J . Sci. Food Agric. 1983, 34, 1330-1340

Substituent Groups Linked by Alkali-labile Bonds to Arabinose and Xylose Residues of Legume, Grass and Cereal Straw Cell Walls and their Fate during Digestion by Rumen Microorganisms

Andrew Chesson, Alex H. Gordon and James A. Lomax

Department of Microbial Biochemistry, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB

(Manuscript received 15 March 1983)

The distribution of alkali labile substituents of the neutral sugar components of hemi- cellulose in unfractionated cell walls of early- and late-cut perennial ryegrass, wheat and barley straw, and white clover, before and after extended incubation in the rumen, was determined by a modified methylation technique. Xylose residues were found to be extensively substituted, with 50% of residues in the Gramineae and 75 % in white clover carrying substituent groups on 0-2 and/or 0 - 3 . Acetyl groups could account for 50-70% of substitutions, the remainder being to unknown cell wall components. The distribution of alkali-labile substituents of xylose was the same in the original plant material as in residues recovered from the rumen, and was not affected by delignification under conditions which preserved the acetyl content of cell walls. Arabinose residues were heavily substituted at 0-5, with 40-60% of residues from the Gramineae, but only 10% of residues from the white clover, carrying sub- stituent groups at this site. Linkages to 0 - 5 of arabinose were preserved during diges- tion and accumulated in residues recovered from the rumen. In all plants examined, the extent of 0-5 substitution strongly correlated with the proportion of total pheno- lics (r = 0.844) and total phenolics minus phenolic acids (r = 0.779) present. The correlation was improved when restricted to the Gramineae (r=0.879 and 0.881 respectively). Within the Gramineae, the increase in the extent of substitution to 0-5 of arabinose correlated well with the increase in the proportion of total material in digested residues (r = 0.899). As correlations were retained or improved when values for phenolic acids were subtracted from total phenolics, and as the proportion of phenolic acids decreased during digestion, it is suggested that linkages to 0-5 of arabinose represent one form of lignin-carbohydrate bonding. The importance of such linkages to the control of polysaccharide degradation and the effect of their cleavage by alkali-treatment is discussed.

1. Introduction

The degradation of cell wall polysaccharides of Gramineae and other plants by rumen micro- organisms can be enhanced substantially by treatment with alkali, particularly with sodium hydroxide.1 This response to basic reagents is well known and has been applied to cereal straws and other crop residues with varying degrees of commercial success for many years.

The identification of the constraints to digestion which are overcome by the addition of alkali has proved difficult. Not only does alkali bring about multiple changes to cell wall structure, but the response shown differs with the phylogenic origin and maturity of the plant material. Many of these changes, notably the loss of acetic and phenolic acid esters, and the partial solubilisation of hemi- cellulose and silica, now appear to be associated phenomena not directly related to the enhance- ment of digestibility.2 The fine structure of cellulose also appears to be unaffected by alkali at application rates normally used for crop residues.2

1330

Rumen digestion of alkPli-labile substituents 1331

Improvements to cell wall digestibility relate to the extent to which phenolic material is solubilised. Release of total phenolics or lignin from cereal straw closely paralleled improvements to cellulose digestibility when straw was treated with levels of sodium hydroxide below 10 g NaOH 100 g-1 straw. At higher application rates there was no additional release of phenolic material and no further improvement in digestibility.2 However, the nature of the protection offered by phenolic polymers remains unclear. It has been widely suggested that a covalent association of wall poly- saccharides, particularly hemicellulose, with polyphenolic material inhibits enzymic attack by microorganisms.3~ 4 Certainly the difficulty in separating lignin from hemicellulose suggests bonding between these polymers, and a number of workers have provided indirect evidence for the presence of covalent linkages.5-7 To function as a major determinant of digestibility, such linkages must also satisfy the criterion of alkali-lability. The recognition by Hartley et a1.89 9 that simple phenolic acids (p-coumaric and ferulic acids), which closely resemble the precursors of lignin itself, were, in part at least, ester-linked to hemicellulose, suggests one possible form of lignin-carbohydrate bonding which could be labile to alkali. Indirect evidence that lignin-carbohydrate linkages re- sembling those to phenolic acids might be present was provided by the direct relationship found between the loss of phenolic acids and the improvement in digestibility which occurred when alkali was applied to cereal straw.2.10 Lignin-carbohydrate and phenolic acid-carbohydrate ester linkages might be expected to show a similar response to basic reagents.

The difficulties involved in the extraction of hemicellulose or lignin-hemicellulose complexes from cell walls without substantially modifying their nature are well recognised. Alkaline extraction methods clearly are inappropriate when the linkages sought are thought to be alkali-labile. To overcome this problem a method for the location of alkali-labile linkages in situ, based on that used by de Belder and Norrmanll for the location of acetyl groups in partially acetylated dextrans which could be applied directly to unmodified plant material, was developed in this laboratory.12 Under this procedure, milled plant samples are first acetalated with methyl vinyl ether to prevent pre- existing free hydroxy groups of sugar residues forming 0-methyl derivatives on subsequent methy- lation. Alkali-labile linkages are hydrolysed under the anhydrous basic conditions of methylation and only the hydroxy groups so generated are methylated. The subsequent hydrolysis and identi- fication of partially-methylated sugar residues thus provides a direct measure of the location and numbers of substituents linked by alkali-labile bonds in the intact cell wall. The results obtained when this method was applied to legume, grass and cereal straw cell walls undergoing degradation in the rumen are now presented.

2. Experimental 2.1. Plant samples

Samples of perennial ryegrass (Lolium pevenne L. cv. Perma), cut in May 1981 (Perma-early) and in October 1981 (Perma-late), and white clover (Trifolium pratense L. cv. Aberystwyth S100) were obtained from trial plots grown by the North of Scotland College of Agriculture. Barley straw (Hordeum vulgare L. cv. Midas) was grown at the Rowett Research Institute and wheat straw (Triticurn sativum L. cv. Hustler) was kindly provided by Mr J. Carr (Uras farm, Catterline, Kincardineshire).

All samljgqs were freeze-dried and ground to pass a 1 mm screen. Straw samples received no further treatment. Grass and clover samples (20 g) were extracted with 700 ml chloroform-methanol (1 : 1 v/v) at room temperature, stirred with iced water for 4 h, centrifuged, and the residue again extracted with 700 ml of chloroform-methanol to provide crude cell wall preparations. Dry weights were then determined.

2.2. Digestibility

Prepared plant samples of approximately 1 g were weighed into nylon bags of 5 pm pore size and suspended in the rumen of a single sheep as described previously.2 Residues recovered from the rumen after 72 h incubation were washed to remove loosely adhering microorganisms, freeze dried

1332 A. Chesson et al.

and weighed. Sub-samples of pooled residues from a number of 72 h incubations were treated either with water or with sufficient IM NaOH to give an application rate of 20 g NaOH 100 8-1 plant residue. Treated residues were left to stand at room temperature overnight and were then washed into nylon bags and resuspended in the rumen for a further 48 h. Residues recovered from this second incubation (subsequently referred to as 120 h incubation) were treated as above.

2.3. Acetalation and methylation Straw, grass and legume samples and their corresponding undigested residues recovered from the rumen (0.5 g) were finely dry milled under liquid nitrogen using a Spex 6700 freezer mill (Spex Industries Inc., Metuchen, NJ, USA).

Methods for the acetalation and subsequent methylation of samples, outlined below, have been fully described elsewhere by Lomax et aL12 Milled samples (10 mg) were suspended in 2 ml dimethyl- sulphoxide (DMSO) and 20 mgp-toluene sulphonic acid and 2 ml methyl vinyl ether (condensed at - 10°C) added. The suspension was left at 15°C for 3.5 h with intermittent mixing. The insoluble acetalated residue was then recovered by centrifuging, washed three times with acetone and dried over PzO5. Dried residues were then dispersed in 1 ml DMSO containing 0.145 mg methyl-8-D- allopyranoside and methylated with freshly prepared methyl sulphinyl sodium and methyl iodide as described by Stellner et a1.,13 Quebrachitol (monomethyl-L-inositol) (0.506 mg) was added to each sample and the methylated polysaccharides hydrolysed by a formolysis-hydrolysis procedure. Following hydrolysis, liberated methyl sugars were converted to their corresponding alditol acetates.

2.4. Separation and identification of partially methylated derivatives Partially methylated alditol acetates were separated by gas-liquid chromatography (g.1.c.) on 50 m x 0.3 mm i.d. SPlOOO W.C.O.T. glass capillary columns (Phase Separations, Clywd, UK) and on a 25 m OV1 W.C.O.T. silica capillary column (Hewlett-Packard, Wokingham, Berkshire, UK). The latter column generally gave a poorer resolution of permethylated alditol acetates, but was found useful both for the confirmation of results obtained with SPlOOO columns and for the separation of specific derivatives, notably 2-0-methyl and 3-0-methyl derivatives of xylose, which co-chromato- graphed on SPlOOO. Carlo-Erba FV 4160 series gas chromatographs (Erba Science (UK) Ltd, Swindon) with on-column injection facilities were used throughout. Other conditions for the separation of partially methylated alditol acetates and for their provisional identification on the basis of their retention coefficients were as described previously.12.14 The peak for 1,5-di-O-acetyl- 2,3,4,6-tetra-O-methylallitol derived from the added standard methyl-8-D-allopyranoside was used as the first retention coefficient standard and as an internal standard for the quantitation of results. Quebrachitol was used as the second retention coefficient 'standard. Mass spectrometry was used to confirm the identities of specific derivatives.

2.5. Other analytical methods Neutral sugars released from plant material by acid hydrolysis15 were converted to their corres- ponding alditol acetateP and separated by g.l.c.l7 The total uronic acid content of hydrolysates was determined by the method of Blumenkrantz and Asboe-Hansen.18 Two known types of alkali- labile substituents of plant cell walls, acetyl groups and phenolic acids, were released from samples and determined by g.1.c. as described previously.2 A measure of the total phenolics present in samples was obtained using the acetyl bromide method.lg Optical density values obtained at 280 nm were related to those produced by a known amount of ferulic acid standard.

2.6. Delignification Plant samples were fully delignified (in terms of acid lignin) by the sodium chlorite method.20 Lactic acid was used in place of acetic acid to avoid any overestimation of acetyl content on sub- sequent analysis.

Rumen digestion of alkali-labile substituents 1333

3. Results 3.1. Digestibility and the effect of alkali The loss of dry matter and loss of glucose residues (a measure of cellulose degradation) from plant samples incubated in the rumen is shown in Table 1. Greatest losses were found on the first incu- bation. Returning 72 h residues to the rumen for a further 48 h produced a limited further loss of dry matter from the cereal straws. Forage species, however, showed additional substantial losses of dry matter and glucose residues. Treatment of 72 h residues with 2Qg NaQH 100g-1 residue increased weight losses when the treated residues were incubated for a further 48 h. As would be expected this effect was most evident with the less digestible cereal straws and the late-cut ryegrass, but all samples showed some response to treatment with alkali. Since glucose residues are not solubilised by NaQH treatment,2 the additional loss of glucose residues indicates that this response was a product solely of enhanced microbial activity.

Table 1. Loss of dry matter (and glucose residues) from grass, straw and legume samples suspended in the rumen of a sheep. Material was incubated for 72 h (1st incubation) and the residue recovered treated with water or 20 g NaOH 100 g-1 sample and reincubated for a further 48 h (2nd incubation)

% Loss of original dry matter (glucose residues)

2nd incubation

Plant sample 1st incubation Water-treated Alkali-treated

Barley straw 27.4 (33.2) 33.9 (34.0) 55.1 (51.4) Wheat straw 31.0 (34.9) 40.3 (48.4) 66.6 (64.8) Late-cut ryegrass 52.9 (66.2) 75.2 (84.8) 89.5 (92.9) Early-cut ryegrass 70.0 (75.2) 85.0 (93.0) 92.2 (99.4) White clover 62.7 (84.3) 80.1 (92.7) 88.8 (94.5)

3.2. Distribution of alkali-labile linkages The location of alkali-labile substituents of the neutral sugar components of hemicellulose was found by acetalation of plant samples followed by methylation analysis. Xylose and arabinose were the major neutral non-cellulosic monosaccharide residues present in all of the samples examined. Recovery of xylose as its partially methylated derivatives was equal to or greater than that indicated by the direct analysis of monosaccharides by the alditol acetate method. A small amount of arabi- nose-containing material in freezer milled samples, however, was found to be DMSQ-soluble and was lost to the analysis. As a result, the recovery of arabinosc was approximately 85 % of that found by the direct analysis of acid hydrolysates.

Xylose residues from all four samples of the Gramineae exhibited a very similar pattern of substitution, with approximately half of all residues substituted on 0-2 or 0-3. In 8-9% of all residues, both positions were found to carry substituted groups (Table 2). The similar results obtained from undigested plant materials and their digested residues indicated that this distribution of substituent groups remained essentially constant throughout the course of digestion. No evidence of the accumulation of any alkali-labile substituent of xylose could be detected.

White clover differed from the grass and straw samples. Xylose residues from this dicotyledon were much more extensively substituted than the grasses, with 20% of residues doubly substituted on 0-2 and 0-3. However, in common with the other plants examined, the extent and distribution of substitution remained constant throughout the course of digestion.

Arabinose residues were also found to carry alkali-labile linked substituent groups, particularly in the 0-5 position (Table 3). Within the Gramineae, the extent of substitution appeared to relate to the degree of lignification, with 60% of arabinose residues from straw carrying one or more

1334 A. Chesson ct al.

Table 2. Distribution of alkali-labile substituents of xyloseiresidues from grass, straw and legume samples before and after incubation in the rumen. Values are

expressed as a fraction of the total xylose in each sample

Position of Original Residue after Residue after Plant sample substitution plant 72 h 120 h

Barley straw

Wheat straw

Late-cut ryegrass

Early-cut ryegrass

White clover

2, 3a 2 3 None

2, 3 2 3 None

2,3 2 3 None

2, 3 2 3 None

2, 3 2 3 None

0.09 0.29 0.13 0.49

0.08 0.26 0.16 0.50

0.08 0.24 0.18 0.50

0.09 0.23 0.19 0.49

0.19 0.32 0.21 0.28

0.08 0.28 0.14 0.50

0.08 0.24 0.18 0.50

0.07 0.26 0.16 0.51

0.09 0.21 0.17 0.53

0.19 0.28 0.24 0.29

0.08 0.27 0.14 0.51

0.09 0.27 0.18 0.46

0.08 0.28 0.14 0.50

0.10 0.32 0.15 0.43

0.20 0.30 0.25 0.25

a Determined as: 1,4,5,-tri-O-acetyl-2,3-di-O-methylxylitol etc.

groups compared to 40 % of residues from early-cut ryegrass; late-cut ryegrass values were inter- mediate. The difference in the extent of substitution between grass and straw samples was largely accounted for by the higher proportion of residues doubly substituted on 0-2 and 0-5 found in the cereal straws. Trace amounts of the 3,5-di-O-methyl derivative of arabinose was also detected in both grass and straw samples. However, as this peak had a similar relative retention time to the 2,3-di-O-methyl derivative of rhamnose (0.930 and 0.934 respectively) and as insufficient was detected to allow confirmation of identity by mass spectroscopy, values have been omitted from Table 3.

Clover again differed from other samples examined, showing a far lower degree of substitution. Residues carrying alkali-labile substituents on 0-5 accumulated during the course of digestion

in the rumen. This was apparent only as a trend in the more lignified cereal straws but was clearly evident in the less lignified grass samples and the white clover.

3.3. Phenolic and acetic acid esters Phenolic (ferulic and p-coumaric) esters formed 1-2% of grass and straw dry matter, but were found only in trace amounts in white clover (Table 4). The overall proportion of phenolic acids in straw and late-cut ryegrass declined during digestion, residues recovered from the rumen showing a reduced phenolic acid content after 12 and 120 h digestion. A substantial loss of phenolic acids accompanied the digestion of early-cut ryegrass. In all of the samples examined, ferulic acid residues were lost more readily than p-coumaric acid residues.

The acetyl content of wheat and barley straw was similar in both digested residues and the original undigested material, while, in the more digestible grass samples, there was evidence of the

Rumen digestion of alkali-labile substituents 1335

Table 3. Distribution of alkali-labile substituents of arabinose residues from grass, straw and legume samples before and after incubation in the rumen. Values are

expressed as a fraction of the total arabinose in each sample

Position of Original Residue after Residue after Plant sample substitution plant 72 h 120 h

Barley straw 2, 5a 5 None

Wheat straw 2, 5 5 None

Late-cut ryegrass 2, 5 5 None

Early-cut ryegrass 2, 5 5 None

White clover 2, 5 5 None

0.25 0.32 0.43

0.29 0.32 0.39

0.05 0.29 0.66

0.05 0.33 0.62

- 0.09 0.91

0.29 0.33 0.38

0.33 0.32 0.35

0.11 0.31 0.58

0.09 0.45 0.46

- 0.29 0.71

0.28 0.34 0.38

0.36 0.35 0.29

0.14 0.36 0.50

0.07 0.48 0.45

- 0.42 0.58

~~~~ ~ ~

a Determined as : 1,3,4-tri-O-acetyl-2,5-di-O-methylarabinitol etc.

accumulation of acetylated residues during digestion (Table 4). The hemicellulose content of these samples also increased. Digestion of white clover was accompanied by a substantial loss of acetyl groups, however, unlike the grasses, legumes are known to contain a large proportion of readily digested acetylated galacturonan.21 The acetyl content of white clover could be considerably reduced by extraction with 5 g litre-1 ammonium oxalate at 100°C for 2 h without a concomitant loss of xylose residues. The uronic acid content of clover fell from 14.8 % of dry matter in the original plant material to 1.2 % of the 72 h residue dry matter. This loss of acetylated galacturonan may have masked an underlying increase in acetyl associated with the hemicellulose fraction as was found for the grasses, since the proportion of hemicellulose and acetyl was slightly higher in the 120 h than the 72 h residue.

Table 4. Phenolic acid and acetyl content of grass, straw and legume samples before and after incubation in the rumen

% Dry matter

Original Residue after 72 h Residue after 120 h

Phenolic Phenolic Phenolic Plant sample acids Acetyl acids Acetyl acids Acetyl

Barley straw 1.89 1.89 1.35 1.86 1.21 1.91 Wheat straw 1.53 2.33 1.77 2.62 1.18 2.60 Late-cut ryegrass 1.53 1.06 1.12 1.49 1.26 1.63 Early-cut ryegrass 2.17 0.83 0.72 0.87 0.22 1.04 White clover 0.14 1.21 0.10 0.74 0.15 0.76

86

1336 A. Chesson et at.

Table 5 provides an indication of the maximum extent to which acetyl and phenolic acids, both known alkali-labile cell wall components, might account for the substitutions to arabinose and xylose detected by acetalation-methylation. In calculating the percentage of total available sites carrying alkali-labile side chains it was assumed that xylose provided two potential siteslresidue (0-2 and 0-3) and arabinose three sites/residue (0-2, 0-3 and 0-5 ) . It was also assumed that all acetyl and phenolic acids detected were associated with arabinose or xylose residues. As is evident from Table 5, even when this assumption is made, approximately 30% of sites carrying alkali- labile linked substituents remained unaccounted for and must represent linkages to other cell wall components.

Table 5. The maximum extent to which the acetyl and phenolic acid content of grass, straw and legumes account for alkali-labile linked substituents of arabinose and xylose residues detected by acetalation-methylation

% Total available sites carrying alkali-labile substituents

Maximum of % total sites attributable to:

Unknown substitu- tions as % total

available sites Plant sample From From Xylosef xylose arabinose arabinose

Acetyl Phenolic groups acids Total

Barley straw Original 72 h

120 h

Wheat straw Original 72 h

120 h

Late-cut Original ryegrass 72 h

120 h

Early-cut Original ryegrass 72h

120 h

Whole clover Original 72 h

120 h

24.5 5.0 29.5 24.5 4.7 29.2 24.5 4.3 28.8

23.8 5.4 29.2 25.1 4.4 29.5 27.7 4.3 32.0

19.1 4.1 23.7 23.5 3.1 26.6 26.4 2.0 28.4

19.8 4.9 24.1 21.6 4.8 26.4 26.8 4 .2 31.0

17.3 1 .9 19.2 41.7 0.6 42.3 44.5 0 .9 45.4

15.2 3.6 18.8 17.2 3.0 20.2 15.0 2.3 17.3

18.6 2.8 20.8 18.6 3 .0 21.6 16.9 1 .8 18.7

9.1 3.2 12.3 11.6 2.1 13.7 14.2 2.6 16.8

9.2 5.8 15.0 12.7 2 .5 15.2 17.9 0 .9 18.8

30.7 0.9 31.6 17.5 0.6 18.1 17.7 0.8 18.5

10.7 9.0

11.5

8.4 7.9

13.3

11.4 12.9 11.6

9 . 1 11.2 12.2

-12.4 24.4 26.9

3.4. Association of arabinose and total phenolics The total phenolic content of the straw, grass and clover samples is shown in Table 6. Straw samples were found to have a higher total phenolic content than the three highly digestible forage species and mature ryegrass a higher content than the early-cut sample. Barley straw, which unexpectedly showed a lower digestibility than wheat straw, was found to have a higher total phenolic content than wheat.

Phenolic material was lost from samples incubated in the rumen at a slower rate than other cell wall components. As a result, a higher proportion of total phenolics was found in all residues recovered from the rumen than in the parent material. Accumulation was limited in the cereal straws bur resulted in a doubling of the phenolic content of residues from the more digestible forage species.

The total phenolic content of the original samples and their residues'from the rumen showed a high correlation (r = 0.844) with the extent of alkali-labile-linked substitution to 0-5 of arabinose (Figure 1). This correlation could be improved if the Gramineae only were considered (v=O.878) suggesting that the relationship between total phenolics and 0-5 substitution of arabinose differed between Grarnineae and dicotyledons. Phenolic acids, unlike total phenolics, did not accumulate during

Rumen digestion of alkali-labile substituents 1337

Table 6. Total phenolic content of grass, straw and legume samples before and after incubation in the rumen

Total phenolics as % dry matter

Residue after Residue after Plant sample Original 72 h 120 h

Barley straw 20.4 22.6 22.7 Wheat straw 17.8 19.2 19.3 Late-cut ryegrass 8.5 10.8 16.5 Early-cut ryegrass 6.8 12.9 16.5 White clover 7.1 11.4 14.5

digestion and no correlation existed with substitution to 0-5 of arabinose. After subtraction of values for phenolic acids from total phenolic values, the correlations with 0-5 of arabinose substi- tution were r=0.779 for all samples and r=0.881 for the Gramineae only. The extent to which total phenolics (or total phenolics minus phenolic acids) acccumulated in 72 h and 120 h residues was also highly correlated with the increase in the proportion of arabinose residues carrying an alkali- labile substituent on 0-5 (r = 0.899).

As the correlations described above suggested bonding between arabinose residues and cell wall phenolics, the effect of delignification on the distribution of alkali-labile substituents was examined.

*,r 2o t

L 7 5

Figure 1. The relationship between the total phenolics content of undigested and digested residues of grass, straw and legume samples and the proportion of arabinose residues carrying alkali-labile substituents on 0-5.-, linear regression curve for all samples b=O.O26x+ 0.094); . . . . ., linear regression curve for Gramineae only y= 0.019x+0.239); (0) Gramineae; (0) White clover. 0

0

0 J

Fraction of arabinose residues containing alkali-labile substituents at 0 - 5

Samples of grass, straw and clover were delignified by the sodium chlorite method before acetalation and methylation. The mild acid conditions of the chlorite reaction favours the retention of alkali- labile linkages, and little loss of acetic acid esters resulted (approximately 7 % loss of acetyl from cereal straws and 5 % from the forage species). However, delignified samples were found to be more soluble in DMSO after freezer-milling, and losses of arabinose at the acetalation stage proved unacceptably high. Therefore it was found necessary to methylate separately both the insoluble fraction and the soluble fraction from acetalation which was normally discarded. Results for each sample were then combined.

1338 A. Chesson et al.

Delignification was found to have no effect on the distribution or extent of xylose substitution and to produce a negligible reduction in arabinose substitution at the 0 - 5 position.

3.5. Solubilisation of total phenolics by alkali Treatment of 72 h digestedIresidues with 20 g NaOH 100 8-1 residue resulted in the solubilisation of a considerable proportion of the phenolic fraction. Approximately half of the total phenolics present in treated cereal straw residues (57% of barley straw and 59% of wheat straw) could be removed by washing residues with water. Total phenolics from forage samples proved even more susceptible to the action of alkali with 67 % removed from late-cut ryegrass and over 80 % from early-cut ryegrass and clover residues.

4. Discussion The direct application of the technique of methylation analysis for the determination of alkali- stable (glycosidic) linkages, to unfractionated plant material and to their extensively degraded residues recovered from the rumen has been reported else~here.~S In the present work the obser- vations were extended to examine alkali-labile linkages to the neutral sugar components of hemi- cellulose in intact and degraded cell walls which are destroyed by conventional methylation analysis.

Both arabinose and xylose residues were found to carry substantial numbers of alkali-labile linked substituent groups. Within the Gramineae, approximately half of all xylose residues were substituted at 0-2 and/or 0-3 (Table 2) and 40-60% of all arabinose residues at 0 - 5 (Table 3). The single legume sample differed from the grass and straw samples, showing a higher proportion of substituted xylose and a considerably lower proportion of substituted arabinose residues. Xylose residues of hardwoods24 and grass cell walls25 are known to possess ester-linked acetyl groups. However, even when the probable presence of acetylated uronic acid residues was disregarded and all cell wall acetyl assumed to be associated with xylose, the presence of acetyl groups could account for only 50-70% of alkali-labile linked substitution to xylose (Table 5). The remaining 30-50 % of sites on xylose residues represented linkages to other, unknown, wall components. Although the proportion of acetyl groups increased in digested residues of forage species there was a similar increase in the proportion of xylose residues (see paper by the same authors in this issue). As a result, the distribution of substituent groups remained constant throughout the course of digestion and no evidence for an increasing proportion of alkali-labile linkages from xylose to acetyl or to unknown cell wall components was found (Table 5).

In marked contrast to xylose residues, there was clear evidence that substituted arabinose residues were preserved during digestion. The extent of substitution to 0-5 of arabinose in undigested and digested residues was closely related to the amount of phenolic material present in cell walls (Figure 1) and the accumulation of 0-5 substituted residues strongly correlated with the increase in the proportion of phenolic material in digested residues. These results suggested a covalent association between the arabinose side chains of straw, grass and legume xylan and phenolic material. Hartley et ~ 1 . ~ 9 9 have reported that some, and possibly all, ferulic acid residues are ester linked to xylan by their carboxy group. However, as the proportion of phenolic acids declined during digestion and as the relationship between substituted arabinose and phenolic material was unaltered when values for phenolic acids were subtracted from values for total phenolics, it seems probable that the bulk of linkages involving 0-5 of arabinose are not to phenolic acids, but involve polyphenolic material. Delignification, however, failed to reduce the extent to which xylose and arabinose residues were substituted. Treatment of samples with sodium chlorite-lactic acid removed only 60-70 % of total phenolics (although samples were fully delignified in terms of acid lignin) and, as shown by the recovery of acetyl groups, favoured the retention of alkali-labile linkages. Under these conditions it is possible that carbohydrate-polyphenolic linkages were retained as linkages to degraded phenolic material. If this is substantiated, delignified cell walls may serve as a useful starting material for the further characterisation of substituents to 0-5 of arabinose.

In the absence of a highly condensed polyphenolic component, the presence of alkali-labile substituents of xylan failed to protect cell wall polysaccharides from attack by rumen micro-

Rumen digestion of alkali-labile substituents 1339

organisms to any extent. Delignified walls, in which alkali-labile substituents are retained, are readily degraded in the rumen at a rate which is not further enhanced by treatment with alkaL2 In intact cell walls the major effect of alkali treatment would seem to be the solubilisation and release of a substantial fraction of the total phenolics initially present. Clearly this process would be aided by cleavage of alkali-labile lignin-carbohydrate linkages. The direct relationship, between the release of phenolic acids and improvements to digestibility observed to follow alkali treatment of cereal straws,2910 may thus reflect the cleavage of lignin-carbohydrate linkages rather than the cleavage of phenolic acid-carbohydrate bonds. However, there is increasing evidence that concentrations of alkali sufficient to release alkali-labile substituents of xylan are also sufficient to bring about some degra*diation of the lignin macromolecule itself.Z6, 27 This partial depolymerisation of lignin may be of greater importance to the solubilisation of lignin, with its consequent effect on the biodegrada- bility of the cell wall, than cleavage of other alkali-labile linkages within the wall.

The present work indicates that the neutral sugar components of hemicellulose are heavily substituted with alkali-labile groups whose identity can be only partly explained by the presence of acetic and phenolic acid esters. Approximately 30% of such substitutions are from unknown cell wall components which may, in part at least, include the polyphenolic fraction of the wall. Work with jute and other vegetable fibres has also suggested that uronic acid residues are involved in bonding to lignin.28 Further work is in progress to detect possible ester linkages from the carboxy group of glucuronic acid to the hydroxyl groups in lignin and the possible effects of such linkages on forage cell wall degradation by rumen microorganisms.

Acknowledgements The authors thank Mr E. W. E. Whitehorn for the determination of acetyl content and Ms Karen Dalgarno for her excellent technical assistance.

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