NMR spectroscopy for structural elucidation of insoluble crystalline mannan from residual seeds of the edible açaí (Euterpe oleracea Mart.) pulp production

NMR spectroscopy for structural elucidation of insoluble crystalline mannan from residual seeds of the edible açaí (Euterpe oleracea Mart.) pulp production

*Ingrid Santos Miguez1,2, Francisco Felipe Bezerra3, Adriane Regina Todeschini4, Carmen Lucia de Oliveira Petkowicz5, Ayla Sant’Ana da Silva1,2

1 Biocatalysis Laboratory, Division of Catalysis, Biocatalysis and Chemical Processes, Instituto Nacional de Tecnologia, RJ, Brazil; 2 Departament of Biochemistry, Institute of Chemistry, Universidade Federal do Rio de Janeiro, RJ, Brazil; 3 Hospital Universitário Clementino Fraga Filho, Instituto de Bioquímica Médica Leopoldo de Meis, UFRJ, RJ, Brazil; 4 Instituto de Biofísica Carlos Chagas Filho, UFRJ, RJ, Brazil; 5 Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, PR, Brazil *ingrid.miguez@pos.iq.ufrj.br

 

Açaí is a “superfruit” endemic to the Amazon Forest that has beneficial effects on health due to its rich nutritional composition. However, a significant portion of açaí weight, 85% of the total fruit, is discarded as waste during edible pulp production. Consequently, Brazil alone generates over 1.6 million tons of açaí seeds annually, which, without proper disposal, leads to environmental concerns due to their accumulation in producing areas. Notably, almost half of the dry mass of this abundant agroindustrial waste comprises mannan, with 90% of this polysaccharide glycosyl residues composed of mannose, suggesting a linear mannan structure1. Therefore, understanding the mannan structure could solve the challenges hindering the industrial conversion of açaí seeds into mannose, a sugar of high market value with significant biological functions. For this, açaí seeds were fractionated to obtain a water-insoluble fraction. NMR analysis of this sample resulted in δ from 1H/13C HSQC spectrum which is in agreement with signals of 1,4-linked β-D-Manp, similar to the δ of mannan from other palm seeds, known to contain linear mannan, and also similar to unbranched β-D-Manp residues of the backbone of low-substituted seed galactomannans. The 13C spectra had six carbon signals assigned to a β-(1→4) D-mannan, as the 1H/13C HMBC confirms the presence of β(1→4) glycosidic linkage between Manp units by the cross peaks at 4.73/78.0 ppm (H-1/C-4) and 3.88/101.8 ppm (H-4/C-1). The compositional characterization of the isolated sample was confirmed by liquid and gas chromatography, however, the molar ratio obtained for Man:Glc:Gal was 81:13:3 and 78:19:1, respectively, suggesting a possible contamination of the mannan with cellulose traces, undetected by NMR due to solvent selected to sample preparation. As cellulose and linear mannan are known to be crystalline polysaccharides, the isolated fraction was evaluated by XRD. Further evaluation via XRD indicated a typical pattern for linear mannan, although a peak corresponding to cellulose Iβ was observed at 22.5° (200). Subsequent, given the difficulty in solubilizing the isolated fraction, it was decided to also analyze the sample by 13C CPMAS ssNMR, for a total assessment of mannan. The identified linear mannan have crystal structures, polymorphs I and II, which were identified in the sample, since the two signals corresponding to carbon 5 of both forms were detected at 70.2 and 70.9 ppm, respectively, with the polymorph I in greater concentration. However, cellulose detection remained elusive, necessitating methodological adjustments. Nevertheless, this unprecedented identification of the linear mannan structure from the açaí seed will allow the establishment of strategies to reduce the crystallinity of the mannan.

Acknowledgments: Serrapilheira Institute, FAPERJ, CAPES, Laboratório de ressonância magnética nuclear de sólidos (UFRJ), Laboratório multiusuário de difração de raios X (UERJ).

References
1. Monteiro, Miguez; et al. Scientific Reports, 9, 10939 (2019).