Note: Descriptions are shown in the official language in which they were submitted.
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E~CTERIPiL TREAlrDENT TO PRESER ~ SII~GE
Field of the Invention
This invention relates to a method of preserving
agricultural products which are used for animal feed after
storage under anaerobic conditions. Specifically, this
invention relates to a method of preserving silage after
storage under anaerobic conditions such that the extent and
rate of digestibility of the silage are improved.
Background of the Invention
The use of silage additives has become a widely
accepted practice throughout much of the agricultural world.
During the ensiling process, aerobic respiration begins
immediately upon chopping of silage. During this early
phase, soluble carbohydrates in the plant tissue are
oxidized and converted to carbon dioxide and water. This
process continues until either the oxygen level is depleted
or the water soluble carbohydrates are exhausted. Under
ideal conditions, with adequate packing and sealing of the
ensiled material, respiration lasts only a few hours. The
growth of microorganisms during this period is limited to
those that are tolerant of oxygen. Typically, this includes
aerobic bacteria, yeasts and molds. These organisms are
generally recognized as being negative to the system because
they metabolize sugar to carbon dioxide, heat, and water.
Another important chemical change that occurs during
this early phase is the breakdown of plant protein by plant
proteases. Proteins are degraded to amino acids and
further metabolized to ammonia and amines. It has been
reported tha~ up to 50% of the total proteins may be broken
down during this process depending on the rate of pH
decline in the silage.
Once anaerobic conditions are established, anaerobic
bacteria proliferate. Enterobacteria and
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heterofermentative lactic acid bacteria are generally the
first populations to become established. These organisms
produce primarily acetic acid, ethanol, lactic acid, and
carbon dioxide from the fermentation of glucose and
fructose. Once the pH begins to decline, there is a marked
increase in the homofermentative lactic acid bacteria
population which produces primarily lactic acid. The rapid
increase in the lactic acid level results in the decline of
the pH to around 4. At this point, the ensiled mass will
generally remain stable throughout storage if undisturbed.
In summary, when the material is initially packed in
an oxygen-limiting structure, such as a covered silo, the
pH is reduced, the residual oxygen is utilized and the
material is said to undergo a lactic acid fermentation.
The material will remain stable and can be stored for many
months in this condition.
When the silage is ready to be fed, the top cover is
removed and the silo is opened for feeding. The material is
then exposed to air and the process is no longer anaerobic.
Microflora in the silage itself or airborne cont~in~nts can
begin to oxidize the acids present. This oxidation causes a
loss in mass or dry matter of the feed and thus causes
feeding losses. In addition, the resultant pH and
temperature increases are objectionable to the animals and
the feed will be refused by the animals after it has begun
to heat. The incidence of aerobic instability observed in
practice depends on the rate at which the ensiled material
is removed from the silo and the length of time that the
material has been ensiled before opening. If the silage is
unloaded slowly then more time is allowed for deterioration
to occur on the surface of the opened silage. Longer
enslling times produce generally more stable silage as the
acid concentrations are higher and all microflora
populations tend to decrease. In general the silage should
be stable for at least five days after opening. This will
allow for adequate time for the silage to be removed.
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Recently it has become known that bacterial inoculants
help preserve silage, including grass si_age, alfalfa silage
and corn silage. For example, inoculation with lactic acid
bacteria during the fermentation phase can be beneficial to
the fermentation process, see e.g. U.S. Patent 4,842,871 of
Hill issued June 27, 198g, as well as the literature
references cited therein. For high moisture alfalfa
stability, this increase is probably due to the inoculants'
enhancing the rate of anaerobic fermentation and pH
decrease. This is beneficial because oxidative losses
caused by aerobic pH-sensitive microflora in the initial
stages are thus avoided. In silages such as whole plant
corn, alfalfa, etc. the inoculant can also have beneficial
effects on the digestibility of the silages by causing an
increase in the availability of the fiber, and/or providing
more nutrients per amount of silage at a faster rate.
Accordingly, it is an objective of the present
invention to develop a bacterial silage inoculant that is
effective both during the initial anaerobic stages and
during the initial aerobic stages when a silo is opened to
air.
It is a further objective of the present invention to
develop a silage inoculant that increases the rate of
digestibility of the silage, thereby making nutrients
available to an animal sooner.
A further objective of the present invention is to
develop a silage inoculant that increases the extent o~
digestibility or the silage, thereby making more nutrients
available to the animal being fed.
30The method and manner of accomplishing each of the
objectives of the present invention as well as others will
become apparent from the detailed description which follows
hereinafter.
35SU ~ RY OF THE INVENTION
In the present invention silage, includlng grass,
alfalfa and/or corn silage, is preserved both during the
=-- =
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initial anaerobic phase of the ensilage process and during
the initial phases of aerobic conditions after a silo is
opened. Preservation is accomplished by mixing certain
facultative bacterial inoculants. The present inoculants
improve the extent and rate of digestibility of silage,
especially alfalfa silage. The inoculants are combinations
of selected strains of Lactobacillus plantarum and
Enterococcus faecium. The present inoculants are compatible
with the other bacteria, and thus do not retard the ensilage
process in any way. Specifically, the inoculants include
TJ1: a combination of Lactobacillus plantarum 347 and
Enterococcus faecium 301, having ATCC number ; ST: a
combination of Lactobacillus plantarum 346 and Lactobacillus
plantarum 347, having ATCC number ; and FS: a
combination of Lactobacillus plantarum 286 and Lactobacillus
plantarum 346, having ATCC number . The
present invention further provides methods of treating
silage which comprise administering to the silage a small
but ensilage preserving effective amount of the present
inoculant prototypes. The inoculants of the present
invention are particularly effective in improving the
digestibility of alfalfa silage.
DETAILED DESCRIPTION OF THE INVENTION
The term "silage" as used herein is intended to include
all types of fermented agricultural products such as grass
silagej alfalfa s~ilage, corn silage, sorghum silage,
fermented grains and grass mixtures, etc. All can be
treated successfully with the inoculants of the present
invention. The present invention is particularly effective
in improving the extent and rate of digestibility of alfalfa
silage.
A surprising aspect of this invention is that only
certain combinations of certain strains of Lactobacillus
plantarum and/or Enterococcus faecium will function
effectively in the present invention. The addition of
Lactobacillus to silage as a general matter is known, see
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s
for example U.S. Patent No. 4,981,705. However, the present
invention is necessarily strain specific with regard to the
Lactobacillus plantarum and Enterococcus faecium. In
particular, the inoculants found to work in the present
invention are: Lactobacillus plantarum 347 in combination
with Enterococcus faecium 301 ("TJ1"), Lactobacillus
plantarium 346 in combination with Lactobacillus plantarum
347 ("ST"), and Lactobacillus plantarum 286 in combination
with Lactobacillus plantarum 346 ("FS"). I~ is to be
understood, however, that applicants' invention, while
species specific, is intended to cover these species and
their genetic equivalents, or the effective mutants thereof,
which demonstrate the desired properties of the named
species and strains. Such genetic equivalents or mutants
thereof are considered to be functionally equivalent to the
parent species. It is well known to those of ordinary skill
in the art that spontaneous mutation is a common occurrence
in microorganisms and that mutations can also be
intentionally produced by a variety of known techniques.
For example, mutants can be produced using chemical,
radioactive, and recombinant techniques.
Regardless of the manner in which mutations or the
genetic equivalents are induced, the critical issue is that
they function to preserve the silage as described for the
2s parent species and/or strain. In other words, the present
invention includes mutations resulting in such minor changes
as, for example, minor taxonomical alterations.
Typical compositions useful for treatment of this
invention may include the present inoculants within the
ranges useful for treating ensilage products, i.e. typically
108-10l4 viable organisms/ton, preferably 109-10ll viable
organisms/ton, more preferably 101C viable organisms/ton. A
mixture of the two strains ranging from about 75~ to about
25% of each strain is preferred. A mixture of about 50% of
each of the two strains per inoculant is particularly
preferred.
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The composition of the present invention can also
include other common silage preservation organisms as, for
example, Propionibacteria, Streptococcus, Lactococcus and
Pediococcus, and certain enzymes from fungi or bacteria,
providing they are in no way antagonistic to the active
organlsms .
Those of ordinary skill in the art will know of other
suitable carriers and dosage forms, or will be able to
ascertain such, using routine experimentation. Further, the
administration of the various compositions can be carried
out using standard techniques common to those of ordinary
skill in the art, i.e. spraying, dusting, etc,
The above disclosure generally describes the present
invention. A more detailed understanding can be obtained by
reference to the following specific examples which are
provided herein for purposes of illustration only and are
not intended to be limiting, unless otherwise specified.
EXAMPLES
In the examples shown in the tables below, the
treatment, preparation and storage were conducted using
standard procedures. The inoculants used in the silage
trials, which were conducted in the years 1992, 1993 and
1994, were compared to a control sample which did not
contain any inoculant. The level of inoculant was 1 x 105
viable organisms per gram of forage in a 50:50 mixture.
This corresponds to 9 x 101~ organisms per ton. Treatments
were applied as a liquid. The prototype inoculants
developed consisted of selected strains of Lactobacillus
plantarum and Enterococcus faecium in the following
combinations: Lactobacillus plantarum strain 347 and
Enterococcus faecium strain 30~1 ("TJ1"); Lactobacillus
plantarum strain 286 and Lactobacillus plantarum strain 346
("FS"); and Lactobacillus plantarum strain 346 and
3s Lactobacillus plantarum strain 347 ("ST").
Prototype combinations were mixed in a 50:50 ratio and
applied on wilted, chopped alfalfa in a liquid form at a
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rate of 1 x 105 cfu/g forage. Treated forage was divided
into equal portions and packed to a stan ard density using a
hydraulic press into 4" x 14" experimental PVC silos. Silos
were sealed at each end with rubber caps held tightly by
metal rings. One end was fitted with a pressure release
valve so that gases could escape and still maintain
anaerobiosis. Experimental silos were stored at 20-25~C for
80-120 days prior to opening to simulate farm silo
conditions.
Experimental silos were opened, silage removed into a
clean container, mixed, and samples taken for microbial,
chemical and digestibility analysis. The remaining silage
was placed in a plastic lined polystyrene cooler, a probe
placed in the center of the silage mass, and temperature
lS measured every 3 hours for one week to determine aerobic
stability. When silage is exposed to air, large losses of
nutrients can occur as the result of aerobic microorganisms'
consuming sugars and fermentation products in the silage.
The sugars are respired to carbon dioxide and water,
producing heat. Besides the loss of highly digestible
portions of the sllage, some aerobic microorganisms produce
toxins which affect an animal's health.
Two measurements were used to determine the stability
of silage upon exposure to air. The hour at which silage
temperature went 1.7~C above ambient temperature was
referred to as the "rot" of the silage. It is a measure of
time after the silage is exposed to air before the aerobic
microorganisms start to grow causing the silage to heat.
Cumulative degree days, or "cumm_ dd" is the integration of
the area between the actual temperature curve and a line
drawn by ambient temperature. It is a measure of the total
amount of heating. Elevated temperatures increase the rate
and amount of protein breakdown and reduce the digestibility
of nitrogen, fiber, and other fractions.
Ammonia nitrogen determination was conducted using
standard procedures involving dissociation of the ammonia
ion by raising the pH, followed by steam distillation of the
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ammonia out of the silage. The amount of ammonia nitrogen
is quantitatively measured by titration. The level of
ammonia nitrogen is an indicator of the rate of
fermentation. The faster the rate of fermentation, the
S lower the activity of proteolytic enzymes, thereby making
more proteins available for an ~n im~1
The fermentation endpoint measurement is pH. A
satisfactory pH for alfalfa silage is less than 4.5. As the
pH decreases, proteolytic activity decreases. The pH
measurements were made with an Orien~ model 70lA pH meter
calibrated with pH 4.0l and 7.00 buffers.
To determine the extent and rate of digestibility,
samples were dried and ground through a 0.Smm Wiley~ mill
screen for digestibility analysis. All samples were scanned
by near infrared radiation spectroscopy (NIRS). Extremes in
spectra were selected on which to run in vitro dry matter
(IVDM) rates and extents of digestibility. IVDM rate of
digestibility was determined using a system designed to
simulate what happens in the rumen. Dried silage samples
are combined with a buffer and rumen fluid containing live
cellulytic mlcroorganisms. As the cellulytic microorganisms
digest the fiber in the silage sample, gas is produced. The
rate of digestibility was defined as the slope of the linear
portion of the curve produced by plotting gas production vs.
time. It was expressed as a percent of a standard to
account for the variation in microbial populations between
batches of rumen fluid. A faster rate of digestibility
means nutrients are being made available to the animal
sooner allowing it to utilize them to produce more milk or
meat. One possibility of how the inoculants are causing
this increase is that they are changing the structure of the
forage, making it more available to the rumen
microorganisms, which in turn convert the forage to energy
for use by the animal. The total volume of gas produced
over a set period of time was referred to as the extent of
digestibility and was also expressed a percent of a
-
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standard. The extent of digestibility is an indicator of
the total amount of nui~-ients made available by the
digestion of the fiber. The IVDM rate and extent of
digestiblities for the extremes were added to the NIRS
5 calibration equation and values for the r~m~ining samples
were predicted based on their spectra.
Tables 1,2 and 3 below summarize the trials conducted.
"Control" indicates uninoculated silage.
Table 1 summarizes the data from a 1992 trial. Table 1
indicates that TJ1, FS and ST all have higher rates of
digestibility than the control silage. Thus, the nutrients
from silages inoculated would be available to an animal
faster than the nutrients from uninoculated silage. TJ1-
and FS-inoculated silages also show lower ammonia nitrogen
lS levels than control silage, thus indicating a faster
fermentation rate leading to lower protein loss. The pH
values were all acceptable (<4.5) with the inoculated
silages having numerically better pH's than the control
silage.
Table 2 summarizes data from 1993 covering seven trials
for pH, rot, cumm_dd, extent of digestion, and rate of
digestion. Five trials were conducted for ammonia nitrogen.
TJ1, FS, and ST all show significantly (P<.1) higher rates
(nutrients available faster) and extents (more nutrients
available) of digestibility and lower (P<.1) ammonia
nitrogen levels (less protein loss) than uninoculated
silage. The inoculants also provide better rot and cumm_dd
values than control silage indicating better aerobic
stability. Better rot values indicates less loss of
nutrients due to aerobic heating. Cumm_dd values were all
very low indicating minim~l total heating. The rot values
were all satisfactory as over 6 days passed before aerobic
microorganisms started growing and causing heating. Cum~_dd
values were very low showing that total heating was minimal.
3s Table 3 summarizes data from five trials conducted for
TJ1 and four trials conducted for ST in 1994. The data
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indicate that TJ1 and ST have higher rates and extents of
digestibility than control silage. Inoculated silages also
had significantly (P<.20) better pH values than control
silage (which had a pH above 4.5).
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Table 1: 1992 Alfalfa Trial Summary
Treatment PHRate of dig.NH3-N (% total N)
Control 4.35 107.9 10.58
FJ1 4.30 123.0 8.88
FS 4.21 126.3 7.82
ST 4.17 124.6
Table 2: 1993 Alfalfa Trial Summary
Treatment pH Rot Cumm dd Extent Rate NH3N
of dig. of dig. (% total N)
1~
Control 4.44 134.8 22.2 80.7 87.3 7.71
TJ1 4.46 146.5 27.5 89.3 95.3 6.66
FS 4.43 157.1 1.6 89.2 92.0 5.36
ST 4.45 160.0 0 88.5 94.8 5.20
Table 3: 1994 Alfalfa Trial Summary
2~ Treatment Rot Cumm dd Extent Rate
of dig. of dig.
Control 152.9 11.4 88.81 87.23
ST 149.4 23.1 94.75 93.39
TJ1 151.8 11.4 92.61 94.21
