Note: Descriptions are shown in the official language in which they were submitted.
CA 02177567 2000-03-13
WO 9i!14395 ~ PCTIU893/1IS93
ANTIBIOTIC BIOMASS ANIMAL FEED COMPOSITIONS
FIELD OF THE INVENTION
The invention relates to animal feed compositions containing antibiotic
biomass.
sgo_uND of z~iE INVENT~aN
In purified form polyene antibiotics are affective antibiotic agents for a
number of uses. Natamycin, also known as pimaricin or tennectin, is an
effective
antifungal agent. Carter, U.S. Patents 4,536,494 and 4,600,706, discloses that
dry
crystalline natamycin can be mixed with feed grain to enhance growth.
U.S. Patent 4,132,777 discloses use of aqueous suspensions of natamycin on
~ 5 animals to control diseases such as ringworm. U.S. Patent 4,950,477
discloses
aqueous aerosol suspensions of pure polyenes, including natamycin, for control
of
various fungal diseases in humans and animals. Canadian Patent 684,259
discloses
feed additives of Candida albicans fermentation product, with or without the
inclusion of an antifungal agent such as nystatin or natamycin. U.K. Patent
2o Application 2,106,498 discloses the use of Smeptomyces SF-1(ASA), NC1B
11738 to
produce a new tetraene and also natamycin. U.S. Patents 4,395,491 and
4,291,053
discloses the use in fodder of salinomyan in its biomass to growth of
ruminants and
pigs.
2 5 ~~~~r.~~
The invention is an animal feed additive composition consisting essentially of
(a) a dried antibiotic biomass containing at least one polyene antibiotic
selected from
the group consisting of amphotericin A and B, virginiamycin, leucomycin,
levorin,
filimarisin, partricin, and trichomycin in admixture with (b) a non-toxic
carrier
3 0 material, the weight ratio of antibiotic to carrier in the admixture being
from 0.167 to
5, wherein said dried biomass prevents fungal attack of feed during storage.
DETAILED DESCRIPT10N OF THE iNVErrrInN
3 5 It has been found that for many uses, particularly in an animal feed
mixture,
it is not necessary to extract and purify the antibiotic from its biomass.
More
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W O 95/14395 PC'TIITS93/11573
specifically, it has been found that the biomass from an antibiotic
fermentation
broth is a superior ingredient in animal feeds, particularly grain based
feeds, such as
poultry feed. Not only does the antibiotic biomass contain biologically
effective
quantities of an antibiotic to protect against fungal mycotoxin production,
but it also
contains significant nutrient values such as protein, trace compounds, etc.,
which are
desirable for maximizing animal growth.
The animal feed compositions comprise an animal feed such as a cracked
grain feed for chickens, turkeys, etc., and a biologically effective amount of
an
antibiotic, which is included within a nonviable antibiotic biomass.
1o Antibiotic biomass is produced by fermentation using a microorganism that
releases an antibiotic. The antibiotic biomass is rendered nonviable by any
suitable
technique that permits the biomass to be used as an ingredient in animal feed.
The
biomass comprises at least one of the following: (1) the cells of the
antibiotic
releasing organism, (2) antibiotic, (3) residual fermentation medium, (4)
fermentation broth (e.g., an aqueousphase which typically includes by-
products).
A biologically effective amount is the amount of biomass required to give
adequate antifungal control and milumize mycotoxin production in the feed.
Adequate control means that the animal feed is protected against mycotoxins
produced by fungi, mold, yeast, etc., over the entire storage period.
2o Animal feed is a foodstuff that is provided to animals, such as chickens,
horses, poultry, swine, turkeys, ruminants (e.g., bovine), pets, etc. The
foodstuff
typically comprises cereal grains (e.g., corn), soybeans, oil seeds, etc.,
which have
been crushed or ground. The antibiotic biomass may be admixed with the
foodstuff
before, during and/or after being crushed or ground to obtain the animal feed.
z5 Stored means any techluque whereby the animal feed is kept in a ready-to-
use form for a prolonged period of time, namely 1 to at least 2 weeks. Storage
may
be in elevators, bins, bags, etc. Dried means that the nonviable antibiotic
biomass,
either sun dried or heat dried, has had its initial moisture content of about
at least
30% by weight, decreased to about 15% or lower, normally about 7%.
3o Antibiotics useful in the antibiotic biomass include natamycin,
amphotericin
A and B, virginiamycin (staphylomycin), leucomycins, levorin, filimarsin,
partricin,
and cabirnicina (Trichomycinc~ ). These antibiotics are disclosed in a number
of
references well known to those skilled in the . art, such as the Antibiotics
Annual. '
Amphotericin is described in Heineman, U.S. Patent 3,126,317. The antibiotic
35 biomass may include functional equivalent derivatives of the antibiotics
that are
produced in the fermentation broth. Although, the fermentation process for
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.WO 95114395 PCT/US93111573
obtaining these antibiotics are known, it was unexpected that
the antibiotic biomass
was a superior feed additive.
The biomass may also contain: inorganic chlorides, sulfates,
phosphates and
combinations thereof as salts of sodium, potassium, calcium,
magnesium, etc; trace
elements, such as boron, cobalt, iron, copper, zinc, manganese,
chromium,
molybdenum; and complex growth factors (e.g., vitamins).
The animal feed compositions are prepared by admixing, i. e.,
blending, etc.,
a biologically effective amount of dried antibiotic biomass
with animal feed at a
concentration of 0.5 to 100 g, typically 5 to 20 g, of antibiotic
biomass per ton (907
to Kg) of feed. The amount of biomass added to the animal feed
depends upon the
environment of the feed. If the environment is favorable for
fungal growth, a
relatively large dosage of the antibiotic may be required to
prevent fungal mycotoxin
contamination, e.g., 50-80 g of antibiotic biomass per ton (907
Kg) of feed.
Favorable environments are those that are warm and moist, possess
a neutral pH,
i5 contain oxygen, and include a substrate that enhances fungi
growth. Since the
amount of antibiotic biomass required to prevent contamination
is less than the
amount of antibiotic biomass required to treat existing fungal
growth, the antibiotic
biomass should be added to the animal feed as soon as it becomes
practicable.
Additional antibiotic may be added to the biomass to increase
its
2 o effectiveness. The added antibiotic may be any pharmaceutically
acceptable
derivative of the antibiotic (e.g., calcium and sodium salts,
and esters of antibiotic).
If desired, the effectiveness of the antibiotic biomass may
be increased by
comminuting or grinding the dried antibiotic biomass to increase
the surface area of
the antibiotic. The comminuted biomass is also more readily
distributed throughout
2 5 the animal feed in a uniform manner.
For some applications it may be desirable to first prepare a
concentrated
premix comprising a compatible non-toxic carrier, such as rice
hulls, limestone,
soybean flour, corn cob fractions, etc., with a relatively large
amount of the
antibiotic biomass, such as 5-20 g of antibiotic per 1-5 pounds
(0.454-2.25 Kg) of the
3 o non-toxic carrier. This premix may then be blended into feed,
along to achieve the
desired concentration of biomass in the animal feed composition.
The antibiotic biomass is produced by any conventional fermentation
process
using a microorganism that produces a desirable antibiotic.
During fermentation,
the antibiotic producing organism culture is introduced into
an aerated nutrient-rich
35 antibiotic production medium. The organisms propagate and produce
an antibiotic.
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WO 95114395 - . PCTIUS93I11573
Depending on fermentation conditions, antibiotic production rates normally are
in
the range of about 0.5 to 1.5 g/L antibiotic per liter of production medium.
One specific technique for obtaining a suitable antibiotic biomass comprises
preparing (e.g., sporulation) and propagating an inoculum comprising an
antibiotic
releasing species that, during fermentation, produces the desired antibiotic.
The
antibiotic releasing species is exposed to a series of predetermined
environments
and/or mediums which may improve the rate at which antibiotic is produced.
Any organism which comprises a natamycin producing Streptomyces species
can be used in accordance with the invention to provide the natamycin biomass.
A
1o preferred Streptomyces species comprise Streptomyces gilvosporeus which has
been
deposited previously with the American Type Culture Collection (ATCC) in
Rockville, Maryland, United States of America, that is registered as ATCC No.
13326.
The biomass is produced by fermentation. Antibiotic producing
I5 microorganisms are propagated and introduced into an aerated nutrient-rich
fermentation medium. The pH of the fermentation broth may be controlled (e.g.,
via addition of an alkali base), in a manner which improves the effectiveness
of the
fermentation process. If desired, the fermentation process may be operated in
a
continuous manner. Depending on fermentation conditions, antibiotic production
2 o yields normally are in the range of about 0.5 to 1.5 g of antibiotic per
liter of
production medium.
One specific technique for obtaining suitable natamycin biomass comprises
preparing and propagating an inoculum comprising a Streptomyces species that,
during fermentation, produces natamycin. The Streptomyces species is exposed
to a
25 series of predetermined environments and/or mediums which improve the rate
at
which natamycin is produced.
By providing an improved environment for the Streptomyces species the yield
of natamycin may be increased to values in the range of about S to 12 g/L. A
biomass having an increased quantity of natamycin can be obtained in a
3 o fermentation process by using the following inoculum propagation and
fermentation
media:
1. A suitable medium for inoculum propagation may be prepared in water (e.g.,
low mineral content water, distilled water, etc.), and comprises:
a. a protein nitrogen source in an amount of from about 2-16 g/L,
s 5 normally about 8 g/L; and
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b. a metabolizable carbon source present in an amount which is
sufficient to avoid total carbon depletion, usually 5-30 g/L of medium,
and normally about 15 g/L;
2. A suitable medium used during fermentation to induce the inoculum to
produce natamycin comprises:
a. about 80-250 g/L of a metabolizable carbon source; and
s
b. a protein nitrogen source containing a high level of protein and trace
ingredients. The protein nitrogen source typically comprises a non-
yeast protein nitrogen component and a yeast protein nitrogen
1o component. These two protein nitrogen components are desirably
present in a ratio ranging, respectively, from about 5:1 to 11:1 based
on protein contents, and for best results about 8:1.
The protein nitrogen source may be supplied from a wide range of sources.
For example, soy protein products (e.g., isolates, flours, meals, etc.), may
comprise
the non-yeast protein nitrogen source (e.g., desirable natamycin biomass
yields are
obtained with a soy protein source comprising 80-95% protein).
The yeast protein nitrogen may also comprise beef extract, protein
hydrolysates (e.g., peptones) and/or yeast (e.g., extracts, autolysates,
etc.).
As discussed above, the production medium must also include a source of
2o carbon which is metabolizable by the Streptomyces species. The carbon
source may
be supplied in any expedient form such as glucose, polysaccharide, corn and
potato
starches, etc.
During fermentation, natamycin may precipitate out as a free crystal, and the
same may be bound to the inside and/or on the outside surface of the cells of
the
organism being fermented. In most fermentations, the natamycin fraction of
total
biomass solids will be in the range of about 5 to 35% natamycin by dry weight
of the
resultant natamycin biomass product.
At the end of the fermentation the biomass, together with the natamycin,
may be physically removed from the fermentation broth. Any solid-liquid
3 o separation technique may be used to separate the natamycin biomass from
any
liquid present (e.g., filtration, centrifugation, etc.). Alternatively, the
fermentation
broth may be concentrated by removal of water, such as by evaporation,
chemical
treatment, etc. The resulting concentrate of natamycin biomass retains all of
the
valuable nutrients and other desirable ingredients that might normally have
been
lost during filtration.
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WO 95114395 ~ ~ ~ ~ ~ ~ ~ PCTIUS93111573
Following concentration, the natamycin biomass is dried by any suitable
technique such as by spray dryer, fluid bed dryer, shelf dryer, etc. The
temperature
used for drying ranges from about 0 to 120 C and depends on the drying
technique
used and the thermal stability of natamycin to a given technique.
The natamycin fermentation, natamycin biomass recovery and treatment
processes generally yield a natamycin biomass which contains about 5 to 35%
natamycin on a dry weight total natamycin biomass basis.
During fermentation, the antibiotic may precipitate out as a free crystal, and
the same may be bound to the inside and/or on the outside surface of the cells
of
1o the organism being fermented. In most fermentations, the antibiotic
fraction of
total biomass solids will be in the range of about 5 to 35% anri'biotic by dry
weight of
the resultant antibiotic biomass product.
At the end of the fermentation the biomass, together with the antibiotic, may
be physically removed from the fermentation broth. Any solid-liquid separation
technique may be used to separate the antibiotic biomass from any liquid
present
(e.g., filtration, centrifugation, etc.). Alternatively, the fermentation
broth may be
concentrated by removal of water, such as by evaporation, chemical treatment,
etc.
The resulting concentrate of antibiotic biomass retains all of the valuable
nutrients
and other desirable ingredients that might normally have been lost during
filtration.
2o Following concentration, the antibiotic biomass is dried by any suitable
technique such as by spray dryer, fluid bed dryer, shelf dryer, etc. The
temperature
used for drying ranges from about 0 to 60 C and depends on the drying
technique
used and the thermal stability of the antibiotic to a given technique.
The antibiotic fermentation, antibiotic biomass recovery and treatment
a5 processes generally yield antibiotic biomass containing about 25 to 35%
antibiotic
on a dry weight total antibiotic biomass basis.
For example, the solids from a fermentation broth containing the antibiotic
may be separated, for example, by centrifuging. The supernate may be discarded
and the biomass divided into panicles. Biomass may be dried in a fluid bed
dryer at
30 70 C until a moisture content of about 7-8% by weight is obtained. The
dried
antibiotic biomasses may be mixed with a chicken feed comprising corn, wheat
and
soybean meal at a rate of about 100 g of biomass per ton (907 Kg) of chicken
feed.
Alternatively, a feed premix may be prepared by mixing about 100 g of the
antibiotic
biomasses with about 1-5 pounds (0.454-2.25 Kg) of rice hulls and/or soybean
flour.
35 The premixes may be admixed with a chicken feed comprising corn, wheat and
soybean meal to obtain an enhanced feed including about 10 to 15 g of
antibiotic
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.WO 95114395 PCT/US93111573
per ton (907 Kg) of feed. The resultant feed is (1) protected from fungi
contamination, (2) permits enhanced chicken growth rates and (3) controls and
prevents diseases in the chicken associated with a fungi infection or
mycotoxin
production.
The antibiotic biomass is compatible with other additives which may be
admixed with the animal feed. Suitable additives include: protein, fat, fiber,
calcium
(e.g., calcium carbonate), phosphorus, trace metals, vitamins, citrus pulp,
grape
hulls, rice hulls, etc. An energy source such as sugar (e.g., lactose) may
already be
present and/or admixed with the added antibiotic biomass. In addition to the
1o energy source, other materials may be used in conjunction with the
antibiotic
biomass. Fish meal, certain vitamins, etc., may be added to reduce the growth
rate
of the fungi. However these materials alone are not sufficient to protect
animal
feed from fungi contamination.
Depending upon the storage conditions, the antibiotic can be expected to be
effective against fungal contamination of the animal feed for several weeks or
months. However, to ensure maximum effectiveness, the feed composition should
be shielded from extended exposure to light, e.g., stored in the dark within a
dry
container.
Any antibiotic in the feed composition consumed by the animal is normally
2 o not absorbed or retained within the animal. As a result, the antibiotic is
generally
not incorporated into the eggs and/or flesh of the animal and does not affect
human
consumption of any animal which has consumed the feed composition. However,
animals which are exposed to the fungi may incorporate the fungi, and in some
cases
toxins thereof, into their eggs and flesh. These fungi may be harmful to
humans
which consume the eggs and/or flesh of contaminated animals and, accordingly,
it is
desirable to prevent any significant exposure of such animals to fungi.
It may be advantageous to introduce the antibiotic biomass in the animal
feed in conjunction with other treatment processes and/or substances. More
importantly, the antibiotic biomass does not preclude using the animal feed in
3 o conjunction with any medication, end use product or event (e.g., the
antibiotic
biomass may be fed to an animal undergoing a certain medical treatment).
~ INDUS IAL PpI,IC RIIITY
The invention provides an enhanced animal feed by adding an antibiotic
~ 35 biomass to the feed. The antibiotic biomass enhances the quality and
nutritional
WO 95114395 ~ ~' ~ ~ ~ ~ ~ PCT/US93II1573
integrity of the feed by reducing, if not eliminating, contamination of the
feed
associated with fungal growth and possible mycotoxin release.
Although the antibiotic within the antibiotic biomass may have some
beneficial effect on the animal, the purpose of the present invention is to
protect the
feed itself from fungal contamination. However, an animal feed containing the
antibiotic which is substantially free from fungal contamination, will enhance
the
r
animal growth rate in a cost effective manner. The antibiotic biomass (1)
enhances
the quality of the feed which enhances the health and, thus, growth rate of
the
animal consuming the feed and (2) permits cost-effective storage of animal
feed for
to a longer period of time without spoilage or waste due to fungal
contamination.
EXAMPLE 1
This example illustrates the preparation of an antibiotic biomass and its
addition to animal feed to produce an antibiotic containing animal feed
composition.
Agar slants were prepared using distilled water to which has been added the
following:
3 g/L yeast extract (Difco 'Bacto" Yeast Extract)
3 g/L malt extract (Difco Malt Extract)
5 g/L peptone (Difco'Bacto" peptone)
10 g/L glucose
15 g/L agar.
The agar was sterilized at about 121 C for about 15 min.
An inoculum medium of the following composition was prepared in distilled
water. The pH was adjusted to about 7.0 with potassium hydroxide.
20 g/L glucose
10 g/L sodium chloride
6 g/L corn steep liquor (PPM (brand), Corn Steep Liquid)
6 g/L peptone (Difco 'Bacto" peptone)
3 o The inoculum medium was sterilized at about 121 C for about 15 min.
Streptomyces gilvosporeus, American Type Culture Collection Registration
No. 13326, was obtained from the American Type Culture Collection as a freeze-
dried spore suspension and used as the culture source. The culture was held on
the
agar slants at about 25 C until the culture sporulated.
The agar slants sporulated heavily within about 10 days and were used after
10-20 days. Spores were scraped off these agar slants into the inoculum medium
to
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WO 95/14395 PCT/US93I11573
achieve a spore suspension concentration of about 108 CFU/mL. About 2 mL of
the spore suspension was added to about 100 mL of the inoculum medium in a 500
mL baffled flask. The inoculum in the baffled flask was incubated
for about 48 hrs.
at about 29 C and agitated at about 200 rpm on a rotary shaker.
After about 48 hrs.
,
about 4 mL of this culture was added to about 200 mL of inoculum
medium in a 1 L
baffled flask, to propagate the inoculum. This inoculum was then
incubated for
about an additional 24 hrs. at about 29 C and agitated at about
200 rpm on a rotary
shaker. The inoculum thus produced was used to inoculate 8 L
of production
medium.
io The natamycin production medium was of the following initial
composition:
19.5 g/L soy protein isolate (ADM,'Profam" 5970)
4.5 g/L yeast extract (Stauffer, Type KAT)
0.2 mL defoamer (Mazu, DF 289)
The production medium was prepared in distilled water in a 14
L fermenter and the
pH was adjusted to about 7.6 with potassium hydroxide. The fermenter
was
sterilized for about 15 min at about 121 C. Glucose was sterilized
separately as a
50% solution in distilled water.
Before inoculation, the production medium was heated to about
29 C and th
e
glucose was added to achieve an initial concentration of glucose
of about 40 g/L.
2o An aeration rate of about 0.3 v/v-min. (volumes of air per volume
of medium
er
p
min) and an agitation rate of about 300 rpm was established for
the fermenter.
The inoculum containing Streptomyces gilvosporeus, (ATCC Registration
No.
13326), was added to the fermentation vessel until the fermentation
vessel had an
inoculum content of about 2% by volume. Glucose was added to
the inoculum after
about 40 hr of fermentation in order to maintain a glucose concentration
of about
20 g/L glucose in the fermentation vessel. This was done by feeding
glucose to the
fermenting vessel at a rate of about 1 g/Lrhr. The agitation
rate of the fermentation
vessel was increased as necessary to maintain a dissolved oxygen
level of about SO%
of air saturation.
3o Solids within the biomass formed may be separated from the
fermentation
broth by centrifugation. The supernate from the centrifugation
may be discarded
and the pellets broken up and dried in a fluid bed dryer (e.g.,
dried at about 70 C to
' obtain a 7-8% moisture content). Alternatively, solids may be
separated from the
fermentation broth by filtration on a Buchner funnel using Whatman
No. 3 filter
' 35 paper. The filter cake from the filtration process may be dried
(e.g., dried to about
30% moisture in a convection oven at about 70 C and then drying
to about 6%
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VVO 95!14395 PCTIUS93111573
moisture in a fluid bed dryer). Typically, a total of about 1-13 g of biomass
is
recovered of which about 8-16% is natamycin. Normally, the antibiotic biomass
is
mixed with conventional fresh cracked corn/wheat/soybean meal (e.g.,
conventional
chicken or turkey feed mix), at a rate of about 100-800 g of product per ton
(907 Kg)
of feed mix..
EX_AM_PLE 2
This example illustrates inhibition of mold growth in an animal feed
composition that contains an antibiotic biomass.
io Commercial -crumbled broiler starter chicken feed was ground in a
laboratory grinder to a uniform particle size of approximately 0.5 mm. Sterile
distilled water added to increase the moisture content of the feed to 15.5%
The
feed was divided into two portions. The control feed was left untreated.
Antibiotic
biomass containing natamycin, similar to that prepared in Example 1, was added
to
i5 the other portion and thoroughly mixed to produce an animal feed
composition that
contained 11 ppm of natamycin.
Five 25 g samples of each portion were added to 250 mL wide-mouth
Erlenmeyer flasks. Each flask was stoppered with a two-hole rubber stopped.
Chmulative oxygen consumption was measured using a Micro-Oxymax respirometer
2 o (Columbus Instruments, Columbus, Ohio).
CUMULATIVE OXYGEN CONSUMPTION (micro-L)
l~a~g~Incuh arinn No BiomassBiomass Added
Day 1 IO 20
25 Day 2 138 132
Day 3 278 269
Day 4 429 391
Day 5 6,722 2,542
Day 6 37,493 14,888
Having described the invention, we now claim the following and their
equivalents.
i
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