Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W~ 931~t72~ i'CT/L'S92/05792
TITLE
NATAMYCIN TREATMENT OF
DRIED VU'HOLE KERNEL, GRAINS
BACKGROUND OF THE INVENTION
The invention relates to a process for treating dried whole
kernel grains with natamycin to control and/or prevent the growth of
yeasts, molds and other fungi.
It is desirable to store whole driedkernel grain under
conditions of minimal microorganism attack, with its attendant formation
of toxins. Fungi attack is particularly troublesome because of the high
propensity of fungi to attack grain and the toxic nature of mycotoxins that
certain fungi may generate. Perhaps the most prevalent and
mycotoxin-producing organisms that attack grain are molds; particularly
~'r~ssa~ium, ~'enicillaum and Aspergillus molds.
Silo storage has not been wholly successful in long term
grain or ensilage starage where mycotoxin production must be limited.
For example, the conventional practice of ensiling is the storage of high
moisture content fodder which is fermented to increase the acidity of the
fodder wherein the acidity controls but does not prevent the formation of
significant quantities of toxins (i.e., toxins which are released by the
metabolic activities of certain fungi). Fermentation has the disadvantage
in that it decreases the food nutrient value of the stored fodder.
z5 The use of antibiotics to control organism growth on
ensilage stored in a silo is not generally practiced because relatively high
concentrations of antibiotics normally would be required for the
prolonged periods associated with the conventional high moisture
environment of silo storage.
3o It has long been recognized that drying the whole kernel
grain before storage is necessary to prevent immediate and virtually total
organism attack of grain which has a high moisture content. However,
drying alone does not provide adequate long-term protection of whole
kernel grain, such as from harvest to harvest.
35 Currently, the generally accepted practice to protect grain
from spoilage while being stored is to treat the grain with propionic acid.
iV~ ~3J01720 Pf.'t'/11S92/~579,.
Propionic acid is sprayed onto the grain normally at the rate of 1-S
pounds per ton of grain. This treatment is not wholly successful,
especially for long-term storage. The propionic acid is volatile and may
evaporate. Further, the propionic acid is corrosive to the handling
s equipment, particularly damaging to pumps unless they are made of
expensive corrosion resistant metal. More importantly, propionic acid
will not effectively control certain species of molds, fungi, yeast, etc.,
that
attack grain in storage.
Natamycin is known generally to be useful as an antifungal
. to agent. Natamycin is a member of the polyene family of antimycotics. This
family is characterized by a macrolide ring of carbon atoms closed by
lactoniZation and pOSSeSSBS a series of conjugated double bonds.
Members of this family can be classified by the number of double bonds
and whether ~or not the compound contains a glycosidically linked
15 carbohydrate. The compound natamycin is a tetraene with a molecular
weight of about 6b6, an empirical formula corresponding generally to
c33~47N013~ ~d contains a glycosidically-linked carbohydrate moiety,
mycosamine. Natamycin has an isoelectric point of pH 6.5. The structure
of natamycin exists in two configurations: the enol-structure and the keto-
20 structure.
It is an object of the invention to provide an improved
process for storing dried whole kernel grain. Particularly, the invention
relates to a process for treating grain with a polyene to reduce the
damage associated with a fungi attack.
25 Although particular emphasis is placed upon using
' natamycin to treat dried whole kernel grain, the present invention is
intended to encompass using any member of the polyene family which is
effective in controlling and/or reducing fungal contamination during
storage of the dried whole kernel gain.
34
SUMMARY OF THE INVENTION
The present invention relates to a process and a product
produced thereby which is obtained by using a polyene antibiotic,
particurally natamycin, to control and/or protect dried whole kernal grain
35 from fungi contamination. The invention also controls and/or prevents
contamination of the whole kernal grain from mycotoxins which may be
CT/US92J05792
~~ 93/01720
_3_ . .
released by the fungi. The invention permits dried whole kernal grain to
be stored for prolonged periods of time while remaining substantially free
from fungal contamination. 'The polyene may be sprayed, admixed, etc.,
with the dried whole kernal grain by any technique which is adequate to
provide the polyene to the whole kernal grain in an effective manner.
DETAILED DlES~RIPTiQN OF THE IhTVEI~T'TI_Ohl
The present invention relates to the treatment of stored
dried whole kernel grain with small quantities of an antibiotic from the
polyene family, such as natamycin. The presence of a polyene inhibits
fungi attack such as the growth of fungi, mold, yeast, etc., and possible
resultant mycotoxin contamination. fine invention . also permits grain to
be stored for longer periods of time and improves the quality of the grain
which is eventually removed from storage ~e.g., the removed grain is
substantially free of fungal contamination).
In accordance with the invention, the term polyene
antibiotic defines and refers to one or more members of the recognized
class of antibiotics, released by fermentation of a species of the
Streptorrayces genus, that are particularly effective against fungi. Also
2o encompassed are the equivalent polyene derivates, such as esters, salts,
and the like. Further, other pharmaceutically acceptable derivatives of
natamycin may used to practice the invention which are effective to
control and/or prevent contamination by the fungi. Generally, the
preferred polyene for use in the present invention comprises natamycin
because natamycin will typically give prolonged antifungal protection at a
relatively low non-toxic dosage, and accordingly, natamycin is economical
for prolonged grain storage.
'Biologically effective amount" defines that the polyene
antibiotic is present in a sufficient quantity and will remain active for a
3o time sufficient to give adequate antifungal control for the contemplated
usage of the stored grain; that is control to adequately preserve the grain
against fungi, mold, yeast, etc., growth and mycotoxin contamination over
the entire storage period.
"Grain" defines and refers to one or more species of grain
that is to be stored dry for a prolonged time, such as from harvest until
spring, for seed grain, or from harvest to harvest for continuous cereal
WO 93/U~?24 : : Pf.'T/g.JS92/OS?9~
_4_
manufacture. Particularly contemplated are monocots gains for use in
human food products and animal feed such as cereal grains, seeds, etc.,
including corn, popcorn, oats, barley, rice, rye, wheat, grass seeds, canola
and sunflower seeds, soy beans, etc.
s "Whole kernel" grain defines and refers to grain that has
not been substantially crushed or otherwise fractured, and that has its hull
generally intact encompassing the grain. Thus the term whole kernel
grain defense and includes grains, such as corn, that is on its growth
substrate such as a corn cob, and/or individual kernels. However, this
to term does not include grain, such as many conventional animal feeds
(e.g., poultry feed), where the individual kernels have been crushed or
fractured to break open the protective kernel hulls:
"Stored" defines any technique whereby the grain is kept in
its dried whole kernel form for a prolonged period of time, namely 1 to 2
15 weeks, months or more. Storage may be in elevators, bins, bags, etc. An
important aspect of the invention is that the storage is for the purpose of
substantially retaining the grain in its initial as-stored condition, without
significant change or contamination by microorganism attack.
'Dried" defines that the grain, either sun dried or heat
2o dried, has had its initial moisture content of at least about 30% by weight
decreased to about 15% or lower, normally about ?%.
'TTon-toxic" defines the concentration of the polyene and
substances used therewith in accordance with the invention for effective
biological protection of the grain. Non-toxic does not include
z5 unacceptable quantities of either toxic chemicals from the polyene or
mycotoxins from fungi infestation.
' The polyene (e.g.,natamycin), is useful at quantities of only
about 5g through at least about 50g per ton, at which quantities it is
biologically effective in controlling fungi attack or infestation while being
3o non-toxic in food products (e.g.,animal feed), which are to be ingested by
animals. and/or humans. Concentrations of natamycin of about 5.5 ppm
through at least about 55 ppm based on the weight of the dried grain to
be treated are non-toxic and biologically effective, for protecting the grain
for 2 months or longer against significant fungal infestation and/or
3s mycotoxin contamination. However, the exact dosage which is sufficient
to be effective against the fungi is dependent upon the specific storage
y ~ Q '~ PCTPUS92/05?92
- 'u~ 93>(DI?20
conditions. If the storage conditions are favorable for the metabolic
activities of the fungi, a relatively large amount of the polyene will be
required. Storage conditions which are warm, moist, possesses a neutral
pH, contain oxygen, include a metabolizable substrate, etc., would require
a relatively large dosage of the polyene to be effective against the
attacking fungi. The amount of the polyene which is necessary to prevent
growth and/or possible toxin contamination by the fungi is less than the
amount which is necessary to treat an existing fungal outbreak.
Therefore, it is advantageous to treat the whole kernel grain immediately
to after drying.
Moreover, the fungi tend to attack any imperfection in the
whole kernel grain which permits the fungi to penetrate into the internal
regions of the kernel (e.g., the interior of the kernel contains starches and
sugars which may be consumed by the fungi). For example, the kernel
may be damaged during drying, transport, etc., in a manner that renders
the kernel susceptible to attack by a fungi. Particularly, the dried whole
kernel rnay contain or develop a crack in the hull which is sufficient to
permit entry of the fungi. Also, the eye of the kernel is especially
susceptible to damage and consequently attack by fungi even though the
2~ hull may not be cracked. Should the fungal attack of stored grain be
extensive it may be necessary to dispose of the entire contents of a
storage vessel (e.g., the presence of fungi and toxins thereof may render
all of the kernels of grain in an elevator unsuitable for any purpose).
The preferred morphology of the polyene is crystalline;
however, any morphological state is acceptable in practice of the
invention which is effective against the fungi. The polyene used in the
' invention may be present in one or more hydrate forms (e.g., mono-, and
tri-hydrates). The tri-hydrate form is advantageous in some aspects of the
invention due to its stability.
Natamycin is an acceptable polyene for practicing the
present invention which per se is essentially insoluble in water. Certain
non-toxic biologically equivalent derivatives of natamycin may be water
soluble. hlatamycin and/or a water-soluble biologically equivalent
derivative can be supplied to the grain in any convenient way which
provides the desired polyene to the dried whole kernels in a manner
su~cient to be biologically effective against the fungi. For example, the
~ 1 ~ 410 2 . Pt.'flUS92/0~'1W2
WO 93101720
-6-
polyene(s) can be applied (e.g.,sprayed, admixed, coated, etc.), onto the
grain as an aerosol, a fog, mist, powder, aqueous suspension, solution, etc.
Faccessive wetting of the dried grain is to be avoided (e.g., moisture
enhances the growth rate of the attacking fungi). Normally the polyene is
supplied while the grain is being delivered or transported to the storage
facility, such as when it is being conveyed into an elevator, bin, bag, etc.
Care must be taken to achieve a polyene application that is distributed
(e.g., uniformly), and remains throughout the grain during storage.
In one aspect of the invention, the polyene is suspended
to within a nons~toxic carrier liquid and sprayed upon the dried whole kernel
grain. Should a non-toxic carrier liquid be used to apply the polyene, the
carrier liquid should generally not be a solvent for the polyene. However,
certain biologically . effective and/or pharmaceutically acceptable
derivatives of the'polyene which may be soluble within the non-toxic
carrier liquid may be used alone and/or in conjunction with natamycin to
practice the invention. Suitable non-toxic carrier liquids comprise at least
one member of the following group: alcohols such as methanol, water,
etc. In addition to the non-toxic carrier liquid, it may be advantageous to
include a non-toxic wetting agent or surfactant in the suspension. The
2o non-toxic wetting agent tends to enhance the effectiveness of the polyene
by permitting the suspension to more readily coat and/or penetrate into
any imperfections of the dried whole kernel grain.
In another aspect of the invention, the polyene may be
comminuted or ground into a powder to increase its surface area. The
?s increased surface area may enhance the effectiveness and ability of the
polyene to be distributed uniformly throughout the dried whole kernel
grain (e.g., relatively small particles of ratamycin may be readily directly
admixed with whole kernel grain for storage).
It was a surprising discovery that if the grain is stored in a
3o generally dark and dry environment, the length of protected storage is
relatively independent of the treatment concentration. For example, if an
amount of natamycin is applied to the whole kernel grain that will give
adequate antifungal protection to the grain stored in a dark and dry
environment for about 2 months, it normally is not necessary to increase
35 the amount of natamycin to be used even for significantly longer storage
times (e.g., from harvest to harvest). For example, it would be expected
PCT/U~92/05792
e~~ 9/01720 ~ y . ~ ~ ~ ~ ~:
-1-
that a suspension comprising natamycin, a non-toxic carrier liguid and a
wetting agent which is sprayed onto grain, that is to be stored in a dark
and dry environment for at least about 2 weeks, is effective to reduce if
not prevent any fungal attack. This, of course, is premised on the
natamycin being chemically stable under the conditions of storage.
In certain aspects of the invention, it may be advantageous
to use the polyene in conjunction with other treatment processes and/or
substances. For example; the polyene suspension discussed above may
include other substances such that a plurality of dried grain treatments
may be implemented simultaneously. More importantly, the polyene
treatment of the invention does not preclude using the treated whole
kernel grain in any subsequent process or end product (e.g., whole.
kernels of grain treated in accordance with the invention may be removed
from storage and processed subsequently into flakes, grist, etc.).
~'Jhile not wishing to be bound by any theory or
explanation, it is believed that the polyene is active or effective against
the fungi, but not against bacteria. One explanation may be that fungi
(but not bacteria) contain ergosterol in their membranes.
In general ~ the fungi will grow; if the environment is
2o appropriate, until contacting the natamycin. The theorized mechanism of
the polyene action is a binding of the polyene molecule and ergosterol
present in the cell membrane of the fungi, mold, yeast, etc. The
complexing with ergosterol is substantiated by the neutralizing effects of
ergosterol addition on the antifungal activities of the polyene against the
25 fungi. A complex between the polyene and the cell membrane of the
fungi is believed to alter membrane permeability since the polyene is a
' relatively large molecule which creates an increased surface pressure
which may tend to induce a reorientation of the ergosterol present in the
membrane, thus altering permeability of the cell and resulting in osmotic
3o shock. This osmotic shock is typically sufficient to interrupt, if not
halt,
the metabolic activities of the fungi (e.g., the polyene may cause irregular
fungi growth, sporulation, etc.).
A significant aspect of this invention is that very small
amounts of a polyene such as natamycin are effective in preventing the
35 excessive contamination of the whole kernel grain by mycotoxins during
storage. This is of particular importance where the grain is to be used in
dV~ X3/01730 P~1'1US92/057~k:~
~~~4~0~
_8_
food products, particularly in human food products such as cereal, where
the polyene compound must be compatible with consumption by the
human and/or animal. The polyenes used in accordance with the
invention are non-toxic to humans and animals. tienerally any poiyene
which is consumed by an animal (e.g., cattle, poultry, swine, etc.) is
normally not retained within the flesh of the animal. As a result, the
polyene does not adversely affect human consumption of any animal
which has consumed a feed containing the polyene. In contrast, animals
which are exposed to the fungi may incorporate the fungi and/or toxins
to thereof into their flesh. Such fungi and/or toxins may be harmful to
humans which consume the flesh of these animals; making it desirable to
use the invention as one technique to prevent and/or control any
significant exposure of these animals to fungi (e.g.,. the polyene treatment
' of whole kernel grain which may be eventually ground for animal feed
may prevent and/or control the exposure of animals and/or humans to
fungi). 'Therefore, one. aspect of the invention permits controlling and/or
preventing fungal attack to whole kernel grain which may be fed to
animals such that any processed animal is not contaminated with fungi,
toxins thereof and/or antifungal agents.
Although a few aspects and embodiments of the invention
have been described in detail, those skilled in the art will readily
appreciate that the present invention embraces many combinations,
equivalents and variations other than those exemplified.
