we describe detailed N-glycoform analysis of all glycoprotein fractions of soybean protein isolate (SPI) separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to disclose structural features of the glycan moieties of more soybean glycoproteins. SPI was fractionated by SDS-PAGE, and the generated protein bands were recovered and subjected to in-gel N-glycan release and labeling using a one-pot method newly developed by our group, followed by detailed analysis by electrospray ionization mass spectrometry (ESI-MS) and online hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS). As a result, we found seven bands mainly containing oligomannose-type glycans; two mainly contain core α1,3-fucosylated glycans, and six have no glycans. This study is the first report that discovers core α1,3-fucosylated N-glycans in bands 1, 2, and 6 and discloses bands 3, 4, 5, and 7 as glycoproteins and their N-glycoforms. Therefore, it can expand our knowledge about soybean protein glycosylation and provide significant structural reference for research of soybean allergens.