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Journal of the Science of Food and Agriculture
J Sci Food Agric 79:1987±1992 (1999)
Ensiling characteristics and ruminal degradation
of Italian ryegrass and lucerne silages treated
with cell wall-degrading enzymes
Yu Zhu,1* Naoki Nishino,1 Yoshiro Kishida2 and Senji Uchida1
1
Division of Animal Science, Faculty of Agriculture, Okayama University, Okayama 700–8530, Japan
Division of Agricultural Production System, Faculty of Agriculture, Okayama University, Okayama 700–8530, Japan
2
Abstract: Two experiments were carried out to investigate the effect of added cell wall-degrading
enzymes at ensiling on the fermentation and in situ degradation of grass and legume silages. Primary
growths of Italian ryegrass (Lolium multi¯orum Lam) and lucerne (Medicago sativa L) were wilted
and ensiled in laboratory-scale silos with or without enzymes. Silages were opened at 2, 5, 15 and
45 days after storage; the fermentation quality and the contents of cell wall components (NDF, ADF,
ADL) were determined. The 45 day silages were subjected to in situ incubation experiments, and the
kinetics of DM and NDF degradation was estimated. The enzyme treatment enhanced the lactic acid
production (P < 0.01) and reduced the pH value (P < 0.01) of both Italian ryegrass and lucerne silages.
The contents of cell wall components, however, were not affected by enzymes, except for NDF of Italian
ryegrass silage. The in situ incubation experiments revealed that added enzymes increased the rapidly
degradable DM (P < 0.01) and appeared to decrease the rate of degradation of DM and NDF of Italian
ryegrass silage. The rapidly degradable DM was not altered when lucerne was treated, but the rate of
degradation of NDF was signi®cantly reduced (P < 0.05). These results suggest that although added cell
wall-degrading enzymes could improve the preservation of grass and legume silages, the effects on
ruminal degradation may be different according to the herbage treated. Enzymatic hydrolysis during
ensilage may be restricted to easily digestible cell walls, leaving relatively less digestible components
that would be retained in treated silages.
# 1999 Society of Chemical Industry
Keywords: cell wall-degrading enzymes; Italian ryegrass; lucerne; silage
INTRODUCTION
As a consequence of added cellulolytic and hemicellulolytic enzymes, sugars will be released from cell
wall components to provide extra substrates for lactic
acid fermentation. This bene®t has been demonstrated
by many workers,1±4 and enzymes of high activity have
been commercialised. However, controversy still exists
about the effect of enzymes on the digestion of organic
matter. Increases in digestibility4±9 might indicate that
the breakdown of cell wall components could facilitate
microbial digestion, while claims have been made that
enzymes would principally hydrolyse easily degradable
components and leave less digestible organic matter in
the treated silages.10,11
If forages were ef®ciently hydrolysed by cell walldegrading enzymes, then the kinetics of organic matter
digestion would be altered. Although several workers
examined the effect of enzymes on the in situ
degradation of silages, the results were variable and
did not reach agreement.9,10,12,13 The response to
enzyme treatment may be different between grasses
and legumes owing to certain differences in their
composition and digestion of cell wall components.
However, information is apparently scarce on the
fermentation and digestion of enzyme-treated silages
prepared from the two forage species.
Two experiments were carried out to study the
ensiling characteristics and in situ degradation of
enzyme-treated silages using wilted Italian ryegrass
and lucerne. The enzymes were composed of two
fungal cellulases derived from Acremonium cellulolyticus
and Trichoderma viride, the former enzyme being
higher in avicelase, carboxymethylcellulase, xylanase
and pectinase than the conventional latter enzyme.14
Wilting was aimed at avoiding an ef¯uent production
in which highly digestible nutrients would be wasted
during storage.
EXPERIMENTAL
Preparation of silages
In experiment 1 the primary growth of Italian ryegrass
* Correspondence to: Yu Zhu, Division of Animal Science, Faculty of Agriculture, Okayama University, Okayama 700–8530, Japan
Contract/grant sponsor: Ministry of Education, Science and Culture of Japan; contract/grant number: 7306013; contract/grant number:
10760157
(Received 24 September 1998; revised version received 22 April 1999; accepted 30 June 1999)
# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50
1987
Y Zhu et al
Item
Composition
Dry matter (g kgÿ1)
Crude protein (g kgÿ1 DM)
Water-soluble carbohydrate (g kgÿ1 DM)
Buffering capacity (meq kgÿ1 DM)
Lactic acid bacteria (log cfu gÿ1)
Table 1. Composition of wilted forages
and activity of enzymes used in silage
preparation
Italian
ryegrass
Lucerne
303
81.0
200
352
4.78
377
175
53.9
493
4.91
Enzyme activity
Avicelase (mmol sugar released gÿ1 minÿ1)
Carboxymethycellulase (mmol sugar released gÿ1 minÿ1)
Xylanase (mmol sugar released gÿ1 minÿ1)
Enzymes
638
9260
2630
Mean values of duplicate analysis.
(Lolium multi¯orum Lam) was harvested at the early
heading stage and wilted for about 2 h to a targeted
300 g kgÿ1 dry matter (DM) content. The herbage was
chopped with a forage cutter to a theoretical 25 mm
length and ensiled in 1 l polyethylene bottles at a rate of
460 g per silo. The enzymes (1:2 mixture of Acremonium and Trichoderma cellulase based on avicelase
activity) were dissolved in the minimum amount of
water and added at 50 mg kgÿ1 just before ensiling.
Triplicate silos for each treatment were opened after 2,
5, 15 and 45 days of storage at room temperature.
In experiment 2 the primary growth of lucerne
(Medicago sativa L) was used at the early blooming
stage. The herbage was wilted for about 3 h and then
chopped to 13 mm length. The procedures of ensiling,
enzyme addition and storage were the same as those in
experiment 1, except that the herbage was ensiled at a
rate of 450 g per silo.
Nylon bag incubation
In situ degradation of DM and NDF was determined
using 45 day silages. The freeze-dried samples were
ground to pass through a 2 mm screen, and triplicate
silages were mixed to a composite for each treatment.
The surface area and pore size of the nylon bags were
110 140 mm2 and 42 mm respectively, and each bag
contained 3 g of composite sample. Three castrated
male goats equipped with permanent rumen cannulae
were used for nylon bag incubation. The goats were
given Italian ryegrass or lucerne hay with concentrates
so as to be fed the same forage as samples incubated.
Bags were removed from the rumen after 3, 6, 12, 24,
48 and 72 h and washed with a domestic washing
machine until running tap water was cleared. The
residues were dried in a forced-air oven at 60 °C for
24 h.
Data and statistical analysis
Analyses
The pH value, lactic acid, volatile fatty acids and NH3N of silage were determined on cold water extracts.15
Viable counts of lactic acid bacteria were measured
using GYP-CaCO3 agar plate.16 The chemical composition of silage was determined on a freeze-dried
sample which was ground to pass through a 1 mm
screen. Total nitrogen (N) was determined by the
standard Kjeldahl procedure. Neutral detergent ®bre
(NDF), acid detergent ®bre (ADF) and acid detergent
lignin (ADL) were determined by the methods of
Goering and Van Soest.17 Water-soluble carbohydrate
(WSC) and buffering capacity were measured according to the methods described by McDonald and
Henderson18 and Playne and McDonald19 respectively.
The activities of avicelase, carboxymethylcellulase
and xylanase of enzyme preparations were determined
at pH 4.50 in 0.1 M acetate buffer. The reaction with
microcrystalline cellulose (Funacel-SF Funakoshi),
carboxymethylcellulose (C-5678 Sigma) or xylan (X0502 Sigma) was conducted at 50 °C for 30 min, and
the sugars released were determined using dinitrosalicylic acid reagent.
1988
The results from the incubation study were ®tted to an
exponential equation with a discrete lag time,20
p = a ‡ b(1 ÿ eÿc(t ÿ t0)), where p is the degradation at
time t, a is the rapidly degradable fraction, b is the
slowly degradable fraction, c is the rate of degradation
and t0 is the degradation lag time. The a fraction for
both DM and NDF was determined as the loss during
the washing described above. The parameters explaining the degradation were estimated by non-linear
regression analysis using Systat (Ver 5.2 for the
Macintosh). The effects of enzymes on silage fermentation, chemical composition and in situ degradation
were statistically analysed by Student's t-test.
RESULTS
The DM contents of Italian ryegrass and lucerne were
both increased above the target (300 g kgÿ1) after
wilting (Table 1). Italian ryegrass had a favourable
property as silage material, with high WSC and low
buffering capacity. The numbers of lactic acid bacteria
were nearly 105 cfu gÿ1 at ensiling for both forages.
Addition of enzymes enhanced the lactic acid
production (P < 0.01) and reduced the pH value
J Sci Food Agric 79:1987±1992 (1999)
Degradation of silages treated with cell wall-degrading enzymes
Figure 1. Changes in lactic acid bacteria count, pH value, lactate and NH3N of Italian ryegrass silage with or without added cell wall-degrading
enzymes.
Figure 3. Changes in lactic acid bacteria count, pH value, lactate and NH3N of lucerne silage with or without added cell wall-degrading enzymes.
(P < 0.01) of Italian ryegrass silage (Fig 1). The
bacterial counts increased to a peak at 5 days after
ensiling and remained almost unchanged until the
fermentation was terminated at 45 days. Although ®nal
pH values were around 4.7, with not more than 32 g
lactate kgÿ1 DM, the proportion of NH3-N was less
than 50 g kgÿ1 N, and no butyrate was found in the
silages. Acetate was detected in all the silages, but the
contents did not exceed 12 g kgÿ1 DM and were not
in¯uenced by enzyme treatment (data not shown).
The contents of NDF and ADF appeared to increase
as the time of storage was prolonged (Fig 2). Addition
of enzymes signi®cantly reduced NDF (P < 0.01) of
Italian ryegrass silage, while the effects on ADF and
ADL were not signi®cant.
The bene®ts of enzymes appeared more pronounced in the treated lucerne silage (Fig 3). The
reduction in pH value and the increase in lactate were
recorded from an early ensiling period, and the ®nal
values at 45 days were both improved (P < 0.01) by the
addition of enzymes. The proportion of NH3-N was
relatively high, with a signi®cant improvement only at
15 days of fermentation (P < 0.05). Acetate was found
in small amounts (<15 g kgÿ1 DM), but butyrate was
not produced in any of the silages (data not shown).
Compared with Italian ryegrass, the content of NDF
was lower and those of ADF and ADL were higher in
Figure 2. Changes in neutral detergent fibre, acid detergent fibre and acid
detergent lignin of Italian ryegrass silage with or without added cell walldegrading enzymes.
Figure 4. Changes in neutral detergent fibre, acid detergent fibre and acid
detergent lignin of lucerne silage with or without added cell wall-degrading
enzymes.
J Sci Food Agric 79:1987±1992 (1999)
1989
Y Zhu et al
3 and 72 h (P < 0.05). The effects of added enzymes on
the kinetics of in situ degradation were not the same as
those for Italian ryegrass silage; the rapidly degradable
DM was not altered, but the amount and rate of
degradation of NDF were signi®cantly reduced
(P < 0.05) when lucerne silage was treated with
enzymes.
DISCUSSION
Figure 5. Ruminal degradation of dry matter and neutral detergent fibre of
Italian ryegrass and lucerne silages with or without added cell walldegrading enzymes.
lucerne, and none of the cell wall components were
affected by enzymes in this study (Fig 4).
Initial degradation of DM was increased (P < 0.01)
when enzyme-treated Italian ryegrass silage was
incubated in the rumen (Fig 5). Although similar
values were found after 6 h, the difference between the
two silages was signi®cant (P < 0.05) at 48 h of
incubation. The effects of added enzymes were much
clearer in the degradation of NDF, and lower values
were obtained at 12, 48 and 72 h (P < 0.05, < 0.01 and
0.05 respectively). The kinetics of in situ degradation
in Italian ryegrass silage was not affected by the
enzyme treatment (Table 2), except for the rapidly
degradable fraction of DM being signi®cantly higher
(P < 0.01). The rate of degradation showed a decrease
in both DM and NDF of enzyme-treated silage.
However, the differences did not reach signi®cance
(P = 0.11 and 0.13 respectively).
Lucerne silage disappeared more quickly than
Italian ryegrass silage, and both DM and NDF
degradation seemed to reach a plateau after 24 h of
incubation. Although the degradation of DM was not
affected by the addition of enzymes, that of NDF was
lowered when enzyme-treated silage was incubated for
The ef®cacy of enzyme addition was shown to relate to
the level of enzyme application and the maturity and
DM contents of treated herbage.10,21 The lower
response in high-DM silages was probably due to less
ef®cient distribution of enzymes, which could be
transported with the aid of water.10 The high DM of
the two forages might therefore have diminished the
effects of enzymes in this study, whereas the two fungal
cellulases were shown to provide much bene®t even
when added to high-DM (300 and 400 g kgÿ1) barley
straw silage.22 The rate of enzyme application
(50 mg kgÿ1) was considerably lower in this study
compared with those reported in other experiments.10,23,24 This was de®ned by manufacturer's
recommendation that a higher level of enzymes would
produce an ef¯uent owing to the higher activity of
Acremonium cellulase than of conventional commercial
cellulases.
Although the enzymes could enhance the lactate
fermentation of both Italian ryegrass and lucerne
silages, the effects appeared greater in the latter silage.
This could be ascribed to the differences in sugar
content and buffering capacity of material crops,
because the reduction of NDF was similar in the two
silages. In sugar-rich forages the additional substrates
released from cell walls may have been only a little help
in organic acid production.
The impact of enzymes on cell wall components was
not clear in either of the two silages. Evidence has
Table 2. Kinetics of ruminal degradation of Italian ryegrass and lucerne silages with or without added cell wall-degrading enzymes
Italian ryegrass
Item
Control
Enzyme-treated
Lucerne
SED
F
Control
Enzyme-treated
SED
F
Dry matter
Rapidly degradable fraction (g kgÿ1)
Slowly degradable fraction (g kgÿ1)
Potential degradation (g kgÿ1)
Rate of degradation (hÿ1)
Lag time (h)
379
435
814
0.047
2.40
405
414
819
0.037
2.53
0.20
1.17
1.00
0.003
0.23
**
NS
NS
NS
NS
372
312
684
0.099
1.56
390
298
688
0.108
1.41
0.68
0.57
0.82
0.006
0.38
NS
NS
NS
NS
NS
Neutral detergent ®bre
Rapidly degradable fraction (g kgÿ1)
Slowly degradable fraction (g kgÿ1)
Potential degradation (g kgÿ1)
Rate of degradation (hÿ1)
Lag time (h)
32.7
692
725
0.042
2.40
42.3
700
742
0.032
2.89
0.31
2.48
2.40
0.004
0.24
NS
NS
NS
NS
NS
110
372
482
0.069
1.99
103
349
452
0.042
2.54
0.57
0.57
0.68
0.007
0.41
NS
*
*
*
NS
Means of three incubation measurements.
NS, not signi®cant.
* P < 0.05, ** P < 0.01.
1990
J Sci Food Agric 79:1987±1992 (1999)
Degradation of silages treated with cell wall-degrading enzymes
shown that added enzymes signi®cantly decrease the
contents of NDF and ADF of silages,10,23,24 while no
signi®cant changes were also reported by several
workers.22,25 When the same enzymes were added to
fresh rhodesgrass at the same level as in this study, the
reduction in NDF and ADF was found regardless of
the storage temperature.26
Although the changes in cell wall contents were
small, in situ degradation was signi®cantly altered by
enzymes in both silages. Results of Italian ryegrass
silage were mostly in agreement with those of Van
Vuuren et al,10 who found that enzyme treatment
increased the rapidly degradable organic matter and
reduced the rate of cellulose degradation in perennial
ryegrass silage. Nadeau et al 13 reported smaller effects
on legume (lucerne) than on grass (cocksfoot) silage,
whereas the response in this study appeared greater
when the legume crop was treated. Unlike the ®ndings
of Hoffman et al,9 both the amount and rate of NDF
degradation were reduced in enzyme-treated lucerne
silage. These disagreements might be due to the
characteristics of added enzymes, because Acremonium
cellulases were shown to have high pectinase activity to
enhance extensive hydrolysis of legume cell walls.27
The amount of NDF degradation was reduced in
lucerne but not in Italian ryegrass silage, suggesting
that a high concentration of lignin in legumes would
interfere with the degradation by both enzymes and
rumen micro-organisms.
It is concluded that although the addition of cell
wall-degrading enzymes could improve the preservation of grass and legume silages, the effects on ruminal
degradation would be different according to forage
species. Enzymatic hydrolysis may be restricted to
easily digestible components, and then relatively less
digestible cell walls would be retained in grass and
legume silages.
ACKNOWLEDGEMENTS
We are grateful to Snow Brand Seed Co for the kind
supply of cell wall-degrading enzymes. This study was
supported in part by Grants-in-Aid for Scienti®c
Research (7306013 and 10760157) from the Ministry
of Education, Science and Culture of Japan.
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J Sci Food Agric 79:1987±1992 (1999)
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