Model studies of ferulate-coniferyl alcohol cross-product formation in primary maize walls: implications for lignification in grasses

Ferulate and diferulates mediate cell wall cross-linking in grasses, but little is known about their cross-coupling reactions with monolignols and their role in lignin formation in primary cell walls. Feruloylated primary walls of maize were artificially lignified and then saponified to release ferulate and diferulates and their cross-products with coniferyl alcohol for analysis by GC-FID, GC-MS, and NMR spectroscopy. Ferulate and 5-5-coupled diferulate had a greater propensity than 8-coupled diferulates to copolymerize with coniferyl alcohol, forming mostly 4-O-beta' and 8-beta' and some 8-O-4' and 8-5' cross-coupled structures. Some 8-beta' structures de-esterified from xylans, but these cross-links were subsequently replaced as 8-coupled diferulates formed stable cross-coupled structures with lignin. Based on the incorporation kinetics of ferulate and diferulates and the predicted growth of lignin, cross-products formed at the onset of lignification acted as nucleation sites for lignin polymerization.     

Cross-linking of maize walls by ferulate dimerization and incorporation into lignin

Cross-linking of xylans and lignin by ferulates was investigated with primary maize walls acylated with 2% ferulate and with ferulate ethyl esters. Peroxidase-mediated coupling of wall ferulate and ethyl ferulate yielded mostly 8-coupled products, including three new dehydrodimers. Significant quantities of 5-5-coupled diferulate formed only within walls, suggesting that matrix effects influence dimer formation. Over 60% of wall ferulate dimerized upon H2O2 addition, suggesting that xylan feruloylation is highly regulated during wall biosynthesis to permit extensive dimer formation at the onset of lignification. During lignification, ferulate and 5-5-coupled diferulate copolymerized more rapidly and formed fewer ether-linked structures with coniferyl alcohol than 8-5-, 8-O-4-, and 8-8-coupled diferulates. The potential incorporation of most ferulates and diferulates into lignin exceeded 90%. As a result, xylans become extensively cross-linked by ferulate dimerization and incorporation to lignin, but only a small and variable proportion of these cross-links is measurable by solvolysis of lignified walls.     

Trapping of p-Coumaryl and Coniferyl Alcohol during Soda-Anthraquinone Treatment: A Means of Estimating Uncondensed β-O-4 Structures in Native Lignin

In most native lignins, at least 50% of the phenylpropane (C(9)) units are involved in β-O-4 linkages. It was recently observed that ethylguaiacol (EG) was efficient at trapping coniferyl alcohol generated from the cleavage of uncondensed β-O-4 dimeric structures during soda-anthraquinone (AQ) or SAQ delignification of sugar maple wood meal. Some of the coniferyl alcohol was transformed to vinylguaiacol and isoeugenol, and the α-carbon atom in all three monomers formed C-C bonds with the C-5 position of EG. In the present research, eucalyptus and sugar cane bagasse meals were also investigated, and the yields of uncondensed β-O-4 structures in the nonsyringyl fraction were quantitated. The estimates of the uncondensed fraction of the lignin in the three samples (assuming S units are 90-95% uncondensed) were in close agreement with results from traditional but more tedious methods such as permanganate oxidation or spectroscopic methods requiring a sample representative of native lignin.     

Analysis of technical lignins by two- and three-dimensional NMR spectroscopy

Modern multidimensional NMR spectroscopic methods were applied to investigate the effects of kraft pulping and oxygen delignification on lignin side-chain structures. In addition to the two-dimensional HSQC measurements, the three-dimensional HSQC-TOCSY technique was utilized to elucidate the (1)H-(1)H and (1)H-(13)C correlations of individual spin systems and thus indicate a certain lignin side-chain structure. Unlike earlier, nonlabeled samples were used for 3D measurements. According to 2D and 3D NMR spectra, most of the structures identified in milled wood lignin (MWL) are still present in technical lignins after kraft pulping and oxygen delignification. Although the main reaction during kraft pulping is the cleavage of beta-O-4 linkages, these structures are still left in spent liquor lignin as well as in residual lignin. The amount of coniferyl alcohol and dihydroconiferyl alcohol end groups, as well as some unidentified saturated end groups, is higher in technical lignins than in MWL. Contrary to our earlier observations, no diphenylmethane structures were observed in any technical lignins. Vinyl aryl ether structures could not be detected in technical lignins either.     

Tetramethylammonium hydroxide (TMAH) thermochemolysis of lignin: behavior of 4-O-etherified cinnamyl alcohols and aldehydes

The thermochemolytic behavior of 4-O-etherified cinnamyl alcohols and aldehydes in lignin was investigated in the presence of tetramethylammonium hydroxide (TMAH) (315 degrees C/4 s), using veratrylglycol-beta-(coniferyl alcohol) ether (1a), veratrylglycol-beta-(sinapyl alcohol) ether (1b), and veratrylglycol-beta-(coniferyl aldehyde) ether (2). The methylated products were monitored with gas chromatography-mass spectrometry. Dimers 1a and 1b provided the coniferyl and sinapyl alcohol dimethyl ethers consisting of three isomers, respectively. Coniferyl alcohol dimethyl ether isomers were also observed in the TMAH thermochemolysis pyrolysates of a bulk dehydrogenation polymer of coniferyl alcohol and a Japanese cedar (Cryptomeria japonica) wood. Coniferyl aldehyde methyl ether was not provided from TMAH thermochemolyses of coniferyl aldehyde, 2, a dehydrogenation polymer of coniferyl aldehyde, and the cedar wood. The former three provided veratryl aldehyde in a large abundance, instead of coniferyl aldehyde methyl ether. Sinapyl aldehyde provided 3,4,5-trimethoxybenzaldehyde in a large abundance and sinapyl aldehyde methyl ether in a trace abundance. The results showed that TMAH thermochemolysis is an effective tool to obtain information on cinnamyl alcohol end groups, but is not applicable to analysis of cinnamyl aldehyde end groups.     

Synthesis and characterization of dehydrogenation polymers in Gluconacetobacter xylinus cellulose and cellulose/pectin composite

To mimic the lignin polymerization process, mats of bacterial cellulose and of a pectin/cellulose composite were used as a host matrix for in vitro polymerization of coniferyl alcohol. A diffusion cell was used to allow the diffusion of both hydrogen peroxide and coniferyl alcohol into the peroxidase impregnated cellulose mats through dialysis membranes. The results indicate that significant polymerization occurs within the mats. The resulting binary and ternary blends were imaged by scanning electron microscopy (SEM) and characterized by chemical means. The presence of pectin induces a better dispersion of the synthetic lignin in the cellulose network and enhances the proportion of alkyl-aryl-ether in the polymer.     

The behavior of deuterium-labeled monolignol and monolignol glucosides in lignin biosynthesis in angiosperms

To examine the behavior of monolignol and monolignol glucosides in lignin biosynthesis, pentadeutero[9-D(2), 3-OCD(3)]coniferyl alcohol and pentadeutero[9-D(2), 3-OCD(3)]coniferin were synthesized and fed to growing Eucalyptus camaldulensis and Magnolia kobus. The differences in the incorporation patterns of these labeled precursors were studied using gas chromatography-mass spectrometry (GC-MS). Both precursors were incorporated into lignin, but the labeled coniferyl alcohol was incorporated more directly, resulting in a high proportion of pentadeutero-labeled guaiacyl and syringyl units in newly formed xylem, while labeled coniferin tended to be incorporated in lignin as tetradeutero units, especially in syringyl lignin in both trees. However, the incorporation efficiencies of the precursors into syringyl lignin were higher in Magnolia than in Eucalyptus, and the ratios of tetradeutero to pentadeutero in guaiacyl lignin were lower in Magnolia than in Eucalyptus when the trees were fed coniferin.     

The effect of in vitro digestion on steryl ferulates from rice (Oryza sativa L.) and other grains

Polished and cargo rice, wild rice, rice bran, corn bran, and wheat bran were subjected to a static in vitro digestion model, to monitor changes in their steryl ferulate content and composition. Free sterols, possible hydrolysis products of steryl ferulates, were also measured. Additionally, steryl ferulate bioaccessibility was calculated as the percentage of steryl ferulates liberated from the grain matrix into the digestive juice. Steryl ferulate content ranged between 6.1 and 3900 μg/g and decreased by 1-63% due to digestion. A parallel increase in free sterols of more than 70% was observed in all samples. Additionally, bioaccessibility of steryl ferulates was found to be almost negligible. These findings suggest that intestinal enzymes immediately hydrolyze steryl ferulates, which are liberated from the grain matrix, and thus they are practically unavailable for absorption in the small intestine. This further indicates that the hydrolysis products of steryl ferulates could be bioactive in the gut.     

Whole Grains and Digestive Health

Whole grains contain all parts of the grain: the endosperm, germ, and bran. Whole grains are rich in fermentable carbohydrates that reach the gut: dietary fiber, resistant starch, and oligosaccharides. Most research that supports the importance of grains to gut health was conducted with isolated fiber fractions, rather than whole grains. Whole grains are an important source of dietary fiber and grain fibers such as wheat, oats, barley, and rye increase stool weight, speed intestinal transit, get fermented to short chain fatty acids, and modify the gut microflora. Wheat bran is particularly effective in increasing stool weight; wheat bran increases stool weight by a ratio of 5:1. In contrast, many novel fibers that are easily incorporated into beverages and foods increase stool weight only on a ratio of 1:1. In vitro fermentation studies with whole grains have been published. Carbohydrates of oat bran (rich in β‐glucan) were consumed by bacteria faster than those of rye and wheat brans (rich in arabinoxylan). Grain fibers were fermented more slowly than inulin, causing less gas production. Wheat is particularly high in fructo‐oligosaccharides, while wheat germ is high in raffinose oligosaccharides. Some in vivo studies show the prebiotic potential of whole grains. Whole grain breakfast cereal was more effective than wheat bran breakfast cereal as a prebiotic, increasing fecal bifidobacteria and lactobacilli in human subjects. Wheat bran consumption increased stool frequency. Thus, the gut enhancing effects of cereal fibers are well known. Limited data exist that whole grains alter gut health.     

The identification of dilignols from dehydrogenation mixtures of coniferyl alcohol and apocynol [4-(1-Hydroxyethyl)-2-methoxyphenol] by LC-ES-MS/MS

A LC-ES-MS/MS method for the identification of dilignols formed by the oxidative cross-coupling of coniferyl alcohol and apocynol has been developed. The identification is based on the generation of ammonium adduct ions [M + NH(4)](+) by electrospray ionization and thereafter the following fragmentation patterns for the selected precursor ions. Fragmentation of several arylglycerol-beta-aryl ether (beta-O-4) and phenyl coumarane (beta-5) model compounds were studied as a reference.     

NMR evidence for benzodioxane structures resulting from incorporation of 5-hydroxyconiferyl alcohol into Lignins of O-methyltransferase-deficient poplars

Benzodioxane structures are produced in lignins of transgenic poplar plants deficient in COMT, anO-methyltransferase required to produce lignin syringyl units. They result from incorporation of 5-hydroxyconiferyl alcohol into the monomer supply and confirm that phenols other than the three traditional monolignols can be integrated into plant lignins.     

Chemical characterization of lignin and lipid fractions in kenaf bast fibers used for manufacturing high-quality papers

The chemical composition of lignin and lipids of bast fibers from kenaf (Hibiscus cannabinus) used for high-quality paper pulp production was studied. Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) of fibers showed a lignin with a high syringyl/guaiacyl ratio (5.4) and minor amounts of p-hydroxyphenyl units. Simultaneously, sinapyl and coniferyl acetates were also identified, indicating that this lignin is partially acetylated. p-Hydroxycinnamic acids were found in only trace amounts. The main lipids identified by GC/MS of extracts from kenaf fibers were series of long-chain n-fatty acids, waxes, n-alkanes, and n-fatty alcohols. Free and esterified sterols and triterpenols, steroid hydrocarbons, and steroid and triterpenoid ketones, as well as steryl glycosides, were also found. Finally, the fate of the main constituents of kenaf fibers in alkaline pulping was also investigated.     

Chemical characterization of Klason lignin preparations from plant-based foods

To analyze the accuracy of the Klason lignin method as applied for the determination of lignin contents in plant based-food products, Klason lignin preparations from curly kale, pears, whole wheat grains, and corn bran were chemically characterized. Characterization included routine ash and protein determinations and the extraction of fat/waxes as well as cutin/suberin depolymerization and extraction of the liberated monomers. Fat/wax and cutin/suberin amounts in the Klason lignin preparations were determined gravimetrically, and their compositions were analyzed by using GC-MS. Typical fat, wax, and cutin (and suberin) constituents such as saturated and unsaturated fatty acids, hydroxy and/or epoxy fatty acids, and phenolic acids were identified in all samples, whereas the detection of long-chain hydrocarbons, alcohols, and ketones, sterols, stanols, and dioic acids was dependent on the sample analyzed. Estimation of the contribution of non-lignin compounds to the Klason lignin contents reduced the noncorrected Klason lignin contents of the insoluble fibers from 28.7% (kale), 22.8% (pear), 14.8% (wheat), and 9.9% (corn) to maximum lignin contents of 6.5% (kale), 16.4% (pear), 4.9% (wheat), and 2.3% (corn). These data demonstrate that certain commonly used statements such as "cereal brans are highly lignified" need to be revised.     

Chemical composition of abaca (Musa textilis) leaf fibers used for manufacturing of high quality paper pulps

The chemical composition of leaf fibers of abaca (Musa textilis), which are commonly used for high-quality paper pulp production, was thoroughly studied. The results revealed that the lignin content was 13.2% of the total fiber. The analysis of abaca fibers by pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS) released predominantly compounds arising from lignin and p-hydroxycinnamic acids, with high amounts of 4-vinylphenol. The latter compound was demonstrated to arise from p-coumaric acid by pyrolysis of abaca fibers in the presence of tetramethylammonium hydroxide, which released high amounts of p-coumaric acid (as the methyl derivative). Products from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) propanoid units, with a predominance of the latter (H:G:S molar ratio of 1.5:1:4.9), were also released after Py-GC/MS of abaca fibers. Sinapyl and coniferyl acetates, which are thought to be lignin monomer precursors, were also found in abaca. The extractives content of the abaca fiber (0.4%) was low, and the most predominant compounds were free sterols (24% of total extract) and fatty acids (24% of total extract). Additionally, significant amounts of steroid ketones (10%), triglycerides (6%), omega-hydroxyfatty acids (6%), monoglycerides (4%), fatty alcohols (4%), and a series of p-hydroxycinnamyl (p-coumaric and ferulic acids) esterified with long chain alcohols and omega-hydroxyfatty acids were also found, together with minor amounts of steroid hydrocarbons, diglycerides, alpha-hydroxyfatty acids, sterol esters, and sterol glycosides.     

Detection and determination of p-coumaroylated units in lignins.

The derivatization followed by reductive cleavage (DFRC) method cleaves alpha- and beta-ethers in lignins but leaves lignin gamma-esters intact. When applied to grasses, which contain p-coumarate esters on their lignins, esterified monolignol derivatives are released. Saturation of the p-coumarate double bond occurs during DFRC, so the released products are 4-acetoxycinnamyl 4-acetoxyphenylpropionates. Synthesis of the esters allowed determination of response factors for the released products. Maize and bamboo lignins released 221 and 38 micromol/g of p-coumarate-derived esters. The sinapyl ester was much more abundant than the coniferyl one. The bamboo and maize lignin S/G ratios in the conjugates were 12 and 38 times greater than those of the normal monomers released by DFRC, evidence of a strong selectivity for acylation of syringyl units. Of three possible biochemical mechanisms for incorporating p-coumarates into lignin, evidence is mounting that the process involves incorporation of preacylated monolignols into the normal lignification process.     

Sinapate dehydrodimers and sinapate-ferulate heterodimers in cereal dietary fiber

Two 8-8-coupled sinapic acid dehydrodimers and at least three sinapate-ferulate heterodimers have been identified as saponification products from different insoluble and soluble cereal grain dietary fibers. The two 8-8-disinapates were authenticated by comparison of their GC retention times and mass spectra with authentic dehydrodimers synthesized from methyl or ethyl sinapate using two different single-electron metal oxidant systems. The highest amounts (481 microg/g) were found in wild rice insoluble dietary fiber. Model reactions showed that it is unlikely that the dehydrodisinapates detected are artifacts formed from free sinapic acid during the saponification procedure. The dehydrodisinapates presumably derive from radical coupling of sinapate-polymer esters in the cell wall; the radical coupling origin is further confirmed by finding 8-8 and 8-5 (and possibly 8-O-4) sinapate-ferulate cross-products. Sinapates therefore appear to have an analogous role to ferulates in cross-linking polysaccharides in cereal grains and presumably grass cell walls in general.     

Differences between lignin in unprocessed wood, milled wood, mutant wood, and extracted lignin detected by 13C solid-state NMR

Solid-state 13C nuclear magnetic resonance (NMR) spectroscopy was applied to intact and isolated loblolly pine wood samples to identify potential structural changes induced by tree age, milling, lignin extraction, or naturally occurring mutations. Special attention was paid to ketone and aldehyde as well as nonpolar alkyl groups, which could be observed at low concentrations (<2 in 1000 C) using improved spinning-sideband suppression with gated decoupling. Carbonyl structures were present in intact wood, and there are more keto groups than aldehydes. Their concentrations increased from juvenile to mature wood and with milling time, whereas extraction did not alter the C=O fraction. Significant amounts of aldehyde and dihydroconiferyl alcohol residues were present in coniferyl aldehyde dehydrogenase-deficient wood, confirming solution-state NMR spectra of the corresponding lignin. These results demonstrate the utility of solid-state NMR as an assay for changes in the lignin structure of genetically modified plants.     

Apoplastic pH and monolignol addition rate effects on lignin formation and cell wall degradability in maize

Monolignol polymerization rate and apoplastic pH and may influence the formation of lignin and its interactions in cell walls. Primary maize walls were artificially lignified by gradual "end-wise" or rapid "bulk" polymerization of coniferyl alcohol at pH 4 or 5.5. Lignification efficiency was greatest for end-wise polymers at pH 5.5 (90-98%), intermediate for bulk polymers formed at either pH (54-82%), and lowest for end-wise polymers at pH 4 (41-53%). End-wise polymers had about 2.2-fold more ether inter-unit linkages and 70% fewer end-groups than bulk polymers. Low pH enhanced the formation of ether linkages in end-wise but not in bulk polymers. Differences in lignin structure did not influence the enzymatic degradability of cell walls, but lowering apoplastic pH from 5.5 to 4.0 during lignification reduced cell wall degradability by 25%. Further studies indicated this pH-dependent depression in degradability was related to cell wall cross-links formed via lignin quinone methide intermediates.     

NMR characterization of lignins isolated from fruit and vegetable insoluble dietary fiber

Compositional information for lignins in food is rare and concentrated on cereal grains and brans. As lignins are suspected to have important health roles in the dietary fiber complex, the confusing current information derived from nonspecific lignin determination methods needs to be augmented by diagnostic structural studies. For this study, lignin fractions were isolated from kiwi, pear, rhubarb, and, for comparison, wheat bran insoluble dietary fiber. Clean pear and kiwi lignin isolates allowed for substantive structural profiling, but it is suggested that the significance of lignin in wheat has been overestimated by reliance on nonspecific analytical methods. Volume integration of NMR contours in two-dimensional (13)C-(1)H correlation spectra shows that pear and wheat lignins have comparable guaiacyl and syringyl contributions and that kiwi lignins are particularly guaiacyl-rich (approximately 94% guaiacyl) and suggest that rhubarb lignins, which could not be isolated from contaminating materials, are as syringyl-rich (approximately 96% syringyl) as lignins from any known natural or transgenic fiber source. Typical lignin structures, including those newly NMR-validated (glycerols, spirodienones, and dibenzodioxocins), and resinols implicated as possible mammalian lignan precursors in the gut are demonstrated via their NMR correlation spectra in the fruit and vegetable samples. A novel putative benzodioxane structure appears to be associated with the kiwi lignin. It is concluded that the fruits and vegetables examined contain authentic lignins and that the detailed structural analysis exposes limitations of currently accepted analytical methods.