Mirko Bunzel

Peroxidase-dependent cross-linking reactions of p-hydroxycinnamates in plant cell walls

AbstractPeroxidases are heavily implicated in plant cell wall cross-linking reactions, altering the properties of the wall and impacting its utilization. Polysaccharide-polysaccharide cross-linking in grasses is achieved by dehydrodimerization of hydroxycinnamate-polysaccharide esters; a complex array of hydroxycinnamic acid dehydrodimers are released by saponification. Ferulates are the major cross-linking agents, but sinapate-ferulate cross-products have been discovered implicating sinapates in a similar role. New dehydrodimers have been authenticated, expanding our knowledge of the chemistry, role, and extent of cross-linking reactions. Ferulate dehydrotrimers have been discovered; whether these trimers truly cross-link three independent polysaccharide chains or only two remains to be determined. Hydroxycinnamates and their dehydrodimers also undergo radical coupling reactions with lignin monomers and possibly oligomers, resulting in lignin-polysaccharide cross-linking in the wall. Both polysaccharide-polysaccharide and lignin-polysaccharide cross-links inhibit the enzymatic hydrolysis of cell walls. The cross-linking process has particular relevance to plant physiology, human and animal nutrition and health, and food technology. Abbreviations: CW – cell wall; DFA – dehydrodiferulic acid (or dehydrodiferulate in context); DSA – dehydrodisinapic acid; TFA – dehydrotriferulic acid; SA – sinapic acid (1S); TA – thomasidioic acid (5C3SS); IDF – insoluble dietary fiber; SDF – soluble dietary fiber; GC-MS – gas chromatography-mass spectrometry; NMR – nuclear magnetic resonance (spectroscopy).

Separation and Detection of Cell Wall-Bound Ferulic Acid Dehydrodimers and Dehydrotrimers in Cereals and Other Plant Materials by Reversed Phase High-Performance Liquid Chromatography With Ultraviolet Detection

Ferulate dehydrodimers and the more recently discovered dehydrotrimers play an important role in the cell wall architecture of plant-based foods and forages. High-performance liquid chromatography methods to determine ferulate dimers often lack specificity; methods for trimers did not exist yet. A method for the determination of 11 cell wall-bound ferulate dehydrodimers and -trimers was developed, including the crucial separation of the di/trimers from the often dominating phenolic monomers. Validation parameters for the basic calibration of the dimers and trimers met our acceptance criteria. However, the matrix calibration revealed that lignin-rich matrices lead to problems with precision and accuracy that likely can be addressed by using a more specific detection, that is, mass spectrometric detection, next to improved sample preparation procedures. The method was used to analyze low-lignin fibers from corn, wheat, and rye grains, wild rice, asparagus, and sugar beet. With the exception of wild rice, the 5-5/8-O-4-, 8-O-4/8-O-4-, and 8-8(aryltetralin)/8-O-4-dehydrotrimers were detected in all analyzed samples, however, often in amounts below the limit of quantitation.

Characterization of high molecular weight coffee fractions and their fermentation by human intestinal microbiota

To investigate the structure and fermentability of high M(r) components of coffee brews by human gut bacteria Arabica coffee samples of three different degrees of roast (light, medium, and dark) were used for drip brew preparations and fractionation by ultrafiltration with different M(r) cut-offs. Total carbohydrates of the fractions ranged from 28.6 g/100 g to 56.7 g/100 g. Galactomannans and arabinogalactans were the main polysaccharides and made up between one-fourth and one-half of the respective coffee fraction. After 24 h of incubation with a human fecal suspension the polysaccharides of all fractions were extensively degraded. A decrease in the absorbance values at 405 and 280 nm, respectively, indicated that also chemically noncharacterized UV-active components such as Maillard reaction products, had been partially degraded or modified by the human gut bacteria. The remainder after 24 h of fermentation still showed antioxidant activity. Bacterial cells belonging to the Bacteroides-Prevotella group increased 2- to 40-fold during fermentation depending on the M(r) range of the fraction and the degree of roast. The production of high amounts of acetate and propionate is in accordance with a role of these bacteria in the degradation of high M(r) components from coffee.

Conversion of Dehydrodiferulic Acids by Human Intestinal Microbiota

Plant cell wall associated dehydrodiferulic acids (DFA) are abundant components of cereal insoluble dietary fibers ingested by humans. The ability of human intestinal microbiota to convert DFA was studied in vitro by incubating 8-O-4- and 5-5-coupled DFA with fecal suspensions. 8-O-4-DFA was completely degraded by the intestinal microbiota of the majority of donors, yielding homovanillic acid, 3-(3,4-dihydroxyphenyl)propionic acid, and 3,4-dihydroxyphenylacetic acid as the main metabolites. The transient formation of ferulic acid and presumably 3-(3-hydroxy-4-methoxyphenyl)pyruvic acid suggests an initial cleavage of the ether bond. In contrast to 8-O-4-DFA, the 5-5-coupled DFA was not cleaved into monomers by any of the fecal suspensions. Only the side chains were hydrogenated and the methoxy groups were demethylated. The cleavage of DFA by human intestinal microbiota, which depended on their coupling type, may affect both the bioavailability of DFA and the degradability of DFA-coupled fiber in the gut.

Characterization and Fermentability of an Ethanol Soluble High Molecular Weight Coffee Fraction

Brews from differently roasted Arabica coffees were shown to contain 8-12% ethanol soluble substances with molecular masses greater than 2 kDa, possibly contributing to their dietary fiber contents. About 13% of these substances were nondigestible carbohydrates, mainly arabinogalactans. The nondigestible high molecular weight ethanol soluble fraction (HESF) of the medium roasted coffee brew was further characterized and subjected to in vitro fermentation with human fecal bacteria. In addition to carbohydrates, HESF contained proteins/peptides (approximately 20%), but the main fraction was composed of structurally unknown Maillard reaction products. From NMR spectroscopy, we conclude that intact caffeic and ferulic acid derivatives were not incorporated into the melanoidins to a significant extent. Stepwise ultrafiltration and gel filtration indicated a large variation in the molecular weights of HESF constituents. Coffee HESF was shown to be less fermentable by fecal bacteria than soluble coffee fiber isolated by the enzymatic-gravimetric methodology, and because of its lower carbohydrate content, less short-chain fatty acids were produced during the fermentation. Total cell counts, destructive chemical analysis, and NMR spectroscopy indicated that coffee carbohydrates are the preferred substrates for colonic microbiota. However, NMR spectra, absorbances at 405 nm, and nonprotein nitrogen contents showed that noncarbohydrate and nonprotein compounds were also utilized to some extent but the bacterial species involved in this degradation remain to be identified.

Influence of Lignification and Feruloylation of Maize Cell Walls on the Adsorption of Heterocyclic Aromatic Amines

Both epidemiological and experimental data indicate that a diet rich in fiber may reduce cancer risk. One possible mechanism is by adsorbing carcinogens and transporting them out of the body without metabolic activation. We investigated the role of fiber lignification and feruloylation on the adsorption of four of the most relevant heterocyclic aromatic amines in food: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and 2-amino-9H-pyrido[2,3-b]indole (AalphaC). Adsorption experiments, under conditions mimicking the small intestine, were carried out using nonlignified and artificially lignified primary maize walls with defined lignin and ferulate/diferulate concentrations and defined lignin compositions. Lignin concentration and composition both influenced the adsorption of heterocyclic aromatic amines, especially the more hydrophobic types. Heterocyclic aromatic amine adsorption increased with lignin concentration. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-9H-pyrido[2,3-b]indole were better adsorbed by guaiacyl-rich lignins, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by syringyl-rich lignins, whereas the adsorption of 2-amino-3-methylimidazo[4,5-f]quinoline was not clearly influenced by lignin composition. Nonlignified cell walls adsorbed lesser amounts of heterocyclic aromatic amines. Variations in cell wall feruloylation had no effect on heterocyclic aromatic amine adsorption.

Peroxidase-catalyzed oligomerization of ferulic acid esters.

Valuable information about possible types of linkages, reaction mechanisms, and sequences for oxidative coupling of phenolic compounds in planta is available from in vitro model systems. Ferulate oligomers were generated in a system using ethyl ferulate, peroxidase, and hydrogen peroxide under various conditions. A molar ferulate/H2O2 ratio of 1:1, an ethanol level of 30% in an aqueous sodium phosphate buffer (pH 6.0), and a reaction time of 10 min were considered to be ideal to produce maximal proportions of ferulate trimers and tetramers from ethyl ferulate as starting material. The dominant trimer and tetramer were each isolated from the reaction mixture and identified as 8-O-4/8-5(cyclic)-dehydrotriferulic acid triethyl ester and 8-5(cyclic)/4-O-5/8-5(cyclic)-dehydrotetraferulic acid tetraethyl ester. The structure of the 8-O-4/8-5(cyclic)-dehydrotriferulic acid triethyl ester revealed that a third ferulate unit is bound to a preformed 8-O-4-diferulate dimer, a surprising reaction sequence considering the dominance of 8-5-coupled dimers among dehydrodiferulates in H2O2/peroxidase-based model reactions. As 4-O-5-coupling is not favored in the dimerization process of ferulates, the main tetramer isolated in this study is probably formed by 4-O-5-coupling of two preformed 8-5(cyclic)-diferulates, a logical step in analogy with reactions occurring in lignin biosynthesis.

Synthesis and identification of 2,5-bis-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-3,4-dicarboxylic acid, an unanticipated ferulate 8–8-coupling product acylating cereal plant cell walls

A new product implicated in cereal grain polysaccharide cross-linking has been authenticated by independent synthesis. Saponification of cereal grain fiber releases the RRRS/SSSR-isomer of 2,5-bis-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-3,4-dicarboxylic acid. The parent ester logically derives from 8-8-coupling of ferulate followed by water addition to one of the incipient quinone methide moieties and internal trapping of the other. The finding adds complexity to the analysis of plant cell wall cross-linking, but provides clues to important polysaccharide cross-linking pathways occurring in planta.

Isolation and characterization of feruloylated arabinoxylan oligosaccharides from the perennial cereal grain intermediate wheat grass (Thinopyrum intermedium).

In comparison to the annual grain crops dominating current agricultural production, perennial grain species require fewer chemical and energy inputs and improve soil health and erosion control. The possibility for producing sustainable grain harvests from marginal land areas is motivating research initiatives to integrate perennial grains into commercial cropping and food processing systems. In this study, the feruloylated arabinoxylans from intermediate wheat grass (Thinopyrum intermedium, IWG), a promising perennial grain candidate in agronomic screening studies, were investigated. Insoluble fiber isolated from IWG whole grain flour was subjected to either mildly acidic (50 mM TFA, 100 °C, 2 h) or enzymatic (Driselase) hydrolysis. The liberated feruloylated arabinoxylan oligosaccharides were concentrated with Amberlite XAD-2, separated with gel chromatography (Sephadex LH-20, water), and purified with reversed-phase HPLC (C18, water-MeOH gradient). Thirteen feruloylated oligosaccharides were isolated (including eight structures described for the first time) and identified by LC-ESI-MS and NMR. Linkage-type analysis via methylation analysis, as well as the monosaccharide and phenolic acid profiles of the IWG insoluble fiber were also determined. IWG feruloylated arabinoxylans have a relatively simple structure with only short feruloylated side chains, a lower backbone substitution rate than annual rye and wheat varieties, and a moderate phenolic acid content.

Nature of Phenolic Compounds in Coffee Melanoidins

Phenolic compounds are incorporated into coffee melanoidins during roasting mainly in condensed form (42-62 mmol/100 g) and also in ester-linked form (1.1-1.6 mmol/100 g), with incorporation levels depending on the green coffee chlorogenic acid content. The phenolic compounds are incorporated in different coffee melanoidin populations, but mainly in those soluble in 75% ethanol (82%), a significant correlation between the amount of phenolic compounds and the amount of protein and color characteristics of the different melanoidin populations being observed. The incorporation of phenolic compounds into coffee melanoidins is a significant pathway of chlorogenic acid degradation during roasting, representing 23% of the chlorogenic acids lost. These account for the nearly 26% of the material not accounted for by polysaccharides and proteins present in coffee melanodins. The cleavage mechanism and the efficiency of alkaline fusion used to release condensed phenolics from coffee melanoidins suggest that the phenolic compounds can be linked to the polymeric material by aryl-ether, stilbene type, and/or biphenyl linkages.