Cell wall composition and degradability of forage stems following chemical and biological delignification

Chemical and biological delignification methods were used to investigate the relationship between the concentration and composition of lignin and degradation of forage cell walls. Stem material from lucerne (Medicago sativa L), smooth bromegrass (Bromus inermis Leyss) and maize (Zea mays L) stalks was treated with alkaline hydrogen peroxide, potassium permanganate, sodium chlorite, sodium hydroxide, nitrobenzene, and the lignolytic fungus Phanerochaete chrysosporium. Klason lignin and esterified and etherified phenolic acids were delermined. Cell wall neutral sugar and uronic acid composition and the extent of in-vitro degradability were measured. Chemical delignification generally removed lignin. but the fungal treatment resulted in the removal of more polysaccharide than lignin. The concentrations of esterfied and etherified p-coumaric and ferulic acids were generally reduced in treated cell walls; chlorite treatment preferentially removing p-coumaric acid whereas nitrobenzene treatment removed more ferulic acid. Syringyl moieties were completely removed from the core lignin polymer by nitrobenzene treatment of forage stems. Alkaline hydrogen peroxide and nitrobenzene were generally the most effective delignification treatments for improving polysaccharide degradability, with the grass species responding similarly to delignification whereas lucerne was somewhat less responsive. Fungal delignification, under these experimental conditions, did not improve cell wall degradability of these forages. Multiple regression and covariate analyses indicated that the lignin components measured were not powerful predictors of cell wall degradability. Neither the concentration nor the composition of the lignin fractions was consistently correlated with degradation. This lack of effect was attributed to the more generalised disruption of the cell wall matrix structure by delignification treatments.     

改良(Ox-Dem)卡伯值：更加准确估计纸浆中残余木素的含量

在现行的卡伯值测定方法之前采用羟汞化—脱汞两段预处理 ,可以将浆中的己烯糖醛酸基和其他干扰测定的碳水化合物衍生结构的基团除去 ,这种改良卡伯值测定方法能够测得纸浆中真正的木素含量     

Effects of Sodium Fertiliser on the Chemical Composition of Grass and Clover Leaves,Stems and Inflorescences

Sodium fertiliser was applied to dairy cow pasture to investigate the effects on the chemical composition of grass and clover leaves, stems and inflorescences. The effect of sodium fertiliser varied between fractions, with grass leaves and stems generally being more responsive than grass inflorescences or clover fractions. The sodium content of all fractions was already high in the control treatment, and was increased to an excessive level by sodium fertiliser in all fractions except grass stems. Grass leaves were most affected. Potassium content was reduced in grass leaves and stems, but not in other fractions. The effects on calcium and magnesium varied between fractions but the K: (Ca+Mg) ratio, the most sensitive index of susceptibility to hypomagnesaemia, was reduced in grass leaves, but increased in other fractions. DM digestibility was decreased in grass and clover leaves by sodium, probably because of the high sodium content. MAD fibre content was also increased in these fractions. Crude protein content was decreased by sodium fertiliser in grass leaves, in particular. Therefore, when applied to pasture which is naturally high in sodium, sodium fertiliser had detrimental effects on pasture macronutrient content. However, beneficial effects were still observed on mineral ratios for plant and animal nutrition. It is concluded that if sodium fertiliser increases herbage sodium concentrations above 5 g kg⁻¹ DM, improvements in mineral ratios that could affect ruminant health may be offset by reductions in the nutritive value of the herbage. It is therefore recommended that the herbage sodium concentration should be known before sodium fertiliser is applied.     

Lignification of switchgrass (Panicum virgatum) and big bluestem (Andropogon gerardii) plant parts during maturation and its effect on fibre degradability

Five cultivars of switchgrass (Panicum virgatum L) and four cultivars of big bluestem (Andropogon gerardii Vitman) were harvested at vegetative, boot and heading stages of maturity. Leaf and stem fractions were analysed for detergent fibre composition and 48-h ruminal in-vitro degradability, ester- and ether-linked non-core lignin phenolic acids, and core lignin composition. Big bluestem leaves contained more neutral detergent fibre than switchgrass, but general composition of the fibre did not differ. Stem fibre of switchgrass had relatively lower levels of cellulose and lignin at the vegetative stage than observed in big bluestem. Esterified and etherified p-coumaric and ferulic acid concentrations were generally higher in switchgrass plant parts. Yield of nitrobenzene oxidation products from core lignin was greater for switchgrass leaves, but very little difference in composition was noted. Leaf tissue contained lower concentrations of all lignin components than stems. Maturation resulted in increased total lignification, but all components did not respond in the same manner. Variation for all measures of lignification seemed to be as great within species as between the grass species. Degradability of fibre declined with maturation. The only species difference was that switchgrass fibre was more degradable at the vegetative stage. Relationships between lignification and fibre degradability were in agreement with some, but not all, previously reported relationships. Concentration of core lignin was only a significant predictor of fibre degradability when the relationship was examined across maturity stages. Within a stage of maturity, lignin composition was more closely related to fibre degradability than was concentration. However, the best predictors of fibre digestibility differed among species, plant part and maturity.     

Biological variability in lignification of maize: Expression of the brown midrib bm2 mutation

The cell wall phenolic components in the internodes of three maize genotypes, namely normal, bm2 and bm3 maize, were determined. The bm2 and bm3 brown midrib mutations lowered the lignin content of the bottom, middle and top internodes to a similar extent. However, unlike bm3, the bm2 trait did not induce a sharp reduction of the level of ester-bound p-coumaric acid in maize internodes. The other main alkali labile phenolic acid, ferulic acid, reached similar levels in the three genotypes. The main difference between bm2 and bm3 mutations occurred in the alkyl aryl ether linked structures of the lignin component. In contrast to bm3 lignins, which are characterised by a low syringyl content, the bm2 lignin had a lower content of guaiacyl units than lignin of normal maize internode. Consequently, the syringyl/guaiacyl molar ratio of bm2 lignin gave higher values (2.7–3.2) than those from either normal (0.9–1.5) or bm3 lignins (0.3). The alkali solubility of lignin was also compared between the three genotypes. Incorporation of the bm3 trait in maize led to a high recovery of alkali soluble lignin whereas the bm2 lignin had a similar solubility to the normal one in 2 M NaOH. The monomeric composition of the alkali soluble lignins was consistent with the non-condensed structures of the in-situ polymer. Although the bm3 and bm2 mutations had different effects on lignification, the modification of the cell wall phenolic level was also found in the bm2 maize stem as previously studied.     

Degradation rate and chemical composition of different types of alkali-treated straws during rumen digestion

The degradation in the rumen and change in chemical composition of untreated and four samples of alkali-treated barley straw were studied in farm-scale treatments with ammonia, ‘dry’ sodium hydroxide and two different aqueous sodium hydroxide systems. The alkali treatments increased the fraction soluble in organic solvents and decreased the lignin content (determined as Klason lignin). The amounts of cellulose and arabinoxylans were not significantly changed. The degree of rumen degradability increased with the order of alkaline treatment given above. The degradation rates of dry matter and the NDF fraction were parallel to each other but varied considerably with the treatments. The two most efficient aqueous alkaline treatments showed a degradation rate four times that of untreated straw. For these two samples the arabinoxylans were solubilised faster than cellulose during the first part of the incubation. In the other samples the degradation of arabinoxylans and cellulose ran almost parallel. During the rumen incubation the lignin content of the straw residues increased for all samples. There was an inverse relationship between the content of p-coumaric and ferulic acids on the degree of digestibility. A relationship between acetyl groups and digestibility was not found.     

Phenolic acids in fresh and retted jute plants (Corchorus capsularis and Corchorus olitorius)

Fresh and retted bark and stem of the Corchorus capsularis and Corchorus olitorius species of jute plant were analysed for their phenolic acid contents. Free and bound phenolic acids were identified and quantitatively determined by means of HPLC. Ferulic and p-coumaric acids were the main components, whereas p-hydroxybenzoic, protocatechuic, vanillic and caffeic acids were present in varying amounts in different jute fractions. The stem contained more free phenolic acids than the bark, and the differences were very pronounced in the retted samples. On the other hand, the bark from all four samples contained more extractable ester-bound phenolic acid than the stem. The stem of fresh C. olitorius and retted bark (fibre) of both species contained more non-extractable bound phenolic acids, in particular p-coumaric acid, than the other fractions.     

Apparent inhibition to digestion by lignin in normal and brown midrib stems

Lignin, a cell wall component, limits digestibility of plant cell walls. Brown midrib (bmr) mutants of forages have lignin with altered chemical composition compared with their normal counterparts. The objectives of this study were to determine if bmr lignin is more inhibitory to digestion than is normal lignin and if bmr has a consistent effect on rate of digestion across species and environments. Extent and rate of in-vitro cell wall digestion of normal and bmr stems of sorghum (Sorghum bicolor (L) Moench, two comparisons), millet (Pennisetum americanum (L) Leeke) and maize (Zea mays L, two comparisons) were determined. Samples were incubated in rumen fluid, and data were fitted with a first-order, nonlinear model to estimate concentrations of potentially digestible neutral detergent fibre (PDNDF), digestion rate of PDNDF, concentration of indigestible residue (IR), and lag time before digestion. The NDF, acid-detergent fibre (ADF), and acid-detergent lignin (ADL) analyses were conducted sequentially on undigested samples. The IR: ADL ratio was 37% greater for bmr than for normal plants, which indicates that bmr lignin inhibits digestion more than normal lignin per unit of lignin. Digestion rate of PDNDF was faster in bmr than in normal counterparts in one of the two sorghum comparisons (difference of 59%) and in the millet comparison (difference of 27%), but in neither maize comparison. The bmr mutants were lower than normal genotypes in NDF (9%) and ADL (47%) concentrations. The PDNDF concentration was 19% greater for bmr than for normal lines. Thus, decreased lignin concentration in bmr mutants increased the extent of NDF digestion but did not consistently increase the rate of digestion.     

不同成熟期蜜桔中酚酸的组成与分布

酚酸具有多种生理功能,在植物中广泛存在.柑橘可能是膳食酚酸的重要来源.采用HPLC-PDA测定了不同成熟期温州蜜桔中八种酚酸的含量,即绿原酸、咖啡酸、对香豆酸、阿魏酸、芥子酸、原儿茶酸、对羟基苯甲酸和香草酸.结果显示,果肉中酚酸含量随成熟度提高而减少,果皮酚酸含量在半成熟期最高;果皮中酚酸含量大大高于果肉;果皮果肉中都以肉桂酸型酚酸为主,苯甲酸型酚酸含量较少;蜜桔果实中绿原酸和阿魏酸是所测八种酚酸中含量最高的.对柑橘中酚酸组成与分布的研究可望应用于食品营养标识、柑橘品种的识别或加工果汗掺假的识别等.     

泔水油制备脂肪酸甲酯的研究

研究了泔水油以甲醇钠和HCl为催化剂的作用下与甲醇反应，生成脂肪酸甲酯过程。在制备脂肪酸甲酯前对泔水油进行预处理。通过对甲醇钠用量、HCl甲醇的用量和反应时间等单因素的考查，应用正交实验得到泔水油制备脂肪酸甲酯实验的最佳反应条件即：甲醇钠用量40mL，盐酸甲醇用量40mL，HCl钠作催化剂时反应时间为20min，HCl甲醇作催化剂时反应时间为15min。     

Effects of sodium hydroxide on cereal straws in relation to the enhanced degradation of structural polysaccharides by rumen microorganisms

The action of sodium hydroxide on the rate and extent of loss of wall polysaccharides from wheat and barley straws incubated in the rumen of the sheep was studied using a nylon bag technique. Cellulose loss reached a maximum in straws treated with 10 g NaOH 100 g^?1 straw. Further increase in amounts of alkali applied resulted in additional solubilisation of straw constituents, particularly xylan, but did not further enhance cellulose degradation. Rates of degradation of isolated straw, cotton and wood celluloses correlated with three of four measures of cellulose crystallinity obtained by X-ray diffraction and infrared analysis. Treatment of cellulose samples with 20 g NaOH 100 g^?1 cellulose did not affect rates of loss or produce detectable changes to cellulose morphology. Loss of acetic acid esters, which accompanied alkali treatment, did not relate to improvements to digestibility. The rate of loss of cellulose from delignified neutral detergent fibre (NDF) was that shown by the isolated straw cellulose, despite the presence of acetylated hemicellulose. Deacetylation of delignified NDF with NaOH or sodium methoxide did not enhance rates of xylose or cellulose loss. In lignified wall material (NDF) the molar ratio acetyl:xylose (approximately 1:3.5) and proportion of the major phenolic acids (p-coumaric and ferulic acids) remained essentially constant throughout the course of degradation in the rumen. It is suggested that loss of phenolic acids, while not directly contributing to digestibility enhancement, may model the susceptibility of ester linkages formed between polysaccharide and polyphenolic material to alkali. The extent of solubilisation of p-coumaric acid produced by NaOH showed a linear relationship with cellulose digestibility. An upper limit of 40% acid detergent lignin and 55% of total phenolics were released by NaOH, the extent of release closely paralleling cellulose digestibility. Phenolic material resistant to alkali remained associated with wall polysaccharides and was not released from nylon bags until over 50% of cellulose (and hemicellulose) had been degraded.     

Effect of ozone and sodium hydroxide treatments on some chemical characteristics of cotton straw

Cotton straw (CS) was treated with ozone or sodium hydroxide, and the effect of treatments on the organic acid and monosaccharide composition and on the chromat-ographic characteristics of alkali lignin was studied. The concentrations of formic and acetic acids in the ozonised CS were 3.98 and 1.36 g 100 g^?1 organic matter (OM) respectively. Oxalic acid was not detected. A high content of uronic acids (9.78 g 100 g^?1 OM) was found in CS. The overall decrease in total monosaccharides in the ozonated CS was very small (5%) with the matrix constituents being principally affected. The 8% sodium hydroxide treatment exerted a very limited effect on the composition of CS cell walls. Ozonation increased the content of soluble carbohydrates from 9.2 to 21.8%; this was contributed mainly by the hemicellulose fraction. Alkali lignin was extracted from CS and was fractionated by gel chromatography. The elution pattern of alkali lignin was very similar for the treated and untreated materials. The three peaks coincided at an average mol. wt of 40 000, as judged from the use of dextran standards.     

Four phenolic acids determined by an improved HPLC method with a programmed ultraviolet wavelength detection and their relationships with lignin content in 13 agricultural residue feeds

BACKGROUND: Lignification‐associated phenolic acids are widely distributed in graminaceous plant cell walls. This study developed a rapid and sensitive reversed‐phase method for the simultaneous quantification of protocatechuic (PRA), vanillic (VA), ferulic (FA) and p‐coumaric (PCA) acids and investigated the relationship between these compounds and lignin contents in 13 fibrous feeds. RESULTS: The phenolic acids were identified at a column temperature of 15 °C in a single run, in which the wavelength was programmed at 260 nm for PRA and VA, then switched to 310 nm for FA and PCA determinations. Satisfactory precision, recovery, and linearity were obtained with this method. Among 13 feeds, PCA was most abundant, followed by FA, VA and PRA. Great variations in phenolic acid and lignin contents were found. FA content was much richer than PCA content in maize and wheat brans, and the highest PCA content occurred in maize stalks. Lignin content was correlated with proportions of FA (r = ? 0.95) and PCA (r = 0.90) in the summed phenolic acids and the PCA:FA ratio (r = 0.91). CONCLUSION: The improved method appears to be useful for simultaneous quantification of target phenolic compounds. Both FA and PCA may be good indicators for plant cell wall lignification associated with feed digestibility. Copyright © 2012 Society of Chemical Industry     

Relationship of cell wall composition to in vitro cell wall digestibility of maize inbred line stems

The phenolic equipment of maize stem tissues was investigated in relation to the feeding value of the detergent fibre components. Sixteen maize inbred lines, including three brown‐midrib 3 mutants and their normal counterparts, were selected for highly divergent in vitro cell wall digestibility. These lines were grown during two years. Maize stems were analysed for detergent fibre concentration, esterified and etherified p‐hydroxycinnamic acids, lignin content and structure and in vitro digestibility. A large genotypic variation was found for neutral detergent fibre, cell wall phenolic composition and cell wall digestibility. Within the normal maize lines the in vitro neutral detergent fibre digestibility (IVNDFD) of stem fractions was negatively correlated with their Klason lignin content. A multiple regression model based on esterified p‐coumaric acid and lignin composition as two explanatory variates accounted for 58% of the IVNDFD variation. In this study, three normal maize inbred lines displaying a lignin content and a cell wall digestibility level close to those observed in the three bm3 lines could be detected, which opens up new breeding avenues. © 2000 Society of Chemical Industry     

Relationship between structural development of cell walls and degradation of tissues in maize stems

Sections of internodes of growing maize stems were used to study the behaviour of cell walls of different tissues during in-vitro degradation with rumen fluid. Tissues with primary cell walls-middle lamellae, at early stages of development, were degraded completely. In specific tissues, newly synthesised secondary walls were highly digestible whereas the primary walls-middle lamellae of these tissues were indigestible. These primary walls-middle lamellae stained positively with acid phloroglucinol but showed no fluorescence. At pollination, when secondary walls were of considerable thickness, these walls were still completely digestible even though they stained intensely with acid phloroglucinol and showed reduced fluorescence. However, at some distance from the cut end of sections, the secondary walls of the elongated tube-like cells of sclerenchyma tissue showed considerable reduction in digestibility. Cross-sectional area and dry weight measurements of different stem tissues revealed the importance of secondary wall digestion of sclerenchyma compared with the thin-walled parenchyma. Chemical treatment with KmnO₄ or NaOH resulted in colourless secondary walls after staining whereas primary walls still reacted positively. It was concluded that very small amounts of phenolic compounds (lignin) located in the primary wall-middle lamella that are not removed by KmnO₄ and NaOH treatment are responsible for the decrease in digestibility of tissues during plant development. Histochemical ‘lignin’ reactions and fluorescence just detect phenolic compounds and cannot be correlated with degradation.     

Biological variability in lignification of maize: Expression of the brown midrib bm3 mutation in three maize cultivars

Cell wall-linked phenolics were investigated in maize internodes located at three positions of the stem (top-middle-bottom). While the lignin content did not change drastically with position, the amount of cell wall-ester linked p-coumaric acid sharply increased from the top to the bottom internodes of the stem. Conversely, the saponified ferulic acid content remained relatively unchanged along the stem. Moreover, the highest syringyl content of the β-O-4-lignin structures was found in the basal (most mature) internode. Therefore, enhanced p-coumaric esterification of the cell wall and preferential deposition of syringyl units in the lignin polymer might indicate an extended maturity stage of the cell wall of maize internodes. The bm3 mutation in the three maize hybrids is expressed by lignin reduction and ester-bound p-coumaric decrease in the mutant lines. Furthermore, all bm3 hybrids synthesised lignin polymers which were characterised by a very low S/G molar ratio (0.16-0.43). This ratio originates from the substantial reduction of the syringyl unit content in β-O-4-lignin structures compared with the normal lignin. The occurrence in the same range of the 5-hydroxyguaiacyl unit in bm3 lignin from each type of hybrids was noteworthy, demonstrating the high heritability of the bm3 mutation at the molecular level. The alkaline solubility of lignin was greater for the three mutant lines compared to the normal cultivars. Furthermore, the alkali-labile fraction of lignin of both normal and mutant lines had a monomeric composition which was consistent with the non-condensed structures of in-situ lignin.     

Organic solvent pretreatment to enhance enzymic saccharification of straw

Enzymic saccharification of wheat straw in the absence of any pretreatment was only about 20 %. Acid hydrolysis did not improve the overall cellulose enzymic breakdown, nor did pretreatment by flash autohydrolysis under pressure or sodium chlorite delignification. When wheat straw was treated with N-methyl-morpholine-N-oxide (MMNO) at J20°C for 20–30 min, this treatment and subsequent enzymic hydrolysis produced carbohydrate conversion of 73%. Similar results could be obtained by treating straw with 0·1 M sodium hydroxide. When MMNO treatment was combined with any other pretreatment, the saccharification yield was greatly enhanced. Nearly quantitative conversion was obtained by swelling straw in 0·1 M sodium hydroxide followed by MMNO treatment. The results show that lignin content of plant material does not represent the limiting factor to enzymic saccharification.     

假木素对卡伯值的贡献及其对漂白的影响

己烯糖醛酸（HUA）和其他碳水化合物衍生结构合称假木素，其对卡伯值有几个单位的贡献。漂前进行酸处理可以除去HUA，随后漂白时可节约漂白剂，奥斯龙漂白段可降低成本15％。     

Roles of structural phenylpropanoids in forage cell wall digestion

Phenolic constituents (lignins and phenolic acids) and carbohydrates are assembled in a tight architecture which differs according to the plant species. During cell wall digestion, the hydrolysis kinetics differ between carbohydrates and seem to depend chiefly on the content and organisation of tissue phenolics. Among the phenylpropanoids, ferulic acid is released more quickly than p-coumaric acid. Lignins remain largely in the cell walls. They also undergo transformations, chiefly solubilisation as lignin-carbohydrate complexes. The limiting effect of lignins on cell wall degradation increases with increasing content. However, their effect on degradation might also depend on qualitative factors such as lignin structure and polymer organisation in walls and tissues. When various grasses (normal and selected genotypes), or grasses and legumes are compared, correlations between certain factors such as lignin uncondensed fraction, syringyl units or phenolic acids contents and cell wall degradation emerge but not clear causal relationship has been shown. Nonetheless, other structural characteristics, related to the alkali reactivity of lignins, seem to have a stronger influence on cell wall degradation. Phenylpropanoids seem to act mainly as a physical and (bio)chemical barrier to the action of the microbial enzymes. In addition, their reactivity as phenolic compounds and their hydrophobicity seem to play a role. Digestion is not limited only by phenolics. The factors that limit glycanolysis—the accessibility, crystallinity and capillary structure of cellulose and the branching of hemicelluloses—seem to have little or no effect on cell wall degradation in vivo. In contrast, other antiquality substances (tannins, cutin and silica), plant antomy, environmental factors, factors modulating microbial growth and animal physiology influence cell wall utilisation. Future research in this field should focus on the effects of phenolic structure and of cell wall and tissue organisation on carbohydrate degradation.     

The in-vitro digestibility and chemical composition of plant parts in white clover,red clover and lucerne during primary growth

White clover (cv. Grasslands Huia), red clover (cv. Norseman) and lucerne (cv. Sabilt) were sampled during primary growth at the flower bud, near full flowering and seeds formed stages in each of 2 years. Each sample was separated into 2–11 plant parts, depending on species and stage of growth, and for each part the in-vitro true dry matter digestibility, digestibility of cell wall and percentages of cell content, nitrogen, water-soluble carbohydrate and lignin were determined. Green leaflets of all three species were consistently high in digestibility. The cell wall fraction of green leaflets was less digestible in red clover than in white clover and lucerne. The digestibility of the upper part of the stem of lucerne and red clover was high at the flower bud stage but declined considerably thereafter, whereas the digestibility of the lower part of the stem was much lower at the flower bud stage and declined relatively little. The green leaf stalks and inflorescences were generally rather less digestible than green leaflets and, in lucerne and red clover, more digestible than stems. In white clover the inflorescence stalks were less digestible than the leaf stalks. The digestibility of plant parts was strongly correlated with the proportion of cell content, and less strongly with the digestibility of cell wall. The proportion of cell content was positively correlated with N%, and the digestibility of cell wall was negatively correlated with the proportion of lignin in cell wall.