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J Sci Food Agric 79 :679–686 (1999)
Journal of the Science of Food and Agriculture
Effects of peptidase inhibitors and other
additives on fermentation and nitrogen
distribution in perennial ryegrass silage
Victor L Ns erekoº and John A Rooke*
Animal Biology Divis ion , SAC , Craibs tone Es tate , Aberdeen , AB21 9YA , UK
Abstract : The eþ ects of formic acid, three concentrations of formaldehyde in a formic acid/
formaldehyde mixture and cysteine-peptidase inhibitors, 1-trans epoxysuccinyl-leucylamido-(4guanidino) butane (E-64), N-ethylmaleimide and cystamine on nitrogen (N) distribution during
ensilage of perennial ryegrass (Lolium perenne) were investigated. A third cut or perennial ryegrass
(163 g dry matter kg—1 and 61 g water-soluble carbohydrate kg—1 dry matter) was ensiled in two silo
sizes ; formaldehyde-treated herbage was ensiled only in larger silos (500–550 g herbage) and cysteinepeptidase inhibitor-treated herbage only in smaller silos (130–150 g herbage). Control silages were
poorly fermented but contained low concentrations of butyric acid and ammonia N indicating little
activity of spoilage bacteria. Formic acid increased peptide N concentrations (P Æ 0.01) in silage from
smaller silos but had little eþ ect on other N constituents ; in the larger silos, formic acid reduced
soluble non protein nitrogen (NPN) and ammonia-N concentrations and increased peptide N concentrations. Increments in formaldehyde reduced silage soluble and ammonia N concentrations (linear
eþ ect ; P Æ 0.001). N-Ethylmaleimide and E-64 reduced soluble NPN concentrations (P Æ 0.05) but had
little eþ ect on other N constituents. Cystamine, however, increased silage peptide N concentrations.
Gel ültration on Sephadex G-25 of silage juice prepared from control and formic acid-treated silages
suggested that most silage peptides were small, with molecular weights of less than 520 Da.
( 1999 Society of Chemical Industry
Keywords : grass silage proteolysis ; peptides ; cysteine peptidase inhibitors
INTRODUCTION
During ensilage, plant endopeptidases degrade
soluble herbage protein to peptides and free amino
acids.1 Silage-soluble nitrogen (N), therefore, consists predominantly of non-protein-N (NPN). As a
consequence, the supplies of silage N and energyyielding substrates to rumen microorganisms may be
poorly synchronised and this may result in rumen
microbial protein synthesis being less efficient than
in animals fed fresh or dried forages.2 Inhibiting
proteolysis during ensilage could improve the utilisation of silage N since the efficiency of rumen
microbial N synthesis can be improved by supplementing silage with protein N rather than NPN.3
However, although formic acid4 and formaldehyde5
restricted proteolysis during ensilage, only formaldehyde subsequently improved silage N utilisation.6h8
Recent attempts to characterise the enzymes involved
in proteolysis during ensilage have revealed that
aspartic- (EC 3.4.23) and cysteine- (EC 3.4.22) endopeptidases were present in perennial ryegrass (PRG)
and that speciüc inhibitors of these classes of peptidases reduced proteolysis in ensiled PRG.9
Several studies have suggested that a considerable
proportion of silage N can be present as peptide
N.10h13 However, little is known about factors that
inýuence silage peptide N concentrations and there
have been no reported attempts to characterise silage
peptides. The objectives of the study were to investigate the eþects of a speciüc cysteine peptidase inhibitor,
1-trans
epoxysuccinyl-leucylamido-(4guanidino) butane (E-64), and two general cysteine
peptidase inhibitors, N-ethylmaleimide14 and
cystamine15 in comparison with formic acid, mixtures of formic acid and formaldehyde and an inoculant of Lactobacillus plantarum on the chemical
composition of ensiled PRG, with particular emphasis on the distribution of silage N. Secondly, gel ültration was used to characterise silage peptides.
º Pres ent addres s : Res earch Center, Agriculture and Agri-Food
Canada, Lethbridge, AB, Canada, T1J 4B1.
* Corres pondence to : John A Rooke, Animal Biology Divis ion,
SAC, Craibs tone Es tate, Aberdeen, AB21 9YA, UK
(Received 3 March 1998 ; revis ed vers ion received 24 July 1998 ;
accepted 24 Augus t 1998 )
MATERIALS AND METHODS
Herbage
Perennial ryegrass (Lolium perenne) was harvested
from experimental plots at MacRobert Farm, Craibstone Estate, Aberdeen as a third cut in September
1992, using a precision chop forage harvester. Grass
( 1999 Society of Chemical Industry. J Sci Food Agric 0022-5142/99/$17.50
679
VL Nsereko, JA Rooke
Table 1. Treatments impos ed on gras s at ens ilage
Treatment
Application rate
Silo s ize
Control
Formic acid
Inoculant
FFL
FFM
FFH
E-64a
N -Ethylmaleimide
Cys tamine dihydrochloride
20 ml kgÉ1
5.4 g formic acid kgÉ1
106 cfu L plantarum gÉ1
2.7 g formic acid and 0.51 g formaldehyde kgÉ1
2.7 g formic acid and 1.02 g formaldehyde kgÉ1
2.7 g formic acid and 1.53 g formaldehyde kgÉ1
22.5 mg kgÉ1 and 106 cfu L plantarum gÉ1
22 g kgÉ1 and 106 cfu L plantarum gÉ1
50 g kgÉ1 and 106 cfu L plantarum gÉ1
150 g and 500 g
150 g and 500 g
150 g and 500 g
500 g
500 g
500 g
150 g
150 g
150 g
a E -64, 1-trans epoxys uccinyl-leucylamido-(4-guanidino) butane
All additives were applied with water at an application rate of 20 ml kgÉ1 FW
was ensiled without wilting in two sizes of silo. The
smaller silos consisted of plastic pipe, 32 mm diameter by 250 mm length, ütted with screw caps
equipped with rubber O-rings and sealed with
acetate-free ýexible sealant. Gas release was achieved
through a 1 mm hole in the screw cap, sealed with
heavy-duty adhesive tape. Larger silos consisted of
plastic pipe, 50 mm diameter by 0.5 m length, sealed
at each end with a rubber bung and acetate-free ýexible sealant. The upper bung was equipped with a
fermentation lock (Boots Pharmaceutical Company,
UK) also sealed into place with ýexible sealant. The
large and smaller silos were packed with 500–550 g
and 130–150 g of grass, respectively. Triplicate silos
were ülled for each treatment and size of silo.
Additives
Additives were prepared on the day of ensilage and
applied to grass in a total of 20 ml kg~1 fresh weight
of herbage (FW). Cystamine, as the dihydrochloride,
and N-ethylmaleimide were applied as solids followed by application of water at 20 ml kg~1 FW ;
these additives were applied as solids in order to
avoid addition of large volumes of water because of
the large quantities of additive applied. Rates of
addition were calculated from the amounts of
cystamine15 and N-ethylmaleimide14 reported to
inhibit cysteine endopeptidases. The peptidase
inhibitors were obtained from Sigma Chemical Co
(Poole, UK). Water alone was applied as a negative
control (Control). The inoculant (Ecosyl, Ecosyl
Products, Billingham, UK) was prepared according
to the manufacturer’s instructions and diluted so that
addition at a rate of 20 ml kg~1 resulted in the application of 106 L plantarum g~1 grass. The amounts of
additives applied and silo sizes are given in Table 1.
Additives were thoroughly mixed with grass before
packing into silos.
Analytical procedures
Silos were opened after 100 days. Dry matter (DM)
concentrations were determined by drying at 80¡C
for 24 h in a forced draft oven. J uice was extracted
from the silages using a hand operated press and cen680
trifuged at 10 000 ] g for 15 min to remove particulate material. Extracts were prepared from fresh
herbage by homogenising 10 g in 100 ml water in a
Waring blender for 1 min, followed by centrifugation
at 10 000 ] g for 15 min. Water-soluble carbohydrate
(WSC) concentrations were measured on dilutions of
fresh herbage extracts or silage juice using the
anthrone procedure.16 Ethanol, lactic acid and volatile fatty acid concentrations (VFA) in silage juice
were measured by HPLC as described previously.17
Total- and soluble-N concentrations in silage juice
and herbage extracts were determined by the Kjeldahl procedure and ammonia-N by autoanalyser
using the phenol hypochlorite procedure (Technicon
method 321-74A). Peptide-N concentrations were
determined on silage juice using the ýuorescamine
peptide assay,18 except that ýuorescence intensities
were determined 30 min after addition of ýuorescamine to suitably diluted silage juice.
For gel ültration, Sephadex G-25 (medium grade,
Sigma Chemical Company, Poole, UK) was packed
into a column (26 mm diameter and 700 mm length ;
Pharmacia LKB Biotechnology, St Albans, UK).
Equal volumes of juice from triplicate silos of control
and formic acid-treated silages were pooled to give
two samples. Trichloroacetic acid (TCA) was then
added (ünal concentrations 40 mg TCA ml~1) and
the extracts centrifuged at 18 000 ] g for 15 min.
The supernatants were stored at [ 20¡C until
required. TCA-soluble silage juice (3.5 ml) was
applied in turn to the column using distilled water as
the eluant at a ýow rate of 60 ml h~1 and 3.75 ml
fractions collected. Trypticase (1 mg ml~1 ; 4 ml) was
also applied to the column as a peptide preparation
of known characteristics.18 Absorbance was determined at 206 nm using a Uvikon 810 spectrophotometer (Kontron Instruments, Zurich, Switzerland) and
ýuorescamine-reactive peptide-N concentrations as
described above.
Statistical analysis
Data were analysed by one-way analysis of variance.
For the smaller silos, control means were compared
with treatment means using Dunnett’s test.19
J Sci Food Agric 79 :679–686 (1999)
Fermentation and nitrogen distribution in ryegrass silage
Orthogonal contrasts were used to identify betweentreatment diþerences for silage made in larger silos.
Contrasts were : control vs other additives ; inoculant
vs all formic acid containing additives ; formic acid vs
formic acid/formaldehyde additives ; linear eþect of
formaldehyde ; quadratic eþect of formaldehyde.
RESULTS AND DISCUSSION
Herbage and fermentation characteristics
The herbage ensiled had a DM content of 163 g kg~1
FW and WSC and N concentrations of 61 and
40 g kg~1 DM, respectively. These low WSC and
high N concentrations were typical of a vegetative
third cut PRG and represented material likely to be
difficult to ensile.
Silages made in the smaller silos (Tables 2 and 3
were generally less well fermented than those made
in larger silos, despite the fact that the same batch of
herbage was used for both silo sizes. This diþerence
in fermentation between silo sizes may have been the
result of a larger surface area to volume ratio in the
smaller silos resulting in more oxygen being trapped
in these silos and allowing the utilisation of WSC by
respiration rather than fermentation, In all silages,
the recovery of fermentation products plus residual
WSC was greater than the WSC concentration in the
ensiled herbage. This anomaly has been noted before
Table 2. The effects of additives on pH and DM (g kgÉ1 FW), WSC and fermentation products (g kgÉ1 DM) in s ilages made in larger s ilos
Treatment a
pH
DM
WSC
TFP
Lactic acid
Formic acid
Acetic acid
Propionic acid
Ethanol
Butyric acid
SED c
Control
Formic
acid
Inoculant
FFL
FFM
FFH
6.1
161
11
81
12
2
41
4
17
5
4.1
176
32
47
20
12
4
1
10
NDd
5.3
167
11
71
27
2
32
3
6
1
4.2
181
24
96
67
10
9
1
8
1
4.4
180
68
51
27
11
5
2
7
1
4.6
181
98
34
11
14
3
2
4
ND
0.11
3.5
10.7
10.9
8.0
1.6
3.3
0.4
2.4
1.0
Significance of contras ts b
1
2
3
4
5
***
***
**
*
***
***
***
***
***
***
***
***
***
NS
***
***
***
***
NS
NS
*
NS
**
NS
NS
NS
NS
*
NS
NS
**
NS
***
***
NS
*
NS
*
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
a For details of treatments s ee Table 1
b Contras ts : 1, Control v additives ; 2, inoculant v other additives ; 3, Formic acid v FFL, FFM and FFH ; 4, Linear effect of formaldehyde ; 5,
quadratic effect of formaldehyde
c SED, Standard error of difference for triplicate s ilos ; NS, not s ignificant ; *, P \ 0.05 ; **, P \ 0.01 ; ***, P \ 0.001
d ND, not detected. TFP, total fermentation products
Table 3. The effects of additives on pH, dry matter (DM, g kgÉ1 FW) and WSC and fermentation products (g kgÉ1 DM) in s ilages made in
s maller s ilos a
Control
pH
DM
WSC
TFP
Lactic acid
Formic acid
Acetic acid
Propionic acid
Ethanol
Butyric acid
Formic acid
5.4
132
15
137
20
2
92
8
10
6
5.3
135
13
136
24
23**
40
4**
35**
11
SED b
L plantarum and
Alone
E -64
N -ethyl maleimide
Cys tamine
5.3
135
12
107
29
4
60
7
7
ND
5.4
133
12
125
22
2
73
6
8
14
5.0
162**
110**
17**
2
3
12*
\ 1**
NDc
ND
5.0
170**
17
89
24
2
54
4**
6
ND
0.22
5.5
2.8
25.9
15.7
2.1
23.1
1.0
3.9
7.3
a For details of treatment s ee Table 1
b SED, s tandard error of difference for triplicate s ilos ; *, P \ 0.05 ; **, P \ 0.01 s ignificantly different from the control
c ND, not detected. TFP, total fermentation products
J Sci Food Agric 79 :679–686 (1999)
681
VL Nsereko, JA Rooke
in silages made from low DM forage containing low
WSC concentrations9,20 and attributed to hydrolysis
of structural carbohydrates during ensilage.
Control silages from both silo sizes were poorly
fermented as indicated by high pH, high acetic acid
concentations and low lactic acid concentrations
(Tables 2 and 3). These fermentation characteristics
were probably a result of low herbage WSC and DM
concentrations. Butyric acid concentrations in both
silo sizes and ammonia-N concentrations, especially
in the larger silos, were low (Tables 4 and 5), suggesting that the activity of spoilage organisms in
these silages was minimal. It is likely that the high
acetic concentrations, particularly in the smaller
silos, were caused by conversion of lactic acid to
acetic acid, when hexose substrates became limiting,
by lactic acid bacteria21 after an initial formation of
lactic acid. The low DM concentrations of control
silaes when compared with some additive-treated
silages probably resulted from losses of volatile
material, which were higher in the control silages,
while determining oven DM concentrations.22
Inoculant treated silages were slightly better fermented than the control silages as indicated by lower
pH and lower concentrations of acetic, butyric and
propionic acids and higher concentrations of lactic
acid (Tables 2 and 3). Despite inoculating the
herbage with 106 cfu L plantarum g~1 grass, acetate
was the dominant acid produced in inoculated silages
and overall fermentation quality was unsatisfactory.
Low herbage WSC and DM concentrations may
have limited the scope for improvement due to inoculation and it was possible for inoculant lactic acid
bacteria to convert lactic to acetic acid when the
hexose supply was limiting.21
When compared with control silages, formic acid
treated silages had higher WSC concentrations and
much lower concentrations of total fermentation products, particularly acetic acid in larger silos (Table
2). In smaller silos (Table 3), the eþects of formic
acid were less marked but acetic acid concentration
was reduced and ethanol concentrations increased.
Generally, when control silages are well preserved,
formic acid has the eþect of restricting the production of lactic and acetic acids and conserving
more of the soluble carbohydrate component.23 This
was the case for silage made in larger silos, but in
smaller silos, the most abundant acid was acetic acid.
When compared with the silages treated with only
formic acid (Table 2), silages treated with the three
concentrations of formaldehyde and with formic acid
had, on average, undergone a more restricted fermentation as indicated by higher average pH
(P \ 0.05) and higher average WSC concentrations
(P \ 0.01). As the amount of formaldehyde applied
Table 4. Total N (g kgÉ1 DM) and s oluble N components of s ilages (g kgÉ1 N) made in larger s ilos
Treatment a
Total N
Soluble N
Peptide N
Ammonia N
SED c
Control
Formic
acid
L plantarum
FFL
FFM
FFH
42
604
92
72
42
463
331
26
41
626
113
80
45
470
177
33
46
378
154
25
44
357
147
23
1.1
16.4
30.6
2.1
Significance of contras ts b
1
2
3
4
5
NS
***
**
***
**
***
**
***
**
***
***
NS
NS
***
NS
***
NS
*
NS
NS
a For treatments s ee Table 1
b Contras ts , 1, Control v additives ; 2, L plantarum v other additives ; 3, Formic acid v FFL, FFM and FFH ; 4, Linear effect of formaldehyde ;
5, quadratic effect of formaldehyde
c SED, Standard error of difference between triplicate s ilos ; NS, not s ignificant ; *, P \ 0.05 ; **, P \ 0.01 ; ***, P \ 0.001
Table 5. Total N (g kgÉ1 DM) and s oluble N cons tituents of s ilages (g kgÉ1 N) made in s maller s ilos a
Control
Total N
Soluble N
Peptide N
Ammonia N
43
724
63
89
Formic acid
44
715
159**
96
SED b
L plantarum
Alone
E -64
N -ethylmaleimide
Cys tamine
45
748
90
102
44
569*
66
92
46*
565*
40
50*
45
618
159**
68
0.9
49.5
26.4
17.4
a For details of treatments s ee Table 1
b SED, s tandard error of difference for triplicate s ilos ; *, P \ 0.05 ; **, P \ 0.01.
Significantly different from the control
682
J Sci Food Agric 79 :679–686 (1999)
Fermentation and nitrogen distribution in ryegrass silage
increased there was a progressive restriction of fermentation as noted by the increases in pH and in
WSC concentrations (linear eþect, P \ 0.001) and
decreases in total fermentation products. Lactic,
acetic and butyric acid concentrations declined with
each increase in formaldehyde concentration ;
however, these relationships were not statistically
signiücant. Other studies24,25 have found that the
bacteriostatic eþects of formaldehyde, which cause a
restriction of fermentation, are enhanced by formic
acid. The silages treated with the medium and high
concentrations of formaldehyde (Table 2) contained
higher concentrations of WSC than those measured
in the herbage prior to ensilage (68 and 99 vs
61 g kg~1 DM, respectively). Similar observations
have been noted by others10,26 and attributed to
hydrolysis of hemicellulose and cellulose to yield
WSC.
Application of E-64 (Table 3) had little or no
eþect on the silage fermentation. N-ethylmaleimide
and cystamine treatment reduced silage pH and
resulted in higher silage DM concentrations when
compared with the control (P \ 0.01) and other
additive-treated silages. The higher DM concentrations observed in N-ethylmaleimide-treated silages
may be explained by the fact that very little fermentation had taken place in these silages and hence
there were minimal losses of volatile components
such as fatty acids and ethanol during oven drying.
The higher DM concentrations in cystamine-treated
silages may be attributed to the weight of additive
applied. Both N-ethylmaleimide and cystamine treatments produced silages with trace concentrations of
butyric acid. N-ethylmaleimide and cystamine treatments reduced the concentrations of total fermentation products (P \ 0.01 and P [ 0.05, respectively).
In addition, N-ethylmaleimide-treated silages contained higher WSC concentrations (P \ 0.01) than
the control but a similar total concentration of fermentation products plus residual WSC. Despite the
low concentration of fermentation products, Nethylmaleimide-treated silages had a lower pH value
(P [ 0.05) than the controls. Unpublished results
from our laboratory have demonstrated that Nethylmaleimide inhibits the growth of the silage
microorganisms, L plantarum and Pediococcus pentosaceus in vitro. The inhibitory eþect of Nethylmaleimide on silage fermentation may therefore
be attributed to both its slightly acidic nature and its
toxicity to silage microorganisms.
N components of silages
Soluble- and ammonia-N
The N components of the large and smaller silos are
presented in Tables 4 and 5, respectively. The
silages resulting from treatment with cystamine contained signiücantly greater concentrations of N than
the control silage because of cystamine-N. For purposes of comparison, the data in Table 5 for
J Sci Food Agric 79 :679–686 (1999)
cystamine-treated
silages
are
corrected
for
cystamine-N assuming that all cystamine-N was
soluble. The poorer fermentation which had taken
place in the smaller silos was also apparent in the N
components of the smaller silos, in that soluble-N
concentrations were higher in the small than larger
silos indicating more extensive proteolysis had taken
place. Inoculant-treatment had no eþect on silage
soluble- or ammonia-N concentrations in either silo
size nor did formic acid-treatment in the smaller
silos. However in the larger silos, formic acidtreatment reduced soluble- and ammonia-N concentrations. The rate of fall of pH is important in
determining the extent of proteolysis in the silo. If
pH is slow to fall, more protein will be broken
down27 as the optimum pH for plant peptidase activity is between 5 and 7.28 In the present experiment,
therefore, the lack of any reduction in proteolysis
when the inoculant and formic acid were applied to
silage made in the smaller silos was consistent with
the inability of the additives to reduce silage pH
below 5.28
Increments in formaldehyde concentration (20,41
and 61 g kg~1 crude protein) decreased total solubleN (linear eþect (P \ 0.001; quadratic P \ 0.05) and
ammonia-N concentrations (linear eþect P \ 0.001,
Table 4). These observations were in accordance
with previous ones.5 Formaldehyde is more eþective
at reducing proteolysis in the presence of formic
acid.25,29,30 The increase in protein preservation
often observed when a combination of formic acid
and formaldehyde is applied to grass at ensilage,
using commercial application rates, may be attributed to the diþering mechanisms by which these
compounds restrict protein breakdown. When
applied together, formaldehyde binds herbage proteins and in doing so, renders the protein undegradable by endopeptidases,25 whereas formic acid
rapidly reduces pH to a value at which proteolysis is
limited.4 In silage, ammonia is predominantly generated by deamination of amino acids by microorganisms.11 Therefore, intensive proteolysis may
occur without any signiücant increase in ammonia
concentrations.23 In the present study, the reduction
in ammonia N concentrations (Table 4) in formic
acid and formaldehyde/formic acid treated silages
may reýect either a reduction in soluble precursors
for ammonia production or of bacterial proteolytic
activity.
E-64 (22.5 mg kg~1 FW) reduced soluble-N concentrations to 79% (P \ 0.05) of those in untreated
controls (Table 5). Wetherall31 ensiled ryegrass with
E-64 (22.5 mg kg~1 FW) and, as a result, solubleand ammonia-N concentrations were reduced to 79%
and 78% of control concentrations. In another study
by the same author, PRG was treated with a higher
concentration of E-64 (44.5 mg kg~1 FW) and
soluble N concentrations were reduced to 86% of the
control with no reduction in ammonia-N concentrations. In the present study, E-64 had no eþect on
683
VL Nsereko, JA Rooke
ammonia-N. Wetherall et al9 concluded that the
reduction in soluble N concentration on application
of E-64 suggested that cysteine-endopeptidases were
present in PRG and involved in proteolysis during
ensilage. The present study supports these conclusions.
Cystamine and N-ethylmaleimide treated silages
contained lower concentrations of soluble-N (Table
5), however this diþerence was only signiücant for
the N-ethylmaleimide treated silages (P \ 0.05). In
addition, treatment with N-ethylmaleimide reduced
silage ammonia-N concentrations. The reductions in
soluble-N concentrations suggest that general
cysteine-peptidase inhibitors may be as eþective as
the speciüc inhibitor, E-64, in reducing proteolysis
during ensilage.
Peptide N
The control silages contained low concentrations of
peptide-N (Tables 4 and 5). These values are in the
same range as reported values (11–207 g kg~1 N)11,32
and similar to concentrations measured in commercial farm silages.13 Of the additive used in the
present study, only the formic acid containing additives and cystamine increased peptide-N concentrations. The lower concentrations of peptide N
(377–443 g peptide-N kg~1 soluble-N) observed in
the formaldehyde-treated silages, when compared
with the silage treated with formic acid alone (715 g
peptide-N kg~1 soluble-N) may be attributed to the
lower concentration of formic acid (2.7 vs 5.4 g kg~1)
applied in conjunction with formaldehyde. It therefore appears that rapid acidiücation of herbage, following the application of formic acid, not only
reduced the breakdown of protein but also that of
peptides. Alternatively, formic acid itself, and not
the H` ions, may have increased peptide N concentrations. A direct eþect of formic acid may explain
why peptide concentrations were increased in the
smaller silos (Table 5) when there was no eþect of
additive on soluble N concentrations. Unlike E-64
and N-ethylmaleimide, which only inhibited proteolysis, cystamine inhibited the breakdown of both
protein and peptides. One explanation for this could
be the presence of both proteolytic and peptidolytic
enzymes in PRG. It has been suggested33 that the
presence of several peptidases would be an advantage
to the senescing plant given the range of structural
complexity of proteins to be degraded.
Gel filtration of silage juice
Trypticase, a pancreatic hydrolysate of casein, which
contains approximately 90% N as peptide-N with an
average molecular weight of 521 Da and chain length
of 3.8 amino acids,18 eluted from Sephadex G-25 in
two main peaks with a third smaller, later eluting
peak which probably consisted of free amino acids
(Fig 1(a)). In comparison, juice from control (Fig
1(b)) and formic acid-treated (Fig 1(c)) silages eluted
684
Figure 1. Elution of (a) trypticas e, (b) juice from control s ilage,
and (c) juice from formic acid-treated s ilage from Sephadex G-25.
Elution was monitored at 206 nm (ÈÈ) or as peptides after
reaction with fluores camine (485 nm ; ·····).
from Sephadex G-25 in two peaks both of which
eluted later than the main peaks found in Trypticase.
The peptides in the silage juice, identiüed by reaction with ýuorescamine, eluted predominantly as a
single peak coincident with the ürst of the two peaks
detected at 206 nm. There was no diþerence between
the control and formic acid-treated silages juices in
the location of the peptide peak. However, the
peptide peak eluted from the formic acid-treated
silage was four times greater in magnitude than that
eluting from the control juice which agrees with the
J Sci Food Agric 79 :679–686 (1999)
Fermentation and nitrogen distribution in ryegrass silage
concentrations of peptide-N measured in the silage
juice. These data suggest that the peptides in silage
were smaller than those found in trypticase (average
molecular weight, 521 Da) and therefore di- and tripeptides and that the eþect of formic acid treatment
was to inhibit the peptidase(s) responsible for further
hydrolysis of these small peptides.
9
10
11
CONCLUSIONS
Proteolysis during ensilage was reduced by treatment
with formic acid, formaldehyde or peptidase inhibitors. The breakdown of peptides to amino acids was
only reduced by high concentrations of formic acid
or cystamine-treatment. Gel ültration of silage
extracts using Sephadex G-25 showed that silage
peptides were di- and tri-peptides.
12
13
14
15
16
17
ACKNOWLEDGMENTS
Financial support from the Ministry of Agriculture
Fisheries and Foods and Zeneca Bio-Products is
gratefully acknowledged. SAC receives ünancial
support from The Scottish Office Agriculture,
Environment and Fisheries Department.
18
19
20
21
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