Jean-Marc Brillouet

Sequential enzymic hydrolysis of potentially aromatic glycosides from grape

Abstract The mechanism of action of α- l -arabinofuranosidase, α- l -rhamnopyranosidase, and β- d -glucopyranosidase on p -nitrophenyl and grape monoterpenyl disaccharide-glycosides has been studied. First, the (1→6) linkage is cleaved by either α- l -arabinofuranosidase or α- l -rhamnosidase, and arabinose, rhamnose, and the corresponding monoterpenyl β- d -glucosides are released. Subsequently, liberation of monoterpenol takes place after action of β- d -glucosidase. The possible use of these glycosidases for the enhancement of the aroma of grape juice and derived beverages is emphasised.

Structural characterization of red wine rhamnogalacturonan II

The pectic polysaccharide rhamnogalacturonan II (RG-II), which accounts for approximately 20% of the ethanol-precipitable polysaccharides in red wine, has been isolated from wine polysaccharides by anion-exchange chromatography. Four fractions enriched with RG-II were obtained and the RG-II then purified to homogeneity by Concanavalin A affinity and size-exclusion chromatographies. The glycosyl-residue compositions of the four RG-IIs are similar; all the RG-IIs contain the monosaccharides (apiose, 2-O-methyl-L-fucose, 2-O-methyl-D-xylose, Kdo, Dha, and aceric acid) that are diagnostic of RG-II. The glycosyl-linkages of the neutral and acidic sugars, including aceric acid, were determined simultaneously by GC-EIMS analysis of the methylated alditol acetates generated from per-O-methylated and carboxyl-reduced RG-II. Two of the RG-IIs contain boron, most likely as a borate di-ester that cross-links two molecules of RG-II together to form a dimer. The dimer contains 3- and 2,3,3-linked apiosyl residues whereas the monomer contains only 3-linked apiosyl residues which suggests that the borate di-ester is located on at least one of the apiosyl residues of RG-II. Although the wine RG-IIs all have similar structures they are not identical since they differ in the length and degree of methyl-esterification of the RG-II backbone and in the presence or absence of borate di-esters. Nevertheless, these studies show that the major structural features of wine and primary cell wall RG-II are conserved.

A Saccharomyces mannoprotein that protects wine from protein haze

Abstract A Saccharomyces cerevisiae yeast mannoprotein which protected wine from protein haze spoilage was isolated from a red wine and purified by a combination of anion exchange, Concanavalin A, cation exchange and gel permeation chromatography. This mannoprotein corresponded to the highest molecular weight colloids in wine and was present at a trace level in the wine. Its apparent molecular weight when compared with pullulan standards was 420 kDa. The carbohydrate part represented 70% of the mannoprotein and consisted of 98% mannose and 2% glucose. Methylation data suggested that there were 2- and 3-linked mannose chains, some present as side chains on a 6-linked mannose backbone. Some of the mannose chains were N -linked to the protein and 60% were O -linked. The protein part represented 30% of the mannoprotein and was dominated by serine, glycine, threonine and alanine.

Structural studies of pectic substances from the pulp of grape berries

Abstract The structure of the neutral sugar side-chains of pectic substances extracted with water (WSP), dilute HCl (HP), and endopectinlyase (AIR/PEL) from an alcohol-insoluble residue (AIR) from the pulp of grape berries was investigated by using chemical methods (methylation, carboxyl-reduction, and periodate oxidation), enzymic degradation with an α- l -arabinofuranosidase, and 13 C-n.m.r. spectroscopy. The side chains of WSP are essentially a type II arabinogalactan, whereas those of HP and AIR/PEL are arabinan-like structures associated with minor proportions of type I and II arabinogalactans. The side chains are ∼80% degraded by the α- l -arabinofuranosidase, indicating that the α- l -arabinofuranosyl residues are mainly 3-linked to the (1→6)-galactan side-chains of the type II arabinogalactan. The three pectic fractions contain minor amounts of proteins rich in hydroxyproline and serine.

Characterization of five type II arabinogalactan-protein fractions from red wine of increasing uronic acid content

Five arabinogalactan-protein conjugates (AGP) were separated from red wine by two successive anion-exchange chromatography steps and further purified to apparent homogeneity by affinity and size-exclusion chromatography. Together they represent more than 40% of total wine polysaccharides, confirming the abundance of AGPs in red wine. The five purified fractions had a common arabinogalactan core with characteristics typical of wine type II AGPs, but differed mainly in their uronic acid content, as evidenced by differences in the strength of their binding to the anion-exchanger. Their uronic acid content and glycosidic linkage composition revealed that the three less acidic AGPs contained from 3 to 7% glucuronic acid, half in terminal non-reducing positions and half in terminal Rhap-(1-->4)-Glc pA-(1-->sequences. The two more acidic AGP-containing fractions contained both glucuronic (6.1 and 13.3%, respectively) and galacturonic (1.9 and 2.3%, respectively) acid in association with 2- and 2,4-linked rhamnose, indicating the presence of AG-rhamnogalacturonan fragments.

Investigation of the structure of a heteroxylan from the outer pericarp (beeswing bran) of wheat kernel

A heteroxylan isolated from the pericarp (beeswing bran) of wheat kernel and purified is shown to be a highly substituted glucuronoarabinoxylan in which 80% of the β-d-xylosyl residues carry one or two substituents. Single terminal α-l-arabinofuranosyl groups and short chains of 2-, 3-, 5-, and 2,3-linked arabinose residues are distributed along the β-(1→4)-linked backbone of the xylan core which also carries residues of glucuronic acid and its 4-O-methyl derivative. Graded acid hydrolysis and degradations with an endo-xylanase and an α-l-arabinofuranosidase indicate that both the arabinan and the xylan moieties are highly branched structures, and this is confirmed by 13C-n.m.r. spectroscopy of the heteroxyaln at various stages of degradation.

Composition of cell walls from cotyledons of Pisum sativum, Vicia faba and Glycine max

Abstract Cell walls from cotyledons of smooth field pea, broad bean and soya bean contain ca 55% pectic polysaccharides associated with 9% cellulose. Arabinose is the major pectic sugar of pea and broad bean walls whereas soya bean pectic polymers are constituted of galactose and arabinose in the ratio (2:1). Galacturonic acid represents ca 20% of the walls. In addition, pea and broad bean cell walls contain, respectively, 12% and 6% of non-starchy and non-cellulosic glucans bearing 4,6-linked and 3-linked glycosyl units. EDTA-soluble acidic pectic substances are distinct rhamnogalacturonans bearing decreasing proportions of interrupting rhamnose from highly interrupted moieties to nearly homogenous homogalacturonans. Pea and broad bean rhamnogalacturonans are associated with arabinose-containing polymers of average DP ca 30–35 whereas soya bean ones have side chains of arabinose and galactose of DP ca 40.

Detection and Partial Characterisation of New Anthocyanin-Derived Pigments in Wine

New anthocyanin-derived red–orange pigments were detected in wine in the course of cross-flow microfiltration experiments. Analysis of the sugar and organic acid released respectively by acid and alkaline hydrolysis showed that the two major products were respectively a glucoside and the corresponding p-coumaroylglucoside. Mass spectrometry indicated molecular masses of 755·5 for the p-coumaroylglucoside and of 447·5 for its aglycone. Therefore, the new pigments are likely to result from condensation reaction between grape anthocyanins (possibly the 3-glucoside and 3-p-coumaroylglucoside of malvidin) and another wine component, which is not a flavanol. This reaction may partici-pate in the mechanism of colour change during ageing of red wines.

Isolation and characterisation of a rhamnogalacturonan II from red wine

Abstract A complex polysaccharide originating from grape was purified from a red wine by size-exclusion and ion-exchange chromatography. This polysaccharide contained 2-O-methylfucose, rhamnose, fucose, 2-O-methylxylose, arabinose, apiose, galactose, galacturonic and glucuronic acids, and thiobarbituric acid assay-positive material, presumably 3-deoxy- d -manno-2-octulosonic acid (Kdo) and 3-deoxy- d -lyxo- 2-heptulosaric acid (Dha). Methylation structural analysis showed that this polysaccharide is a typical rhamnogalacturonan II (RG-II), a structurally complex pectic polysaccharide that has been isolated previously from the primary cell walls of sycamore. Red wine RG-II presented discrete differences from previously described RG-IIs, a greater richness in uronic acids and some additional glycosyl linkages, which might be related to the pattern of degradation of grape-wall pectic substances by endogenous pectinolytic enzymes after pressing the berries and subsequent fermentation to wine.

New investigations of the structure of grape arabino-galactan-protein

The structure of an arabinogalactan-protein (AGP) isolated from grape juice was studied by methylation analysis, n.m.r. spectroscopy, and interactions with peanut lectin, after specific degradation with purified enzymes and/or Smith degradation. AGP appeared to be homogeneous with a weight-average molecular weight of 110,000. Treatment of AGP with arabinofuranosidase released 88% of the arabinose and left GP1. Hydrolysis of GP1 with an endo-(1----6)-beta-D-galactanase removed 50% of the galactose and left GP2. Smith degradation of GP1 gave a 3-linked galactan that still contained 3,6-linked residues. Endogalactanase- and Smith-degraded GP1, but not AGP and GP1, reacted strongly with peanut lectin. Thus, AGP is a 3-linked galactan cross-linked at positions 6. The core also carries, at positions 6, 6-linked galactan chains heavily 3-substituted with arabinofuranose residues.