8

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Research Article
Received: 16 December 2009 Revised: 15 October 2010 Accepted: 9 November 2010 Published online in Wiley Online Library: 24 December 2010
(wileyonlinelibrary.com) DOI 10.1002/jsfa.4255
Comparative evaluation of laboratory-scale
silages using standard glass jar silages
or vacuum-packed model silages
Sandra Hoedtke and Annette Zeyner

Abstract
BACKGROUND: The objective of this study was to compare the fermentation variables of laboratory-scale silages made in glass
preservingjars (GLASS) andvacuum-packedplastic bags (Rostockmodel silages, ROMOS). Silages werepreparedfromperennial
ryegrass (freshandwilted, 151 g kg
1
and286 g kg
1
dry matter (DM), respectively) andremoistenedcoarsely groundrye grain
(650 g kg
1
DM) either with or without the addition of a lactic acid bacteria inoculant (3 10
5
colony forming units (cfu) g
1
,
LAB). Quintuplicate silos were opened on days 2, 4, 8, 49 and 90.
RESULTS: Silage pH (P = 0.073), acetic acid content (P = 0.608) and ethanol content (P = 0.223) were not inuenced by the
ensiling method. The contents of DM (P < 0.001) and propionic acid (P = 0.008) were affected by the ensiling method, but
mean differences were only marginal. In ROMOS the concentration of lactic acid was increased (P = 0.007) whereas butyric acid
was produced less (P = 0.001) when compared to GLASS. This suggested slightly better ensiling conditions for ROMOS.
CONCLUSIONS: ROMOS represents a reasonable alternative to glass jar silages and opens the possibility for further
investigations, e.g. studying the impact of packing density as well as the quantitative and qualitative analysis of fermentation
gases.
c 2010 Society of Chemical Industry
Keywords: laboratory-scale silage; glass jar; vacuum-packing; inoculant; perennial ryegrass; rye grain
INTRODUCTION
Rapid and cost-effective evaluation of the improvement of the
foragepreservation, as well as determiningtheefcacyof additives
(acids, enzymes or bacterial inoculants) requires studies of the
ensiling process at the laboratory scale. Although there are only
a limited number of publications, it has been assumed that
small-scale silos provide a reliable prediction of the farm-scale
fermentation process.
13
Model silages have been used since
the beginning of the 20th century, comprising different types of
xed-volume vessels like test tubes,
4
porcelain containers,
5
milk
bottles
6
and glass jars
7
with different capacities ranging from50 g
to several kilograms. In Germany, following the recommendations
of the German Agricultural Society (DLG), glass preserving jars
are commonly used for studies on the fermentation process
as well as for monitoring and evaluation of additives.
8
Despite
being widely used,
911
the glass jar silage method has several
disadvantages: Pre- and post-treatment are costly and time-
consuming and require considerable extended storing space for
the jars. Depending on the operator there is a marked inuence
on the packing density of the forage inside the glass and a high
variability within one vessel and between replications.
Besides xed-volume vessels, silages made in plastic bags
have also been examined. However, method procedures are
heterogeneous and often a detailed description is not provided.
Jones,
12
for instance, described silages of different herbages made
in polyethylene bags using a suction pump with a carbon dioxide
lled bag. More recent studies report on sealed plastic bags using
a controlled vacuum of 0.2 bar.
13
The use of a household vacuum
sealer
14,15
and a single-chamber vacuum-packaging machine
16
for sealing polyethylene bag silos have been described, but no
information about the experimental design or the level of vacuum
was given in those studies. Hence, unlike ensilage trials with
silo containers like glass jars, results of plastic bag silages are
difcult to compare, depending on the experimental design.
Moreover, no comparison with glass jar silages was made in
those studies. Johnson et al.
17
studied varying packing densities
in vacuum-packed plastic bag silos by applying different initial
vacuum settings and additionally compared results with glass jar
silages. They concluded that silages made in plastic bags provide
a reasonable alternative for glass vessels. However, it seems to be
a shortcoming that the vacuum needed to achieve equal packing
densities in glass jars and plastic bags was only approximate
and that the packing density of the forage decreased during the
fermentation process.
The disadvantages of the glass jar ensiling system made clear
that there is a demand for an adequate alternative for studying

Correspondence to: Annette Zeyner, Universit at Rostock, Agrar- und

Umweltwissenschaftliche Fakult at, Professur f ur Ern ahrungsphysiologie und
Tierern ahrung, Justus-von-Liebig-Weg 8, 18059 Rostock, Germany.
E-mail: annette.zeyner@uni-rostock.de
Universit at Rostock, Agrar- und Umweltwissenschaftliche Fakult at, Professur
f ur Ern ahrungsphysiologie und Tierern ahrung, 18059 Rostock, Germany
J Sci Food Agric 2011; 91: 841849 www.soci.org c 2010 Society of Chemical Industry
8
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www.soci.org S Hoedtke, A Zeyner
the silage fermentation process. Ensiling in plastic pouches is a
favourable method, as it does not have the common drawbacks of
glass jar silages. Conversely, moreconsistent packingdensities and
a higher throughput of samples can be achieved using plastic bag
silages. Toour knowledge, apart fromthestudies of Johnsonet al.
17
there is a lack of informationabout comparisons of glass jar vessels
and vacuum-packed plastic bags used for ensiling. Therefore, the
objective of the present study was to establish Rostock model
silages (ROMOS) as a method for model silages made in vacuum-
packed plastic bags using the standardized method of glass jar
silages as a reference. Both model silage procedures were run
under identical conditions using the fermentation pattern and
aerobic stability as criteria of comparison.
MATERIALS ANDMETHODS
Study design
Silages were made in glass jars (GLASS) and plastic bags (ROMOS)
using fresh perennial ryegrass as harvested (FPR), wilted perennial
ryegrass (WPR) and remoistened coarsely ground rye grain (RRG).
GLASS and ROMOS were carried out with all plant materials under
similar conditions, with particular attention to identical packing
densities. Silages were made either with the addition of a lactic
acid bacteria inoculant (+LAB) or without additive (LAB) in ve
replications each. Silos were opened on days 2, 4, 8, 49 and
90. Variables used to evaluate ensiling methods were pH value
(all storage durations), fermentation products (2-day and 49-day
silages to characterize the early fermentation process and the
stabilized silage) and aerobic stability (49-day silages).
Plant material
Perennial ryegrass (Lolium perenne) was harvested from a pure
sward (fourth cut) at the early owering stage using a cutter-bar
type mower. Silages were made from fresh and wilted material
with dry matter (DM) contents of 151 and 286 g kg
1
, respectively.
The chopped grass (approximately 4 cm) was ensiled in glass jars
(GLASS) as well as polyethylene bags (ROMOS) without (control)
and with the addition of a lactic acid bacteria (LAB) inoculant
(Lactobacillus plantarum, 3 10
5
cfu g
1
, BIO-SIL

, Dr Pieper
Technologie- und Produktentwicklung, Wuthenow, Germany).
Rye grains (Secale cereale) with a DM content of 881 g kg
1
were coarsely ground in a chopper (mesh size 4 mm). The coarsely
groundrye grains were remoistenedby adding deionizedwater to
a calculated DM content of 650 g kg
1
. Model silages were made
in GLASS as well as in ROMOS without additive (control) or with
addition of LAB inoculant applied at the rate mentioned above.
Glass jar silages (GLASS)
Glass jars (1.5 L volume) were washed and sterilized (180

C, 8 h)
before use. The jar was lled with herbage according to DLG
recommendations,
8
at a preferable pore volume of 4 L kg
1
DM.
For silages made of FPR (151 g kg
1
DM), 681 g were packed into
the glass jar and compressed by hand with the aid of a rod,
representing a nal packing density of 0.45 g cm
3
. Respectively
513 g of WPR (286 g kg
1
DM) were lled into the jars, resulting in
a packing density of 0.34 g cm
3
. As there are no recommended
lling quantities established for RRG, this material was packed into
the entire jar, only providing 0.5 cm of unlled space to the lid.
Finally, a sample weight of 1500 g was determined for RRG which
resulted in a packing density of 1 g cm
3
. Jars were closed with a
rubber-lined lid that was xed using metal clips. Glass jars of all
plant materials and treatments were stored in a tempered room
at 25

C and opened on days 2, 4, 8, 49 and 90.

Vacuum-packed Rostock model silages (ROMOS)
For ROMOS vacuum-packed plastic bag silages a vacuum sealer
(V.300, LAVA vacuum-package, Bad Saulgau, Germany) was used.
Four-hundred grams of FPR or WPR as well as RRG were placed in
polyethylenebags(PA-PE20/70, 200 mm300 mm, LAVAvacuum
package, Bad Saulgau, Germany). The bags had a gas permeability
at 23

Cand0%relativehumidityof 50, 150and10 cm

3
m
2
d
1
for
oxygen, carbon dioxide and nitrogen, respectively.
18
The required
vacuum for ROMOS was obtained by a volumetric measurement
of the air-evacuated plastic bags. According to the sample weight,
the vacuumhadtobe set soas torealize a bagvolume of 889 (FPR),
1176 (WPR) and 400 (RRG) cm
3
in order to obtain a corresponding
packing density comparable to GLASS of 0.45, 0.34 and 1 g cm
3
,
respectively. Silage material was pre-compressed by hand before
the bags were air-evacuated and heat-sealed.
In order to prevent deformation of ROMOS bags, adhesive tape
was wrapped around the sealed polyethylene bags. Thereby care
had to be taken that the shape was stabilized without the packing
density being increased. To avoid bloating, the wrapped bag was
punctured with an injection needle (2 50 mm, Rometsch GmbH,
Heilbronn, Germany), which was disinfected after each bag with
70% ethanol, and put immediately into a second bag (PA-PE
20/70, 300 mm 500 mm, LAVA vacuum package, Bad Saulgau,
Germany) which was air-evacuated with the same vacuum and
sealed at once to maintain anaerobic conditions. ROMOS silages
were stored under the same conditions and opened on the same
days as GLASS.
Chemical analyses
A representative sample of the chopped herbage and coarsely
ground rye grains was freeze-dried and milled to 1 mm mesh size
(Brabender, Duisburg, Germany). Of the lyophilized samples, DM
was determined by oven drying at 105

C for 3 h. Ashing followed

at 600

C for 5 h in a mufe furnace. Crude protein (N 6.25) was

analysed with Kjeldathermand Vapodest (Gerhardt, K onigswinter,
Germany).
19
Neutral detergent bre (exclusive residual ash) and
acid detergent bre (exclusive residual ash) were determined by
wet chemical analyses
20
and crude bre
21
using a FOSS analyser
(Fibertec 2010, Rellingen, Germany). Water-soluble carbohydrates
(WSC) were analysed as monomeric and dimeric sugars as well
as fructans in water extracts (1 h at 25

C) by high-performance
liquid chromatography (HPLC) (HPX-87C, Biorad, Hercules, CA,
USA) according to Menge-Hartmann et al.,
22
with a ow rate of
0.65 mL min
1
at refractive index detector (column temperature
80

C). For determination of starch an enzymatic procedure

using amylase (Thermamyl 120, Novo Nordisk A/S, Denmark)
was chosen.
23
Concentration of glucose was measured by HPLC
with the conditions mentioned before and the starch content
was calculated by considering the previously determined water-
soluble carbohydrate content (glucose). Buffering capacity was
analysed by titration with lactic acid (0.1 mol L
1
) to a pH of 4.0.
24
Silage DM was determined by drying to a constant weight
(105

C, 17.5 h). The pH value was measured potentiometrically

each day the silage was opened, using a calibrated pH analyser
with glass and reference electrodes (precision 0.01, temperature
compensation 070

C). For this purpose silage extracts were pre-

pared with 50 g silage and 200 mL deionized water. Fermentation
products were analysed in the ltrated extracts (2-day and 49-day
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silages). Lactic acid was determined by HPLC (Aminex HPX-87H,
Biorad) with a ow rate of 0.60 mL min
1
at the UV detector.
Short-chain fatty acids and ethanol were quantitatively separated
by gas chromatography (GC-14A, CLASS-VP, Shimadzu, Kyoto,
Japan). Nitrogenwas usedas carrier gas at a pressure of 1 kg cm
2
.
The temperature of the injector and ame ionization detector was
kept constant at 190

C each; the temperature of the column oven

was programmed at 110

C during the rst 1.5 min, increasing to

170

C at a rate of 12

C min
1
thereafter.
Aerobic stability
Aerobic stability was measured at day 49 of ensiling by means of
temperature rise.
25
Silage samples representing 100 g DM were
put into plastic containers (1.25 L capacity), of which the base as
well as the lid were provided with a hole of 1 cm diameter. A
thermocouple was xed in the geometrical centre of the sample
and the closed test container was put into a cylinder made of
Styrofoam. Temperature was measured at intervals of 6 h for
7 days and recorded by data logger software (Version 4.2, PS
ES Electronics Services, Nieuwendijk, Netherlands). Silages were
considered as aerobically unstable if the temperature of the silage
sample and the room temperature (set at 20

C) differed by more
than 3

C.
Statistical analysis
Results wereanalyzedbySPSS15.0computer program(SPSS15.0
c
for Windows; SPSS Inc., Chicago, IL, USA). Analysis of variance
(ANOVA) was performed to investigate the effects of the main
factors method (GLASS and ROMOS), silage material (FPR, WPR
and RRG), inoculation (LAB and +LAB), and storage duration
(for pH: 2, 4, 8, 49 and 90 days; for fermentation products: 2 and
49 days) as well as the respective interactions. Results fromANOVA
were givenas meanpooledSD, whereby pooledSDis the square
root of mean square residue. Multiple comparison of means was
done by applying the StudentNewmanKeuls test. For most
variables there was a signicant (P < 0.05) interaction between all
included main factors. Therefore, regarding each silage material,
means (SD) were additionally calculated for GLASS and ROMOS
within the individual steps of the other factors (inoculation
storage duration) and compared by paired Students t-tests.
Differences of means for aerobic stability between GLASS and
ROMOS were also analysed by paired Students t-test. The level of
signicance was preset at P < 0.05.
RESULTS
The WSC of the perennial ryegrass before wilting and the coarsely
ground rye grain before remoistening were 105 and 58 g kg
1
DM, respectively (Table 1). Comparatively favourable ensiling
conditions were observed with the perennial ryegrass, with a
buffering capacity (BC) of 69 g lactic acid 1 Kg
1
DM(WSC/BC ratio
1.5). Due to a rather high content of fructans and a BC of only
15 g lactic acid 1 Kg
1
, rye grains showed a WSC/BC ratio of 3.9,
suggesting excellent ensilability characteristics.
The technique of model silages had a signicant inuence
on the DM (P < 0.001), whereas there was only a marginal
difference between overall mean values of GLASS with 343 g kg
1
DM and ROMOS with 346 g kg
1
DM (Table 2) without biological
relevance. While within treatments there were differences of DM
between ensiling methods, these did not seem to follow any
pattern and were considered to be random in all silage materials
Table 1. Chemical characterization of perennial ryegrass and rye
grains used for ensiling
Perennial ryegrass Rye grains
Dry matter (g kg
1
) 151 881
Crude ash (g kg
1
DM) 114 18
Crude protein (g kg
1
DM) 131 94
Neutral detergent bre (g kg
1
DM) 457 141
Acid detergent bre (g kg
1
DM) 269 34
Crude bre (g kg
1
DM) 244 29
WSC (g kg
1
DM) 105 58
Starch (g kg
1
DM) ND 692
Buffering capacity (g LA 1 Kg
1
DM) 69 15
DM, dry matter; LA, lactic acid; ND, not detected; WSC, water-soluble
carbohydrates.
(Tables 3 and 4). However, fermentation losses were signicantly
higher (P = 0.014) in ROMOS than in GLASS, being an average
of 12.9 g kg
1
and 10.2 g kg
1
sample weight, respectively (data
not shown). There was a method silage interaction on the
DM (P = 0.003) and multiple interactions except for method
inoculation x storage duration (P = 0.084).
The pHof silages was not inuencedby the method(P = 0.073),
being 4.43 for GLASS and 4.40 for ROMOS, but was affected
by the silage material (P < 0.001), inoculation (P < 0.001) and
storageduration(P < 0.001). Withinsingletreatments andstorage
durations of the silage material differences of pH (P < 0.05) were
observed only randomly and were considered to be generally
low (Table 5). Interactions were observed for method silage
(P < 0.001), method storage duration (P < 0.001) and all
multiple interactions except method silage inoculation
storage duration (P = 0.370).
Lactic acid was the primary acid in GLASS as well as in
ROMOS; however, the overall lactic acid content was signicantly
higher (P < 0.007) in ROMOS (64.3 g kg
1
DM) than in GLASS
(58.6 g kg
1
DM). This was especially the case for FPR and WPR in
both inoculation treatments, as vacuum-packed silages showed
predominantly higher lactic acid contents at days 2 and 49 of
opening (Table 3). However, signicant differences between the
ensiling methods were only found at few storage durations or
inoculation treatments and can thus be seen as randomized.
ROMOS silages of RRG showed lower lactic acid contents than
GLASSsilages, but differences (P < 0.05) couldbeobservedinonly
some treatments. Apart from the storage duration (P = 0.800),
silage material (P < 0.001) and inoculation (P < 0.001) had
signicant effects on the lactic acid content. Interactions between
the ensiling method and the plant material, inoculation and
storage duration as well as method silage storage and silage
inoculation storage duration were observed.
Whiletherewas nosignicant effect of theensilingtechniqueon
the acetic acid content (P = 0.608), the silage material (P < 0.001)
as well as inoculation(P < 0.001) andstorageduration(P < 0.001)
inuenced the formation of acetic acid. There was a method
inoculation storage duration andsilage inoculation storage
duration interaction. The average propionic acid content differed
signicantly (P = 0.008) between GLASS and ROMOS, although
differences were only marginal. Moreover, of all prepared silages,
propionic acid was only present in FPR (+LAB and LAB) and WPR
(LAB) at day 49 of opening (Table 3), and only in FPR (LAB)
was a signicant difference (P < 0.05) observed. There were
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Table 2. Mean and pooled SD of DM, pH value and content of fermentation products and P-values for variance factors silage, method, inoculation
and storage duration and the respective interactions potentially inuencing DM, pH value and concentrations of fermentation products
DM
(g kg
1
) pH
LA
(g kg
1
DM)
AA
(g kg
1
DM)
PA
(g kg
1
DM)
BA
(g kg
1
DM)
Ethanol
(g kg
1
DM)
Method GLASS 343b 4.43 58.6b 7.5 0.9a 16.6a 9.1
ROMOS 346a 4.40 64.3a 7.7 0.6b 13.6b 9.4
Silage FPR 134c 4.40b 87.5a 12.8a 1.2a 25.3a 11.9a
WPR 265b 4.52a 80.0b 8.9b 1.1b 20.1b 8.7b
RRG 634a 4.32c 16.9c 1.2c ND ND 7.2c
Inoculation LAB 341b 4.71a 48.9b 6.7b 1.2a 22.1a 10.1a
+LAB 347a 4.12b 74.1a 8.5a 0.3b 8.1b 8.4b
Storage duration 2 days 355a 4.52a 61.7 8.9a ND ND 7.8b
4 days 350b 4.32c NA NA NA NA NA
8 days 346c 4.34c NA NA NA NA NA
49 days 337d 4.39b 61.2 6.4b 1.5 30.3 10.7a
90 days 334e 4.49a NA NA NA NA NA
Pooled SD 4.55 0.118 11.5 1.90 0.71 4.97 1.58
P-value
Method <0.001 0.073 0.007 0.608 0.008 0.001 0.223
Silage <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Inoculation <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Storage duration <0.001 <0.001 0.800 <0.001 <0.001 <0.001 <0.001
Method silage 0.003 <0.001 <0.001 0.897 0.093 0.001 0.012
Method inoculation 0.986 0.610 0.019 0.292 <0.001 0.001 0.011
Method storage duration 0.065 <0.001 0.019 0.225 0.008 0.001 0.019
Method silage storage duration 0.002 0.015 <0.001 0.223 0.093 0.001 <0.001
Method inoculation storage duration 0.084 0.034 0.057 0.025 <0.001 0.001 0.134
Silage inoculation method 0.038 <0.001 0.057 0.249 <0.001 0.027 <0.001
Silage inoculation storage duration <0.001 <0.001 <0.001 0.005 <0.001 <0.001 <0.001
Methodsilage inoculationstorage duration 0.007 0.370 0.269 0.067 <0.001 0.027 0.029
AA, acetic acid; BA, butyric acid; GLASS, glass jar silage; FPR, fresh perennial ryegrass; LA, lactic acid; LAB, without additive; +LAB, with actic acid
bacteria inoculant; NA, not analysed; ND, not detected; PA, propionic acid; ROMOS, vacuum-packed Rostock model silage; RRG, remoistened coarsely
ground rye grain; WPR, wilted perennial ryegrass.
Entries followed by different letters indicate signicantly different means (P < 0.05).
interactions except for method silage and method silage
storage duration.
Butyric acid was detected only in 49-day silages for both
inoculation treatments of FPR and WPR, but in none of the silages
of RRG. Theformationof butyricacidwas inuencedbytheensiling
method (P = 0.001), with higher average contents in GLASS than
in ROMOS, and by the silage material (P < 0.001), inoculation
(P < 0.001) and storage duration (P < 0.001). Interactions were
observed.
Ethanol occurred in all model silages and its content was inu-
enced signicantly by the silage material, inoculation treatment
and storage duration (P < 0.001 each), whereas the technique
of model silages had no effect on the formation of ethanol
(P = 0.223). There were interactions except for method inocu-
lation storage duration.
Aerobic deterioration was found in ROMOS of FPR with LAB
inoculant as well as in GLASS of WPR without additive. In those
treatments four of ve repetitions were aerobically stable and in
only one sample of each treatment the temperature rose by more
than 3

C above the reference room temperature of 20

C after
1.8 and 2.0 days, respectively, resulting in mean values of 6.0 days
of aerobic stability (Fig. 1). The silages of RRG with addition of
LAB inoculant showed the lowest aerobic stability in both GLASS
(mean 4.8 days) andROMOS (mean 5.5 days) silages. Nevertheless,
in all treatments no signicant differences between GLASS and
ROMOS silages were veried (P > 0.05).
DISCUSSION
The aim of this study was to compare the model silage systems
of glass jars and vacuum-packed ROMOS as an alternative for
commonly used xed-volume silo vessels. Minor measurable
effects of the ensiling technique (GLASS and ROMOS) on the DM
were considered to be irrelevant since only sporadic signicant
differences between the ensiling methods were observed with
single treatments. Signicantly higher DM losses were found
in plastic bags compared to glass jars, but were thought not
to be of biological signicance. Unless the fermentation is not
entirely homolactic,
26
fermentation losses are likely to take place.
As ROMOS showed losses in the range of GLASS, air-evacuated
plastic bags consequently full the demands of common airtight
silo vessels.
The silages of FPR showed a rise in pH from day 49 to day 90
in both GLASS and ROMOS. A rise in pH indicates badly preserved
silages, with either enterobacteria or clostridia dominating the
fermentation. Presumably they are made from crops which
contain low levels of fermentable carbohydrates, which show
only insufcient numbers of epiphytic lactic acid bacteria or which
are ensiled too wet,
2
the latter being probably the case for the
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J Sci Food Agric 2011; 91: 841849 c 2010 Society of Chemical Industry wileyonlinelibrary.com/jsfa
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wileyonlinelibrary.com/jsfa c 2010 Society of Chemical Industry J Sci Food Agric 2011; 91: 841849
8
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7
Evaluation of silages using standard glass jar silages or vacuum-packed model silages www.soci.org
Table 5. pH values of fresh and wilted perennial ryegrass and
remoistened coarsely ground rye grain ensiled without and with
addition of a LAB inoculant at different storage durations
Storage LAB +LAB
duration
(days) GLASS ROMOS GLASS ROMOS
FPR
2 4.50a 0.02 4.47b 0.02 4.24 0.03 4.28 0.02
4 4.43 0.09 4.39 0.02 4.18 0.09 4.16 0.04
8 4.24 0.04 4.21 0.03 4.09 0.03 4.06 0.03
49 4.56 0.04 4.46 0.05 4.25 0.19 4.39 0.27
90 4.93 0.35 4.78 0.25 4.70 0.35 4.64 0.56
WPR
2 4.76a 0.06 4.66b 0.2 4.31 0.04 4.29 0.05
4 4.68a 0.04 4.64b 0.02 4.24a 0.03 4.16b 0.04
8 4.89a 0.02 4.64b 0.03 4.19a 0.05 4.10b 0.03
49 5.19a 0.07 4.72b 0.07 4.30a 0.06 4.13b 0.04
90 5.13a 0.04 4.74b 0.03 4.42a 0.04 4.27b 0.03
RRG
2 5.45b 0.02 5.52a 0.3 3.91 0.05 3.92 0.04
4 4.50b 0.12 4.94a 0.07 3.77a 0.01 3.79b 0.01
8 4.84b 0.06 5.25a 0.05 3.78 0.01 3.78 0.01
49 4.36b 0.03 4.66a 0.04 3.85 0.01 3.83 0.02
90 4.32b 0.05 4.44a 0.02 3.76a 0.01 3.73b 0.01
FPR, fresh perennial ryegrass; GLASS, glass jar silages; LAB, without
additive; +LAB, with lactic acid bacteria inoculant; ROMOS, Rostock
model silages; RRG, remoistenedcoarsely groundryegrain; WPR, wilted
perennial ryegrass.
Mean values and standard deviation () of quintuplicate silages
followed by different letters differ at P < 0.05 between GLASS and
ROMOS within each silage material, inoculation treatment and storage
duration (n = 5).
poor-quality silages of FPR. None of the untreated silages of
FPR and WPR reached the DM-dependent critical pH value for
anaerobic stability
27
within 90 days of ensilage (4.09 and 4.44 for
FPR and WPR, respectively). The silages of RRG reached the critical
pH value of 5.38 after just day 2 of ensiling. It could be shown
that the overall pH decline was rapid in both ensiling methods,
with most of the decrease occurring in the rst 2 days of ensilage,
showing similar runs of the pH curve (Table 5).
Despite low lactic acid contents compared to ryegrass silages,
silagesof RRGcouldbeevaluatedaswell fermented. High-moisture
maize silages (700750 g kg
1
DM) should contain 520 g kg
1
lactic acid to ensure a sufcient acidication.
28
No references were
found for silages of remoistened coarsely ground rye grains, but
taking the information of the high moisture maize silages as a
guideline, at a DM of 650 g kg
1
, the average lactic acid content
of RRG (16.9 g kg
1
DM) lies within the recommended range. In
well-preserved silages the content of acetic acid should amount
to only 1040 g kg
1
DM.
29
All silages of FPR and WPR as well
as of RRG did not exceed this level irrespective of the ensiling
method. It is known that propionic acid is mostly found in wet
silages (<250 g kg
1
DM),
28
as was the case in FPR at 49 days
in both inoculation treatments, but it was also observed in the
silages without additive of WPR after 49 days of ensilage. The
silages of RRG contained no propionic acid, probably due to the
high DM content. The ensiling method had a signicant effect on
the concentration of butyric acid whereas the overall mean was
higher in GLASS than in ROMOS. This could be taken as a sign
0
1
2
3
4
5
6
7
8
9
-LAB +LAB -LAB +LAB -LAB +LAB
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FPR WPR RRG
Figure 1. Aerobic stability of glass jar and ROMOS silages made of fresh
and wilted perennial ryegrass and remoistened rye grist. Silages of fresh
perennial ryegrass (FPR), wilted perennial ryegrass (WPR) and remoistened
coarsely ground rye grain (RRG) were prepared without additive (LAB)
or with lactic acid bacteria inoculant (+LAB) in glass jars () and vacuum-
packed plastic bags () and opened at day 49. Changes in temperature
were logged at 6 h intervals, and silages were considered to be aerobically
unstable when the temperature was more than 3

C above the ambient

temperature. Columns indicate mean values of quintuplicate silages, with
standard deviations represented by vertical bars.
of more favourable fermentation conditions in the plastic bags,
as furthermore it was observed that the breakdown of lactic acid
duringtheensilageperiodwas higher inglass jar silages of FPRand
WPR, mostly inthe non-inoculatedtreatments. Insilages of RRGno
butyric acid could be observed, suggesting a good fermentation
quality of this material.
In vacuum-packed inoculated ROMOS silages of FPR and in the
glass jar silages of WPR without additive, one of ve replicates
were not aerobically stable as they showed slightly increased
temperatures (between 3 and 5

C) for a period of 30 h and

42 h respectively. The lower aerobic stability of the inoculated
treatments of the RRG silages (Fig. 1) could be attributed to
increased levels of ethanol, which is seen as an indicator for yeast
activity.
2,28
In addition, fermentation losses anda pHincrease after
7 days were observed in the aerobically instable silages.
When using plastic pouches instead of glass jars for model
silages, air tightness and thus gas permeability is an important
criterion for maintaining anaerobic conditions. The plastic bags of
ROMOS used in this study completely meet the demands of the
quality standard for silo foil. According to guidelines of the DLG
30
for oxygen permeability of silo foils a maximum of 250 cm
3
m
2
d
1
should not be exceeded. With permeability to oxygen of just
50 cm
3
m
2
d
1
, ROMOS bags are even better than the recently
developed oxygen barrier lm, with an oxygen permeability of
70 cm
3
m
2
d
1
.
31
However, studies of Ashbell et al.
32
indicate
that oxygen permeability through the plastic bag is not a crucial
factor for maintaining acceptable silage quality, so long as the bag
is intact and the inside atmosphere contains volatile fatty acids at
levels of 1520 g kg
1
DM. The authors tested different types of
commercial plastic bags and found that permeability to oxygen
ranged from 43 to 504 cm
3
m
2
d
1
, with good-quality silages in
all tested materials.
A problem that is likely to occur when ensiling in plastic
pouches is a decreasing packing density during storage. As gases
accumulate during fermentation, plastic bags can bloat, resulting
in loosening of the fermenting herbage.
17,33
Those difculties
were overcome by wrapping the inside bag with adhesive tape
J Sci Food Agric 2011; 91: 841849 c 2010 Society of Chemical Industry wileyonlinelibrary.com/jsfa
8
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8
www.soci.org S Hoedtke, A Zeyner
to maintain shape. The use of a bigger outside bag guaranteed
gathering of all gases produced without bloating the inside bag.
Compared to xed-volume vessels like glass jars, ensiling in
plastic bags enables the user to modify the sample size in
accordance with the amount of material needed for analyses.
Studies on the fermentation process of lucerne in vacuum-packed
plastic bags were made with sample sizes as small as 150 g.
34
Others have reported ensiling maize ranging from 50 to 600 g
material.
16
Although pH was equal among sample sizes, the 50 g
samples were found to differ from the bigger sample sizes by the
fermentationproleanda minimumsamplesizefor maizeof 200 g
was suggested. Later studies with different grass species proved
that with such herbages vacuum-sealed plastic bags can be used
with samples as small as 250 g.
35
However, it should be noted
that sampling is often a large source of error in these methods.
36
Therefore we suggest using a sample size of at least 400 g, as it
has been found to be a suitable amount to achieve good-quality
model silages.
It is well known that the initial packing density of forage has
an effect on the fermentation process.
37,38
The V.300 vacuum
sealer or a similar device offers the possibility for studies with
varying packing densities as the vacuum can be modied within
a wide range. Furthermore, by heat sealing different types of
wrapping foils can be evaluated. Microbial processes during
fermentation result in measurable physical variables, among
them the production and composition of silo gas.
39
Applying
the ROMOS technique also provides the possibility of qualitative
and quantitative analyses of fermentation gases.
CONCLUSIONS
The present study provided evidence that ensiling conditions
in vacuum-packed ROMOS are similar to those in conventional
glass jar silages using pH value, fermentation pattern as well as
aerobic stability of model silages as criteria. Differences between
the methods were generally only marginal. Whether ensiling
conditions are more favourable in plastic pouches than in glass
jars should be brought into question, although the lactic acid
content was signicantly higher in ROMOS than in GLASS, and
also the production of butyric acid was reduced in the plastic bags
compared to the conventional model silage vessels.
ACKNOWLEDGEMENTS
The authors are grateful to Dr Edda MOtt for her support over the
course of the study. We would also like to thank Dr Lisa Dittmann
for her helpful advice on statistics.
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