Changes in the alkaline-labile phenolic compounds of
wheat straw cell walls as affected by SO
2treatment
and passage through the gastro-intestine of sheep
E. Yosef, D. Ben-Ghedalia
*Metabolic Unit, Institute of Animal Science, ARO, The Volcani Center, Bet Dagan 50250, Israel
Received 28 October 1998; received in revised form 1 June 1999; accepted 15 October 1999
Abstract
Sheep were fed two rations based on untreated (WS) and SO2-treated (SO2-WS) wheat straw, and the effect of chemical treatment and passage through the gastro-intestine on the composition and degradation of ester and ether-linked cell wall (CW) phenolics was studied. The SO2 treatment reduced the content of total ferulic acid (FA) andp-coumaric acid (PCA) by 35% while tripling the level of vanillin and increasing by 40% the concentration of protocatechuic acid. In WS most of the phenolic compounds were CW-bound, but 37% of the vanillic and 88% of the protocatechuic acids were in the alcohol soluble (AS) fraction. The solubilising effect of the treatment was expressed in releasing the phenolics from the CW mainly as AS-lignins. Most of the FA (62%) was ether-linked, whereas most of the PCA (78%) was ester-linked in the CW of WS. The other minor components were either entirely or mostly, etheri®ed units. The SO2treatment was more effective in cleaving the ester than the ether bonds of the cinnamic acids. Ester-linked FA was more extensively degraded in the rumen than ester-linked PCA. Ester-linked FA and PCA were more extensively degraded in the rumen than the respective linked compounds. Nevertheless, substantial amounts of ether-linked FA, PCA and other phenolics were removed from CW in the rumen, most likely as oligo-lignols. Phenolic compounds were determined in rumen liquor of sheep fed the WS and WS-SO2 rations. FA was not detected and PCA was at a very low (20±40mM) concentration.
Phenyl-propanoic acid (PPA) was the major monomeric phenolic compound detected, at concentrations of 580 and 380mM in the rumen of WS and WS-SO2 sheep, respectively. It is suggested that hydrogenation of PCA and combined hydrogenation and demethoxylation of FA were responsible for the production of PPA in the rumen.#2000 Elsevier Science B.V. All rights reserved.
Keywords: p-Coumaric acid; Ferulic acid; SO2-treated wheat straw; Digestibility; Sheep
83 (2000) 115±126
*Corresponding author. Tel.:972-3-960-5113; fax:972-3-960-4023.
E-mail address: danielbg@actcom.co.il (D. Ben-Ghedalia).
1. Introduction
Wheat straw is a graminaceaous lignocellulose and as such, it is richer than conventional forages in cinnamic acids (Bourquin and Fahey, 1994). These compounds have been extensively researched by several groups worldwide, in order to characterize their chemical role as constituents and bridging units between the matrix components of wheat straw cell walls (Scalbert et al., 1985; Kondo et al., 1992; Lam et al., 1992; Lawther et al., 1996; Sun et al., 1997). Surprisingly, there is very little information on the biodegradational features of ester and ether-linkedp-coumaric (PCA) and ferulic (FA) acids of wheat straw cell walls (CW), based on in situ (Bourquin and Fahey, 1994) or in vivo (Kerley et al., 1988) studies. This fact is probably associated with the low nutritional value of wheat straw. However by using the SO2 treatment it was shown that wheat straw can be converted into a highly productive, concentrate-like feed (Ben-Ghedalia et al., 1988). In vivo studies on the biodegradational features of ester and ether-linkedp-coumaric and ferulic acids of wheat straw CWare important because of the confusion existing in the literature regarding their role as biodegradation barriers or inhibitors (Besle et al., 1994). It was suggested that cinnamic acids interfere with the attachment of rumen bacteria to CW, resulting in a reduction in CW degradation (Varel and Jung, 1986; Akin et al., 1988). However with mixed rumen micro¯ora, the addition of PCA and FA failed to decrease the disappearance rate of structural carbohydrates (Besle et al., 1988). Jung and Casler (1990) pointed that esteri®ed ferulic acid was greater in CWof the high in vitro dry matter digestibility (IVDMD) genotypes of smooth bromegrass, supporting the positive correlation found between FA concentration and NDF fermentability of grasses by Buxton and Russell (1988). A year later, Jung et al. (1991) working on a model system in vitro, concluded that the concentration of esteri®ed phenolic acids was negatively correlated with IVDMD and that FA was more inhibitory to IVDMD than PCA. According to Akin et al. (1993), growth of pure cultures of Ruminococcus,
SelenomonasandButyrivibriospecies was limited by ester-linked feruloyl andp-coumaroyl groups. Notwithstanding, McSweeney et al. (1998) working on a tropical grass (Heteropogon contortus), have shown that high levels of cinnamoyl esterases are characteristic of many strains and species of ®ber-degrading rumen bacteria and fungi. Accordingly, Grabber et al. (1998) have demonstrated that ferulate substitution of xylans in primary CWof maize, did not affect CW hydrolysis. Free monomeric phenolics in SO2-treated wheat straw constitute a very small proportion of the total solubilised phenolic materials (Yosef et al., 1994). Nevertheless, a digestibility study on model substrates such as untreated and SO2-treated wheat straw, may provide some decisive answers to the points of disagreement mentioned above. The objective of this study was to follow up the changes in ester and ether-linked cinnamic acids of wheat straw CW as affected by SO2treatment and passage through the gastro-intestine of sheep; these particular research issues are uncovered in the literature.
2. Materials and methods
2.1. The straw
Wheat straw, chopped to pass through a 6 mm screen, either untreated or treated with gaseous SO2 at 40 g kgÿ1 at 708C for 72 h, as described by Miron and Ben-Ghedalia
(1987), was used in this study. The pH of the SO2-WS was raised from 3 to 5 by the addition of a technical solution of concentrated ammonium hydroxide, to enrich the total crude protein content of the straw and to make it acceptable to the animals.
2.2. Feeding trial
Four two-year-old Merino rams, of 60 kg average weight, cannulated in the rumen and at the duodenum, were allocated randomly to two feeding groups: (i) SO2-treated wheat straw (SO2-WS); and (ii) untreated wheat straw (WS), in a 22 change over design. The rams were kept in metabolism cages in an air-conditioned animal house (238C) to allow sampling of rumen liquor, duodenal digesta and total feces collection. The daily ration, containing 700 g straw dry matter (DM) was divided into 12 portions and delivered by automatic feeders at 2 h intervals, to create steady-state conditions. The rations were supplemented with N (50% contributed by a protein source, soybean meal (90 g), and 50% by NPN) to reach a total N content of 2%, and with a mineral±vitamin mixture to meet the maintenance requirements of the animals. Sampling of rumen contents and duodenal digesta (4 days) and total collection of feces (10 days), were started after 15 days of adaptation to the rations. Six days prior to and during the digesta sampling, Cr2O3 impregnated paper was given twice daily at 8 : 00 and 20 : 00 h via the rumen cannula. The whole rumen digesta and duodenal digesta was sampled four times per day at different intervals, 25 ml per sampling, composited to one sample per ram. The sample of rumen digesta was centrifuged (2000g for 10 min) and the upper liquid phase was stored at ÿ208C until further processing. Samples of feces and duodenal digesta were taken from the daily total collection, composited proportionately to one sample per ram, and stored atÿ208C. Samples of feces and duodenal digesta were freeze-dried, ground to
pass a 1 mm screen and stored atÿ208C under nitrogen.
2.3. Analytical procedures
2.3.1. Preparation of CW material
Cell walls were prepared by extracting ground (1 mm) WS and WS-SO2, the respective duodenal digestas and feces with 80% ethanol, according to Theander and Westerlund (1986). One gram of sample was added to 100 ml of 80% ethanol, at room temperature for 24 h with occasional shaking. The alcohol insoluble residues (cell wall) were recovered by ®ltration through ®lter paper (Whatman No. 41), washed 2 with 80% ethanol and once with acetone, allowed to air-dry under a hood and
freeze dried. The cell walls were ball milled and served for the determination of linked phenolic compounds. Filtrates were used for determination of alcohol soluble phenolic compounds.
2.3.2. The determination of alcohol-soluble phenolic compounds
to extract the free aromatic compounds; this phase was concentrated under nitrogen and dried over Na2SO4 prior to silylation. One milliliter of this organic solution was transferred to a silylation amber tube, evaporated to dryness and trimethylsilylated (TMS) at room temperature, for 24 h, with a 1 : 1 mixture of pyridine (GC grade):N-methyl-N -trimethyl silyltri¯uoro-acetamide (MSTFA). The phenolic compounds were quanti®ed by GLC (Hewlett±Packard 5890 II), on a megabore column type HP-5 Ultra 2 (5% phenyl± 95% methyl±silicone±fused silica), (0.25mm ®lm thickness; 30 m0.33 mm ID) and FID detector. The temperature was programmed from 120 to 2608C (108C/min).
2.3.3. Determination of ester and ether-linked phenolic compounds
The procedure is based on subjecting CW or whole material to hydrolysis by 1 N NaOH at room temperature for determining the ester-linked phenolic acids and in parallel to 4 N NaOH at 1708C for measuring total ester plus ether-linked phenolic acids. Ether-linked phenolic acids were calculated as the difference between total and ester-Ether-linked phenolic acids.
2.3.3.1. Ester-linked phenolic acids. Samples of straw, duodenal digesta, feces and the respective CW preparations were ball milled and placed (200 mg) in glass tubes with teflon-lined caps. N NaOH solution (10 ml) was added to each tube under a nitrogen stream. Samples were extracted at 398C, for 24 h in the dark with occasional shaking, according to Jung and Shalita-Jones (1990). Veratryl aldehyde (0.5 mg) was added, as GC internal standard. Samples were subsequently centrifuged for 20 min at 2000g and washed with 5 ml of water. The combined supernatants were acidified to pH 2.6 with concentrated phosphoric acid, to allow the precipitation of polymeric compounds. The acidified sample was re-centrifuged at 2000gand the supernatant was transferred to a new tube. Ethyl acetate was added to extract the free aromatic compounds. One milliliter of the ethyl acetate phase was silylated and analyzed by GLC as described above (Section 2.3.2).
2.3.3.2. Ether-linked phenolic compounds. Ball milled samples (100 mg) were placed in stainless-steel tubes with 8 ml of 4 N NaOH, under nitrogen stream, according to Iiyama et al. (1990). The tubes were kept at 1708C, for 2 h with occasional shaking. At the end of the reaction, 1 ml of veratryl aldehyde solution in 1 N NaOH (1 mg/ml) was added, as GC internal standard. Samples were subsequently centrifuged for 20 min at 2000g and washed 3x with 5 ml of water. The combined supernatants were acidified to pH 2.6 with concentrated phospheric acid, to allow the precipitation of polymeric compounds. The phenolic compounds were recovered from supernatants, extracted by ethyl acetate and quantified by GLC system as described in Section 2.3.2.
2.3.4. Determination of phenolic compounds in rumen liquor
Rumen liquor was centrifuged at 30 000g for 60 min, at 48C and the supernatant was used for the determination of free monomeric phenolic compounds. The supernatants were acidi®ed with 1 N H3PO4to pH 2, to allow the precipitation of polymeric aromatic material by centrifugation (Pometto and Crawford, 1988). Ethyl acetate was added to the supernatant, to extract the free aromatic compounds; this phase was concentrated under
nitrogen and dried over Na2SO4 prior to silylation. The silylation of samples for GC analysis was as described above for the alcohol-soluble phenolics.
The phenolic compounds were identi®ed and quanti®ed by GC-MS system, on a 5% phenyl±95% methyl±silicone±fused silica capillary column (0.25mm ®lm thickness; 50 m0.25 mm ID). The temperature was programmed from 120 to 2608C (108C/ min). Detection was accomplished with MS (MS-VG Autospec-High Resolution Magnetic Sector Instrument) in the electron impact mode (70 eV) and chemical ionization (reagent gas CH4). Peak identi®cation in electron impact mode was by matching the spectra with those of standard mass spectra stored in the computer library (Yosef et al., 1994).
2.4. Statistical analysis
Values of the digestibility of phenolic compounds were analyzed statistically by standard ANOVA (Little and Hills, 1978).
3. Results and discussion
Determination of CW ester and ether-linked phenolic acids is based on the differential effect of the combination of alkali concentration and temperature, on those linkages. Esteri®ed phenolic acids are easily liberated from cell walls by 1 N NaOH at room temperature, whereas phenolic benzyl aryl ethers are released by alkali at higher temperature and completely cleaved by 4 N NaOH at 1708C (Iiyama et al., 1990). This procedure has been widely applied during the last decade and provides a better alternative to the saponi®cation/alkaline nitrobenzene oxidation method, as the recovery of the released cinnamic acids is much higher (Lam et al., 1990). It is widely accepted that in CW of grasses and graminaceaous plants, most of the FA (60±90%) is ether-linked, whereas the smaller part is either ester or both ester and ether-linked only (Scalbert et al., 1985; Kondo et al., 1994; Lawther et al., 1996). However, data based on the saponi®cation/alkaline nitrobenzene oxidation procedure reported in earlier studies, show that the proportion of ester to ether-linked FA in tall fescue is 1786/55 mg kgÿ1DM (Jung et al., 1983) and in wheat straw 1209/282 mg kgÿ1 NDF (Kerley et al., 1988). Unfortunately, Kerley et al. (1988) is the only study reporting in vivo digestibility data of ether and ester-linked phenolic acids in wheat straw; which means that there is a lack of relevant published data for comparison.
protocatechuic acids were not CW bound, but found in the alcohol soluble lignin fraction (ASL). The major proportion of all the other components was CW bound. Excluding the PCA which was almost unaffected by the treatment, the solubilising effect of SO2was exerted on all the other CW bound phenolics which were released mostly as ASL and not as monomers. This ®nding is in accord with our previous data showing that the major part of the lignin solubilised by SO2 is released as lignins and oligolignols but not as monomeric phenolics (Yosef et al., 1994; Yosef and Ben-Ghedalia, 1999).
The concentration and distribution of ester and ether-linked phenolics in CW of wheat straw are shown in Table 2. The concentrations of FA and PCA, 11.2 and 9.06 mg gÿ1 CW, respectively are within the range of published values for untreated wheat straw (Provan et al., 1994). Most of the FA (62%) was ether-linked, whereas most of the PCA
Table 1
Concentration of phenolic compounds in whole straw solubilised by 4 N NaOH at 1708C (mg/g straw) and their distribution (%) in cell walls (CW), alcohol soluble lignins (ASL) and alcohol soluble monomers (ASM) Phenolic compound Untreated wheat straw SO2-treated wheat straw
Concentration Distribution in Concentration Distribution in CW ASL ASM CW ASL ASM Ferulic acid 9.63 98.6 0.83 0.57 6.30 63.2 23.9 12.9
p-Coumaric acid 7.91 97.3 0.63 2.07 5.05 88.5 5.35 6.15 Syringic acid 2.13 91.5 6.57 1.93 2.58 73.3 24.0 2.70 Syringaldehyde 2.51 90.4 8.77 0.83 2.73 79.5 19.8 0.70 Vanillic acid 0.92 63.0 30.4 6.60 1.07 62.6 27.1 10.3 Vanillin 1.59 86.8 8.81 4.40 5.30 58.1 40.8 1.10 Protocatechuic acid 2.69 11.9 85.9 2.20 3.75 30.1 69.1 0.80
p-Hydroxybenzoic acid 0.50 94.0 ± 6.00 0.73 78.1 16.4 5.50
p-Hydroxybenzaldehyde 1.42 97.1 ± 2.90 0.54 96.2 ± 3.80
Table 2
Concentration of phenolic compounds solubilised by 4 N NaOH at 1708C from cell walls of straw (mg/g CW) and their distribution (%) as ester (Es) and ether (Et) linked moieties
Phenolic compound Untreated wheat straw cell walls SO2-treated wheat straw cell walls
Concentration Distribution in Concentration Distribution in
Esa Et Esa Et
Ferulic acid 11.2 38.2 61.8 5.53 18.6 81.4
p-Coumaric acid 9.06 77.5 22.5 6.21 66.2 33.8 Syringic acid 2.29 4.40 95.6 2.63 0.50 99.5 Syringaldehyde 2.67 7.00 93.0 3.01 7.40 92.6 Vanillic acid 0.71 21.4 78.6 0.83 22.4 77.6 Vanillin 1.62 19.7 80.3 4.28 3.90 96.1 Protocatechuic acid 0.38 0 100 1.57 0 100
p-Hydroxybenzoic acid 0.55 0 100 0.79 0 100
p-Hydroxybenzaldehyde 1.62 0 100 0.72 0 100
aReleased by 1 N NaOH at 39
8C, for 24 h.
(78%) was ester-linked in the CW of the untreated straw. The pattern of ester and ether linkages found for the cinnamic acids in this paper, is in accord with earlier publications, attributing FA the bridging role within the CW-matrix in terms of: heteroxylan-ester FA ether-lignin structure (Scalbert et al., 1985; Iiyama et al., 1990). PCA is regarded as a lignin component, esteri®ed mostly to the Cgof the phenyl propanoid units of lignin in monocot CW (Nakamura and Higuchi, 1976; Lam et al., 1992). The other minor components are either entirely or mostly, etheri®ed units and are regarded as a part of lignin polymers.
The SO2treatment decreased the concentrations of FA and PCA in CW of wheat straw by 51 and 32%, respectively, and concomitantly increased the content of vanillin by 164%. This effect could be the result of solubilisation of CW components as well as in situ decomposition of C9 to C7 units within the lignin polymer. The SO2treatment was more effective in cleaving the ester bonds of the cinnamic acids. Therefore, the proportion of ether bonds was higher in the CW of the treated than in those of the untreated WS.
The extensive degradation in the rumen and disappearance from the GI of FA and PCA as shown in Table 3, raises the question regarding the fate of these compounds in the rumen. Concentrations of phenolic compounds in the rumen are shown in Table 6. FA was not detected and PCA was very low in rumen liquor. Phenyl-propanoic acid (PPA) was the major phenolic compound detected, at concentrations of 580 and 380mM in the rumen of WS and SO2-WS sheep, respectively. The rumen is a highly reductive environment as fermentation of carbohydrates to VFA yields hydrogen; thus the propensity of the rumen to hydrogenate double bonds of unsaturated compounds is well known. That hydrogenating capacity could also be expressed in reductive demethylation and dehydroxylation; processes which have been both documented to occur with cinnamic acids in the rumen (Martin, 1982). These routes are regarded as responsible for the production of PPA in the rumen from both FA and PCA (Lowry et al., 1993). In support to the above-mentioned, it was found that PPA concentration in hepatic portal venous blood is correlated with the intake of FA and PCA, suggesting that PPA is derived from dietary FA and PCA (Cremin et al., 1995). Thus, FA and PCA once suspected of being inhibitory to rumen microorganisms are extensively degraded, reduced and modi®ed in the rumen,
Table 3
Quantities (g/day) of total phenolic compoundsapresent in the food, ¯owing to the duodenum and excreted in
the feces of sheep fed wheat straw (WS) and SO2-treated wheat straw (SO2-WS) diets
Phenolic compound Treatment Quantities Digested (%)b In food In duo In feces In rumen In GI Ferulic acid WS 6.70 3.67 2.53 45.2 a 62.2 a
SO2-WS 4.57 1.43 1.23 68.7 b 73.1 b
SEM 3.70 1.76
p-Coumaric acid WS 5.50 4.19 2.96 23.8 a 46.2 SO2-WS 3.66 1.90 1.57 48.1 b 57.1
SEM 4.30 1.94
Syringic acid WS 1.58 1.65 1.01 ÿ4.4 a 36.1 SO2-WS 1.96 1.45 1.30 26.0 b 33.9
SEM 5.05 3.72
Syringaldehyde WS 1.92 1.69 0.93 12.0 a 51.8 SO2-WS 2.14 1.24 0.77 42.1 b 64.2
SEM 2.27 1.68
Vanillic acid WS 0.60 0.53 0.26 11.7 a 57.1 SO2-WS 0.81 0.51 0.33 37.0 b 58.8
SEM 3.23 2.81
Vanillin WS 1.13 0.79 0.48 30.1 a 57.3 SO2-WS 3.83 1.63 1.50 57.4 b 60.8
SEM 5.28 2.52
Protocatechuic acid WS 1.89 0.50 0.29 73.5 84.9 SO2-WS 2.72 0.74 0.19 72.8 93.1
SEM 14.3 2.46
aSolubilised by 4 N NaOH at 170
8C.
bValues in the same column with different letters are signi®cantly different,p< 0.05.
Table 4
Degradation of cell wall ester-linked phenolic compounds in the rumen of sheep fed untreated (WS) and SO2
-treated wheat straw (SO2-WS) diets
Phenolic compound
Treatment Quantities (mg/day) Degraded in the rumen (%)a
SEM In food At duo
Ferulic acid WS 2526 402 84.1
SO2-WS 573 101 82.3 1.18
p-Coumaric acid WS 4155 2302 45.2 a
SO2-WS 2153 811 62.3 b 2.79
Syringaldehyde WS 127 84.6 33.2
SO2-WS 130 75.8 41.9 5.98
Vanillic acid WS 309 46.6 84.9 a
SO2-WS 131 38.2 70.9 b 1.54
Vanillin WS 201 50.2 75.1 a
SO2-WS 102 55.7 45.5 b 1.43 aValues in the same column with different letters are signi®cantly different,p< 0.05.
Table 5
Degradation of cell wall ether-linked phenolic compounds in the rumen of sheep fed untreated (WS) and SO2
-treated wheat straw (SO2-WS) diets
Phenolic compound
Treatment Quantities (mg/day) Degraded in the rumen (%)a
SEM In food At duo
Ferulic acid WS 4049 1508 62.8 a
SO2-WS 2301 995 56.8 b 0.76
p-Coumaric acid WS 1216 936 23.0
SO2-WS 1091 874 19.9 9.51
Syringic acid WS 1287 764 40.6 a
SO2-WS 1351 884 34.6 b 1.99
Syringaldehyde WS 1456 856 41.2
SO2-WS 1443 667 53.8 5.93
Vanillic acid WS 113 93.4 17.3 a
SO2-WS 371 115 69.0 b 2.86
Vanillin WS 761 302 60.3
SO2-WS 2116 969 54.2 1.72
Protocatechuic acid WS 222 181 18.5 a
SO2-WS 812 161 80.2 b 4.53
p-Hydroxybenzoic acid WS 321 137 57.3
SO2-WS 410 160 61.0 1.96
p-Hydroxybenzaldehyde WS 949 59.6 93.7 a
giving rise to PPA which is considered as a growth factor for some rumen cellulolytic bacteria (Hungate and Stack, 1982; Stack and Cotta, 1986).
In summary, the SO2treatment reduced the concentrations of cinnamic acids in wheat straw. The lignin solubilising effect of the treatment was expressed mainly in releasing oligomeric but not monomeric phenolics. Extensive degradation of FA and PCA occurred in the rumen. FA was undetectable and PCA content was very low in rumen liquor. PPA, most likely the degradation product of both FA and PCA, was the major monomeric phenolic compound in the rumen.
Acknowledgements
The authors appreciate with thanks the helpful advice and assistance of Dr. J. Miron.
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