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Low Permeability to Oxygen of a New Barrier Film Prevents Butyric Acid
Bacteria Spore Formation in Farm Corn Silage
G. Borreani1 and E. Tabacco
Dipartimento di Agronomia, Selvicoltura e Gestione del Territorio, University of Torino, Grugliasco (Torino), Italy
ABSTRACT
The outgrowth of Clostridium spore-forming bacte-
ria causes late blowing in cheeses. Recently, the role of
air diffusion during storage and feed-out and the role
of aerobic deterioration has been shown to indirectly
favor butyric acid bacteria (BAB) growth and to deter-
mine the presence of high concentrations of BAB spores
in farm tank milk. A new oxygen barrier (OB) film was
tested and compared with conventional polyethylene
(ST). The objective was to verify whether the OB film
could prevent BAB spore formation in whole-crop corn
silage during storage on 2 commercial farms with differ-
ent potential silage spoilage risks. Two bunkers (farms
1 and 2) were divided into 2 parts along the length so
that half the feed-out face would be covered with ST
film and the other half with OB film. Plastic net bags
with freshly chopped corn were buried in the upper
layer and in the central part (CORE) of the bunkers.
The silos were opened in summer and fed out at differ-
ent removal rates (19 vs. 33 cm/d). Herbage at ensiling,
silage at unloading, and silage after air exposure (6 and
15 d) were analyzed for pH, nitrate, BAB spores, yeasts,
and molds. The BAB spores in herbages at ensiling
were 2.84 log10 most probable number (MPN)/g, with
no differences between treatments or farms. Nitrate
was below the detection limit on farm 1 and exceeded
2,300 mg/kg of fresh matter on farm 2. At unloading,
the BAB spores in the ST silage on farm 1 were greater
than 5 log10 MPN/g, whereas in the CORE and the OB
silages, they were approximately 2 log10 MPN/g. The
ST silage had the greatest pH (5.89), the greatest mold
count (5.07 log10 cfu/g), and the greatest difference
between silage temperature and ambient temperature
(dTsection−ambient). On farm 2, the ST silage had the great-
est concentration of BAB spores (2.19 log10 MPN/g), the
greatest pH (4.05), and the least nitrate concentration
compared with the CORE and the OB silages. Pooled
data on BAB spores collected from aerobically deterio-
rated samples showed a positive relationship with pH,
mold count, and dTsection−ambient and a negative relation-
ship with nitrate concentration. A high concentration
of BAB spores (>5 log MPN/g) was associated with vis-
ible spoilage, high pH values (>5.00), high mold counts
(>5 log cfu/g), high dTsection−ambient, and nitrate below
1,000 mg/kg of fresh matter. We concluded that the use
of a film with reduced oxygen permeability prevented
the outgrowth of BAB spores during conservation and
feed-out, and it could improve the microbiological qual-
ity of corn silage by eliminating the fractions of silage
with high BAB spore concentrations.
Key words: butyric acid bacteria, corn silage, oxygen
barrier film Silostop, aerobic deterioration
INTRODUCTION
The multiplication of spore-forming bacteria of the
genus Clostridium causes off-flavors and excessive gas
formation in either sweet cheeses such as Gruyère,
Gouda, Edam, and Emmental or hard cheeses with
long ripening times, such as Cheddar, Grana Padano,
Beaufort, and Parmigiano Reggiano (Le Bourhis et
al., 2005). This problem is frequently associated with
Clostridium tyrobutyricum (Klijn et al., 1995). These
bacteria, called butyric acid bacteria (BAB), are able
to convert lactic acid into butyric acid, hydrogen, and
carbon dioxide at a relatively low pH. Recently, several
commercial Grana Padano cheese makers have had
problems with late blowing (from 15 to 35% of the total
production) in some periods of the year, even though
BAB spore concentration in the milk was lower than 2
log10 most probable number (MPN)/L (Colombari et al.,
2001; G. Borreani and E. Tabacco; personal communi-
cation). Corn silages are known to be an important con-
tamination source of BAB spores in raw milk through
dung contamination during milking (Stadhousers and
Spoelstra, 1990). The growth of clostridia in silage can
take place during the acidification phase, the storage
phase, and, as demonstrated by Jonsson (1991), on
exposure of the material to air. The nitrate content of
forages can play an important role in preventing BAB
activity during fermentation when concentrations
are greater than 1,000 mg/kg of fresh matter (FM;
Spoelstra, 1983).
J. Dairy Sci. 91:4272–4281
doi:10.3168/jds.2008-1151
© American Dairy Science Association, 2008.
4272
Received March 5, 2008.
Accepted June 22, 2008.
1
Corresponding author: giorgio.borreani@unito.it
Although clostridia are strict anaerobic microorgan-
isms, some early studies have shown that BAB spore
formation can be promoted by air penetration in silage
(Ohyama et al., 1970; Kwella and Weissbach, 1991).
Corn is often considered a good forage for ensiling be-
cause of its relatively high DM content, low buffering
capacity, and usually adequate levels of fermentable
sugars (McDonald et al., 1991). Corn silage cores usually
contain less than 3 log10 MPN/g of BAB spores (Colom-
bari et al., 1999, 2001; Vissers et al., 2007). However,
in the early 1980s French studies pointed out a large
heterogeneity in clostridia spore counts in whole-crop
corn silage stored in horizontal silos (Corrot, 1986). This
author found low clostridia spore contamination levels
(approximately 2 log MPN/g) in the core of the silos but
high contamination levels (>5 log10 MPN/g) in the pe-
ripheral zones of the silos, which were more exposed to
air. Recently, Vissers et al. (2006, 2007) emphasized the
role of aerobic deterioration in corn silage at the farm
level in relation to the presence of high concentrations
of BAB spores in tank milk. Air diffusion during stor-
age and feed-out indirectly favors BAB growth. When
oxygen penetrates the silage, yeasts begin assimilating
lactic acid, other fermentation products, and residual
sugars, with an increase in temperature and pH (Pahl-
ow et al., 2003). Thus, oxidation processes consume
inhibitory substances and favor deterioration activity.
As clearly described by Jonsson (1989), the growth of
C. tyrobutyricum during oxygen penetration of silage
can be explained by the maximal biological activity
that takes place in the aerobic-anaerobic interface. The
aerobic microorganisms consume oxygen close to the
surface, and the aerobic-anaerobic zone moves toward
the surface and anaerobiosis is restored in the deeper
parts. Clostridium tyrobutyricum and other anaerobes
can grow and multiply in this ecosystem in microniches
with less inhibitory activity (Jonsson, 1989). When
the pH rises, other less acid-tolerant clostridia, such
as Clostridium sporogenes, can also grow (Cato et al.,
1986).
Vissers et al. (2007) hypothesized that the outgrowth
of BAB spores could probably be prevented by limiting
the penetration of oxygen or inhibiting the detrimen-
tal effect of oxygen penetration during conservation
and feed-out. A recent alternative sealing system
(Silostop-125 μm, IPM, Mondovì, Italy) has been devel-
oped that uses a new plastic formulation that is more
impermeable to oxygen, which reduces spoilage and
DM losses in the peripheral area of a silo (Borreani et
al., 2007).
The aim of this work was to verify whether this new
oxygen barrier film could prevent BAB spore formation
in whole-crop corn bunker silos during conservation,
during feed-out, and on air exposure. The tests were
carried out on 2 commercial farms with different po-
tential silage spoilage risks.
MATERIALS AND METHODS
Crop and Ensiling
Two trials were carried out on 2 commercial farms
at Saluzzo (Cuneo; 44°40′ latitude, 7°32′ longitude, 325
m above sea level; farm 1) and at Frossasco (Torino;
44°75′ latitude, 7°23′ longitude, 290 m above sea level;
farm 2) in 2005 and 2006 on corn silage in bunker silos
to study the effect of 2 types of plastic sheeting used to
seal the silos (standard polyethylene vs. a new concept
oxygen barrier film, Silostop-125 μm). The 2 sealing
treatments were 1) a single sheet of 180-μm-thick
(6 and 8 m wide for farm 1 and farm 2, respectively)
black-on-white polyethylene (ST); 2) a single sheet of
125-μm-thick (6 and 8 m wide for farm 1 and farm 2,
respectively) black-on-white coextruded polyethylene-
polyamide film with an enhanced oxygen barrier (OB).
The silages sealed with ST and OB films were also com-
pared with silages from the core of the bunker (CORE).
The bunkers (filled with approximately 200 and 1,100
t of fresh silage for farm 1 and farm 2, respectively)
were divided into 2 parts along the length; half was
covered with ST film, and half was covered with OB
film to allow silage sampling at the same time for the 2
treatments. Approximately 2 m of the plastic sheeting
was placed on the side wall and turned on the top sur-
face of the silos at the end of filling, to have an overlap
of approximately 40 cm of the 2 sheets in the middle of
the silos. The 2 plastic sheets were held to the silage
with tires and gravel bags near the side walls. When
the silos were being filled, 8 plastic net bags (4 for each
treatment) with well-mixed fresh material (approxi-
mately 7 kg of fresh weight per bag) were subsampled
for preensiling analyses, weighed, and buried in the
upper layer of the bunker in 2 sections 10 m apart.
The bags were placed so as to be representative of the
peripheral 40 cm of the stored silage. Four more bags
were weighed and buried in the central part of each
bunker (1.5 m from the top). The crop characteristics,
bunker characteristics, and sampling procedures are
described in detail in Borreani et al. (2007).
The silos were opened for summer consumption,
and when the feed-out face reached a distance of 0.5
m from the bags, they were removed from the silos for
analyses. Each bag was subsampled and analyzed for
DM concentration (3 replicates) and chemical and mi-
crobiological parameters (2 replicates). The remaining
silage was used for deterioration trials, which were per-
formed in the laboratory by placing approximately 3 kg
of silage from each bag of each treatment in duplicate
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OXYGEN BARRIER FILM AND BUTYRIC ACID BACTERIA
Journal of Dairy Science Vol. 91 No. 11, 2008
20-L polystyrene boxes. These were allowed to deterio-
rate aerobically at room temperature. A single layer of
aluminum cooking foil was placed over each container
to prevent drying and contamination, but also to allow
air penetration. The room temperature and the tem-
perature from each silage were measured each hour
by a data logger. Samples for pH and microbiological
analyses were taken from each container after 6 and 15
d. Nitrate content and pH were monitored daily after
the temperature increased by more than 2°C above the
ambient temperature.
Sample Preparation and Analyses
The preensiled material, the silage at unloading,
and the silage after air exposure were subsampled and
analyzed for DM content, pH, yeast and mold counts,
BAB spore concentration, and nitrate content.
Dry matter content was determined by oven-drying
at 80°C for 24 h and was corrected for volatile losses
during drying. Herbage and silage extracts were pre-
pared by adding 270 mL of deionized water to 30 g of
sample and homogenizing for 4 min in a laboratory
Stomacher blender (Seward Ltd., London, UK). The
sample suspension was used for pH and nitrate mea-
surements.
Nitrate content was determined by semiquantita-
tive analysis, using Merckoquant test strips (Merck,
Darmstadt, Germany) with a detection limit of 100 mg
of NO3–/kg (MacKown and Weik, 2004). Twenty ran-
domly selected samples were also analyzed for nitrate
concentration by ion chromatography. The extraction
media were filtered through Whatman no. 1 paper, then
through a 0.45-μm syringe filter, and finally through
a Dionex On Guard RP syringe filter (Dionex Corp.,
Sunnyvale, CA) before analysis with the flow-injection
procedure. Nitrate in the solution was measured by us-
ing a Dionex-500 Ion Chromatograph (Dionex Corp.)
equipped with a Dionex Ion Pac AS4A-SC analytical
column and a Dionex Ion AG4A-SC guard column (Di-
onex Corp.) with an eluent of 1.8 mM carbonate and
1.7 mM bicarbonate. The resulting regression equation
between the data obtained from ion chromatography
(mg/kg) and the data obtained from the semiquantita-
tive strips (mg/kg) was
Nitrate (strips) = 0.997 × nitrate
(ion chromatography),
with an adjusted r2 of 0.948 and a root mean square
error of 24. The data from the semiquantitative Mer-
ckoquant test strips were therefore used for the data
discussion in this paper.
For the microbial counts, 30 g of sample was trans-
ferred into sterile homogenization bags, suspended
1:10 (wt/vol) in peptone physiological salt solution
(PPS: 1 g of neutralized bacteriological peptone and
9 g of sodium chloride per liter), and homogenized
for 4 min in a laboratory Stomacher blender (Seward
Ltd.). Serial dilutions were prepared, and mold and
yeast numbers were determined by using the pour
plate technique with 40.0 g/L of yeast extract glucose
chloramphenicol agar (YGC agar, Difco, West Molesey,
Surrey, UK) after incubation at 25°C for 3 and 5 d for
yeast and mold, respectively. Mold and yeast colony-
forming units were enumerated separately, based on
their macromorphological features. The mean count of
the duplicate subsamples was recorded for total yeasts
and molds on plates that yielded 10 to 100 cfu per Petri
dish.
The BAB spore concentration was determined by
the MPN procedure. A serial 10-fold dilution was
prepared in Ringer’s solution (Oxoid Ltd., Hampshire,
UK). Tubes from each dilution step containing 9 mL
of sterilized reinforced Clostridium medium (RCM,
Merck) supplemented with sodium lactate (10 mL/L)
and agar (1.5 g/L) were each inoculated with 1 mL of
diluted sample. The tubes were heated in a water bath
for 10 min at 80°C to inactivate the vegetative cells and
to trigger the germination of spores. The tubes were
sealed with paraffin and incubated for 7 d at 37°C. A
tube scored positive if it exhibited abundant gas forma-
tion after incubation.
Temperature can be useful as a heating index re-
lated to the aerobic deterioration because increases in
temperature are clearly linked to yeast activity and
DM loss, and could help alert farmers to the onset of
aerobic deterioration. The difference between silage
temperature and ambient temperature was defined
as dTsection−ambient. During aerobic deterioration, hours
with t > 35°C and hours with nitrate <1,000 mg/kg of
FM were also calculated to better describe the optimal
temperature for BAB spore germination in the absence
of inhibitory conditions (Spoelstra, 1983; Jonsson,
1989).
Statistical Analysis
All microbial counts were log10-transformed to obtain
log-normal distributed data. To calculate averages, the
values below the detection level (detection levels: 30
BAB spores/g, 10 yeast/g, and 10 mold/g) were assigned
a value corresponding to half the detection level (i.e.,
15 BAB spores/g, 5 yeast/g, and 5 mold/g). The micro-
bial counts, pH, nitrate contents, and dTsection−ambient
were analyzed via ANOVA by using the general linear
model of the Statistical Package for the Social Sciences
Journal of Dairy Science Vol. 91 No. 11, 2008
BORREANI AND TABACCO
4274
(version 11.5; SPSS Inc., Chicago, IL). Significant dif-
ferences between means were considered significant at
P < 0.05. When the calculated values of F were sig-
nificant, the Duncan multiple range test (P < 0.05) was
used to interpret any significant differences among
the mean values. The number of hours with t > 35°C
and the number of hours with nitrate <1,000 mg/kg of
FM were analyzed for their statistical significance via
the Kruskal-Wallis nonparametric independent group
comparison of SPSS and, because equal variances were
not assumed, Tamhane’s T2 post hoc test (P < 0.05) was
applied to interpret any significant differences among
the mean values.
Pooled data on BAB spore concentrations col-
lected from aerobically deteriorated samples were
regressed on pH, mold count, nitrate concentration,
and dTsection−ambient as the independent variables. Lin-
ear and quadratic regressions were compared by using
the stepwise selection procedure of SPSS to select the
best regression model at P < 0.05. The best equation for
each parameter was selected by using the coefficient
of determination and root mean square error. All the
reported determination coefficients (r2) were adjusted
for degrees of freedom.
RESULTS
Tables 1 and 2 list the BAB spore, yeast, and mold
concentrations; pH; and nitrate concentration in fresh
corn before ensiling, from the CORE, and from the pe-
ripheral area of the bunker silos on farm 1 and farm 2,
respectively. The BAB spore concentrations were 2.67
and 3.01 log10 MPN/g for farm 1 and farm 2, respectively.
The yeast concentration ranged from 6.26 to 7.48 log10
cfu/g, and the mold concentration ranged from 6.36 to
7.54 log10 cfu/g, with no differences between herbages
from the CORE and the peripheral areas (OB and ST)
of the 2 silos. The nitrate concentration was below the
detection limit on farm 1, whereas it exceeded 2,300
mg/kg of FM on farm 2.
Tables 3 and 4 show the BAB spore, yeast, and
mold concentrations; pH; nitrate concentration; and
dTsection−ambient in silage from the 2 bunkers at unload-
ing after 261 and 325 d of conservation for farm 1 and
farm 2, respectively. On farm 1, the BAB spores, mold,
and pH were greater (P < 0.001, P < 0.001, and P =
0.001, respectively) in the ST silage than in the OB
and the CORE silages. The BAB spore concentration in
the ST silage was greater than 5 log10 MPN/g, whereas
BAB spores from the CORE and the OB silages were
approximately 2 log10 MPN/g. The silage sealed with
the ST film had the greatest pH (5.89) and the greatest
mold count (5.07 log10 cfu/g), but no differences were
observed between silage in the CORE and silage sealed
with the OB film. The dTsection−ambient was also greater
(P = 0.027) in silage sealed with the ST film than in
the CORE and the OB silages. On farm 2, significant
differences between the silage sealed with the ST and
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OXYGEN BARRIER FILM AND BUTYRIC ACID BACTERIA
Journal of Dairy Science Vol. 91 No. 11, 2008
Table 1. Farm 1: Butyric acid bacteria (BAB) spores, molds, yeasts, pH, and nitrate at ensiling of herbages from the core and from the
peripheral areas, sealed with an oxygen barrier and standard polyethylene films
Item CORE1
Plastic film2
SE P-valueOB ST
BAB spores, log10 most probable number/g 2.76 2.76 2.48 0.105 0.506
Molds, log10 cfu/g 6.36 6.47 6.44 0.034 0.429
Yeasts, log10 cfu/g 7.15 7.19 7.48 0.148 0.657
pH 5.96 6.02 5.97 0.028 0.634
Nitrate concentration, mg/kg of fresh matter <30 <30 <30 —3—
1CORE = silage in the core of the bunker.
2OB = silage under the oxygen barrier film; ST = silage under the polyethylene film.
3Statistical analysis not performed.
Table 2. Farm 2: Butyric acid bacteria (BAB) spores, molds, yeasts, pH, and nitrate at ensiling of herbages from the core and from the
peripheral areas sealed with an oxygen barrier and standard polyethylene films
Item CORE1
Plastic film2
SE P-valueOB ST
BAB spores, log10 most probable number/g 3.01 3.02 2.98 0.165 0.995
Molds, log10 cfu/g 7.28 7.19 7.54 0.084 0.134
Yeasts, log10 cfu/g 6.36 6.28 6.26 0.038 0.543
pH 5.89 5.89 5.91 0.010 0.629
Nitrate concentration, mg/kg of fresh matter 2,408 2,388 2,363 62.7 0.965
1CORE = silage in the core of the bunker.
2OB = silage under the oxygen barrier film; ST = silage under the polyethylene film.
OB films were found in BAB spore concentration (P
< 0.001), pH (P = 0.038), nitrate concentration (P =
0.013), and dTsection−ambient (P = 0.018). The ST silage
had an average BAB spore concentration of 2.19 log10
MPN/g, which was greater than the values of 1.19
and 1.29 log10 MPN/g of the CORE and the OB silage,
respectively. The greatest pH value was observed in
the ST silage, with a value of 4.05 compared with 3.81
and 3.85 for the CORE and the OB silage, respectively.
The ST silage also had the least nitrate concentration
and the greatest dTsection−ambient, whereas no differences
were observed between the silage sealed with OB film
and the CORE silage.
Tables 5 and 6 show the BAB spore concentrations,
yeast and mold concentrations, pH, nitrate concentra-
tion, dTsection−ambient, and number of hours with a tem-
perature greater than 35°C of samples from the CORE
and from the peripheral area of the silages after 6 and
15 d of air exposure. On farm 1, significant differences
among the 3 treatments were observed for BAB spores
(P < 0.001), molds (P < 0.001), and pH (P = 0.022) after
6 d of air exposure. The BAB spores were greater in
the ST and OB silages than in the CORE silage. Mold
counts of 5.85 log10 cfu/g were observed in the OB silage,
and this value was lower than those observed for the
CORE and the ST silages. All silages were deteriorated
greatly after 15 d of air exposure. Only mold counts
were slightly lower in silage from the CORE than in
silages from the peripheral areas.
On farm 2, significant differences between the ST and
OB silages were observed for BAB spores (P = 0.008),
mold counts (P = 0.020), pH (P = 0.004), nitrate con-
centration (P = 0.042), dTsection−ambient (P = 0.008), and
hours with a temperature greater than 35°C during air
exposure (P = 0.010) after 6 d of aerobic deterioration,
whereas no differences were observed between the
CORE and the OB silages. After 15 d of air exposure,
significant differences were observed between the ST
and OB silages in BAB spores (P < 0.001), nitrate con-
centration (P = 0.008), dTsection−ambient (P = 0.016), hours
with a temperature greater than 35°C (P = 0.002), and
number of hours at which the nitrate concentration
Journal of Dairy Science Vol. 91 No. 11, 2008
BORREANI AND TABACCO
4276
Table 3. Farm 1: Butyric acid bacteria (BAB) spores, molds, yeasts, pH, nitrate, and temperature difference between silage section and
ambient temperature (dTsection−ambient) at unloading of silages after 261 d of conservation from the core and from the peripheral areas sealed
with an oxygen barrier and standard polyethylene films
Item CORE1
Plastic film2
SE P-valueOB ST
BAB spores, log10 most probable number/g 2.09b2.24b5.04a0.363 <0.001
Molds, log10 cfu/g 1.35b1.55b5.07a0.479 <0.001
Yeasts, log10 cfu/g 3.05 <1.00 <1.00 —3—
pH 3.60b3.99b5.89a0.280 0.001
Nitrate concentration, mg/kg of fresh matter <30 <30 <30 — —
dTsection−ambient, °C −0.3b0.9b13.6a2.56 0.027
a,bMeans in the same row with different superscripts differ (P < 0.05).
1CORE = silage in the core of the bunker.
2OB = silage under the oxygen barrier film; ST = silage under the polyethylene film.
3Statistical analysis not performed.
Table 4. Farm 2: Butyric acid bacteria (BAB) spores, molds, yeasts, pH, nitrate, and temperature difference between silage section and
ambient temperature (dTsection−ambient) at unloading of silages after 325 d of conservation from the core and from the peripheral areas sealed
with an oxygen barrier and standard polyethylene films
Item CORE1
Plastic film2
SE P-valueOB ST
BAB spores, log10 most probable number/g 1.19b1.29b2.19a0.222 <0.001
Molds, log10 cfu/g 1.16 1.46 1.39 0.089 0.559
Yeasts, log10 cfu/g 1.61 <1.00 1.66 —3—
pH 3.81b3.85b4.05a0.039 0.038
Nitrate concentration, mg/kg of fresh matter 2,250a2,025a1,688b98.5 0.013
dTsection−ambient, °C −1.1b−0.1b2.0a0.52 0.018
a,bMeans in the same row with different superscripts differ (P < 0.05).
1CORE = silage in the core of the bunker.
2OB = silage under the oxygen barrier film; ST = silage under the polyethylene film.
3Statistical analysis not performed.
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OXYGEN BARRIER FILM AND BUTYRIC ACID BACTERIA
Journal of Dairy Science Vol. 91 No. 11, 2008
Table 5. Farm 1: Butyric acid bacteria (BAB) spores, molds, yeasts, pH, nitrate, average temperature difference between silage section and ambient temperature (dTsection−ambient)
during air exposure, and number of hours with temperature over 35°C after 6 and 15 d of air exposure of samples from the core and from the peripheral areas sealed with an
oxygen barrier (OB) and standard polyethylene (ST) films
Item
Air exposure2 (6 d) Air exposure2 (15 d)
CORE1OB ST SE P-value CORE OB ST SE P-value
BAB spores, log10 most probable number/g 3.20b5.64a6.00a0.306 <0.001 5.12 6.35 6.79 0.338 0.084
Molds, log10 cfu/g 7.54a5.85b7.75a0.253 <0.001 7.16b8.63a8.49a0.224 0.001
Yeasts, log10 cfu/g 1.96 <1.00 <1.00 —2— 6.71 6.77 5.52 0.223 0.051
pH 6.79b6.69b7.30a0.142 0.022 6.85 6.77 7.49 0.169 0.211
Nitrate concentration, mg/kg of fresh matter <30 <30 <30 — — <30 <30 <30 — —
dTsection−ambient, °C 14.9 18.3 21.2 2.32 0.587 13.8 13.5 16.2 2.26 0.868
Hours with t > 35°C 32 72 80 — 0.322 148 261 296 — 0.117
a,bMeans in the same row for the same air exposure period with different superscripts are different (P < 0.05).
1CORE = silage in the core of the bunker.
2OB = silage under the oxygen barrier film; ST = silage under the polyethylene film.
2Statistical analysis not performed.
Table 6. Farm 2: Butyric acid bacteria (BAB) spores, molds, yeasts, pH, nitrate, average temperature difference between silage section, and ambient temperature (dTsection−ambient)
during air exposure, and number of hours with temperature greater than 35°C after 6 and 15 d of air exposure of samples from the core and from the peripheral areas sealed
with an oxygen barrier and standard polyethylene films
Item
Air exposure2 (6 d) Air exposure2 (15 d)
CORE1OB ST SE P-value CORE OB ST SE P-value
BAB spores, log10 most probable number/g 1.28b1.14b2.14a0.164 0.008 1.34b1.76b3.07a0.215 <0.001
Molds, log10 cfu/g 1.15b1.78b2.81a0.271 0.020 1.89 3.24 4.74 0.444 0.087
Yeasts, log10 cfu/g <1.00 <1.00 3.13 —3— 1.30 3.40 1.17 0.587 0.210
pH 3.81b3.84b4.08a0.041 0.004 3.83b5.43ab 6.71a0.411 0.014
Nitrate concentration, mg/kg of fresh matter 2205b2137b1519a133.2 0.032 2,160a2,057a1,052b181.2 0.008
dTsection−ambient, °C −0.9b1.6b9.9a1.60 0.008 0.3b2.8b13.6a2.126 0.016
Hours with t > 35°C 0b0b57a— 0.010 0b6b179a— 0.002
Hours with NO3 < 1,000 mg/kg of fresh matter 0 0 0 — — 0b0b54a— 0.037
a,bMeans in the same row for the same air exposure period with different superscripts are different (P < 0.05).
1CORE = silage in the core of the bunker.
2OB = silage under the oxygen barrier film; ST = silage under the polyethylene film.
3Statistical analysis not performed.
was below 1,000 mg/kg of FM (P = 0.037). The BAB
spore concentration of 3.07 log10 MPN/g observed in the
ST silage was greater than the values of BAB spores
of 1.34 and 1.76 log10 MPN/g observed in the CORE
and in the OB silage, respectively. The ST silage also
had the greatest values for pH (6.71) and dTsection−ambient
(13.6°C), and the least value for nitrate concentration
(1,052 mg/kg of FM). Even after 15 d of air exposure, no
differences were observed between the OB and CORE
silages.
Figure 1 reports the BAB spore concentration data
observed during the aerobic deterioration trials re-
gressed on pH, mold count, nitrate concentration, and
dTsection−ambient. The pH, mold count, and dTsection−ambient
showed a positive relationship with BAB spore con-
centrations, whereas nitrate concentration was nega-
tively related. All equations had adjusted coefficients
of determination greater than 0.70 and low root mean
square errors.
DISCUSSION
In corn silage, BAB spores seem to be crucial in
contaminating the rations and the bulk milk when
Journal of Dairy Science Vol. 91 No. 11, 2008
BORREANI AND TABACCO
4278
Figure 1. Aerobic deteriorated silages (pooled data of farms 1 and 2): butyric acid bacteria (BAB) spore concentrations in relation to pH,
mold count, nitrate concentration, and differences between silage temperature and ambient temperature (dTsection−ambient). Regressions ob-
tained were a) BAB spores [log10 most probable number (MPN)/g] = 1.26 pH − 3.55 [r2 adj. = 0.75; root mean square error (RMSE) = 1.06]; b)
BAB spores (log10 MPN/g) = 0.727 molds + 0.01 (r2 adj. = 0.87; RMSE = 0.781); c) BAB spores (log10 MPN/g) = −0.0193 nitrate + 5.55 (r2 adj.
= 0.85; RMSE = 0.818); and d) BAB spores (log10 MPN/g) = 0.244 dT + 1.21 (r2 adj. = 0.72; RMSE = 1.13).
silage is subjected to aerobic deterioration (Vissers et
al., 2007), because corn silage is not a good substrate
for BAB outgrowth when a good anaerobic condition is
achieved during storage. By conducting trials on com-
mercial farms, the present study clarifies some ques-
tions recently raised by Vissers et al. (2007) regarding
whether the limitation of oxygen penetration can pre-
vent the outgrowth of BAB spores during conservation
and feed-out. The present study investigated all the
steps from corn silage harvesting to feed-out on farm
and the following aerobic deterioration tests in labora-
tory conditions, through a method that permitted the
outgrowth of BAB spores in the same silage material
to be traced from the beginning to the end of the trial
(Borreani et al., 2007). This limited the sampling vari-
ability that is typical of trials conducted on commercial
farm silages.
The primary habitat of BAB spores is soil and decay-
ing plant and animal products (Cato et al., 1986). The
presence of BAB spores in agricultural soil varies to a
great extent and depends on the degree of OM degra-
dation. Data collected in the Po plain (Italy) showed a
mean content of 3.20 log10 MPN/g in soil that had not
received any organic fertilization for more than 20 yr,
a content of 4.20 log10 MPN/g in the soil of dairy farms
with a low livestock stocking rate, and a content of 4.85
log10 MPN/g in soil regularly fertilized with animal
slurry and manure at a rate of more than 50 t of FM/
ha (Borreani et al., 2002). The BAB spore concentra-
tions in slurries and manures ranged from 2.30 to 4.70
log10 MPN/g, depending on the stocking conditions and
animal contamination during production (Östling and
Lindgren, 1991). Butyric acid bacteria spores do not
belong to the epiphytic microflora of crops, because
this anaerobic genus on green plants is generally lower
than 2.00 log10 MPN/g (Östling and Lindgren, 1991).
Its presence in silage crops is generally due to soil and
residual manure contamination during harvest. In
our trial, the BAB spore concentrations of corn after
chopping ranged from 2.48 to 3.02 log10 MPN/g, with
few variations between treatments and farms. Lin et
al. (1992) reported a BAB spore concentration of 1.97
log10 MPN/g in standing corn crops, and this increased
to 2.88 log10 MPN/g in preensiled forage after chopping.
In preensiled grass and legumes, BAB spores were re-
ported to be in the range of less than 1.70 log10 MPN/g
(Rammer et al., 1994) to 4.36 log10 MPN/g (Davies et al.,
1996). When strict anaerobic conditions were ensured
during the storage of silage (i.e., in the core of the silo),
the number of BAB spores decreased from 2.76 and 3.01
log10 MPN/g at ensiling to 2.09 and 1.19 log10 MPN/g at
unloading for farm 1 and farm 2, respectively. The data
observed from the CORE silage were consistent with
those of Cotto (1983), who, in a survey of 1953 corn
silages, found an average BAB spore concentration of
2.23 log10 MPN/g, and they were also in the range of
values observed by Corrot (1986), who reported an av-
erage BAB spore concentration of 1.0 to 3.0 log10 MPN/g
in the core of 20 surveyed corn silages, which had a
greater homogeneity than in the peripheral areas. The
decrease in BAB spore concentration during the stor-
age period was more evident in the silage of farm 2, and
this could be related to the high nitrate concentration
of this silage at ensiling. Bester and Claassens (1970)
showed the important role of nitrites in stimulating
spore germination of Clostridium butyricum and C.
tyrobutyricum. Ando (1980) reported that nitrites in-
duced germination of Clostridium perfrigens spores by
acting directly on a component of the spore cortex. The
same author reported that sodium nitrite could block
the outgrowth stage, cell division stage, or even both
when added as a meat-curing agent. Spoelstra (1983)
reported that nitrate, probably because of its reduced
nitrite and nitric oxide products, prevented clostridial
growth during the silage acidification phase.
The lower oxygen permeability of the OB film than
the ST film reduced the detrimental effect of oxygen
permeation during storage and feed-out, especially on
farm 1. Significant differences in BAB spore concentra-
tions between the silages sealed with the ST and OB
films were observed at unloading. Vissers et al. (2007)
suggested that the growth of BAB spores requires suf-
ficient amounts of oxygen penetration into the silo, and
this requires time. Gas permeation models showed that,
during storage, oxygen can penetrate up to a depth of
0.2 m from the top of a silage sealed with polyethylene
sheets (McGechan and Williams, 1994). As reported
in Borreani et al. (2007), silages in the 2 bunker silos
in this experiment were conserved for more than 8 mo
before feed-out. Furthermore, the 2 silos were opened
in midsummer, when temperatures are known to in-
crease the oxygen permeability of the film used to cover
the silo. In this context, the 180-μm-thick polyethylene
ST film could not prevent permeation of oxygen in the
peripheral area of the silo during the storage period,
because its permeability was 990 cm3/m2 per 24 h at
23°C and, if the film was heated to 50°C by summer
temperatures, it could increase to more than 3,000
cm3/m2 per 24 h (G. Borreani and E. Tabacco; personal
communication). These values were 14 to 7.5 times
greater than the oxygen permeability values of the
125-μm-thick OB film, which were reported to be 70
and 400 cm3/m2 per 24 h at 23 and 50°C, respectively
(Borreani et al., 2007). Douglas and Rigby (1974) dem-
onstrated that the low tension of oxygen stimulates a
few spores of C. butyricum to outgrow and replicate,
and this outgrowth is then followed by the germination
of the other spores in the medium. They suggested that
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OXYGEN BARRIER FILM AND BUTYRIC ACID BACTERIA
Journal of Dairy Science Vol. 91 No. 11, 2008
the few cells that are initially able to metabolize can
produce reduced NAD, which reduces the oxygen ten-
sion to a noninhibitory level for the remaining spores.
The results from farm 1 demonstrated that high con-
centrations of BAB spores can occur already during
the storage period in peripheral areas (to a depth of
0.4 m from the top), especially when low amounts of
oxygen can penetrate the silo through the ST polyeth-
ylene cover for several months. A high concentration of
BAB spores in the peripheral areas sealed with the ST
polyethylene film was associated with visible spoilage,
high pH values (>5.00), high mold counts (>5 log cfu/g),
and a dTsection−ambient of 13.6°C with a silage tempera-
ture greater than 35°C. Furthermore, by analyzing the
fermentation profiles of the silages from farm 1, as
reported by Borreani et al. (2007), it is possible to state
that silage with an average BAB spore concentration of
5 log10 MPN/g (with values ranging from 4.1 to 8.2 log10
MPN/g) contained a limited amount of butyric acid
(less than 0.2% DM) and ammonia nitrogen (11% total
nitrogen). Similar results were reported by Lafrenière
(2007), who found BAB spore concentrations greater
than 4 log10 MPN/g during feed-out of silage, with bu-
tyric acid below the detection level. When clostridial
activity took place during the anaerobic phase of silage
fermentation, similar BAB spore concentrations could
be observed (Pahlow et al., 2003) but were associated
with high concentrations of butyric acid (>2.5% DM),
acetic acid (>4% DM), and ammonia nitrogen (>20%
total nitrogen; McDonald et al., 1991). On farm 2, a
significant difference in BAB spore concentrations be-
tween the silages sealed with the ST and OB films was
observed at unloading, but to a lesser extent because
of a nitrate concentration greater than 1,000 mg/kg of
FM, which prevented BAB spore formation during the
conservation period.
To better understand the role of air on BAB out-
growth during silage aerobic deterioration, silage
samples from the bags buried in the bunker silos of
the 2 farms were deteriorated in laboratory trials un-
til d 15. This time period was chosen to represent the
average age of a silage in the peripheral areas at risk
of air exposure when a feed-out rate of 0.7 to 1.4 m/
wk is adopted. This removal rate range was suggested
from data from a survey on commercial farms in Italy,
where feed-out rates of 0.5 to 1.5 m/wk were observed
in more than 70% of the surveyed farms (Tabacco and
Borreani, 2002). During feed-out, air can penetrate the
peripheral areas of a silo to up to 4 m from the feed-out
face (Parsons, 1991), especially when the sealing cover
is not weighted down or is weighted only with tires.
Silo face removal rates of 1.07 and 1.50 m/wk were
recommended by Pitt and Muck (1993) and by Vissers
et al. (2007) for the United States and the Netherlands,
respectively, to avoid extended aerobic spoilage of the
silage.
Silages from the core and the peripheral areas of the
farm 1 bunker had already resulted in deterioration
after 6 d of air exposure. Furthermore, these silages
had temperatures greater than 35°C for more than 70
h. Butyric acid bacteria spore concentrations greater
than 5 log10 MPN/g were associated with high pH val-
ues and mold counts, with no differences between the
ST and the OB silages. This means that a silage with
poor aerobic stability, such as that observed on farm
1 (Borreani et al., 2007), quickly undergoes spoilage
and BAB outgrowth when exposed to air, with no ef-
fect attributable to the film that was used for sealing.
The extent of mold development during air exposure
greatly influenced the BAB spore concentration by
reducing the inhibitory conditions of BAB outgrowth.
This is particularly related to the increases in silage
pH and temperature and to the decrease in nitrate
concentration, as shown by the regression equations in
Figure 1. Bester and Claassens (1970) reported a pH
of approximately 5.5 and temperatures of 24 to 40°C
as optimal conditions for BAB spore germination. Re-
sidual nitrate in silage seems to be a key factor in pre-
venting BAB outgrowth during aerobic deterioration,
because an increase in BAB spore concentration was
not observed until the amount of nitrate decreased be-
low 1,000 mg/kg of FM, as observed on farm 2. After 15
d of air exposure, the nitrate concentrations in silage
sealed with the ST film showed a high range of varia-
tion, from 90 to 2,250 mg/kg of FM. In silage samples in
which the nitrate concentration fell below 1,000 mg/kg
of FM after air exposure, the BAB spore concentrations
reached 3.97 log10 MPN/g. The nitrate concentration of
1,000 mg/kg of FM was very close to the values reported
by Spoelstra (1983) as being an inhibitory factor for
clostridial growth during the anaerobic phase of grass
silage fermentation.
CONCLUSIONS
The results obtained in this study indicate that the
use of a film with an oxygen permeability lower than
100 cm3/m2 per 24 h can prevent the outgrowth of BAB
spores during silage conservation. Furthermore, the
key roles of nitrate and mold development were shown
to have a great influence on BAB spore outgrowth. This
new OB film could contribute to improving the microbi-
ological quality of whole-corn silage by eliminating the
small fractions of silage with high BAB spore concen-
trations, especially on farms under critical conditions
or with inadequate amounts of silage removed daily.
Journal of Dairy Science Vol. 91 No. 11, 2008
BORREANI AND TABACCO
4280
ACKNOWLEDGMENTS
The authors wish to thank Bartolomeo Forzano (In-
dustria Plastica Monregalese S.p.A., Mondovì, Italy)
for providing the coextruded barrier plastic films (Si-
lostop) used in the experiment. We would also like to
thank Serenella Piano (Dipartimento di Agronomia,
Selvicoltura e Gestione del Territorio, University of
Turin, Turin, Italy) for the chemical and microbiologi-
cal analyses. Sergio Francia and Pierangelo Franco are
gratefully acknowledged for having made their bunker
silos available. This work was funded by the Regione
Piemonte, Assessorato Qualità, Ambiente e Agricol-
tura, years 2005 to 2008 project “Influenza della zona
di produzione e del tipo di gestione aziendale sulla
qualità del Grana Padano D.O.P. piemontese.” The 2
authors contributed equally to the work described in
this paper.
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