The interactive effects of enriched sources of Aspergillus ferulic acid esterase and Trichoderma

Transcription

1 The interactive effects of enriched sources of Aspergillus ferulic acid esterase and Trichoderma xylanase on the quantitative release of hydroxycinnamic acids from oat hulls P. Yu 1, J. J. McKinnon 1, D. D. Maenz 1, 2, V. J. Racz 1, 2, and D. A. Christensen 1 1 Department of Animal and Poultry Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5A8 ( yupe@sask.usask.ca); 2 Prairie Feed Resource Centre Inc., 51 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5A8. Received 26 April 2001, accepted 18 February Yu, P., McKinnon, J. J., Maenz, D. D., Racz, V. J. and Christensen, D. A The interactive effects of enriched sources of Aspergillus ferulic acid esterase and Trichoderma xylanase on the quantitative release of hydroxycinnamic acids from oat hulls. Can. J. Anim. Sci. 82: Oat hulls contain relatively high amounts of hydroxycinnamic acids, mainly ferulic (4-hydroxy-3-methoxycinnamic) and p-coumaric acids (4-hydroxy-cinnamic), which are inhibitory to cell wall biodegradability by rumen microorganisms. In this paper, a study of the interactive effects of enriched sources of Aspergillus ferulic acid esterase (A-FAE) and Trichoderma xylanase (T-XYL) at different levels on the quantitative release of ferulic acid and p-coumaric acid from oat hulls was carried out. The results show that relative to A-FAE alone, the combined action of A-FAE and T-XYL was superior in causing the release of ferulic acid [up to 41.0% (± 2.1%)], indicating that T-XYL is important in acting with A-FAE in the degradation of feruloyl-polysaccharides of oat hulls. There was no effect of A-FAE alone, but a significant effect of A-FAE in combination with T-XYL on the release of p-coumaric acid from oat hulls. However, there was no extensive release of p-coumaric acid [(maximum release of 9.0% (± 0.7%)] by A-FAE in the presence of T-XYL, indicating a specificity of A-FAE for feruloyl groups, which only efficiently releases ferulic acid and not p-coumaric acid from oat hulls. This study suggests that A-FAE with T-XYL has an interactive effect to be able to break the ester linkage between ferulic acid and the attached sugar, releasing a significant proportion of the ferulic acid from oat hulls. This action, which causes disruption of crosslinks, has the potential to improve hydrolysis of the remaining polysaccharides by rumen microorganisms, which, in turn, would improve rumen degradability of oat hulls. Key words: Ferulic acid esterase, oat hulls, hydroxycinnamic acids, biodegradation Yu, P., McKinnon, J. J., Maenz, D. D., Racz, V. J. et Christensen, D. A Interaction de l estérase de l acide férulique d Aspergillus et de la xylanase de Trichoderma sur la quantité d acides hydroxycinnamiques libérée par la bale d avoine. Can. J. Anim. Sci. 82: La bale d avoine renferme passablement d acides hydroxycinnamiques, surtout de l acide férulique (4-hydroxy-3-méthoxycinnamique) et de l acide p-coumarique (4-hydroxy-cinnamique), qui entravent la dégradation de la paroi cellulaire par les microorganismes du rumen. Les auteurs ont tenté de voir comment une concentration variable d estérase de l acide férulique d Aspergillus (A-FAE) et de xylanase de Trichoderma (T-XYL) agit sur la quantité d acide férulique et d acide p-coumarique libérée par la bale d avoine. Selon les résultats obtenus, une fois combinés, l A-FAE et le T-XYL libèrent plus d acide férulique [jusqu à 41,0 (± 2,1 %)] que l A-FAE seule, signe que le T-XYL joue un rôle important en intervenant avec l A-FAE dans la dégradation des féruloyl-polysaccharides présents dans la bale d avoine. Si l A-FAE seule n agit pas sur la libération de l acide p-coumarique par la bale d avoine, son action est importante une fois combinée au T-XYL. En présence de T-XYL, l A-FAE n entraîne toutefois pas la libération d une grande quantité d acide p-coumarique (maximum de 9,0 (± 0,7) %). On en déduit que l A-FAE agit spécifiquement sur les groupes féruloyle, qui permettent la libération d acide férulique mais pas d acide p-coumarique dans la bale. Les résultats donnent à penser qu en interagissant avec le T-XYL, l A-FAE réussit à briser le pont ester qui fixe l acide férulique à son sucre, libérant ainsi une quantité importante de cet acide dans la bale d avoine. Cette action perturbe les ponts intercaténaires, ce qui pourrait améliorer l hydrolyse des autres polysaccharides par les microorganismes du rumen, donc aboutir à une meilleure dégradation de la bale d avoine. Mots clés: Estérase de l acide férulique, bale d avoine, acides hydroxycinnamiques, biodégradation 251 Oat hulls comprise 26 to 31% of the weight of the oat seed, and are an agricultural byproduct of the oat milling process. Nutritionally, oat hulls contain approximately 4% crude protein, 1.5% lipid, 37% hemicellulose, 35% cellulose, 6% lignin, 19% neutral detergent insoluble protein (% of total crude protein) and 6% acid detergent insoluble protein (% of total crude protein) (Thompson et al. 2000). The poor nutritional value of oat hulls is a direct result of highly lignified, rigid, cell wall structures that comprise the hull. As such, they are only suitable for ruminant feed. However, because of the enormous quantities available (excess of t per annum) in western Canada (Racz V. J., McKinnon, J. J., personal communication), it is economically important to attempt to improve the nutritional qualities of this byproduct.

2 252 CANADIAN JOURNAL OF ANIMAL SCIENCE Oat hulls contain relatively high amounts of hydroxycinnamic acids, mainly ferulic acid (3-methoxy-4-hydroxycinnamic acid) and p-coumaric acid (4-hydroxycinnamic acid) (Garleb et al. 1987, 1991) at levels of 0.4 to 0.5% and 0.3 to 0.4%, respectively (Garleb et al. 1991; Yu et al. 2000, 2002). They are covalently cross-linked to polysaccharides by ester bonds and to components of lignin, mainly by ether bonds (Scalbert et al. 1985; Borneman et al. 1991). These cross-links are a barrier to biodegradation and limit cell-wall degradability by rumen microorganisms (Graham and Aman 1984; Bohn and Fales 1989; Hartley and Ford 1989; Borneman et al. 1996; Brezillon et al. 1996). It is believed that these hydroxycinnamic acids are among the factors most inhibitory to the biodegradability of plant cell wall polysaccharides (Borneman et al. 1990, 1991). Ferulic acid esterase has been shown to break the ester linkage between ferulic acid and the attached sugar and release ferulic acid from plant cell wall materials (Faulds and Williamson 1995; Bartolomé and Gómez-Cordovés 1999), a process that is likely to improve their rumen biodegradability. This provides a potential biotechnology to improve nutritional value of byproduct feeds with high lignocellulose content, such as oat hulls. There are several sources of ferulic acid esterases, each with specificity on the release of hydroxycinnamic acids. Faulds and Williamson (1994) found that ferulic acid esterase from Aspergillus niger could release ferulic and sinapic acids (3,5-dimethoxy-4-hydroxycinnamic acid), but not p-coumaric or caffeic acids (3, 4-dihydroxycinnamic acid). This indicates that this ferulic acid esterase is active only on methyl esters of certain hydroxycinnamic acids, including ferulic and sinapic esters, but not p-coumaric or caffeic esters. This also shows that only certain substitutions on the aromatic ring can be tolerated and this gives specificity to this enzyme. In contrast, ferulic acid esterase from Pseudomonas fluorescens subsp. Cellulosa is effective on all these methyl hydroxycinnamic esters (Faulds and Williamson 1994), including ferulic acid and p-coumaric acid. Previous research (Yu et al. 2002) has shown that release of ferulic acid and p-coumaric acid by enriched sources of Aspergillus ferulic acid esterase (A-FAE) was dependent upon particle size ( 250 µm) of oat hulls and the presence of an enriched source of Trichoderma xylanase (T-XYL). Several other studies have also shown that efficient release of ferulic acid and/or p-coumaric acid is not only dependent on substrate particle size (Borneman et al. 1991), but also on the presence of other cell wall degrading enzymes such as xylanase (Faulds and Williamson 1995; Bartolomé and Gómez-Cordovés 1999). The objective of this study was to further investigate the interactive effects of enriched sources of A-FAE and T-XYL at different levels on the quantitative release of ferulic and p-coumaric acids from oat hulls. MATERIALS AND METHODS Oat Hulls and Particle Size Oat hulls were obtained from Can-Oat Milling Ltd. at Martinsville, Saskatchewan (Canada) and were ground to pass through a 250-µm-pore-size mesh screen in a centrifuge mill (Christie Norris Model). Enzymes and Activity Assays Enriched sources of Aspergillus ferulic acid esterase (A-FAE) and Trichoderma xylanase (T-XYL) were obtained from Finnfeeds International (UK). The A-FAE activity was determined by measuring the rate of hydrolysis of methyl ferulate (Apin Chemicals Limited, UK) by HPLC using the modified methods from Faulds and Williamson (1995) and Kroon and Williamson (1996). The 100 µl methyl ferulate substrate (1 mm final concentration) and appropriate amount of the esterase in a final volume of 0.5 ml were incubated in a 1.5 ml eppendorf tube at 37 C for 30 min in a water bath. Reaction was stopped by adding 200 µl glacial acetic acid. Samples were centrifuged ( g, 15 min) and then filtered (0.45 µm syringe filters); the supernatant was pipetted into a glass HPLC vial (1 ml volume), and the amount of ferulic acid was determined. One unit (U) of A-FAE activity was defined as the amount of enzyme releasing 1 µmol ferulic acid min 1 at ph 6.0 at 37 C. The T-XYL activity was estimated by measuring the release of reducing sugars (Miller 1959) from 1% (wt/vol) soluble oat spelt xylan (Sigma, X-0627) (Kellet et al. 1990) and expressed as xylose equivalents [Sigma-D (+), X-3877]. One U of activity was defined as the amount of enzyme releasing 1 µmol sugar min 1 at ph 4.8 at 37 C. All assays were performed in triplicate and replicated three times (each time a new enzyme preparation was used), with blanks to correct for background in enzyme and substrate samples. The activities of A-FAE and T-XYL were (± 368) U ml 1 and (± ) U ml 1, respectively. Chemical Hydrolysis Total alkali-extractable ferulic acid and p-coumaric acid contents in oat hull samples (10 mg) were determined by adding 1 M NaOH solution (0.55 ml) followed by incubation at 37 C for 24 h. After centrifugation ( g, 15 min), the supernatant was collected, acidified with acetic acid to ph and extracted five times with equal volumes of ethyl acetate. The organic solutions were combined and evaporated to dryness in an evaporator unit under N 2. The residue was dissolved in 1 ml of methanol/water (50:50, vol/vol) filtered through a 0.45 µm filter and 10 µl samples were analyzed in quadruplicate by HPLC (Faulds and Williamson 1995; Kroon and Williamson 1996). All assays were replicated three times to allow for calculation for means and standard deviation. Enzymic Hydrolysis Enzymic hydrolyses were carried out in 0.1 M MOPS buffer (3-[N-morpholino] propane-sulfonic acid), ph 6.0, in a thermostatically controlled shaking incubator (45 rpm) at 37 C. Oat hulls (10 mg) were incubated with different amounts of A-FAE and/or T-XYL in a final volume of 0.55 ml. After a 24 h incubation, the reaction was stopped by adding 200 µl glacial acetic acid. The samples were centrifuged ( g, 15 min), the supernatant extracted into ethyl acetate five times and pooled, dried samples were taken up in 50%

3 YU ET AL. ENZYMIC RELEASE OF HYDROXYCINNAMIC ACIDS FROM OAT HULLS 253 methanol and filtered through a 0.45 µm filter and 10 µl samples were analyzed in quadruplicate by HPLC (Faulds and Williamson 1995; Kroon and Williamson 1996). All assays were replicated four times. HPLC Conditions Ferulic acid and p-coumaric acid from oat hulls were analyzed by HPLC [a Beckman chromatograph equipped with a 126 programmable solvent module, a 507 autosampler (System Gold) and a RF-551 PC spectrofluorometric detector]. A mobile phase [3.1% methanol, ph 9.5, 20 mm K 2 HPO 4 (Sigma, P-3786)] was applied to a PRP 1 column (Hamilton, PRP-1, Reversed Phase, mm, ph 1 13, Particle Size 5 µm). The flow was 1 ml min 1, with detection at 280 nm excitation and 428 nm emision, with a response time of 1.5 s, 4 range and high sensitivity. The temperature of the column was maintained at room temperature. Ferulic acid and p-coumaric acid contents of the samples were quantified using external standards of ferulic acid (Sigma, F-3500) and p-coumaric acid (Sigma, C-9008). For this procedure, standard solutions (between 2 and 100 µmol) were subjected to the ethyl acetate extraction procedure described above. Calibration curves were calculated based on the linear correlation between concentration of standards and the height of the ferulic acid and p-coumaric acid peaks. The percentage release of ferulic acid and p-coumaric acid were calculated as: 100 enzyme release of ferulic acid or p-coumaric acid/total alkali-extractable ferulic acid or p-coumaric acid. One hundred % ferulic acid and p-coumaric acid were defined as the content of total alkali-extractable ferulic acid and p-coumaric acid in oat hulls. Statistical Analysis Effects of Aspergillus Ferulic Acid Esterase Alone on Release of Hydroxycinnamic Acids Analysis of variance for the effects of A-FAE alone on the release of hydroxycinnamic acids from oat hulls was conducted using the GLM procedure of SAS (SAS, Institute, Inc. 1989) with the following model: Y ik = µ + E1 i + e ik where Y ik is the dependent variable under examination (% ferulic acid and % p-coumaric acid), µ is the overall mean, E1 i the A-FAE effect and e ik the error term. Treatment means were compared using the Student- Newman-Keuls (SNK) test (Steel and Torrie 1980). Significance was declared at P < Effects of A-FAE levels on the release of ferulic and p-coumaric acids from oat hulls were also evaluated by regression analysis with the GLM procedure of SAS, using the following additive model. Where possible, the model was reduced in complexity by excluding non-significant quadratic effects (P > 0.10). Before tests of significance in regression analysis, the UNIVARIATE procedure of SAS was used to test residuals for normality. Y i = α +β 1 + β e i where Y i is the dependent variable under examination (% ferulic acid and % p-coumaric acid), α is the intercept, is the A-FAE effect (in U/assay), β 1 is the linear effect of A- FAE, β 2 is the quadratic effect of A-FAE and e i is the residual error term. Effects of Aspergillus Ferulic Acid Esterase and Trichoderma Xylanase on Release of Hydroxycinnamic Acids Analysis of variance for the effects of A-FAE and T-XYL on the release of hydroxycinnamic acids from oat hulls was conducted using the GLM procedure of SAS with following model: Y ijk = µ + E1 i + E2 j + (E1 E2) ij + e ijk where Y ijk is the dependent variable under examination (% ferulic acid and % p-coumaric acid), µ is the overall mean, E1 i is the A-FAE effect (i = 1 4), E2 j is the T-XYL effect (i = 1 3), (E1 E2) ij is the average effect of the interaction of A-FAE T-XYL, and e ijk is the error term. Treatment means were compared using the Student- Newman-Keuls (SNK) test (Steel and Torrie 1980). Significance was declared at P < Effects of A-FAE and T-XYL levels on the release of ferulic acid and p-coumaric acid from oat hulls were also evaluated by regression analysis with the GLM procedure of SAS, using the following additive model. Where possible, the model was reduced in complexity by excluding non-significant quadratic effects (P > 0.10). Y i = α + β 1 + β 2 + β 3 ( ) + β β e i where Y i is the dependent variable under examination (% ferulic acid and % p-coumaric acid), α is the intercept, the A-FAE effect (in U/assay), is the T-XYL effect (in U/assay), β 1 is the linear effect of A-FAE, β 2 is the linear effect of T-XYL, β 3 is the interaction of A-FAE and T- XYL, β 4 is the quadratic effect of A-FAE, β 5 is the quadratic effect of T-XYL and e i is the residual error term. RESULTS Effects of Aspergillus Ferulic Acid Esterase Alone on Release of Hydroxycinnamic Acids The effects of an enriched source of A-FAE at levels ranging from 3.1 mu to U/assay on the release of ferulic acid and p-coumaric acid from oat hulls after a 24 h incubation are presented in Fig. 1. Total alkali-extractable ferulic and p-coumaric acids in the oat hulls were 3.83 ± 0.69 and 5.21 ± 0.66 mg µg 1, respectively. The results indicate that increasing A-FAE concentration resulted in an increase (P < 0.01) in the release of ferulic acid, but not (P > 0.05) p-coumaric acid. The maximum release of ferulic was 4.7 (± 2.3) % at an A-FAE concentration of U/assay (Fig. 1). Polynomial regression results show that A-FAE alone had both linear (P < 0.001) and quadratic effects (P < 0.001) on release of ferulic acid from oat hulls.

4 254 CANADIAN JOURNAL OF ANIMAL SCIENCE Fig. 1. Enzymic release of ferulic acid (FA) and p-coumaric acid (PCA) from oat hulls with a particle size 250 µm by different levels of an enriched source of Aspergillus ferulic acid esterase (A-FAE). The predictive equation for release of ferulic acid from oat hulls by A-FAE alone was: FA (%) = (A-FAE) (A-FAE) 2, (R 2 = 0.76, RSD = 0.67, P < 0.001), where, A-FAE is expressed in U/assay. Effects of Aspergillus Ferulic Acid Esterase and Trichoderma Xylanase on Release of Hydroxycinnamic Acids The effects of T-XYL alone at levels ranging from 0 to U/assay on the release of ferulic and p-coumaric acids from oat hulls after a 24 h incubation were tested. The results show no detectable release of ferulic and p-coumaric acids. The effects of A-FAE at four concentrations (12.5 mu, 800 mu, 2 U and U/assay) on the release of ferulic and p-coumaric acids from oat hulls after a 24 h incubation in the presence of T-XYL at concentrations (1 U, 256 U and 4096 U/assay) are given in Table 1. In contrast to the results of exp. 1, in which A-FAE alone failed to release any high amount of ferulic acid, A-FAE increased (P < 0.01) both ferulic acid and p-coumaric acid release in the presence of T-XYL (Table 1). Statistical results showed that A-FAE and T-XYL had significant effects (P < 0.01) on the release of ferulic and p-coumaric acids. Averaged over A-FAE levels, increasing T-XYL concentration from 1 U to 256 U to 4096 U/assay resulted in an increase (P < 0.01) of ferulic acid release from 5.3 to 15.5 to 36.5% (SEM = 0.40), respectively, but only a relatively slight increase (P < 0.01) of p-coumaric acid release from 2.3 to 4.9 to 6.3% (SEM = 0.17), respectively. However, averaged over T-XYL levels, increasing A-FAE concentration from 12.5 mu to 800 mu to 2 U to U/assay resulted in only a slight increase in both ferulic and p-coumaric acids release from 17.5 to 21.9% (SEM = 0.46) and from 4.6 to 6.6% (SEM = 0.19), respectively. Polynomial regression was used to evaluate the relationship between A-FAE and T-XYL levels and the release of ferulic and p-coumaric acids. Statistical analysis results show that in the additive model (Y i = α + β 1 + β 2 + β 3 ( ) + β β e i ), linear effects of A-FAE (β 1 ) (P < 0.01) and T-XYL (β 2 ) (P < 0.001), quadratic effects of T-XYL (β 5 2 ) (P < 0.001) on the release of ferulic acid from oat hulls were significant. The interaction of A-FAE T-XYL [β 3 ( )] tended to be significant (P = ). However, the quadratic effect of A-FAE (β 4 2 ) was not significant (P > 0.1). Therefore, the additive model was reduced in complexity by excluding non-significant effects (β 4 2 ). The predictive equation for release of ferulic acid from oat hulls by A-FAE and T-XYL was: FA (%) = (A-FAE) (T-XYL) (A-FAE) (T- XYL) (T-XYL) 2, (R 2 = , RSD = 2.14, P < 0.001) where, A-FAE and T-XYL are expressed in U/assay. For the release of p-coumaric acid from oat hulls, in the additive model (Y i = α + β 1 + β 2 + β 3 ( ) + β β e i ), linear effects (P < 0.001) of A-FAE (β 1 ) and T-XYL (β 2 ), and quadratic effects (P < 0.001) of A-FAE (β 4 2 ) and T-XYL (β 5 2 ) were significant. A-FAE T-XYL interaction [β 3 ( )] was not significant (P > 0.1). The predictive equation for release of p-coumaric acid from oat hulls by A-FAE and T-XYL was:

5 YU ET AL. ENZYMIC RELEASE OF HYDROXYCINNAMIC ACIDS FROM OAT HULLS 255 Table 1. Influence of an enriched source of Trichoderma xylanase (T-XYL: 1 U, 256 U and 4096 U/assay) on the release of p-coumaric acid and ferulic acid z from oat hulls (10 mg, particle size 250 µm) by an enriched source of Aspergillus ferulic acid esterase (A-FAE: 12.5 mu, 800 mu, 26 U and U/assay) after a 24 h incubation y Enzyme level used A-FAE (mu or U/assay) 12.5 mu 800 mu 26 U U T-XYL (U/assay) SEM x Release of hydroxycinnamic acids from oat hulls % of total PCA w 1.71fg 5.38c 6.63b 2.85ef 4.42d 7.57b 1.01g 2.70ef 2.03fg 3.63de 7.01b 9.04a (s.e.) ±0.75 ±0.96 ±0.43 ±0.33 ±0.64 ±1.00 ±0.19 ±0.27 ±0.64 ±0.22 ±0.69 ±0.69 % of total FA w 3.78g 14.96d 33.62c 5.59fg 12.13e 38.06b 4.35g 17.42d 33.34c 7.46 f 17.54d 40.95a (s.e.) ±1.29 ±1.75 ±1.75 ±1.65 ±0.33 ±1.49 ±0.58 ±1.02 ±2.55 ±1.42 ±1.24 ±2.11 Statistical significance v of linear and quadratic effects of A-FAE and T-XYL level on the releases of hydroxycinnamic acids Linear effect Interaction effect Quadratic effect FAE XYL FAE XYL FAE XYL % of total PCA ** ** ** ** % of total FA ** ** + ** z Total alkali-extractable ferulic and p-coumaric acids in the oat hulls were and µg mg 1, respectively. y Enzymic hydrolyses were carried out in 0.1 M MOPS buffer (MOPS=3-[N-morpholino] propane-sulfonic acid), ph 6.0, in a thermostatically controlled shaking incubator at 37 C. x SEM = standard error of means. w PCA = p-coumaric acids; FA = ferulic acid. v excluded from the model; + = P < 0.1; * = P < 0.05; ** = P < a g Means with the different letter in the same row are significantly different at P < 0.05 (SNK test). PCA (%) = (A-FAE) (T-XYL) (A-FAE) (T-XYL) 2, (R 2 = , RSD = 1.02, P < 0.001) where, A-FAE and T-XYL are expressed in U/assay. DISCUSSION The enzyme preparations used in this study were enriched sources of A-FAE and T-XYL supplied by Finnfeeds International. While it is not possible to state that these are pure sources of the respective enzymes, we are confident that there is no detectable cross contamination. For example, testing the A-FAE preparation for reducing sugar release resulted in no detectable release of reducing sugar (results not shown). Testing the T-XYL preparation for ferulic acid release resulted in no detectable release of ferulic acid (results not shown). As such, we are confident that we are dealing with relatively pure sources of these enzymes. Hydroxycinnamic-acid-containing plant cell walls are resistant to rumen microbial degradation (Akin et al. 1983; Akin and Rigsby 1987; Borneman et al. 1991). Rumen degradability of oat hulls is extremely low, mainly due to their contents of hydroxycinnamic acids, mainly ferulic and p-coumaric acids (Garleb et al. 1987, 1991). Although rumen fungi possess some enzymes allowing the fungi to partially degrade some types of phenolic-containing tissue (e.g. xylem) (Borneman et al. 1992), to date, rumen bacteria have been reported to possess no or little ferulic acid and p-coumarol esterase activity (Borneman et al. 1991). Enzymatic studies using material containing ferulic acid and p-coumaric acid as substrates show that three major fiberdegradating rumen bacteria (F. succinogenes, B. fibrisolvens and R. flavefaciens) produced little feruloyl and no p-coumaroyl esterase activity (Borneman and Akin 1990). Therefore, it is likely that using ferullic acid and/or p-coumaroyl esterases to pre-treat hydroxycinnamic-containing plant materials such as oat hulls may provide rumen microorganisms an advantage in the degradation of such materials. The present and previous investigations (Yu et al. 2000, 2002) have shown that a ferulic acid esterase, A-FAE, had no detectable activity toward oat hulls that were ground to pass through a 1-mm screen, but had low levels of activity toward very finely ground (250 and 100 µm) oat hulls, suggesting that substrate size and/or accessibility impedes enzyme activity. The results of the present study show that Trichoderma xylanase alone was unable to release free ferulic and p-coumaric acids from oat hulls. These results are in agreement with those of Faulds and Williamson (1995) and Bartolomé et al. (1997a), who reported that Trichoderma viride xylanase alone did not release any free ferulic acid from wheat bran and barley spent grain. However, both Faulds and Williamson (1995) and Bartolomé et al. (1997a) found that Trichoderma viride xylanase alone could release low-molecular-mass feruloylated material such as O-β-Dxylopyranosyl-(1 4)-O-[5-O-(trans-feruloyl)-α-L-arabinofuranosyl]-(1 3)-O-β-D-xylopyranosyl-(1 4)-D-xylopyr anose (FAXXX); O-[5-O-(trans-feruloyl)-α-L-arabinofuranosyl]-(1 3)-O-β-D-xylopyranosyl-(1 4)-D-xylopyranosem (FAXX), as seen for hydrolysis of wheat bran and barley spent grain. In the experiment of Faulds and Williamson (1995), some unknown feruloylated materials were released that could not be identified. These researchers suggested that the soluble feruloylated oligosaccharides appear to be converted into free ferulic acid only when FAE

6 256 CANADIAN JOURNAL OF ANIMAL SCIENCE is present. Ferulic acid esterase has a greater specificity for short xylo-oligomers and that synergism occurs between xylanase and ferulic acid esterase in the degradation of large feruloyl-polysaccharides (MacKenzie and Bilous 1988; Bartolomé et al. 1995; Faulds and Williamson 1995; Faulds et al. 1995; Bartolomé et al. 1997a, b). This is supported by our experimental results, that the action of enriched sources of A-FAE in combination with T-XYL releases substantially more free ferulic acid from oat hulls than A-FAE alone. This suggests that T-XYL may be particularly important in acting synergistically with A-FAE to release ferulic acid. A likely hypothesis is that the actions of T-XYL result in a feruloyl-oligosaccharide substrate, which can be readily degraded by A-FAE. The present study also shows that A-FAE could not extensively release p-coumaric acid from oat hulls in both the presence and absence of T-XYL, indicating a specificity of A-FAE that only acts on the release of ferulic acid but not p-coumaric acid from oat hulls. To increase the release of p-coumaric acid, it is likely that a specific p-coumaroyl esterase is required. This is an area that needs further investigation. The significance of the present results is that A-FAE activity is enhanced by T-XYL leading to the release of ferulic acid from oat hulls. We hypothesize that in the presence of other cell wall degrading enzymes such as cellulases and glucanases, this interactive activity of A-FAE and T-XYL will lead to an increase in the release of reducing sugar from oat hulls, thus enhancing the utilization of oat hulls by the animal. It is likely that A-FAE with T-XYL acts to free carbohydrates and liberate reducing sugars from oat hulls, which are ester linked to ferulic acid and are not otherwise available for hydrolysis by other enzymes. Theoretically, an increase in the release of ferulic acid from oat hulls should also result in an opening of the lignocellulose structure of oat hulls, facilitating the attack by other cell wall degrading enzymes, thus resulting in high release of reducing sugar. High release of ferulic acid and reducing sugars from oat hulls would be expected to alter the physical and chemical properties of the cell walls and make them more accessible to rumen microorganisms, thus resulting in higher biodegradation. ACKNOWLEDGMENTS The authors are grateful to Dr. Paul A. Kroon (Food Molecular Biochemistry Department, Institute of Food Research, Norwich, UK) and Dr. Andrew A. Olkowski (Department of Animal and Poultry Science, University of Saskatchewan) for technical assistance and helpful discussion, to Finnfeeds International (UK) for supplying the enzymes, and to the Canadian Adaptation and Rural Development Program (CARD) in Saskatchewan, Can-Oat Milling Ltd., Saskatchewan Wheat Pool and FinnFeeds International for financial support. Akin, D. E. and Rigsby, L. L Mixed fungal populations and lignocellulose tissue degradation in the bovine rumen. Appl. Environ. Microbiol. 53: Akin, D. E., Gordon, G. L. R. and Hogan, J. P Rumen bacterial and fungal degradation of Digitaria pentzii grown with or without sulfur. Appl. Environ. Microbiol. 46: Bartolomé, B. and Gómez-Cordovés, C Barley spent grain: release of hydroxycinnamic acids (ferulic and p-coumaric acids) by commercial enzyme preparations. J. Sci. Food Agric. 79: Bartolomé, B., Faulds, C. B., Tuohy, M., Hazlewood, G. P., Gilbert, H. J. and Williamson, G Influence of different xylanases on the activity of ferulic acid esterase on wheat bran. Biotechnol. Appl. Biochem. 22: Bartolomé, B., Faulds, C. B. and Williamson, G. 1997a. Enzymic release of ferulic acid from barley spent grain. J. Cereal Sci. 25: Bartolomé, B., Faulds, C. B., Kroon, P. A., Waldron, K., Gilbert, H. J., Hazlewood, G. and Williamson, G. 1997b. An Aspergillus niger esterase (ferulic acid esterase III) and a recombinant Pseudomonas fluorescens subsp. Cellulosa esterase (XylD) from barley and wheat cell walls. Appl. Environ. Microbiol. 63: Bohn, P. J. and Fales, S. L Cinnamic acid-carbohydrate esters: an evaluation of a model system. J. Sci. Food Agric. 48: 1 7. Borneman, W. S. and Akin, D. E Lignocellulase degradation by rumen fungi and bacteria: ultrastructure and cell wall degrading enzymes. Pages in D. E. Akin, L. G. Ljungdahl, J. R. Wilson, and P. J. Harris, eds. Microbial and plant opportunities to improve lignocellulose utilization by ruminants. Elsevier Science Publishing Co Inc., New York, NY. Borneman, W. S., Akin, D. E. and van Eseltine, W. P Effect of phenolic monomers on ruminant bacteria. Appl. Environ. Microbiol. 52: Borneman, W. S., Hartley, R. D., Morrison, W. H., Akin, D. E. and Ljungdahl, L. G Feruloyl and p-coumaroyl esterases from anaerobic fungi in relation to plant cell wall degradation. Appl. Microbiol. Biotechnol. 33: Borneman, W. C., Ljungdahl, L. G., Hartley, R. D. and Akin, D. E Isolation and characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC 2. Appl. Environ. Microbiol. 57: Borneman, W. C., Ljungdahl, L. G., Hartley, R. D. and Akin, D. E Purification and partial characterization of two feruloyl esterases from the anaerobic fungus Neocallimastix strain MC-2. Appl. Environ. Microbiol. 58: Brezillon, C., Kroon, P. A., Faulds, C. B., Brett, G. M. and Williamson, G Novel ferulic acid esterases are induced by growth of Aspergillus niger on sugar-beet pulp. Appl. Microbiol. Biotechnol. 45: Faulds, C. B. and Williamson, G Purification and characterization of a ferulic acid esterase (FAE III) from Aspergillus niger: specificity for the phenolic moiety and binding to microcrystalline cellulose. Microbiology. 140: Faulds, C. B. and Williamson, G Release of ferulic acid from wheat bran by a ferulic acid esterase (FAE III) from Aspergillus niger. Appl. Microbiol. Biotechnol. 43: Faulds, C. B., Relat, M. C., Williamson, G., Hazlewood, G. P. and Gilbert, H. J Specificity of an esterase (XYLD) from Pseudomonas fluorescens subsp. Cellulosa. Biochim. Biophys. Acta. 1243: Garleb, K. A., Fahey, G. C. and Lewis, Jr., S. M Chemical composition and digestibility of fibre fractions of certain by-product feedstuffs fed to ruminants. J. Anim. Sci. 66: Garleb, K. A., Bourquin, L. D., Hsu, J. T., Wagner, G. W., Schmidt, S. J. and Fahey, Jr., G. C Isolation and chemical analysis of nonfermented fiber fractions of oat hulls and cottonseed hulls. J. Anim. Sci. 69: Graham, H. and Aman. P A comparison between degradation in vitro and in sacco of constituents of untreated and ammonia treated barley straw. Anim. Feed Sci. Technol. 10:

7 YU ET AL. ENZYMIC RELEASE OF HYDROXYCINNAMIC ACIDS FROM OAT HULLS 257 Hartley, R. D. and Ford, C. W Pages in N. G. Lewis and M. G. Paice, eds. Phenolic constituents of plant cell walls and wall biodegradability, Plant Cell Wall Polymers, Biogenesis and Biodegradation. ACR Symp. Ser. 399, American Chemical Society, Washington, DC. Kellet, L. E., Poole, D. M., Ferreira, L. M. A., Durrant A. J., Hazlewood G. P. and Gilbert, H. J Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. Cellulosa contains identical cellulose-binding domains and are encoded by adjacent genes. Biochem. J. 272: Kroon, P. A. and Williamson, G Release of ferulic acid from sugar-beet pulp by using arabinanase, arabinofuranosidase and an esterase from Aspergillus niger. Biotechnol. Appl. Biochem. 23: MacKenzie, C. R. and Bilous, D Ferulic acid esterase activity from Schizophyllum commune. Appl. Environ. Microbiol. 54: Miller, G. L Use of dinitrosalicylic reagent for determination of reducing groups. Anal. Chem. 31: Scalbert, A., Monties, B., Lallemand, J. Y., Guittet, E. and Rolando, C Ether linkage between phenolic acids and lignin fractions from wheat straw. Phytochemistry 24: SAS Institute, Inc User s guide: Statistics. Version 6, SAS Institute, Inc, Cary, NC. Steel, R. G. and Torrie, J. H Principles and procedures of statistics. McGraw-Hill, New York, NY. Thompson, R. K., Mustafa, A. F., McKinnon, J. J., Meanz, D. D. and Rossnagel, B Genotypic differences in chemical composition and ruminal degradability of oat hulls. Can. J. Anim. Sci. 80: Yu, P., Maenz, D. D., McKinnon, J. J. and Racz, V. J Enzymic release of ferulic acid from oat hulls by Aspergillus ferulic acid esterase. Can. J. Anim. Sci. 80: Yu, P., Maenz, D. D., McKinnon, J. J., Racz, V. J. and D. A. Christensen Release of ferulic acid from oat hulls by Aspergillus ferulic acid esterase and Trichoderma xylanase. J. Agric. Food Chem: 50:

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