Effect of cysteinyl caffeic acid, caffeic acid, and L-dopa on the oxidative cross-linking of feruloylated arabinoxylans by a fungal laccase

To study a way to covalently link arabinoxylans and proteins using a fungal laccase from the fungus Pycnoporus cinnabarinus, the effect of cysteinyl caffeic acid on the cross-linking of wheat arabinoxylans was investigated by means of capillary viscometry and RP-HPLC of alkali labile phenolic compounds. Cysteinyl caffeic acid provoked a delay in gelation and in the consumption of the esterified ferulic acid on arabinoxylans. When reacting free ferulic acid and cysteinyl caffeic acid with laccase, the ferulic acid consumption and the dehydrodimers production were also diminished. These results suggest that cysteinyl caffeic acid is oxidized while reducing the semiquinones of ferulic acid produced by laccase. Thus, ferulic acid could not be oxidized into dimers until all cysteinyl caffeic acid was consumed, preventing the cross-linking of feruloylated arabinoxylan chains. A similar mechanism is proposed in the case of caffeic acid and of L-Dopa.     

Hydroxycinnamic acids and ferulic acid dehydrodimers in barley and processed barley

Hydroxycinnamic acid content and ferulic acid dehydrodimer content were determined in 11 barley varieties after alkaline hydrolysis. Ferulic acid (FA) was the most abundant hydroxycinnamate with concentrations ranging from 359 to 624 microg/g dry weight. p-Coumaric acid (PCA) levels ranged from 79 to 260 microg/g dry weight, and caffeic acid was present at concentrations of <19 microg/g dry weight. Among the ferulic acid dehydrodimers that were identified, 8-O-4'-diFA was the most abundant (73-118 microg/g dry weight), followed by 5,5'-diFA (26-47 microg/g dry weight), the 8,5'-diFA benzofuran form (22-45 microg/g dry weight), and the 8,5'-diFA open form (10-23 microg/g dry weight). Significant variations (p < 0.05) among the different barley varieties were observed for all the compounds that were quantified. Barley grains were mechanically fractionated into three fractions: F1, fraction consisting mainly of the husk and outer layers; F2, intermediate fraction; and F3, fraction consisting mainly of the endosperm. Fraction F1 contained the highest concentration for ferulic acid (from 77.7 to 82.3% of the total amount in barley grain), p-coumaric acid (from 78.0 to 86.3%), and ferulic acid dehydrodimers (from 79.2 to 86.8%). Lower contents were found in fraction F2, whereas fraction F3 exhibited the lowest percentages (from 1.2 to 1.9% for ferulic acid, from 0.9 to 1.7% for p-coumaric acid, and <0.02% for ferulic acid dehydrodimers). The solid barley residue from the brewing process (brewer's spent grain) was approximately 5-fold richer in ferulic acid, p-coumaric acid, and ferulic acid dehydrodimers than barley grains.     

Peroxidase-mediated cross-linking of a tyrosine-containing peptide with ferulic acid

The tyrosine-containing peptide Gly-Tyr-Gly (GYG) was oxidatively cross-linked by horseradish peroxidase in the presence of hydrogen peroxide. As products, covalently coupled di- to pentamers of the peptide were identified by LC-MS. Oxidative cross-linking of ferulic acid with horseradish peroxidase and hydrogen peroxide resulted in the formation of dehydrodimers. Kinetic studies of conversion rates of either the peptide or ferulic acid revealed conditions that allow formation of heteroadducts of GYG and ferulic acid. To a GYG-containing incubation mixture was added ferulic acid in small aliquots, therewith keeping the molar ratio of the substrates favorable for hetero-cross-linking. This resulted in a predominant product consisting of two ferulic acid molecules dehydrogenatively linked to a single peptide and, furthermore, two ferulic acids linked to peptide oligomers, ranging from dimers to pentamers. Also, mono- and dimers of the peptide were linked to one molecule of ferulic acid. A mechanism explaining the formation of all these products is proposed.     

Content of phenolic acids and ferulic acid dehydrodimers in 17 rye (Secale cereale L.) varieties

The contents of pnenolic acids and ferulic acid dehydrodimers were quantified by HPLC analysis after alkaline hydrolysis in kernels of 17 rye (Secale cereale L.) varieties grown in one location in Denmark during 1997 and 1998. Significant variations (P < 0.05) with regard to the concentration of the analyzed components were observed among the different rye varieties and also between different harvest years. However, the content of phenolic acids in the analyzed rye varieties was narrow compared to cereals such as wheat and barley. The concentration of ferulic acid, the most abundant phenolic acid ranged from 900 to 1170 microgram g(-1) dry matter. The content in sinapic acid ranged from 70 to 140 microgram g(-1) dry matter, p-coumaric acid ranged from 40 to 70 microgram g(-1) dry matter, and caffeic, p-hydroxybenzoic, protocatechuic, and vanillic acids were all detected in concentrations less than 20 microgram g(-1) dry matter. The most abundant ferulic acid dehydrodimer 8-O-4 -DiFA was quantified in concentrations from 130 to 200 microgram g(-1) dry matter followed by 8,5 -DiFA benzofuran form (50-100 microgram g(-1) dry matter), 5,5 -DiFA (40-70 microgram g(-1) dry matter), and 8,5 -DiFA (20-40 microgram g(-1) dry matter).     

Enzymatic binding of the pollutant 2,6-xylenol to a humus constituent

Dimeric and polymeric hybrid oliogmers were obtained by the reaction of 2,6-xylenol and syringic acid in the presence of an enzyme from the fungus Trametes versicolor. Three hybrid dimers were formed in an assay at pH 4.5, while at pH 6.5, the formation of additional higher polymers with a phenyl-ether bond (tail to head linkage) was detected. The major product was identified as 3,5-dimethyl-3′,5′-dimethoxydiphenoquinone (4,4′), and it amounted to approximately 45% of the applied 2,6-xylenol after 3.5 hr incubation at pH 4.5. About 8% of the 2,6-xylenol was transformed to 4,4′-dihydroxy-3,5-dimethyl-3′,5′-dimethoxydiphenol and 22% to 3-methoxy-5-(2′,6′-dimethylphenoxy)-benzoquinone (1,2).     

Cell wall fractions isolated from outer layers of rye grain by sequential treatment with alpha-amylase and proteinase: structural investigation of polymers in two ryes with contrasting breadmaking quality

Recent studies have indicated that some structural features of arabinoxylans, the major cell wall polysaccharides, might be potential quality markers in the selection of rye breeding materials. To specify the most appropriate characteristics, the differences in the structure of cell wall components were studied in two ryes with high and low breadmaking qualities. Two cell wall fractions were isolated from the outer layers of the grain (pooled shorts and bran fractions) by a consecutive water extraction with alpha-amylase (WE-A) and proteinase K (WE-P). Polysaccharides predominated in the WE-A fraction (approximately 64%, mainly arabinoxylans). By contrast, the WE-P fraction contained mostly protein (approximately 63%), and its level of polysaccharides was relatively low (approximately 18%). The 1H NMR and sugar analysis of the ammonium sulfate precipitated subfractions revealed that the WE-A was built of four arabinoxylan populations with marked structural differences (arabinose-to-xylose ratios, Ara/Xyl, of approximately 0.3, 0.5, 0.8, and 1.2). Instead, the arabinoxylans present in the WE-P were generally enriched in disubstituted xylopyranosyl residues. The ratio of phenolic components to arabinose residues in the WE-P fraction (indicated by 1H NMR) and the proportion of polymers with the highest molecular weights in the WE-A fraction (revealed by HPSEC) distinguished well two ryes with diverse breadmaking qualities. Much less obvious differences between both ryes were observed in the ratio of amide I to amide II band intensities of FTIR spectra for the WE-P and in the level of phenolic acids and ferulic acid dehydrodimers for both cell wall preparations.     

Effect of storage on wall-bound phenolics in green asparagus

The cell walls of green asparagus spears have been analyzed for their phenolic and carbohydrate composition as modified by postharvest storage. Esterified phenolic components were released by sequential alkaline hydrolysis and identified and quantified by diode array HPLC. Significant quantities of ferulic acid (FA) and its derivatives were found to increase at least 3-fold during storage, particularly in walls from the lower parts of the stem, where accompanying changes in sugar composition were also observed. In fresh asparagus, >60% of the total FA was in the form of diferulic acid, and this increased to approximately 70% after 3 days of storage. The main FA dehydrodimers were 8-8-, 8-O-4-, and 8-5-diferulates. These have been detected in other monocotyledonous and dicotyledonous plants, but as a smaller proportion of the total FA. The possible roles phenolic esters might have in relation to the mechanical, textural, and wound-response properties of asparagus spears are discussed.     

Influence of cross-linked arabinoxylans on the postprandial blood glucose response in rats

Viscous dietary fibers are well established to reduce the blood glucose response to a meal. In this study, arabinoxylans, the most abundant dietary fiber in most cereals, were extracted under alkaline conditions and cross-linked by using laccase. Cross-linking of the arabinoxylans led to gel formation and increased in vitro viscosity almost 100-fold after drying and rehydration. To determine the ability of these cross-linked arabinoxylans to blunt the postprandial blood glucose curve of a meal, arabinoxylans, either native or cross-linked, and either prehydrated or not, were fed to rats as part of a meal, and blood glucose was monitored at intervals after the meal. Cellulose, a nonviscous fiber, served as a control. Cross-linked, but not native, arabinoxylans significantly reduced the area under the blood glucose time curve 5-9% relative to cellulose, indicating that they remained viscous within the gastrointestinal tract, and thus likely provide the health benefits found with other viscous fibers.     

Dehydrotriferulic and Dehydrodiferulic Acid Profiles of Cereal and Pseudocereal Flours

Dehydrooligomers of ferulic acid cross‐link polysaccharides such as arabinoxylans and pectic polysaccharides in cereal and certain pseudocereal grains, affecting physiological effects of these fiber components and their physicochemical properties during food processing. An HPLC‐MS method for the analysis of eight diferulic acids and five triferulic acids in low‐lignin samples such as cereal grains and pseudocereals was developed and validated. This method was applied to the analysis of ester‐linked diferulates and triferulates in maize, popcorn, wheat, rye, oats, barley, buckwheat, and amaranth, giving a complete profile of this set of diferulates and triferulates in cereals and pseudocereals. Triferulic acid contents of the cereal flours are roughly 1/10 of the diferulic acid contents, ranging between 23 (oats) and 161 (popcorn) μg/g of flour, with lower amounts for the pseudocereal flours (1–3 μg/g of flour). Dominating trimers are either the 5‐5/8‐O‐4‐ and/or the 8‐O‐4/8‐O‐4‐regioisomers with lower proportions of 8‐8cyclic/8‐O‐4‐, 8‐5noncyclic/8‐O‐4‐, and 8‐5noncyclic/5‐5‐triferulic acids. A unique diferulate pattern was found for buckwheat, with more than 90% of the dimers being 8‐5‐coupled. Amaranth contains an unusually high proportion of 8‐8cyclic‐diferulate, with 27% of the total dimers, whereas oats and barley show comparably high proportions (23%) of the 8‐8tetrahydrofuran diferulate.     

Effects of Laccase and Ferulic Acid on Wheat Flour Doughs

The effects of a laccase from the fungus Pycnoporus cinnabarinus on the mixing of a wheat flour dough with or without added ferulic acid (FA) were studied. Laccase reduced dough time‐to‐peak and accelerated dough breakdown in comparison with the control. Its effect was enhanced with FA. The water extractability of arabinoxylans (AX) increased during mixing of a dough free of added laccase, especially with exogenous FA. At the same time, the extractability of FA decreased during mixing. Added FA may have competed with endogenous AX feruloyl esters, inhibiting partly oxidative gelation. Laccase decreased AX extractability by chain cross‐linking through oxidative dimerization of feruloyl esters. FA and, moreover, FA plus laccase, increased the oxidation of sulfhydryl (SH) groups. FA and, even more, FA in combination with laccase, increased the rate of protein depolymerization during mixing. FA and the products of FA laccase oxidation participated in a redox reaction involving SH groups. A coupling reaction involving enzymatically generated feruloyl radicals and thiol radicals generated through the mechanical breakdown of inter‐chain disulfide bonds might explain these results.     

Regioselective dimerization of ferulic acid in a micellar solution.

Dehydrodimers of hydroxycinnamates play an important role in the cross-linking of plant cell walls. An aqueous solution of quaternary ammonium salts with a long aliphatic chain is known to spontaneously organize itself into micelles with the ionic part at the outer sphere. It is shown that regioisomeric ferulic acid dehydrodimers can be obtained in one step from trans-ferulic acid after attachment to these micelles and using the biomimetic peroxidase-H2O2 system. The surfactant hexadecyltrimethylammonium hydroxide yielded trans-4-(4-hydroxy-3-methoxybenzylidene)-2-(4-hydroxy-3-methoxyphenyl)-5-oxotetrahydrofuran-3-carboxylic acid (25%), (E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid (21%), and trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (14%), whereas the surfactant tetradecyltrimethylammonium bromide gave 4-cis, 8-cis-bis(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-2,6-dione (18%) as the main product. The use of micelles appears to be not only a new way to synthesize regioisomeric ferulic acid dehydrodimers but may also help to understand the regiospecificity of dimeric hydroxycinnamate formation in vivo.     

Enzymatic oxidative treatments of wheat bran layers: effects on ferulic acid composition and mechanical properties

Enzymatic treatments known to induce the gelation of feruloylated arabinoxylans solutions were applied to tissue strips isolated from peripheral layers of wheat grain to tentatively produce in situ arabinoxylan reticulation. The treatments by horseradish peroxidase (HRP) and manganese dependent peroxidase (MnP) induced a dimerization of ferulic acid (FA) in wheat bran with concomitant decrease of arabinoxylan solubility. Similar results were obtained, but to a lesser extent, by simple incubation of bran strips in water, suggesting the action of endogenous peroxidases. The fact that these treatments proved to be ineffective on the isolated aleurone layer and pericarp suggested that dimerization occurred mostly at the aleurone-pericarp interface. In addition, the MnP system generated a consumption of monomer and dimer of ferulic acid in the pericarp, perhaps due to their incorporation into lignin. Micro-mechanical tests using DMTA were performed on isolated tissue strips and showed that oxidation of wheat bran increased their mechanical strength (increase of stress and strain to rupture).     

Stability of Oat Avenanthramides

The three main oat avenanthramides, N‐(4′‐hydroxy)‐(E)‐cinnamoyl‐5‐hydroxyanthranilic acid ( Bp ), N‐(4′‐hydroxy‐3′‐methoxy)‐(E)‐cinnamoyl‐5‐hydroxyanthranilic acid ( Bf ), and N‐(3′,4′‐dihydroxy)‐(E)‐cinnamoyl‐5‐hydroxyanthranilic acid ( Bc ), and their corresponding cinnamic acids, p‐coumaric ( P ), ferulic ( F ), and caffeic ( C ), were investigated for stability to pH, temperature, and UV‐light treatment. The retention of the avenanthramides after processing of oat‐based food products was also analyzed. The avenanthramide Bc and the cinnamic acid C were sensitive to alkali and neutral conditions, especially in combination with heat treatment, whereas the other compounds studied were more stable. The cinnamic acids but not the avenanthramides were isomerized when irradiated with UV‐light. The avenanthramides were restored after processing of oat‐based products.     

Effects of Arabinoxylans on Activation of Murine Macrophages and Growth Performance of Broiler Chicks

Arabinoxylans occur in a wide variety of agricultural products and may contribute a significant portion of human dietary fiber intake. Corn hulls and banana peels are potential sources of arabinoxylans with isolation yields of ≈40 and 10% when extracted with dilute alkali. A broiler chick growth study was performed to determine the effect of extracted corn hull arabinoxylan on performance and attachment of Salmonella, as a representative of an enteric pathogen, to the ileum. Ability of arabinoxylans to activate a macrophage cell line as an immune stimulator was determined by respiratory burst assay. Corn hull arabinoxylan tended to increase body weight gain and reduced attachment of Salmonella to ileal tissue in broiler chicks undergoing mild heat stress. Arabinoxylans from corn hulls and banana peels showed positive oxidative burst in macrophage cells. Collectively, these data indicate the two arabinoxylans have the potential to be used as health‐promoting dietary supplements.     

Solubilization of Arabinoxylans from Isolated Water-Unextractable Pentosans and Wheat Flour Doughs by Cell-Wall-Degrading Enzymes

Water-unextractable pentosans (WUP) isolated from the flours of three wheat cultivars (Apollo, Soissons, Thésée) were treated with enzymes to solubilize the arabinoxylans. The water-unextractable arabinoxylans from the three cultivars had similar susceptibility to solubilization by enzymes: Grindamyl S 100 (GS100), a commercial preparation for baking, rich in pentosanase activities that originated from an Aspergillus niger culture; and three endoxylanases (E1, E2, E3), an arabinofuranosidase (Af), a β- glucanase (βG), and a ferulate esterase (FAE) purified from GS100. A cellulase (C) and a pure endoglucanase (eG) from Trichoderma reesei were also used. GS100 was able to solubilize high molecular weight arabinoxylans (HMWAX) from WUP that markedly enhance the viscosity of the reaction mixture supernatants. The endoxylanase E1 was responsible for this solubilizing activity of GS100, whereas E2 and E3 made only a very low contribution. Combining E1 with FAE led to a limited increase in the arabinoxylan-solubilizing effect. Also, enzymes hydrolyzing cellulose and β-glucans slightly improved the arabinoxylan solubilization from WUP when combined with GS100 or E1, but produced arabinoxylans of lower intrinsic viscosity. Similar effects of the enzymes were observed on arabinoxylan solubilization when applied to dough instead of isolated WUP.     

Feruloylated Wheat Bran Arabinoxylans: Isolation and Characterization of Acetylated and O–2‐Monosubstituted Structures

Arabinoxylan structures vary based on the degree and pattern of substitution of the β‐(1→4)‐linked d ‐xylopyranose backbone with α‐l ‐arabinofuranose units, acetyl groups, uronic acids, and feruloylated side chains. Substitution differences affect arabinoxylans’ physicochemical and physiological characteristics. Wheat bran arabinoxylans were hydrolyzed with GH10 and GH11 endo‐1,4‐β‐xylanases, and feruloylated oligosaccharides were isolated and purified (Amberlite XAD‐2 isolation, Sephadex LH‐20 gel permeation chromatography, and preparative reversed‐phase HPLC). The pure, isolated compounds were structurally characterized via liquid chromatography–electrospray ionization–mass spectrometry and one‐dimensional and two‐dimensional NMR analyses. In addition to the well‐known products of endo‐xylanase hydrolysis (xylotriose and xylobiose O–3‐substituted with a 5‐O‐trans‐feruloyl‐α‐arabinofuranosyl unit on the middle and nonreducing xylose residue, respectively), novel structural features, including O–2‐monosubstitution of xylose adjacent to a xylose carrying feruloylated arabinose, were observed. Additionally, a simultaneously acetylated and feruloylated oligosaccharide has been isolated and tentatively characterized. Oligosaccharides esterified with caffeic acid were also isolated, but these were proven to result, at least in part, as artifacts of the enzymatic hydrolysis.     

FT‐Raman Spectra of Unsoaked and NaOH‐Soaked Wheat Kernels,Bran, and Ferulic Acid

The sodium hydroxide (NaOH) test for determining wheat color class depends on the observation that on soaking in NaOH, red wheat turns a darker red and white wheat turns straw yellow. To understand the mechanism of this test, Raman spectra of wheat bran, wheat starch, ferulic acid, and whole kernels of wheat, before and after NaOH soak, were studied. The major observable components in the whole kernel were that of starch, protein, and ferulic acid, perhaps esterified to arabinoxylan and sterols. When kernels are soaked in NaOH, spectral bands due to ferulic acid shift to lower energy and show a slightly reduced intensity that is consistent with deprotonation of the phenolic group and extraction of a portion of the ferulic acid into solution. Other phenolic acids, alkyl resorcinols, and flavonoids observed in the NaOH extracts of wheat by HPLC were not observed in the Raman spectra. Wheat bran accounts for most of the ferulic acid in the whole kernel, as indicated by the increased intensity of the doublet at 1,631 and 1,600 cm^‐1 in the bran. The intense starch band at 480 cm^‐1 in whole kernel wheat was nearly absent in the wheat bran.     

Identification of 4-O-5'-coupled diferulic acid from insoluble cereal fiber

The extracts of saponified cereal fibers of whole grains of corn (Zea mays cv. microsperma KOERN.), wheat (Triticum aestivum L.), spelt (Triticum spelta L.), and rice (Oryza sativa L.) were investigated for dehydrodimers of ferulic acid using gas-liquid chromatography (GLC) with mass spectrometric detection (GLC-MS) and flame ionization detection (GLC-FID). In addition to the 8,5'-, 8, 8'-, 5,5'-, and 8-O-4'-coupled diferulic acids previously identified from other plant materials the 4-O-5'-coupled diferulic acid (E)-3-[4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy]-4-hydroxy-5-methoxyci nnamic acid (4-O-5'-DFA) was identified in all fibers investigated. This new diferulate was authenticated by comparison of its mass spectrum and its relative GLC retention time with those of the synthesized compound. Semiquantitative determination of 4-O-5'-DFA showed that it is present at 8-30 microg/g, approximately 70-100 times lower concentrations than the sum of 8,5'-coupled diferulic acids, the major diferulic acids in the investigated fibers.     

Novel oxidations of (+)-catechin by horseradish peroxidase and laccase

Horseradish peroxidase (HRP; EC 1.11.1.7) catalyzed the H(2)O(2)-dependent oxidative coupling of (+)-catechin 1 to form three different biphenyl C-C dimers 2-4, whereas Rhus vernicifera laccase catalyzed the formation of two new catechin-hydroquinone adducts 5 and 6. Spectroscopic evidence showed that HRP dimers were linked through position 8 of the A-ring of one catechin moiety to C-5' of ring B in 2 and 4 and to C-2 of ring C in 3. The unusual catechin dicarboxylic acid dimer 4 was obtained by ortho cleavage of the E-ring. Hydroquinone served as both a shuttle oxidant and a reactant by coupling at C-2' and C-5' of the catechin B-ring during laccase oxidations. HRP and laccase oxidation products were compared to D,L-alpha-tocopherol and (+)-catechin for their abilities to inhibit iron-induced lipid peroxidation in rat brain homogenates and Fe(3+)-ADP/NADPH in rat liver microsomes, as measured by the intensity of thiobarbituric acid reactive substance. All metabolites exhibited anti-lipid peroxidation with IC(50) values approximately 2-8 times higher than those of standard compounds. Characteristic reaction products may prove to be novel markers for (+)-catechin antioxidant reactions in living systems.     

Enzymatic solubilization of arabinoxylans from native, extruded, and high-shear-treated rye bran by different endo-xylanases and other hydrolyzing enzymes

The overall objective of this research was to find a new way to valorize rye bran, by producing a gellifier from the enzymatic solubilization of arabinoxylans (AX). The effects of three pure endo-xylanases from Aspergillus niger (Xyl-1), Talaromyces emersonii (Xyl-2), and Bacillus subtilis (Xyl-3) and of Grindamyl S100 (GS100), a commercial enzyme preparation containing a Xyl-1 type endo-xylanase, were tested on rye bran to study the solubilization of water-unextractable arabinoxylans (WUAX). Eight different extrusion-treated rye brans were also used as substrates to find the best physical treatment to facilitate enzymatic arabinoxylan (AX) solubilization. Arabinoxylans were better solubilized from the bran extruded at high temperature using Xyl-3. This enzyme was then tested in combination with pure (1,4)-beta-d-arabinoxylan arabinofuranohydrolase (AXH) and endo-beta-d-glucanase or ferulic acid esterase (FAE), from A. niger. Only beta-glucanase in combination with Xyl-3 improved the AX extraction, but it did not have a marked effect on the viscosity of the extracts. Xyl-3 was then tested on a high-shear-treated rye bran, and results were compared to those obtained with the high-temperature-extruded rye bran. The high-shear treatment did not improve the bran AX enzymatic solubilization. The combination of FAE with Xyl-1 or Xyl-3 did not improve the AX extraction from untreated and high-shear-treated rye bran. Finally, to study the gelation capacity of the enzymatically solubilized AX, the effect of the hydrogen peroxide/horseradish peroxidase (H(2)O(2)/POD) was tested on the Xyl-3 high-temperature-extruded bran extracts. Solubilized AX did not gel in the presence of the oxidizing system.