Functional improvements in β-conglycinin by edible bioconjugation with carboxymethyl dextran

β-Conglycinin was conjugated with carboxymethyl dextran (CMD) by the Maillard reaction to improve its function. The β-conglycinin-CMD conjugate was purified by dialysis. Conjugation was confirmed by SDS-PAGE with CBB and PAS staining. Composition of the β-conglycinin-CMD was β-conglycinin:CMD = 1:2.7 (molar ratio) which was confirmed by BCA method and phenol sulfuric acid method. Solubility of β-conglycinin in the range of pH 2.0–7.0 was much improved by conjugation with CMD. Emulsifying property of β-conglycinin at pH 7 and in presence of salt was improved by conjugation with CMD. Immunogenicity of β-conglycinin was reduced by conjugation with CMD. Conjugation method performed in this study was considered to be valuable in that it can be used in food processing.


Introduction
β-Conglycinin is a major soy protein, which represents about 30% of total protein in soybean seeds. β-Conglycinin has high nutritional value and its various functional properties such as emulsifying (Stone and Campbell 1980;Yamauchi et al. 1982;Tang 2017), foaming (Sirison et al. 2021) and gelling properties (Renkema et al. 2001) are well known. Because β-conglycinin contains saccharide chains, β-conglycinin has high affinity to water and contributes to high emulsion stability. In addition to functional properties, it was clarified that β-conglycinin has serum lipid-lowering (Ma et al. 2013) and antiobesity effects (Wanezaki et al. 2020). Although β-conglycinin is a useful protein, it is also known that β-conglycinin is a major antigen of soybean allergy (Cordle 2004;Ogawa et al. 2000;Shan et al. 2021). Soybean is recognized as one of 8 major food allergens in Japan. In addition, solubility and emulsifying properties of β-conglycinin decrease in the acidic pH range. Hence, it is strongly desirable to develop a new method that would lower the allergenicity of β-conglycinin and improve functional properties. We 1 3 Vol:. (1234567890) have been studying the neoglycoconjugates of protein to achieve this. Protein conjugation can simultaneously achieve reduced allergenicity and improved functional properties (such as thermal stability, solubility, and emulsifying ability) while maintaining the physiological functions of proteins (Hattori 2002).
In the present study, we conjugated carboxymethyl dextran (CMD) to β-conglycinin so as to cover the epitopes of β-conglycinin which would lead to reduced immunogenicity, improved solubility and emulsifying property. CMD is an anionic polysaccharide with homogenous sequence and molecular weight. CMD is made by modifying dextran which is composed of D-glucose by β-1,6 bond. CMD shows low antigenicity and immunogenicity. In this study, we used dextran of about 10 kDa which is rather low molecular weight among dextrans. Since CMD is an acidic polysaccharide, conjugation of CMD would bring the shift in pI value and add hydrophilicity and lead to shielding of epitopes.
For conjugation between β-conglycinin and CMD, we used the Maillard reaction. The Maillard reaction is naturally occurring reaction in food and safe reaction. Since the Maillard reaction is a safe reaction, the conjugates between proteins and saccharides are considered to be applicable to actual food. In the present report, we will describe on the preparation of the β-conglycinin-CMD conjugate and improvements in solubility, emulsifying property and immunogenicity of β-conglycinin.
Purification of β-conglycinin β-Conglycinin was isolated from defatted soybean seeds which was a gift from Nisshin OilliO Corporation (Tokyo Japan). Crude β-conglycinin was obtained according to the method of Nagano et al. (1992) and purified by ion-exchange chromatography using a Q Sepharose Fast Flow column (2.5 ID × 50 cm, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The column was equilibrated with 35 mM sodium phosphate buffer (pH 7.6) in advance. Crude β-conglycinin was applied to the column and eluted by a 0.1-0.5 M NaCl linear gradient in a 35 mM sodium phosphate buffer (pH 7.6), and eluted by a 0.5 M NaCl in a 35 mM sodium phosphate buffer (pH 7.6) at a flow rate of 5.0 ml/ min. Eluted protein was detected by the absorbance at 280 nm. After dialysis and lyophilization, purified β-conglycinin was obtained. Purity of β-conglycinin was confirmed by SDS-PAGE.

Preparation of carboxymethyl dextran (CMD)
Dextran was carboxymethylated by applying the method of carboxymethylation for the starch that has been described previously (Hattori et al. 1994). In brief, 1 g of dextran was dissolved in 4.7 mL of 15% monochloroacetic acid solution containing 0.7 g of sodium hydrochloide and then incubated at 40 °C for 48 h. The reaction mixture was neutralized to pH 6.5 with acetic acid after cooling to room temperature. After dialysis against distilled water and lyophilization, carboxymethyl dextran (CMD) was obtained. The degree of modification was determined by hydrochloride-methanol titration (Smith 1967), and it was clarified that about 19 carboxy groups were attached one molecule of dextran.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was carried out using the method of Leammli (1970) with a 15% separating gel and 4% stacking gel. Electrophoresis was carried out at 20 mA constant current, and the gels were stained with CBB and Schiff reagent. Periodic acid Schiff (PAS) staining was carried out under the following conditions: 12.5% trichloroacetic acid was used for fixing proteins in the gel, and 1% orthoperiodic acid was used for oxidation of 1, 2-glycolic groups of carbohydrates. The gel was stained in Schiff reagent in the dark and then washed with 0.5% potassium pyrosulfite.

Preparation of the β-conglycinin-CMD conjugate
The β-conglycinin-CMD conjugate was prepared by the Maillard reaction. At First, β-conglycinin (250.0 mg) and CMD (681.7 mg) was solubilized in 6 M GdnHCl at a protein concentration was 0.5%. The ratio of Lys in β-conglycinin to carboxymethyl residue in CMD was 1:10. And the mixture was dialyzed against distilled water and lyophilization was carried out. Subsequently this lyophilized mixture was incubated at 60 °C at a relative humidity of 79% for 0, 1, 3, 5 or 7 d.
Purification of the β-conglycinin-CMD conjugate After the Maillard reaction, the reaction mixture was dispersed in distilled water at a concentration of 5.0 mg/mL and salted out by adding ammonium sulphate (70% saturation) in 1 h. After standing still for overnight, and then centrifuge at 12,000 rpm at 4 °C for 30 min, supernatant was removed. The precipitate was redissolved in distilled water and dialyzed against distilled water and lyophilized. Obtained lyophilized sample was dissolved in 6 M GdnHCl and dialyzed against 0.1 M imidazole buffer at pH 7.6 and dialyzed against 0.1 M imidazole buffer containing 700 μL/L mercaptoethanol with 100 kDa MWCO dialysis membrane.
Measurement of CMD content in the β-conglycinin-CMD conjugate CMD content in the β-conglycinin-CMD conjugate was quantitated by phenol sulfuric acid method (Dubois et al. 1956) by using CMD as standard.

Evaluation of solubility
Samples (β-conglycinin and the β-conglycinin-CMD conjugate) were stirred in 0.1 M imidazole buffer (pH 2.0-8.0) for 1 h and centrifuged at 18,000 rpm at 4 °C for 20 min. Absorbance of the supernatant was measured at 280 nm.

Evaluation of the emulsifying property
Emulsifying property of the β-conglycinin-CMD conjugate was evaluated by the turbidimetric method (Pearce and Kinsella 1978). Samples were dissolved in a McIlvaine buffer at pH 7.0 or in the buffer containing 0.2 M NaCl. Concentration of β-conglycinin was adjusted to 0.5 mg/mL.
To prepare an oil-in-water emulsion, 2 mL of a protein solution and 0.5 mL of corn oil were homogenized by a Polytron PTA-7 (Kinematica, Switzerland) homogenizer at 24,000 rpm at room temperature for 1 min. A 100 μL aliquot was immediately taken from the bottom of the homogenized emulsion and 50-fold diluted with 0.1% SDS solution, the absorbance was measured at 500 nm by a spectrophotometer. The emulsifying activity was calculated as follows.
[A = A 500 , L =10 -2 m (light path), φ= 0.2 (oil-phase volume fraction)] Immunization Female BALB/c mice at 6 weeks of age (the number of mice was n = 7) were immunized intraperitoneally with β-conglycinin or β-conglycinin-CMD (100 μg as protein) emulsified in Freund's complete adjuvant (Difco Laboratory, MI, USA). 2 weeks after the primary immunization, the mice were boostered with 100 μg of protein emulsified with Freund's incomplete adjuvant (Difco Laboratory). Blood samples were collected from mice seven days after the secondary immunization and stored at 4 °C for 24 h to form a clot. Antisera were collected from each blood sample after clot formation. Mice were sacrificed by cervical dislocation. This study was performed in conformance with the guidelines for the care and use of experimental animals established by the ethics committee of Tokyo University of Agriculture and Technology (R03-186, July 29th, 2021).

Enzyme-Linked immunosorbent assay (ELISA)
β-conglycinin or β-conglycinin-CMD conjugate dissolved in PBS at a protein concentration of 0.01% (100 μL) was added to wells of a polystyrene microtitration plate (Maxisorp, Nunc, Roskilde, Denmark), and the plate was incubated at 4 °C overnight to coat the well with each antigen. After the removal of the solution, each well was washed three times with 200 μL of PBS-Tween (PBS containing 0.05% Tween 20). 125 μL of 1% OVA/PBS solution was added to each well, and the plate was incubated at 25 °C for EAI m 2 ∕g = 2T∕ C T = 2.3 A∕L 2 h, and then the plate was washed three times with 200 μL of PBS-Tween. A 100 μL of diluted antiserum was added and incubated at 25 °C for 2 h, and then the plate was washed three times with 200 μL of PBS-Tween. A 100 μL of alkaline phosphate-labeled rabbit anti-mouse immunoglobulin (Dako A/S Denmark) diluted with PBS-Tween was added to each well, and the plate was incubated at 25 °C for 2 h. After three washings, 100 μL of 0.1% sodium p-nitrophenyl phosphate disodium/diethanolamine hydrochloride buffer (pH 9.8) was added to each well, and the plate was incubated at 25 °C for 30 min. After the addition of 5 M sodium hydroxide solution (20 μL) to each well to stop the reaction, the absorbance at 405 nm was measured with a microplate reader (iMark microplate reader, Bio Rad Laboratories,Inc., California, USA).

Statistical analysis
In the evaluation of solubility, emulsifying property and immunogenicity of β-conglycinin and the β-conglycinin-CMD conjugate, statistical analysis was performed on the obtained results by Student's T-test.

Results and discussion
Preparation and characterization of the β-conglycinin-CMD conjugate β-conglycinin and CMD were conjugated by the Maillard reaction. Formation of the β-conglycinin-CMD conjugate was evaluated by SDS-PAGE (Fig. 1a, and  b). As shown in Fig. 1a and b, high molecular weight species on the upper part of separation gel that were stained by PAS staining emerged as the reaction time increased. And the positions of these bands stained by CBB and PAS were the same and the results indicate that the Maillard reaction products were made. At day 7, the bands were deepest in color which was considered to be progress of the Maillard reaction. However, the sample after 7 days of the Maillard reaction showed low solubility. So, we adopted reaction time to be 5 days. 158.5 mg of the β-conglycinin-CMD conjugate was obtained from 250.0 mg of β-conglycinin and 681.7 mg of CMD. The yield was 17.0%. Saccharide content in the β-conglycinin-CMD conjugate was 14.4%, and the ratio of β-conglycinin to CMD in the conjugate was calculated to be 1:2.7 (molar ratio). The yield on the protein basis was 54.3%. SDS-PAGE pattern of the β-conglycinin-CMD conjugate after purification with dialysis membrane of 100 kDa MWCO is shown in Fig. 1c and d. High molecular weight bands were observed which were considered to be the β-conglycinin-CMD conjugate. Future tasks are improvement in reaction efficiency and establishment of the method to remove unreacted protein.

Improvement in solubility of β-conglycinin by conjugation with CMD
Influence of pH on the solubility of the β-conglycinin-CMD conjugate was evaluated (Fig. 2). The results show that improvements in solubility of the β-conglycinin-CMD in the range of pH 2.0-7.0. In particular, the β-conglycinin-CMD conjugate showed much higher solubility than β-conglycinin at pH 2.0, 4.0, 5.0 and 7.0. These results indicate that the solubility of β-conglycinin was improved by the increase in anionic polar group and hydrophilicity by conjugation with CMD.
Solubility of β-conglycinin is lost around pH 5.0 because isoelectric point of β-conglycinin is near. Although not to the great degree, significant improvement in the solubility was achieved by conjugation with CMD. Improvement in the solubility is considered to be brought about by addition of net charge. More improvement in the solubility at pH 5.0 is expected by adding more net charge. Improvement in emulsifying property of β-conglycinin by conjugation with CMD Emulsifying property of the β-conglycinin-CMD conjugate was evaluated by turbidity method at pH 7.0 and in the presence of salt. Emulsifying ability of the conjugate compared to that of β-conglycinin was evaluated on the basis of the emulsifying activity index (EAI). EAI value of the conjugate was 1.5-fold higher than that of β-conglycinin at pH 7.0 (Fig. 3a).
Emulsifying property of β-conglycinin and β-conglycinin-CMD at pH 7.0 in the presence of 0.2 M NaCl was also evaluated (Fig. 3b). Emulsifying property of the conjugates in the presence of 0.2 M NaCl was 5.8-fold higher than that of β-conglycinin. Improvement in emulsifying property of β-conglycinin after conjugation with CMD was considered to be brought about by addition of hydrophilicity and ion-exchanging ability of CMD. Nagasawa et al. (1996) revealed that increase in polysaccharide content and net charge by conjugation with acidic polysaccharides was effective to improve the emulsifying property of bovine β-lactoglobulin. Their findings indicate that addition of hydrophilicity and net charge is important for the emulsifying property of β-lactoglobulin bioconjugates. In the case of this study, addition of hydrophilicity and net charge by conjugation with CMD is considered to be important for improved emulsifying property of β-conglycinin.
Reduced immunogenicity of β-conglycinin by conjugation with CMD In the experiment for evaluating immunogenicity, mixture of anti-β-conglycinin antisera was used as a standard serum. Standard curve was made with the standard serum and the relative concentration of antibody in the tested antisera was calculated. In Fig. 4, vertical axis indicates the relative concentration of antibody. Immunogenicity of the β-conglycinin-CMD conjugate was evaluated by noncompetitive ELISA in BALB/c mice (Fig. 4a). Immunogenicity of β-conglycinin was dramatically reduced by conjugation with CMD. In addition, the emergence of novel immunogenicity was not observed after conjugation Thick arrows indicate the boundaries between the stacking (upper) and separating (lower) gels with CMD (Fig. 4b). Conjugation with CMD was considered to be an effective method to reduce immunogenicity of β-conglycinin without inducing novel immunogenicity.

Conclusion
In this study, we prepared the β-conglycinin-CMD conjugate with modified function by the Maillard reaction. By this conjugation, solubility of β-conglycinin was improved and the emulsifying property of β-conglycinin at pH 7.0 and in the presence of NaCl were improved. Immunogenicity of β-conglycinin was reduced by this conjugation. Since the conjugation method used in this study is a safe method, this method is very valuable in that it would be applicable for food processing.   Anti-β-conglycinin (a) and anti-β-conglycinin-CMD (b) responses were evaluated by non-competitive ELISA. A significant difference (*: p < 0.05) was determined by Student's T-test