Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol83.Issue2.Feb2000:
Animal Feed Science and Technology
83 (2000) 115±126
Changes in the alkaline-labile phenolic compounds of
wheat straw cell walls as affected by SO2 treatment
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) and p-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 SO2 treatment 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 ether-linked compounds. Nevertheless, substantial amounts of etherlinked FA, PCA and other phenolics were removed from CW in the rumen, most likely as oligolignols. 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±40 mM) concentration. Phenylpropanoic acid (PPA) was the major monomeric phenolic compound detected, at concentrations of
580 and 380 mM 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
*
Corresponding author. Tel.: 972-3-960-5113; fax: 972-3-960-4023.
E-mail address: danielbg@actcom.co.il (D. Ben-Ghedalia).
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 1 2 4 - 8
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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-linked p-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-linked p-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,
Selenomonas and Butyrivibrio species was limited by ester-linked feruloyl and p-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 SO2 treatment 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
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
117
(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 2 2 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 (2000 g 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 at ÿ208C. Samples of feces and duodenal digesta were freeze-dried, ground to
pass a 1 mm screen and stored at ÿ208C 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
Veratryl aldehyde was added as an internal standard to alcohol extracts from straw,
duodenal digesta and feces, prepared as described above. The extracts were acidi®ed with
1 N H3PO4 to pH 2, to allow the precipitation of polymeric aromatic material by
centrifugation (Pometto and Crawford, 1988). Ethyl acetate was added to the supernatant,
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E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
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-Ntrimethyl 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), (0.25 mm ®lm thickness; 30 m 0.33 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. Etherlinked phenolic acids were calculated as the difference between total and ester-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 2000 g 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 2000 g and 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 2000 g 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 30 000 g 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 H3PO4 to 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
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
119
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 (0.25 mm ®lm thickness;
50 m 0.25 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 kgÿ1 DM
(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.
The concentrations of phenolic compounds solubilised by 4 N NaOH at 1708C in
whole straw, are shown in Table 1. The levels of 9.63 and 7.91 mg gÿ1 for total FA and
PCA, respectively, in untreated wheat straw, are close to those reported by Provan et al.
(1994) for wheat varieties. The SO2 treatment reduced the contents of FA and PCA by
35% while concomitantly tripling the level of vanillin and increasing by 40% the
concentration of protocatechuic acid. We are unaware whether the treatment effect was
mediated through the conversion of the former to the later ones, or by modi®cation of FA
and PCA while enhancing the release of vanillin and protocatechuic acid from the CW, or
by both pathways. In the untreated straw, 37% of the vanillic and 88% of the
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E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
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
Ferulic acid
p-Coumaric acid
Syringic acid
Syringaldehyde
Vanillic acid
Vanillin
Protocatechuic acid
p-Hydroxybenzoic acid
p-Hydroxybenzaldehyde
Untreated wheat straw
SO2-treated wheat straw
Concentration
Concentration Distribution in
9.63
7.91
2.13
2.51
0.92
1.59
2.69
0.50
1.42
Distribution in
CW
ASL
ASM
98.6
97.3
91.5
90.4
63.0
86.8
11.9
94.0
97.1
0.83
0.63
6.57
8.77
30.4
8.81
85.9
±
±
0.57
2.07
1.93
0.83
6.60
4.40
2.20
6.00
2.90
6.30
5.05
2.58
2.73
1.07
5.30
3.75
0.73
0.54
CW
ASL
ASM
63.2
88.5
73.3
79.5
62.6
58.1
30.1
78.1
96.2
23.9
5.35
24.0
19.8
27.1
40.8
69.1
16.4
±
12.9
6.15
2.70
0.70
10.3
1.10
0.80
5.50
3.80
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 SO2 was
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 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
Concentration
Distribution in
a
Ferulic acid
p-Coumaric acid
Syringic acid
Syringaldehyde
Vanillic acid
Vanillin
Protocatechuic acid
p-Hydroxybenzoic acid
p-Hydroxybenzaldehyde
a
11.2
9.06
2.29
2.67
0.71
1.62
0.38
0.55
1.62
Es
Et
38.2
77.5
4.40
7.00
21.4
19.7
0
0
0
61.8
22.5
95.6
93.0
78.6
80.3
100
100
100
Released by 1 N NaOH at 398C, for 24 h.
5.53
6.21
2.63
3.01
0.83
4.28
1.57
0.79
0.72
Distribution in
Esa
Et
18.6
66.2
0.50
7.40
22.4
3.90
0
0
0
81.4
33.8
99.5
92.6
77.6
96.1
100
100
100
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
121
(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 Cg of 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 SO2 treatment 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 SO2 treatment 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 degradation of total individual phenolic compounds in the rumen and their
disappearance from the gastro-intestine (GI) of sheep is shown in Table 3. If we exclude
the protocatechuic acid, which is a minor, largely non-CW, soluble compound (see Table
1), the highest degradation values in the rumen and disappearance from the GI, were
found with the FA. This phenomenon could be explained by the fact that FA is bound to
the hydrophilic, more accessible part of the matrix, namely to the arabino-furanosyl unit
of the heteroxylan, whereas the other compounds are a part of the less accessible,
hydrophobic lignin (Mosoni et al., 1994; Jacquet et al., 1995). However, PCA and the
other phenolic compounds regarded as lignin constituents, disappeared from the GI also
at substantial rates. The detailed degradation pattern in terms of ester and ether-linked
compounds is presented in Tables 4 and 5. There was no consistent effect of the SO2
treatment on the degradation of ester-linked phenolics in the rumen. However, esterlinked FA was more extensively degraded in the rumen than ester-linked PCA. This
®nding is in accord with Mosoni et al. (1994) who showed that ferulate esters were more
rapidly degraded than p-coumarate esters in wheat CW, and could be explained by the
fact that feruloyl esterases of rumen microorganisms are more active than p-coumaroyl
esterases (Bornemann et al., 1990). The hydrophobicity of the location of PCA could also
be associated with the above-mentioned. Comparing data of Tables 4 and 5, ester-linked
FA and PCA were more extensively degraded in the rumen than the respective etherlinked compounds. Whereas, there is information on cinnamoyl esterase activity of rumen
microorganisms (Bornemann et al., 1990; McSweeney et al., 1998), we are unaware of
published data on the existence of ether-cleaving enzymes in the rumen. Nevertheless,
Table 5 shows that substantial amounts of ether-linked FA, PCA and other phenolic
aldehydes and acids, were removed from the CW of untreated and SO2-treated WS in the
rumen. This phenomenon has probably little to do with direct cleavage of ether linkages
within the lignin molecule in the rumen, and more likely with removal from the CW of
oligo-lignols bound to the heteroxylans via the FA ester anchor (Cornu et al., 1994).
Indeed, our previous publications show that the major route of lignin degradation in the
rumen works through the release of oligomeric phenolics (Ben-Ghedalia et al., 1994;
Yosef and Ben-Ghedalia, 1999).
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E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
Table 3
Quantities (g/day) of total phenolic compoundsa present 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
Digested (%)b
Quantities
In food
In duo
In feces
In rumen
In GI
Ferulic acid
WS
SO2-WS
SEM
6.70
4.57
3.67
1.43
2.53
1.23
45.2 a
68.7 b
3.70
62.2 a
73.1 b
1.76
p-Coumaric acid
WS
SO2-WS
SEM
5.50
3.66
4.19
1.90
2.96
1.57
23.8 a
48.1 b
4.30
46.2
57.1
1.94
Syringic acid
WS
SO2-WS
SEM
1.58
1.96
1.65
1.45
1.01
1.30
ÿ4.4 a
26.0 b
5.05
36.1
33.9
3.72
Syringaldehyde
WS
SO2-WS
SEM
1.92
2.14
1.69
1.24
0.93
0.77
12.0 a
42.1 b
2.27
51.8
64.2
1.68
Vanillic acid
WS
SO2-WS
SEM
0.60
0.81
0.53
0.51
0.26
0.33
11.7 a
37.0 b
3.23
57.1
58.8
2.81
Vanillin
WS
SO2-WS
SEM
1.13
3.83
0.79
1.63
0.48
1.50
30.1 a
57.4 b
5.28
57.3
60.8
2.52
Protocatechuic acid
WS
SO2-WS
SEM
1.89
2.72
0.50
0.74
0.29
0.19
73.5
72.8
14.3
84.9
93.1
2.46
a
b
Solubilised by 4 N NaOH at 1708C.
Values in the same column with different letters are signi®cantly different, p < 0.05.
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 380 mM 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,
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
123
Table 4
Degradation of cell wall ester-linked phenolic compounds in the rumen of sheep fed untreated (WS) and SO2treated wheat straw (SO2-WS) diets
Phenolic
compound
Treatment
Ferulic acid
WS
SO2-WS
2526
573
402
101
p-Coumaric acid
WS
SO2-WS
4155
2153
2302
811
Syringaldehyde
WS
SO2-WS
127
130
WS
SO2-WS
WS
SO2-WS
Vanillic acid
Vanillin
a
Quantities (mg/day)
In food
At duo
Degraded in
the rumen (%)a
SEM
84.1
82.3
1.18
45.2 a
62.3 b
2.79
84.6
75.8
33.2
41.9
5.98
309
131
46.6
38.2
84.9 a
70.9 b
1.54
201
102
50.2
55.7
75.1 a
45.5 b
1.43
Values 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 SO2treated wheat straw (SO2-WS) diets
Phenolic
compound
Treatment
Ferulic acid
Quantities (mg/day)
In food
At duo
WS
SO2-WS
4049
2301
1508
995
WS
SO2-WS
1216
1091
WS
SO2-WS
Syringaldehyde
Vanillic acid
Degraded in
the rumen (%)a
SEM
62.8 a
56.8 b
0.76
936
874
23.0
19.9
9.51
1287
1351
764
884
40.6 a
34.6 b
1.99
WS
SO2-WS
1456
1443
856
667
41.2
53.8
5.93
WS
SO2-WS
113
371
93.4
115
17.3 a
69.0 b
2.86
WS
SO2-WS
761
2116
302
969
60.3
54.2
1.72
WS
SO2-WS
222
812
181
161
18.5 a
80.2 b
4.53
p-Hydroxybenzoic acid
WS
SO2-WS
321
410
137
160
57.3
61.0
1.96
p-Hydroxybenzaldehyde
WS
SO2-WS
949
369
59.6
139
93.7 a
62.3 b
1.66
p-Coumaric acid
Syringic acid
Vanillin
Protocatechuic acid
a
Values in the same column with different letters are signi®cantly different, p < 0.05.
124
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
Table 6
Concentrations of phenolic compounds (mM) in rumen liquor of sheep fed untreated (WS) and SO2-treated
wheat straw (SO2-WS) diets
Phenolic compound
WSa
SO2-WSa
SEM
Phenylpropanoic acid
Phloretic acid
p-Hydroxyphenylacetic acid
p-Coumaric acid
Vanillin
p-Hydroxybenzoic acid
580 a
8.46
15.5
21.0 a
42.2 a
13.6
380 b
16.5
18.0
39.2 b
3.83 b
10.2
26.5
2.53
1.62
3.47
6.88
2.14
a
Values in the same row with different letters are signi®cantly different, p < 0.05.
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 SO2 treatment 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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83 (2000) 115±126
Changes in the alkaline-labile phenolic compounds of
wheat straw cell walls as affected by SO2 treatment
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) and p-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 SO2 treatment 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 ether-linked compounds. Nevertheless, substantial amounts of etherlinked FA, PCA and other phenolics were removed from CW in the rumen, most likely as oligolignols. 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±40 mM) concentration. Phenylpropanoic acid (PPA) was the major monomeric phenolic compound detected, at concentrations of
580 and 380 mM 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
*
Corresponding author. Tel.: 972-3-960-5113; fax: 972-3-960-4023.
E-mail address: danielbg@actcom.co.il (D. Ben-Ghedalia).
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 1 2 4 - 8
116
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
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-linked p-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-linked p-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,
Selenomonas and Butyrivibrio species was limited by ester-linked feruloyl and p-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 SO2 treatment 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
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
117
(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 2 2 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 (2000 g 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 at ÿ208C. Samples of feces and duodenal digesta were freeze-dried, ground to
pass a 1 mm screen and stored at ÿ208C 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
Veratryl aldehyde was added as an internal standard to alcohol extracts from straw,
duodenal digesta and feces, prepared as described above. The extracts were acidi®ed with
1 N H3PO4 to pH 2, to allow the precipitation of polymeric aromatic material by
centrifugation (Pometto and Crawford, 1988). Ethyl acetate was added to the supernatant,
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E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
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-Ntrimethyl 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), (0.25 mm ®lm thickness; 30 m 0.33 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. Etherlinked phenolic acids were calculated as the difference between total and ester-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 2000 g 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 2000 g and 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 2000 g 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 30 000 g 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 H3PO4 to 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
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
119
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 (0.25 mm ®lm thickness;
50 m 0.25 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 kgÿ1 DM
(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.
The concentrations of phenolic compounds solubilised by 4 N NaOH at 1708C in
whole straw, are shown in Table 1. The levels of 9.63 and 7.91 mg gÿ1 for total FA and
PCA, respectively, in untreated wheat straw, are close to those reported by Provan et al.
(1994) for wheat varieties. The SO2 treatment reduced the contents of FA and PCA by
35% while concomitantly tripling the level of vanillin and increasing by 40% the
concentration of protocatechuic acid. We are unaware whether the treatment effect was
mediated through the conversion of the former to the later ones, or by modi®cation of FA
and PCA while enhancing the release of vanillin and protocatechuic acid from the CW, or
by both pathways. In the untreated straw, 37% of the vanillic and 88% of the
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E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
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
Ferulic acid
p-Coumaric acid
Syringic acid
Syringaldehyde
Vanillic acid
Vanillin
Protocatechuic acid
p-Hydroxybenzoic acid
p-Hydroxybenzaldehyde
Untreated wheat straw
SO2-treated wheat straw
Concentration
Concentration Distribution in
9.63
7.91
2.13
2.51
0.92
1.59
2.69
0.50
1.42
Distribution in
CW
ASL
ASM
98.6
97.3
91.5
90.4
63.0
86.8
11.9
94.0
97.1
0.83
0.63
6.57
8.77
30.4
8.81
85.9
±
±
0.57
2.07
1.93
0.83
6.60
4.40
2.20
6.00
2.90
6.30
5.05
2.58
2.73
1.07
5.30
3.75
0.73
0.54
CW
ASL
ASM
63.2
88.5
73.3
79.5
62.6
58.1
30.1
78.1
96.2
23.9
5.35
24.0
19.8
27.1
40.8
69.1
16.4
±
12.9
6.15
2.70
0.70
10.3
1.10
0.80
5.50
3.80
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 SO2 was
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 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
Concentration
Distribution in
a
Ferulic acid
p-Coumaric acid
Syringic acid
Syringaldehyde
Vanillic acid
Vanillin
Protocatechuic acid
p-Hydroxybenzoic acid
p-Hydroxybenzaldehyde
a
11.2
9.06
2.29
2.67
0.71
1.62
0.38
0.55
1.62
Es
Et
38.2
77.5
4.40
7.00
21.4
19.7
0
0
0
61.8
22.5
95.6
93.0
78.6
80.3
100
100
100
Released by 1 N NaOH at 398C, for 24 h.
5.53
6.21
2.63
3.01
0.83
4.28
1.57
0.79
0.72
Distribution in
Esa
Et
18.6
66.2
0.50
7.40
22.4
3.90
0
0
0
81.4
33.8
99.5
92.6
77.6
96.1
100
100
100
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
121
(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 Cg of 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 SO2 treatment 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 SO2 treatment 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 degradation of total individual phenolic compounds in the rumen and their
disappearance from the gastro-intestine (GI) of sheep is shown in Table 3. If we exclude
the protocatechuic acid, which is a minor, largely non-CW, soluble compound (see Table
1), the highest degradation values in the rumen and disappearance from the GI, were
found with the FA. This phenomenon could be explained by the fact that FA is bound to
the hydrophilic, more accessible part of the matrix, namely to the arabino-furanosyl unit
of the heteroxylan, whereas the other compounds are a part of the less accessible,
hydrophobic lignin (Mosoni et al., 1994; Jacquet et al., 1995). However, PCA and the
other phenolic compounds regarded as lignin constituents, disappeared from the GI also
at substantial rates. The detailed degradation pattern in terms of ester and ether-linked
compounds is presented in Tables 4 and 5. There was no consistent effect of the SO2
treatment on the degradation of ester-linked phenolics in the rumen. However, esterlinked FA was more extensively degraded in the rumen than ester-linked PCA. This
®nding is in accord with Mosoni et al. (1994) who showed that ferulate esters were more
rapidly degraded than p-coumarate esters in wheat CW, and could be explained by the
fact that feruloyl esterases of rumen microorganisms are more active than p-coumaroyl
esterases (Bornemann et al., 1990). The hydrophobicity of the location of PCA could also
be associated with the above-mentioned. Comparing data of Tables 4 and 5, ester-linked
FA and PCA were more extensively degraded in the rumen than the respective etherlinked compounds. Whereas, there is information on cinnamoyl esterase activity of rumen
microorganisms (Bornemann et al., 1990; McSweeney et al., 1998), we are unaware of
published data on the existence of ether-cleaving enzymes in the rumen. Nevertheless,
Table 5 shows that substantial amounts of ether-linked FA, PCA and other phenolic
aldehydes and acids, were removed from the CW of untreated and SO2-treated WS in the
rumen. This phenomenon has probably little to do with direct cleavage of ether linkages
within the lignin molecule in the rumen, and more likely with removal from the CW of
oligo-lignols bound to the heteroxylans via the FA ester anchor (Cornu et al., 1994).
Indeed, our previous publications show that the major route of lignin degradation in the
rumen works through the release of oligomeric phenolics (Ben-Ghedalia et al., 1994;
Yosef and Ben-Ghedalia, 1999).
122
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
Table 3
Quantities (g/day) of total phenolic compoundsa present 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
Digested (%)b
Quantities
In food
In duo
In feces
In rumen
In GI
Ferulic acid
WS
SO2-WS
SEM
6.70
4.57
3.67
1.43
2.53
1.23
45.2 a
68.7 b
3.70
62.2 a
73.1 b
1.76
p-Coumaric acid
WS
SO2-WS
SEM
5.50
3.66
4.19
1.90
2.96
1.57
23.8 a
48.1 b
4.30
46.2
57.1
1.94
Syringic acid
WS
SO2-WS
SEM
1.58
1.96
1.65
1.45
1.01
1.30
ÿ4.4 a
26.0 b
5.05
36.1
33.9
3.72
Syringaldehyde
WS
SO2-WS
SEM
1.92
2.14
1.69
1.24
0.93
0.77
12.0 a
42.1 b
2.27
51.8
64.2
1.68
Vanillic acid
WS
SO2-WS
SEM
0.60
0.81
0.53
0.51
0.26
0.33
11.7 a
37.0 b
3.23
57.1
58.8
2.81
Vanillin
WS
SO2-WS
SEM
1.13
3.83
0.79
1.63
0.48
1.50
30.1 a
57.4 b
5.28
57.3
60.8
2.52
Protocatechuic acid
WS
SO2-WS
SEM
1.89
2.72
0.50
0.74
0.29
0.19
73.5
72.8
14.3
84.9
93.1
2.46
a
b
Solubilised by 4 N NaOH at 1708C.
Values in the same column with different letters are signi®cantly different, p < 0.05.
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 380 mM 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,
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
123
Table 4
Degradation of cell wall ester-linked phenolic compounds in the rumen of sheep fed untreated (WS) and SO2treated wheat straw (SO2-WS) diets
Phenolic
compound
Treatment
Ferulic acid
WS
SO2-WS
2526
573
402
101
p-Coumaric acid
WS
SO2-WS
4155
2153
2302
811
Syringaldehyde
WS
SO2-WS
127
130
WS
SO2-WS
WS
SO2-WS
Vanillic acid
Vanillin
a
Quantities (mg/day)
In food
At duo
Degraded in
the rumen (%)a
SEM
84.1
82.3
1.18
45.2 a
62.3 b
2.79
84.6
75.8
33.2
41.9
5.98
309
131
46.6
38.2
84.9 a
70.9 b
1.54
201
102
50.2
55.7
75.1 a
45.5 b
1.43
Values 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 SO2treated wheat straw (SO2-WS) diets
Phenolic
compound
Treatment
Ferulic acid
Quantities (mg/day)
In food
At duo
WS
SO2-WS
4049
2301
1508
995
WS
SO2-WS
1216
1091
WS
SO2-WS
Syringaldehyde
Vanillic acid
Degraded in
the rumen (%)a
SEM
62.8 a
56.8 b
0.76
936
874
23.0
19.9
9.51
1287
1351
764
884
40.6 a
34.6 b
1.99
WS
SO2-WS
1456
1443
856
667
41.2
53.8
5.93
WS
SO2-WS
113
371
93.4
115
17.3 a
69.0 b
2.86
WS
SO2-WS
761
2116
302
969
60.3
54.2
1.72
WS
SO2-WS
222
812
181
161
18.5 a
80.2 b
4.53
p-Hydroxybenzoic acid
WS
SO2-WS
321
410
137
160
57.3
61.0
1.96
p-Hydroxybenzaldehyde
WS
SO2-WS
949
369
59.6
139
93.7 a
62.3 b
1.66
p-Coumaric acid
Syringic acid
Vanillin
Protocatechuic acid
a
Values in the same column with different letters are signi®cantly different, p < 0.05.
124
E. Yosef, D. Ben-Ghedalia / Animal Feed Science and Technology 83 (2000) 115±126
Table 6
Concentrations of phenolic compounds (mM) in rumen liquor of sheep fed untreated (WS) and SO2-treated
wheat straw (SO2-WS) diets
Phenolic compound
WSa
SO2-WSa
SEM
Phenylpropanoic acid
Phloretic acid
p-Hydroxyphenylacetic acid
p-Coumaric acid
Vanillin
p-Hydroxybenzoic acid
580 a
8.46
15.5
21.0 a
42.2 a
13.6
380 b
16.5
18.0
39.2 b
3.83 b
10.2
26.5
2.53
1.62
3.47
6.88
2.14
a
Values in the same row with different letters are signi®cantly different, p < 0.05.
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 SO2 treatment 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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