Saul Assunção Bicca1,2, Céline Poncet-Legrand1, Pascale Williams1, Julie Mekoue NGuela1,2, Thierry Doco1, Aude Vernhet1
1 UMR SPO, INRAE, Université Montpellier, SupAgro, France
2 Lallemand, SAS, 19 rue de Briquetiers, France
Email contact: bicca.saul[@]gmail.com
The aim was to study the impact of structural features in the polysaccharide moiety of mannoproteins on their interaction with polyphenols and the formation of colloidal aggregates.
To this end, mannoproteins fractions were extracted from four different yeast strains: a commercial enological strain (MP-com), the wild-type BY4742 strain (MP-WT) and its mutants ΔMnn4 (MP-Mnn4) and ΔMnn2 (MP-Mnn2). The Mnn4p and Mnn2p are responsible for mannosyl-phosphorylation and branching of the N-glycosylation backbone1. Enzymatic extraction was performed using a commercial Endo-b-1,3-Glucanase of Trichoderma sp. (E-LAMSE, Megazym)2. Mannoprotein fractions were thoroughly characterized by composition of their polysaccharide and protein moieties, branching degree, net charge, molecular weight distribution, static and dynamic molecular parameters3. Their interactions with seed tannins and a pool of red wine polyphenols and the formation of colloidal aggregates were studied in model solutions at different polyphenol/mannoprotein ratios through Dynamic Light Scattering (DLS). Model solutions were followed during one month. The number and size distribution of colloidal aggregates was determined by Nanoparticle Tracking Analysis (NTA).
The four Mannoprotein had broad and high molecular weight distributions, as well as similar protein, polysaccharide mass % and amino acid composition. However, they showed different proportions of mannose and glucose and the structural characterization of the polysaccharide moiety confirmed the expected differences between MP-WT, MP-Mnn2, and MP-Mnn4. DLS and NTA experiments indicated a two-step interaction process between seed tannins and mannoproteins: an immediate formation of colloidal aggregates (150-300 nm), followed by a very progressive evolution related to a reversible aggregate flocculation. The number, dispersity and extent of flocculation were dependent on the tannin/MP ratio. However, no notable differences were evidenced between the four MP fractions. With the polyphenol pool of red wine, neither DLS nor NTA experiments were able to evidence the formation of colloidal aggregates. This does not mean that interactions do not exist4,5.
Although the mannoproteins used had different polysaccharide compositions, structures, and properties, no difference in terms of colloidal behavior when in solution with tannins or wine polyphenols was evidenced by the methods applied. Thus, neither the absence of mannosyl phosphate groups (MP-Mnn4) nor the absence of branching of the outer chains of the N-glycosylated carbohydrate structures (MP-Mnn2) seems to play a determining role in the colloidal behavior of mannoproteins in the presence of seed tannins or red wine polyphenols.
1. Corbacho, I., Olivero, I. & Hernández, L. M. A genome-wide screen for Saccharomyces cerevisiae nonessential genes involved in mannosyl phosphate transfer to mannoprotein-linked oligosaccharides. Fungal Genetics and Biology 42, 773–790 (2005).
2. Schiavone, M. et al. A combined chemical and enzymatic method to determine quantitatively the polysaccharide components in the cell wall of yeasts. FEMS Yeast Research 14, 933–947 (2014).
3. Lopez-Torrez, L., Nigen, M., Williams, P., Doco, T. & Sanchez, C. Acacia senegal vs. Acacia seyal gums - Part 1: Composition andstructure of hyperbranched plant exudates. Food Hydrocolloids 51, 41–53 (2015).
4. Mekoue Nguela, J., Poncet-Legrand, C., Sieczkowski, N. & Vernhet, A. Interactions of grape tannins and wine polyphenols with a yeast protein extract, mannoproteins and β-glucan. Food Chemistry 210, 671–682 (2016).
5. Poncet-legrand, C., Doco, T., Williams, P. & Vernhet, A. Inhibition of grape seed tannin aggregation by wine mannoproteins: Effect of polysaccharide molecular weight. American Journal of Enology and Viticulture 58, 87–91 (2007).