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,3
,4
* Department of Agriculture, The University of Reading, Reading RG6 6AR United Kingdom;
and
FMS Division, Institute of Food Research, Norwich NR4 7UA United Kingdom; and
and
Agriculture and Agri-Food Canada, Research Centre, Lethbridge, AB, Canada T1J 4B1
3 Correspondence:
Agriculture and Agri-Food Canada, Research Centre, P.O. Box 3000, Lethbridge, AB, T1J 4B1 Canada (phone: +1-403-317 3427; fax: +1-403-317 2182; E-mail:
colombattod{at}agr.gc.ca).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Cellulose • Enzymes • Fermentation • Rumen • Xylan
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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As an alternative to costly in vivo trials, several in vitro studies have been conducted to examine the effects of enzymes on the degradation of feedstuffs. However, the complexity of these feeds makes it difficult to identify which feed fractions are most influenced by enzymic action. The use of purified xylans and cellulose will minimize this complexity and provide an alternative way to evaluate the mode of action of enzymes.
Our hypothesis was that fibrolytic enzymes would increase the rate of fermentation of cellulose and xylan during the incubation period with ruminal fluid by enhancing the release of reducing sugars during the preincubation period. The present study was undertaken to examine the effect of adding a commercial enzyme product on: a) the release of reducing sugars from cellulose and xylan, b) the profile of the main enzymic activities during incubation, and c) the rate and extent of in vitro fermentation of these pure substrates.
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Materials and Methods |
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Substrates and Enzyme Product
The substrates used were microcrystalline cellulose (CE, Avicel PH-101, Fluka Chemicals, Seelze, Germany), xylan from oat spelt (XYL, X-0627, Sigma Chemicals, Dorset, U.K.) and a mixture (CEXYL, 1:1 wt/wt) of the two. The enzyme product used, Liquicell 2500, was manufactured by Specialty Enzymes and Biochemicals Co. (Fresno, CA) and originated from Trichoderma reesei. The enzyme was extensively characterized prior to use (Colombatto, 2000). At pH 5.5 and 39°C, it contained 14,864, 1,699, 2.6, 45.5, and 1.4 units (µmol•min-1•mL of product-1) of xylanase (EC 3.2.1.8), endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91), ß-D-glucosidase (EC 3.2.1.21), and 
-L-arabinofuranosidase (EC 3.2.1.55) activity, respectively. It contained no detectable amounts of ß-D-xylosidase (EC 3.2.1.37) activity.
Release of Reducing Sugars
Triplicate 50-mg samples of each substrate were weighed into 15-mL plastic test tubes. The enzyme product was added at four levels: 0, 0.51, 2.55, and 5.10 µL/g of substrate DM (control, 1x, 5x and 10x, respectively). The lowest level of enzyme was equivalent to levels used in vivo and had previously been shown to positively alter alfalfa stem fermentation in vitro (Colombatto, 2000). Citrate-phosphate buffer (pH 6.8) was added to the tubes in order to achieve a 50-mM final buffer concentration. The enzyme product was dissolved in distilled water to give a final solution volume of 5 mL before adding to the tubes. Sodium azide (0.1 mg/mL) was added to each tube to prevent microbial growth. Treatments were placed at 20°C for 20 h, after which the samples were immediately analyzed for reducing sugars using the dinitrosalicylic acid method (DNS, Miller, 1959). The absorbance was read at 540 nm using a spectrophotometer (Lambda 15, Perkin Elmer, Beaconsfield, U.K.) and the value converted to reducing sugar equivalents using a standard curve of glucose. The experiment was replicated on two occasions.
Enzyme Activities Throughout the In Vitro Incubation
One hundred milligrams of each substrate (CE, XYL, CEXYL) was weighed into separate Hungate tubes (Bellco Glass Inc., Vineland, NJ) with eight replicates per treatment. The enzyme was added to each tube 20 h prior to inoculation with ruminal fluid at three levels: 0, 0.51, and 2.55 µL/g of substrate DM (control, 1x, and 5x, respectively). Enzyme was added in distilled water to give a final volume of 0.2 mL. Three hours later, 8 mL of anaerobic buffer (Goering and Van Soest, 1970) was added and the tubes were stored at room temperature (24°C) for 20 h. Ruminal fluid was collected 5 h postfeeding from a ruminally-cannulated steer that was offered ad libitum access to alfalfa hay. Whole ruminal contents were strained through four layers of cheesecloth under a continuous CO2 stream. The fluid was transferred to prewarmed Thermos flasks and immediately transported to the laboratory. Two milliliters of ruminal fluid was added to each tube using an Aqueous Minipet dispenser (Bel-Art Products, Pequannock, NJ). Inoculation was complete within 45 min of the fluid being collected. Tubes were capped and a needle was inserted to prevent fermentation gases from inhibiting fermentation, after which the tubes were stored at 39°C with sporadic shaking. At 0, 6, 18 and 48 h postincubation, 1.0-mL samples were taken from the liquid fraction of two tubes per treatment for enzyme activity determinations. The samples were immediately centrifuged (Spectrafuge 16M, Labnet Int. Inc., Woodbridge, NJ) at 16,000 x g for 5 min at room temperature and supernatants were stored at -15°C until further analysis. Volatile fatty acid analyses were carried out on 1.0-mL samples obtained at 6, 18, and 48 h. These samples were acidified with 25% (wt/vol) metaphosphoric acid, applied at a 1:5 (vol/vol) ratio, and frozen at -15°C until analyzed. Tubes from the 6- and 18-h incubations were further processed to obtain feed particle-associated (FPA) fractions, as described by Wang et al. (2001). The tube contents were centrifuged (20,000 x g, 20 min, 4°C) and the supernatants discarded. The resulting pellets were placed in individual sealed plastic bags together with 6 mL of 0.1 M citrate-phosphate buffer (pH 6.0) and processed for 90 s in a Stomacher 400 laboratory blender (Seward Medical Ltd., London, U.K.). The processed samples were centrifuged (20,000 x g; 30 min; 4°C) and the supernatant stored at -15°C until analysis for enzyme activity.
Enzyme activities in liquid and FPA fractions were determined at pH 6.0 and 39°C following the procedures of Wood and Bhat (1988). Endoglucanase, exoglucanase, ß-D-glucosidase, xylanase, ß-D-xylosidase, and -L-arabinofuranosidase were determined. Solutions or suspensions (10 mg/mL) of oat spelt xylan, low-viscosity carboxymethylcellulose (C-5678, Sigma Chemicals), and Avicel were used as substrates for xylanase, endoglucanase, and exoglucanase determination, respectively. Undiluted solutions of enzymes (40 to 50 µL) were incubated in duplicate for 120, 240, or 360 min for xylanase, endoglucanase, and exoglucanase determination, respectively. The enzymatic reaction was terminated by adding DNS reagent and absorbance was read at 530 nm using a MRX-HD plate reader (Dynatech Laboratories Inc., Chantilly, VA). The absorbance values were converted to reducing sugars with standard xylose or glucose curves developed under identical conditions. Blanks, substrate alone (i.e., no enzyme), and enzyme alone (i.e., no substrate) were also included to correct for substrate autolysis and sugars present in the enzyme formulation, respectively. One unit of activity was defined as the amount of enzyme required to release 1 nmol of xylose or glucose equivalent in 1 min, under the conditions of the assay.
For glycosidase activity determinations, stock solutions (1 mM) of p-nitrophenyl (p-NP) derivatives were used. Substrates were p-NP-ß-D-glucopyranoside (Sigma N-7006), p-NP-ß-D-xylopyranoside (Sigma N-1895), and p-NP--L-arabinofuranoside (Sigma N-3641). Undiluted solutions of enzymes (12.5 to 20.0 µL) were incubated with buffer and substrate at 39°C and pH 6.0 for 180 min and the reaction was terminated by the addition of 0.4 M glycine NaOH buffer (pH 10.8). Release of p-nitrophenol was determined colorimetrically at 420 nm. One unit of enzyme activity was defined as the amount of enzyme required to release 1 nmol p-nitrophenol in 1 min, under the conditions of the assay.
In Vitro Gas Production
The Reading pressure technique (RPT, Mauricio et al., 1999) was used. Approximately 0.5 g of DM of each substrate were weighed in triplicate and added to 125-mL serum flasks (Wheaton Scientific, Millville, NJ). The enzyme was applied at the same levels as described for the reducing sugar assay and dissolved in distilled water. After 3 h, 90 mL of anaerobic buffer was added and the flasks were stored overnight at room temperature (20°C). Ruminal fluid was obtained prefeeding (0700) from a nonlactating cow that was fed grass hay (first series) or grass silage plus straw (second series). Ruminal fluid was strained through two layers of muslin under CO2 and kept at 39°C in a water bath. The temperature of the flasks was raised to 39°C prior to inoculation with 10 mL of ruminal fluid. Inoculation of all flasks was complete within 1 h of the ruminal fluid being obtained. Incubation proceeded for 96 h at 39°C. Head-space gas produced (GP) from substrate fermentation was measured as pressure at 2, 4, 6, 8, 10, 12, 15, 19, 24, 30, 36, 48, 72, and 96 h of incubation, as described by Mauricio et al. (1999). Pressure values, corrected for the amount of substrate OM incubated and gas released from negative controls (ruminal fluid only and ruminal fluid plus enzymes at the three addition levels), were used to generate volume estimates using the quadratic equation reported by Mauricio et al. (1999). Estimates of the rate of gas production were calculated from the values obtained at each measurement interval. The experiment was replicated twice in time.
Values for each treatment, averaged across replicates in both gas runs, were fitted to the model proposed by France et al. (1993) to obtain estimates of the lag phase before gas production started (L), the time at which half of the asymptote gas volume was produced (T/2), and fractional rates of digestion (µ) at different time points. Estimated values were obtained using the maximum likelihood program (MLP, Ross, 1987). The equation Y = A [1 - eb(T - t) - c (vt - vT)] was transformed and fitted in the functional form G = A - BQt Z(vt) where G is gas (mL) accumulated at time (t), A is asymptotic gas pool value (mL), B = A(bt + cvT) has no biological meaning, T is the lag time (h) prior to the start of gas production, Q = e-b and Z = e-c, where c (h-1/2) and b(h-1) are constants. The combined rates of gas production (µ) are time-dependent and are calculated as µ = b + c/(2
t), where t is incubation time.
Statistical Analyses
The experiments were analyzed using the MIXED procedures of SAS (SAS Inst., Inc., Cary, NC). Data for the reducing sugar release were analyzed with a model that included substrate, enzyme level (0, 0.51, 2.55, and 5.10 µL/g DM substrate), and their interaction as fixed effects, and experimental run (replication in time) as a random effect. Data were also analyzed separately for each substrate. The enzymatic activities during the in vitro incubation with ruminal fluid were analyzed separately for each activity and incubation time. Separate analyses by incubation time were carried out to separate possible effects attributable to the exogenous enzymes (short incubation times) or to the ruminal microbes (longer incubation times). The model included substrate, level of enzyme (0, 0.51, and 2.55 µL/g DM substrate), and their interaction as fixed effects. The VFA profiles were analyzed as described for the enzymatic activities. Differences among means of the enzymic activities and VFA were evaluated for significance using a LSD test. A LSD test was used since the main objective was to compare the control to the enzyme treatments, and trend analysis would not have been meaningful with only three treatment levels.
Since our objective was to determine differences between enzyme and control treatments for each substrate, the cumulative in vitro gas production was analyzed separately for each incubation time and substrate. The model included enzyme level (0, 0.51, 2.55, and 5.10 µL/g DM substrate) as a fixed effect and experimental run as a random effect. Linear and quadratic contrasts were performed. Rates of gas production were derived from the cumulative values, and analyzed separately for each time and substrate. Contrasts between rates of gas production in the control and enzyme treatments were performed. The parameters obtained with the France et al. (1993) model were analyzed using a model that included substrate, enzyme level, and their interaction as fixed effects. In all cases, significance was declared at the 5% probability level.
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Results |
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). Moreover, the release of reducing sugars was positively related to the level of enzyme applied. With CE, only the highest level of enzyme addition increased (P < 0.001) the release of reducing sugars. With XYL, the highest level of enzyme addition released 0.85 mg of glucose equivalents/mL, corresponding to 4.25 mg sugars from the 50 mg of XYL originally incubated. The same level of enzyme released 2.40 of mg sugars from 50 of mg CEXYL, 56% of that released from XYL alone.
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). At the endpoint (48 h), xylanase activity was greatest (P < 0.05) at the 1x level in CE, but decreased (P < 0.05) by the same amount in CEXYL. The addition of enzyme at 5x increased (P < 0.05) the initial (0 h) endoglucanase activity in both XYL and CEXYL, but not in CE.
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). In XYL and CEXYL, differences (P < 0.05) were still observed after 6 h, but only at the highest level of enzyme addition. At 48 h of incubation, the addition of enzyme was found to reduce (P < 0.05) ß-D-glucosidase activity in CEXYL, whereas there was a small increase in ß-D-xylosidase activity in XYL with 5x at 6 h. Enzyme addition reduced (P < 0.05) endpoint ß-D-xylosidase activity in CEXYL. At 6 h, -L-arabinofuranosidase activity was increased (P < 0.05) at 5x in XYL, and this effect was maintained through 48 h of incubation. No differences (P > 0.05) were observed in CE and CEXYL during the first 6 h of incubation. Consistent with the findings for ß-D-glucosidase and ß-D-xylosidase, endpoint -L-arabinofuranosidase was reduced (P < 0.05) by enzyme addition to CEXYL.
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). In contrast, xylanase activity was increased (P < 0.05) by the highest level of enzyme after 18 h of incubation in all substrates. Endoglucanase activity essentially followed the same trend as xylanase activity, except that no differences were detected between the enzyme-treated XYL and control samples after 18 h of incubation.
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). At 18 h, treating XYL with the highest enzyme level reduced (P < 0.05) ß-D-glucosidase activity. ß-D-Xylosidase activity was unchanged in CE and XYL at 6 h, but decreased (P < 0.05) in the treated CEXYL samples. At 18 h of incubation, ß-D-xylosidase activity was negligible. -L-Arabinofuranosidase activity increased (P < 0.05) at 6 h in XYL treated at 5x, but remained unchanged in CE and decreased (P < 0.05) in CEXYL. At 18 h of incubation, small but significant (P < 0.05) reductions in (-L-arabinofuranosidase activity were found when substrates were treated with enzyme at 5x.
Volatile Fatty Acids
The VFA profiles are shown in Table 4. Averaged across treatments, total VFA concentrations at 6 h were higher (P < 0.05) in the XYL samples than in CEXYL or CE. After 48 h of incubation, however, the trend was reversed with the XYL samples containing the lowest concentration.
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At 6 h of incubation, the acetate:propionate ratio was affected (P < 0.05) by substrate (CE > CEXYL > XYL) and by level, with the control samples having a higher (P < 0.05) acetate:propionate ratio than the enzyme treatments. At endpoint, the acetate:propionate ratio was increased (P < 0.05) by enzyme addition at 5x in the CE and CEXYL samples; however, the increments are unlikely to be of biological importance.
In Vitro Gas Production
Addition of the enzyme product increased (P < 0.05) GP from the fermentation of the substrates (Table 5). A quadratic response of GP to enzyme level was found in all substrates, but with the exception of treated XYL, no differences were detected among 1x, 5x, and 10x after 96 h of incubation. However, addition of enzyme increased (P < 0.05) GP after 30, 36, and 48 h of incubation of CE, whereas with XYL and CEXYL the increase (P < 0.05) in GP occurred at an earlier stage (6 h) of incubation (data not shown). When the rates of GP were examined, the three substrates showed clear differences in their release profiles (Figure 2). With CE, the maximal rate was attained between 30 and 36 h of incubation, whereas although the XYL samples showed two peaks in the controls (10 and 19 h postinoculation), only one was found with the enzyme-treated samples (at 10 h). The CEXYL samples also showed two distinctive peaks: the first one between 4 and 10 h of incubation and the second at 24 to 36 h of incubation (Figure 2).
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. There was an effect (P < 0.05) of substrate (CE > CEXYL > XYL) and enzyme addition (all enzyme treatments > control) on the potential GP predicted by the model. The T/2 was only affected (P < 0.05) by the substrate (CE > CEXYL > XYL). The fractional rates of digestion (µ) showed that, averaged across treatments, XYL was fermented more rapidly (P < 0.05) than the other substrates at 6 h, 12 h, and at T/2. Moreover, enzyme addition increased (P < 0.05) µ at 6 h postincubation.
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Discussion |
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Studies have demonstrated that an enzyme x feed interaction is required prefeeding for a benefit in digestion to be observed (Lewis et al., 1996). Some authors have suggested that this is possibly due to the creation of a stable enzyme-feed complex (Kung et al., 2000), whereas others have indicated the possibility of alteration in the fiber structure (Nsereko et al., 2000) or the release of reducing sugars, which should help increase the establishment and action of the rumen microbial populations. Wang et al. (2001) found that enzymes applied 24 h before interaction with ruminal fluid in continuous culture increased the release of reducing sugars from barley silage, but not from alfalfa hay. In our study, the addition of enzyme enhanced the release of reducing sugars from both xylan and a mixture of cellulose and xylan. Release of sugars from the mixture amounted to 56% of those released from xylan alone, indicating that release of reducing sugars from cellulose was negligible. Probable reasons for this were the low exoglucanase and ß-D-glucosidase activities present in the commercial product or that the incubation conditions (pH 6.8, 20°C) inhibited the enzymatic action, despite the long incubation time (20 h).
However, lack of reducing sugar production does not necessarily mean that the enzymes were ineffective against cellulose. Mechanical action of cellulases on cellulose has been detailed (Kerley et al., 1988; Klyosov, 1990). One of the most important phenomena is the dispersion of cellulose, brought about by the adsorption of cellulases to cellulose defects (disturbances in the crystalline structure), followed by their penetration into the interfibrillar space. Banka et al. (1998) purified a low-molecular-weight (11.4 kDa) protein from the culture supernatant of Trichoderma reesei, which caused nonhydrolytic disruption of filter paper without release of reducing sugars. Those authors hypothesized that the action of this protein, named fibril-forming protein, seemed to be the breakdown of hydrogen bonds, leading to a loosening of the cellulose structures. These processes may result in the weakening of crystalline cellulose, creating new sites for faster microbial attachment and fermentation.
In agreement with previous reports (Williams and Strachan, 1984; Williams et al., 1989; Michalet-Doreau et al., 2001), the activity levels found in the FPA fractions were higher than the levels found in the liquid fraction. Moreover, the activity levels in the FPA fraction were expressed in terms of nmol•min-1•mL of supernatant-1, and are therefore likely to be a significant underestimate.
The enhancement of fibrolytic activity, mainly xylanase, found in the present study agrees with in vitro data reported by Morgavi et al. (2000b). Hristov et al. (2000) found that direct infusion of an enzyme additive into the rumen of growing heifers increased xylanase activity by 56% and endoglucanase activity by 20%. In the present study, the marked increase in xylanase activity in the liquid fraction up to 6 h postincubation obtained with the addition of enzyme suggests that this particular activity was resistant to degradation by the ruminal fluid, which concurs with previous results (Hristov et al., 1998; Morgavi et al., 2000b; 2001). However, the exogenous endoglucanase activity in the liquid fraction appeared to be less stable than xylanase, with increases observed only at 0 h in the XYL and CEXYL treatments at the highest level of enzyme addition. Lower stability of endoglucanases compared to xylanases from Trichoderma species has been reported (Morgavi et al., 2000b). Likewise, the initial ß-D-glucosidase activity level in the liquid fraction was enhanced by enzyme addition in all substrates, and this enhancement persisted after 6 h in the 5x-treated XYL and CEXYL samples. It is relevant to point out that glycosidases are generally present in very high quantities in the feed itself (Wallace and Hartnell, 2001); thus, added enzymes may not have any influence on total activities when applied to feedstuffs.
The extra enzymes present in the treated substrates may have two possible origins. The increase in enzyme activities in the liquid fraction at the beginning of the fermentation probably originated from the exogenous enzyme applied, whereas later increases in the liquid fraction and the higher activities found in the FPA fractions could be derived from an increase in the fibrolytic bacterial population. This is in agreement with Wang et al. (2001), who found that the addition of enzymes increased the number of cellulolytic bacteria almost 10-fold and that xylanase activity increased in both the liquid and FPA bacterial fractions.
The increase in the rate and extent of GP with enzyme addition indicates an increase in the fermentability of the substrates. Moreover, increasing the enzyme level from 1x to 5x increased the rate and extent of GP, but enzyme addition at 10x did not produce further improvements. Positive quadratic responses to enzyme addition have been reported in vitro (Colombatto et al., 2002) and in vivo (Beauchemin et al., 1995; Lewis et al., 1999). A probable reason for such a response has been proposed by Morgavi et al. (2000c), who found that elevated enzyme levels decreased the attachment of the rumen bacterium Fibrobacter succinogenes to pure cellulose. The authors hypothesized that the enzyme product competed with F. succinogenes for available binding sites on cellulose. Morgavi et al. (2000c) also found that the same product stimulated adhesion to corn silage and alfalfa hay, suggesting that the nature of responses in complex vs. pure substrates could be different. However, that study was conducted with only one ruminal species and may not truly reflect ruminal conditions where competition with other cellulolytic organisms is normally high (Mosoni et al., 1997). Recently, Nsereko et al. (2002) reported that the addition of incremental levels of an enzyme product (derived from T. longibrachiatum) to a diet fed to dairy cows stimulated the numbers of total viable bacteria in a quadratic manner, which may help explain the observed negative effects of excessive enzyme levels on in vivo trials.
As expected, the gas production profiles differed widely between the substrates examined. Xylan was the substrate most readily fermented by the ruminal microbes, followed by the mixture of cellulose and xylan, and then by cellulose alone. This is clearly evidenced by both the observed rate of gas release curves (Figure 2) and by the parameters of the France et al. (1993) model, where the lag phase was reduced and the fractional rate of gas production at 6 h was increased by enzyme addition (Table 6). This finding is further supported by the fact that averaged across enzyme levels, initial activities in the xylan "diet" were higher than that with the other substrates, as were the VFA concentrations. Analysis of the rate of gas production suggests that purified xylans contained two distinct fermentable pools, which tended to combine when exogenous enzymes were applied. The latter underlines one of the most likely modes of action of exogenous enzymes in ruminant diets: an increase in the rate of fermentation and, probably, degradation of feedstuffs in the rumen, compared with untreated diets. The RPT technique used was capable of detecting such subtle changes, which identifies it as a suitable technique for the initial selection of potential exogenous enzyme additives.
The increase in the rate of cellulose fermentation in the early stages of fermentation, albeit small and therefore difficult to quantify, supports the hypothesis that subtle changes in the cellulose structure by enzyme action allowed the rumen microbes to obtain earlier access to fermentable substrates. This was accompanied by an increase in ß-D-glucosidase activity in the FPA fraction, suggesting a more rapid utilization of cellobiose and probably other cellooligosaccharides.
The fact that the final gas production with CE was similar to that obtained with XYL, despite the differences in the release of reducing sugars, suggests that the release of reducing sugars during the pretreatment period may not always be the key to improving the subsequent ruminal digestion of feedstuffs. Other mechanisms (i.e., disruption of cellulose structure) are clearly also involved.
Under the conditions of the present study, it appears that the fermentation of pure cellulose, pure xylan, or a mixture of both were all enzyme-limited. These results were obtained using enzyme levels equivalent to application rates used in vivo (Beauchemin et al., 1995; Kung et al., 2000). Moreover, improvements in rate of gas production and OM degradation of alfalfa stems treated with the same enzyme product at the same rates as those applied here have been reported previously (Colombatto et al., 2000), in contrast with Wallace et al. (2001), who suggested that excessive enzyme levels were required to affect the fermentation of forages in vitro.
Overall, these findings indicate that the responses observed when enzymes are added to ruminant diets are not due to a single effect; rather, they are the result of a combination of pre- and postfeeding mechanisms. Although they might not fully represent natural feedstuffs, purified substrates are a helpful tool for examining the mode of action of exogenous enzyme products.
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Footnotes |
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2 D. Colombatto acknowledges the FOMEC Program, Facultad de Agronomia, Universidad de Buenos Aires, Argentina, for a Ph.D. scholarship. BBSRC (U.K.) is also acknowledged for financial support.
4 Present address: Institute National de la Recherche Agronomique, Centre Clermont-Theix, 63122 Saint-Genès-Champanelle, France.
Received for publication July 8, 2002. Accepted for publication December 9, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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) or treated with fibrolytic enzymes at 1x (•), 5x (
), and 10x (
). Data are least squares means ± SED. An asterisk indicates a difference between control and enzyme-treated substrates (P < 0.05).