Ether-linked Ferulic Acid Research Articles

The maize low-lignin brown midrib3 mutant shows pleiotropic effects on photosynthetic and cell wall metabolisms in response to chilling

Maize (Zea mays L.) is one of the major cereal crops in the world and is highly sensitive to low temperature. Here, changes in photosynthetic and cell wall metabolisms were investigated during a long chilling exposure in inbred line F2 and a low-lignin near-isogenic brown midrib3 mutant (F2bm3), which has a mutation in the caffeic acid O-methyltransferase (COMT) gene. Results revealed that the plant biomass was reduced, and this was more pronounced in F2bm3. Photosynthesis was altered in both lines with distinct changes in photosynthetic pigment content between F2bm3 and F2, indicating an alternative photoprotection mechanism between lines under chilling. Starch remobilization was observed in F2bm3 while concentrations of sucrose, fructose and starch increased in F2, suggesting a reduced sugar partitioning in F2. The cell wall was altered upon chilling, resulting in changes in the composition of glucuronorabinoxylan and a reduced cellulose level in F2. Chilling shifted lignin subunit composition in F2bm3 mutant to a higher proportion of p-hydroxyphenyl (H) units, whereas it resulted in lignin with a higher proportion of syringyl (S) residues in F2. On average, the total cell wall ferulic acid (FA) content increased in both genotypes, with an increase in ether-linked FA in F2bm3, suggesting a greater degree of cross-linking to lignin. The reinforcement of the cell wall with lignin enriched in H-units and a higher concentration in cell-wall-bound FA observed in F2bm3 as a response to chilling, could be a strategy to protect the photosystems.

The release and catabolism of ferulic acid in plant cell wall by rumen microbes: A review.

Ferulic acid (FA) is one of the most abundant hydroxycinnamic acids in the plant world, especially in the cell wall of grain bran, in comparison with forage and crop residues. Previous studies noted that FA was mainly linked with arabinoxylans and lignin in plant cell walls in ester and ether covalent forms. After forages were ingested by ruminant animals or encountered rumen microbial fermentation in vitro, these cross-linkages form physical and chemical barriers to protect cell-wall carbohydrates from microbial attack and enzymatic hydrolysis. Additionally, increasing studies noted that FA presented some toxic effect on microbial growth in the rumen. In recent decades, many studies have addressed the relationships of ester and/or ether-linked FA with rumen nutrient digestibility, and there is still some controversy whether these linkages could be used as a predicator of forage digestibility in ruminants. The authors in this review summarized the possible relationships between ester and/or ether-linked FA and fiber digestion in ruminants. Rumen microbes, especially bacteria and fungi, were found capable of breaking down the ester linkages within plant cell walls by secreting feruloyl and p-coumaroyl esterase, resulting in the release of free FA and improvement of cell wall digestibility. The increasing evidence noted that these esterases secreted by rumen microbes presented synergistic effects with xylanase and cellulase to effectively hydrolyze forage cell walls. Some released FA were absorbed through the rumen wall directly and entered into blood circulation and presented antioxidant effects on host animals. The others were partially catabolized into volatile fatty acids by rumen microbes, and the possible catabolic pathways discussed. To better understand plant cell wall degradation in the rumen, the metabolic fate of FA along with lignin decomposition mechanisms are needed to be explored via future microbial isolation and incubation studies with aims to maximize dietary fiber intake and enhance fiber digestion in ruminant animals.

Effect of lignin linkages with other plant cell wall components on in vitro and in vivo neutral detergent fiber digestibility and rate of digestion of grass forages

The objective of this study was to correlate in vitro and in vivo neutral detergent fiber (NDF) digestibility (NDFD) with the chemical composition of forages and specific chemical linkages, primarily ester- and ether-linked para-coumaric (pCA) and ferulic acids (FA) in forages fed to dairy cattle. The content of acid detergent lignin (ADL) and its relationship with NDF does not fully explain the observed variability in NDFD. The ferulic and p-coumaric acid linkages between ADL and cell wall polysaccharides, rather than the amount of ADL, might be a better predictor of NDFD. Twenty-three forages, including conventional and brown midrib corn silages and grasses at various stages of maturity were incubated in vitro for measurement of 24-h and 96-h NDFD. Undigested and digested residues were analyzed for NDF, acid detergent fiber (ADF), ADL, and Klason lignin (KL); ester- and ether-linked pCA and FA were determined in these fractions. To determine whether in vitro observations of ester- and ether-linked pCA and FA and digestibility were similar to in vivo observations, 3 corn silages selected for digestibility were fed to 6 ruminally fistulated cows for 3 wk in 3 iso-NDF diets. Intact samples and NDF and ADF residues of diet, rumen, and feces were analyzed for ester- and ether-linked pCA and FA. From the in vitro study, the phenolic acid content (total pCA and FA) was highest for corn silages, and overall the content of ester- and ether-linked pCA and FA in both NDF and ADF residues were correlated with NDF digestibility parameters, reflecting the competitive effect of these linkages on digestibility. Also, Klason lignin and ADL were negatively correlated with ether-linked ferulic acid on an NDF basis. Overall, esterified FA and esterified pCA were negatively correlated with all of the measured fiber fractions on both a dry matter and an NDF basis. The lignin content of the plant residues and chemical linkages explained most of the variation in both rate and extent of NDF digestion but not uniformly among forages, ranging from 56 to 99%. The results from the in vivo study were similar to the in vitro data, demonstrating the highest total-tract aNDF digestibility (70%; NDF analysis conducted with α-amylase and sodium sulfite) for cows fed the corn silage with the lowest ester- and ether-linked pCA content in the NDF fraction. In this study, digestibility of forage fiber was influenced by the linkages among lignin and the carbohydrate moieties, which vary by hybrid and species and most likely vary by the agronomic conditions under which the plant was grown.

The Effect of Drought on Lignin Content and Digestibility of Tifton-85 and Coastal Bermudagrass (<i>Cynodon dactylon</i> L.) Hays Produced in Georgia

Digestibility of “Tifton 85” Bermudagrass has been noted to be higher than most other Bermudagrass cultivars. However, the superior digestibility of Tifton-85 has not been verified based on samples from producers, nor is it known how water availability might affect this comparison. Recent past weather conditions in Georgia allowed this comparison. Much of Georgia was in severe drought in 2007 and 2008. In contrast, there was less/no drought in 2006 and 2009. In each of these years, producers submitted a substantial number of Tifton-85 and Coastal forage samples to our laboratory for lignin and “Digestible Neutral Detergent Fiber (dNDF48)” analyses. Over all years, Tifton-85 had lower lignin content than coastal. However, Tifton-85 had significantly lower lignin content only in drought free 2006 and 2009, whereas the lignin content of Coastal was unaffected by drought in 2007 and 2008. The lignin of Tifton-85 increased during these two drought years. Despite this, the dNDF48 for Tifton-85 was significantly higher than coastal in all four years, suggesting that drought had hardly any effect on the digestibility of Tifton-85. Apparently, the type of lignin in Tifton-85 is different from that in coastal. Higher dNDF48 for Tifton-85 has been attributed to its lower concentrations of ether-linked ferulic acid than in Coastal. Decreased ether bonding in lignin results in higher digestion.

In situ rumen digestibility of ester-linked ferulic and p-coumaric acids in crop stover or straws in comparison with alfalfa and Chinese wild ryegrass hays

Phenolic acids are widely distributed in graminaceous plant cell walls. Five Charolais×Nanyang crossbred steers, fitted with rumen fistulas, were used as experimental animals. Nylon bags containing corn stover, wheat straw, rice straw, alfalfa hay and Chinese wild ryegrass, commonly used forages on dairy farms in China, were incubated in the rumen for 6, 12, 24, 36, 48 and 72h. Unlike the observations for alfalfa hay, ruminal disappearance of ester-linked ferulic (FAest) and p-coumaric (PCAest) acids in Chinese wild ryegrass and crop stover and/or straw was dependent on the incubation time (P<0.001), and the disappearance of FAest was much greater than that of PCAest. The rapidly degradable fractions (a) of FAest and PCAest were all lower than the insoluble but potentially degradable fractions (b) in all forages except alfalfa hay. The degradation rate (c) of fraction b for both phenolic acid types was highest in alfalfa hay, followed by Chinese wild ryegrass hay, crop straw and/or stover (P<0.001). Consequently, effective degradability (ED) ranked as: alfalfa hay>Chinese wild ryegrass>corn stover>rice straw>wheat straw (P<0.001). Contents of lignin (sa), ether-linked ferulic acid (FAeth) and ether-linked p-coumaric acid (PCAeth) in the forages were negatively correlated with a, b, c and ED values of forage FAest and PCAest degradation (P<0.05). In contrast with alfalfa hay, soluble FAest and PCAest fractions were lower in crop straw and/or stover and Chinese wild ryegrass, and the insoluble phenolic acid fraction can be potentially degraded in the rumen. The degradation of FAest and PCAest in the rumen was dependent on forage FAeth and PCAeth contents, as well as lignin (a) content, in the forages.

In situ ruminal degradation of phenolic acid, cellulose and hemicellulose in crop brans and husks differing in ferulic and p-coumaric acid patterns

SUMMARYLignification-associated phenolic acids are widely distributed in graminaceous plant cell walls. Nylon bags containing maize bran, wheat bran, millet husk and rice husk were incubated in the rumens of five Charolais (♂) × Nanyang (♀) crossbred steers for 6, 12, 24, 36, 48 and 72 h. The in situ ruminal disappearance of ester-linked phenolic acids linearly increased in the brans with increasing incubation time, and the disappearance was greater for ester-linked ferulic acid (FAest) than for ester-linked p-coumaric acid (PCAest). The disappearances of FAest and PCAest were positively correlated with disappearances of neutral detergent fibre (NDF), cellulose and hemicellulose. The effective degradabilities of NDF, cellulose and hemicellulose in the brans were markedly greater than the effective degradabilities of these components in the husks, and were negatively correlated with the contents of Lignin (sa), ether-linked ferulic acid, PCAest and ether-linked p-coumaric acid in both the cereal brans and husks. These findings suggested that breeding forage crops with modified phenolic acid contents could represent an alternative strategy to promote further increases in fibre digestibility of cereal residue feeds for ruminant animals.

The potential to increase digestibility of tropical grasses with a fungal, ferulic acid esterase enzyme preparation

This study examined effects of an enzyme preparation (Depol 740L, Biocatalyst, Pontypridd, UK) containing high esterase (32 U/ml) activity on in vitro digestibility and ruminal in situ degradability of three tropical grass hays. The enzyme was applied at 0, 0.5, 1, 2 and 3 g/100 g dry matter (DM) to ground (1 mm), 12-week regrowths of Pensacola bahiagrass (BAH), Coastal bermudagrass (C-B), and Tifton 85 bermudagrass (T-85) hays in a 3 × 5 factorial experiment replicated thrice. Twenty-four hours later, samples were incubated in triplicate for 6, 24 and 48 h in buffered ruminal fluid in two ANKOM Daisy II incubators The enzyme was also applied at similar rates to ground (4 mm) samples of hays and incubated in duplicate in each of two cows for 0, 3, 6, 9, 12, 24, 48, 72, 96 and 120 h. Enzyme application enhanced (P<0.05) release of WSC from the hays. The 6 and 24 h in vitro dry matter digestibility (IVDMD) of the hays increased linearly (P<0.05) as the enzyme application rate increased. Incremental enzyme addition increased (P<0.05) release of free ester and ether-linked ferulic acid from BAH and T-85, respectively, as well as ester-linked p-coumaric acid from T-85 and BAH. The 48-h IVDMD of C-B increased linearly (P=0.05) as enzyme application increased (487, 484, 489, 506, 510 g/kg) but that of T-85 and BAH did not. The 6-h neutral detergent fiber (NDF) digestibility (IVNDFD) of T-85 increased linearly (P<0.05) with increasing enzyme addition (41, 34, 29, 61, 72 g/kg), whereas that of C-B or BAH did not. In vitro 24-h IVNDFD of hays increased linearly (P<0.01) as enzyme application increased. Incremental enzyme addition increased (P<0.05) the in situ washout fraction of all hays, and the potentially degradable fraction, the extent of degradability, and the effective degradability of C-B. Enzyme application increased (P<0.05) the degradation rate of T-85 and decreased (P<0.01) the lag phase of C-B. This enzyme enhanced in vitro digestion and ruminal in situ degradation of hays, but the pattern and extent of improvement was forage specific.

Quantitative determination of hydroxycinnamic acids in wheat, rice, rye, and barley straws, maize stems, oil palm frond fiber, and fast-growing poplar wood.

A new method has been developed for the quantitative determination of hydroxycinnamic acids participating in ester or ether linkages to the cell wall polymers. The method is based on mild alkaline hydrolysis followed by acid hydrolysis or mild alkaline hydrolysis, which partially removed esterified phenolic acids, and high-temperature concentrated alkaline treatment, which cleaved both the ester and ether linkages. It was found that traditional mild alkaline hydrolysis and acid hydrolysis released only part of the ester- and ether-linked phenolic acids, respectively. Approximately half (44.0-47.9%) of the total ester-linked p-coumaric acid and 18.2-32.6% of the total esterified ferulic acid remained ester-linked to the mild alkali-soluble lignin polymers, and 55.0-72.0% of the total ether-linked p-coumaric acid and 37.5-53.8% of the total ether-linked ferulic acid remained ether-linked to the solubilized lignin molecules after the acid hydrolysis. To correct this, a second mild alkaline hydrolysis of the alkali-soluble lignin preparations and acid hydrolysis of the solubilized lignin fractions, obtained from the first acid hydrolysis of the cell wall materials, was investigated. On the basis of this new method, a majority of the cell wall p-coumaric acid (55.8-81.5%) was found to be ester-linked to cell wall components, mainly to lignin, and about half of the cell wall ferulic acid is etherified through its phenolic oxygen to the cell wall lignin component, whereas the remainder is esterified to the cell wall hemicelluloses and/or lignin in different plant materials.

Effect of enzyme or microbial treatment of bermudagrass forages before ensiling on cell wall composition, end products of silage fermentation and in situ digestion kinetics

Tifton 85 bermudagrass (T85) and Coastal bermudagrass (CBG) established on adjacent plots and managed similarly were harvested after 3 or 6 weeks of regrowth and used to investigate the effects of fibrolytic enzymes or microbial inoculant treatment before ensiling on nutrient composition and recovery, cell wall chemistry and digestion. Prior to ensiling T85 had higher concentrations of neutral detergent fiber (NDF) and acid detergent fiber (ADF), similar concentrations of total lignin, and greater ( p < 0.05) in vitro and in situ dry matter (DM) and NDF disappearances when compared with CBG. Coastal bermudgrass had higher ( p < 0.05) concentrations of acid-insoluble lignin and ether-linked ferulic acid (monomers and dimers), and lower concentrations of glucose and mannose than T85. Treatment of bermudagrass forages with microbial inoculant decreased ( p < 0.05) concentrations of NDF, hemicellulose, butyrate, lactate, cell walls and acid-insoluble lignin, and increased ( p < 0.05) concentrations of ammonia, total volatile fatty acids (VFA) and acetate in silages. Treatment of bermudagrass forages with fibrolytic enzymes had no effect on silage fiber concentration, cell wall carbohydrate fraction or concentration of p-coumaric and ferulic acids, but increased the concentration of butyrate. Among silages, T85 had higher ( p < 0.05) in vitro and in situ dry matter DM and NDF disappearance and higher ( p < 0.05) potentially digestible fractions and smaller ( p < 0.05) indigestible fractions of DM and NDF than CBG. Treatment of bermudagrass forages with fibrolytic enzymes had no effect on in vitro or in situ DM or NDF disappearance of silages. Treatment of bermudagrass forages with microbial inoculant increased in situ DM disappearance at 72 h of incubation ( p < 0.10) and the potentially digestible fraction of DM ( p < 0.05) of silages. Although treatment of bermudagrass forages with microbial inoculant had no effect on silage in vitro or in situ NDF disappearance at 48 h of incubation, it increased in situ NDF disappearance at 72 h ( p < 0.05) and the potentially digestible fraction of NDF. It is concluded that the greater cellulose content of cell walls with the same or less lignin in T85, and the greater concentration of ether-linked ferulic acid in CBG explain the greater digestibility of T85 when compared with CBG at similar stages of maturity. Treatment of bermudagrass forage at ensiling with microbial inoculants may have more potential than extracts of fibrolytic enzymes in improving silage fiber digestion.

Ether-linked ferulic acid amides in natural and wound periderms of potato tuber

The occurrence of ether-linked ferulic acid amides (feruloyltyramine and/or feruloyloctopamine) in suberin-enriched samples of natural and wound periderms of potato tubers was established by thioacidolysis and desulphuration, followed by GC-mass spectrometry of diagnostic dihydroferuloyltyramine. Analysis of samples premethylated in the presence of diazomethane showed that the major part of the ether bonds involved the ferulic moiety of the amides; 20% of the amides released upon thioacidolysis were nevertheless attached to cell walls through the tyramine phenolic group. In addition, dicovalently linked ferulic acid amides were evidenced. These bridges were twice as abundant in the wound than in the natural periderm.

Brown midrib mutant maize with reduced levels of phenolic acids ether-linked to the cell walls

Cell wall composition and rumen degradability of leaf blade, leaf sheath and stem of normal and brown midrib mutant 3 (bmr3) maize ( Zea mays L.) phenotypes were determined in this study. Concentrations of ether-linked phenolic acids were determined by acid and alkaline hydrolysis of whole cell wall materials. The bmr3 maize had higher rumen degradability and lower contents of cellulose, lignin, ferulic and p-coumaric acids esterified and/or etherified to the cell walls, and a lower ratio of xylose to arabinose residues as compared with the normal phenotype. Cellulose degradability was inversely correlated with the acetyl bromide lignin, but not with the acid detergent lignin or Klason lignin. The contents of ether-linked ferulic and p-coumaric acids, whether determined by acid or by alkaline hydrolysis, were inversely correlated with degradability. The bmr3 mutation is characterized not only by a lower lignin content, but also by a reduced level of total ether-linked ferulic acid which is thought to play an important role in cross-linking lignin and cell wall polysaccharides.

Comparison of phenolic acids in lignin fractions from forage grasses before and after digestion by sheep

Three lignin fractions, dioxane-soluble lignin, alkali lignin and acidolysis lignin, were isolated from Italian ryegrass and orchardgrass hay harvested at the heading stage and from faeces of sheep fed on them and analysed for ester-and ether-linked p-coumaric (PCA) and ferulic acid (FA). Chemical analysis and IR spectra confirmed that the lignin fractions obtained were composed mostly of lignin components and accompanied by little carbohydrate. A comparison between the hay and faecal lignin fractions indicated that ester-linked PCA and FA and ether-linked FA were removed or degraded from the grass lignins during sheep digestion. The faecal dioxane-soluble lignin which was released from the grass cell walls by sheep digestion showed the lowest levels of the bound phenolic acids. The results suggested that removal or degradation of bound phenolic acids by sheep digestion occurred more extensively in the lignin fraction solubilized from the grass cell walls than in the insoluble lignin fraction that remained in the grass cell walls.

An approach to the estimation of ferulic acid bridges in unfractionated cell walls of wheat internodes

A modification of a published procedure has been developed to allow the estimation of ester—ether bridges involving hydroxycinnamic acids in unfractionated walls of grasses. The modified procedure involves the use of a soluble catalyst, chlorotris-(triphenylphosphine) rhodium, for the conversion of the propenoic to propanoic sidechains, and lithium triethylborohydride for reduction of ester-linked propanoic acids. The reaction conditions for hydrogenation are severe and tests with a synthetic feruloyl-arabinoxylan showed that significant cleavage of ester-linked hydroxycinnamic acid results. Nevertheless, it was shown that 40% of ether-linked ferulic acid in whole internode walls is involved in ester—ether bridges. If these values are corrrected, based on the loss of feruloyl ester-linkages during the hydrogenation reaction, the proportion of ester—ether-linked ferulic acid in the walls approaches 100%. On the other hand, the occurrence of ester—ether bridges through p-coumaric acid appears to be low.

Comparison of characteristics of soluble lignins from untreated and ammonia-treated wheat straw

Wheat straw ( Triticum aestivum, cultivar ‘Hachimankomugi’) was treated with aqueous ammonia (ammonia level: 30 g kg −1 straw) at 25 °C for 36 days and then air dried. Soluble lignins were isolated with 90% aqueous dioxane from ether-extracted residues of the ammonia-treated and untreated wheat straw samples without ultragrinding and their chemical and physical properties were compared. The ammonia-treated wheat straw showed a higher dry matter digestibility (determined by a nylon bag technique) and yielded a larger quantity of dioxane-soluble lignin than the untreated straw. IR spectra indicated the presence of guaiacyl- and syringylpropane units in the two lignins. The two soluble lignins showed similar molecular weight distributions. The lignin preparation from the ammonia-treated straw contained less p-coumaric acid and much more ferulic acid than that from the untreated straw. Most of the p-coumaric acid in both soluble lignins was present in the ester-linked form, whereas ferulic acid was predominantly in the ether-linked form. The lignin from the ammonia-treated straw was much more abundant in ether-linked ferulic acid than that from the untreated straw. The results provided evidence of the solubilization of the ferulic acid ether-rich lignin fraction from wheat straw by ammonia treatment. Mechanisms of the solubilization of wheat straw lignin and improvement of biodegradability of wheat straw by ammonia treatment were discussed in relation to possible ferulic acid cross-linkages in the cell walls.

CELL WALL-BOUND p-COUMARIC AND FERULIC ACIDS IN ITALIAN RYEGRASS

Cell wall-bound p-coumaric and ferulic acids were determined for stems of Italian ryegrass (Lolium multiflorum Lam.) sampled at different stages of growth. The greater part of the detected p-coumaric acid was linked to cell wall polymers through ester linkages. No definite relationship was found between the accumulation of p-coumaric acid esters and plant maturation. Some part of the detected ferulic acid was simultaneously esterified and etherified to cell wall polymers. Such ester- and ether-linked ferulic acid augmented with increasing maturity, suggesting that the number of ferulic acid cross-links in the cell wall matrices of ryegrass increases with plant maturation.Key words: p-coumaric acid, ferulic acid, Italian ryegrass, cell wall, phenolic acids