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Animal Feed Science and Technology


141 (2008) 326338

Effect of enzyme extracts isolated from white-rot


fungi on chemical composition and in vitro
digestibility of wheat straw
M.A.M. Rodrigues a, , P. Pinto b , R.M.F. Bezerra b , A.A. Dias b ,
C.V.M. Guedes a , V.M.G. Cardoso a , J.W. Cone c ,
L.M.M. Ferreira a , J. Colaco a , C.A. Sequeira a
a

CECAV, Universidade de Tras-os-Montes e Alto Douro, Department of Animal Science,


Apartado 1013, 5001-801 Vila Real, Portugal
b CETAV, Universidade de Tr
as-os-Montes e Alto Douro, Departament of Biological and Environmental
Engineering, Apartado 1013, 5001-801 Vila Real, Portugal
c Animal Nutrition Group, Department of Animal Sciences, Wageningen University, The Netherlands
Received 30 March 2007; received in revised form 5 June 2007; accepted 12 June 2007

Abstract
A series of in vitro experiments were completed to evaluate the potential of enzyme extracts,
obtained from the white-rot fungi Trametes versicolor (TV1, TV2), Bjerkandera adusta (BA) and
Fomes fomentarius (FF), to increase degradation of cell wall components of wheat straw. The studies
were conducted as a completely randomized design and analysed using one-way ANOVA. Enzyme
activities of the extracts, previously obtained from a liquid culture medium, were characterized in terms
of laccase and peroxidase for ligninolytic activity. Carboxymethyl cellulase (CMCase) and avicell
digesting cellulase (Avicelase) were used for cellulolytic enzyme assays. Wheat straw samples were
incubated with enzyme extracts in a citrate buffer (pH 5.0) in a forced air oven at 25 C for 6 days. In
Abbreviations: A, estimated asymptotic gas production; ADFom, ADF expressed exclusive of residual ash; B,
time of incubation at which half of the asymptotic gas production (A) has been formed; BA, Bjerkandera adusta;
C, sharpness of the switching characteristic for the gas production profile; DM, dry matter; FF, Fomes fomentarius;
MnP, manganese peroxidase; IVNDFD, in vitro NDF digestibility; NDFom, NDF not assayed with stable amylase
expressed exclusive of residual ash; R, fractional rate of substrate fermentation; Rmax G, maximum rate of gas
production; TRmax G, time at which maximum rate of gas production is reached; TV, Trametes versicolor
Corresponding author. Tel.: +351 259 350 422; fax: +351 259 350 482.
E-mail address: mrodrigu@utad.pt (M.A.M. Rodrigues).
0377-8401/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2007.06.015

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327

vitro NDF digestibility (IVNDFD), and the rate and extent of NDF fermentation, without and after
incubation with the white-rot enzyme extracts, were determined using a gravimetric microbiological
method and a gas production technique, respectively. Results from cell wall chemical composition
showed that TV2 and BA enzyme extracts decreased NDF concentration (P<0.05) and that TV1 had
higher activity (P<0.05) towards cellulose. There was an increase in IVNDFD (P<0.05), resulting
from treatment of wheat straw with enzyme extracts from BA, TV1 and TV2, reaching a difference
of 13% for TV2 (P<0.05), versus the non-treated straw control. Treatment with enzyme extract from
TV2 caused increased gas production (P<0.05) after the first 20 h of incubation, and also increased
the maximum rate of gas production, thus enhancing fermentation kinetics. This study indicates
that enzyme extracts from white-rot fungi can be used to develop new approaches to overcome low
digestibility of some plant cell walls. Utilization of different substrates to produce enzyme extracts
can lead to production of viable ligninolytic complexes which could improve the nutritive value of
fibrous feeds.
2007 Elsevier B.V. All rights reserved.
Keywords: White-rot fungi; In vitro digestibility; Wheat straw

1. Introduction
The structural complexity of plant cell wall is responsible for the partial hydrolytic
enzymatic inaccessibility to cell wall components, limiting the extent of rumen fermentation.
Considering the existing intermolecular cross-links (e.g., ionic, hydrogen, covalent bonds),
cell wall polysaccharide digestion requires both hydrolytic enzymes and enzymes capable
of cleaving the bonds within the cross-linked matrix.
Utilization of exogenous fibrolytic enzymes was first reported in the early 1960s
(Burroughs et al., 1960; Rust et al., 1965). More recent developments in the enzyme industry
have led to re-examination of the role of these enzymes, and several comprehensive reviews
have been published (e.g., McAllister et al., 2001; Beauchemin et al., 2003; Krause et al.,
2003). According to these reviewers, use of enzymes is considered a promising method
to increase milk production, animal performance and feed digestion. Nevertheless, most
commercially available enzyme products are produced for non-feed applications and are
mainly cellulases and xylanases (Beauchemin et al., 2003).
Available data on ligninolytic enzymes is scarce, and most research was with fungi
colonization of cereal straws where digestibility increases were reported (Agosin et al.,
1987; Kamra and Zadrazil, 1988). In fact, fungi in general, but more specifically the
wood-decaying basidiomycetes of white-rot type, are the most efficient microorganisms
in depolymerization and lignin mineralization. In the last decade, crop residue colonization
by white-rot fungi has been studied using solid-state fermentation, with positive in vitro
digestibility results but with a loss of dry matter (DM) compromising the efficacy of utilization of these fungi (Zadrazil et al., 1991; Karunanandaa et al., 1995; Karunanandaa and
Varga, 1996a,b; Jalc et al., 1999; Jalc, 2002). More recently, Gangwar et al. (2003) verified
that wheat straw inoculated with ligninolytic fungi had higher rumen in sacco degradability
versus the control. The methodology used was, once again, based on colonization with
fungi.

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The selective property of white-rot fungi to degrade lignin compounds is mainly due
to the extracellular ligninolytic enzymes in which laccase, lignin peroxidase and manganese peroxidase are considered to be key (Arora et al., 2002). In recent years, laccase
and manganese peroxidase have shown enormous biotechnological potential as they can
be used in a wide variety of lignin degradation applications (Ren and Buschle-Diller,
2007).
The purpose of this research was to evaluate effects of ligninolytic enzymatic complexes,
in which laccase activity is predominant and extracted from different white-rot fungi, on
in vitro digestibility of wheat straw cell walls in order to assess the influence of different
enzymatic activities, mainly those related to lignin oxidation, on enhancement of cell wall
digestion.

2. Materials and methods


2.1. Fungal strains
Four fungal strains were used to obtain the enzymatic extracts, being Trametes versicolor
(TV1, TV2), Bjerkandera adusta (BA), and Fomes fomentarius (FF). Fungi were collected
from the north of Portugal and were isolated by Gomes (2004). Fungi were maintained on
potato dextrose agar (PDA) plates at 4 C and periodically subcultured.
2.2. Production of enzyme extracts
Enzymatic extracts were obtained from a liquid culture medium containing 4.5 g of wheat
straw with 99 ml of citrate buffer 50 mM, adjusted to pH 4.5, and 1 ml of nutrient solution
prepared as by Dias et al. (2003). Incubations were in 250 ml Erlenmeyer flasks containing
the culture media and two 10 mm agar plugs removed from each isolated fungus. Flasks
were put on an orbital shaker (Certomat H-S; B. Braun, Germany) set at 100 rpm and 28 C,
for 15 days.
2.3. Determination of enzymatic activities
Enzymatic activities were determined at 25 C using a Helios UVvis spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA). Ligninolytic activities were
monitored as previously described (Dias et al., 2003) using 0.10.4 ml of culture samples
and the respective buffered substrate in 1.5 ml total reaction volume. Briefly, laccase was
measured following oxidation of 2.0 mM 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulphonic
acid) (ABTS) at 420 nm. Manganese peroxidase was measured following formation of
Mn(III)tartrate complex at 238 nm.
For cellulolytic enzyme assays, activities of carboxymethyl cellulase (CMCase) and Avicell digesting cellulase (Avicelase) were measured according to the IUPAC-Biotechnology
Commission procedures (Wood and Bhat, 1988). The reducing sugars released were determined by dinitrosalicyclic acid (DNS) or the Somogy-Nelson method, using glucose as a
standard (Bezerra and Dias, 2004).

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329

2.4. Wheat straw incubation with enzymatic extracts


From the culture media used to produce the enzymatic extracts, 8 ml of solution were
collected and incubated in 250 ml Erlenmeyer flasks with 150 ml citrate buffer (pH 5.0) and
11 g of wheat straw. The incubation procedure used a micro filter (0.2 m, FP 30/0.2 CA-S,
Schleicher & Schuell, Dassel, Germany). Duplicates were put in a forced air oven at 25 C
for 6 days. Wheat straw residues were obtained after filtration through paper filter. Samples
were immediately dried in a forced air oven at 60 C, ground to pass a 1 mm screen (Retsch,
Cutting mill, model SM1, Haan, Germany) and stored in airtight flasks at room temperature
for later chemical analysis.
2.5. Chemical analyses
Samples were analysed for ash (no. 942.05, AOAC, 1990). Neutral detergent fibre
(NDFom), acid detergent fibre (ADFom) and lignin(sa) fractions by the detergent procedures of Robertson and Van Soest (1981) and Van Soest et al. (1991). Sodium sulfite
and heat stable amylase were not added to the ND. The concentration of hemicellulose
was calculated as the difference between NDFom and ADFom completed sequentially on
the same sample, while that of cellulose as the difference between ADFom and lignin(sa).
The concentrations of volatile fatty acids (VFA) in the fermentation medium after gas
production were analysed in a gasliquid chromatograph (Shimadzu GC-141 B, Kyoto,
Japan) equipped with a flame-ionization detector and a capillary column (SUPELCO Nukol,
0.25 mm i.d. 30 m 0.25 m), using pivalic acid as the internal standard (Czerkawski,
1976).
2.6. In vitro incubations with rumen uid
Two cows, weighing 480 42 kg and fitted with ruminal cannula (Bar Diamond Inc.,
Parma, ID, USA), were used to collect rumen fluid. Cows were fed a diet of maize silage
(0.70), a standard concentrate (0.25) and meadow hay (0.05), offered in equal proportions
at 0800 and 1600 h. Cows had free access to water and mineralvitamin blocks. Rumen
fluid samples were drawn 2 h after the morning feeding into pre-warmed insulated flasks
previously filled with CO2 . Rumen fluid was strained through four layers of cheesecloth
and kept at 39 C under CO2 .
Rumen fluid used to measure gas production was mixed (1:2, v/v) with an anaerobic
buffer/mineral solution (Cone et al., 1996). All laboratory handling was under continuous
flushing with CO2 . Samples (400 mg) were accurately weighed into 250 ml serum bottles
(Schott, Mainz, Germany) and incubated in 60 ml buffered rumen fluid. Each sample was
incubated in duplicate in three series, completed on different days. Gas production was
recorded every 20 min for 72 h using a fully automated system (Cone et al., 1996). After
incubation, the ash content of the residues was determined in order to calculate organic
matter (OM) fermented.
In vitro NDF digestibility was determined according to Tilley and Terry (1963) as modified by Marten and Barnes (1980), but replacing the pepsin/HCl digestion by extraction
with ND at 100 C as described by Goering and Van Soest (1970). Each sample was incu-

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bated in duplicate in three series, completed on different days. Fermentations were in 50 ml


centrifuge tubes containing 250 mg of sample and 25 ml of buffered rumen fluid solution.
Immediately after addition of buffer solution, flasks were closed with rubber stoppers fitted
to a Bunsen valve. After 48 h of incubation, tubes were removed and the NDF contents
determined.
Gas curves were fitted by iteration to a mono-phasic model as described by Groot et al.
(1996) as
Y=

A
1 + (B/t)C

where A = estimated asymptotic gas production; B = time of incubation at which half of the
asymptotic gas production has been formed; C = sharpness of the switching characteristic
for the profile.
Maximum rate of gas production (Rmax G) and the time at which maximum rate of gas
production is reached (TRmax G) were calculated according to Yang et al. (2005) as
Rmax G =
and

A(BC )C(TRmax G(C1) )


2

(1 + (BC )(TRmax G(C) ))




C1
TRmax G = B
C+1

1/C

Fractional rate of substrate fermentation (R) was calculated using the equation of Groot
et al. (1996) as
R=

CTRmax G(C1)
BC + TRmax GC

All data were expressed in terms of ml of gas/g OM fermented.


2.7. Statistical analysis
Data were analysed with the GLM procedure of SAS (1999) as a completely randomized
design experiment using one-way ANOVA.
The statistical model used for the one-way ANOVA was Yij = + i + ij ; where Y is the
response variable; i are the levels of main factor (i = 15); is the overall mean; is the
effect of main factor; is the experimental error.
When significant (i.e., P<0.05) differences were obtained using least square means procedures of SAS (1999) to compare means at the 5% level of confidence using a multiple
comparison t-test.

3. Results
Enzymatic activities of the extracts obtained from cultivation of fungi are in
Fig. 1. In the case of BA, manganese peroxidase (MnP) had higher (P<0.05) activity

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331

Fig. 1. Characterization of ligninolytic activities of the fungi extracts. [Vertical bars represent standard deviation.].

(0.017 0.0118 U/ml) than laccase, while in TV1 the highest (P<0.05) activity was for
laccase (0.144 0.0698 U/ml). With the exception of BA, laccase activities were higher
(P<0.05) than MnP activities, and both BA and FF had much lower activities for all
enzymatic extracts than did TV1 and TV2.
All fungi extracts had endoglucanase (CMCase) and exoglucanase (Avicelase) activities
(Fig. 2) with higher (P<0.05) values for CMCase, in which FF had higher (P<0.05) values
(0.34 0.098 U/ml).
Cell wall chemical composition data (Table 1) show that TV2 and BA enzymatic
extracts decreased NDFom (P<0.05). It is also evident that TV1 had higher activity

Fig. 2. Characterization of cellulolytic activities of the fungi extracts. [Vertical bars represent standard deviation.].

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Table 1
Cell wall composition (g/kg DM) and in vitro gas production constants of samples after treatment with fungi
extractsa
Control

Chemical composition
NDFom
ADFom
Lignin(sa)
Cellulose
Hemicellulose

964 c
615 c
104
511 bc
349 c

In vitro gas production


A (ml/g OM)
242.7 bc
B (h)
23.9
C
1.8
6.36 b
Rmax G (ml/h)
11.8 d
TRmax G (h)
R (/h)
0.034

Enzyme extracts

S.E.M.

BA

TV1

TV2

FF

956 b
623 cd
105
518 c
333 b

964 c
605 b
101
504 b
359 d

958 b
622 cd
105
517 c
336 b

964 c
630 d
111
520 c
334 b

1.5
2.9
3.7
4.2
2.6

257.8 bc
21.3
1.8
7.68 bc
10.7 cd
0.039

270.0 c
22.6
1.8
7.59 bc
10.7 cd
0.036

307.5 d
22.5
1.7
8.84 c
9.01 b
0.037

238.6 b
19.8
1.9
7.66 bc
10.3 bc
0.042

8.67
2.08
0.09
0.005
0.72
0.0046

Means in the same row with different letters differ significantly (P<0.05). S.E.M.: standard error of the mean.
a NDFom, NDF not assayed with stable amylase expressed exclusive of residual ash; ADFom, ADF expressed
exclusive of residual ash; Lignin(sa): lignin determined by solubilisation of ADF with sulphuric acid; A: estimated
asymptotic gas production; B: time of incubation at which half of the asymptotic gas production has been formed;
C: sharpness of the switching characteristic for the profile; Rmax G: maximum rate of gas production; TRmax G:
time at which maximum rate of gas production is reached, R: fractional rate of fermentation.

(P<0.05) towards cellulose than the other enzymatic extracts, which is consistent with
data on the cellulolytic activity of endoglucanase. The TV1 enzymatic extract did not
affect hemicellulose concentration, while the other three extracts had a pronounced effect
(P<0.05).

Fig. 3. Variation in the IVNDFD of wheat straw treated with enzymatic extracts. Different letters (ac) denote
a difference (P<0.05) with a one-way analysis of variance (S.E.M. = 5.12). [Vertical bars represent standard
deviation.].

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333

Fig. 4. Volatile fatty acid (acetate, propionate, butyrate, iso-valeric, valeric) production after in vitro incubation with
rumen fluid of wheat straw treated with enzymatic extracts (mmol/l). [Vertical bars represent standard deviation.].

There was an increase (P<0.05) in in vitro NDF digestibility (IVNDFD) from treatment
of wheat straw with enzymatic extracts from BA, TV1 and TV2, reaching a difference of
13% in the case of TV2 (Fig. 3).
Enzymatic extracts increased the amount of fermentable carbohydrates after the first
20 h of incubation, thereby increasing total gas production. Cumulative gas production of
enzymatic treatment TV2 was continuously higher than by all the other enzymatic extracts
(data not shown). Table 1 shows that the maximum gas production and maximum rate of
gas production of TV2 were 27 and 40% higher (P<0.05) than control values. In addition,
the time at which maximum rate of gas production was reached was lower (P<0.05) for
TV2.
Although there were no differences between the VFA concentrations (Fig. 4), acetate
production for the enzymatic treatments numerically varied from 40.0 mmol/l for the control
BA and FF to 46 mmol/l for TV1 and TV2 treatments.

4. Discussion
The enzymatic extracts had laccase and MnP activities that were lower than those
obtained by other authors using basidiomycetous fungi grown on wheat straw mediums.
Arora et al. (2002) presented values for laccase and MnP in T. versicolor of about 2.40 and
2.00 U/ml, respectively. More recently, Kapich et al. (2005), using a culture medium containing wheat straw incubated with T. versicolor, reported that F. fomentarius had an MnP
activity of 0.17 U/ml and B. adusta a laccase activity of 0.22 U/ml. Differences normally

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reported in the literature are mainly because these enzymes are produced in different growth
mediums and due to utilization of different strains of fungi cultivated during different periods of incubation. These differences are well described in Songulashvili et al. (2007) who,
evaluating 18 strains of basidiomycetes in submerged fermentation of mandarin peelings,
reported ligninolytic activities for T. versicolor that varied from 17.1 to 20.4 U/ml for laccase and 0.06 to 0.71 U/ml for MnP. In addition, variable results for Fomes fomentarium,
with values ranging from 2.28 to 8.97 U/ml for laccase and 0 to 0.03 U/ml for MnP, were
also obtained. In the same study, values of enzymatic activities varied considerably when
the same fungi were cultivated with wheat grain residues from ethanol production.
Regarding cellulolytic enzyme assays, a common feature is the higher CMCase activity
in relation to Avicelase (Valaskova and Baldrian, 2006). Tanaka et al. (1999) reported values
of CMCase 80-fold higher than Avicelase in T. versicolor cultivated in wood-containing
cultures. Our results suggest an average 15-fold difference in all enzymatic extracts, probably because we utilized enzyme extracts and, once again, a determinant influence of fungi
strains should be stressed.
Research on the utilization of white-rot fungi to improve degradation of straws have
mainly focused on utilization of a specific number of fungi species and strains. With the
exception of Zadrazil (1985), who examined approximately 200 strains of white-rot fungi,
and Capelari and Zadrazil (1997) who screened 72 species and strains of tropical fungi,
most results have used Phanerochaete chrysosporium, Cyathus stercoreus and Pleurotus
spp. Our experiment deals with straw biodegradability using a different approach and fungi
species not previously assayed. In fact, utilization of these enzymatic extracts is not common
in the treatment of fibrous feeds.
Our results do not seem to suggest high variability in the potential for white-rot fungi to
alter the structure of cell wall components as reported elsewhere (Jung et al., 1992; Camarero
et al., 1994; Karunanandaa and Varga, 1996a,b). Only small cell wall composition changes
occurred, mainly in the hemicellulose fraction. The decrease in hemicellulose is normally
attributed to initial consumption of carbohydrates during the mycelial fungi growth. In
our study, xylanolytic activity was not measured, but it seems that this fraction may have
been preferentially degraded by the enzymatic extracts. Similarly, Karunanandaa and Varga
(1996a) noted that, during fungal colonization with Cyathus stercoreus, hemicellulose was
degraded in preference to cellulose, and Jalc et al. (1996), studying six fungi species, verified
that, among individual NDF components, hemicellulose was reduced to the greatest extent.
Nevertheless, Jung et al. (1992) reported that while treatment of oat straw with five white-rot
fungi induced a reduction in NDF concentration for some treatments, no changes occurred
in the composition of NDF, except for lignin(sa) concentration.
The enzymatic activity of endo and exoglucanase indicated that cellulose could be
degraded to some extent, but our results showed that there was no effect. Experiments
of Jalc (2002) showed that cellulose degradation is also dependent on the fungal treatment,
noting that when 13 species of fungi were examined with wheat straw, the cellulose content
was only reduced with 6 species.
Lignin(sa) concentration was not affected by treatment with enzymatic extracts. The
capacity to alter lignin composition seems to depend of the fungi species. However, Jalc et
al. (1997) found that NDF and ADF were reduced in wheat straw treated with different fungi.
However, while three of the treatments led to a large proportional loss of hemicellulose and

M.A.M. Rodrigues et al. / Animal Feed Science and Technology 141 (2008) 326338

335

lignin, two of the others had higher losses in cellulose and hemicellulose. It is possible that
lignin decomposition would increase if the incubation period was extended. However, the
enzymatic activity of extracts is not sustained for long periods precluding incubation stages
as long as those normally presented for white-rot colonization.
In spite of the lack of chemical composition changes in cell wall components, the enzymatic extracts seemed to affect cell wall structure. Higher IVNDFD, as well as differences
in gas production of TV2 extracts, support this assumption. We believe that this enhanced
capacity of TV2 is related to its ligninolytic activities, and to the synergic effect that might
have been established with the other enzymes. Although most white-rot fungi secrete at least
two ligninolytic enzymes (Tuor et al., 1995), others apparently secrete only one. Our results
show that FF and BA are essentially MnP producers, while both T. versicolor strains showed
a typical laccase/MnP producing pattern, with TV2 having a lower relationship between the
concentrations of these two enzymes. Similar synergic effects were observed by Camarero
et al. (1994), who showed preferential degradation of lignin moieties by Pleurotus eryngii and Phanerochaeta chrysosporium, and considered that the decrease in the amount of
lignin units could be due to action of laccase and MnP, both of which could easily oxidize
these phenolic structures. Synergistic effects between enzymes are considered essential for
efficient degradation of cell wall polysaccharides, and examples of such relationships have
also been found by De Vries et al. (2000) and Bhat and Hazlewood (2001).
The effect exerted by the enzymatic extracts, especially from the TV2 basidiomycete,
in the in vitro fermentation, increasing total gas production and the maximum rate of fermentation, indicates that cell wall structure must have been modified, as the values were
expressed in ml of gas produced per g of OM fermented. Furthermore, the time needed to
attain maximum rate of gas production was numerically lower for all the fungi treatments,
suggesting increase of a more rapidly fermentable fraction within the cell walls.
Probable changes in the integrity of cell walls are also supported by the VFA concentrations. The tendency to higher acetate production in fermentation of the fungi wheat straw
substrates corresponds to an improvement of fibre degradation, either due to an effect in the
microbial population or in the metabolic pathways of substrate degradation.
The capacity for white-rot fungi to degrade fibrous components was expected. Indeed,
Agosin et al. (1986) reported increased degradability of cell wall when fungi treated wheat
straw was ruminally fermented in situ, and Jung et al. (1992) reported an increase in in vitro
DM digestibility in oat straw treated with fungi, while Karunanandaa and Varga (1996a)
found that rice straw NDF digestibility increased from 0.480 to 0.570 in a 30-day culture
with white-rot fungi. However, our data show that these results can also be obtained using
enzymatic extracts, with the advantage that DM losses are substantially decreased. Jalc
(2002), in an extensive review on straw degradation by white-rot fungi, noted that medium
DM loss is normally about 260 g/kg with values varying from 120 to 660 g/kg. Our results
(data not shown) indicate a much lower loss of about 20 g/kg.

5. Conclusions
The low nutritive value of straws has been widely demonstrated. Thus, treatments that
might increase ruminal degradation of NDF are of great importance because increased NDF

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digestibility may reduce physical fill in the rumen, thereby allowing higher voluntary feed
intake. This study showed that utilization of enzymatic extracts isolated from white-rot
fungi enhanced in vitro degradation of wheat straw cell walls. This effect seems to depend
on synergistic effects between the enzymes. Results also indicate that this type of treatment
may promote attack of ligninocellulosic substrates by enzymes, thereby avoiding excessive
polysaccharide consumption. Further study is needed to examine effects of ligninolytic
enzymes in cell wall degradation, as well as analysing the type and characteristics of several
types of enzyme mixtures.

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