Notes

A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography

Homoexopolysaccharides (HoEPS) such as α-glucans and β-fructans are synthesized by lactic and acetic acid bacteria. Methylation analysis is an important and well-established tool for the structural analysis of these polysaccharides, however, multiple steps are required for polysaccharide derivatization. Because ultrasonication during methylation and the conditions during acid hydrolysis may influence the results, we investigated their role in the analysis of selected bacterial HoEPS. The results reveal that ultrasonication is crucial for water insoluble α-glucan to swell/disperse and deprotonate prior to methylation whereas it is not necessary for water soluble HoEPS (dextran and levan). Complete hydrolysis of permethylated α-glucans requires 2 M trifluoroacetic acid (TFA) for 60/90 min at 121 °C while levan is hydrolyzed in 1 M TFA for 30 min at 70 °C. Nevertheless, levan was also detectable after hydrolysis in 2 M TFA at 121 °C. Thus, these conditions can be used to analyze a levan/dextran mixture. However, size exclusion chromatography of permethylated and hydrolyzed levan showed degradation and condensation reactions at harsher hydrolysis conditions. Application of reductive hydrolysis with 4-methylmorpholine-borane and TFA did not lead to improved results. Overall, our results demonstrate that conditions used for methylation analysis have to be adjusted for the analysis of different bacterial HoEPS.

Yeast cell walls (CWs) may adsorb red wine polyphenols with a significant impact on wine quality. This adsorption is mainly attributed to physicochemical interactions with mannoproteins. However, the mannoprotein structural features that promote polyphenol adsorption are not clearly established yet. The impact of mannosylphosphorylation and mannan backbone branching on polyphenol adsorption by yeast was studied through adsorption isotherms with Saccharomyces cerevisiae BY4742 wild-type and mnn4 and mnn2 mutants (EUROSCARF). Polymeric and oligomeric tannins and derived pigments were preferentially adsorbed by the yeasts and their corresponding CWs. Mannoprotein N-glycosyl phosphorylation appeared as the key groupments involved in polyphenol adsorption by CWs. When dealing with whole yeasts, behaviors were different, due to the coupled phenomena of polyphenol adsorption at cell wall level and their diffusion inside the yeast cytoplasm. The key role of phosphorylation observed in this work is of interest in the modulation of wine color and astringency by yeasts.

Burkholderia cenocepacia is an opportunistic pathogen isolated from cystic fibrosis patients where it causes infections that are extremely difficult to treat with antibiotics, and sometimes have a fatal outcome. Biofilm is a virulence trait of B. cenocepacia, and is associated with infection persistence and increased tolerance to antibiotics. In biofilms exopolysaccharides have an important role, conferring mechanical stability and antibiotic tolerance. Two different exopolysaccharides were isolated from B. cenocepacia H111 biofilms: a water-soluble polysaccharide rich in rhamnose and containing an L-Man residue, and a water-insoluble polymer made of glucose, galactose and mannose. In the present work, the product encoded by B. cenocepacia H111 bepA-L gene cluster was identified as the water-insoluble exopolysaccharide, using mutant strains and NMR spectroscopy of the purified polysaccharides. It was also demonstrated that the B. cenocepacia H111 wild type strain produces the water-insoluble exopolysaccharide in pellicles, thus underlining its potential importance in in vivo infections.

Plant wastes are the prime choice of feedstock for producing new chemicals and materials required for the transition toward a biobased economy. Plant biomass is mainly constituted by plant cell walls (PCWs), primarily composed of polysaccharides presenting a wide diversity of glycosyl residues content and linkages, leading to different structures and physicochemical properties. Thus, the effective use of plant biomass requires a comprehensive understanding of PCW polysaccharides through characterizing methods that can guide the selection of suitable industrial applications well-matched with each biomass source's properties. In this context, this chapter discusses consolidated analytical methodologies for PCW polysaccharides characterization grouped into three categories: (i) glycosyl residue composition analysis, (ii) structural analysis, and (iii) complementary analysis. Furthermore, a critical discussion of their main advantages and limitations is provided to aid the reader in identifying the most appropriate methods for different biomass samples.

Lupin is among the most promising plant-based food protein sources due to its high protein and fibre content. The fibre fraction, especially from seed coats, is often seen as low in value (discarded as waste or as animal feed) and greater knowledge on its composition and structure are crucial to increase its usefulness. However, only one model of lupin fibre structure exists in the literature. Our sample of Finnish-grown narrow-leafed lupin seed consisted of fibre (43.3%), protein (31.3%), fat (8.1%) and starch (0.2%). According to the sugar analysis, rhamnogalacturonan-I, with branches of arabinan and galactan, constituted the main pectin population in the fibre fraction. A revised model of the overall fibre structure is proposed. At concentrations of 0.75% and 1.0%, both unrefined and defatted flour of whole lupin seeds produced stable suspensions and oil-in-water emulsions, demonstrating their application as potential emulsifiers. This study presents the knowledge and opportunity to support sustainability through the utilization of whole lupin seed for future industrial applications.

While they have many properties of interest in enology, the structure-function relationships of mannoproteins and the part played by their polysaccharide moiety are not yet well understood. Mannoproteins (MP) extracted with β-glucanase from a laboratory yeast strain (WT), two of its mutants (Mnn2 with unbranched N-glycosylated chains and Mnn4 without mannosyl-phosphorylation), and an enological strain (Com) were purified and thoroughly characterized. The protein moiety of the four MPs had the same amino acid composition. Glycosyl-linkage and net charge analyses confirmed the expected differences in mutant strain MPs. MP-Com had the highest mannose/glucose ratio followed by MP-WT/MP-Mnn4, and MP-Mnn2 (13.5 > 5.6 ≈ 5.2 > 2.2). The molar mass dependencies of R_g, R_h, and [η], determined through HPSEC-MALLS-QELS-Viscosimetry, revealed specific conformational properties of mannoproteins related to their nature of highly branched copolymers with two branching levels. It also clearly showed structural differences between MP-Com, MP-WT/Mnn4, and MP Mnn2, and differences between two populations within the four mannoproteins.