Functional improvements in β-conglycinin by preparing bioconjugates with carboxymethyl cellulose

Functional improvements in β-conglycinin by preparing bioconjugates with carboxymethyl cellulose | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Functional improvements in β-conglycinin by preparing bioconjugates with carboxymethyl cellulose Yui Hataishi, Aya Tanaka, Misaki Ishizuka, Hibine Mizobuchi, Tadashi Yoshida, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4120241/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Nov, 2024 Read the published version in Cytotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract β-Conglycinin was conjugated with carboxymethyl cellulose (CMC) by using water-soluble carbodiimide to improve its function. Two kinds of CMC differing in average molecular weight (about 1 kDa and 90 kDa) were used to investigate the relationship between molecular weight of conjugated saccharide and saccharide content in the conjugates and degree of functional changes in β-conglycinin. The β-conglycinin-CMC conjugates were purified by dialysis using a dialysis membrane whose molecular weight cutoff is 100 kDa. Composition of the β-conglycinin-low molecular weight (LMW) CMC and β-conglycinin-high molecular weight (HMW) CMC was β-conglycinin:CMC = 1:3.3 and 1:2.1 (weight ratio) respectively which was confirmed by BCA method and phenol sulfuric acid method. Conjugation was confirmed by SDS-PAGE with CBB. Solubility of β-conglycinin in the range of pH4.0-7.0 was much improved by conjugation with both LMW and HMW CMC. Emulsifying property of β-conglycinin at pH5.0 and pH7.0 was much improved by conjugation with HMW CMC and greater improvement was achieved by conjugation with LMW CMC. Immunogenicity of β-conglycinin was decreased by conjugation with LMW CMC. β-conglycinin protein conjugation solubility emulsifying property reduced immunogenicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 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 anti-obesity 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 ; 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 have been studying about neoglycoconjugates of proteins to achieve this (Hattori 2002 ). Protein conjugation can simultaneously achieve reduced allergenicity and improved functional properties (such as thermal stability, solubility, and emulsifying property) while maintaining the physiological functions of proteins. In the present study, we conjugated carboxymethyl cellulose (CMC) to β-conglycinin so as to cover the epitopes of β-conglycinin which would lead to reduced immunogenicity and improve solubility and emulsifying property. The concept underlying this study is to reduce allergenicity by shielding the epitope with a low allergenic substance and to improve functional properties such as emulsifying property by binding hydrophilic substances. CMC is an anionic polysaccharide composed of D-glucose by b-1,4 bond (Du et al. 2009 ). It has excellent thickening, water absorbing, and water retaining properties, and is used in a wide range of applications as food additives, feed additives, cosmetics, thickeners, binders and water absorbing and water retaining agents (Kennedy et al. 1995 ). In this study, we used two types of CMC; low molecular weight (1 kDa) CMC (LMW CMC) and high molecular weight (90 kDa) CMC (HMW CMC). Since CMC has carboxymethyl groups and acidic polysaccharide, conjugation of CMC would add hydrophilicity, bring the shift in pI value and lead to shielding of epitopes. We used 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to bind CMC to β-conglycinin. This water-soluble carbodiimide activates carboxy groups to form amide bonds with primary amino groups. Carboxy groups of CMC and free amino groups in b-conglycinin reacts. EDC is characterized by the fact that it is not incorporated into the reaction product and that it can react in water. By the conjugation with saccharide by EDC, functional improvements in a protein would be expected. In the present report, we will describe on the preparation of the β-conglycinin-CMC conjugates and improvements in solubility, emulsifying property and immunogenicity of β-conglycinin. Materials and methods Materials Low molecular weight (LMW) carboxymethyl celluloses (CMC) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and high molecular weight (HMW) CMC was purchased from Sigma Aldrich Co. (St. Luis, USA). Molecular weight of LMW CMC was about 1 kDa and HMW CMC was about 90 kDa. Purification of the β-conglycinin β-Conglycinin was isolated from defatted soybean flakes which were gifts 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 x 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 against distilled water with cellulose membrane (MWCO: 14,000) (Viskase Companies, Inc., IL, USA) and lyophilization with ALPHA 1–2 LDplus lyophilizer (Martin Christ, Osterode, Germany), purified β-conglycinin was obtained. As for dialysis, the ratio of the internal fluid to the external fluid was 1:10, and the external fluid for dialysis was changed 5 times every 3 h. Purity of β-conglycinin was evaluated by SDS-PAGE. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS-PAGE was carried out using the method of Laemmli ( 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. Preparation of the β-conglycinin-CMC conjugate The β-conglycinin-CMC conjugate was prepared by cross-linking reaction with EDC. At First, β-conglycinin and CMC was solubilized in 6M GdnHCl at a protein concentration of 0.1%. After the mixture was dialyzed against 0.9% NaCl solution (pH7.0) at 4°C, EDC dissolved in 0.9% NaCl solution (pH7.0) was added. In the conjugation reaction, 100.0 mg of β-conglycinin and 166.0 mg of LMW CMC were used for preparation of the β-conglycinin-LMW CMC conjugate and 100.0 mg of β-conglycinin and 133.0 mg of HMW CMC were used for preparation of the β-conglycinin-HMW CMC conjugate. 92.0 mg of EDC in 1.0 mL in 0.9% NaCl was gradually added. Reaction ratio was amino groups in b-conglycinin : carboxymethyl groups in CMC : EDC was 1:10:10 (molar ratio). After stirring for 24 h at 4°C, 2M acetate buffer added in the reaction regent to stop the reaction. Purification of the β-conglycinin-CMC conjugate After conjugation, the reaction mixture was dialyzed against distilled water at 4°C to remove EDC and unreacted CMC using a dialysis membrane whose molecular weight cutoff is 100 kDa. After buffer was changed every 3 h 5 times and lyophilization was carried out. Chemical analysis of the β-conglycinin-CMC conjugate s CMC content in the β-conglycinin-CMC conjugate was quantitated by phenol sulfuric acid method (Dubois et al. 1956 ) by using CMC as a standard. Protein content in the β-conglycinin-CMC conjugate was quantitated with BCA Protein Assey Kit (TAKARA, Kusatsu, Japan) by using bovine serum albumin as standard. Evaluation of solubility Samples (β-conglycinin, the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates) were stirred in distilled water. Concentration of β-conglycinin was adjusted to 1 mg/mL. After adjusting pH of the protein solution to 3.0–7.0, the solutions were centrifuged at 72,500 x g at 4°C for 20 min. Protein content of the supernatant was measured by BCA method. Evaluation of the emulsifying property Emulsifying property of the β-conglycinin-CMC conjugates was evaluated by the turbidimetric method (Pearce and Kinsella 1978 ). Samples were dissolved in a McIlvaine buffer at pH 3.0, 5.0, 7.0 or in the buffers 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 50 µL aliquot was taken immediately (0 min) and after 10, 30, 60 and 120 min from the bottom of the homogenized emulsion and 100-fold diluted with 0.1% SDS solution, the absorbance was measured at 500 nm by a spectrophotometer. The emulsion stability was evaluated by the absorbance at 500 nm of the diluted emulsion at 30 min after emulsification. The emulsifying activity index (EAI) was calculated as follows. EAI (m 2 /g) = 2T/φC T = 2.3 A/L (A = A 500 , L = 10 − 2 m (light path), φ = 0.2 (oil-phase volume fraction)) Immunization Through the experiments for evaluation of immunogenicity, sample solutions were made as follows. At first, excessive amount of each sample (β-conglycinin, the β-conglycinin-LMWCMC and β-conglycinin-HMWCMC conjugates) was solubilized in 6M GdnHCl. Then the sample in 6 M GdnHCl was dialyzed against PBS. According to protein concentration of dialyzed solution measured by BCA method, solution was diluted with PBS to adjust to desired concentration. Female BALB/c mice at 5 weeks of age (the number of mice was n = 7) were immunized intraperitoneally 3 times a week (0, 3, 6 day) with sample solutions (100 µg as protein/100 µL) adsorbed to Alum (100 µL) adjuvant (InvivoGen, San Diego, CA, USA). Blood samples were collected from mice seven days after the final immunization and stored at 4°C for 24 h to form a clot (Yoshida et al. 2022 ). 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 (R05-165, July 18th, 2023). Enzyme-Linked Immunosorbent Assay (ELISA) Each sample solutions at a protein concentration of 0.01% (100 µL) were 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% ovalbumin/PBS solution was added to each well, and the plate was 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 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 with 200 µL of PBS-Tween, 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 was performed on the obtained results by Tukey-Kramer multiple range test. Results and discussion Preparation and characterization of the b-conglycinin-CMC conjugates b-conglycinin and CMC were conjugated by cross-linking reaction with EDC, and the formation of b-conglycinin-CMC conjugate was confirmed by SDS-PAGE. As shown in Fig. 1 , new bands appeared on the border of separation gel and stacking gel and on the top of stacking gel. And the CBB stained bands of b-conglycinin disappeared by conjugation. Purification of the b-conglycinin-CMC conjugates was carried out by dialysis using 100 kDa cutoff membrane. 242.8 mg of the β-conglycinin-LMW CMC conjugate was obtained from cross-linking reaction using 100.0 mg of β-conglycinin and 166.0 mg of LMW CMC which was 91.3% yield. 227.3 mg of the β-conglycinin-HMW CMC conjugate was obtained from cross-linking reaction using 100.0 mg of β-conglycinin and 133.0 mg of HMW CMC which was 97.6% yield. Chemical analysis indicated the weight ratio of b-conglycinin to LMW CMC and HMW CMC were about 1:3.3 and 1:2.1 respectively. Improvement in solubility of b-conglycinin by conjugation with CMC Influence of pH on solubility of the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates was evaluated (Fig. 2 ). The results show that improvements in solubility of both β-conglycinin-LMW CMC and β-conglycinin-HMWCMC conjugates in the range of pH 4.0–7.0. Meanwhile, at pH 3.0, solubility of the β-conglycinin-HMW CMC decreased. These results indicate that solubility of β-conglycinin was improved by the increase in anionic polar group and hydrophilic group by conjugation with CMC. Solubility of b-conglycinin decreased around pH 5 because the pH value is close to isoelectric point. In the case of conjugates, isoelectric point shifted to further acidic side and decrease in solubility around pH 5 did not occur. When compared between two conjugates, improvements in solubility were more effective by conjugation with LMW CMC. This difference might be due to the contents of saccharide as anionic polar group and hydrophilic group. Improvement in emulsifying property of b-conglycinin by conjugation with CMC Emulsifying property of the β-conglycinin-CMC conjugates was evaluated by the turbidimetric method at various pHs and in the presence of salt. The effect of pH on emulsifying ability of the conjugates compared to β-conglycinin was evaluated on the basis of the emulsifying activity index (EAI) and emulsion stability. EAI value and emulsion stability value of the conjugates were higher than these of β-conglycinin at pH 5.0 and pH 7.0 (Fig. 3 ). When compared numerically to b-conglycinin, EAI values of the b-conglycinin-HMW CMC conjugate increased 4- to 5-fold, and that of the b-conglycinin-LMW CMC conjugate further increased 8- to 10-fold. Emulsifying property of the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates in the presence of 0.2 M NaCl was also evaluated (Fig. 4 ). At pH 5.0 and pH 7.0, emulsifying property of the conjugates in the presence of 0.2 M NaCl was higher than that of β-conglycinin and as high as that in the absence of NaCl. When compared numerically to b-conglycinin, the EAI values of the b-conglycinin-HMW CMC conjugate increased 5- to 10-fold, and that of the b-conglycinin-LMW CMC conjugate further increased 10- to 30-fold in the presence of 0.2 M NaCl. Since 0.2 M NaCl is similar to salt concentration in food (Hayabuchi et al. 2020 ), the conjugates obtained in this study are valuable for food application. These results indicate that increase in acidic polysaccharide content was effective to improve the emulsifying property of b-conglycinin by conjugation with CMC. Nagasawa et al. ( 1996 ) also revealed that polysaccharide content had a positive correlation with emulsifying property of bovine b-lactoglobulin-carboxymethyl dextran conjugates. Their findings indicate that addition of hydrophilicity and negative charge from acidic polysaccharides was important for emulsifying property of b-lactoglobulin bioconjugates. Similarly, improvement in emulsifying property of b-conglycinin after conjugation with CMC was considered to be brought about by addition of negative charge and hydrophilicity of CMC. Reduced immunogenicity of b-conglycinin by conjugation with CMC In the experiment to evaluate immunogenicity of the β-conglycinin-CMC conjugates, mixture of anti-b-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. 5 , vertical axis indicates the relative concentration of antibody. Immunogenicity of the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates was evaluated by noncompetitive ELISA in BABL/c mice after immunization with Alum adjuvant (Fig. 5 a). Results were expressed as relative IgG amount as compared with mixture of anti-β-conglycinin antisera. Immunogenicity of β-conglycinin was significantly reduced by conjugation with LMW CMC. Shielding of epitopes of β-conglycinin was achieved by conjugation with CMC. And effective shielding was considered to be achieved by conjugation with LMW CMC, which cross-linked with b-conglycinin more effectively than HMW CMC. These results indicate that immunogenicity of b-conglycinin was effectively reduced when higher content of saccharide was conjugated. In addition, the emergence of novel immunogenicity was not observed after conjugation with LMW and HMW CMC (Fig. 5 b, c). Conjugation with CMC was considered to be an effective method to reduce immunogenicity of β-conglycinin without inducing novel immunogenicity. Concluding remarks In this study, we prepared two kinds of β-conglycinin-CMC conjugates differing in average molecular weight of CMC by means of water-soluble carbodiimide. By conjugation, solubility of β-conglycinin was improved and emulsifying property of β-conglycinin in the acidic pH region and in the presence of NaCl was also improved. Immunogenicity of β-conglycinin was reduced by this conjugation. Especially, conjugation with LMW CMC led to a remarkably high functionality and low immunogenicity. Conjugation method used in this study is very valuable for food processing. We hope that our study contributes to the development of new food material with high functionality. Abbreviations CBB Coomassie brilliant blue CMC carboxymethylcellulose EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide MWCO molecular weight cut off HMW high molecular weight LMW low molecular weight SDS-PAGE sodium dodecyl sulfate poly acrylamide gel electrophoresis. Declarations Conflict of Interest Statement The authors have no conflicts of interest to declare. Author Contribution YH, AT, MI, HM: Data curation, Writing manuscript, TY, MH: Conceptualization, Writing manuscript. Acknowledgments We thank Nisshin OilliO Corporation (Tokyo Japan) for presenting defatted soybean flakes. 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Biosci Biotechnol Biochem, 86:390-396. https://doi.org/1093/bbb/zbab220 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 21 Nov, 2024 Read the published version in Cytotechnology → Version 1 posted Editorial decision: Revision requested 17 Sep, 2024 Reviews received at journal 17 Sep, 2024 Reviewers agreed at journal 08 Aug, 2024 Reviews received at journal 11 Jun, 2024 Reviewers agreed at journal 03 Jun, 2024 Reviewers invited by journal 02 Jun, 2024 Submission checks completed at journal 18 Mar, 2024 Editor assigned by journal 18 Mar, 2024 First submitted to journal 18 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4120241","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":280957971,"identity":"4c3ff59b-0958-4764-9619-2f6d4bc9ff7f","order_by":0,"name":"Yui Hataishi","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yui","middleName":"","lastName":"Hataishi","suffix":""},{"id":280957972,"identity":"6451c158-c8cf-4a6a-a8b1-5d4b1f515784","order_by":1,"name":"Aya Tanaka","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Aya","middleName":"","lastName":"Tanaka","suffix":""},{"id":280957973,"identity":"4900fc22-e173-407a-ab20-9872a9788e5f","order_by":2,"name":"Misaki Ishizuka","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Misaki","middleName":"","lastName":"Ishizuka","suffix":""},{"id":280957974,"identity":"2f60c6c0-177a-4fce-b101-ee732a1eae2b","order_by":3,"name":"Hibine Mizobuchi","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hibine","middleName":"","lastName":"Mizobuchi","suffix":""},{"id":280957975,"identity":"e7057949-8b4c-4724-a646-a8844f4ecac2","order_by":4,"name":"Tadashi Yoshida","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Tadashi","middleName":"","lastName":"Yoshida","suffix":""},{"id":280957976,"identity":"d1646209-a8cc-4c4a-8a40-1edd71345963","order_by":5,"name":"Makoto Hattori","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYFACNhBhgyKUQFjLAYY0IIMZrpgoLYdRtOAHuu3HEh9/qDgvz9/Af0zi5w8GIIPh2QN8WszOpB02OHDmtuGMA8xskj0JDEAGQ7oBXi0H0tskDrbdTjBgYGaT4ElgYNzAwJAmgVfL+ecgLefAWiT/JDDYE9ZyI+0YUMsBsBZpoC2JRGh5lmxw5kyy4YzDzMbWMmkSyTMOE/LL+TTDBxUVdvL87Y0Pb76xsbHtb+9Je4BPCwIwg0mgk5h50ojTgQTYj5GsZRSMglEwCoY1AAATaERlFyq9dwAAAABJRU5ErkJggg==","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":true,"prefix":"","firstName":"Makoto","middleName":"","lastName":"Hattori","suffix":""}],"badges":[],"createdAt":"2024-03-18 05:48:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4120241/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4120241/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10616-024-00664-9","type":"published","date":"2024-11-21T15:57:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53112997,"identity":"e9fcf18e-4459-4a48-aff5-c454434d109e","added_by":"auto","created_at":"2024-03-20 18:13:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":241490,"visible":true,"origin":"","legend":"\u003cp\u003eConfirmation of conjugation between β-conglycinin and CMC.\u003c/p\u003e\n\u003cp\u003eM: molecular weight marker, 1: β-conglycinin, 2: β-conglycinin-LMW CMC conjugate, 3: β-conglycinin-HMW CMC conjugate\u003c/p\u003e\n\u003cp\u003eA thick arrow indicates the boundary between the stacking (upper) and separating (lower) gels.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4120241/v1/62b77c17328bbae6b93ea903.jpg"},{"id":53113001,"identity":"3560220f-ba4e-48f0-acd3-240987fc4272","added_by":"auto","created_at":"2024-03-20 18:13:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":201145,"visible":true,"origin":"","legend":"\u003cp\u003eSolubility of the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates.\u003c/p\u003e\n\u003cp\u003e●, β-conglycinin; △ , β-conglycinin-LMW CMC; 〇, β-conglycinin-HMW CMC.\u003c/p\u003e\n\u003cp\u003eFor each sample, means not followed by the same letter are significant different at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 level of significance, according to Tukey-Kramer multiple range test.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4120241/v1/0738e866eac2c162c2f65c09.jpg"},{"id":53112998,"identity":"59786e49-bef4-45c1-a0e5-3471feac9de2","added_by":"auto","created_at":"2024-03-20 18:13:56","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":262841,"visible":true,"origin":"","legend":"\u003cp\u003eEmulsifying property of the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates at various pHs.\u003c/p\u003e\n\u003cp\u003e(a) EAI; (b) Emulsion stability 30 minutes after emulsification.\u003c/p\u003e\n\u003cp\u003e●, β-conglycinin; △ , β-conglycinin-LMW CMC; 〇, β-conglycinin-HMW CMC. \u0026nbsp;\u0026nbsp;For each sample, means not followed by the same letter are significant different at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 level of significance, according to Tukey-Kramer multiple range test.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4120241/v1/8a4787a22f3ff9b57cac88a1.jpg"},{"id":53113000,"identity":"ff069c6b-0779-45ba-a653-046adbb9ebb7","added_by":"auto","created_at":"2024-03-20 18:13:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":261082,"visible":true,"origin":"","legend":"\u003cp\u003eEmulsifying property of the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates in the presence of 0.2 M NaCl.\u003c/p\u003e\n\u003cp\u003e(a) EAI; (b) Emulsion stability 30 minutes after emulsification.\u003c/p\u003e\n\u003cp\u003e●, β-conglycinin; △ , β-conglycinin-LMW CMC; 〇, β-conglycinin-HMW CMC. For each sample, means not followed by the same letter are significant different at \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 level of significance, according to Tukey-Kramer multiple range test.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4120241/v1/c7a0b7231599e1ca39fa9574.jpg"},{"id":53113002,"identity":"cb6f46a5-aa4d-4f65-a57d-3fbd619590bb","added_by":"auto","created_at":"2024-03-20 18:13:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":318598,"visible":true,"origin":"","legend":"\u003cp\u003eImmunogenicity of the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates in BALB/c mice.\u003c/p\u003e\n\u003cp\u003eAnti- β-conglycinin (a), anti- β-conglycinin-LMW CMC (b) and anti- β-conglycinin-HMW CMC responses (c) were evaluated by non-competitive ELISA. \u0026nbsp;Markers indicate individual data and bar graphs indicate average values. A significant difference (*:\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) was determined by Tukey-Kramer multiple range test.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4120241/v1/06d1b912c4262f78a45c02e1.jpg"},{"id":69834989,"identity":"644066b1-bb8d-4f4c-9be9-aae70286cbff","added_by":"auto","created_at":"2024-11-25 16:11:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1749608,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4120241/v1/8fbd4635-cd69-4fe6-bd3d-c12b3d0e2e7f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eFunctional improvements in β-conglycinin by preparing bioconjugates with carboxymethyl cellulose\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eβ-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 \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Yamauchi et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Tang \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), foaming (Sirison et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and gelling properties (Renkema et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) 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. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and anti-obesity effects (Wanezaki et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although β-conglycinin is a useful protein, it is also known that β-conglycinin is a major antigen of soybean allergy (Cordle \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Shan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 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 have been studying about neoglycoconjugates of proteins to achieve this (Hattori \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Protein conjugation can simultaneously achieve reduced allergenicity and improved functional properties (such as thermal stability, solubility, and emulsifying property) while maintaining the physiological functions of proteins.\u003c/p\u003e \u003cp\u003eIn the present study, we conjugated carboxymethyl cellulose (CMC) to β-conglycinin so as to cover the epitopes of β-conglycinin which would lead to reduced immunogenicity and improve solubility and emulsifying property. The concept underlying this study is to reduce allergenicity by shielding the epitope with a low allergenic substance and to improve functional properties such as emulsifying property by binding hydrophilic substances.\u003c/p\u003e \u003cp\u003eCMC is an anionic polysaccharide composed of D-glucose by b-1,4 bond (Du et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). It has excellent thickening, water absorbing, and water retaining properties, and is used in a wide range of applications as food additives, feed additives, cosmetics, thickeners, binders and water absorbing and water retaining agents (Kennedy et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). In this study, we used two types of CMC; low molecular weight (1 kDa) CMC (LMW CMC) and high molecular weight (90 kDa) CMC (HMW CMC). Since CMC has carboxymethyl groups and acidic polysaccharide, conjugation of CMC would add hydrophilicity, bring the shift in \u003cem\u003epI\u003c/em\u003e value and lead to shielding of epitopes.\u003c/p\u003e \u003cp\u003eWe used 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to bind CMC to β-conglycinin. This water-soluble carbodiimide activates carboxy groups to form amide bonds with primary amino groups. Carboxy groups of CMC and free amino groups in b-conglycinin reacts. EDC is characterized by the fact that it is not incorporated into the reaction product and that it can react in water.\u003c/p\u003e \u003cp\u003eBy the conjugation with saccharide by EDC, functional improvements in a protein would be expected. In the present report, we will describe on the preparation of the β-conglycinin-CMC conjugates and improvements in solubility, emulsifying property and immunogenicity of β-conglycinin.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials \u003c/h2\u003e \u003cp\u003eLow molecular weight (LMW) carboxymethyl celluloses (CMC) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and high molecular weight (HMW) CMC was purchased from Sigma Aldrich Co. (St. Luis, USA). Molecular weight of LMW CMC was about 1 kDa and HMW CMC was about 90 kDa.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePurification of the β-conglycinin\u003c/b\u003e \u003c/p\u003e \u003cp\u003eβ-Conglycinin was isolated from defatted soybean flakes which were gifts from Nisshin OilliO Corporation (Tokyo, Japan). Crude β-conglycinin was obtained according to the method of Nagano et. al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) and purified by ion-exchange chromatography using a Q Sepharose Fast Flow column (2.5 ID x 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\u0026ndash;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 against distilled water with cellulose membrane (MWCO: 14,000) (Viskase Companies, Inc., IL, USA) and lyophilization with ALPHA 1\u0026ndash;2 LDplus lyophilizer (Martin Christ, Osterode, Germany), purified β-conglycinin was obtained. As for dialysis, the ratio of the internal fluid to the external fluid was 1:10, and the external fluid for dialysis was changed 5 times every 3 h. Purity of β-conglycinin was evaluated by SDS-PAGE.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)\u003c/h3\u003e\n\u003cp\u003eSDS-PAGE was carried out using the method of Laemmli (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1970\u003c/span\u003e) 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.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of the β-conglycinin-CMC conjugate\u003c/h2\u003e \u003cp\u003eThe β-conglycinin-CMC conjugate was prepared by cross-linking reaction with EDC. At First, β-conglycinin and CMC was solubilized in 6M GdnHCl at a protein concentration of 0.1%. After the mixture was dialyzed against 0.9% NaCl solution (pH7.0) at 4\u0026deg;C, EDC dissolved in 0.9% NaCl solution (pH7.0) was added. In the conjugation reaction, 100.0 mg of β-conglycinin and 166.0 mg of LMW CMC were used for preparation of the β-conglycinin-LMW CMC conjugate and 100.0 mg of β-conglycinin and 133.0 mg of HMW CMC were used for preparation of the β-conglycinin-HMW CMC conjugate. 92.0 mg of EDC in 1.0 mL in 0.9% NaCl was gradually added. Reaction ratio was amino groups in b-conglycinin : carboxymethyl groups in CMC : EDC was 1:10:10 (molar ratio). After stirring for 24 h at 4\u0026deg;C, 2M acetate buffer added in the reaction regent to stop the reaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePurification of the β-conglycinin-CMC conjugate\u003c/h2\u003e \u003cp\u003eAfter conjugation, the reaction mixture was dialyzed against distilled water at 4\u0026deg;C to remove EDC and unreacted CMC using a dialysis membrane whose molecular weight cutoff is 100 kDa. After buffer was changed every 3 h 5 times and lyophilization was carried out.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChemical analysis of the β-conglycinin-CMC conjugate\u003c/b\u003e \u003cem\u003es\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCMC content in the β-conglycinin-CMC conjugate was quantitated by phenol sulfuric acid method (Dubois et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1956\u003c/span\u003e) by using CMC as a standard. Protein content in the β-conglycinin-CMC conjugate was quantitated with BCA Protein Assey Kit (TAKARA, Kusatsu, Japan) by using bovine serum albumin as standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of solubility\u003c/h2\u003e \u003cp\u003eSamples (β-conglycinin, the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates) were stirred in distilled water. Concentration of β-conglycinin was adjusted to 1 mg/mL. After adjusting pH of the protein solution to 3.0\u0026ndash;7.0, the solutions were centrifuged at 72,500 x g at 4\u0026deg;C for 20 min. Protein content of the supernatant was measured by BCA method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of the emulsifying property\u003c/h2\u003e \u003cp\u003eEmulsifying property of the β-conglycinin-CMC conjugates was evaluated by the turbidimetric method (Pearce and Kinsella \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). Samples were dissolved in a McIlvaine buffer at pH 3.0, 5.0, 7.0 or in the buffers containing 0.2 M NaCl. Concentration of β-conglycinin was adjusted to 0.5 mg/mL.\u003c/p\u003e \u003cp\u003eTo 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 50 \u0026micro;L aliquot was taken immediately (0 min) and after 10, 30, 60 and 120 min from the bottom of the homogenized emulsion and 100-fold diluted with 0.1% SDS solution, the absorbance was measured at 500 nm by a spectrophotometer. The emulsion stability was evaluated by the absorbance at 500 nm of the diluted emulsion at 30 min after emulsification. The emulsifying activity index (EAI) was calculated as follows.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eEAI (m\u003csup\u003e2\u003c/sup\u003e/g)\u0026thinsp;=\u0026thinsp;2T/φC\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003cp\u003eT\u0026thinsp;=\u0026thinsp;2.3 A/L\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e(A\u0026thinsp;=\u0026thinsp;A\u003csub\u003e500\u003c/sub\u003e, L\u0026thinsp;=\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e m (light path), φ\u0026thinsp;=\u0026thinsp;0.2 (oil-phase volume fraction))\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunization\u003c/h2\u003e \u003cp\u003eThrough the experiments for evaluation of immunogenicity, sample solutions were made as follows. At first, excessive amount of each sample (β-conglycinin, the β-conglycinin-LMWCMC and β-conglycinin-HMWCMC conjugates) was solubilized in 6M GdnHCl. Then the sample in 6 M GdnHCl was dialyzed against PBS. According to protein concentration of dialyzed solution measured by BCA method, solution was diluted with PBS to adjust to desired concentration.\u003c/p\u003e \u003cp\u003eFemale BALB/c mice at 5 weeks of age (the number of mice was n\u0026thinsp;=\u0026thinsp;7) were immunized intraperitoneally 3 times a week (0, 3, 6 day) with sample solutions (100 \u0026micro;g as protein/100 \u0026micro;L) adsorbed to Alum (100 \u0026micro;L) adjuvant (InvivoGen, San Diego, CA, USA). Blood samples were collected from mice seven days after the final immunization and stored at 4\u0026deg;C for 24 h to form a clot (Yoshida et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 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 (R05-165, July 18th, 2023).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e \u003cp\u003eEach sample solutions at a protein concentration of 0.01% (100 \u0026micro;L) were added to wells of a polystyrene microtitration plate (Maxisorp, Nunc, Roskilde, Denmark), and the plate was incubated at 4\u0026deg;C overnight to coat the well with each antigen. After the removal of the solution, each well was washed three times with 200 \u0026micro;L of PBS-Tween (PBS containing 0.05% Tween 20). 125 \u0026micro;L of 1% ovalbumin/PBS solution was added to each well, and the plate was incubated at 25\u0026deg;C for 2 h, and then the plate was washed three times with 200 \u0026micro;L of PBS-Tween. A 100 \u0026micro;L of diluted antiserum was added and incubated at 25\u0026deg;C for 2 h, and then the plate was washed three times with 200 \u0026micro;L of PBS-Tween. A 100 \u0026micro;L 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\u0026deg;C for 2 h. After three washings with 200 \u0026micro;L of PBS-Tween, 100 \u0026micro;L of 0.1% sodium \u003cem\u003ep\u003c/em\u003e-nitrophenyl phosphate disodium/diethanolamine hydrochloride buffer (pH 9.8) was added to each well, and the plate was incubated at 25\u0026deg;C for 30 min. After the addition of 5 M sodium hydroxide solution (20 \u0026micro;L) 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 was performed on the obtained results by Tukey-Kramer multiple range test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and characterization of the b-conglycinin-CMC conjugates\u003c/h2\u003e \u003cp\u003eb-conglycinin and CMC were conjugated by cross-linking reaction with EDC, and the formation of b-conglycinin-CMC conjugate was confirmed by SDS-PAGE. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, new bands appeared on the border of separation gel and stacking gel and on the top of stacking gel. And the CBB stained bands of b-conglycinin disappeared by conjugation. Purification of the b-conglycinin-CMC conjugates was carried out by dialysis using 100 kDa cutoff membrane. 242.8 mg of the β-conglycinin-LMW CMC conjugate was obtained from cross-linking reaction using 100.0 mg of β-conglycinin and 166.0 mg of LMW CMC which was 91.3% yield. 227.3 mg of the β-conglycinin-HMW CMC conjugate was obtained from cross-linking reaction using 100.0 mg of β-conglycinin and 133.0 mg of HMW CMC which was 97.6% yield. Chemical analysis indicated the weight ratio of b-conglycinin to LMW CMC and HMW CMC were about 1:3.3 and 1:2.1 respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImprovement in solubility of b-conglycinin by conjugation with CMC\u003c/h2\u003e \u003cp\u003eInfluence of pH on solubility of the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates was evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results show that improvements in solubility of both β-conglycinin-LMW CMC and β-conglycinin-HMWCMC conjugates in the range of pH 4.0\u0026ndash;7.0. Meanwhile, at pH 3.0, solubility of the β-conglycinin-HMW CMC decreased. These results indicate that solubility of β-conglycinin was improved by the increase in anionic polar group and hydrophilic group by conjugation with CMC. Solubility of b-conglycinin decreased around pH 5 because the pH value is close to isoelectric point. In the case of conjugates, isoelectric point shifted to further acidic side and decrease in solubility around pH 5 did not occur. When compared between two conjugates, improvements in solubility were more effective by conjugation with LMW CMC. This difference might be due to the contents of saccharide as anionic polar group and hydrophilic group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImprovement in emulsifying property of b-conglycinin by conjugation with CMC\u003c/h2\u003e \u003cp\u003eEmulsifying property of the β-conglycinin-CMC conjugates was evaluated by the turbidimetric method at various pHs and in the presence of salt. The effect of pH on emulsifying ability of the conjugates compared to β-conglycinin was evaluated on the basis of the emulsifying activity index (EAI) and emulsion stability.\u003c/p\u003e \u003cp\u003eEAI value and emulsion stability value of the conjugates were higher than these of β-conglycinin at pH 5.0 and pH 7.0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When compared numerically to b-conglycinin, EAI values of the b-conglycinin-HMW CMC conjugate increased 4- to 5-fold, and that of the b-conglycinin-LMW CMC conjugate further increased 8- to 10-fold.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEmulsifying property of the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates in the presence of 0.2 M NaCl was also evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At pH 5.0 and pH 7.0, emulsifying property of the conjugates in the presence of 0.2 M NaCl was higher than that of β-conglycinin and as high as that in the absence of NaCl. When compared numerically to b-conglycinin, the EAI values of the b-conglycinin-HMW CMC conjugate increased 5- to 10-fold, and that of the b-conglycinin-LMW CMC conjugate further increased 10- to 30-fold in the presence of 0.2 M NaCl. Since 0.2 M NaCl is similar to salt concentration in food (Hayabuchi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), the conjugates obtained in this study are valuable for food application.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results indicate that increase in acidic polysaccharide content was effective to improve the emulsifying property of b-conglycinin by conjugation with CMC.\u003c/p\u003e \u003cp\u003eNagasawa et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) also revealed that polysaccharide content had a positive correlation with emulsifying property of bovine b-lactoglobulin-carboxymethyl dextran conjugates. Their findings indicate that addition of hydrophilicity and negative charge from acidic polysaccharides was important for emulsifying property of b-lactoglobulin bioconjugates. Similarly, improvement in emulsifying property of b-conglycinin after conjugation with CMC was considered to be brought about by addition of negative charge and hydrophilicity of CMC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eReduced immunogenicity of b-conglycinin by conjugation with CMC\u003c/h2\u003e \u003cp\u003eIn the experiment to evaluate immunogenicity of the β-conglycinin-CMC conjugates, mixture of anti-b-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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, vertical axis indicates the relative concentration of antibody. Immunogenicity of the β-conglycinin-LMW CMC and β-conglycinin-HMW CMC conjugates was evaluated by noncompetitive ELISA in BABL/c mice after immunization with Alum adjuvant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Results were expressed as relative IgG amount as compared with mixture of anti-β-conglycinin antisera. Immunogenicity of β-conglycinin was significantly reduced by conjugation with LMW CMC. Shielding of epitopes of β-conglycinin was achieved by conjugation with CMC. And effective shielding was considered to be achieved by conjugation with LMW CMC, which cross-linked with b-conglycinin more effectively than HMW CMC. These results indicate that immunogenicity of b-conglycinin was effectively reduced when higher content of saccharide was conjugated. In addition, the emergence of novel immunogenicity was not observed after conjugation with LMW and HMW CMC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c). Conjugation with CMC was considered to be an effective method to reduce immunogenicity of β-conglycinin without inducing novel immunogenicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eConcluding remarks\u003c/h2\u003e \u003cp\u003eIn this study, we prepared two kinds of β-conglycinin-CMC conjugates differing in average molecular weight of CMC by means of water-soluble carbodiimide. By conjugation, solubility of β-conglycinin was improved and emulsifying property of β-conglycinin in the acidic pH region and in the presence of NaCl was also improved. Immunogenicity of β-conglycinin was reduced by this conjugation. Especially, conjugation with LMW CMC led to a remarkably high functionality and low immunogenicity. Conjugation method used in this study is very valuable for food processing. We hope that our study contributes to the development of new food material with high functionality.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCBB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCoomassie brilliant blue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCMC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarboxymethylcellulose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1-ethyl-3-(3-dimethylaminopropyl)carbodiimide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMWCO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emolecular weight cut off\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHMW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh molecular weight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLMW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elow molecular weight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDS-PAGE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esodium dodecyl sulfate poly acrylamide gel electrophoresis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYH, AT, MI, HM: Data curation, Writing manuscript, TY, MH: Conceptualization, Writing manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank Nisshin OilliO Corporation (Tokyo Japan) for presenting defatted soybean flakes.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data underlying this article are available in the article and also from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCordle CT (2004) Soy protein allergy: Incidence and relative severity. J Nutr 134:1213S-1219S. https://doi.org/1093/jn/134.5.1213S\u003c/li\u003e\n\u003cli\u003eDu, B, Li J., Zhang, H, Huang, L, Chen, P, Zhou, J (2009) Influence of molecular weight and degree of substitution of carboxymethylcellulose on the stability of acidified milk drinks. Food Hydrocolloids 23:1420-1426. https://doi.org/10.1016/j.foodhyd.2008.10.004\u003c/li\u003e\n\u003cli\u003eDubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350-356. https://doi.org/10.1021/ac60111a017\u003c/li\u003e\n\u003cli\u003eHayabuchi H, Morita R, Ohta M, Nanri A, Matsumoto H, Fujitani S, Yoshida S, Ito S, Sakima A, Takase H, Kusaka M, Tsuchihashi T (2020) Validation of preferred salt concentration in soup based on a randomized blinded experiment in multiple regions in Japan\u0026mdash;influence of umami (L-glutamate) on saltiness and palatability of low-salt solutions. Hypertension Res 43:525\u0026ndash;533. https://doi.org/1038/s41440-020-0397-1\u003c/li\u003e\n\u003cli\u003eHattori M (2002) Functional improvements in food proteins in multiple aspects by conjugation with saccharides: Case studies of β-lactoglobulin-acidic polysaccharides conjugates. Food Sci Technol Res: 8:291-299. https://doi.org/10.3136/fstr.8.291\u003c/li\u003e\n\u003cli\u003eKennedy JF, Phillips GO, Williams PA, Piculell JL (1995) Cellulose and cellulose derivatives: Cellucon'93 proceedings: Physico-chemical aspects and industrial applications. Woodhead Publishing Ltd., Cambridge\u003c/li\u003e\n\u003cli\u003eLaemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. https://doi.org/10.1038/227680a0\u003c/li\u003e\n\u003cli\u003eMa D, Taku K, Zhang Y, Jia M, Wang Y, Wang P (2013) Serum lipid-improving effect of soyabean β-conglycinin in hyperlipidaemic menopausal women. Br J Nutr 110:1680-1684. https://doi.org/10.1017/S0007114513000986\u003c/li\u003e\n\u003cli\u003eNagano T, Hirotsuka M, Mori, H, Kohyama K, Nishinari K (1992) Dynamic viscoelastic study on the gelation of 7 S globulin from soybeans. J Agric Food Chem 40:941-944. https://doi.org/10.1021/jf00018a004\u003c/li\u003e\n\u003cli\u003eNagasawa K, Ohgata K, Takahashi K, Hattori M (1996) Role of the polysaccharide content and net charge on the emulsifying properties of β-lactoglobulin-carboxymethyl dextran conjugates. J Agric Food Chem 44:2538-2543. https://doi.org/10.1021/jf960150m\u003c/li\u003e\n\u003cli\u003ePearce KN, Kinsella JE (1978) Emulsifying properties of proteins: evaluation of a turbidimetric technique. J Agric Food Chem 26:716-723. https://doi.org/10.1021/jf60217a041\u003c/li\u003e\n\u003cli\u003eRenkema JMS, Knabben JHM, van Vliet T (2001) Gel formation by β-conglycinin and glycinin and their mixtures. Food Hydrocolloids 15:407-414. https://doi.org/10.1016/S0268-005X(01)00051-0\u003c/li\u003e\n\u003cli\u003eShan D, Yu H, Lyu, B, Fu H (2021) Soybean β-Conglycinin: Structure Characteristic, Allergenicity, Plasma Lipid-Controlling, Prevention of Obesity and Non-alcoholic Fatty Liver Disease. Curr Protein Peptide Sci 22:831-847. https://doi.org/2174/1389203722666211202151557\u003c/li\u003e\n\u003cli\u003eStone MB, Campbell AM (1980) Emulsification in systems containing soy protein isolates, salt and starch. J Food Sci 45:1713-1716. https://doi.org/10.1111/j.1365-2621.1980.tb07595.x\u003c/li\u003e\n\u003cli\u003eSirison J, Ishii T, Matsumiya K, Samoto M, Kohno M, Matsumura Y (2021) Comparison of surface and foaming properties of soy lipophilic protein with those of glycinin and β-conglycinin. Food Hydrocolloids 112:106345-56. https://doi.org/10.1016/j.foodhyd.2020.106345\u003c/li\u003e\n\u003cli\u003eTang C (2017) Emulsifying properties of soy proteins: A critical review with emphasis on the role of conformational flexibility. Crit Rev Food Sci Nutr 57:2636-2679. https://doi.org/10.1080/10408398.2015.1067594\u003c/li\u003e\n\u003cli\u003eWanezaki S, Saito S, Inoue N, Tachibana N, Shirouchi B, Sato M., Yanagita T., Nagao K (2020) Soy β-conglycinin peptide attenuates obesity and lipid abnormalities in obese model OLETF rats. J Oleo Sci 69:495-502. https://doi.org/10.5650/jos.ess20010\u003c/li\u003e\n\u003cli\u003eYamauchi F, Ogawa Y, Kamata Y, Shibasaki K (1982) Emulsifying properties of soybean β-conglycinin and glycinin: Evaluation by turbidimetry. Biosci Biotechnol Biochem 46:615\u0026ndash;621. https://doi.org/10.1271/bbb1961.46.615\u003c/li\u003e\n\u003cli\u003eYoshida T, Tanemura M, Shimizu A, Oyon, Tanaka H, Kurokawa S, Takahashi K, Hattori M (2022) Functional improvements in β-lactoglobulin by preparing edible conjugate with microbial transglutaminase. Biosci Biotechnol Biochem, 86:390-396. https://doi.org/1093/bbb/zbab220\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cytotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cyto","sideBox":"Learn more about [Cytotechnology](http://link.springer.com/journal/10616)","snPcode":"10616","submissionUrl":"https://submission.nature.com/new-submission/10616/3","title":"Cytotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"β-conglycinin, protein conjugation, solubility, emulsifying property, reduced immunogenicity","lastPublishedDoi":"10.21203/rs.3.rs-4120241/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4120241/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eβ-Conglycinin was conjugated with carboxymethyl cellulose (CMC) by using water-soluble carbodiimide to improve its function. Two kinds of CMC differing in average molecular weight (about 1 kDa and 90 kDa) were used to investigate the relationship between molecular weight of conjugated saccharide and saccharide content in the conjugates and degree of functional changes in β-conglycinin. The β-conglycinin-CMC conjugates were purified by dialysis using a dialysis membrane whose molecular weight cutoff is 100 kDa. Composition of the β-conglycinin-low molecular weight (LMW) CMC and β-conglycinin-high molecular weight (HMW) CMC was β-conglycinin:CMC = 1:3.3 and 1:2.1 (weight ratio) respectively which was confirmed by BCA method and phenol sulfuric acid method. Conjugation was confirmed by SDS-PAGE with CBB. Solubility of β-conglycinin in the range of pH4.0-7.0 was much improved by conjugation with both LMW and HMW CMC. Emulsifying property of β-conglycinin at pH5.0 and pH7.0 was much improved by conjugation with HMW CMC and greater improvement was achieved by conjugation with LMW CMC. Immunogenicity of β-conglycinin was decreased by conjugation with LMW CMC.\u003c/p\u003e","manuscriptTitle":"Functional improvements in β-conglycinin by preparing bioconjugates with carboxymethyl cellulose","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-20 18:13:51","doi":"10.21203/rs.3.rs-4120241/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-18T00:18:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-17T10:14:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110701647893331041054415728804624685374","date":"2024-08-08T06:54:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-11T14:09:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59848422654763413449436620203081276584","date":"2024-06-03T04:04:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-03T02:13:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-18T14:30:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-18T14:30:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cytotechnology","date":"2024-03-18T05:47:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cytotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cyto","sideBox":"Learn more about [Cytotechnology](http://link.springer.com/journal/10616)","snPcode":"10616","submissionUrl":"https://submission.nature.com/new-submission/10616/3","title":"Cytotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"93bba563-3637-4a4c-9d2b-80909bf30100","owner":[],"postedDate":"March 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T16:03:47+00:00","versionOfRecord":{"articleIdentity":"rs-4120241","link":"https://doi.org/10.1007/s10616-024-00664-9","journal":{"identity":"cytotechnology","isVorOnly":false,"title":"Cytotechnology"},"publishedOn":"2024-11-21 15:57:55","publishedOnDateReadable":"November 21st, 2024"},"versionCreatedAt":"2024-03-20 18:13:51","video":"","vorDoi":"10.1007/s10616-024-00664-9","vorDoiUrl":"https://doi.org/10.1007/s10616-024-00664-9","workflowStages":[]},"version":"v1","identity":"rs-4120241","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4120241","identity":"rs-4120241","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}