1073Investigating covalent interactions between 11S glycinin and genistein following thermal treatment

E Smith1*, L Condict1 , J Ashton2, S Kasapis1

1School of Science, RMIT University, Bundoora West Campus, Plenty Road, Melbourne, VIC 3083, Australia
2Sanitarium Development and Innovation, Sanitarium Health and Wellbeing Company, Cooranbong, NSW 2265, Australia

Soy-derived products have been a staple in human diets for centuries owing to their nutritive values including high protein and isoflavone content. Typically, these food products are subject to thermal processing which may modify functional properties and promote chemical interactions with other components in the system such as isoflavones1,2. Soy proteins and isoflavones have been reported to interact non-covalently however, the impact of thermal treatment on the nature of these interactions has not been thoroughly explored3,4. In the present work, the effect of thermal treatment on the molecular interactions between glycinin (11S) and genistein (GNE) at 95oC was explored utilising a variety of spectroscopic techniques and in-silico analysis.

The nature of interactions occurring between 11S and GNE were probed using UV-vis absorbance and MALDI-TOF MS analysis. UV-vis revealed a considerable decrease in the absorption spectrum alongside a spectral shift following ligand addition and heat exposure, suggesting a covalent interaction may have occurred. This was confirmed with MALDI-TOF MS analysis, where a mass shift on the acid group was observed, corresponding to the addition of 6 covalently linked molecules of GNE. These covalent additions resulted in minor increases in both alpha helix and beta sheet components of 11S, as confirmed by both FTIR and circular dichroism (CD) analyses. In silico analysis revealed the presence of only one binding site under ambient conditions. However, following a simulated thermal treatment which induced changes to protein structure, six distinct binding locations were revealed, in agreement with benchtop experiments. Quantum mechanics calculations demonstrated the possibility of covalent bonds, likely formed via an oxidative (quinone) pathway and Michael addition, in each of the proposed binding pockets. These were formed with arginine, alanine and glutamine residues. These findings provide a molecular level explanation of thermally induced interactions in soy protein systems and may assist industry, in developing protocols/procedures to control these effects for specific bio- or techno-functionality.

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  2. Lakemond, C. M.; de Jongh, H. H.; Hessing, M.; Gruppen, H.; Voragen, A. G. Heat denaturation of soy glycinin: influence of pH and ionic strength on molecular structure. Journal of Agricultural and Food Chemistry2000, 48 (6), 1991-1995.
  3. Smith, E.; Condict, L.; Ashton, J.; Kasapis, S. Molecular interactions between soybean glycinin (11S) and genistein using spectroscopic and in silico analyses. Food Hydrocolloids2023, 139 , 108523.
  4. Smith, E.; Condict, L.; Ashton, J.; Kasapis, S. Molecular interactions between soybean glycinin (11S) and genistin: Impact of glycosylation. Food Hydrocolloids 2025, 165, 111266.