Subunits Of Β-conglycinin Research Articles

Influence of lecithin on the digestibility and immunoreactivity of β-conglycinin

A growing body of evidence suggests that protein structure can be uniquely altered by lipids. However, the role of lecithin on the digestibility and immunoreactivity of allergenic protein remains poorly understood. Therefore, we investigated the impact of different protein/lecithin (P:L) ratios (5:1, 10:1, 20:1, 30:1 and 40:1) on the digestion properties, peptide profiles and immunoreactivity of β-conglycinin in vitro simulated gastrointestinal digestion. Results revealed that lecithin addition increased the degree of hydrolysis and digestion rate of β-conglycinin during digestion in a dose-dependent manner, which may be attributed to the enhancement of pepsin activity. Additionally, the addition of lecithin contributed to the dispersion and homogeneity of β-conglycinin digestion products. When the ratio of lecithin to β-conglycinin was 1:10, the obtained β-conglycinin digestion products exhibited the highest DH value (23.80 %) and the lowest antigenicity (52.69%) in the intestinal digestion. Peptidomics and immunoinformatic analysis further confirmed that PL-10 disrupted 18/30, 15/44, and 37/75 epitopes in the α, α′, and β subunits of β-conglycinin, respectively. These results suggested that lecithin changed the cleavage preferences of β-conglycinin, and promoted the disruption of allergic epitopes, specifically for the β subunit. The study can contribute to our understanding of the role of lecithin in the digestion properties and immunoreactivity of allergenic protein.

A molecular genetic toolkit for the differential expression analysis of soybean β-conglycinin subunit genes

The majority of proteins in soybean seeds are storage ones, including β-conglycinin and glycinin, which are necessary for seed germination. At the same time, they are the most valuable soy proteins used in the food industry, since their subunit composition and proportion of total protein can affect the quality of the resulting food product. β-conglycinins are trimers with different composition of subunits which are designated as α', α, β and encoded by the CG-1, CG-3, and CG-4 genes, respectively. The PCR analysis employed a model soybean cultivar ‘Sentyabrinka’. A complementary DNA synthesized from the RNA isolated from seeds of the studied cultivar served as a template. The in silico created pairs of primers for CG-1, CG-3, and CG-4 gene transcripts were used. As the result of PCR and the analysis of the obtained electrophoregrams, optimal annealing temperatures of primers for the CG-1, CG-3 and CG-4 genes were selected, at which only the characteristic fragment was observed. Thus, a molecular genetic toolkit has been developed for a comprehensive study of the qualitative and quantitative composition of soybean protein and can be used for further analysis of differential expression of genes responsible for the synthesis of β-conglycinin subunits.

Comparative study of interaction and complex formation of Tremella fuciformis polysaccharide with whey protein, lentil protein, and soy protein isolates

The electrostatic interactions and complex formation of Tremella fuciformis polysaccharide (TPS) with whey protein (WPI), lentil protein (LPI), and soy protein (SPI) isolates were studied. The molecular size and components in each protein isolate complexed with TPS were characterized, along with the structural changes in the protein components, induced by TPS complexation. The stability of emulsions prepared with protein:TPS complexes was also evaluated (pH 4.5). Compared to WPI, the LPI and SPI protein components exhibited more negative charges and larger molecular sizes. Independent of the isolate type, pHc of all protein:TPS systems remained unchanged and pHɸ1 shifted to higher pHs as the protein/TPS ratio increased; the pHopt of WPI:TPS and SPI:TPS occurred at higher values as the protein/TPS ratio increased, while the pHopt of LPI:TPS system was less dependent on the biopolymer mixing ratio, probably due to the lower solubility of LPI. Protein components such as β-LG in WPI, convicilin, vicilin, and basic legumin in LPI, and α’ subunits of 7S β-conglycinin and acidic subunits of 11S glycinin in SPI might be the main components interacting with TPS for complex formation. Different proteins exhibited distinct trends in changing their secondary structural elements when interacting with TPS. At pH 4.5, a high proportion of TPS increased the stability of protein emulsion, regardless of the protein used. The WPI:TPS emulsions with low content of TPS were highly destabilized due to the formation of insoluble WPI:TPS complex coacervates, a phenomenon not observed in emulsions stabilized by the LPI:TPS or SPI:TPS systems.

Generation of New β-Conglycinin-Deficient Soybean Lines by Editing the lincRNA lincCG1 Using the CRISPR/Cas9 System.

Soybean β-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in β-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean β-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the β-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans-acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and β-subunits of soybean β-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1, and consequently, the β-conglycinin-deficient phenotype associated with the lincCG1-gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and β-conglycinin-deficient homozygous mutant lines were obtained in the T1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein β-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the β-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

Roles of soybean β-conglycinin subunit fractions in fibril formation and the effects of glycinin on them

This research is aimed at exploring the fibrillation of soybean β-conglycinin (7S) subunits (αα′ and β) at 90 °C and pH 2.0, as well as the effects of glycinin (11S) on them. Fibril formation kinetics was monitored by measuring changes in thioflavin T (Th T) fluorescence intensity of the proteins. This analysis showed that αα′ subunits had a higher fibrillation capacity but slower fibril formation rate than the β subunit. Glycinin accelerated the rate of fibrillation of the αα′ and β subunits but weakened the fibril structure. The size of the fibrils first decreased and then increased with increasing heating time. Circular dichroism and intrinsic fluorescence spectroscopy analysis indicated that the β-sheet structure and hydrophobic interactions were important for the generation of ordered fibrils. Shear viscosity and transmission electron microscopy analysis suggested that the αα′ subunits formed relatively rigid fibrils that led to a high solution viscosity. In contrast, β subunit fibrils became unstable after prolonged heating, forming shorter fibrils (fragments) that tended to aggregate, which led to a lower solution viscosity. This research also suggested that the extension region of αα′ subunits played an important role in fibrillation, and the presence of the highly hydrophobic basic polypeptides of glycinin might be the main reason for the inhibition of soy protein fibrillation. Our analysis of soy protein fibrillization at the subunit level provides insights that might enhance the future application of soy protein fibrils as functional ingredients in foods.

Mathematical modeling of optimal coagulant dosage for tofu preparation using MgCl2

To explore the association between the optimal coagulant for tofu and the components of soybeans,30 different kinds of soybeans were selected, and tested for their optimal coagulant MgCl2 content. The optimal amount of coagulant was taken as the dependent variable, and the soybean Composition were taken as independent variables for the correlation analysis. The results showed that there was a positive correlation between the optimal coagulant content and the content of histidine, 7S β-conglycinin, B1aB1bB2B3B4 of 11 s glycincin, and α'-subunit of 7S β-conglycinin, negative correlation with lysine. The regression formula is y = -1.186 + 3.457*B1aB1bB2B3B4 + 2.304*7S + 0.351*histidine − 0.084*lysine + 4.696*α', and the model is validated to be within 10 % of the error value and has a high degree of confidence. This study provides theoretical support for realizing the green production of traditional soybean products.

Mutation of storage protein gene using CRISPR/Cas9 removed α′-subunit of β-conglycinin in soybean seeds

Mutation of storage protein gene using CRISPR/Cas9 removed α′-subunit of β-conglycinin in soybean seeds

Effects of soybean β-conglycinin subunits composition on its emulsifying properties and the mechanism

In the soybean β-conglycinin system, subunit composition has an important influence on its emulsification properties. To elucidate the mechanism of subunit action in the emulsification process, the separated and purified β-subunit and the mixture α- and α′-subunits (referred to αα′-subunit) were remixed in varying proportions, and then the physicochemical properties and structural properties of the subunit emulsifying system were characterized. The results demonstrated that when the ratio of αα'-subunit to β-subunit was 5:5, the subunit aqueous dispersion system exhibited a minimum particle size of 47.58 nm, a maximum solubility of 87.21%, and the emulsification stability and emulsification activity were also optimal. Scanning electron microscope (SEM) images revealed excellent cross-linking between αα′-subunit and β-subunit, forming a protein network structure that provided a structural basis for its emulsification. The emulsion with a subunit ratio of 5:5 exhibited the highest fraction of interfacial protein adsorption of 23.43% and the largest interfacial protein concentration of 11.79 mg/m2. Inter-subunit adsorption and emulsion formation were primarily driven by hydrophobic interactions, with the β-subunit exhibiting a higher affinity for adsorption than the αα′-subunit. The action mechanism of the subunits in the emulsion formation was proposed as a “three-stage” process, including establishing an initial weak protein network structure within the aqueous dispersion, followed by developing an emulsion interface layer and ultimately culminating in forming an emulsion gel network structure. Elucidating the emulsifying mechanism at the subunit level is crucial for enhancing the theoretical framework of soybean protein emulsification.

Influence of pH and ionic strength on the physicochemical and structural properties of soybean β-conglycinin subunits in aqueous dispersions

Understanding the impact of pH and ionic strength on the physicochemical and structural properties of soy proteins at subunit level is essential for design and fabrication of many plant-based foods. In this study, soybean β-conglycinin and its subunit fractions αα′ and β were dispersed in solutions with different pH values (3.7, 7.6, and 9.0) at low (5 mM NaCl) and high (400 mM NaCl) ionic strengths, respectively. The solubility, rheology, particle size, zeta potential, microstructure, secondary structure, and tertiary structure of the different dispersions were analyzed using a range of analytical methods. The β-conglycinin, αα′- and β-subunits aggregated near the isoelectric point (pH 3.7). Increasing the ionic strength led to the assembly of more homogeneous units. An increase in ionic strength at pH 7.6 and pH 9.0 led to electrostatic screening, which promoted dissociation of the aggregates. The β-subunit showed a greater sensitivity to pH and ionic strength than the αα′-subunits. Based on the evidence from a range of analytical methods, the highly hydrophilic extension region of the αα′-subunits played an important role in determining the stability of the β-conglycinin dispersions under different environmental conditions. Moreover, the N-linked glycans appeared to impact the conformation and aggregation state of the β-conglycinin.

Simultaneous detection of glycinin and β-conglycinin in processed soybean products by high-performance liquid chromatography-tandem mass spectrometry with stable isotope-labeled standard peptides

Glycinin and β-conglycinin are the two main allergic proteins in soybean. Due to their complex structures and lack of protein standards, it is difficult to achieve quantitative determination of these proteins in soybeans. In this study, an HPLC-MS/MS method was developed for the simultaneous determination of five subunits of glycinin (G1, G2, G3, G4, and G5) and three subunits of β-conglycinin (α, α', and β) in processed soybean products based on 8 specific peptides and their stable isotope-labeled peptides. Here, each specific peptide was derived from one of the above 8 subunits. When soy protein was extracted and digested with trypsin, 8 specific peptides, and corresponding stable isotope-labeled peptides were analyzed by HPLC-MS/MS. The linear range for the specific peptides was between 3.2 and 1000 ng/mL (R2 > 0.9955). The recoveries of added peptides ranged from 83.4% to 117.8%, and the intra-day precisions (% CV) were below 17.4%. The limit of quantification of each subunit of glycinin and β-conglycinin in processed soybean products (in terms of protein amount) was between 15.1 and 156.1 g/g. This method was successfully applied to the analysis of 8 subunits of glycinin and β-conglycinin in 68 different processed soybean products, which provides technical support for processed product quality evaluation and monitoring soybean processing technology.

Structure, physicochemical and functional properties of plant-based SPI films from different soybean varieties deficient in β-conglycinin subunits

This study discusses the effects of different β-conglycinin subunit compositions on the processing properties of plant-based soy protein isolate (SPI) films. SPI from different soybean varieties, deficient in different β-conglycinin subunits (Wild, α'-lack, α-lack, and (α + α')-lack) were studied. The effects of α and α′ subunits on the structure and properties of SPI films were analyzed by circular dichroism, intrinsic emission fluorescence, and surface properties. α′-lack films had the lowest water solubility and water vapor transmission coefficient, as well as the best thermal stability and optical properties. This study demonstrates that eliminating the α′ subunit affects microstructure and rheological properties of SPI films by changing the internal structural conformation of the protein. This study also provides a theoretical basis for targeted improvements in the mechanical properties, water resistance, and thermal stability of the films.

In Silico Analysis of Protein of Milk, Soybean, and Kefir as Anti- Thrombotic Bioactive Peptide

Currently, Cardiovascular Diseases (CVDs) cases are increasing significantly in Indonesia and the world. WHO reported that in 2019-related CVD patients accounted for 85%, which increased to 87% in 2021. The cases increased significantly by 2%, which is a significant number as it covered the world population. Stroke is a disease affected by platelets undergoing thrombolysis by the α-thrombin (IIa) receptor with coagulation factors on the heart blood vessel. Chemical or synthesis drugs to treat thrombolytics such as heparin are already available, but they would be harmful if misused. In this study, we focused on the activity, interaction, and prediction of the physicochemical of anti-thrombotic peptides. We found that k-casein from kefir has the highest activity of 0.037 A based on BIOPEP analysis with 17 peptide libraries and molecular docking visualization analysis using PyMol, PyRx, and Discovery Studio for screening the best peptide. ToxinPred and AlgPred showed there are no potentially harmful peptides, but the protein variant of β-conglycinin subunit can be an allergen known to the immune system IgE. In conclusion, the GPR peptide has the highest binding affinity, with a total energy of -6.8 kcal/mol.

In Silico Approach of Glycinin and Conglycinin Chains of Soybean By-Product (Okara) Using Papain and Bromelain.

This study explores utilization of a sustainable soybean by-product (okara) based on in silico approach. In silico approaches, as well as the BIOPEP database, PeptideRanker database, Peptide Calculator database (Pepcalc), ToxinPred database, and AllerTop database, were employed to evaluate the potential of glycinin and conglycinin derived peptides as a potential source of bioactive peptides. These major protein precursors have been found as protein in okara as a soybean by-product. Furthermore, primary structure, biological potential, and physicochemical, sensory, and allergenic characteristics of the theoretically released antioxidant peptides were predicted in this research. Glycinin and α subunits of β-conglycinin were selected as potential precursors of bioactive peptides based on in silico analysis. The most notable among these are antioxidant peptides. First, the potential of protein precursors for releasing bioactive peptides was evaluated by determining the frequency of occurrence of fragments with a given activity. Through the BIOPEP database analysis, there are several antioxidant bioactive peptides in glycinin and β and α subunits of β-conglycinin sequences. Then, an in silico proteolysis using selected enzymes (papain, bromelain) to obtain antioxidant peptides was investigated and then analyzed using PeptideRanker and Pepcalc. Allergenic analysis using the AllerTop revealed that all in silico proteolysis-derived antioxidant peptides are probably nonallergenic peptides. We also performed molecular docking against MPO (myeloperoxidases) for this peptide. Overall, the present study highlights that glycinin and β and α subunits of β-conglycinin could be promising precursors of bioactive peptides that have an antioxidant peptide for developing several applications.

Structure and rheology of foams stabilized by different soybean varieties deficient in β-conglycinin subunits trimers

The need to replace animal protein with plant-based protein for foam formation is growing rapidly based on a variety of factors including sustainable environmental development, biosecurity, and limited option for vegetarians, which decreases the overall status of foam. The structure and rheology of foams made from soy protein isolate (SPI) deficient in β-conglycinin subunits (α′-lack SPI, α-lack SPI, and (α+α′)-lack) were explored in this study. Comparison of microstructure and the foaming characteristics of α′-lack SPI with those of Wild, α-lack SPI, and (α+α′)-lack SPI. Similarly, the microstructure demonstrated that different compositions of β-conglycinin subunits have potential influence on the secondary structure and density of SPI. The α′-lack SPI structure was closest and orderly arranged. The microstructure and foaming properties of α′-lack SPI were better than other SPI, which indicated that the α subunit in α′-lack SPI could generate fine bubbles with high stability. The interfacial shear rheological index of α′-lack SPI was also highest, so subunits may be related to the firmness of the air-water interface, foam rheology was shown to correlate not only to interfacial tension but also to the protein solutions' interfacial elasticity. (α+α′)-lack SPI has the lowest subunit content, foaming ability, and foam stability. This work was helpful to understand the air-liquid behavior of different subunit deficient SPI in β-conglycinin. It lays the foundation for the production of “plant active agents” with superior foaming properties based on soy protein.

Structure, Physicochemical and Functional Properties of Plant-Based SPI Films from Different Soybean Varieties Deficient in Β-Conglycinin Subunits

Structure, Physicochemical and Functional Properties of Plant-Based SPI Films from Different Soybean Varieties Deficient in Β-Conglycinin Subunits

Insights into the mechanism on Glucono-delta-lactone induced gelation of soybean protein at subunit level

To elucidate the gelation process and gel properties of coagulation in soybean protein at subunit level, the dispersions of subunits (αα′- and β-subunit) of β-conglycinin and polypeptides (acidic and basic polypeptides) of glycinin were coagulated by Glucono-delta-lactone (GDL), and the gelation processes were monitored with a dynamic rheometer. The rheological properties, microstructure, water distribution, and secondary structure of the gels were determined as well. The results showed that the β-subunit and basic polypeptide coagulated faster, while αα′-subunit and acidic polypeptide developed firmer and finer gels, who had stronger ability of water holding and contained higher content of β-sheet structure. The GDL-induced subunits/polypeptides gel network were dominated by hydrophobic interactions and disulfide bonds. It is proposed that the mechanism on gelation of soybean protein at subunit level as: with acidification of GDL, the basic polypeptide and β-subunit initially aggregate and form a proto-gel matrix. Subsequently, with the developing of gelation, α- and α′-subunits, especially acidic polypeptide, who is the last one to coagulate, complements to form finer and firmer network, and finally grows together to a mature gel. Besides the gelation rate, the higher content of β-sheet secondary structure and better water holding ability of acidic polypeptide and αα′-subunits would also contribute to the firmness of gel. The elucidation of the gelation mechanism at subunit level would support innovation of gelling technique and benefit the soybean industry.

Saltiness-Enhancing Peptides Isolated from the Chinese Commercial Fermented Soybean Curds with Potential Applications in Salt Reduction.

Salt is very important for human health and food seasoning. Recently, several peptides isolated from natural food products have been reported exhibiting a salty taste or a saltiness-enhancing function. In this investigation, taste-active peptides occurring in commercial Chinese fermented soybean curd were isolated and identified using ultrafiltration, gel permeation chromatography, ion-exchange chromatography, and nano-LC/Q-TOF MS/MS. The salty taste-enhancing function of the target fractions was confirmed by both a rat taste cell model and/or human sensory evaluation. Four decapeptides were found as taste-active compounds. Among them, peptide E (EDEGEQPRPF) was the most potent saltiness-enhancing peptide: 0.4 mg/mL in 50 mmol/L NaCl solution could increase its salty perception equivalent to the salt level of 63 mmol/L NaCl reference solution. The sequence of the peptide has been found in the α'-subunit of β-conglycinin [Glycine max]. The remaining peptides V (VGPDDDEKSW), DD (DEDEQPRPIP), and DG (DEGEQPRPFP) showed umami and kokumi tastes as well as a weak saltiness-enhancing sensation. These findings suggest that the decapeptide EDEGEQPRPF could be a possible alternative to partially reduce the amount of sodium intake without compromising for saltiness.

Development of soybean experimental lines with enhanced protein and sulfur amino acid content

Soybean is the preferred protein source for both poultry and swine feed. However, this preferred status is being challenged due to competition from alternative feed ingredients. To overcome this, it becomes necessary for breeders to develop soybean cultivars that contain higher protein and better nutritional composition. In this study, we have developed experimental soybean lines that not only contain significantly higher amounts of protein but also improved sulfur amino acid content. This objective was achieved by crossing a O-acetylserine sulfhydrylase (OASS) overexpressing transgenic soybean line with elevated levels of sulfur amino acid content (CS) with a high protein Korean soybean cultivar (Lee 5). Introgression of high protein and overexpression of OASS was monitored in the experimental lines at each successive generation (F2-F6) by measuring protein content and OASS activity. The average protein content of transgenic CS and Lee 5 seeds were 34.8 % and 44.7 %, while in the experimental soybean lines the protein content ranged from 41.3 %–47.7 %, respectively. HPLC and inductively coupled plasma-mass spectrometry analyses revealed that all the experimental lines developed in this study contained significantly higher amounts of sulfur containing amino acids and elemental sulfur in the seeds. The sulfur amino acid (cysteine + methionine) content of the experimental lines ranged from 1.1 % to 1.26 % while the parents Lee 5 and CS had 0.79 % and 1.1 %, respectively. SDS-PAGE and western blot analysis demonstrated that the accumulation of Bowman-Birk protease inhibitor and lunasin, two sulfur amino acid rich peptides, were elevated in experimental soybean lines. High-resolution 2D-gel electrophoresis and Delta2D gel analysis validated that an overall increase in the different subunits of 7S β-conglycinin and 11S glycinin were mainly responsible for the observed increase in the total amount of protein in experimental lines.

Preparation of soybean β-conglycinin epitope antibody and its preliminary application in frozen surimi detection

Food allergy has become one of the food safety issues in the world. As a common source of food allergy, soybean poses a dietary risk to allergic individuals due to the presence of β-conglycinin, a main allergenic protein in soybean. Therefore, an efficient and sensitive detection method using highly specific antibody materials is necessary for the detection of allergens. In this study, we have used soybean major allergen β-conglycinin as research object, using analysis software such as DNAStar subroutine Protean, SOPMA server, etc. Three main subunits (α, α′ and β subunits) of β-conglycinin were sequenced. After a series of predictions, screenings and validations, we have obtained the most active epitope peptide with the sequence SNLNFLAFGINAENNQ from the β subunit. This epitope peptide was used to prepare rabbit-derived polyclonal antibodies. Using this antibody, we developed a quantum-dot-based fluorescent lateral flow immunoassay (LFIA) to detect β-conglycinin in frozen surimi. In this detection method, relevant conditions were optimized, and the detection limit was found to be 1.6 mg/g. In conclusion, the epitope peptide SNLNFLAFGINAENNQ was synthesized and using this epitope peptide as the immunogen, rabbit-derived polyclonal antibodies were prepared. A QD-based fluorescent LFIA detection method was developed for a simple, sensitive and specific detection of soybean major allergen β-conglycinin in surimi. This method is beneficial to the healthy development of the frozen surimi industry.

Immunoglobulin E and immunoglobulin G cross-reactive allergens and epitopes between cow milk αS1-casein and soybean proteins

Some infants allergic to cow milk-based formula are also sensitive to soybean-based formula. This paper aimed to explore the association of IgE and IgG cross-reactivity between αS1-casein in cow milk (CM) and soybean proteins. The IgE and IgG cross-reactive allergens and epitopes were identified using sera from infants allergic to CM or mice monoclonal antibodies. The AA sequence alignment was performed using bioinformatics software. Finally, the digestion and heating stability of the cross-reactive allergen were explored by sodium dodecyl sulfate (SDS)-PAGE and Western blotting. The results showed that the IgE and IgG cross-reactive allergen was α subunit of β-conglycinin named Gly m Bd 60K. The IgE and IgG epitopes were the sequences at AA 319-341 and AA 164-182. No intact Gly m Bd 60K allergen could be observed after 2 min in simulated gastric fluid by SDS-PAGE. Heating did not change IgE and IgG cross-reactivity by Western blotting. Therefore, the existence of cross-reactivity between CM αS1-casein and soybean proteins possibly contributes to the frequently observed cosensitization for these allergens in cow milk-allergic patients. The same IgE- and IgG-binding epitopes of cross-reactive allergens may provide important information for elucidation of the association between IgG and IgE antibody generation.