Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Title: Reduction of malodour in soiled animal litter
FIELD OF THE INVENTION
The present invention relates to an animal litter material
s comprising an enzyme capable of preventing, inhibiting and/or
reducing development of malodour emanating from the litter
material when soiled with urine or other wastes from humans or
animals. The invention also relates to a process for producing
such litter material and a method for reducing formation of
to malodours in soiled lfitter materials.
BACKGROUND OF THE INVENTION
The control of odour in animal litter has been a continuing
problem as such litter materials most frequently are used
is indoors. Also most often the litter is not changed immediately
after an animal, e.g. a cat, has relieved itself in the litter
material, and such soiled litter may remain indoors in the open
box for prolonged periods of time at ambient temperature, thus
facilitating development of foul odours. Especially feline urine.
2o may develop a foul odour.
Suggested solutions include the use of bacteriostats such
as: halogenated aromatic hydrocarbons (U.S. Pat. No.
4,494,482, Arnold, issued Jan. 22, 1985); soluble salts of
transition metals of Group Ib or Group Ilb of the periodic
z5 table of elements, especially zinc, which are taught as both
bacteriostats and urease inhibitors (U. S. Pat. No. 4,494,481,
Rodriguez et al., issued Jan. 22, 1985 and U.S. Pat. No.
4,736,706, Lang, issued Apr. 12, 1988); boron containing
compounds which are claimed to be urease inhibitors (U. S. Pat.
3o Nos. 4,949,672 and 5,176,108, Ratcliff et al. and Jenkins et
al., issued Aug. 21, 1990 and Jan. 5, 1993 respectively);
sodium bisulfite complexes (U.S. Pat. No. 5,267,531, Appel et
al., issued Dec. 7, 1993); and sodium or potassium bicarbonate
(U.S. Pat. Nos. 5,303,676 and 5,421,291, Lawson and Lawson et
35 al., issued Apr. 19, 1994 and Jun. 6, 1995 respectively).
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Other approaches to controlling odour include the use of
absorbents for odour such as cyclodextrin and polycarboxylate
polymers (U.S. Pat. Nos. 4,727,824; 4,844,010; 4,881,490; and
4,883,021, Ducharme et al., issued Mar. 1, 1988; Jul. 4, 1989;
s Nov. 21, 1989; and Nov. 28, 1989 respectively).
Still another approach of "covering up" the bad odour
involves using encapsulated perfumes (U. S. Pat. No. 4,407,231,
Colbom et al. issued Oct. 4, 1983). Thus, many commercial
animal litter products contain a fragrance to mask the malodour
to and to provide a freshness impression. Many of these
fragrances are developed with human aesthetic preference in
mind, apparently without consideration of the effect to the
animal. Thus, many perfumes used in commercial animal litter
compositions contain significant amounts of ingredients that
is are repulsive to animals e.g. cats. On the other hand,
commercially available products which claim control of animal
behavior, such as cat repellent and cat attractant products,
contain only the purported active ingredients without
consideration to human aesthetics.
zo Many types of materials are used as animal litter. Clay and
various cellulosic materials are commonly used, as disclosed in
the above patents and additional disclosures of materials that
can be used are fond in U.S. Pat. Nos.: 5,064,407, Peiffer,
issued Nov. 12, 1991; 5,100,600, Keller et al., issued Mar. 31,
2s 1992; 5,207,389, Hall et al., issued May 4, 1993; 5,209,186,
Dewing, issued May 11, 1993; and 5,229,348, Ivie, issued Jul.
20, 1993. Preferred animal litter materials are those that
"clump" to permit ready removal of the material that has been
contacted by, e.g., urine and/or feces, such as U.S. Pats. Re.
30 33,983, Hughes, issued Jul. 7, 1992 and 5,193,489, Hardin,
issued Mar. 16, 1993. All of the above patents are
incorporated herein by reference.
It is well established that malodour may be caused by a
number of compounds including Volatile Surphuric compounds
3s (VSC), nitrogen containing compounds and short fatty acids.
A source of nitrogen containing compounds, such as ammonia,
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is urine, faeces and blood giving a bad smell known by most
people from soiled animal litters.
The bad smell coming from soiled animal litter is at least
partly a consequence of growth of bacteria, especially
Escherichia coli, Enterococcus spp. and Proteus spp. present on
the skin in the perineum (the region between the anus and the
external sexual organs). All strains of Proteus spp. form the
enzyme urease during their metabolism. Urease has the ability to
rapidly break down urea (constituting about 2% of human urine)
to into ammonia causing an unpleasant odour. Also in WO 98/27261 an
animal litter is described incorporating odor absorbing
materials such as cyclodextrins or zeolites and antimicrobial
agents or urease inhibitors such as metallic salts or various
organic compounds. The litter may also contain a protease enzyme
for digestion of excretions.
DESCRIPTION OF DRAWINGS
Figure 1, in which Z = bactericidal activity in log cfu/ml; X =
mg/1 Curvularia verruculosa haloperoxidase; Y = mM Hz02, shows a
2o response surface plot for the antibacterial activity in
artificial urine of a haloperoxidase (rHP) against a mixed
culture of E. faecalis, E. coli and P. mirabilis adhering to
CTMP.
SUi~iARY OF THE INVENTION
A solid liquid absorbing animal litter material is provided,
comprising an effective amount of an oxidoreductase (EC 1.-. .
enzyme that inhibits the formation of odour from soils
present in the litter material. The oxidoreductase preferably
3o has at least one attribute selected from the group consisting
of antimicrobial activity, urease inhibition activity,
oxidation of malodorous compounds activity and/or mixtures
thereof, but preferably the oxidoreductase will possess all
properties.
The invention also encompass in a additional aspects a process
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for reduction of malodour in soiled animal litters comprising
contacting the soiled animal litter with an effective amount of
oxidoreductase, the use of oxidoreductases for reduction of
malodour in animal litters and a process for preparing an
s improved animal litter comprising the step .of adding an
oxidoreductase to the animal litter composition.
DETAILED DESCRIPTION OF THE INVENTION
The animal litter composition of the invention comprises,
to besides from oxidoreductases conventional litter material such
as described infra.
CONVENTIONAL ANIMAL LITTER MATERIALS:
is CONVENTIONAL BASIC SOLID LIQUID ABSORBING MATERIAL:
Any solid liquid (moisture) absorbing material suitable for
use, e.g., as an animal litter is suitable for use in the
present invention. Suitable examples include minerals,
typically clay such as kaolinites, montmorillonites, or
2o bentonites ; fly ash as obtained f rom the burning of coal ; but
also absorbing fibrous materials or webs, like paper,
cellulosic webs, or polymeric fibrous webs; wood chips;
alfalfa; bark; straw; sand; pelletized absorbing litter
materials (e. g. sawdust or polyurethane foam); and the like,
as including mixtures thereof. Other examples of suitable solid
absorbing litter materials are disclosed in U.S. Pat. No.
3,921,581, issued Nov. 25, 1975 to Brewer, incorporated herein
by reference.
3o In one absorbent composition, there is a major amount of a
cellulosic material, e.g., a cereal or grain hull, or peanut
hulls, along or preferably in admixture with, a second
cellulose material comprised of plant pulp, either vegetable or
fruit pulp. The cellulosic material, hulls and pulp are ground
3s to a desirable particle size and admixed with a minor amount of
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a suitable binder, up to about 20o by weight. Suitable binders
are the carbohydrates, protein or mixtures thereof, such as
flour and starch from plant sources, and the synthetic binders
disclosed hereinafter. The cellulosic hull materials will
s generally be obtained from cereal grain sources such as corn,
rice, wheat, oats and the like, soybean, sunflower and cotton
seeds or peanut hulls. The plant pulp materials are generally
obtained from vegetable sources such as beets, tomato, apple,
grape or citrus pulp generally obtained for citrus fruits such
to as oranges, lemon, lime, grapefruit and the like. The
carbohydrate binders are generally flours and starches from
cereal grains such as corn, rice, wheat, oats and the like.
Protein such as gluten found in wheat flour, or protein from
bean or seed sources such as soybean or flaxseed and the like
is also provide suitable binders.
Other materials that can be used for litters,include clay or
clay-like materials. Their ability to absorb, or adsorb,
moisture makes them excellent candidates for litters. Suitable
litters include specific clays such as Georgia White clay,
2o attapulgite, bentonite, kaolinite, halloysite, montmorillonite,
smeetite, vermiculite, hectorite, diatomaceous earth, Fuller's
earth, fossilized plant materials, expanded perlites, gypsum
and other equivalent litter materials known to those skilled in
the art. Preferred clays are those having water expanding
2s crystal lattices, such as bentonite, i.e., montmorillonite.
The clay particles can be comminuted. That is, they are
pelletized or formed into particles which have a size varying
from about 200 mesh USS (0.075 mm) to about 3~ mesh (5.6 mm) ,
preferably from about 60 mesh (0.25 mm) to about 4 mesh (4.75
3o mm) .
A desirable property, which is characteristic of certain
natural earths which may be used as litter, is the tendency to
"clump". Clumping is a tendency, marked in certain earths and
less marked or absent in others, for the earth particles to
35 adhere firmly to each other when wet to form a mass having
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sufficient physical integrity to enable it to be removed from
the remainder of the particles without undue crumbling or loss
of peripheral litter material. The liquid with which the
litter has been wet is entrained in the clump and is removed
s with it. Where the earth has good clumping properties
substantially all of the liquid can be retained in the clump
and the portion of the earth which remains after the removal of
the clump can be completely dry. This property provides a
means for removing urine from used litter which, in conjunction
to with the physical removal of feces, results in a residue of
relatively uncontaminated litter, with diminished levels of
undesired odours. The litter can then be replenished with
fresh litter. This represents an economical use of litter in
comparison with the complete replenishment of the litter.
15 The present invention is especially good when the litter has
clumping properties, which further limits the amount of odor
that is created. If the litter has only poor or medium
clumping properties, or if better clumping is required, one can
use techniques such as are found in U.S. Pat 5,193,489. The
20 litter can be an earth, for example, such as a montmorillonite
or other smectite, suitably in the alkaline earth metal form,
an attapulgite, a palygorskite or a sepiolite. U.S. Pat. No.
5,014,650 relates to litter comprising a porous, inert solid
substrate, such as a clay, containing a cellulosic ether in an
z5 amount sufficient to agglomerate the animal urine deposited on
the litter to form a gelled agglomerate having sufficient
mechanical integrity to be conveyed from the litter box as a
discrete entity. Additional polymers disclosed to be useful in
the litter include polyvinyl alcohol, xanthan gum, gum acacia
3o and various water-soluble polysaccharides. U.S. Pat. No.
4,676,196 describes an absorbing non-clay litter material
comprising a mix of particulate litter materials which are
caused to agglomerate to form noncompacted particles of a
required size by tumbling in the presence of a moistened binder
35 comprising starches, gums such as guar gum or glues. The US
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5,193,489 patent provides an animal litter comprising a
particulate earth substrate in admixture with a water soluble
or dispersible polysaccharide selected from the galactomannan
gums, said polysaccharide being present in an amount sufficient
s to increase the inherent clumping ability of the earth. A
galactomannan is a polysaccharide mainly or wholly consisting
of mannose and galactose, and preferably comprising a chain of
mannose units bearing galactose side-chains. It is alleged
that the galactomannans can be selected to be effective at
to relatively low concentrations and to give a fast clumping
response. Vegetable based gums are usually marketed in a
number of grades ranging from the relatively impure base gum,
through purified gums from which some extraneous vegetable
matter has been removed to derivatised gums which have been
is treated chemically to alter their characteristics in some way.
The US 5,193,489 patent teaches that the gums are preferably
relatively purified and can be derivatised, e.g. by reaction
with propylene oxide to form the hydroxy propyl ether, to
augment their hydrophilic character so as to be particularly
zo effective. The gum can also be treated to reduce its
alkalinity in aqueous dispersion or solution e.g. by the
inclusion therein of a relatively weak organic or inorganic
acid for example one having a pK value in aqueous solution of
at least 4Ø
z5 Preferred galactomannan gums are guar gum or derivative
thereof. The concentrations of cellulose ethers specifically
disclosed to be effective in U.S. Pat. No. 5,014,650 range
from 0.3% upwards with some failures at 0.5% by weight. The
galactomannans used according to the US 5,193,489 patent are
3o alleged to give effective clumping at concentrations down to
0.05% by weight of the earth (dry weight) or below in certain
instances and are preferably used in from 0.02% to 1% by weight
although any larger quantities, for example up to 2.5% or more
by weight may also by used. Such litters are mixed with
35 particles of the polysaccharide. The earth preferably has a
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particle size mainly, for example at least 95% by weight, in
the range of about 10 mesh (1.7 mm) to 140 mesh (0.11 mm),
preferably about 18 mesh (1 mm) to about 100 mesh (0.15 mm).
The polysaccharide preferably has a similar size range. The
s presence of the polysaccharide in particulate form is alleged
to encourage swift dissolution or dispersion in liquid, in
comparison with gum which might have been deposited onto the
earth particles from solution, and therefore to encourage a
quick clumping response. Pelletized litter materials (e. g.,
to sawdust or polyurethane foam) typically have particle sizes in
the range from about 1 mm to about 1.3 cm, preferably from
about 2.5 mm to about 1 cm.
The basic litter material can be any of the art recognized
materials, with those that have the ability to clump being
is preferred.
CONVENTIONAL MATERIALS FOR INHIBITION OF ODOUR FORMATION:
Besides from the oxidoreductase of the invention the litter may
also optionally if desired contain conventional materials that
2o inhibit the formation of undesirable odors such as described in
WO 98/27261, typically conventional urease inhibitor and/or
antimicrobial agents if desired. Although an oxidoreductase
usually have both urease inhibiting and antimicrobial
properties conventional urease inhibitors and/or antimicrobial
2s agents may if desired be incorporated in the animal litter
composition of the invention if additional effect is desired.
Metal salts
Such urease inhibitor and/or antimicrobial agent may be a metal
3o salt, such as described in WO 98/27261 pp 6 and 7 in the
section "Metallic salts" hereby incorporated in the present
application by reference.
Urease inhibitors
35 Other conventional materials may serve as an urease inhibitor
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and/or suppresser such as described in VJO 98/27261 pp 7-12 in
the section "Urease inhibitors" hereby included by reference.
Antimicrobials
s Conventional organic antimicrobials may also be used in the
present invention. It is preferable to use a broad spectrum
antimicrobial, e.g., one that is effective on both bacteria
(both gram positive and gram negative) and fungi. A limited
spectrum antimicrobial, e.g., one that is only effective on a
to single group of microorganisms, e.g., fungi, can be used in
combination with a broad spectrum antimicrobial or other
limited spectrum antimicrobials with complimentary and/or
supplementary activity. A mixture of broad spectrum
antimicrobials may also be used.
is Antimicrobials useful in the present invention can be
bactericidal and/or bacteriostatic and/or fungicidal and/or
fungistatic and/or a virucidal effect, wherein
The term "bactericidal" is to be understood as the
antimicrobial being capable of killing bacterial cells.
2o The term "bacteriostatic" is to be understood as the
antimicrobial being capable of inhibiting bacterial growth,
i.e. inhibiting growing bacterial cells.
The term "fungicidal" is to be understood as the
antimicrobial being capable of killing fungal cells.
as The term "fungistatic" is to be understood as the
antimicrobial being capable of inhibiting fungal growth, i.e.
inhibiting growing fungal cells.
The term "virucidal" is to be understood as the
antimicrobial being capable of inactivating virus.
3o Also in the context of the present invention the term
"inhibiting growth of microbial cells" is intended to mean
that the cells are in the non-growing state, i.e., that they
are not able to progate.
Suitable preferred antimicrobials and suitable amounts for
3s its use in animal litter compositions are described in WO
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98/27261 pp. 13 to 15 in the section "Antimicrobials"
incorporated herein by reference. Here it is also noted that in
general, it is desirable to limit, or exclude, materials that
can have adverse effects. Therefore, it is desirable to
s exclude the more toxic metals and those elements such as boron,
that can have adverse effects on the environment (e. g. boron
can adversely affect citrus crops).
pH Control Materials and Proteases
to Other conventional materials known to inhibit the formation of
odour include materials with pH activity. Especially useful
materials are acidic materials that neutralize the amine
molecules that are typically created by bacteria. Polymeric
carboxylate materials like polyacrylic acid are useful for this
is purpose.
Protease enzymes can be useful in preventing odour by digesting
excretions in such a way that non-odorous products are created.
They can also reduce odour by destroying other enzymes that
break down excretions. The typical proteases are disclosed
2o hereinafter in the section enzymes.
ODOR ABSORBING MATERIALS:
The animal litter compositions of the invention may contain an
effective, i.e., odour-absorbing, amount of various odour
a5 absorbing materials. For the purpose of the present invention,
the liquid absorbing litter materials, such as clay, saw dust,
and the like, as described supra, are not considered to be
odour absorbing materials, except when specifically noted,
because the novel development of this invention, I.e adding an
30 oxidoreductase to the animal litter composition has the purpose
of improving the malodour absorbing/neutralising capability of
the litter compositions which already contain these liquid
absorbing litter materials.
35 Odour-absorbing materials control undesirable odour by various
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means to reduce the concentration, or availability, of the
malodorous molecules in the ambient air, thus reducing or
eliminating the undesirable smell in the air. Such materials
include, for example, cyclodextrins, zeolites, activated
s carbon, kieselguhr, chelating agents, chitin, alkali metal
carbonates and bicarbonates, pH buffered materials such as
carboxylic acids, and the like. Preferred materials are those
which absorb primary amines. Especially preferred are
cyclodextrin and/or zeolite, disclosed herein to provide odour
to control benefits. Some partially neutralised hydrogel forming
odour absorbing gelling materials, such as polyacrylate gelling
material and acrylate grafted starch gelling material (vide
infra), are also useful in the present invention to control
certain ammonia-type odours. These materials also function as
is fluid absorbing materials.
Zeolite Odour-Absorbing Agent
In general terms, traditional zeolites comprise an aluminate/
silicate framework, with associated cations, M, providing
20 overall electrical neutrality. Empirically, the zeolite
framework can be represented as
xAlOz ~ ySiOz
25 and the electrically neutral zeolite as
x/n M ~ xA102 ~ ySi02 ~ zH20
wherein: x and y are each integers, M is a cation and n is the
3o charge on the ration. As noted by the empirical formula,
zeolites may also comprise waters of hydration (zH20). M can
be a wide variety of rations, a . g . , Na+, K+, NHq+,
alkylammonium, heavy metals, and the like.
Zeolites which may suitably be included in the animal litter
35 composition of the invention is described in WO 98/27261 pp 16
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to 17 incorporated herein by reference.
Cyclodextrins
A preferred odour absorbing material which may be included in
s the animal litter composition of the invention is uncomplexed
cyclodextrin. As used herein, the term "cyclodextrin" includes
any ~ of the known cyclodextrins such as unsubstituted
cyclodextrins containing from six to twelve glucose units.
Examples of suitable cyclodextrins may be found in WO 98/27261
to pp 17 to 21 incorporated herein by reference.
Activated Carbon Odour-Absorbing Agent
The carbon material which may be employed herein is the
material well known in commercial practice as an adsorbent for
is organic molecules and/or for air purification purposes. Carbon
suitable for use herein is available from a variety of
commercial sources under trade names such as CALGON Type "CP6",
Type "PCB", Type "SGL", Type "CAL", and Type "0L." Often, such
carbon material is referred to simply as "activated" carbon or
20 "activated" charcoal. Typically, it is available in the form
of extremely fine, dusty particles (e. g., 0.1-300 microns)
having large surface areas (about 200 to several thousand
M2/g). It is to be understood that any of the "air purifying"
or "activated" carbons of commerce can be used in the practice
2s of this invention.
If the zeolites herein are optionally used in conjunction
with the activated carbon, it is preferred (for aesthetics
reasons) to coat the carbon with the zeolite using a binder.
Other Odour-Absorbing Agents
Other odour-absorbing agents which may be included as part of
the composition of the invention includes kieselguhr, chelating
agents, chitin, pH buffered materials, and the like.
3s
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PERFUMES:
Perfume is usually important in animal litter materials to
disguise foul odours emanating from soil in the litter. The use
of desirable, refreshing perfume ingredients to formulate a
s "refreshing perfume", can make the undesirable odours more
palatable to the owners. Also, use of appropriate perfume
ingredients can influence the animals' behaviour. For example,
the right perfume ingredients in an "attractant perfume" can
attract e.g. a cat to its litter box, its toys, scratching
to post, etc., and the right perfume in a "deterrent perfume" can
influence the cat to stay away from objects such as furniture
that it likes to use as a scratching post.
The refreshing perfume compositions typically, and
preferably, contain ingredients with odour characteristics
is which are preferred by humans in order to provide a fresh
impression and deodorising benefit. Preferably, the perfume
ingredients are selected predominantly from two groups of
ingredients, namely, (a) volatile ingredients having a boiling
point (BP) at normal pressure of less than about 260°C, and
zo more preferably less than about 250°C, and (b) ingredients
having significantly low detection threshold.
Nonlimiting examples of preferred volatile perfume
ingredients and preferred amounts in animal litter materials
are described in WO 98/27261 pp. 22 to 27 in the sections
z5 "PERFUME" and "Perfume in animal litter" incorporated herein by
reference.
Cyclodextrin/Perfume Inclusion Complexes
Perfume/cyclodextrin inclusion complexes known to the art which
3o are a useful component in the litter material of the invention
may be found in the prior art as well as suitable amounts for
litter materials, perfume cyclodextrin ratios, suitable complex
sizes and other relevant parameters, e.g. in WO 98/27261 pp. 27
to 29 in the section "Cyclodextrin/Perfume Inclusion
35 Complexes", incorporated herein by reference, which also refer
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to the disclosures Atwood, J.L., J.E.D. Davies & D.D.
MacNichol, (Ed.): Inclusion Compounds, Vol. III, Academic
Press (1984), especially Chapter 11, Atvood, J.L. and J.E.D.
Davies (Ed.): Proceedings of the Second International Symposium
s of Cyclodextrins Tokyo, Japan, (July, 1984), and J. Szejtli,
Cyclodextrin Technology, Kluwer Academic Publishers (1988),
said publications incorporated herein by reference.
Pressure-Activated or Moisture-Activated Perfume or Enzyme
to Microcapsules
Microcapsules suitable for affixation to the animal litter of
the invention such as disclosed in U.S. Pat. 4,407,231, Colbom
et al, issued Oct. 4, 1983, or WO 97/24179 or WO 99/01500, all
incorporated herein by reference, may be useful to the
is invention as a delivery system of fragrances or other active
components such as the oxidoreductases of the invention or
other enzymes. Such microcapsules, its use, amounts in litter
materials, capsule sizes and other relevant capsule parameters
are described in WO 98/27261 pp. 29 to 30 in the sections
20 "Pressure-Activated Perfume Microcapsules" and "Moisture -
Activated Cellular Perfume Microcapsules" incorporated herein
by reference.
AQUEOUS CARRIER:
as Aqueous solutions are preferred in the present invention for
the preparation of the animal litter and/or freshening the
animal litter. The preferred aqueous carrier of the present
invention is water. The water which is used can be distilled,
deionized, or tap water. Water containing a small amount of
30 low molecular weight monohydric alcohols, e.g., ethanol,
methanol, and isopropanol, or polyols, such as ethylene glycol
and propylene glycol, can also be useful. However, the
volatile low molecular weight monohydric alcohols such as
ethanol and/or isopropanol should be limited since these
35 volatile organic compounds can contribute both to flammability
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problems and environmental pollution problem. If small amounts
of low molecular weight monohydric alcohols are present in the
composition of the present invention due to the addition of
these alcohols to such things as perfumes and as stabilizers
s for some preservatives, it is preferred that the level of
monohydric alcohol be less than about 10%, preferably less than
about 5%, more preferably less than about 3%, by weight of the
composition used to prepare the animal litter.
to SOLUBILIZING AID:
The aqueous composition used to form the animal litter of the
present invention can optionally, but preferably, contain a
solubilizing aid to solubilize any excess hydrophobic organic
materials, especially the perfume, and also optional
is ingredients which can be added to the composition, e.g., insect
repelling agent, antioxidant, etc., that are not readily
soluble in the composition. A suitable solubilizing aid is
surfactant or wetting agent. In a spray product, it is
preferred that the surfactant is a non-foaming or low-foaming
zo surfactant. Suitable surfactants are nonionic surfactants,
cationic surfactants, amphoteric surfactants, zwitterionic
surfactants, and mixtures thereof, preferably nonionic
surfactants and cationic surfactants, and mixtures thereof.
Anionic surfactants are not preferred when the preferred urease
2s inhibiting transition metal ions are present, because they can
form water-insoluble salts with the metal ions. Suitable
surfactants can be emulsifiers and/or detersive surfactants.
Mixtures of emulsifiers and detersive surfactants are also
preferred. The surfactant may suitably a conventional
3o surfactant or mixtures thereof and in amounts and combinations
such as described in WO 98/27261 pp. 32 to 34 in the section
"SOLUBIZING AID" and incorporated herein by reference.
BINDING AID:
35 A suitable method of affixing solid powder active ingredients,
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such as uncomplexed cyclodextrin powder, perfumelcyclodextrin
complex, perfume microcapsules, and enzymes is to form a slurry
of said ingredients in a liquid carrier, preferably water, and
then the slurry is sprayed onto the basic solid liquid
s absorbing material. The slurry can also be separately packaged
in a suitable spray dispenser for spraying onto litter
particles by an animal's owner. The enzymes such as
oxidoreductases may also suitably be separately sprayed onto
the animal litter material as an enzyme concentrate or as a
to slurry of amorphous or crystalline enzyme protein. Also the
enzyme may be in a conventional granular form and mixed with
the basic solid liquid absorbing material before spraying the
slurry onto this mixture.
The slurry can optionally include a suitable binding agent in
is an effective amount to help affix (improve the affixation) the
solid powder active ingredients to the basic solid liquid
absorbing material when the slurry is sprayed thereon. More
particularly, suitable binding agents will function to form a
bond between the solid powder or microcapsules and exterior
2o surfaces of the basic solid liquid absorbing material which is
strong enough to affix and hold at least a major portion of the
solid powder active ingredients onto the substrate during
handling, such as shaking, pouring and the like, as is
encountered in packaging procedures. Suitable binding agents
2s and amounts for use in this invention is described in WO
98/27261 p. 34 in the section "BINDING AID" and incorporated
herein by reference.
SUSPENSION AID:
3o A suspension aid is optionally used to suspend solid powder
active ingredients, such as uncomplexed cyclodextrin powder,
perfume cyclodextrin complex, perfume microcapsules and
enzymes, so that they are fairly evenly dispersed to form -a
relatively evenly distributed layer upon the basic solid liquid
3s absorbing material's surface when the slurry is sprayed
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thereon. More particularly, for commercial preparation of the
animal litter composition of this invention, the suspension
agent should be present in a sufficient amount to provide
suspension stability for the slurry (e. g., prevent separation,
s or settling, of the microcapsules and/or enzyme granules and
particles in the slurry), so that the slurry can be pumped and
sprayed in metered amounts over a period of time. That is, the
suspension agent preferably provides a stably suspended slurry
for a length of time, e.g., at least about 12 hours, more
to preferably for greater than about 24 hours. The suspension
agent should be dispersible in the slurry composition, and
preferably thickens the slurry to a viscosity of at least about
100 centipoise.
Preferred materials for use as the suspension agent are
is polyacrylic acids, such as are available from B. F. Goodrich as
Carbopol (e. g., neutralized Carbopol 94 1) and from Rohm & Haas
as Acrysol, which are very effective (in the neutralized form)
in stably suspending microcapsules of the slurry at levels as
low as about 0.01% to about 1%, more preferably from about
20 0.05% to about 0.2%, by weight of the slurry.
Other preferred materials are particulate clays, such as
smectite clay. A preferred clay suspending agent is calcium
bentonite clay, available from Southern Clay Products under the
trade name Bentolite-.C L. The clay suspension agent is
25 preferably present at a level of from about 0. 1 % to about I
0%, more preferably from about 0. 3 % to about 5%, by weight of
the slurry.
The suspension agent must desirably be selected from
materials which are compatible with the suspended actives and
30 other ingredients. Thus materials which can substantially form
complex with cyclodextrin should not be used to suspend
uncomplexed cyclodextrin and/or cyclodextrin/perfume complex
powder, and polyacrylates and ion-exchangeable clays should not
be used in the presence of water soluble, heavy transition
3s metal ions, such as zinc ions. Also the suspension agent
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should be dispersible in the liquid carrier, with water being
the most preferred liquid carrier.
CLEANING INGREDIENTS:
s Other cleaning components are the typical detergent surfactants
at a level of from about 0.001% to about 1%, preferably from
about 0.005% to about 0.5%, more preferably from about 0.05% to
about 0.3% by weight of the composition. Detersive surfactants
utilized can be of the anionic, nonionic, zwitterionic,
to ampholytic or cationic type or can comprise compatible mixtures
of these types. Detergent surfactants useful herein are
described in U.S. Patent 3,664,961, Norris, issued May 23,
1972, U.S. Patent 3,919,678, Laughlin et al., issued December
30, 1975, U.S. Patent 4,222,905, Cockrell, issued September 16,
is 1980, and in U.S. Patent 4,239,659, Murphy, issued December 16,
1980. All of these patents are incorporated herein by
reference.
Detergent builders can optionally be included in the
2o compositions herein to assist in controlling mineral hardness.
Inorganic as well as organic builders can be used. Builders
are typically used to assist in the removal of particulate
soils. The level of builder, when present in the compositions,
will typically comprise from about 0.1% to about 5%, more
z5 typically about 0.5% to about 1%, by weight, of detergent
builder.
Examples of suitable and preferred inorganic P-containing
builders, nonphosphorous builders including silicates and
3o carbonate builders, water-soluble, nonphosphorus organic
builders such as, carboxylate or polycarboxylate builders,
citrate builders, fatty acid builders and succinate builders
are given in the art eg. in WO 98/27261 p. 38-40 in the section
"CLEANING INGREDIENTS, INCLUDING ENZYMES" incorporated herein
35 by reference, which also refers to the relevant documents on
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19
useful builder materials all incorporated by refererence: U.S.
Patents 3,159,581; 3,213,030; 3,422,021; 3,400,148 and
3,422,137; U.S. Patent No. 4,605,509, U.S. Patent 4,664,839,
issued May 12, 1987 to H. P. Rieck; DE-A-3,417,649 and DE-A-
3,742,043; German Patent Application No. 2,321,001 published on
November 15, 1973; U.S. Patent 3,128,287, issued April 7, 1964;
U.S. Patent 3,635,830, issued January 18, 1972; U.S. Patent
4, 663, 071, issued to Bush et al, on May 5, 1987; U. S. Patents
3,923,679; 3,835,163; 4,158,635; 4,120,874 and 4,102,903; U.S.
to Patent 4,566,984, Bush, issued January 28, 1986; European
Patent Application 86200690.5/0,200,263, published November 5,
1986; U.S. Patent 3,308,067, Diehl, issued March 7, 1967; U.S.
Patent 3,723,322; U.S. Patent 4,144,226, issued March 13, 1979
to Crutchfield et al; U.S. Patent 4,246,495, issued March 27,
is 1979 to Crutchfield et al.
OXIDOREDUCTASE ENZYMES
The enzymes to be included in the animal litter composition
oxidoreductases in accordance with the invention. However other
zo enzyme types with completely different characteristics may be
included such as proteases as known from the prior.
The oxidoreductase in the context of the present invention may
be any oxidoreductase or combination of different
zs oxidoreductases or combination of oxidoreductases with other
enzymes, which facilitates solution of the devised problems in
animal litter compositions, primarily contamination and/or
infection risks in soiled litter and development of foul odors
in the soiled litter, by killing and/or inhibiting
3o microorganisms, inhibitions of urease and/or oxidizing odorous
components. One particularly advantageous feature of using
oxidoreductases in animal litter compositions is that
oxidoreductases will inhibit development of malodour and
infection on all levels, i.e. (1) oxidoreductases will inhibit
35 or kill microorganisms responsible for malodour development or
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infection, (2) even if some microorganisms survives and
proliferate oxidoreductases will inhibit urease enzyme of such
microorganisms from converting urea into malodorous compounds
and (3) even if such malodorous compounds are developed
s oxidoreductases will oxidize such compounds into less odorous
compounds. Another advantageous feature of using
oxidoreductases in animal litter compositions is that
oxidoreductases are biodegradable biological compound
(proteins). In that respect it is surprising that
to oxidoreductases will work in animal litter compositions, which
have a completely different chemical composition than other
composition in which oxidoreductases have been used. Yet
another advantageous feature of using oxidoreductases in animal
litter compositions is that oxidoreductases may be formulated
is into protective granules which may enable a slow release of the
oxidoreductase so that the oxidoreductase is release from the
granule when needed, e.g. upon wetting the animal litter
composition. Methods of granulating enzyme are known to the art
(vide infra) such as fluid bed granulation, mixer granulation,
2o prilling and/or extrusion providing enzyme granules having one
or more protective or release controlling coatings. Also
oxidoreductases may be immobilised on a hygroscopic carrier,
which will actively bring oxidoreductases and malodorous
compound dissolved e.g. in urine together. Also as moisture is
z5 a common condition for microbial proliferation this may ensure
that moisture and microorganisms are brought in contact with
oxidoreductases in the animal litter composition. Accordingly,
when reference is made to "an oxidoreductase" this will in
general be understood to include combinations of one or more
oxidoreductases.
It is to be understood that oxidoreductase variants
(produced, for example, by recombinant techniques) are included
within the meaning of the term "oxidoreductase".
The term "oxidoreductase", as used herein, denotes an
enzyme classified as EC 1.-. . according to Recommendations
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(1992) of the Nomenclature Committee of the International Union
of Biochemistry and Molecular Biology, Academic Press, Inc.,
1992, i.e. any enzyme classified as EC 1.1 (acting on the CH-OH
group of donors) , EC 1.2 (acting on the aldehyde or oxo group
of donors), EC 1.3 (acting on the CH-CH group of donors), EC
1.4 (acting on the CH-NHz group of donors), EC 1.5 (acting on
the CH-NH group of donors), EC 1.6 (acting on NADH or NADPH),
EC 1.7 (acting on other nitrogenous compounds as donors), EC
1.8 (acting on a sulfur group of donors) , EC 1.9 (acting on a
to heme group of donors), EC 1.10 (acting on diphenols and related
substances as donors), EC 1.11 (acting on a peroxide as
acceptor), EC 1.12 (acting on hydrogen as donor), EC 1.13
(acting on single donors with incorporation of molecular oxygen
(oxygenases), EC 1.14 (acting on paired donors with
is incorporation of molecular oxygen), EC 1.15 (acting on
superoxide radicals as acceptor), EC 1.16 (oxidizing metal
ions), EC 1.17 (acting on -CHZ- groups), EC 1.18 (acting on
reduced ferredoxin as donor), EC 1.19 (acting on reduced
flavodoxin as donor), and EC 1.97 (other oxidoreductases).
2o Preferably the oxidoreductase used in the composition of
the invention is an isolated and/or purified enzymes obtained
from fermenting a suitable microorganism.
Preferred oxidoreductases in the context of the invention
are any peroxidase belonging to the classification group EC
25 1.11.1.-, any laccase belonging to EC 1.10.3.2, any catechol
oxidase belonging to EC 1.10.3.1, any bilirubin oxidase
belonging to EC 1.3.3.5 or any monophenol monooxygenase
belonging to EC 1.14.99.1 or any oxidases belonging to EC
1.1.3. .
35
Laccase and laccase related enzymes
Preferred laccase enzymes and/or laccase related enzymes
are enzymes of microbial origin. The enzymes may be derived
from plants, bacteria or fungi (including filamentous fungi and
yeasts) .
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Suitable examples from fungi include a lactase derivable
from a strain of Aspergillus, Neurospora, e.g., N. crassa,
Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus,
Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia,
s e.g., R. solani, Coprinus, e.g., C. cinereus, C. comatus, C.
friesii, and C. plicatilis, Psathyrella, e.g., P. condelleana,
Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M.
thermophila, Schytalidium, e.g., S. thermophiluin, Polyporus,
e.g., P. pinsitus, Phlebia, e.g., P. radita (WO 92/01046), or
to Coriolus, e.g., C. hirsutus (JP 2-238885).
Suitable examples from bacteria include a lactase
derivable from a strain of Bacillus.
A lactase derived from Coprinus, Myceliophthora,
Polyporus, Scytalidium or Rhizoctonia is preferred; in
is particular a lactase derived from Coprinus cinereus,
Myceliophthora thermophila, Polyporus pinsitus, Scytalidium
thermophilum or Rhizoctonia solani.
The lactase or the lactase related enzyme may furthermore
be one which is producible by a method comprising cultivating a
2o host cell transformed with a recombinant DNA vector which
carries a DNA sequence encoding said lactase as well as DNA
sequences encoding functions permitting the expression of the
DNA sequence encoding the lactase, in a culture medium under
conditions permitting the expression of the lactase enzyme, and
2s recovering the lactase from the culture.
Determination of Lactase Activity (LACU)
Lactase activity, preferable laccases derivable from a
strains of Polyporus, may be determined from the oxidation of
3o syringaldazin under aerobic conditions. The violet colour
produced is photometered at 530 nm. The analytical conditions
are 19 mM syringaldazin, 23 mM acetate buffer, pH 5.5, 30°C, 1
min. reaction time.
1 lactase unit (LACU) is the amount of enzyme that
35 catalyses the conversion of 1.0 .mole syringaldazin per minute
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at these conditions.
Determination of Lactase Activity (LAMU)
Lactase activity may be determined from the oxidation of
syringaldazin under aerobic conditions. The violet colour
produced is measured at 530 nm. The analytical conditions are
19 mM syringaldazin, 23 mM Tris/maleate buffer, pH 7.5, 30°C, 1
min. reaction time.
1 lactase unit (LAMU) is the amount of enzyme that
to catalyses the conversion of 1.0 mole syringaldazin per minute
at these conditions.
The use of Laccases for incorporation in animal litter
compositions benefits from the fact that laccases may utilize
abundant molecular oxygen directly as oxidant.
Peroxidases and Compounds possessing Peroxidase Activity
Compounds possessing peroxidase activity may be any
peroxidase enzyme comprised by the enzyme classification (EC
1.11.1.7), or any fragment derived there from, exhibiting per
oxidase activity. In the context of this invention, compounds
possessing peroxidase activity comprise peroxidase enzymes and
peroxidase active fragments derived from cytochromes,
haemoglobin or peroxidase enzymes.
Preferably, the peroxidase employed in the method of the
z5 invention is producible by plants (e. g. horseradish or soybean
peroxidase) or microorganisms such as fungi or bacteria.
Some preferred fungi include strains belonging to the
subdivision Deuteromycotina, class Hyphomycetes, e.g., Fusari-
um, Humicola, Trichoderma, Myrothecium, Verticillum, Arthromy-
3o ces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or
Dreschlera, in particular Fusarium oxysporum (DSM 2672),
Humicola insolens, Trichoderma resii, Myrothecium verrucaria
(IFO 6113), Verticillum alboatrum, Verticillum dahlie,
Arthromyces ramosus (FERM P-7754), Caldariomyces fumago,
35 Ulocladium chartarum, Embellisia alli or Dreschlera halodes.
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Other preferred fungi include strains belonging to the
subdivision Basidiomycotina, class Basidiomycetes, e.g.,
Coprinus, Phanerochaete, Coriolus or Trametes, in particular
Coprinus cinereus f, microsporus (IFO 8371), Coprinus
s macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or
Trametes (previously called Polyporus), e.g., T. versicolor
(e.g. PR4 28-A).
Further preferred fungi include strains belonging to the
subdivision Zygomycotina, class Mycoraceae, e.g., Rhizopus or
to Mucor, in particular Mucor hiemalis.
Some preferred bacteria include strains of the order
Actinomycetales, e.g. Streptomyces spheroides (ATTC 23965),
Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum
verticillium ssp. verticillium.
is Other preferred bacteria include Bacillus pumilus (ATCC
12905), Bacillus stearothermophilus, Rhodo~bacter sphaeroides,
Rhodomonas palustri, Streptococcus lactis, Pseudomonas
purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11).
Further preferred bacteria include strains belonging to
2o Myxococcus, e.g. , M. virescens.
The peroxidase may furthermore be one which is producible
by a method comprising cultivating a host cell transformed with
a recombinant DNA vector which carries a DNA sequence encoding
said peroxidase as well as DNA sequences encoding functions
z5 permitting the expression of the DNA sequence encoding the
peroxidase, in a culture medium under conditions permitting the
expression of the peroxidase and recovering the peroxidase from
the culture.
Particularly, a recombinantly produced peroxidase is a
3o peroxidase derived from a Coprinus sp., in particular C.
macrorhizus or C. cinereus according to w0 92/16634.
Haloperoxidases such as chromo-, bromo- and/or
iodoperoxidases are particularly suitable and preferred for
incorporation in animal litter compositions. Haloperoxidases
3s form a class of enzymes which are able to oxidize halides (C1-,
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Br-, I-) in the presence of hydrogen peroxide or a hydrogen
peroxide generating system to the corresponding hypohalous acids
according to:
s HzOz + X- + H+ - > H20 + HOX
If a convenient nucleophilic acceptor is present, a
reaction will occur with HOX and a halogenated compound will be
formed.
to There are three types of haloperoxidases, classified
according to their specificity for halide ions:
chloroperoxidases (E.C. 1.11.1.10) which catalyse formation of
hypo-chlorit from chloride ions, hypo-bromit from bromide ions
and hypo-iodit from iodide ions; bromoperoxidases which catalyse
is formation of hypo-bromit from bromide ions and hypo-iodit from
iodide ions; and iodoperoxidases (E. C. 1.11.1.8) which solely
catalyze the formation of hypoiodit from iodide ions. However,
hypoiodit undergoes spontaneous disproportionation to iodine and
thus, iodine is usually the observed product of the reaction.
zo These hypo-halit compounds may subsequently react with other
compounds forming halogenated compounds. Haloperoxidases have
been isolated from various organisms: mammals, marine animals,
plants, algae, a lichen, fungi and bacteria (for reference see
Biochimica et Biophysica Acta 1161, 1993, pp. 249-256). It is
zs generally accepted that haloperoxidases are the enzymes
responsible for the formation of halogenated compounds in
nature, although other enzymes may be involved.
Haloperoxidases have been isolated from many different
fungi, in particular from the fungus group dematiaceous hypho
3o mycetes, such as Caldariomyces, e.g., C. fumago, Alternaria,
Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera,
Ulocladium and Botrytis (see US Patent No. 4,937,192).
According to the present invention a haloperoxidase
obtainable from Curvularia, in particular C. verruculosa is
preferred such as C. verruculosa CBS 147.63 or C. verruculosa
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CBS 444.70. Curvularia haloperoxidase and recombinant production
hereof is described in WO 97/04102.
Haloperoxidases have also been isolated from bacteria such
as Pseudomonas, e.g., P. pyrrocinia (for reference see The
Journal of Biological Chemistry 263, 1988, pp. 13725-13732) and
Streptomyces, e.g., S. aureofaciens (for reference see Struc-
tural Biology 1, 1994, pp. 532-537).
Bromide peroxidase has been isolated from algae (see US
Patent No. 4,937,192).
to In a preferred embodiment the haloperoxidase is derivable
from Curvularia sp., in particular C. verruculosa and C.
inaequalis.
In a preferred embodiment the haloperoxidase is a
vanadium haloperoxidase derivable from a strain of Curvularia
is inaequalis such C. inaequalis CBS 102.42 as described in WO
95/27046, e.g. a vanadium haloperoxidase encoded by the DNA
sequence of WO 95/27046, figure 2 all incorporated by
reference.
In another preferred embodiment the haloperoxidase is a
zo vanadium haloperoxidase derivable from a strain selected from
Drechslera hartlebii, Dendryphiella salina, Phaeotrichoconis
crotalarie and Geniculosporium sp. The vanadium haloperoxidase
is more preferably derivable from Drechslera hartlebii (DSM
Acc. No. DSM 13444), Dendryphiella salina (DSM Acc. No. DSM
2s 13443), Phaeotrichoconis crotalarie (DSM Acc. No. DSM 13441)
and Geniculosporium sp. (DSM Acc. No. DSM 13442) such as
described in the co-pending Danish patent applications PA2000
00628, PA 2000 00627, PA2000 00625 and PA2000 00626 all
incorporated by reference.
Determination of Peroxidase Activity (POXU)
One peroxidase unit (POXU) is the amount of enzyme which
under the following conditions catalyze the conversion of 1
.mole hydrogen peroxide per minute:
0.1 M phosphate buffer pH 7.0, 0.88 mM hydrogen peroxide, 1.67
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27
mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS)
and 3 0°C .
The reaction is followed for 60 seconds (15 seconds after
mixing) by the change in absorbance at 418 nm, which should be
s in the range 0.15 to 0.30.
For calculation of activity is used an absorption
coefficient of oxidized ABTS of 36 mM-1 cm-1 and a stoichiometry
of one .mole Hz02 converted per two mole ABTS oxidized.
to A suitable amount of oxidoreductase to be incorporated in
an animal litter composition will generally depend on the
oxidoreductase, but typically an amount between about 0.01 to
about 1000 mg enzyme protein per kg composition, preferably
0.1-100 mg/kg, e.g. 0.1-50 mg/kg or 0.2-10 mg/kg will be
is suitable.
The action, e.g. antimicrobial action, oxidation etc., of an
oxidoreductase requires the presence of an oxidising agent
which acts as an electron donor. A suitable choice of oxidizing
zo agent depends on the type of oxidoreductase. If the
oxidoreductase is a laccase or a laccase related enzyme the
oxidizing agent may be molecular oxygen available from the
atmosphere. If the oxidoreductase is a peroxidase or a compound
having peroxidase activity the oxidizing agent is suitably a
z5 peroxo compound in particular hydrogen peroxide or a source of
hydrogen peroxide (a hydrogen peroxide precursor) which provide
for in situ production of hydrogen peroxide, e.g., percarbonate
or perborate compounds or a peroxycarboxylic acid or a salt
thereof, or it may be a hydrogen peroxide generating enzyme
3o system, such as an oxidase and its substrate. Useful oxidases
may be, a glucose oxidase, a glycerol oxidase or an amino acid
oxidase. An example of an amino acid oxidase is given in V~10
94/25574. It may be advantageous to use enzymatically generated
hydrogen peroxide, since this source results in a relatively
3s low concentration of hydrogen peroxide under the biologically
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28
relevant conditions. Low concentrations of hydrogen peroxide
result in an increase in the rate of peroxidase-catalysed
reaction. The oxidizing agent in this case may suitably be
present in the animal litter composition in an amount
s corresponding to levels from 0.001-500 millimole/kg, particu-
larly to levels from 0.01-100 millimole/kg such as 0.01-25
millimole/kg.
Other useful components which may be included in the animal
to litter compositions to further increase the action of the
oxidoreductase are enhancers or oxidoreductase enhancing
agents. It is believed that these compounds act as intermediate
electron acceptors which may be oxidized by the oxidising agent
to form a reactive radical in a reaction catalysed by the
is oxidoreductase and that this reactive radical have potent
antimicrobial properties. Accordingly enhancers which may be
used in the animal litter compositions to enhance the
antimicrobial effect include organic enhancers and inorganic
enhancers. Various organic enhancers acting as electron donors
ao for oxidoreductases for various purposes are known to the art
(e.g. from WO 94/12620, WO 94/12621, WO 95/01626 and WO
96/00179) and may suitably be employed in accordance with this
invention.
One group of preferred organic enhancers is phenolic
z5 compounds (alkylsyringates) of the formula:
Formula I
OB
A ~ ~ OH
OC
wherein the letter A in said formula denotes be a group such as
30 -D, -CH=CH-D, -CH=CH-CH=CH-D, -CH=N-D, -N=N-D, or -N=CH-D, in
which D is selected from the group consisting of -CO-E, -SOZ-E,
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29
-N-XY, and -N'-XYZ, in which E may be -H, -OH, -R, or -OR, and
X and Y and Z may be identical or different and selected from -
H and -R; R being a C1-C16 alkyl, preferably a C1-Ce alkyl, which
alkyl may be saturated or unsaturated, branched or unbranched
and optionally substituted with a carboxy, sulpho or amino
group; and B and C may be the same or different and selected
f rom CmHzm+1, where m = 1 , 2 , 3 , 4 or 5 .
In the above mentioned formula A may be placed meta to
the hydroxy group instead of being placed in the para-position
to as shown.
In particular embodiments of the invention the enhancer
is selected from the group having the formula:
Formula II
OMe
A
OH
O
OMe
in which A is a group such as -H, -OH, -CH3, -OCH3, -O (CHZ) "CH3,
where n = 1, 2, 3, 4, 5, 6, 7 or 8.
2o Such enhancers may suitably be present in the animal
litter composition in amount between 0.00001-500 millimole/kg,
preferably 0.0001-5 millimole/kg, e.g. 0.001-0.050
millimole/kg.
z5 Another preferred group of well performing organic
enhancers comprises a -CO-NOH- group and have the following
formula:
Formula III
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0\\
TB
A-N
\0 H
in which A is:
s Formula IV
R3 R2
R4
R5 ~R6
and B is the same as A, or B is H, or C1-C16 branched or
unbranched alkyl wherein said alkyl may contain hydroxy, ether
to or ester groups, and R2, R3, R4, R5 and R6 are H, OH, NH2,
COOH, S03H, C1-C12 branched or unbranched alkyl, aryl, N02, CN,
C1, CF3, NOH-CO-phenyl, Cl-C6-CO-NOH-A, CO-NOH-A, COR12,
phenyl-CO-NOH-A, OR7, NR8R9, COOR10, or NOH-CO-R11, wherein R7,
R8, R9, R10 and R11 are C1-C12 branched or unbranched alkyl or
is acyl. Within this group of enhancers particularly preferred
enhancers are selected from the group consisting of
4-nitrobenzoic acid-N-hydroxyanilide;
4-methoxybenzoic acid-N-hydroxyanilide;
N,N'-dihydroxy-N, N'-diphenylterephthalamide;
2o decanoic acid-N-hydroxyanilide;
N-hydroxy-4-cyanoacetanilide;
N-.hydroxy-4-acetylacetanilide;
N-hydroxy-4-hydroxyacetanilide;
N-hydroxy-3-(N'-hydroxyacetamide)acetanilide;
zs 4-cyanobenzoic acid-N-hydroxyanilide;
N-hydroxy-4-nitroacetanilide; and
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31
N-hydroxyacetanilide.
These enhancers may suitably be present in the animal
litter composition in concentrations from 1 to 1000
micromole/kg, preferably from 5 to 500 micromole/kg.
The enhancer may also be one of the compounds disclosed in
WO 96/18770 such as N-hydroxy compounds, in particular
aliphatic, cycloaliphatic, heterocyclic or aromatic compounds
containing NO-, N(OH) -, or N(OH) (R1) , especially N-hydroxy
benzotriazol (HOBT), Violuric acid, or N-hydroxyacetanilide
to (HAA) .
In a preferred embodiment of the invention the enhancer is
a compound of the general formula (V):
Formula V
R1
R2
/ X]
R3
H
R4
wherein R1, R2, R3, R4 are individually selected from the group
consisting of hydrogen, halogen, hydroxy, formyl, carboxy and
salts and esters thereof, amino, nitro, C1-Clz alkyl, C1-C6
2o alkoxy, carbonyl(C1-C1z alkyl), aryl, in particular phenyl,
sulpho, aminosulfonyl, carbamoyl, phosphono, phosphonooxy, and
salts and esters thereof , wherein the R1, R2, R3, R4 may be
substituted with R5, wherein RS represents hydrogen, halogen,
hydroxy, formyl, carboxy and salts and esters thereof, amino,
nitro, C1-C12 alkyl, C1-C6 alkoxy, carbonyl (C1-C12 alkyl) , aryl,
in particular phenyl, sulpho, aminosulfonyl, carbamoyl,
phosphono, phosphonooxy, and salts and esters thereof, [X]
represents a group selected from (-N=N-) , (-N=CR6-)m, (-CR6=N-
mi ( -CR6=CR'- ) m, ( -CR6=N-NR'- ) , ( -N=N-CHR6- ) , ( -N=CR6-NR'- ) , ( -
3o N=CR6-CHR'-) , (-CR6=N-CHR'-) , (-CR6=CR'-NRe-) , and (-CR6=CR'-CHRB-
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32
), wherein R6, R', and R8 independently of each other are
selected from H, OH, NHz, COOH, S03H, C1_6-alkyl, NOz, CN, C1,
Br, F, CH20CHj, OCH3, COOCH3; and m is 1 or 2.
In a more preferred embodiment of the invention the
s enhancer is a compound of the general formula (VI):
Formula VI
R1
R2
R3
4 ~OH
wherein R1, RZ, R3, R4 are individually selected from the group
to consisting of hydrogen, halogen, hydroxy, formyl, carboxy and
salts and esters thereof, amino, nitro, C1-C12 alkyl, C1-C6
alkoxy, carbonyl(C1-C12 alkyl), aryl, in particular phenyl,
sulpho, aminosulfonyl, carbamoyl, phosphono, phosphonooxy, and
salts and esters thereof, wherein the R1, R2, R3, R4 may be
15 substituted with R5, wherein RS represents hydrogen, halogen,
hydroxy, formyl, carboxy and salts and esters thereof, amino,
nitro, C1-C12 alkyl , C1-C6 alkoxy, carbonyl (C1-C12 alkyl ) , aryl ,
in particular phenyl, sulpho, aminosulfonyl, carbamoyl,
phosphono, phosphonooxy, and salts and esters thereof.
2o The enhancer may also be a salt or an ester of formula V
or VI.
Further preferred enhancers are oxoderivatives and N-
hydroxy derivatives of heterocyclic compounds and oximes of
oxo- and formyl-derivatives of heterocyclic compounds, said
25 heterocyclic compounds including five-membered nitrogen-
containing heterocycles, in particular pyrrol, pyrazole and
imidazole and their hydrogenated counterparts (e. g.
pyrrolidine) as well as triazoles, such as 1,2,4-triazole; six-
membered nitrogen-containing heterocycles, in particular mono-,
3o di- and triazinanes (such as piperidine and piperazine),
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33
morpholine and their unsaturated counterparts (e. g. pyridine
and pyrimidine); and condensed heterocycles containing the
above heterocycles as substructures, e.g. indole,
benzothiazole, quinoline and benzoazepine.
s Examples of preferred enhancers from these classes of
compounds are pyridine aldoximes; N-hydroxypyrrolidinediones
such as N-hydroxysuccinimide and N-hydroxyphthalimide; 3,4
dihydro-3-hydroxybenzo[1,2,3]triazine-4-one; formaldoxime
trimer (N,N',N " -trihydroxy-1,3,5-triazinane); and violuric
to acid (1,3-diazinane-2,4,5,6-tetrone-5-oxime).
Still further enhancers which may be applied in the
invention include oximes of oxo- and formyl-derivatives of
aromatic compounds, such as benzoquinone dioxime and
salicylaldoxime (2-hydroxybenzaldehyde oxime), and N-
ls hydroxyamides and N-hydroxyanilides, such as N-
hydroxyacetanilide.
Preferred enhancers are selected from the group
consisting of 1-hydroxybenzotriazole; 1-hydroxybenzotriazole
hydrate; 1-hydroxybenzotriazole sodium salt; 1-
2o hydroxybenzotriazole potassium salt; 1-hydroxybenzotriazole
lithium salt; 1-hydroxybenzotriazole ammonium salt; 1-
hydroxybenzotriazole calcium salt; 1-hydroxybenzotriazole
magnesium salt; and 1-hydroxybenzotriazole-6-sulphonic acid.
A particularly preferred enhancer is 1-
z5 hydroxybenzotriazole.
All the specifications of N-hydroxy compounds above are
understood to include tautomeric forms such as N-oxides
whenever relevant.
In particular, the enhancer of the invention may be the
3o corresponding N-oxyl free radical to any of the compounds
disclosed in WO 96/18770 such as TEMPO (2,2,6,6
tetramethylpiperidinoxyl).
These organic enhancers may suitably be present in the
litter composition in concentrations from 1 to 1000
35 micromole/kg, preferably from 5 to 500 micromole/kg.
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34
Inorganic enhancers may also be relevant. Especially when
using haloperoxidases for animal litter compositions presence
of inorganic halide ions such as chloride, bromide and/or
iodide may enhance the antimicrobial effect of the
s haloperoxidase. Suitable ranges of chloride ions are 0.05 - 500
millimole/kg and suitable ranges of bromide and/or iodide ions
are 0.01 - 100 millimole/kg.
We have further observed that an improved anti-
io microbial or preservation effect may be obtained using an
ammonium enhancer, preferably in combination with a halide
enhancer or an organic enhancer. The ammonium enhancer may be
compounds of the formula:
is Formula VII
R1
HN
R2
wherein the substituent groups R1 and R2 may be identical or
different. R1 and R2 may suitably be any of the following
groups: hydrogen, halide, sulphate, phenyl, a straight or
2o branched chain alkyl having from 1 to 14 carbon atoms, or a
substituted straight or branched alkyl group having from 1 to 14
carbon atoms where the substituent group is located at C1-C14 and
represent any of the following radicals: hydroxy, halogen,
formyl, carboxy, carboxy esters, carboxy salts, carbamoyl,
z5 sulfo, sulfo esters, sulfo salts, sulfamoyl, nitro, amino,
phenyl, C1-CS-alkoxy, carbonyl-C1-CS-alkyl, aryl-C1-CS-alkyl.
Where R1 and/or R2 includes groups selected from carbamoyl,
sulfamoyl, and amino groups these groups may furthermore be un-
substituted or substituted once or twice with a substituent
3o group R3, Where R1 and/or R2 includes a phenyl group it may
furthermore be unsubstituted or substituted with one or more
substituent groups R3. Where R1 and/or R2 includes groups
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selected from C1-CS-alkoxy, carbonyl-C1-CS-alkyl, and aryl-C1-CS-
alkyl these groups may be saturated or unsaturated, branched or
unbranched, and may furthermore be unsubstituted or substituted
with one or more substituent groups R3. R3 represents any of
s the following groups: halogen, hydroxy, formyl, carboxy,
carboxy esters, carboxy salts, carbamoyl, sulfo, sulfo esters,
sulfo salts, sulfamoyl, nitro, amino, phenyl, aminoalkyl,
piperidino, piperazinyl, pyrrolidin-1-yl, C1-CS-alkyl, C1-CS-
alkoxy. Where R3 includes groups selected from carbamoyl,
to sulfamoyl, and amino these groups may furthermore be
unsubstituted or substituted once or twice with hydroxy, C1-CS-
alkyl, C1-CS-alkoxy. Where R3 includes phenyl this group may
furthermore be substituted with one or more of the following
groups: halogen, hydroxy, amino, formyl, carboxy, carboxy
is esters, carboxy salts, carbamoyl, sulfo, sulfo esters, sulfo
salts, and sulfamoyl. Where R3 includes groups selected from C1-
CS-alkyl, and C1-CS-alkoxy these groups may furthermore be
saturated or unsaturated, branched or unbranched, and may
furthermore be substituted once or twice with any of the
2o following radicals: halogen, hydroxy, amino, formyl, carboxy,
carboxy esters, carboxy salts, carbamoyl, sulfo, sulfo esters,
sulfo salts, and sulfamoyl. R1 and R2 may also suitably
together a group -B-, in which B represents any of the
following groups: (-CHR3-N=N-), (-CH=CH-)n or (-CH=N-)n in
as which groups n-represents an integer of from 1 to 3 and R3 is a
substituent group as defined, supra. (It is to be understood
that if the above mentioned formula comprises two or more R3-
substituent groups, these R3-substituent groups may be the same
or different).
3o As used herein, the ammonium enhancer may be in their
cationic form.
In a preferred embodiment R1 is hydrogen.
In another preferred embodiment R1 is hydrogen and R2
is an alcohol (amino alcohol), e.g., ethanol amine.
35 In a further preferred embodiment the ammonium
enhancer is an ammonium salt, i . a . any ammonium salt known in
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36
the art: e.g., diammonium sulphate, ammonium chloride, ammonium
bromide, or ammonium iodide.
The ammonium enhancer may suitably be present in the
litter composition of the invention in a concentration
s corresponding to an ammonium concentration in the range of from
0.01-1000 millimole/kg, preferably in the range of from 0.05-500
millimole/kg.
Other enzymes
to Conventionally other enzymes have been disclosed as desirable
components for the purpose of cleaning and for destroying
animal excretions, including regurgitated. Enzymes other than
ureases can be used to destroy the soils, such as hydrolase
and/or a combination of hydrolases. In a preferred embodiment,
15 the hydrolase to be used is selected from the group consisting
of glucosidases, i.e. cellulases (endoglucanases,
cellobiohydrolases, ~i-glucosidases), hemicellulases (xylanases,
mannanases, xylan acetyl esterases)), pectinases (arabinanases,
a-arabino-furanosidases, galactanases, pectin lyases, pectin
zo methyl esterases, polygalacturonases, rhamnogalacturonan acetyl
esterases, rhamnogalacturonases), amylases; proteases, and
lipases.
The hydrolases to be used may be selected according to the
z5 properties or a combination of several hydrolases having
different enzyme activities may be used.
Nonlimiting examples of specific enzymes useful in animal
litter compositions are: 1,2-1,3-a-D-mannan mannohydrolase,
30 1,3-(3-D-xylan xylanohydrolase, 1,3-(3-D-glucan glucanohydrolase,
1,3(1,3;1,4)-a-D-glucan 3-glucanohydrolase, 1,3(1,3;1,4)-(3-D-
glucan 3(4)-glucanohydrolase, 1,3-1,4-a-D-glucan 4-
glucanohydrolase, 1,4-a-D-glucan glucanehydrolase, 1,4-a-D-
glucan glucohydrolase, 1,4-(1,3:1,4)-(3-D-glucan 4-
3s glucanohydrolase, 1,4-(3-D-glucan glucohydrolase, 1,4-(3-D-xylan
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37
xylanohydrolase, 1,4-(3-D-mannan mannanohydrolase, 1,5-a-L-
arabinan 1,5-a-L-arabinanohydrolase, 1,4-a-D-glucan
maltohydrolase, 1,6-a-D-glucan 6-glucanohydrolase, 2,6-~3-D-
fructan fructanohydrolase, a-Dextrin 6-glucanohydrolase, a-D-
galactoside galactohydrolase, a-D-glucoside glucohydrolase, a-
D-mannoside mannohydrolase, acylneuraminyl hydrolase,
Aerobacter-capsular-polysaccharide galactohydrolase,
(3-D-fructofuranoside fructohydrolase, (3-D-fucoside
fucohydrolase, (3-D-fructan fructohydrolase,
to (3-D-galactoside galactohydrolase, (3-D-glucoside glucohydrolase,
(3-D-glucuronoside, glucuronosohydrolase, (3-D-mannoside
mannohydrolase, ~3-N-acetyl-D-hexosaminide N-acetylhexosamino
hydrolase, cellulose-sulfate sulfohydrolase, collagenase,
dextrin 6-a-D-glucanohydrolase, glycoprotein-
phosphatidylinositol phosphatidohydrolase, hyaluronate 4-
glycanohydrolase, hyaluronoglucuronidase, pectin
pectylhydrolase, peptidoglycan N-acetylmuramoylhydrolase,
phosphatidylcholine 2-acylhydrolase, phosphatidylcholine 1-
acylhydrolase, poly(1,4-a-D-galacturonide), poly(1,4-(N-
2o acetyl-(3-D-glucosaminide))-glycanohydrolase, proteases,
sucrose a-glucosidase, triacylglycerol acylhydrolase,
triacylglycerol protein-acylhydrolase.
A particularly useful hydrolytic enzyme for incorporation
z5 in an animal litter composition is any enzyme having
proteolytic activity. Thus, the enzyme may be a proteolytic
enzyme of plant origin, e.g. papain, bromelain, ficin, or of
animal origin, e.g. trypsine and chymotrypsine, or of microbial
origin, i.e. bacterial or fungal origin or from yeasts. It is
3o to be understood that any mixture of various proteolytic enzyme
may be applicable in an animal litter composition.
In a preferred embodiment of the invention, the proteoly-
tic enzyme is a serine-protease, a metallo-protease, or an
aspartate-protease. A serine protease is an enzyme which
3s catalyzes the hydrolysis of peptide bonds, and in which there
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38
is an essential serine residue at the active site. They are
inhibited by diisopropylfluorophosphate, but in contrast to
metalloproteases, are resistant to ethylene diamino tetraacetic
acid (EDTA) (although they are stabilized at high temperatures
s by calcium ions). They hydrolyze simple terminal esters and are
similar in activity to eukaryotic chymotrypsin, also a serine
protease. A more narrow term, alkaline protease, covering a
sub-group, reflects the high pH optimum of some of the serine
proteases, from pH 9.0 to 11.0 The serine proteases usually
to exhibit maximum proteolytic activity in the alkaline pH range,
whereas the metallo-proteases and the aspartate-proteases
usually exhibit maximum proteolytic activity in the neutral and
the acidic pH range, respectively.
A sub-group of the serine proteases are commonly desig-
15 nated as subtilisins. A subtilisin is a serine protease
produced by Gram-positive bacteria or fungi. The amino acid
sequence of a number of subtilisins have been determined,
including at least six subtilisins from Bacillus strains,
namely, subtilisin 168, subtilisin BPN, subtilisin Carlsberg,
2o subtilisin DY, subtilisin amylosacchariticus, and
mesentericopeptidase, one subtilisin from an actinomycetales,
thermitase from Thermoactinomyces vulgaris, and one fungal
subtilisin, proteinase K from Tritirachium album. A further
subgroup of the subtilisins, subtilases, have been recognised
2s more recently. Subtilases are described as highly alkaline
subtilisins and comprise enzymes such as subtilisin PB92
(MAXACAL~, Gist-Brocades NV), subtilisin 309(SAVINASEm, NOVO
NORDISK A/S), and subtilisin 147 (ESPERASE~, NOVO NORDISK A/S).
In the context of this invention, a subtilisin variant or
3o mutated subtilisin protease means a subtilisin that has been
produced by an organism which is expressing a mutant gene
derived from a parent microorganism which possessed an original
or parent gene and which produced a corresponding parent
enzyme, the parent gene having been mutated in order to produce
3s the mutant gene from which said mutated subtilisin protease is
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39
produced when expressed in a suitable host. These mentioned
subtilisins and variants thereof constitute a preferred class
of proteases which are useful in the method of the invention.
An example of a useful subtilisin variant is a variant of
s subtilisin 309 (SAVINASE~) wherein, in position 195, glycine is
substituted by phenylalanine (G195F or l9sGly to 195Phe) .
Conveniently, conventional fermented commercial proteases
are useful. Examples of such commercial proteases are Alcalase~
(produced by submerged fermentation of a strain of Bacillus li
to cheniformis), Esperasem (produced by submerged fermentation of
an alkalophilic species of Bacillus), Rennilase~ (produced by
submerged fermentation of a non-pathogenic strain of Mucor mie-
hei), Savinase~ (produced by submerged fermentation of a gen-
etically modified strain of Bacillus), e.g. the variants
is disclosed in the International Patent Application published as
WO 92/19729, and Durazyrri (a protein-engineered variant of Sa-
vinase~). Also Everlase° and Kannase~ are useful. All the men-
tioned commercial proteases are produced and sold by Novo Nor-
disk A/S, DK-2880 Bagsvaerd, Denmark. Further useful commercial
2o proteases are MAXATASE~ from International Bio-Synthetics, Inc.
(The Netherlands)and proteases made by Genencor International,
Inc., according to one or more ofthe following patents:
Caldwell et al, U.S. Patent Nos. 5,185,258, 5,204,015 and
5,244,791, e.g. Properase~.
25 Other preferred serine-proteases are proteases from
Nocardiopsis, Aspergillus, Rhizopus, Bacillus alcalophilus, B.
cereus, N. natto, B. vulgatus, B. mycoide, and subtilins from
Bacillus, especially proteases from the species Nocardiopsis
sp. and Nocardiopsis dassonvillei such as those disclosed in
3o the International Patent Application published as WO 88/03947,
especially proteases from the species Nocardiopsis sp., NRRL
18262, and Nocardiopsis dassonvillei, NRRL 18133. Yet other
preferred proteases are the serine proteases from mutants of
Bacillus subtilins disclosed in the International Patent
35 Application No. PCT/DK89/00002 and in the International Patent
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Application published as WO 91/00345, and the proteases
disclosed in EP 415 296.
Another preferred class of proteases are the metallo-pro-
teases of microbial origin. Conveniently, conventional fer-
5 mented commercial proteases are useful. Examples of such a
commercial protease is Neutrase~ (Zn) (produced by submerged
fermentation of a strain of Bacillus subtilis), which is
produced and sold by Novo Nordisk A/S, DK-2880 Bagsvaerd,
Denmark.
to Other useful commercial protease enzyme preparation are
Bactosol~ WO and Bactosol~SI, available from Sandoz AG, Basle,
Switzerland; Toyozyme°, available from Toyo Boseki Co. Ltd.,
Japan; and Proteinase K~ (produced by submerged fermentation of
a strain of Bacillus sp. KSM-K16), available from Kao
is Corporation Ltd., Japan.
Still other proteases include Protease A (see European
Patent Application 130,756, published January 9, 1985);
Protease B (see European Patent Application Serial No.
87303761.8, filed April 28, 1987, and European Patent
zo Application 130,756, Bott et al, published January 9, 1985)
Another hydrolytic enzyme which may be useful for
incorporation in animal litter is a microbial lipase. As such,
the lipase may be selected from yeast, e.g. Candida, lipases,
25 bacterial, e.g. Pseudomonas or Bacillus, lipases; or fungal,
e.g. Humicola or Rhizomucor, lipases. More specifically,
suitable lipases may be the Rhizomucor miehei lipase (e. g.
prepared as described in EP 238 023), Thermomyces lanuginosa
lipase e.g. prepared as described in EP 305 216 (available from
3o Novo Nordisk under the trade name LipolaseTM), Humicola
insolens lipase, Pseudomonas stutzeri (eg. ATCC 19.154) lipase,
Pseudomonas cepacia lipase, Candida antarctica lipase A or B,
or lipases from rGPL, Absidia blakesleena, Absidia corymbifera,
Fusarium solani, Fusarium oxysporum, Penicillum cyclopium,
35 Penicillum crustosum, Penicillum expansum, Rhodotorula
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41
glutinis, Thiarosporella phaseolina, Rhizopus microsporus,
Sporobolomyces shibatanus, Aureobasidium pu11u1ans, Hansenula
anomala, Geotricum penicillatum, Lactobacillus curvatus,
Brochothrix thermosohata, Coprinus cinerius, Trichoderma
s harzanium, Trichoderma reesei, Rhizopus japonicas or
Pseudomonas plantari. Other examples of suitable lipases may be
variants of any one of the lipases mentioned above, e.g. as
described in WO 92/05249 or WO 93/11254. Also suitable lipase
enzymes for usage herein include those described in Japanese
to Patent Application 53,20487, laid open to public inspection on
February 24, 1978. This lipase is available from Amano
Pharmaceutical Co. Ltd., Nagoya, Japan, under the trade name
Lipase P "Amano," herinafter referred to as "Amano-P." Other
commercial lipases include Amano-CES, lipases ex Chromobacter
is viscosum, e.g. Chromobacter viscosum var. lipolyticum NRRLB
3673, commercially available from Toyo Jozo Co., Tagata, Japan;
and further Chromobacter viscosum lipases from U.S. Biochemical
Corp., U.S.A. and Disoynth Co., The Netherlands, and lipases ex
Pseudomonas gladioli.
2o Nonlimiting examples of amylases useful for incorporation
in animal litter include Bacillus amylases, e.g. Bacillus
stearothermophilus amylase, Bacillus amyloliquefaciens amylase,
Bacillus subtilis amylase or Bacillus licheniformis amylase
(e. g. as available from Novo Nordisk under the trade name
z5 Termamyl°), or Aspergillus amylases, e.g. Aspergillus niger or
Aspergillus oryzae amylase. Other examples of suitable amylases
may be variants of any one of the amylases mentioned above,
e.g. as described in US 5,093,257, EP 252 666, WO 91/00353, FR
2,676,456, EP 285 123, EP 525 610, PCT/DK93/00230. Also the
3o amylase RAPIDASE~, Novo Industries may be suitable.
Another useful hydrolytic enzyme is a "cellulase" or
"cellulolytic enzyme" which refers to an enzyme which catalyses
the degradation of cellulose to glucose, cellobiose, triose and
other cello-oligosaccharides. Preferably, the cellulase is an -
35 endoglucanase, more preferably a microbial endoglucanase,
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42
especially a bacterial or fungal endoglucanase. Examples of
bacterial endoglucanases are endoglucanases derived from or
producible by bacteria from the group of genera consisting of
Pseudomonas or Bacillus lautus.
s The cellulase or endoglucanase may be an acid, a neutral
of an alkaline cellulase or endoglucanase, i.e. exhibiting
maximum cellulolytic activity in the acid, neutral of alkaline
range, respectively. Accordingly, a useful cellulase or
endoglucanase is an acid cellulase or endoglucanase, preferably
to a fungal acid cellulase or endoglucanase, more preferably a
fungal acid cellulase or endoglucanse enzyme with substantial
cellulolytic activity at acidic conditions which is derived
from or producible by fungi from the group of genera consisting
of Trichoderma, Actinomyces, Myrothecium, Aspergillus, and
is Botrytis.
A preferred useful acid cellulase or endoglucanase is
derived from or producible by fungi from the group of species
consisting of Trichoderma viride, Trichoderma reesei,
Trichoderma longibrachiatum, Myrothecium verrucaria, Asper-
2o gillus niger, Aspergillus oryzae, and Botrytis cinerea.
Another useful cellulase or endoglucanase is a neutral or
alkaline cellulase or endoglucanse, preferably a fungal neutral
or alkaline cellulase or endoglucanse, more preferably a fungal
alkaline cellulase or endoglucanase with substantial
as cellulolytic activity at alkaline conditions which is derived
from or producible by fungi from the group of genera consisting
of Aspergillus, Penicillium, Myceliophthora, Humicola, Irpex,
Fusarium, Stachybotrys, Scopulariopsis, Chaetomium, Mycogone,
Verticillium, Myrothecium, Papulospora, Gliocladium, Cephalo-
3o sporium and Acremonium.
A preferred alkaline cellulase or endoglucanase is
derived from or producible by fungi from the group of species
consisting of Humicola insolens, Fusarium oxysporum, Myce-
liopthora thermophile, or Cephalosporium sp., preferably from
3s the group of species consisting of Humicola insolens, DSM 1800,
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43
Fusarium oxysporum, DSM 2672, Myceliopthora thermophila, CBS
117.65, or Cephalosporium sp., RYM-202.
Examples of xylanases useful in the method of the present
invention include enzymes having xylanolytic activity which are
s produced or producible by a strain selected from the group of
species consisting of Humicola insolens (see e.g. WO 92/17573),
Aspergillus aculeatus (an enzyme exhibiting xylanase activity,
which enzyme is immunologically reactive with an antibody
raised against a purified xylanase derived from Aspergillus
to aculeatus, CBS 101.43, see e.g. WO 94/21785), Bacillus pumilus
(see e.g. WO 92/03540), Bacillus stearathermophilus (see e.g.
WO 91/18976, WO 91/10724), Bacillus sp. AC13 (especially the
strain NCIMB 40482, see e.g. WO 94/01532), Trichoderma
longibrachiatum and Chainia sp. (see e.g. EP 0 353 342 A1),
is Thermoascus aurantiacus (see e.g. US patent 4,966,850),
Trichoderma harzianum and Trichoderma reseei (see e.g. US
patent 4,725,544), Aureobasidium pullulans (see e.g. EP 0 373
107 A2), Thermomyces lanuginosus (see e.g. EP 0 456 033 A2),
Bacillus circulans (WO 91/18978), Aspergillus oryzae (see e.g.
2o SU 4610007), Thermomonospora fusca (see e.g. EP 0 473 545 A2),
Streptomyces lividans (see e.g. WO 93/03155), Streptomyces
viridosporus (see e.g. EP 496 671 A1), Bacillus licheniformis
(see e.g. JP 9213868) and Trichoderma longibrachiatum [see
W.J.J. van den Tweel et al.(Eds.), "Stability of
z5 Enzymes",Proceedings of an International Symposium heeld in
Maastricht, The Netherlands, 22-25 November 1992, Fisk,R.S. and
Simpson, pp.323-328]; or from the group of genera consisting of
Thermotoga (see e.g. WO 93/19171), Rhodothermus (see e.g. WO
93/08275), Dictyoglomus (see e.g. WO 92/18612) and Streptomyces
30 (see e.g. US patent 5,116,746). Other examples of suitable
xylanases may be variants (derivatives or homologues) of any
one of the above-mentioned enzymes having xylanolytic activty.
A useful pectinase may be an enzyme belonging to the
enzyme classes polygalacturonases (EC3.2.1.15), pectinesterases
35 (EC3.2.1.11), pectin lyases (EC4.2.2.10) and hemicellulases
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44
such as endo-1,3-b-xylosidase (EC 3.2.1.32), xylan 1,4-b-
xylosidase (EC 3.2.1.37) and a-L-arabinofuranosidase (EC
3.2.1.55). A suitable source organism for pectinases may be
Aspergillus niger.
In a preferred embodiment, the hydrolase enzymes) is/are
produced by a strain of the fungus Aspergillus aculeatus,
preferably Aspergillus aculeatus, CBS 101.43. It is known that
this strain produces an enzyme composition comprising
pectolytic and a range of hemicellulolytic enzyme activities.
to The hydrolase(s) are present in the cleaning composition
in an amount from about 0.01 to about 5000 ~g protein/g of
composition, preferably from about 1 to about 500 ~.g protein/g
of animal litter composition.
Enzyme-polyethylene glycol conjugates are also preferred. Such
polyethylene glycol (PEG) derivatives of enzymes, wherein the
PEG or alkoxy-PEG moieties are coupled to the protein molecule
through, e.g., secondary amine linkages. Suitable
derivatization decreases immunogenicity, thus minimizes
2o allergic reactions, while still maintains some enzymatic
activity. An example of protease-PEG's is PEG-subtilisin
Carlsberg from B. lichenniformis coupled to methoxy-PEGs
through secondary amine linkage, and is available from Sigma-
Aldrich Corp., St. Louis, Missouri.
PROCESS
Animal litter compositions according to the present invention
can be prepared as follows. The calculated amounts of actives,
i.e., enzymes and other antimicrobial and/or urease inhibitor,
odour absorbing actives, and optional ingredients, e.g.,
perfume microcapsules, binders, and the like, are dissolved
and/or suspended, in appropriate amounts of liquid carrier,
preferably water, enough to sufficiently uniformly distribute
the actives over the solid absorbing litter material. All
actives are preferably incorporated in the same solution and/or
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slurry. However, the enzymes may more preferably be applied
onto the solid absorbing litter material separately, e.g., by
spraying it, sequentially to the animal litter, preferably with
a drying step in between, as these biological molecules may be
s sensitive to other active ingredients in solution. The
enzymes) may be applied as a fermentation broth, a
concentrated enzyme solution, or a slurry of amorphous and/or
crystalline enzyme such as described in WO 91/09943 included
herein by reference. In another more preferred embodiment of
to the invention the enzymes) are added to and mixed with the
animal litter compositions in the form of a conventional enzyme
granule. Such granules may be prepared by methods known to the
art, e.g. as
is - Spray dried products, wherein a liquid enzyme containing
solution is atomized in a spray drying tower to form small
droplets which during its way down the drying tower dries up
to form an enzyme containing particulate material. Very
small particles can be produced this way (Michael S. Showell
20 (editor); Powdered detergents; Surfactant Science Series;
1998; vol. 71; page 140-142; Marcel Dekker).
Layered products, wherein the enzyme is coated as a layer
around a preformed core particle, wherein an enzyme
z5 containing solution is atomized, typically in a fluid bed
apparatus wherein the preformed core particles are
fluidized, and the enzyme containing solution adheres to the
core particles and dries up to leave a layer of dry enzyme
on the surface of the core particle. Particles of a desired
3o size can be obtained this way if a useful core particle of
the desired size can be found. This type of product is
described in eg WO 97/23606
- Another type of product is known wherein an absorbing core
35 particle is applied, and rather than coating the enzyme as a
layer around the core, the enzyme is absorbed onto and/or
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46
into the surface of the core. Such a process is described in
WO 97/39116.
- Extrusion or pelletized products, wherein an enzyme
containing paste is pressed to pellets or under pressure is
extruded through a small opening and cut into particles
which is subsequently dried. such particles usually have a
considerable size because of the material in which the
extrusion opening is made (usually a plate with bore holes)
to sets a limit on the allowable pressure drop over the
extrusion opening. Also very high extrusion pressures when
using a small opening increases heat generation in the
enzyme paste which is harmful to the enzyme. (Michael S.
Showell (editor); Powdered detergents; Surfactant Science
is Series; 1998; vol. 71; page 140-142; Marcel Dekker)
- Prilled products, wherein an enzyme powder is suspended in
molten wax and the suspension is sprayed, eg through a
rotating disk atomizer, into a cooling chamber where the
2o droplets quickly solidify (Michael S. Showell (editor);
Powdered detergents; Surfactant Science Series; 1998; vol.
71; page 140-142; Marcel Dekker).
Mixer granulation products, wherein an enzyme containing
25 liquid is added to a dry powder composition of conventional
granulating components. The liquid and the powder in a
suitable proportion is mixed in and as the moisture of the
liquid is absorbed in the dry powder, the components of the
dry powder will start to adhere and agglomerate and
3o particles will build up forming granules comprising the
enzyme. Such a process is described in 4,106,991 (NOVO
NORDISK) and related documents EP 170360 Bl (NOVO NORDISK),
EP 304332 B1 (NOVO NORDISK), EP 304331 (NOVO NORDISK), WO
90/09440 (NOVO NORDISK) and WO 90/09428 (NOVO NORDISK).
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47
Also these granular enzyme products may be prepared as so-
called co-granules, wherein an oxidoreductase may be granulated
together in one granule with an enhancer such as described in
WO 95/33039.
s Also in the cases where the actives are not totally
compatibles, such as in the case of cyclodextrin odour control
active and an organic urease inhibitor which can form complex
with the cyclodextrin, or in the case of a water-insoluble
organic antimicrobial which is more effectively soluble in a
to solvent such as alcohol (and such organic antimicrobial can
form complex with the cyclodextrin), it is preferably that the
incompatible actives are distributed. For some absorbing clay
litter materials, since the water of the aqueous solutions can
be absorbed quickly into the clay particles, the drying steps)
i5 may not be necessary.
For materials with low solubility the water is preferably
heated to from about 40°C to about 90°C, preferably from about
50°C to about 80°C, more preferably from about 60°C to
about
75°C, to permit the use of the minimum amount of water, thus
20 lowering the time and/or heat needed to dry the litter
material. Such poorly soluble materials, e.g., beta-
cyclodextrin, can also preferably be used in as a finely
divided powder and be suspended in a suitable amount of liquid
carrier (e. g., preferably, water) into a mobile and sprayable
z5 slurry, typically at a liquid carrier-to-powder ratio of from
about 1:1 to about 10:1, more preferably from about 2:1 to
about 5:1. Similarly, the perfume/cyclodextrin complex is
preferably added as a slurry. It is convenient to use the
slurry used to form the complex and add any uncomplexed
3o cyclodextrin to that slurry, the excess cyclodextrin helping to
make more efficient use of the perfume.
The amount of water necessary will vary with the kind of
actives and/or absorbent litter material used and typically is
in the range of from about 5 ml to about 500 ml, preferably
35 from about 8 ml to about 250 ml, more preferably from about 12
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48
ml to about 150 ml, per kg of solid absorbing litter material.
The amount of solution is preferably just sufficient to
distribute the actives on the absorbing litter material. This
insures quantitative deposition of the actives and eliminates
s the necessity of handling excess water. The solid absorbing
litter material is then dried at ambient conditions, or in an
oven at a temperature of from about 50°C to about 95°C. This
procedure lends itself extremely well to a continuous process,
whereby metered flows of solid absorbing litter material and
to solution (or solutions) and/or slurry (or slurries) of actives
are contacted with one another (e. g., by spraying the actives
in liquid carriers), and the litter material is subsequently
dried on a perforated conveyor belt and/or in an air-dry
tunnel. The amount of actives, e.g., enzymes and/or other
is antimicrobial/urease inhibitor, odour absorbing actives,
perfume, and the like, to be used depends on its effectiveness,
its cost and its toxicity. However in a preferred embodiment
the drying temperature is kept low, eg. under 70°C or under
60°C or under 50°C to prevent heat denaturation of the enzymes.
zo Accordingly the application of enzyme to the solid litter
composition may very well be performed in a fluid bed type of
equipment.
T/fi v+-"v~~r.
zs In general, the more water soluble materials which are added
to the animal litter, replenishment compositions, etc., to
control the formation of odor and/or absorb odor are desirable
to promote spreading. Also, the less toxic materials are more
desirable, since animals, and cats, especially, will tend to
30 lick off any litter that sticks to their fur. Mixtures of the
above compounds can be used. Mixtures can be more effective
and, by limiting the amount of any one material, can be less
toxic.
3s Industrial Process - An animal litter in accordance with the
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49
present invention is preferably formed by spraying metered
quantities of slurries onto a quantity of absorbent particles
to affix the activities onto at least some of these absorbent
particles in substantially intact form. The solutions and/or
s slurries can be pumped with a conventional metering pump and
sprayed through a plurality of conventional spray nozzles onto
an evenly distributed bed of absorbent particles being moved,
as by a conveyor belt, past the spray nozzles. The moving bed
of absorbent particles is preferably fairly thin, for example
to about i~" ( 1 . 3 cm) to about 1 l;~" ( 3 . 8 cm) , so that a
significant number of the litter particles have affixed
actives, and to aid in homogeneous dispersion of the particles
with affixed actives in packaging.
All percentages, ratios, and parts herein, in the
is specification, examples, and claims are by weight and are
approximations unless otherwise stated.
The following are non-limiting examples of the instant
composition.
Methods
Determination of haloperoxidase activity
Color reagent:
z5 2.98 g potassium bromide is dissolved in a mixture of 2 ml
0.2 % Phenol Read in 96 % EtOH and 48 ml 0.3 M TRIS buffer pH
7.0
Vanadate solution:
18.4 mg sodium orthovanadate is dissolved in 10 ml de-
3o ionized water.
Hydrogen peroxide solution:
0.1 ml 30 % hydrogen peroxide is added to 9.9 ml de-ionized
water.
When performing the assay, the enzyme sample is pre-
35 incubated with an amount of the Vanadate solution for a
specified period of time and then specified amounts of hydrogen
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peroxide and the Color Rreagent is added and the activity is
monitored by measuring the absorbance at 595 nm (the color
changes from read to bluish violet).
s Determination of smell:
Evaluation of smell was performed by a test panel of three
trained persons.
EXAMPLES
to
Eample 1
Experiments were performed using 10 ml. of artificial urine with
pH 6.0 (Urea (300 mM), calcium sulphate (2 mM), magnesium
sulphate (3.5 mM), potassium chloride (60 mM), Triton X-100 and
i5 sodium chloride (130 mM)). Initially, hydrogen peroxide was
added to a final concentration of 1 mM and then recombinant
haloperoxidase derived from Curvularia verruculosa CBS 147.63
(rHP) described in WO 97/04102 (Novo Nordisk) was added to the
desired concentration. The experiments were initiated by
2o addition of urease type III from Jack Beans (Sigma U1500).
Samples were incubated on a shaking water bath at 25°C in closed
vessels. Urease activities in the samples was evaluated at
given time.intervals by monitoring pH (Radiometer PHM 85, pH
meter) and production of ammonia monitored by the development of
z5 the characteristic malodour.
Results:
HALOPEROXIDASE UREASE PH SMELL PH
(MG/L) (U/ML) AT 15 MIN AT 15 MIN AT 45 MIN
0 10 8.5 strong 8.8
0.1 10 8.4 strong 8.7
0.5 10 6.2 none 6.2
1.0 10 6.0 none 6.0
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51
This example clearly illustrates that the system of halo
peroxidase/hydrogen is capable, in a dose dependent manner, of
completely inactivating urease and thereby prevent the
s development of malodour from the samples.
EXAMPLE 2
The anti-microbial activity of Cuvularia verruculosa
to haloperoxidase was evaluated in artificial urine against
Escherichia coli (Accession No. DSM 1576), Enterococcus
faecalis (Accession No. DSM 2570) and Proteus mirabilis
(Accession No. DSM 788) adhering to pulp material.
The anti-microbial activity was determined as reduction in
15 living bacterial cells (bactericidal activity) by use of
impedance measurements (Malthus).
The detection times measured by the Malthus instrument were
converted to cfu/ml (colony forming units per milliliter) by a
calibration curve. Either direct or Indirect Malthus
2o measurements were used when enumerating total survival cells
(Malthus Flexi M2060, Malthus Instrument Limited). By the
direct measurements, the cell metabolism was determined by
conductance measurements in the growth substrate. The Malthus-
method is based on the methods described in Johnston and Jones,
2s (1995), Journal of Microbiological Methods 21, p. 15-26 and
Johansen et al. (1995), Journal of Applied Bacteriology 78, p.
297-303.
By the indirect measurements, 3 ml of growth medium was trans-
ferred to the outer chamber of the indirect Malthus cells, and
30 0.5 ml of sterile KOH (0.1 M) was transferred to the inner
chamber. The cell suspensions were after enzyme treatment
transferred to the outer chamber of the Malthus cell. As cells
are growing in the outer chamber they produce CO2 which will
dissolve in the KOH in the inner chamber and thereby change the
35 conductance of the KOH. The amount of COz formed by the
respiring cells surviving the enzyme treatment was used for
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52
estimating the number of viable cells. 4dhen the conductance
change is measurable by the Malthus, a detection time (dt) will
be recorded. The dt~s were converted to colony counts by use of
a calibration curve relating cfu/ml to dt.
s The strains were grown in Brain Heart Infusion (BHI) (Oxoid CM
225) until stationary growth phase (30°C, 20 hours), diluted in
peptone water and inoculated to chemo-thermo-mechanical-pulp
CTMP at the final cell concentration of approximately 104
cfu/O.Olg CTMP. The CTMP was inoculated with either mono
to cultures or a mixed culture of the three strains.
The anti-microbial activity of the haloperoxidase (was
determined at 35°C for 30 minutes, by adding the haloperoxidase
to the bacterial cells adhering to the CTMP, together with
hydrogen peroxide (0.5 mM) and artificial urine. The number of
is living cells was determined by transfer of the CTMP to Malthus
cells.
The activity of the haloperoxidase (0-2 mg enzyme protein/1
urine) was evaluated in artificial urine without electron-
donor, thus the enzyme activity was initiated by the Cl- in the
ao urine .
The haloperoxidase caused a total kill of the three test
organisms; E. faecalis, E. coli and P. mirabilis.
Furthermore the anti-microbial activity of the haloperoxidase
was unchanged in artificial urine compared to a buffer system
z5 (pH 6). A total kill of the mixed culture was found at
haloperoxidase concentrations above 1.5 mg/1 and a hydrogen
peroxide concentration above 0.75 mM. Figure 1, in which Z=
bactericidal activity in log cfu/ml; X - mg/1 Curvularia
verruculosa haloperoxidase; Y - mM H202, shows a response
3o surface plot for the antibacterial activity in artificial urine
of a haloperoxidase (rHP) against a mixed culture of E.
faecalis, E. coli and P. mirahilis adhering to CTMP.
