HPLC-MS Evaluation of Mannuronic and Guluronic Acid in Bacterial Alginate from Azotobacter vinelandii and the Effect on Glyceollin Induction and Accumulation in Soybeans

Ojokoh Eromosele *

Department of Food Technology, Akanu Ibiam Federal Polytechnic Unwana, P.M.B. 1007, Afikpo, Ebonyi State, Nigeria.

*Author to whom correspondence should be addressed.


Aims: The objective of this study was to investigate the eliciting efficacy of bacterial alginate which was extracted from Azotobacter vinelandii in the induction of glyceollins and accumulation in soybeans.

Methodology: The preparative high performance liquid chromatography (HPLC) was used to detect, separate and purify the alginate after extraction and ultra performance liquid chromatography – mass spectrometry (UPLC-MS) was used to detect the molecular weights of both the extracted alginate and the induced glyceollins in soybeans.

Results: Bacterial alginate induced glyceollins synthesis in soybean seeds in high quantity. The optimal conditions of elicitation were as follows: concentration of bacterial alginate 60 µL presoaked in the sterile water for 5h, incubated at a controlled temperature of 30℃ and in the dark for 4 days. Alginate induction ability exhibited glyceollin-inducing activities of 1.775 (mg/g dry weight) while the control had no record of glyceollin induction.  The results demonstrated that a larger molecular weight or a higher G/M ratio might correlate with a higher glyceollin-induction activity.

Conclusion: In conclusion; alginate could be introduced as relatively safe and efficient elicitor of high glyceollin accumulation in soybeans. The method for bacterial alginate extraction and its application in soybean seeds for glyceollin induction may be of practical use in the food.

Keywords: Glyceollins, alginate, elicitation, accumulation and Azotobacter vinelandii

How to Cite

Eromosele, O. (2023). HPLC-MS Evaluation of Mannuronic and Guluronic Acid in Bacterial Alginate from Azotobacter vinelandii and the Effect on Glyceollin Induction and Accumulation in Soybeans. Asian Food Science Journal, 22(10), 80–91. https://doi.org/10.9734/afsj/2023/v22i10675


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Toomer OT, Oviedo EO, Ali M, Patino D, Joseph M, Frinsko M, Vu T, Maharjan P, Fallen B, Mian R. Current agronomic practices, harvest & post-harvest processing of soybeans (Glycine max)—A Review. Agronomy. 2023;13(2):427.

Jenkins DJ, Mirrahimi A, Srichaikul K, Berryman CE, Wang L, Carleton A, Abdulnour S, Sievenpiper JL, Kendall CW, Kris- Etherton PM. Soy protein reduces serum cholesterol by both intrinsic and food displacement mechanisms. J Nutr. 2010;140:2302–2311.

Boue SM, Carter CH, Ehrlich KC, Cleveland TE. Induction of the soybean phytoalexins coumestrol and glyceollin by Aspergillus. J Agric Food Chem. 2000; 48:2167–2172.

Salvo VA, Boue SM, Fonseca JP, Elliott S, Corbitt C, Collins-Burow BM, Curiel TJ, Srivastav SK, Shih BY, Carterwientjes C. Antiestrogenic glyceollins suppress human breast and ovarian carcinoma tumorigenesis. Clin Cancer Res. 2006;12:7159–7164.

Yoon EK, Jeong YT, Li X, Song-Cui PD, Kim YH, Yong D.K, Chang HW, Lee SH, Hwang SL. Glyceollin improves endoplasmic reticulum stress-induced insulin resistance through CaMKKAMPK pathway in L6 myotubes. J Nutr Biochem. 2013;24:1053–1061

Wood CE, Boue SM, Collins-Burow BM, Rhodes LV, Register TC, Cline JM, Dewi FN, Burow, ME. Glyceollin-elicited soy protein consumption induces distinct transcriptional effects as compared to standard soy protein. J Agric Food Chem. 2012;60:81–86.

Fett, W.F., Zacharius, R.M. Bacterially-induced glyceollin production in soybean cell suspension cultures. Plant Sci Lett. 1982; 24:303–309

Moesta P, Grisebach H. Effects of biotic and abiotic elicitors on phytoalexin metabolism in soybean. Nature. 1980;49:710–711.

Yoshikawa M, Yoshikawa M. Divers mode of action of biotic and abiotic phytoalexin elicitors. Nature. 1978; 275:546–547.

Pitta Alvarez SI, Spollansky TC, Giulietti AM. The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida. Enzyme Microb Technol. 2000; 26:252–258.

Stintzi A, Weber H, Reymond P, Browse J, Farmer EE. Plant defense in the absence of jasmonic acid: the role of cyclopentenones. Proc Natl Acad Sci USA. 2001;98:12837–12842.

Sto¨ssel P. Regulation by sulfhydryl groups of glyceollin accumulation in soybean hypocotyls. Planta. 1984;160:314–319.

Angelova Z, Georgiev S, Roos W. Elicitation of plants. Biotechnol biotechnol equip 2004;20:72–83. Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol. 2006; 55:373–399

Nagasawa N, Mitomo H, Yoshii F, Kume T. Radiation-induced degradation of sodium alginate. Polym Degrad Stabil. 2000; 69:279–285.

Zhang Y, Liu H, Yin H, Wang W, Zhao X, Du Y. Nitric oxide mediates alginate oligosaccharides-induced root development in wheat (Triticum aestivum L.). Plant Physiol Biochem. 2013;71:49–56.

Holtan S, Zhang Q, Strand WI, Skja°k-Braek G. Characterization of the hydrolysis mechanism of polyalternating alginate in weak acid and assignment of the resulting MG-oligosaccharides by NMR spectroscopy and ESI–mass spectrometry. Biomacromolecules. 2006;7:2108–2121.

Jochum M, Bakry R, Wartusch I, Huck CW, Engelhardt H, Bonn GK. Analysis of carbohydrates using different quaternized polystyrene-divinylbenzene particles and pulsed amperometric detection. Chromatographia. 2002; 56:263–268.

Scherz H, Huck CW, Bonn GK. Electrophoresis carbohydrates. encyclopedia of analytical. Science. 2005;28:433–445.

Kawada A, Hiura N, Shiraiwa M, Tajima S, Hiruma M, Hara K, Ishibashi A, Takahara H. Stimulation of human keratinocyte growth by alginate oligosaccharides, a possible cofactor for epidermal growth factor in cell culture. FEBS Lett. 1997;408:43–46.

Kawada A, Hiura N, Tajima S, Takahara H. Alginate oligosaccharides stimulate VEGF-mediated growth and migration of human endothelial cells. Arch Dermatol Res. 1999; 291:542–547.

Akimoto C, Aoyagi H, Dicosmo F, Tanaka H. Synergistic effect of active oxygen species and alginate on chitinase production by Wasabia japonica cells and its application. J Biosci Bioeng. 2000 ;89:131–137.

Chandı´a NP, Matsuhiro B, Mejı´as E, Moenne A. Alginic acids in Lessonia vadosa: partial hydrolysis and elicitor properties of the polymannuronic acid fraction. J Appl Phycol. 2004; 16:127–133.

Iwasaki KI, Matsubara Y. Purification of pectate oligosaccharides showing root-growth-promoting activity in lettuce using ultrafiltration and nanofiltration membranes. J Biosci Bioeng. 2000; 89:495–497.

Ma LJ, Zhang Y, Bu N, Wang SH. Alleviation Effect of alginate-derived oligosaccharides on vicia faba root tip cells damaged by cadmium. Bull Environ Contam Toxicol. 2010;84:161–164

Natsume M, Kamo Y, Hirayama M, Adachi T. Isolation and characterization of alginate-derived oligosaccharides with root growth-promoting activities. Carbohydr Res. 1994;258:187–197.

Zhang S, Tang W, Jiang L, Hou Y, Yang F, Chen W, Li X. Elicitor activity of algino-oligosaccharide and its potential application in protection of rice plant (Oryza saliva L.) against Magnaporthe grisea. Biotechnol Biotechnol Equip. 2015; 29:1–7

Jia H, Shi B, Eromosele O, Liang P, Li J. Effects of alginate oligosaccharides on the accumulation of glyceollins in soybean. China Agric Sci. 2012;45:1576–1586.

Schu¨rks N, Wingender J, Flemming HC, Mayer C. Monomer composition and sequence of alginates from Pseudomonas aeruginosa. Int J Biol Macromol. 2002;30:105–111.

Sime WJ. Alginates in: Harris, P., (Eds.) Food Gels, Elsevier Applied Science Publication, London. 1990;53-78.

Moe ST, Draget KI, Skjik-Brzk G, Smidsrod O. Alginates. In: Stephen AM, (Eds.), Marcel Dekker Food Polysaccharides and Their Applications. E-Publishing Inc., New York. 1995;245-286.

Saude NHC, Lange D, Beunard P, Dhulster D, Guillochon AM, Caze M, Morcellet, Junter GA. Alginate production by Azotobacter vinelandii in a membrane bioreactor. Process Biochemistry. 2002;38: 273-278.

Ertesvag HF, Erlien GS, Braek BHA, Rehm Valla S. Biochemical properties and substrate specificities of a recombinantly produced Azotobacter vinelandii alginate lyase. Journal of Bacteriology. 1998;180:3779-3784.

Skjak-Braek G, Grasdalen H, Larsen L. Monomer sequence and acetylation pattern in some bacterial alginates. Carbohydrate Research. 1986;154:239-250.

Brivonese AC, Sutherland IW. Polymer production by a mucoidstrain of Azotobacter Vinelandii in batch culture. Applied Microbiology and Biotechnology. 1989;30:97–102.

Moresi MI, Sebastiani Wiley DE. Experimental strategy to assess the main engineering parameters characterizing sodium alginate recovery from model solutions by ceramic tubular ultrafiltration membrane modules. Journal of Membrane Science. 2009;26:441-452.

Cheong HG, Paul WS, Lai WC. Alginates as a useful natural polymer for microencapsulation and therapeutic applications. Carbohydrate Polymers. 2011;88,1–12.

Eromosele O, Shi B, Liang P. Induction of phytochemical glyceollins accumulation in soybean following treatment with biotic elicitor (Aspergillus oryzae). J Funct Foods. 2013;5:1039–1048

Clementi F, Crudele MA, Parente E, Mancini M, Moresi M. Production and characterization of alginate from Azotobacter vinelandii. Journal of the Science of Food and Agriculture. 1999;79( 4):602-610.

Byrd MS, Sadovskaya I, Vinogradov E, Lu H, Sprinkle AB, Richardson SH, Ma L, Ralston B, Parsek M R, Anderson E M, Lam JS, Wozniak DJ. Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production. Molecular Microbiology. 2009;73(4):622–638.

Conti E, Flaibani A, O’Regan M, Sutherland I W. Alginate from Pseudomonas fluorescens and P.putida: production and properties. Microbiology. 1994;140: 1125–1132.

Chaki T, Kakimi H, Shibata A, Baba T. Detection of alginate oligosaccharides from mollusks. Biosci Biotechnol Biochem 2006;70:2793–2796.

Zhang Z, Yu G, Zhao X, Liu H, Guan H, Lawson A.M, Chai W. Sequence analysis of alginate-derived oligosaccharides by negative-ion electrospray tandem mass spectrometry. J Am Soc Mass Spectrom. 2006;17:621–630.

Ku¨pper FC, Mu¨ller DG, Peters AF, Kloareg B, Potin P. Oligoalginate recognition and oxidative burst play a key role in natural and induced resistance of sporophytes of Laminariales. J Chem Ecol. 2002;28:2057–2081.

Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol. 2004;55:373–399.

Hai D., Huang Y, Tang Y. Genetic and metabolic engineering of isoflavonoid biosynthesis. Appl Microbiol Biotechnol. 2010; 86:1293–1312.

Landin S, Graham MY, Graham TL. Lactofen induces isoflavone accumulation and glyceollin elicitation competency in soybean. Phytochemistry. 2003;62:865–874.

Peng Q, Zhang M, Gao L, Eromosele O, Qiao Y, Shi B. Effects of alginate oligosaccharides with different molecular weights and guluronic to mannuronic acid ratios on glyceollin induction and accumulation in soybeans. Journal of food science and technology. 2018;55:1850-8.