Note: Descriptions are shown in the official language in which they were submitted.
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The present invention relates to anti-fungal bacterial
strains, in particular anti-fungal bacterial strains of
Bacillus subtilis and of Serratia, for use in preserving
animal feedstuff compositions, including silages and hay.
Animal feedstuffs include forages such as cereal
grains, whole cereal crops, grasses, legumes, rice, alfalfa
and hay. The forage is usually stored as a dried material,
or in the form of a silage produced from these materials by
fermentation processes. This fermentation to produce silage
is generally conducted in an oxygen-free environment and in
the presence of acid-producing bacteria. The dried
material or the silage is taken for use as feedstuff as
needed.
When a forage is to be stored as a dried material, it
is necessary that the material be dried to varying degrees
prior to storing in order to minimize the colonization of
harmful microbes naturally present. For example, hay in the
field carries a complex population of fungi, yeasts,
actinomycetes and bacteria which could colonize during
storage and lead to, in extreme cases, spontaneous
combustion. Also, the development of mould or fungus can
lead to palatability and health problems for both animals
and humans. The rate of microbial development varies
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depending upon the moisture content, and for baled hay a
moisture content of about 15% is a generally preferred upper
limit where microbial activity is reduced to a tolerable
level. In North America, in order to achieve this low level
of moisture content, the usual practise is to allow a period
of field wilting or drying prior to baling.
Silage is the fermentation product of crops, such as
those listed hereinbefore, brought about by native lactic
acid-producing bacteria present on the crop at harvesting.
Lactic acid fermentation can be improved by the addition of
selected lactic acid-producing bacteria. Such bacteria
include selected lactic acid-producing bacterial strains of
the genus Lactobacillus, the genus Streptococcus and the
genus Pediococcus.
It is important that a state of anaerobiosis be
attained in order to obtain good silage, and therefore, the
crop is usually stored and permitted to ferment in a sealed
container or silo. The state of anaerobiosis, however, can
be difficult to achieve rapidly, and is governed by the
degree to which air comes into contact with the preserving
material in the silo. The exposure of silage to air, in
particular oxygen in the air, results in spoiling of the
silage, and the degree of spoiling will depend on the
aerobic stability of the silage. Also, silage material can
spoil after having been removed from the silo prior to
animal feeding depending upon this elapsed time interval.
In these instances, the spoiling is primarily due to yeast
or mould contamination of the ensiled material.
Fungal growth in forage can be modified or prevented by
the addition of chemical fungicides, such as ammonia or
organic acids or their salts, prior to storing, or by
various physical means including longer wilting periods in
the field, the use of driers, reduction of the oxygen
concentration, and/or alteration of pH.
A bacterial strain of the species Bacillus subtilis
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has been disclosed in SU 751382, dated July 30, 1980, to
display fungicidal activity in fodder specifically against
StachYbotris alternans and Dendrodochium toxicum. US
4266028, dated May 5, 1981, discloses the preparation of the
anti-fungal antibiotic prodigiosin by cultivation of the
bacterium Serratia marcescens R-2.
It would be required that any specific biological
control agent for use in silage or dried forage would have
to operate under a complex set of changing conditions and
compete with the natural microflora that are present.
Surprisingly, we have now found bacterial strains that
can be used for effectively controlling the growth of fungi,-
actinomycetes and bacteria in forage. These bacterial
strains may be mixed with lactic acid-producing bacterial
strains for use in producing and preserving silage. In
particular, we have found bacterial strains of the species
Bacillus subtilis and of the genus Serratia that exhibit
both anti-fungal and anti-microbial properties enabling the
biological control of several moulds and bacteria in forage.
Therefore, it is an object of the present invention to
better prepare and preserve silage by treatment of a
suitable material to be ensiled with a mixture of an
anti-fungal bacterial strain and a lactic acid-producing
bacterial strain.
It is a further object of the present invention to
provide anti-fungal bacterial strains that may be used in
permitting hay or other dry forage to be baled or stored
(preserved) at significantly higher moisture content than
normal.
It is yet a further object of the present invention to
provide said bacterial strains that may be formulated into
compositions for use in treating forage.
Accordingly, in one aspect of the present invention
there is provided an animal feedstuff composition comprising
a forage and an anti-fungal effective amount of an
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anti-fungal bacterial strain selected from the group
consisting of Bacillus subtilis FB260 and an anti-fungal
strain of the genus Serratia.
Preferably, said anti-fungal bacterial strain is of the
species Serratia rubidaea. More preferably, said
anti-fungal Serratia rubidaea bacterial strain is Serratia
rubidaea FB299.
Preferably, said forage of said animal feedstuff
composition is hay.
In a further aspect of the present invention said
animal feedstuff composition further comprises a lactic
acid-producing bacterial strain of a genus selected from the
group consisting of the genus Lactobacillus, the genus
Streptococcus and the genus Pediococcus in a lactic
acid-producing effective amount to effect fermentation to
produce silage. Preferably, said lactic acid-producing
strain is of the genus Lactobacillus. More preferably, said
lactic acid-producing strain is of the species Lactobacillus
plantarum. Yet more preferably, said lactic acid-producing
strain is Lactobacillus plantarum MTD 1, a culture of which
is deposited at the National Collections of Industrial And
Marine Bacteria (NCIMB), Aberdeen, Scotland, accession
number NCIMB 40027.
In yet a further aspect of the present invention there
is provided a silage composition comprising silage and an
anti-fungal effective amount of an anti-fungal bacterial
strain selected from the group consisting of Bacillus
subtilis FB260 and an anti-fungal strain of the genus
Serratia. Preferably, said anti-fungal bacterial strain is
of the species Serratia rubidaea. More preferably, said
anti-fungal bacterial strain is Serratia rubidaea FB299.
In yet a further aspect of the present invention there
is provided a bacterial composition of use in the
preparation and preservation of silage, said composition
comprising bacterial cells of an anti-fungal bacterial
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strain selected from the group consisting of Bacillus
subtilis FB260 and an anti-fungal strain of the genus
Serratia; and bacterial cells of a lactic acid-producing
strain of a genus selected from the group consisting of the
genus Lactobacillus, the genus Streptococcus and the genus
Pediococcus. Preferably, said anti-fungal bacterial strain
is of the species Serratia rubidaea; and said lactic
acid-producing strain is of the species Lactobacillus
Plantarum. More preferably, said anti-fungal bacterial
strain is selected from the group consisting of Bacillus
subtilis FB260 and Serratia rubidaea FB299. Yet, more
preferably, said lactic acid-producing strain is
Lactobacillus plantarum MTD 1, a culture of which is
deposited at the National Collections of Industrial And
Marine Bacteria (NCIMB), Aberdeen, Scotland, accession
number NCIMB 40027.
Preferred characteristics of said anti-fungal bacterial
strain of use in said animal feedstuff composition, said
silage composition and said bacterial composition of the
invention, in addition to having fungicidal activity,
include: the ability to grow at pH values from about 4.0 to
about 8.0; the ability to grow at temperatures from about
15C to about 50C, preferably from about 25C to about
50C; the ability to use the carbohydrates glucose, sucrose,
melibiose, raffinose, cellobiose, and xylose as a carbon
source; weak production of protease; aerobic and
spore-forming, gram positive bacterial cells; facultative
non spore-forming, gram negative bacterial cells; and
osmo-tolerance shown by the ability to grow at sodium
chloride concentrations from about 1% to about 15%.
Any lactic acid-producing strain of the genus
Lactobacillus, the genus Streptococcus or the genus
Pediococcus that is suitable for making silage may be used
in said animal feedstuff composition and said silage
composition as defined hereinbefore. Bacterial strains
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suitable include lactic acid-producing strains of the
species Lactobacillus plantarum, Lactobacillus amylophilus,
Lactobacillus caseii, Lactobacillus acidopholus,
Lactobacillus curvatis, Lactobacillus brevis, Streptococcus
lactis, Streptococcus thermophilus, Streptococcus faecium,
and Streptococcus faecalis.
In addition to said bacterial strains used in said
animal feedstuff and said silage and said bacterial
compositions, as defined hereinbefore, cells of other lactic
acid-producing bacterial strains and suitable for use in
silage may also be present that are strains of the genus
Lactobacillus or the genus Streptococcus different from said
strains of said compositions as defined hereinbefore.
In the compositions of the present invention, where
appropriate, bacterial cells of said anti-fungal bacterial
strain and said lactic acid-producing bacterial strain are
suitably present in relative proportions within the range
between 1:9 and 9:1. Preferably they are present in
substantially equal proportions.
In yet a futher aspect of the present invention there
is provided the novel bacterial strain FB260 of Bacillus
subtilis and the novel bacterial strain FB299 of Serratia
rubidaea.
The anti-fungal bacterial strains according to the
present invention may be prepared in any required quantity
by fermenting a sample of said strain under suitable
conditions in an appropriate medium. Such conditions and
media are well known in the art. The media will, for
example, generally contain a nitrogen source (e.g. fish meal
or tryptone) and a carbohydrate source such as starch or
glucose. Suitable conditions include a temperature in the
range from about 20C to about 40C, and an approximately
neutral pH. Fermentation may be conveniently carried out in
batches, typically for periods of 1-2 days, or using
continuous culture. The living biomass of the bacterial
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strain may be obtained from the fermentation liquor by
concentration, for example by centrifugation or micro or
ultra-filtration, followed by addition of any desired and
appropriate formulating agents. Formulating agents which
may be useful include, for example, surface active agents,
e.g., wetting agents, solid diluents, dispersing agents and
W stabilisers. If desired, solid formulations may be
prepared by known methods.
In yet a further aspect of the present invention there
is provided a method for the preparation or preservation of
an animal feedstuff composition as described hereinbefore,
said method comprising treating forage with an effective
amount of bacterial cells of an anti-fungal bacterial strain
as hereinbefore defined. Preferably the forage is hay.
In still yet a further aspect of the present invention
there is provided a method for the preparation and
preservation of silage comprising treating a suitable
forage with an effective amount of the bacterial composition
of the invention as hereinbefore defined. The bacterial
composition can be applied as a liquid or a particulate
solid. When it is to be applied as a liquid the carrier
will be water whilst when it is to be applied as a solid the
carrier will be a solid material. Generally compositions to
be applied as solids will be supplied to users as complete
formulations including the carriers. However, when the
composition is to be applied as a liquid, the user will
generally be supplied with the appropriate bacterial cells
to be suspended in an appropriate volume of water before
use.
When the bacterial composition of the invention is to
be applied as a solid, any suitable material may be used as
a carrier in said composition. Examples of suitable
carriers include cereals such as ground corn cobs, ground
barley and wheat and other materials such as clay, chalk,
magnesite and talc.
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In addition to carriers and bacteria the compositions
for use in the preparation of silage will generally contain
further materials added for a variety of reasons. Any
further material added may be included as another component
of the bacterial composition of the invention. Further
materials include nutrients (which can be sugars such as
sucrose, lactose, etc); growth factors which are necessary
for the growth of some bacteria (including yéast extract,
corn steep liquor, vitamins and amino acids); materials
added to protect the viability of the bacteria;
anti-oxidants; materials to assist with oxygen uptake; and
oils and other materials to reduce dusting tendencies of the
additives or improve adhesion to crops. The further
materials are not however essential components of the silage
compositions of the invention.
The relative proportions of carrier and bacteria
included in a composition for use in the preparation of
silage depend upon the particular bacterial strain used and
upon its activity and viability. A particularly suitable
bacterial composition to be applied as a solid will include
896 g of a solid carrier such as ground corn cobs (maize
grits). When the bacterial cells are to form part of a
liquid additive for application, they will be suspended by
the user in a sufficient amount of water. The concentration
of living cells, as defined by the number of Colony Forming
Units (CFU), may be used as a guide for determining
application rates. As a guide bacterial cells may be
applied as a liquid suspension or solid additive in
concentrations in the range from about 1 x 105 CFU to about
1 x 108 CFU per gram of forage treated depending upon the
nature and condition of the forage.
In the preparation of the animal feedstuff, silage and
bacterial compositions of the invention, the bacteria are
usually dried by suitable means, e.g. freeze dried or spray
dried, or a living culture is resuspended in water, and are
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then mixed with other components by conventional
blending/mixing procedures.
The preparation of the animal feedstuff and silage
compositions according to the present invention is generally
carried out by applying, such as by spraying, the bacterial
cells of the appropriate bacterial strain or bacterial
composition as hereinbefore defined to high-moisture corn
silage naturally infested or liable to infestation by
various microbes.
The living biomass (bacterial cells) undergo their life
cycle providing the anti-fungal and anti-microbial
activities required to control spoilage.
The novel bacterial strains and the bacterial
composition according to the present invention may be used
in the preparation and preservation of a wide variety of
silage and hay that are prone to infestation by moulds.
Specific examples of commercially important forage to be
protected by the invention are alfalfa, legumes,
high-moisture corn grains, corn silage, grain silage, grass
silage, vegetable silages and alfalfa hay.
A further advantage of the present invention is to
provide bacterial strains, and feedstuff, silage and
bacterial compositions that are acceptable under appropriate
feedstuffs legislation with no undesirable effects on
livestock and no by-products therefrom, and produce no
residues in said by-products.
A yet further advantage is to provide bacterial strains
and said compositions of the present invention that are
safer for operators to use and cause no corrosion or damage
to machinery.
The following Examples additionally illustrate the
invention, but the scope of the invention is not limited to
the embodiments shown therein.
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EXAMPLE 1
The following example illustrates the isolation of
Serratia rubidaea strain FB299 and Bacillus subtilis strain
FB260.
A sample of hay was obtained from St. Valerien, Quebec,
Canada, and a sample of silage was obtained from "Ferme
Brioeniman" also in Quebec. Isolation of FB29g and FB260 is
carried out by transferring 0.1 g of the hay or silage (1 g
of corn) sample, respectively, into 10 ml 0.05% peptone and
performing serial dilutions to 104 to 107, depending on how
turbid the sample appeared. 0.1 ml of the dilution higher
than the one desired is plated using the spread plate
method, onto Nutrient, Yeat-malt, Sabouraud-dextrose and L-S
Differential Media. The plates are examined after 5 days
incubation at room temperature. The colonies are selected
and purified on the medium of isolation and are incubated
until growth at room temperature. Slides are made of the
chosen colonies, gram stained and observed under the
microsocope (at a magnification of lOOOX using the oil
immersion lens) for further characterization. Bacterial
isolates are transferred to nutrient agar slants and stored
at 4C prior to use.
Table 1 lists the morphological and microbiological
characteristics of the strains Bacillus subtilis FB260 and
Serratia rubidaea FB299.
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TABLE 1
MORPHOLOGY AND MICROBIOLOGICAL CHARA'CTERIZATION
FB260: Aerobic and spore-forming bacterium, gram positive
individidual cells, colonial morphology typical of
Bacillus species
growth observed at pHs varying from 4.5 to 8.0
qrowth observed at temperatures varying from 25 to
50C
growth observed at final NaCl concentration varying
from 1 to 15%
the str~in can use, as a carbon source, glucose,
sucrose, melibiose, rafinose, cellobiose and xylose
the strain is a weak producer of proteases, but a
strong producer of amylases and ~ylanases.
the strain has shown anti-fungal effect against many
moulds particularly against Penicillium sp, Absidia
sp, Aspergillus niger and Aspergillus flavus.
FB299: Facultative and non spore-forming bacterium, gram
negative individidual cells,
growth observed at pHs varying from 4.0 to 8.0
growth observed at temperatures varying from 25 to
50C
growth observed at final NaC1 concentration varying
~rom 1 to 10%
2S the strain san use, as a carbon source, glucose,
sucrose, melibiose, rafinose, cellobiose and xylose
the strain has shown anti-fungal effect aqainst many
moulds particularly against Penicillium sp, Absidia
sp and Aspergillus niger and Aspergillus flavus.
A
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EXAMPLE 2
This example illustrates the application of bacterial
strains FB260 and FB299 for preserving alfalfa hay.
500 g of alfalfa hay were weighed out in a plastic
bucket on a scale. Each sample was placed in a separate
pile on a plastic sheet. There were 8 replications per
treatment so 8 piles were arranged at a time. Each pile was
sprayed with 10 ml of the appropriate treatment strain using
SpotGUN-Lurmark spray guns. Hay was rearranged in each pile
and proceeded to spray a further 10 ml onto each sample.
This was done so that a better coverage of the alfalfa was
obtained. The samples were then bagged individually in
nylon mesh bags by packing the alfalfa tightly as a "bale",
wiring the bags shut and labelling them. This procedure was
followed for each treatment, 1 through 3 resulting in 24
"bales" of hay at each moisture level 40% and 28%. The
bales were piled in two piles, according to the moisture
content, on the floor in an environmental chamber set at
26C and 70% relative humidity. After two weeks time a
single bale from each moisture level was opened and checked
for moisture content using a microwave oven and scale. The
experiment was stopped and mould spoilage assessment was
conducted. For statistical testing the +/- rating system
used in assessing the visual moulds were converted to
discrete values based on the following system. A minus (-)
was assigned a value of 0%. A plus (+) was assigned a value
of 33%. A double plus (++) was assigned a value of 66% and
finally a triple plus (+++) was assigned a value of 99%.
These percentages were based on the maximum amount of moulds
growth a sample could incur before being classified into the
next highest rating. The results are listed in Table 2 and
show that on the 28% moisture hay, both FB299 and FB260
provided for good control of moulds. On the 40% moisture
hay, FB299 showed particularly good control of moulds.
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TABLE 2 133549~
I. 28% Moisture Level:
Presence of Moulds on Each "Bale"
1 2 3 ~ 5 6 7 8
Treatment
l.Control + +T+ ++ TT+ ++ ++ +++ +++
2.FB260+ + + ~+ ++ + + +
3.FB299+++ ++ ++ + + _ ++ +
TREATMENT
Control FB299 FB260
Mean Percentage 78 49 ` 41
of Visual Moulds
II. 40% Moisture Level:
Presence of Moulds on Each "Bale"
1 2 3 4 5 6 7 8
Treatment
l.Control +++ +++ +++ +++ +++ +++ +++ +++
2.FB260 +++ + +++ + +++ +++ ++ +++
3.FB299 - ++ + + - ++ +++ +++
. TREATMENT
Control FB260 FB299
Mea~ Percentage 99 78 49
of Visual Moulds
~ Visual Moulds Legend :
- : No Moulds Growth
+ : Slight Moulds Growth
++ : Fair Amount of Moulds Growth
+++ : Lots of Moulds Growth
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EXAMPLE 3
The following example shows a comparison of the
application of FB260 or FB299 alone or in combination with
Lactobacillus plantarum (Ecosyl, trade mark) to ensiled
high-moisture corn grains.
2 kg of high-moisture corn grain for each sample was
weighed out and placed in oxygen-impermeable polyethylene
bags. 100 ml of the appropriate treatment was poured into
the sample bag which was then sealed shut and inverted
rapidly 10 times to ensure complete coverage. The final
concentration of living cells ranged from 1 x 105 to 1 x 108
CFU/g of corn. The test included 3 replicates for each of 4
sampling times to give 12 bags per treatment. Nine of the 12
samples belonging to each treatment were placed in separate
75-L galvanized steel garbage bins and covered with a 20 kg
sandbag to mimic a silo's pressure. Finally the lids were
shut on the 10 "silos". The remaining 3 samples per
treatment represented time 0 and were placed in a fridge to
halt any activity before the samples could be visually
analyzed and their supernatants collected the next day.
After-7 days at room temperature, the remaining replicates
of each treatment were placed in an environmental chamber at
25.2C and 70% relative humidity for the remaining 8 days.
At each sampling time, the appropriate samples were visually
assessed for mould spoilage and washed with water to collect
volatile acids, water-soluble carbohydrates, etc for
analytical purposes.
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Treatments were conducted according to the following:
Treatment 1 : Control
Treatment 2 : FB260
Treatment 3 : FB299
Treatment 4 : Lactobacillus plantarum
Treatment 5 : Lactobacillus Plantarum + FB260
Treatment 6 : Lactobacillus plantarum + FB299
The results are listed in Table 3 and show that under
the conditions described above, treament #5 showed less
moulds contamination than the control when silage was tested
after 15 days. For aerobic stability, treatments #3 and #6
showed both good control of moulds and less temperature
increase after 5 days of air exposure. In respect of pH,
good values (< 4.2) were observed with both treatments #5
and #6 as compared with the control. Each of FB260 and FB299
work well in combination with Lactobacillus plantarum to
lead to a better silage than the Lactobacillus plantarum
strain alone.
TABLE 3
20 TREATMENT VISUAL MOULDS pH AEROBIC
STABILITY
1 77% 4.35 77%(29.7C)
2 55% 4.35 66%(29.3C)
3 55% 4.32 33%(23.7C)
4 55% 4.21 55%(26.7C)
22% 4.16 66%(26.7C)
6 44% 4.14 44%(25.0C)
* : Tests conducted after 15 day-old silage
**: Tests conducted after the ensilage material was exposed to
air for 5 days, at room temperature. The values in
percentages are mould spoilage when the values between
brackets are averaged temperatures.
