U.S. patent application number 15/482366 was filed with the patent office on 2017-07-27 for rapid acting lactobacillus strains and their use to improve aerobic stability of silage.
This patent application is currently assigned to PIONEER HI-BRED INTERNATIONAL, INC.. The applicant listed for this patent is PIONEER HI-BRED INTERNATIONAL, INC.. Invention is credited to Elizabeth HARMAN, William RUTHERFORD, Brenda SMILEY.
Application Number | 20170208836 15/482366 |
Document ID | / |
Family ID | 52693041 |
Filed Date | 2017-07-27 |
United States Patent
Application |
20170208836 |
Kind Code |
A1 |
HARMAN; Elizabeth ; et
al. |
July 27, 2017 |
RAPID ACTING LACTOBACILLUS STRAINS AND THEIR USE TO IMPROVE AEROBIC
STABILITY OF SILAGE
Abstract
A method for treating silage to enhance the aerobic stability by
increasing the fermentation and stabilization of silage by
inhibiting growth of microorganisms selected from yeasts, molds and
spore-forming bacteria and permitting earlier aerobic exposure is
disclosed. The method comprises treating silage or feed with a
composition comprising Lactobacillus buchneri strain LN7125, or
Lactobacillus brevis strain LB5328, or Lactobacillus brevis strain
LB7123, and mixtures or a mutant thereof which retains the silage
preservative activity of LN7125, LB5328, or LB7123, or the
antimicrobial components produced thereby. The strains of
Lactobacillus buchneri and Lactobacillus brevis disclosed in the
invention have been purified and isolated and have been found to
improve aerobic stability of silage allowing earlier aerobic
exposure post ensiling than is presently practiced
Inventors: |
HARMAN; Elizabeth; (Alleman,
IA) ; RUTHERFORD; William; (Grimes, IA) ;
SMILEY; Brenda; (Granger, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIONEER HI-BRED INTERNATIONAL, INC. |
Johnston |
IA |
US |
|
|
Assignee: |
PIONEER HI-BRED INTERNATIONAL,
INC.
Johnston
IA
|
Family ID: |
52693041 |
Appl. No.: |
15/482366 |
Filed: |
April 7, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14200231 |
Mar 7, 2014 |
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15482366 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23K 30/18 20160501;
A23Y 2220/13 20130101; C12R 1/24 20130101; A23Y 2220/67 20130101;
C12N 1/20 20130101; C12R 1/225 20130101 |
International
Class: |
A23K 30/18 20060101
A23K030/18; C12R 1/24 20060101 C12R001/24; C12R 1/225 20060101
C12R001/225 |
Claims
1. A sealed storage unit containing a fermented silage composition
comprising: a silage quality preserving amount of the bacteria
Lactobacillus buchneri LN7125, or Lactobacillus brevis LB5328, or
Lactobacillus brevis LB7123, and mixtures thereof, silage, and a
suitable carrier, wherein said composition is characterized by
having dry matter loss of the silage reduced in seven days after
the composition is made, and wherein said sealed storage unit
opened after seven days of anaerobic fermentation results in a
silage mixture having increased aerobic stability as compared to
the aerobic stability of a silage without said bacteria as measured
by dry weight loss.
2. The sealed storage unit containing the fermented silage
composition of claim 1 wherein the composition contains from about
10.sup.1 to about 10.sup.11 viable organisms per gram wet weight of
silage.
3. The sealed storage unit containing the fermented silage
composition of claim 1 wherein the composition contains from about
10.sup.2 to about 10.sup.7 viable organisms per gram wet weight of
silage.
4. The sealed storage unit containing the fermented silage
composition of claim 1 wherein the composition contains from about
10.sup.3 to about 10.sup.6 viable organisms per gram wet weight of
silage.
5. The sealed storage unit containing the fermented silage
composition of claim 1 wherein the silage is opened at twenty-nine
(29) days.
6. The sealed storage unit containing the fermented silage
composition of claim 1 wherein the silage is opened at twenty-eight
(28) days.
7. The sealed storage unit containing the fermented silage
composition of claim 1 wherein the silage is opened at fourteen
(14) days.
8. The sealed storage unit containing the fermented silage
composition of claim 1 wherein the silage is opened at seven (7)
days.
9. The sealed storage unit containing the fermented silage
composition of claim 1 wherein the carrier is liquid.
10. The sealed storage unit containing the fermented silage
composition of claim 1 wherein the carrier is solid.
11. The sealed storage unit containing the fermented silage
composition of claim 1 further comprising calcium carbonate,
starch, or cellulose.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 14/200,231, filed Mar. 7, 2014, which is
incorporated herein in its entirety by reference.
FIELD OF THE INVENTION
[0002] The invention relates to compositions and methods of
treating animal feed and preserving silage to enhance aerobic
stability.
BACKGROUND OF THE INVENTION
[0003] The ensiling process is a method of moist forage
preservation and is used worldwide. Silage accounts for more than
200 million tons of dry matter stored annually in Western Europe
and the United States alone. The process involves natural
fermentation, where lactic acid bacteria ferment water soluble
carbohydrates to form organic acids under anaerobic conditions.
This causes a decrease in pH which then inhibits detrimental
microbes so that the moist forage is preserved.
[0004] Aerobic instability is the primary problem in silage
production. Traditionally, the recommendation has been to allow
silage to ferment for at least thirty (30) days before feeding to
aid in increased silage digestibility. Even before storage units
are open for feedout, silage can be exposed to oxygen because of
management problems (i.e., poor packing or sealing). Under these
types of aerobic conditions, rapid growth of yeast and mold cause
silages to heat and spoil, decreasing its nutritional value.
Feeding a crop that has not been properly fermented can lower dry
matter intake (DMI), decrease milk production, and cause digestive
upsets. Allowing time for adequate fermentation creates a more
palatable and digestible feed for optimum DMI and milk
production.
[0005] Aerobic instability can be a problem even in inoculated
silage that has undergone what would traditionally be considered a
"good" fermentation: a rapid pH drop, and a low terminal pH. The
yeast organisms which contribute to instability in these conditions
however may be those which are tolerant of acid conditions and can
metabolize the lactic acid produced by lactic acid bacteria during
fermentation.
[0006] It is possible to use both chemical and biological additives
in making silage to promote adequate fermentation patterns
especially under sub-optimal conditions. Typical chemical additives
are most often organic acids and biological additives comprise
bacterial inoculants and enzymes. Bacterial inoculants have
advantages over chemical additives because they are safe, easy to
use, non-corrosive to farm machinery, they do not pollute the
environment and are regarded as natural products.
[0007] Production of silage inoculant strains and the ensiling
process is complex and involves interactions of numerous chemical
and microbiological processes. Different strains of even the same
species do not have identical properties and vary in their
fermentation and production characteristics. Further, different
silages and different methods of ensiling present a variety of
different needs. A continuing need exists in the art for improved
compositions and methods to improve the aerobic stability of silage
and increase the efficient production of ensiled animal feed.
[0008] The present invention provides novel strains of L. buchneri
and L. brevis and superior combinations thereof for use as silage
inoculants.
SUMMARY OF THE INVENTION
[0009] Embodiments of the invention include compositions for use as
silage inoculants comprising silage quality preserving amounts of
heterofermentative lactic acid bacteria species and mixtures or a
mutant thereof, and a suitable carrier. The heterofermentive lactic
acid bacteria compositions, isolated and purified, improve the
aerobic stability of ensiled forage, increasing the fermentation
and stabilization of silage to permit earlier aerobic exposure.
Such compositions may include, but are not limited to,
Lactobacillus buchneri strain LN7125 (hereafter LN7125), having
Patent Deposit No. NRRL B-50733, or Lactobacillus brevis strain
LB5328 (hereafter LB5328), having Patent Deposit No. NRRL B-50731,
or Lactobacillus brevis strain LB7123 (hereafter LB7123), having
Patent Deposit No. NRRL B-50732, and mixtures or a mutant thereof
which retains the silage preservative activity of LN7125, LB5328,
or LB7123, and carrier. Such compositions may comprise about
10.sup.1 to about 10.sup.11 viable organisms per gram wet weight of
silage optionally about 10.sup.2 to about 10.sup.7 viable organisms
per gram wet weight of silage, for example about 10.sup.3 to about
10.sup.6 viable organisms per gram wet weight of silage. The
carrier in the compositions of the embodiments may be a liquid or a
solid, such as, but not limited to, calcium carbonate, starch, and
cellulose.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The present invention now will be described more fully
hereinafter with reference to the accompanying tables, in which
some, but not all embodiments of the inventions are shown. Indeed,
these inventions may be embodied in different modifications and
other embodiments of the inventions set forth herein will come to
mind to one skilled in the art to which these inventions pertain
having the benefit of the teachings presented in the foregoing
descriptions and the associated drawings. Therefore, it is to be
understood that the inventions are not to be limited to the
specific embodiments disclosed and that modifications and other
embodiments are intended to be included within the scope of the
appended claims. Although specific terms are employed herein, they
are used in a generic and descriptive sense only and not for
purposes of limitation.
[0011] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which embodiments of the invention
pertain. Many methods and materials similar, modified, or
equivalent to those described herein can be used in the practice of
the embodiments of the present invention without undue
experimentation, the preferred materials and methods are described
herein. In describing and claiming the embodiments of the present
invention, the following terminology will be used in accordance
with the definitions set out below.
[0012] Units, prefixes, and symbols may be denoted in their SI
accepted form. Numeric ranges recited within the specification are
inclusive of the numbers defining the range and include each
integer within the defined range.
[0013] The article "a" and "an" are used herein to refer to one or
more than one (i.e., to at least one) of the grammatical object of
the article. By way of example, "an element" means one or more
elements.
[0014] As used herein, "animal performance" means the yield of
meat, milk, eggs, offspring, or work.
[0015] As used herein, "ensiling" or "ensiled" refers to an
anaerobic fermentation process used to preserve forages, immature
grain crops, and other biomass crops for feed and biofuels. In some
embodiments, the process of ensiling comprises the steps of
contacting forage with a microbial inoculant and storing the
mixture in an anaerobic condition. In certain embodiments, the
process of ensiling comprises the steps of storing forage in
anaerobic condition in a manner so as to exclude air. Forage,
having been inoculated with the microbial inoculant described
elsewhere herein, is also packed and stored in a manner so as to
exclude air. The moisture content of forage can be about 50% to
about 80%, depending on the means of storage, the amount of
compression, and the expected moisture loss during storage.
Ensiling can occur in silos, silage heaps, silage pits, silage
bales, or any other method appropriate for ensiling the chosen
plant material. Plant material with the microbial inoculant
described elsewhere herein can be ensiled for any amount of time
appropriate to produce silage at the desired maturity stage. In
some embodiments, ensiling occurs for about 7, about 15, about 20,
about 25, about 30, about 35, about 40, about 41, about 42, about
43, about 44, about 45, about 46, about 47, about 48, about 49,
about 50, about 55, about 60, about 65, about 70 days, about 4
months, about 8 months, about 12 months, about 18 months, or about
24 months or any time period deemed suitable by the practitioner.
The ensiling process can take place at any ambient temperature, for
example at an ambient temperature from 0-45.degree. C. The
temperature of the plant material being ensiled may, however,
increase above 45.degree. C. Mature silage can be used for animal
feed, frozen and stored for a later use, or added to a biogas
generator for the production of biogas.
[0016] As used herein, "functional mutant" means a bacterial strain
directly or indirectly obtained by genetic modification of, or
using, the referenced strain(s) and retaining at least 50% of the
activity of the referenced strain. The genetic modification can be
achieved through any means, such as but not limited to, chemical
mutagens, ionizing radiation, transposon-based mutagenesis, or via
conjugation, transduction, or transformation using the referenced
strains as either the recipient or donor of genetic material.
[0017] As used herein, the term "heterofermentative lactic acid
bacteria species" shall be interpreted to include, but not limited
to, leuconostocs, some lactobacilli, oenococci, and weissella
species. Heterofermenters produce lactic acid, ethanol, acetic acid
and carbon dioxide, with the proportions depending upon the
substrates available.
[0018] As used herein, the term "homofermentative lactic acid
bacteria species" shall be interpreted to include, but not limited
to, some lactobacilli and most species of enterococci, lactococci,
pediococci, streptococci, tetragenococci, and vagococci that
ferment hexoses by the Embden-Meyerhof (E-M) pathway.
Homofermentative denotes that lactic acid is the principal
metabolite without the production of carbon dioxide. For each six
carbon sugar molecule, homofermentative lactic acid bacteria will
produce two molecules of lactic acid.
[0019] As used herein, "isolated" means removed from a natural
source including, but not limited to, uninoculated silage or other
plant material.
[0020] As used herein, "microbial inoculant" refers to a
composition comprising at least one bacterial culture and a
suitable carrier. A "combination microbial inoculant" comprises at
least 2, at least 3, at least 4, at least 5, at least 6, at least
7, or more bacterial cultures and a suitable carrier. Bacterial
cultures comprise at least one bacterial strain and may comprise
multiple bacterial strains, including for example, at least 2, at
least 3, at least 4, at least 5, at least 6, at least 7, or more.
Bacterial cultures useful in the methods and compositions disclosed
herein include, but are not limited to, LN7125, LB5328, or
LB7123.
[0021] As used herein, "pre-ensiled plant material" includes, but
is not limited to, grasses, maize, alfalfa, wheat, ryegrass,
cereals, oil seeds, sorghum, sunflower, barley and mixtures thereof
prior to fermentation. All of which can be treated successfully
with the inoculants of the embodiments of the present invention.
The inoculants of the embodiments of the present invention are also
useful in treating high moisture corn (HMC).
[0022] As used herein, "oilseeds" includes, but is not limited to
sunflower, canola, soy, and mixtures thereof.
[0023] As used herein, "purified" means that a bacterial species or
strain is substantially separated from, and enriched relative to:
yeasts, molds, and/or other bacterial species or strains found in
the source from which it was isolated.
[0024] The term "silage" as used herein is intended to include all
types of fermented agricultural products, including but not limited
to, grass silage, alfalfa silage, wheat silage, legume silage,
sunflower silage, barley silage, whole plant corn silage (WPCS),
sorghum silage, fermented grains and grass mixtures, etc.
[0025] As used herein, the term "strain" or "strain(s)" shall be
interpreted to include, but not limited to, any mutant or
derivative of the various bacterial strains disclosed herein, for
example, L. buchneri strain LN7125, (Patent Deposit No. NRRL
B-50733), or L. brevis strain LB5328, (Patent Deposit No. NRRL
B-50731), or L. brevis strain LB7123, (Patent Deposit No. NRRL
B-50732) which retains the functional activity of improving aerobic
stability of forage as described and defined by the methods and
examples disclosed herein.
[0026] Several microorganisms have been isolated and purified which
improves the aerobic stability of ensiled forage, increasing the
fermentation and stabilization of silage to permit earlier aerobic
exposure. Specific strain(s) of the species L. buchneri or L.
brevis have been shown to enhance aerobic stability of silage by
not only reducing lactic acid levels but also by producing a
substance which is inhibitory to microorganisms that contribute to
causing aerobic instability in silage. While not wishing to be
bound by any one theory, it is likely that a combination of
metabolites is responsible for this effect. Furthermore, the
metabolism of L. buchneri or L. brevis is believed to produce both
acetic acid and propionic acid, both of which are known to inhibit
the growth of yeast and molds.
[0027] The primary goal of ensiling forages is to conserve the
maximum amount of original dry matter, nutrients and energy in the
crop for feeding at a later time. The process can be characterized
by four general phases of silage fermentation.
[0028] Upon sealing in the storage unit, the first phase is
aerobic, when oxygen is still present between plant particles and
the pH is 6.0 to 6.5. These conditions allow for continued plant
respiration, protease activity and activity of aerobic and
facultative aerobic microorganisms.
[0029] The second phase is fermentation, which lasts several days
to several weeks after the silage becomes anaerobic. Lactic acid
bacteria grow and become the primary microbial population thereby
producing lactic and other organic acids, decreasing the pH to 3.8
to 5.0.
[0030] The third phase is stable with few changes occurring in the
characteristics of the forage so long as air is prevented from
entering the storage unit.
[0031] The final phase is feedout, when the silage is ultimately
unloaded and exposed to air. This results in reactivation of
aerobic microorganisms, primarily yeast, molds, bacilli and acetic
acid bacteria which can cause spoilage.
[0032] Management techniques that can be used to help prevent this
condition, include but are not limited to, using care to pack the
silage well during the ensiling process, compaction, sealing, rapid
filling, face management and, also, using care in removing silage
for feeding to minimize the aeration of the remaining silage.
[0033] The susceptibility of silage to aerobic deterioration is
determined by physical, chemical, and microbiological factors.
Management (compaction, unloading rates) largely effects the
movement of oxygen into silage. During feedout, air can penetrate
up to 1 m behind the silage face so that exposure to oxygen is
prolonged. Fermentation acids and pH inhibit the rate of microbial
growth but spoilage rates are affected also by microbial numbers
and the rate of aerobic microbial growth on available
substrates.
[0034] Lactic acid bacteria (LAB) are present as part of the normal
microflora on growing plants. LAB can be classified as one of two
types depending upon their primary metabolic end products;
homofermentative which produce only lactic acid from the metabolism
of glucose and heterofermentative which produce lactic acid,
ethanol, acetate and CO.sub.2. The occurrences of these types are
quite variable in both type and number, crop to crop and location
to location.
[0035] Silage inoculants comprising principally homofermentative
lactic acid bacteria have become the dominant additives in many
parts of the world. Their function is to promote rapid and
efficient utilization of a crop's water soluble carbohydrates
resulting in intensive production of lactic acid and a rapid
decrease in pH, thus minimizing dry matter losses. Inoculants may
also improve animal performance. However, homofermentative
inoculants often have a negative effect on aerobic stability due to
the conservation of readily available substrates for spoilage
organisms.
[0036] The concept of heterofermentative lactic acid bacteria in an
inoculant has gained recent favor. The idea is that increased
levels of undissociated volatile fatty acids, such as acetate, may
inhibit other microbes that initiate aerobic deterioration.
Heterofermenters produce lactic acid, ethanol, acetic acid and
carbon dioxide, with the proportions depending upon the substrates
available. The acetate produced may inhibit deleterious organisms
in the silage. Additionally, heterofermenters, such as
Lactobacillus buchneri, are capable of metabolizing lactic acid to
acetate and 1,2 propanediol under anaerobic conditions. With such
mechanisms, one sixth of the carbon is lost to carbon dioxide
during fermentation of glucose and one third of the lactic acid
carbon is lost during anaerobic conversion to acetic acid. However,
a small loss of 1% or perhaps up to 2% of the dry matter is easily
offset by much larger losses by that spoilage action of aerobic
microorganisms. Concerns with heterofermentative lactic acid
bacteria include, but are not limited to, effects on animal
performance as well as the identification of appropriate strains
useful for the procedure. Different strains of even the same
species do not have identical properties and vary in their
fermentation characteristics.
[0037] Nilson (Arch Microbiol. (1956) 24: 396-411) found that the
predominant LAB in silage are Streptococci and Lactobacilli with L.
plantarum being the most frequent species. Gibson et. al (J. Gen.
Micro. (1958) 24: 60-70) reported that L. plantarum and L.
acidophilus were the dominant components of the homofermentative
flora. Beck (Landwirtschaftliche Forschung. (1972) 27: 55-63)
showed that even in grass silage where the epiphyte population was
dominated by heterofermentative LAB, by day four of the ensiling
process, 85% of the organisms present were homofermentative.
Langston et. al. (USDA Technical Bulletin No. 1187 (1958)) has
shown that the 69% of the isolates in mature silage were
homofermentative. A shift is sometimes noted toward
heterofermentative LAB in mature silage owing to their tolerance to
low pH and high acetate concentrations. Szigeti (Acta Almentaria.
(1979) 8: 25-40) found that the LAB flora at extremely low pH
consisted mainly of L. plantarum and L. brevis. Grazia and Suzzi
(J. Appl. Bacteriol. (1984) 56: 373-379) have shown that a strong
sensitivity to pH 3.6 was observed among the heterofermentative
LAB.
[0038] A review of the silage process and the use of inoculants can
be found in Weinberg, Z N G. and Muck, R E. (1996) FMS Microbiology
Rev. 19:53-68, Wilkinson, J. M. and Davies, D. R. (2012) Grass and
Forage Science 68:1-19, and Muck, Richard E. (2013) Agricultural
and Food Science 22:3-15 the disclosures of which is incorporated
herein by reference.
[0039] In embodiments of the present invention, the inhibition of
organisms responsible for spoilage is accomplished by treating the
silage with organisms of the species L. buchneri or L. brevis,
especially the strain(s) LN7125, LB5328, or LB7123 or with
compositions comprising LN7125, LB5328, or LB7123 or closely
related organisms, and as well by treatment with effective mutants
or equivalents of LN7125, LB5328, or LB7123 and compositions
comprising same.
[0040] An embodiment of the invention is a microbial inoculant
comprising Lactobacillus species that will alter the fermentation
and enhance stabilization of silage to allow earlier aerobic
exposure post ensiling than is presently practiced. Currently it is
the industry standard to recommend to allow a minimum of thirty
(30) days and preferably sixty (60) days for inoculated silages to
remain under anaerobic conditions to achieve the maximum benefit of
the inoculant's ability to preserve and enhance aerobic stability
of the stored forage. Often producers are unable to permit their
silage to remain unopened for the recommended length(s) of time due
to their individual limitations of available silage for feeding. An
embodiment of the invention to set a target of less than thirty
(30) days for anaerobic fermentation, in a sealed silage structure,
with a Lactobacillus strain with or without a lactic acid bacteria
(LAB) combination. A sealed silage structure may have a target for
anaerobic fermentation of at least 29, 28, 27, 26, 25, 24, 23, 22,
21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7
days.
[0041] An embodiment of the invention is a biologically pure
culture of L. buchneri strain LN7125, having Patent Deposit No.
NRRL B-50733, or L. brevis strain LB5328, having Patent Deposit No.
NRRL B-50731, or L. brevis strain LB7123, having Patent Deposit No.
NRRL B-50732.
[0042] A method of the embodiments is a method of treating animal
feed or silage, comprising administering a silage inoculant
comprising LN7125, LB5328, or LB7123 to the feed or silage at about
1.times.10.sup.3 to 1.times.10.sup.6 CFU/g of feed or silage.
Additionally, another method of the embodiments is a method of
improving animal performance, comprising feeding the animal the
animal feed that has been inoculated with the silage inoculants as
described in the other embodiments.
[0043] A further embodiment is a silage inoculant, comprising
viable cultures of a homofermentive lactic acid bacteria and a
heterofermentive lactic acid bacteria, see for example, U.S. Pat.
No. 6,403,084. Additional embodiments include animal feed or silage
comprising this silage inoculant.
[0044] Embodiments of the invention include methods for treating
silage by inhibiting the growth thereon of spoilage organisms
selected from yeasts, molds and spore-forming bacteria, which
comprises: adding to the silage a spoilage organism inhibiting
amount of the compositions of the embodiments. The silage to be
treated by the methods of the embodiments may be made from a
variety of plant sources, including but not limited to, grass,
maize, alfalfa, wheat, rye grass, cereals, oil seeds, sorghum,
sunflower and barley. The compositions of the embodiments may also
be added to the silage upon storage. The silage may be ensiled in a
variety of ways, including in the form of a bale, a bag, a bunker,
a stave silo, or a pile. The methods of treating silage using the
compositions of the embodiments include adding to the silage a
silage quality preserving amount of LN7125, LB5328, or LB7123.
[0045] Embodiments of the invention further include silage
comprising a silage quality preserving amount of LN7125, LB5328, or
LB7123 or a silage quality preserving amount of a mutant
thereof.
[0046] The silage included in the embodiments provides methods of
treating silage for animal feed with the silage inoculant of the
present invention, as well as the treated animal feed or silage
itself. Often, the animal feed or silage will be whole plant corn
silage (WPCS) or high moisture corn (HMC). The embodiments also
provide a method of improving animal performance by feeding the
inoculated silage. Containers comprising the silage inoculant of
the present invention and a carrier are also included.
[0047] An embodiment of the invention is a method for improving
aerobic stability of silage while also enhancing plant fiber
digestion in an animal by feeding an effective amount of silage
that has been inoculated with LN7125, LB5328, or LB7123 combined
with a ferulate esterase-producing bacterial strain or a functional
mutant thereof and a suitable carrier. Methods of using such
ferulate esterase producing strains is disclosed in U.S. Pat. No.
7,799,551, herein incorporated by reference. The ferulate esterase
strain may be, for example, a Lactobacillus strain or a functional
mutant thereof, such as a Lactobacillus strain selected from the
group consisting of L. buchneri, L. plantarum, L. brevis, L.
reuteri, L. alimentarius, L. crispatus, and L. paralimentarius.
Such strains may include, for example, those selected from the
group consisting of L. buchneri, strain LN4017 (Patent Deposit No.
PTA-6138), L. plantarum, strain LP678 (Patent Deposit No.
PTA-6134), L. plantarum, strain LP3710 (Patent Deposit No.
PTA-6136), L. plantarum, strain LP3779 (Patent Deposit No.
PTA-6137), L. plantarum, strain LP7109 (Patent Deposit No.
PTA-6139), L. brevis, strain LB1154 (Patent Deposit No. NRRL
B-30865), L. buchneri, strain LN4888 (Patent Deposit No. NRRL
B-30866), L. reuteri, strain LR4933 (Patent Deposit No. NRRL
B-30867), L. crispatus LI2127 (Patent Deposit No. NRRL B-30868), L.
crispatus, strain LI2350 (Patent Deposit No. NRRL B-30869), L.
crispatus, strain LI2366 (Patent Deposit No. NRRL B-30870),
Lactobacillus species unknown, strain UL3050 (Patent Deposit No.
NRRL B-30871), and mixtures thereof (See U.S. Pat. No. 7,799,551).
Such compositions may include about 10.sup.1 to about 10.sup.10
viable organisms of the bacterial strains or functional mutants
thereof per gram of a pre-ensiled plant material. Optionally, they
may include from about 10.sup.2 to about 10.sup.7 viable organisms
of the bacterial strains or functional mutants thereof, for example
from about 10.sup.3 to about 10.sup.6 viable organisms of the
bacterial strains or functional mutants thereof per gram of a
pre-ensiled plant material.
[0048] The composition that is fed to the animal may be treated
with an effective catalytic amount of the ferulate esterase
producing bacterial strain or functional mutant thereof, as is
readily determinable by those skilled in the art in animal
husbandry. Animals that are benefited by embodiments of the present
invention are mammals and birds, including but not limited to
ruminant, equine, bovine, porcine, caprine, ovine and avian
species, e.g., poultry.
[0049] The compositions which are used in the embodiments of the
invention may be in either liquid or dry form and may comprise
additional bacterial strains. In solid treatment forms, the
composition may comprise mixed bacterial culture comprising LN7125,
LB5328, or LB7123 together with a carrier.
[0050] The carrier may be in the nature of an aqueous or nonaqueous
liquid or a solid. In solid forms, the composition may comprise
solid carriers, solid diluents or physical extenders. Examples of
such solid carriers, solid diluents or physical extenders include
maltodextrin, starches, calcium carbonate, cellulose, whey, ground
corn cobs, and silicone dioxide. Liquid carriers may be solutions,
without limitation, in the form of emulsifiable concentrates,
suspensions, emulsion including microemulsions and/or
suspoemulsions, and the like which optionally can be thickened into
gels. In short, the carrier may be organic or an inorganic physical
extender. The solid composition can be applied directly to the
forage in the form of a light powder dusting, or if it is disbursed
in a liquid carrier, it can successfully be sprayed on the
forage.
[0051] 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.
[0052] Another embodiment of the invention is the combination of
LN7125, LB5328, or LB7123 with other specific bacterial species in
the proper ratio to provide both an increase in fermentation and
stabilization of silage or animal feed as well as an enhanced
aerobic stability upon exposure of the silage or feed to air to
allow for early aerobic exposure. The silage inoculant can be an
isolated and purified combination of at least one viable strain of
the homofermentative lactic acid bacteria Lactobacillus plantarum
combined with the heterofermentive bacteria of LN7125, LB5328, or
LB7123. In some embodiments, the silage inoculant will comprise at
least 2 to 10 strains of homofermenter and/or heterofermenter.
Exemplary strains of L. plantarum include at least one of LP286,
LP287, LP329, LP346, LP347, or functional mutants thereof (see, for
example, U.S. Pat. No. 6,403,084). Exemplary strains of L. buchneri
which could be combined with LN7125, LB5328, or LB7123 include
LN1391, LN4637, LN4750, or functional mutants thereof. The silage
inoculant optionally comprises at least one viable strain of
Enterococcus faecium, such as, but not limited to, strains EF301,
EF202, or functional mutants thereof. The number of viable
homofermentive bacteria and heterofermentive bacteria in the
inoculant are present in a ratio of from about 1:5 to about 1:15.
In some embodiments the ratio is about: 1:6 to 1:14, 1:7 to 1:13,
1:8 to 1:12, 1:9 to 1:11, or 1:10.
[0053] Methods of using mixed cultures for improving either
fermentation or aerobic stability of silage are disclosed in U.S.
Pat. No. 6,403,084, which is herein incorporated by reference.
[0054] An embodiment of the invention is a composition for use as a
silage inoculant comprising LN7125, LB5328, or LB7123 or a
functional mutant thereof and a suitable carrier. In an embodiment
of the invention the composition contains from about 10.sup.1 to
about 10.sup.10 viable organisms of the bacterial strain or
functional mutant thereof per gram of a pre-ensiled plant material.
In a further embodiment of the invention the composition contains
from about 10.sup.2 to about 10.sup.7 viable organisms of the
bacterial strain or functional mutant thereof per gram of a
pre-ensiled plant material. In yet a further embodiment the
composition contains from about 10.sup.3 to about 10.sup.6 viable
organisms of the bacterial strain or functional mutant thereof per
gram of a pre-ensiled plant material.
[0055] Materials that are suitable for ensiling or storage,
according to the methods of the invention, are any which are
susceptible to aerobic spoilage. The material will usually contain
at least 25% by weight dry matter. Such materials include, but are
not limited to, rye or traditional grass, maize, including high
moisture corn, whole plant corn, alfalfa, wheat, legumes, cereals,
oil seeds, sorghum, sunflower, barley or other whole crop cereals.
The silage storage management includes, but is not limited to, in
bales (a form particularly susceptible to aerobic spoilage), oxygen
limiting bags, bunkers, upright stave silos, oxygen limiting silos,
bags, piles or any other form of storage which may be susceptible
to aerobic spoilage.
[0056] The activity associated with this invention may be found in
other strains of L. buchneri, in other species of Lactobacillus,
e.g. L. kefir, L. parakefir and L. parabuchneri, L. brevis, L.
sake, L. curvatus, other species of homofermentative lactic acid
bacteria and possibly also in other genera. This can be established
by routine experimentation, on the basis of the information
herein.
[0057] As used herein, the term "strain" or "strain(s)" shall be
interpreted to include any mutant or derivative of the various
bacterial strains disclosed herein, for example, L. buchneri strain
LN7125, (Patent Deposit No. NRRL B-50733), or L. brevis strain
LB5328, (Patent Deposit No. NRRL B-50731), or L. brevis strain
LB7123, (Patent Deposit No. NRRL B-50732) which retains the
functional activity of improving aerobic stability of forage as
described and defined by the methods and examples disclosed
herein.
[0058] The LN7125, LB5328, or LB7123 microorganism of the
embodiments was purified and isolated from corn or the feces of
corn-fed sheep. After much experimentation it was discovered from
testing a collection of isolates.
[0059] After purification and isolation of the specific strain,
taxonomic studies were done to identify the strain. It was
identified as L. buchneri or L. brevis and given the prototype
number LN7125, LB5328, or LB7123. According to the invention, these
strain(s), compositions comprising these strain(s), or the factors
produced by these strain(s), are used to treat forage
materials.
[0060] Embodiments of the present invention are further defined in
the following Examples. It should be understood that these
Examples, while indicating certain embodiments of the invention,
are given by way of illustration only. From the above discussion
and these Examples, one skilled in the art can ascertain the
essential characteristics of this invention, and without departing
from the spirit and scope thereof, can make various changes and
modifications of the embodiments of the invention to adapt it to
various usages and conditions. Thus, various modifications of the
embodiments of the invention, in addition to those shown and
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims.
[0061] The disclosure of each reference set forth herein is
incorporated herein by reference in its entirety.
DEPOSITS
[0062] The Lactobacillus buchneri strain LN7125, Lactobacillus
brevis strain LB5328 and Lactobacillus brevis strain LB7123 were
deposited on Mar. 14, 2012, with the Agricultural Research Service
(ARS) Culture Collection, housed in the Microbial Genomics and
Bioprocessing Research Unit of the National Center for Agricultural
Utilization Research (NCAUR), under the Budapest Treaty provisions.
The strain(s) were given Patent Deposit No. NRRL B-50733, Patent
Deposit No. NRRL B-50731, and Patent Deposit No. NRRL B-50732,
respectively. The address of NCAUR is 1815 N. University Street,
Peoria, Ill., 61604. The deposit(s) will irrevocably and without
restriction or condition be available to the public upon issuance
of a patent. However, it should be understood that the availability
of a deposit does not constitute a license to practice the subject
invention in derogation of patent rights granted by government.
[0063] Applicant(s) will meet all the requirements of 37 C.F.R.
.sctn..sctn.1.801-1.809, including providing an indication of the
viability of the sample when the deposit(s) is made. Each deposit
will be maintained without restriction in the NRRL Depository,
which is a public depository, for a period of 30 years, or 5 years
after the most recent request, or for the enforceable life of the
patent, whichever is longer, and will be replaced if it ever
becomes nonviable during that period. The deposits will irrevocably
and without restriction or condition be available to the public
upon issuance of a patent. However, it should be understood that
the availability of a deposit does not constitute a license to
practice the subject invention in derogation of patent rights
granted by government action.
[0064] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this disclosure pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0065] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
EXAMPLES
Example 1: Rapid Acting Lactobacillus Strains for Improving Silage
Aerobic Stability in Corn
[0066] Studies were performed to develop a microbial inoculant
comprising Lactobacillus species that can increase the fermentation
and stabilization of whole plant corn silage to allow for early
opening (aerobic exposure) at less than thirty (30) days
post-ensiling.
Strain Selection:
[0067] Heterofermentative lactic acid bacterial cultures (252
isolates), taken from Pioneer Hi-Bred's microbial culture
collection, were grown in De Man Rogosa Sharpe broth (MRS broth;
Difco.TM. Lactobacilli MRS; Becton Dickinson and Company, Sparks,
Md. 21152 USA), prepared as described by the manufacturer, for 24
to 48 hours. An aliquot of cell suspension was transferred to an
extract of whole plant corn forage broth (1:10 mixture of dried
ground corn forage in water, autoclaved, 0.2-micron filter
sterilized then 0.5% glucose added) and grown for 40 hours at 37
C.
[0068] After an initial screening process, five isolates were
selected for field testing in 2011 to evaluate their ability to
enhance the aerobic stability of ensiled whole plant corn forage.
Strains were discovered and identified from corn or the feces of
corn fed sheep samples taken in the United States. In 2012, three
of the isolates were repeated in whole plant corn and in
combination with commercial homofermentative strains LP286 and
LP329.
Field Testing:
Test Strain Characteristics:
TABLE-US-00001 [0069] 16S rDNA MRS gas Strain Species ID Source
production LB7123 Lactobacillus brevis Sheep feces fed WPCS, gas
positive Polk City, IA, 2008 LB4616 Lactobacillus brevis HMC,
Dallas Center, 1997 gas positive LB5328 Lactobacillus brevis WPCS,
IA, 1995 gas positive LB31 Lactobacillus brevis WPCS, Kersey, CO,
1993 gas positive LN7125 Lactobacillus WPCS, Polk City, IA, 2008
gas positive buchneri
Field Testing:
2011 Corn Harvest:
[0070] Three hybrids (P1162, 33M16, DeKalb 61669vt3) of whole plant
corn forage were individually harvested Sep. 1, 2011 at the
Livestock Nutrition Center in Sheldahl, Iowa at a dry matter range
of 33-37%.
[0071] 2012 Corn Harvest:
[0072] Three hybrids (P90115XR, P1162XR, P1395XR) of whole plant
corn forage were individually harvested Sep. 21, 2012 at the
Livestock Nutrition Center in Sheldahl, Iowa at a dry matter range
of 33-37%.
2011 Corn Silage Treatments:
TABLE-US-00002 [0073] Commercial Pioneer .RTM. brand Experimental
Test Whole Plant Corn Inoculants Strains 11A44 LB31 1132 LB4616
11C33 LB5328 11CFT LB7123 LN7125
2012 Corn Silage Treatments:
TABLE-US-00003 [0074] Commercial Pioneer .RTM. brand Experimental
Test Whole Plant Corn Inoculants Strains & Combinations 11A44
LB5328 1132 LB7123 11C33 LN7125 11CFT LB5328 + LP286 + LP329 LB7123
+ LP286 + LP329 LN7125 + LP286 + LP329
Inoculation:
[0075] In 2011, individual experimental test strains were grown and
supplied as fresh grown culture. In 2012, individual experimental
test strains were grown, lyophilized in-house and supplied as dry
powder culture. Commercial and experimental lyophilized products
were suspended in water then all treatments were adjusted to a
standard concentration of 4.54.times.10.sup.7. Treatments
suspensions were applied using a 10-cc syringe at a rate of 1.0
ml/lb of forage. The application dose for all treatments was
1.times.10.sup.5 CFU/g forage.
Silos:
[0076] PVC silos were filled with 160 kg DM/m.sup.3 of whole plant
corn forage and air infused for 24 hours as described below based
on opening days.
TABLE-US-00004 Silo 24 hr Air Opening Infusion Day Day 7 0 14 7 28
14 60 45
[0077] Aerobic Stability:
[0078] The method of Honig (Proc. Of the Eurobac. Conf., P.
Lingvall and S. Lindgren (ed.) (12-16 Aug. 1986) Swed. Univ. of
Agric. Sci. Grass and Forage Report No. 3-1990. Pp. 76-81. Uppsala,
Sweden.) used for measuring aerobic stability. Aerobic dry matter
losses (DML) were estimated from the rise in temperature after
exposure to air as described by Honig.
Results and Discussion
Aerobic Stability
[0079] Treatments with heterofermentative lactobacillus decrease
the aerobic dry matter loss and increase the time to heating.
Differences were observed between strains over time.
2011--Corn Silage (Table 1)
[0080] Opening at days 7 and 14 resulted in statistical differences
between treatments LB5328, LB7123 and LN7125 and uninoculated
control silage which held until day 60 when numerical effects were
observed.
[0081] Treatment with LB5328, LB7123 and LN7125 also resulted in a
statistical improvement over the current commercial inoculants
(11A44, 11C33 and 11CFT) when evaluated at early opening times.
These three strains show marked improvement over other selected
heterofermentative strains (LB31 and LB4616).
[0082] Two Lactobacillus brevis (LB7123 & LB5328) and one
Lactobacillus buchneri (LN7125) selected from these studies were
efficacious in improving aerobic stability of whole plant corn
silage when opened prior to day 30 (days 7 and 14). Obvious
differences from control and less efficacious strains were noted.
The three strains were advanced for inclusion in the 2012 whole
plant corn silage trials to be tested individually and in
combination with current commercial homofermentative strains LP286
and LP329.
2012--Corn Silage (Table 2)
[0083] Opening on days 7 and 14, resulted in biological and
statistical differences between single strain treatment LB7123 and
uninoculated control silage. The differences at day 28 & 60
were numerically but not statistically better between LB7123 and
control.
[0084] There was a continuing trend for the combination treatments
of LB5328+, LB7123+, and LN7125+ responding positively on day 7 and
14. Day 7 opening resulted in a numerical improvement in aerobic
DML over control while the combination treatments were
significantly better than control at day 14. All three combination
treatments maintained the improvement (30-40%) over control.
Similar improvement in aerobic dry matter losses were observed with
11A44 and 11CFT. The commercial products did not seem to be
actively improving DML on days 7, 14 or 28; however, 11A44 and
11CFT had a positive impact on aerobic stability at day 60, as
observed in previous research trials.
2011 & 2012 Combined Corn Silage Studies--(Table 3)
[0085] In general, the performance of single strain treatments
LB5328, LB7123 and LN7125 over two years of whole plant corn silage
trials (6 studies, 24 silos/tmt), was consistent in reducing
aerobic dry matter loss over uninoculated control silage and
current commercial products at openings before 28 days.
[0086] Treatments LB7123 and LN7125 were statistically better than
control and commercial treatments at day 7 and 14. LN7123, LN7125
and LN7125 also demonstrated statistical differences from control
by day 14. By day 28 & 60, these three single strains were
numerically better than control and showed aerobic stability
equivalent to commercial products 11A44, 11C33 and 11CFT.
Summary
[0087] Because of the reduced aerobic dry matter loss afforded by
these strains and the combinations with homofermentors, repeatable
improvements in dry matter losses are observed at early opening of
ensiled whole plant forages providing an economic advantage to the
producer using specifically selected L. buchneri or L. brevis
inoculants.
TABLE-US-00005 TABLE 1 Effect of Lactobacillus buchneri and
Lactobacillus brevis on dry matter losses upon exposure to air in
whole plant corn silage ensiled for various lengths of time. DMLoss
- Days post-ensiling 2011 7 14 28 60 Control 3.36.sup.a 5.73.sup.a
4.93.sup.a 4.37.sup.ab 1132 2.74.sup.a 5.22.sup.a 2.91.sup.bc
5.77.sup.a 11A44 3.71.sup.a 5.76.sup.a 3.19.sup.abc 5.39.sup.ab
11C33 2.99.sup.a 5.39.sup.a 4.26.sup.ab 4.57.sup.ab 11CFT
2.29.sup.ab 4.75.sup.ab 3.14.sup.abc 4.39.sup.ab LB31 2.09.sup.abc
5.41.sup.a 3.52.sup.abc 5.09.sup.ab LB4616 2.12.sup.abc
4.41.sup.abc 3.17.sup.abc 5.04.sup.ab LB5328 1.95.sup.abc
3.23.sup.bc 2.40.sup.c 3.88.sup.ab LB7123 0.94.sup.bc 3.32.sup.bc
2.67.sup.bc 3.95.sup.b LN7125 0.51.sup.bc 2.99.sup.c 2.14.sup.c
3.95.sup.b
TABLE-US-00006 TABLE 2 Effect of Lactobacillus buchneri,
Lactobacillus brevis and combinations with homofermentors on dry
matter losses upon exposure to air in whole plant corn silage
ensiled for various lengths of time. DMLoss - Days post-ensiling
2012 7 14 28 60 Control 2.91.sup.ab 4.66.sup.a 2.90.sup.b
2.95.sup.a 1132 2.94.sup.ab 4.17.sup.ab 2.97.sup.b 2.35.sup.ab
11A44 3.60.sup.a 3.60.sup.abcd 2.95.sup.b 1.03.sup.b 11C33
2.99.sup.ab 3.89.sup.abc 4.09.sup.ab 2.70.sup.a 11CFT 3.46.sup.a
4.45.sup.a 4.72.sup.a 1.55.sup.ab LB5328 2.91.sup.ab 3.37.sup.abcd
3.89.sup.ab 2.39.sup.ab LB7123 0.95.sup.c 2.54.sup.de 2.77.sup.b
2.52.sup.a LN7125 2.20.sup.b 3.39.sup.abcd 3.32.sup.ab 2.28.sup.ab
LB5328 + LP286 + LP329 2.76.sup.ab 2.80.sup.bcde 2.82.sup.b
2.02.sup.ab LB7123 + LP286 + LP329 2.20.sup.b 1.85.sup.e
3.31.sup.ab 1.86.sup.ab LN7125 + LP286 + LP329 2.03.sup.bc
2.70.sup.cde 2.50.sup.b 1.74.sup.ab
TABLE-US-00007 TABLE 3 Effect of Lactobacillus buchneri and
Lactobacillus brevis on dry matter losses upon exposure to air in
whole plant corn silage ensiled for various lengths of time. 2011
& 2012 DMLoss - Days post-ensiling Combined 7 14 28 60 Control
3.10.sup.ab 5.02.sup.a 3.77.sup.ab 3.66.sup.a 11A44 3.76.sup.a
4.72.sup.a 2.98.sup.ab 3.31.sup.a 11C33 2.99.sup.ab 4.67.sup.a
4.17.sup.a 3.63.sup.a 11CFT 2.87.sup.ab 4.44.sup.ab 3.77.sup.ab
3.03.sup.a LB5328 2.43.sup.b 3.56.sup.bc 3.00.sup.ab 3.66.sup.a
LB7123 0.94.sup.c 2.82.sup.c 2.72.sup.b 3.20.sup.a LN7125
1.35.sup.c 3.08.sup.c 2.64.sup.b 3.33.sup.a
Example 2: Rapid Acting Lactobacillus Strains for Improving Silage
Aerobic Stability in Grasses
Strain Selection:
[0088] Heterofermentative lactic acid bacterial cultures (252
isolates), taken from Pioneer Hi-Bred's microbial culture
collection, were grown in De Man Rogosa Sharpe broth (MRS broth;
Difco.TM. Lactobacilli MRS; Becton Dickinson and Company, Sparks,
Md. 21152 USA), prepared as described by the manufacturer, for 24
to 48 hours. An aliquot of cell suspension was transferred to an
extract of whole plant corn forage broth (1:10 mixture of dried
ground corn forage in water, autoclaved, 0.2 micron filter
sterilized then 0.5% glucose added) and grown for 40 hours at 37
C.
[0089] Strains were discovered and identified from corn or the
feces of corn fed sheep samples taken in the United States. In
2012, three of the isolates were tested in European rye grass, and
in 2013 only two isolates were tested as single strains and in
combination with current commercial homofermentative strains LP286
and LP329.
Experimental Test Strain Characteristics:
TABLE-US-00008 [0090] 16S rDNA MRS gas Strain Species ID Source
production LB7123 Lactobacillus brevis Sheep feces fed WPCS, gas
positive Polk City, IA, 2008 LB5328 Lactobacillus brevis WPCS, IA,
1995 gas positive LN7125 Lactobacillus WPCS, Polk City, IA, 2008
gas positive buchneri
Field Testing:
2012 Grass Harvest:
[0091] European rye grass harvested around Buxtehude, Germany on
May 22, 23, & 24 2012 at a dry matter range of 33-49%.
2013 Grass Harvest:
[0092] European rye grass harvested around Buxtehude, Germany on
Jun. 3, 4 & 6 2013 at a dry matter range of 36-46%.
2012 European Rye Grass Silage Treatments:
TABLE-US-00009 [0093] Commercial Pioneer .RTM. Experimental Test
brand Grass Inoculants Strains 11A44 LB5328 11G22 LB7123 11GFT
LN7125
2013 European Rye Grass Silage Treatments:
TABLE-US-00010 [0094] Commercial Pioneer .RTM. Experimental Test
brand Whole Plant Corn Strains & Inoculants Combinations 11A44
LB7123 11GFT LN7125 LB7123 + LP286 + LP329 LN7125 + LP286 +
LP329
Inoculation:
[0095] In 2012 & 2013, individual experimental test strains
were grown, lyophilized in-house and supplied as dry powder
culture. Commercial and experimental lyophilized products were
suspended in water then all treatments were adjusted to a standard
concentration of 4.54.times.10.sup.7. Treatments suspensions were
applied using a 10-cc syringe at a rate of 1.0 ml/lb of forage. The
application dose for all treatments was 1.times.10.sup.5 CFU/g
forage.
Silos:
[0096] PVC silos were filled with 100 kg DM/m.sup.3 of grass and
air infused for 24 hours as described below based on opening
days.
TABLE-US-00011 Silo 24 hr Air Opening Infusion Day Day 7 0 14 7 28
14 60 45
[0097] Aerobic Stability: The method of Honig (Proc. Of the
Eurobac. Conf., P. Lingvall and S. Lindgren (ed.) (12-16 Aug. 1986)
Swed. Univ. of Agric. Sci. Grass and Forage Report No. 3-1990. Pp.
76-81. Uppsala, Sweden.) used for measuring aerobic stability.
Aerobic dry matter losses (DML) were estimated from the rise in
temperature after exposure to air as described by Honig.
Results and Discussion:
Aerobic Stability
[0098] Treatments with heterofermentative lactobacillus decreased
the aerobic dry matter loss and increased the time to heating.
Differences were observed between strains over time.
2012--Grass Silage (Table 1)
[0099] Opening grass silos at day 14, 28 and 60 resulted in
differences between single strain treatments LB5328, LB7123 and
LN7125 and uninoculated control silage. These three treatments at
days 7, 14 and 28 were numerically better than control and
commercial products.
[0100] The commercial products did not appear to reduce aerobic DML
on days 7 or 14; however, by day 28 11A44 and 11G22 were
significantly improved over control. By day 60, with the exception
of 11GFT, all treatments were statically improved over control.
2013--Grass Silage (Table 2)
[0101] Single strain treatments LN7125 and LB7123 resulted in a
considerable reduction in the aerobic dry matter loss when compared
to uninoculated control across all opening days tested. The
combination of LB7123 and the L plantarum strains was effective at
reducing aerobic dry matter losses across all days while the L.
plantarum combinations with LN7125 was not statistically different
than the uninoculated control.
[0102] The commercial products had little effect on the aerobic dry
matter loss until day 90 post-ensiling. Treatment with 11A44 was
more effective at reducing the dry matter losses than was the
combination product 11G22.
Summary
[0103] Because of the reduced aerobic dry matter loss afforded by
these strains and the combinations with homofermentors, repeatable
improvements in dry matter losses were observed at early opening of
ensiled grass forages providing an economic advantage to the
producer using specifically selected L. buchneri or L. brevis
inoculants.
TABLE-US-00012 TABLE 1 2012 Effect of Lactobacillus buchneri and
Lactobacillus brevis on dry matter losses upon exposure to air in
European grass silage ensiled for various lengths of time. 2012
European DMLoss - Days post-ensiling Grass 7 14 28 60 Control
2.76.sup.ab 7.10.sup.a 6.5.sup.a 3.30.sup.a 11A44 2.27.sup.abc
4.3.sup.ab 2.72.sup.b 1.18.sup.b 11G22 3.25.sup.a 6.35.sup.a
2.31.sup.b 0.00.sup.b 11GFT 2.5.sup.ab 6.05.sup.a 6.07.sup.a
3.42.sup.a LB5328 1.68.sup.abc 2.41.sup.b 2.72.sup.b 0.77.sup.b
LB7123 0.29.sup.c 2.47.sup.b 1.58.sup.b 0.01.sup.b LN7125
0.75.sup.bc 2.34.sup.b 2.02.sup.b 0.11.sup.b .sup.abcwithin a day,
values with different superscript differ p .ltoreq. 0.05.
TABLE-US-00013 TABLE 2 2013 Effect of Lactobacillus buchneri and
Lactobacillus brevis alone and in combination with Lactobacillus
plantarum on dry matter losses upon exposure to air in European
grass silage ensiled for various lengths of time. 2013 European
DMLoss - Days post-ensiling Grass 7 14 28 90 Control 7.11.sup.a
6.46.sup.ab 6.08.sup.ab 1.77.sup.a 11A44 3.54.sup.abc 2.63.sup.bcd
0.22.sup.c 0.10.sup.a 11GFT 5.77.sup.ab 7.56.sup.a 7.44.sup.a
1.46.sup.a LB7123 0.00.sup.c 0.23.sup.d 0.00.sup.c 0.00.sup.a
LN7125 1.25.sup.ab 1.35.sup.cd 0.11.sup.c 0.00.sup.a LN7125 + LP286
+ LP329 6.18.sup.a 5.24.sup.abc 3.52.sup.bc 0.17.sup.a LB7123 +
LP286 + LP329 0.44.sup.c 1.08.sup.cd 2.58.sup.bc 0.46.sup.a
.sup.abcdwithin a day, values with different superscript differ p
.ltoreq. 0.05.
[0104] Having illustrated and described the principles of the
embodiments of the present invention, it should be apparent to
persons skilled in the art that the embodiments of the invention
can be modified in arrangement and detail without departing from
such principles. Thus, the invention encompasses all alternate
embodiments that fall literally or equivalently within the scope of
these claims.
[0105] It is understood that various preferred embodiments are
shown and described above to illustrate different possible features
of the invention and the varying ways in which these features may
be combined. Apart from combining the different features of the
above embodiments in varying ways, other modifications are also
considered to be within the scope of the invention.
[0106] All publications and published patent documents cited in
this specification are incorporated herein by reference to the same
extent as if each individual publication or published patent
document was specifically and individually indicated to be
incorporated by reference.
* * * * *