Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITE ANIMAL LITTER MATERIAL AND METHODS
FIELD OF THE INVENTION
This invention generally relates to the field of domestic animal disease
detection and
more particularly to composite animal litter materials and methods.
BACKGROUND OF THE INVENTION
Domestic animals such as cats are susceptible to various diseases, ailments
and
conditions, which are not only arduous and painful for the animal itself but
also a source
of concern and stress for animal owners. While animal owners nurture, watch
over and
bestow affection on their pets, they must balance this attention with other
responsibilities. Convenience is thus an important factor when taking care of
a domestic
animal. While owners may be devoted and considerate to their pets, they may
lack the
sophistication to diagnose animal diseases, ailments and conditions.
Convenient, simple
and effective means to inform pet owners of the presence of diseases, such as
urinary
infections, are desired so that appropriate steps can be taken to reverse,
mitigate or
avoid serious illness in the animal.
For example, feline urinary tract disease can be a serious condition for cats.
In feline
urinary tract disease, crystals of magnesium ammonium phosphate can
precipitate in the
cat's urinary tract and cause obstruction. If untreated, the obstruction can
lead to intense
pain and can often be fatal within days. In some cases, upon observing feline
urinary
tract disease symptoms - such as bloody urine and urination discomfort and
straining -
cat owners often consult their veterinarian who may be able to provide
treatments, which
may be expensive. However, many cats with feline urinary tract disease do not
show any
obvious symptoms, which is why this disease has been referred to as a "silent
killer".
Early detection of feline urinary tract disease is therefore of paramount
importance in
facilitating treatment, lessening the likelihood of severe complications or
aggravations,
and reducing the cost of treatment.
Some methods of early detection are known. Early detection may be possible by
occult
blood testing, allowing cat owners to treat the problem of feline urinary
tract disease by
changing the cats' diets. However, some known occult blood testing techniques
present
various disadvantages concerning the complexity and inconvenience of the
tests. For
instance, cats are creatures of routine and will often resist urine sample
gathering.
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Also known are some detection agents that may be combined with animal litter
or used
as animal litter to allow daily assessment of the animal's health.
It is known to use diagnostic agents, incorporated into test strips, beads or
particles, for
detection purposes. Usually, such test strips consist of an absorbent carrier
made from
fibrous or non-woven material, in the simplest case filter paper, which is
coated or
impregnated with the detection reagents. Components of the detection reagent
may be a
chromogen as an indicator, an oxidizing agent such as a hydroperoxide as an
activator
of the indicator. The oxidizing agent, which is sometimes also called a
sensitizer or an
accelerator, increases the sensitivity of detection. Standard additional
components are,
apart from a surface-active agent (wetting agent), thickening agents which
prevent the
bleeding of the wetted test field, pigments, complex-forming agents and/or
other
stabilizers for the chromogen and/or the hydroperoxide.
Similarly, various analytical methods are presently available for detecting
the presence
of "peroxidatively active substances" in samples such as urine, fecal
suspensions, and
gastrointestinal contents. Hemoglobin and its derivatives are typical of such
peroxidatively active substances because they behave in a manner similar to
the
enzyme peroxidase. Such substances are also referred to herein as
pseudoperoxidases.
Peroxidatively active substances are enzyme-like in that they catalyze the
redox reaction
between peroxides and benzidine, o-tolidine, 3,3',5,5'-tetramethyl benzidine,
2,7-
diaminofluorene or similar benzidine-type indicator substances, thereby
producing a
detectable response such as a color change. Most methods for determining the
presence of occult blood in test samples rely on this pseudoperoxidase
activity.
A benzidine-type indicator responds in the presence of hydrogen peroxide and
peroxidase by changing its light absorptive capability.
Providing a reliable occult blood detection system in animal litter itself
also has many
problems and challenges. For example, the test indicator material should be
stable when
exposed to a wide variety of ambient conditions, be they dry or humid, and
over a wide
range of temperatures. Such stability is quite often difficult to achieve.
A further problem with many known test indicators is that pet owners are
insufficiently
observant or sophisticated to appreciate the positive indication, such as a
color change,
before the indicator decays. Many known indicators do not stay at the changed
color for
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a sufficient period of time to allow pet owners to reliably recognize the
indicated health
issue.
An additional problem with various detection reagents mixed with animal litter
is that the
test reagents give off sufficient scent such that cats, which have an
extraordinary sense
of smell, recognize the odor change in their litter and thus tend to shy away
from the
litter. As will be appreciated, this not only defeats the purpose of a
convenient detector
but can also cause unwanted excretory mishaps. Thus, test reagents with
significant,
offensive or upsetting odors - both to the user and the cat - have many
disadvantages.
A further problem with known detection reagents is poor shelf life stability,
particularly if
combined with an animal litter for storage as a single mixture. Poor stability
leads to
disadvantages in the ability to store, transport, display, purchase and use
the detection-
litter combination.
Detection materials that are merely coated over the surface of a carrier
material also
have various disadvantages that may relate to poor shelf-life stability, low
in-use stability
and lifetime, and insufficient color change visibility.
Some other animal litters or detection additives are difficult or complicated
to
manufacture requiring multiple steps of impregnation, drying, coating,
material cutting,
etc.
Known materials and methods for detection of feline urinary tract disease have
involved
one or more of the above deficiencies. There is a need for a technology that
overcomes
at least some of these deficiencies.
Clays and other mineral compositions such as diatomaceous earth are
environmentally
friendly, naturally abundant and economic. Even though many types of clay are
known
for their liquid absorbing properties, their use is often restricted due to
their colloidal,
dispersive properties in water. The use of clays in combination with other
ingredients
such as polymers is known.
Nanocomposites can be prepared by numerous techniques. The most common
technique involves ion exchange of the cations located in the interlayer
spacing of the
clays using cationic surfactants (cationic molecules bearing C8 - C30
aliphatic chains).
This technique was first reported by Okada et al. (Mat. Res. Soc. Proc., 1990,
171, 45-
50) and subsequently by Pinnavaia et al. (U.S. Pat. No. 6,261,640; U.S. Pat.
No.
6,414,069, and U.S. Pat. No. 6,261,640).
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Techniques for increasing the interlayer spacing between the phyllosilicates
making up
the clays have been described by Beall et al. (U.S. Pat. No. 6,228,903 and
U.S. Pat. No.
5,760,121); Lan et al. (U.S. Pat. No. 6,399,690); Qian et at. (U.S. Pat. No.
6,407,155);
Zilg et at. (U.S. Pat. No. 6,197,849), Ross et al. (U.S. Pat. No. 6,521,690);
Barbee et at.
(US20020169246 Al); Ishida (U.S. Pat. No. 6,271,297); Powell et al. (U.S. Pat.
No.
6,730,719); Knudson et al. (US20020165305 Al); Lorah et al. (US20030060555
Al);
Fischer et al. (U.S. Pat. No. 6,579,927) and Bagrodia et al. (U.S. Pat. No.
6,586,500).
Nanocomposites have also been prepared using physico-chemical techniques such
as
extrusion, lyophilization, and ultrasonic wave treatments, as disclosed by
Torkelson et al.
(WO 2004043663); Lee et al. (US20030134942 Al); Nobuyoshi (JP 02-203936), and
McClelland et at. (CA 2,352,502).
Hybrid organic-inorganic gels for use in cosmetic or pharmaceutical
compositions have
been disclosed by Lahanas et al. (U.S. Pat. No. 6,042,839); Udagawa (JP 09-
187493);
Collin et al. (EP 1327435 Al); and Chevalier et al. (EP 1203789 Al). However,
these
gels have not been reported as detectors for feline urinary tract disease
applications.
Starches have also been reported as being used as components in nanocomposite
materials. Hydroxyapatite reinforced starch/ethylene-vinyl alcohol copolymer
composites
have been reported by Reis et al. (J. Adv. Polym. Technol. 1997, 16, 263).
Calcined
kaolin/thermoplastic starch composites have been disclosed by DeCarvalho et
at.
(Carbohydr. Polym. 2001, 45 (2), 189-194). Montmorillonite/thermoplastic
starch hybrids
have been described by Park et al. (Macromolecular Materials and Engineering,
2002,
287(8), pp. 553-558, J. of Mat. Sci, 2003, 38 (5), pp. 909-915) and McGlashan
et at.
(Polymer International, 2003, 52(11), PP 1767-1773). However, these starch
containing
nanocomposite materials were not reported as exhibiting feline urinary tract
disease
detection properties.
The use of chitosan in nanocomposite materials has also been reported.
Cationic
chitosan, intercalated in montmorillonite, has been disclosed by Darder et al.
(Chemistry
of materials, 2003, 15 (20), PP 3774-3780). A butyl-acrylate-graft chitosan
montmorillonite nanocomposite, has been reported by Li et at. (Radiation
physics and
chemistry, 2004, 69(6) APR, PP 467-471). The use of xanthan and scleroglucan
in
nanocomposite materials has also been reported.
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Takahiro et al (Japanese Patent No. 01-296933), Marx (U.S. Pat. No. 4,615,923)
and
Brander et al (U.S. Pat. No. 6,376,034) describe inorganic additives
(kieselguhr, clays,
diatomaceous earth) added to biodegradable superabsorbents. However, none of
these
patents teach additives made from organic components.
In summary, there is a need for a technology that provides animal litter that
enables the
detection of conditions such as feline urinary tract disease and overcomes at
least some
of the drawbacks of what is already known.
SUMMARY OF THE INVENTION
The present invention responds to the above need by providing a composite
material
allowing detection of certain conditions.
In one aspect of the present invention, there is provided a composite litter
material for
detecting a disease when contacted by feline urine, the composite litter
material
comprising: an absorptive polymer material forming a solid matrix; exfoliated
clay
embedded and dispersed within the solid matrix; a chromogenic indicator
provided within
the solid matrix; and an oxidizing agent distributed and stabilized within the
solid matrix
such that the oxidizing agent is available and responsive to peroxidase or
pseudoperoxidase activity in the urine to activate the chromogenic indicator.
In an optional aspect, the absorptive polymer material is a polysaccharide. In
another
optional aspect, the polysaccharide comprises alpha-linkages. In another
optional
aspect, the exfoliated clay comprises exfoliated phyllosilicates. In another
optional
aspect, the exfoliated phyllosilicates are dispersed throughout the entire
solid matrix. In
another optional aspect, the chromogenic indicator is cationic and the
exfoliated clay
comprises sheets having anionic surface characteristics. In another optional
aspect, the
chromogenic indicator is a benzidine-based compound. In another optional
aspect, the
chromogenic indicator corresponds to the following formula:
R1 R2 R2 R1
HN NH
R5 / RB
R4 R3 R3 R4
R6 R5
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wherein R1, R2, R3 and R4 are the same or different and represent hydrogen,
halogen, a lower alkyl or alkoxy group containing 1 to 4 carbon atoms, a (Cl -
C4)-dialkylamino group, an acetylamino group, a nitro group or an aromatic
group
which may be substituted; and
wherein R5 and R6 are the same or different and represent water-soluble
groups,
hydroxyl group, amino group, acidic group, disulfonyl group, ether group,
halogen, and a lower alkyl or alkoxy group containing 1 to 4 carbon atoms, a
(Cl
-C4)-dialkylamino group, an acetylamino group or a nitro group.
In another optional aspect, the chromogenic indicator is 3,3',5,5'-
tetramethylbenzidine
(TMB). In another optional aspect, the chromogenic indicator is 3,3',5,5'
tetramethylbenzidine dihydrochloride (TMB-HC).
In another optional aspect, the oxidizing agent is a hydroperoxide. In another
optional
aspect, the oxidizing agent is cumene hydroperoxide. In another optional
aspect, the
oxidizing agent is diisopropylbenzene dihydroperoxide.
In another optional aspect, the absorptive polymer material comprises polyols
having
polymer backbones and the hydroperoxide is stabilized along the polymer
backbones.
In another optional aspect, the composite material is provided in the form of
discrete
pellets. In another optional aspect, each pellet has a disk shape, a lozenge
shape, a
wafer shape or a cat head shape. In another optional aspect, each pellet has a
diameter
between about 1 mm to about 10 mm and a thickness between about 0.5 mm and
about
3 mm.
In another optional aspect, the composite material further comprises an
impregnated
stabilizer composition. In another optional aspect, the stabilizer composition
comprises a
color enhancer.
In another aspect of the present invention, there is provided a cat litter for
detecting a
feline disease when contacted by feline urine, the cat litter comprising: a
particulate filler
material; composite pellets mixable with the particulate filler material, each
of the
composite pellets comprising:an absorptive polymer material forming a solid
matrix;
exfoliated clay embedded and dispersed within the solid matrix; a chromogenic
indicator
provided within the solid matrix; and an oxidizing agent distributed and
stabilized within
the solid matrix such that the oxidizing agent is available and responsive to
peroxidase
or pseudoperoxidase activity in the urine to activate the chromogenic
indicator.
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In an optional aspect, the particulate litter material comprises clumping or
non-clumping
clay. In another optional aspect, the particulate litter material and the
composite pellets
are sized and mixed to as to form a substantially homogeneous mixture.
In yet another aspect of the present invention, there is provided a method for
detecting
or diagnosing a feline disease, the method comprising:
allowing urine to contact a composite material comprising an absorptive
polymer
material forming a solid matrix, exfoliated clay embedded and dispersed within
the solid matrix, a chromogenic indicator provided within the solid matrix and
an oxidizing agent distributed and stabilized within the solid matrix such
that the
oxidizing agent is available and responsive to peroxidase or pseudoperoxidase
activity in the urine to activate the chromogenic indicator;
detecting or diagnosing the feline disease by determining whether the
chromogenic indicator has been activated as evidenced by a color change.
The method may further comprise providing the composite material pre-mixed
with a
conventional cat litter material and allowing a cat to urinate thereon.
The method may further comprise providing the composite material as part of a
testing
kit.
In still another aspect of the present invention, there is provided a method
of
manufacturing a composite animal litter material for detecting a feline
disease,
comprising:
preparing a mixture comprising an absorptive polymer and water;
distributing exfoliated clay, a chromogenic indicator and an oxidizing agent
to
within the mixture to produce the composite animal litter material.
In an optional aspect of the method, a solution comprising the exfoliated clay
is added to
the mixture during the preparation thereof.
In another optional aspect of the method, a chromogenic solution comprising
the
chromogenic indicator, a solvent and the oxidizing agent is prepared and
introduced into
the mixture.
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In another optional aspect of the method, the chromogenic solution and the
mixture are
combined by extrusion, pressure agglomeration, tumble growth agglomeration or
matrix
melt formation, or a combination thereof.
In another optional aspect of the method, the chromogenic solution is buffered
at a pH
between about 1.5 and about 7. The chromogenic solution may also be buffered
at a pH
allowing solubility in the absence of alcohol.
In another optional aspect of the method, the chromogenic solution and the
mixture are
combined by extrusion. The method may comprise: providing an extrusion
apparatus
having an upstream section, a mid section and a downstream section; feeding
the
mixture into the upstream section of the extrusion apparatus; feeding the
chromogenic
solution into the mixture at the downstream section of the extrusion
apparatus, to form a
combined blend; extruding, cutting and drying the combined blend to form the
composite
material.
In another optional aspect of the method, the mid section is operated at a
higher
temperature than the upstream and downstream sections.
In another optional aspect of the method, the temperature of the mid section
is about 60
to about 80 C higher than either one of the upstream or downstream sections;
optionally,
the upstream section is operated at a temperature between about 25 C and
about 40 C,
the mid section is operated at a temperature between about 80 C and about 130
C, and
the downstream section is operated at a temperature between about 25 C and
about
40 C; and optionally, the upstream section is operated at a temperature of
about 30 C,
the mid section is operated at a temperature of about 110 C, and the
downstream
section is operated at a temperature of about 40 C.
In another optional aspect of the method, it further comprises homogenizing
the
combined blend. Thus can be done by extending the downstream section or adding
another extruder. In another optional aspect of the method, the extrusion
apparatus
comprises a twin screw extruder. The extrusion apparatus may include a first
extruder
having an inlet and an outlet, and a second extruder coupled to the first
extruder to
receive the combined blend from the outlet of the first extruder. The first
extruder may be
a twin-screw extruder. The second extruder may be a single screw extruder.
In another optional aspect of the method, the pH of the chromogenic solution
is acidic. In
another optional aspect of the method, the chromogenic solution further
comprises an
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amount of alcohol sufficient for solubilization. In another optional aspect of
the method,
the exfoliated clay is exfoliated bentonite and is added at less than 1 % w/w
with respect
to the mixture. In another optional aspect of the method, the absorptive
polymer is wheat
starch or corn starch. In another optional aspect of the method, the combined
blend,
after exiting the extruder, is dried at about 50 C.
Further aspects, embodiments and advantages of the present invention will be
further
understood upon reading of detailed description and the appended drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig I is a cut view schematic representation of a composite material pellet
according to
an embodiment of the present invention.
Fig 2 is a graph showing the elementary analysis of a granule of composite
material
containing wheat starch and exfoliated bentonite by Energy-dispersive X-ray
spectroscopy.
Fig 3 is a graph of absorption of pellets versus soaking time.
Fig 4 is a photograph MET of wheat starch with 1 % exfoliated bentonite.
Fig 5 is a photograph MET of wheat starch with 1% exfoliated bentonite (small
particles)
and chromogenic compound (large particles).
While the invention will be described in conjunction with example embodiments,
it will be
understood that it is not intended to limit the scope of the invention to such
embodiments. On the contrary, it is intended to cover all alternatives,
modifications and
equivalents as may be included as described by the present description and
appended
claims.
DETAILLED DESCRIPTION
The present invention provides a composite material for use in connection with
animal
litter and having a structure for detection of certain abnormalities in
excretions and
aqueous solutions such as urine.
Some preferred embodiments described herein pertain to the composite
material's use
in connection with detecting feline urinary tract disease from peroxidase or
pseudoperoxidase activity in feline urine, i.e. in peroxidatic assays.
However, it should
be understood that the composite material may be used in other applications
for
detection purposes. For instance, in some embodiments, the composite material
is used
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in connection with detecting elevated glucose levels which can be useful in
diagnosing
diabetes, via a glucose oxidase-peroxidase system. In this regard, in the
presence of
glucose oxydase (Aspergillus niger), glucose in aqueous solution is oxidized
into
gluconic acid by dissolved oxygen with the formation of hydrogen peroxide,
which is then
dosed by an indicating enzymatic reaction (Horseradish) which oxidizes the
chromogen
which changes color.
In one embodiment of the present invention, as illustrated in Figure 1, the
composite
material is provided in the general form of a pellet. By providing a plurality
of discrete
pellets, the composite material may be easily packaged and may also be pre-
mixed with
conventional animal litter.
Referring to Figure 1, each composite pellet 10 comprises an absorptive
polymer
material 12 forming a solid matrix; exfoliated clay 14 embedded and dispersed
within the
solid matrix; a chromogenic indicator 16 provided in the matrix; and an
oxidizing agent
18 distributed and stabilized within the solid matrix such that the oxidizing
agent 18 is
available and responsive to peroxidase or pseudoperoxidase activity in the
feline urine to
activate the chromogenic indicator 16.The composite material may also be
referred to
herein as a "nanocomposite material".
The absorptive polymer material 12, which forms a solid matrix, preferably
comprises a
polysaccharide, which provides polysaccharide chain backbones in addition to
the
general polysaccharide matrix. More particularly, polysaccharides that are
suitable for
use in the nanocomposite material may be selected from the following non-
limited group:
starches, modified starches, amylopectin, modified amylopectin, amylose,
modified
amylose and mixture thereof. Amongst these polysaccharides, starch is
frequently
chosen as a polysaccharide for use in the agglomerated particle. Non-limiting
examples
of such starches are starch granules, pregelatinized starches, glass-like
starches, waxy
starches, anionic starches, cationic starches, fractionated starches, cross-
linked
starches, hydroxyalkylated starches, alkylated starches and mixture thereof.
Starch that
is suitable for the present invention may be obtained from many sources,
including but
not limited to wheat, maize, buckwheat, potato, cassaya, sorghum, millet, oat,
arrowroot,
barley, beans, peas, rice, rye, waxy starches and mixture thereof. A commonly
used
starch is wheat starch. Naturally occurring starch is usually organized in a
semi-
crystalline, water insoluble pattern, which is sometimes referred to as a
"starch granule".
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The form of these starch granules is characteristic of their botanical origin,
and their
mean particle size may range from about 1 pm to about 60 pm.
The polymer material 12 absorbs urine with which it comes into contact. The
specific
polymer and its modification and form may be selected to allow sufficient
internal
diffusion of the urine on contact and sufficient urine retention to facilitate
the
chromogenic response over time. The exemplary absorption properties shown in
Fig 3
were sufficient for the present invention, though many other absorption rates
may also
be used.
The exfoliated clay 14 is dispersed throughout the solid polymer matrix. It
should be
noted that at least a portion of the clay incorporated into the composite
material is
exfoliated and thus it is possible to include some non-exfoliated clay as
well. It should
also be understood that the "exfoliated clay" may include fully exfoliated,
partially
exfoliated, and/or semi-exfoliated clays. Preferably, all of the clay within
the composite
material is substantially exfoliated clay, dispersed throughout the composite
material.
Depending on the quatity of exfoliated clay and the characteristics of other
compounds
included in the composite material, the exfoliated clay may promote binding
with the
chromogenic indicator 16 and may improve dispersion of the exfoliated clay 14
and the
chromogenic indicator 16 throughout the composite material in an efficient and
substantially homogeneous manner. Improved dispersion of the chromogenic
indicator
leads to improved detection functionality of the composite material and
homogeneity of
the material in general leads to improved structural properties of the
composite material.
In one optional aspect, the exfoliated clay may be selected and prepared in
order to
have anionic surface characteristics, the chromogenic indicator is selected to
be cationic,
and the exfoliated clay and chromogenic indicator are proportioned such that
within the
solid matrix they may ionically bond.
In one preferred aspect, the clay is a crystalline alkali metal phyllosilicate
which is
exfoliated. The clay source can be either natural or synthetic. Examples of
clays include,
but are not limited to smectites, hectorites, bentonites, montmorillonites,
LaponitesTMcelites, illites and mixtures thereof. One preferred clay is
bentonite, which is a
montmorillonite type clay. Bentonite is essentially made from colloidal
hydrated
aluminum phyllosilicates and contains varying amounts of iron, alkali, and
alkaline earth
metals. Calcium, sodium, potassium and magnesium bentonites are preferred. Ion-
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exchanged benionites can also be used in the preparation of polysaccharide-
phyllosilicate composite materials according to embodiments of the present
invention.
The composite material of the present invention comprises an inorganic portion
that
includes exfoliated clay. The inorganic portion may also include other mineral
components. The inorganic portion may interact physicochemically with the
chromogen.
The typical inorganic portion content ranges up to about 40%. The particle
size of the
inert inorganic portion may range from about 10 pm to about 150 pm. Water
adsorbents,
such as molecular sieves, zeolites, clays, silicates, silica gel, insoluble
salts and mixture
thereof may preferably be used. Among this class, swelling clays may also be
used. Non
limiting examples of inorganic substances are calcium sulfate, silica gel,
zeolites and
mixtures thereof. Clinoptilolite is a good source of zeolites. Non-limiting
examples of
swelling clays are smectites, hectorites, bentonites, montmorillonites,
LaponitesTM
celites, illites and mixture thereof. Bentonite has been found to be quite
suitable.
The chromogenic indicator 16 allows the oxidizing agent 18 to activate it when
the
oxidizing agent 18 is triggered by the presence of peroxidase or
pseudoperoxidase
activity. In one preferred embodiment, the chromogenic indicator 16 is an
electron donor,
i.e. a reducing agent that changes color upon losing an electron.
In an optional aspect, the chromogenic indicator 16 is homogeneously dispersed
throughout the solid polymer matrix, assisted by the preparation method and
the
presence of exfoliated clay. Thus, the chromogenic indicator is present not
only at the
exterior surface of a given composite pellet, but also in a neighboring sub-
surface region
that can be rapidly exposed to urine that absorbs into the pellet. Especially
when the
solid polymer matrix is glassy or substantially transparent, the presence of
the
chromogenic indicator in a sub-surface region allows it to be readily visible
when color
change occurs and also avoids exposure to the air. In some aspects, the
chromogenic
indicator may also be bound with the exfoliated clay 14 by being adsorbed on
the
exfolitated clay sheets in the polysaccharide solid matrix. The chromogenic
indicator 16
may be intercalated between the exfoliated clay 14.
In one preferred embodiment of the composite pellet 10, the chromogenic
indicator 16
may be a compound as shown in Formula I:
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R1 R2 R2 R1
R2N NR2
I
P4 P3 R3 R¾
Formula I
In Formula 2A, R1, R2, R3 and R4 may be the same or different and may be
hydrogen,
halogen, a lower alkyl or alkoxy group containing 1 to 4 carbon atoms, a (Cl -
C4)-
dialkylamino group, an acetylamino group, a nitro group or an aromatic group
which may
be substituted. In one preferred embodiment, the chromogenic indicator 16 may
be a
compound as shown in Formula II:
R, R2 R2 R1
HN NH
R5 Re
R4 R3 R3 R4
R6 R5
Formula II
In Formula II, R1, R2, R3 and R4 may be the same or different and represent
hydrogen,
halogen, and a lower alkyl or alkoxy group containing 1 to 4 carbon atoms, a
(Cl -C4)-
dialkylamino group, an acetylamino group, a nitro group or an aromatic group
which may
be substituted; R5 and R6 are the same or different and represent water-
soluble groups
as hydroxyl groupl, amino group, acidic group, disulfonyl group, ether group,
halogen,
and a lower alkyl or alkoxy group containing 1 to 4 carbon atoms, a (Cl -C4)-
dialkylamino group, an acetylamino group or a nitro group.
Thus, a water soluble benzidine-type indicator of Formula II, responds in the
presence of
hydrogen peroxide and peroxidese by changing its light absorptive capability,
which is
due to the chemical transformation to the compound shown in Formula III:
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WO 2010/133001 PCT/CA2010/000789
R1 R2 R2 R1
N N
R5 R5
R4 R3 R3 R4
R5 R 5
Formula III
Several different types of benzidene chromogenic indicators 16 may be used in
embodiments of the present invention.
The oxidizing agent 18 can be stabilized, by ascorbic acid for example, in the
polymer
matrix, is reactive to peroxidase or pseudo-peroxidase activity and is able to
activate the
chromogenic indicator. In one preferred embodiment, the oxidizing agent 18
comprises a
hydroperoxide. The hydroperoxide may be, for example, cumene hydroperoxide
which is
sutable for detection of hemoglobin and also can have some reactivity to
elevated
glucose levels, thus for urinary tract disease and diabetes. The hydroperoxide
may also
be, for example, diisopropylbenzene dihydroperoxide which has high selectivity
to
detection of hemoglobin and thus of urinary tract disease. In some
embodiments, both of
these hydroperoxides could be used in the same composite material, or in
different
pellets, the former for more general disease detection and the later for more
specific
detection of urinary tract disease.
In one embodiment of the present invention, the composite pellet 10 comprises
both
organic and inorganic components. The composite pellets 10 may indeed be
composed
of mainly renewable resources and are cost-efficient.
In an optional embodiment of the present invention, the composite pellet 10
may further
comprise a surface-active agent, if necessary, other auxiliary agents such as
thickeners,
stabilizers, pigments, a buffer system and/or complex-forming agents. Each of
such
additives may be used in pellets for particular applications.
In another optional embodiment of the present invention, the composite
material may
also comprise a color enhancer. The color enhancer should be chosen depending
on the
particular components of the composite material, in particular the chromogenic
indicator.
The color enhancer may comprise 5-isoquinoline sulfonic acid.
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WO 2010/133001 PCT/CA2010/000789
The chromogenic indicator and the exfoliated clay particles may be
incorporated into the
polymer matrix and sized so as to have sizes and distributions as shown in the
photographs of the Figs.
The composite pellet 10 is illustrated as having a diameter D which may
optionally be
from about 1 mm to about 10 mm, and preferably in the range of about 5 mm. The
composite pellet 10 may have a thickness between about 0.5 mm and about 3 mm,
and
preferably in the range of about 1 mm. Each pellet 10 may therefore have a
disk shape,
a lozenge shape, cat head shape or a wafer shape. This generally flattened
shape
allows a greater surface area per pellet which leads both to improved
absorption of the
feline urine and also improved color visibility. The composite pellets 10 are
also able to
retain the urine better than porous two-dimensional filter paper, surface
coatings or non-
absorptive beads, for example. It should be understood, however, that the
pellets may
be produced to have a variety of other shapes and sizes, as desired.
The composite pellets 10 are discrete and may be provided as a separate
additive to cat
litters, when the detection function is desired. The pellets 10 may also be
provided pre-
mixed directly with conventional cat litter materials, thus forming part of
the packaged
litter formula for sale. Conventional cat litters may be clay-based, cellulose-
based or
another suitable type of animal litter.
The composite pellets 10 of the present invention may be discretely blended
with cat
litter materials or with other materials or products for animal occult blood
detection
applications such as various hygiene articles. The composite pellets may be
pre-blended
with a polymer having absorbance sufficient for a desired purpose or
application of a
particular composite material. The pellets may also be mixed with fluff pulp
and other
components in the manufacturing process.
Composite pellets may also be produced with various components in order to
detect
different feline diseases which are indicated by a predetermined
characteristic of the
cat's urine such as a particular pH range and/or blood in the cat's urine.
Feline diseases
which may be thus detected include feline urinary tract diseases, such as
feline
urological syndrome or feline lower urinary tract disease and/or cystitis
(bladder
infection).
The embodiments of the present invention have various features and allow
several
advantages. Composite material embodiments allow accurate testing for feline
urinary
CA 02737489 2011-03-16
WO 2010/133001 PCT/CA2010/000789
tract disease and also allow safe, convenient and reliable storage,
transportation, and
end-use by the pet owner. The composite pellets retain their peroxidatic
activity and do
not develop a color change during storage. The composite material is also
stable when
exposed to ambient conditions for several days and provide a positive color
response
that does not deteriorate significantly for at least 6 hours subsequent to
exposure to
urine having occult blood. The composite pellets remain stable when exposed to
ambient conditions and provide a positive color response that also remains
stable for a
sufficient time for the color response to be observed. The composite pellets
are
convenient, stable to substantially avoid spontaneous oxidation, and ready-to-
use. The
composite pellets can be used as aggregate to be conveniently homogeneously
mixed
with conventional cat box filler to provide a safe and reliable occult blood
test for feline
urinary tract disease. The composite material may also be used in various
other fields
and applications for detection of a condition, disease, ailment, cycle, or
generally the
presence of a given chemical in a fluid. The composite pellets, alone or in
combination
with the cat litter, can be produced so as to not have offensive odors that
may dissuade
the cat from properly using the litter. The pellets can be mixed with
conventional cat box
filler and still show adequate stability over time despite changing weather or
light
conditions. The composite material may also be triggered by as few as 100 red
blood
cells per microliter of cat urine. The pellets may also be produced to have a
specific color
in their non-reacted state. For instance, the non-reacted color may be chosen
to provide
various indications, such as an orange color which is found to be the
registered trade
mark logo color for Le Groupe Intersand Canada Inc. The non-reacted color may
also be
chosen to obtained better contrast between reacted pellets, non-reacted
pellets and the
litter material.
In various preparation techniques, separate solutions and mixtures may be
prepared and
then combined together to produce the end-product composite material.
In one aspect, a first stage of the process includes mixing water and the
polysaccharides
which then undergo processing to ensure that the solid matrix of the composite
material
will be "gellified" and avoid breaking or crumbling. The processing of the
polysaccharide
mixture is usually performed at elevated temperatures, for example around 110
C. The
exfoliated clay and the chromogenic indicator may be added to the mixture at
various
points in processing the polysaccharide mixture. The bentonite is preferably
added at the
beginning of processing the polysaccharide mixture. The peroxide is preferably
added
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WO 2010/133001 PCT/CA2010/000789
near the end of processing the polysaccharide mixture at a point when the
mixture is
brought back to a lower temperature, for example around 40 C. This order of
processing
and addition allows preparation of a gellified, hard, solid solid polymer
matrix, while
avoiding unwanted pre-mature reaction of the peroxide triggered by elevated
temperature, which could lead to deactivation of the end-material and
dangerous
processing conditions.
In one optional method for manufacturing the composite material, a chromogenic
solution is prepared containing a chromogen compound susceptible to
peroxidatic
oxidation and clay in a suitable solvent that permits the exfoliation of the
clay and the
high solubility of chromogen under high shear mixing. When the chromogen has
high
water-solubility, such as TMB hydrochloride, water can be preferably used as
the
solvent. In addition, a buffered substrate mixture may be separately prepared
containing
a buffer of pH between about 6 and 8, preferably water or a combination of
water with
alcohol, a hydroperoxide and a polysaccharide polymer. This mixture is usually
highly
viscous due to the polysaccharide polymer. Upon mixing of the solution and
mixture
described above by extrusion or other process to form "nanocomposite
particles", the
chromogen, oxidizing agent and the exfoliated clay are dispersed throughout
the
polymer matrix. One difficulty with this technique, however, is that when some
compounds (e.g. hydroperoxides) and methods (e.g. extrusion) are used, the
desired
temperature for processing the polysaccharide mixture can lead to the
degradation or
reaction of the hydroperoxide. Thus, for such embodiments, the polysaccharide
mixture
should be handled carefully at reduced temperatures, using stabilizing
additives, or
choosing oxidizing agents that do not react at the polysaccharide processing
conditions,
such that the oxidizing agents remain active in the final composite material
product.
In another optional method for manufacturing the composite material, which
avoids some
of the disadvantages mentioned above, a solution is prepared containing a
chromogen
compound, a solvent and an oxidizing agent. The solution may also include a
color
enhancer such as lepidine, a buffering compound such as citric acid, a
stabilizer such as
ascorbic acid, a pH-increasing agent such as sodium hydroxide, surfactants and
other
additives. The solution may be prepared and tailored to the particular
absorptive polymer
and processing method to be used. A mixture is also prepared containing an
absorptive
polymer, a solvent and exfoliated clay. The clay is thus not in contact with
the active
agents, namely the chromogen and the peroxide as this stage. The solvent for
the
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WO 2010/133001 PCT/CA2010/000789
mixtrue is usually water, in particular when the absorption polymer is a water-
soluble
polysaccharide such as various starches. The solution and the mixture are then
combined together to form the composite material. The solution is preferably
added to
the mixture once the mixture has undergone heat and shear treatment and has
cooled to
a sufficiently low temperature such that the chromogen and particularly the
oxidizing
agent do not degrade or deactivate due to high temperature. In one exemplary
method,
the mixture is formed in an upstream section of an extruder and the solution
is
introduced at a point near the exit of the extruder such that the
polysaccharide and
exfoliated clay mixture have formed a gelled matrix and the components in the
solution
are quickly dispersed within the gelled matrix prior to exiting the extruder.
More
regarding this will be explained and understood with reference to the
examples.
The solution may be buffered between about 1.5 and about 8 pH. It has been
observed
that a chromogenic solution having a pH of 1.7 enabled production of reactive
composite
pellets.
Process conditions may influence morphology and efficiency of the components
of the
composite pellets. The composite material of the present invention may be
formed by
pressure agglomeration, tumble growth agglomeration or matrix melt formation.
In order to obtain composite pellets, the polysaccharide and the inorganic
exfoliated clay
may be uniformly blended together before they will be bound to each other. An
agglomerating agent or a binder may optionally be mixed with other components.
Non-
limiting examples of suitable binders are gelling polysaccharides, such as
sodium
carboxymethyl cellulose.
Matrix melt formation may be used to form uniform and semi-uniform composites.
In
matrix melt formation, the polysaccharide component of the composite material
may be
partially molten (for semi uniform) or totally molten (for uniform) and act as
matrix
material. Extrusion is an efficient way to melt a polysaccharide, such as
described by
Canadian patent No. 2,308,537 (Huppe et al) or Canadian patent No. 2,462,053
(Thibodeau et al) for example.
Regarding manufacturing techniques, operating with high pH solutions
containing
chromogenic compound, the addition of sodium hydroxide can cause the
appearance of
precipitated material that can decrease the homogeneity of the solution. The
use of an
alcohol can alleviate this issue, but for certain operating conditions and
processes it is
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WO 2010/133001 PCT/CA2010/000789
preferable to avoid using alcohols. Thus, the extrusion was preferably done
with a
solution containing no alcohol and prepared to have acidic pH between about
1.5 and
about 7. When TMB chromogenic indicator was used, it dictated the pH of the
solution
since it is quite acidic. However, depending on the particular components of
the
composite material, different pHs and processing conditions may be
appropriate.
"Peroxidatic activity" refers to the ability of catalytic substances to drive
the reaction of
hydroperoxides with colorless chromogenic electron donors which become
fluorescent or
visibly colored after oxidation.
"Pseudoperoxidatic activity" refers to the ability of an endogenous peroxidase
or a non-
peroxidase catalytic substance to drive the reaction of hydroperoxidases with
colorless
chromogenic electron donors which become fluorescent or visibly colored after
oxidation.
Certain transition metals and their ions and hemoproteins are known to have
pseudoperoxidatic activity. Basophils, neutrophils, eosinophils and mast cells
synthesize
endogenous peroxidase which can be visualized at the ultrastructural level in
the
secretory apparatus of immature cells. Red blood cells and hematin containing
compounds have iron as part of their heme groups, which can catalyze the
oxidation of
chromogenic electron donors. This pseudoperoxidatic activity can be inhibited
with
strong H202 solutions, sodium azide and methanol-H202 solutions.
"Peroxidatic assay" refers to any test or procedure that is based on a
peroxidatic activity,
as defined above, to generate a signal which can be detected or measured to
evidence
the presence of analyte or the amount present.
"Spontaneous oxidation" refers to the spontaneous production of visible color
of a
chromogenic electron donor in the absence of peroxidase enzyme.
"Chromogenic electron donor" refers to a compound which undergoes an easily
observed change in color upon oxidation by an oxidizing agent such as a
hydroperoxide.
Examples of these are: benzidine, 3,3'-dimethylbenzidine (o-tolidine, OTD),
3,3'-
dimethoxybenzidine (o-dianisidine, oDAD), 3,3'-diaminobenzidine (DAB),
3,3',5,5'-
tetramethylbenzidine (TMB), 3,3', 5,5' tetramethylbenzidinedihydrochloride,
3,3'-
diethylbenzidine, 2,7-diaminofluorene (DAF), o-phenylenediamine (OPD), N,N-
diethylphenylenediamine (DEPDA), N,N-dimethylphenylenediamine (DMPDA), 2,2'-
azino-di (3-ethyl-benzthiazoline sulfonate) (ABTS), 3-methyl-2-
benzothiazolinone
hydrazone hydrochloride (MBTH), aminoethyl carbazole (AEC), and 4-chloro-1-
naphthol
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WO 2010/133001 PCT/CA2010/000789
(4-CN). Chromogenic compounds that are soluble in water, such as TMB
hydrochloride,
may be preferred.
"Hydro peroxide" refers to compounds of the general formula, ROOH, wherein the
R
group is an aryl, alkyl, or acyl group (organic hydroperoxide), or hydrogen
atom
(hydrogen peroxide).
"Storage lifetime" of a substrate-chromogen particles is the time interval
after initial
preparation of the indicator particles in which (a) the absorbance of said
particles in the
400-800 nm wavelength range remains below 0.05 and (b) the peroxidatic
activity of said
solution is at least about 80% of its initial value, which corresponds to the
stability limit by
certain analyses.
"Nanocomposite(s)" refers to materials comprising a nanoscale dispersion of
phyllosilicates in a polymer network. Typical phyllosilicate nanocomposites
are exfoliated
nanocomposites, intercalated nanocomposites and semi-exfoliated
nanocomposites.
"Phyllosilicates" or "sheet-like silicates", in general refers to materials
with parallel
slicate-based sheets. Phyllosilicates can be aluminosilicates having a
thickness of about
1 nm, while having a length and a width ranging from 300 to 500 nm.
Phyllosilicates are
a major constituent of clays. Size, composition and shape of phyllosilicates
will vary
depending on the clay sources. Phyllosilicates typically have the general
molecular
formula: A1203.4 Si02.H20.
"Intercalated nanocomposites", also referred to as "sandwich nanocomposites",
refers to
nanocomposites composed of repeating and alternating phyllosilicate-polymer
layers.
Within theses intercalated nanocomposites, the spacing between the
phyllosilicate layers
is increased to provide for the insertion of a polymer.
"Exfoliated nanocomposites", also referred to as "phyllosilicate dispersions",
refers to
nanocomposites comprising delaminated phyllosilicates which are dispersed
throughout
a polymeric network.
"Semi-exfoliated nanocomposites", refers to nanocomposites comprising
partially
exfoliated clays. Within these semi-exfoliated nanocomposites, clays are
delaminated
into smaller units comprising from about 45 to 70 phyllosilicate blocks. Clays
usually
comprise units having from about 85 to 140 phyllosilicate blocks as defined by
Chenu et
al. (Comptes Rendus de I'Acadmie des Sciences, Srie 2, 1990,310 (7 srie 2),
PP. 975-
CA 02737489 2011-03-16
WO 2010/133001 PCT/CA2010/000789
980). Within these semi-exfoliated nanocomposites, the smaller phyllosilicate
units are
dispersed uniformly throughout the polymer.
In the context of certain embodiments of the composite material, intercalation
is a
preliminary step to the exfoliation of the clay. During manufacture of some
embodiments
of the composite material, the clay is mostly but not completely exfoliated.
The degree of
exfoliation will depend on the particular process and the process conditions
that are
used.
"Polysaccharide" refers to polymers comprising a backbone comprising at least
90% of
monosaccharide repeating units and/or derivatized monosaccharide repeating
units.
Non-limiting examples include starches, modified starches, amylopectin,
modified
amylopectin, amylose, modified amylose, chitosan, chitin, guar gum, modified
guar gum,
locust bean gum, tara gum, konjac gum, konjac flour, fenugreek gum, mesquite
gum,
aloe mannans, cellulose, modified cellulose (representative examples include
carboxyalkylated cellulose and carboxymethyl cellulose), oxidized
polysaccharides,
sulfated polysaccharides, cationic polysaccharides, pectin, arabic gum, karaya
gum,
xanthan, kappa, iota or lambda carrageenans, agar-agar and alginates. Non-
limiting
examples of mannose-based polysaccharides include guar gum, tara gum, locust
bean
gum, konjac, mesquite gum, and fenugreek extracts.
In addition, various organic and inorganic components described and referred
to herein
may be used to produce different embodiments of the composite material. It
should also
be noted that the components of embodiments of the composite material may be
used in
various proportions and concentrations. Though some particular exemplary
concentrations are described in the examples, it should be noted that the
components
may optionally be used in concentrations in the range of 5 wt% with respect
to the
exemplary concentrations, and preferably in the range of 2 wt% with respect
to the
exemplary concentrations. In addition, exfoliated bentonite may be preferably
used in a
sufficiently low concentration and under conditions so as to avoid forming a
gelled
material that is non-fluid and thus difficult to introduce into the
polysaccharide mixture,
for instance about 0.5 wt% bentonite with respect to the total mixture.
EXAMPLES
Example 1
Several "recipes" are known and used for detecting blood in urine or faeces.
However,
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the substrate that receives the chemically reactive agents also plays a
significant role. In
fact, it was found that certain solution compositions that were impregnated
into paper
supports (e.g. Whatman n 4) resulting in paper-based indicators giving a
sufficient color
change response, were not effective solutions when applied substrates
containing
bentonite or other minerals. Such bentonite-containing substrates are notably
less
neutral than paper substrates, requiring adjustment of the composition of
solutions to be
dispersed into the bentonite-containing solutions.
Various solutions used in the following examples were developed and selected.
It should
be understood that these solutions are exemplary tests which were used in
connection
with an extrusion preparation method, and should not be seen as limiting.
Example 2
Equipment : Coperion TM twin screw extruder, model KSK25 WILE TM with 25 mm
diameter, UD = 40, 10 barrels.
Extrusion conditions:
Extruder temperature profile
T C
Barrel 2-3 34
Barrel 4 110
Barrel 5-6 110
Barrel 7-8 40
Barrel 9-10 40
Screw speed was 125 RPM.
Products:
Corn starch C* Gel 03420TM introduced into the extruder. Water was injected at
the
second barrel on a basis of 21.3% relative to the total mass.
The following solution was prepared and introduced at the ninth barrel:
Aqueous solution
Product TMB Cumene hydro- Lepidine Citric NaOH pH
Hydrochloride peroxide acid
(HC)
% 0,41 0,32 0,12 4,48 1,48 4,2
The extruded mixture is dried at 50 C. The product already absorbs one time
its own
weight of liquid after five contact seconds, which ensures effective urine
absorption in
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WO 2010/133001 PCT/CA2010/000789
short timeframe, facilitating contact with the reactive agents. In addition,
the studies
show that the product can absorb large amounts of liquid, approximately four
times its
own weight over an hour of contact time, which enables intense and durable
coloration
of the pellets and prolonged reactivity in case multiple urine applications
occur. See Fig
3 showing absorption versus time for different types of pellets.
In the presence of urine containing haemoglobin, a light bluish localised
coloration
appeared.
Example 3
The same installation as in Example 2 was used. The temperature profile in the
extruder
was the following:
Extruder temperature profile
T C
Barrel 2-3 34
Barrel 4 110
Barrel 5-6 110
Barrel 7-8 40
Barrel 9-10 40
Corn starch C* Gel 03420TM (CargillTM) was used.
The following solution was prepared:
Aqueous solution
Product TMB Cumene Lepidine Citric NaOH Ascorbic Tensio- pH
HC HP acid Acid active
% 0,33 0,26 0,18 3,61 1,19 0,03 0,41 4,7
The same operating conditions as in Example 2 were used. The product that was
obtained was clear yellow and reacted when put in contact with blood.
Example 4
The same installation as in Example 2 was used. The temperature profile in the
extruder
was the following:
Extruder temperature profile
T C
Barrel 2-3 35
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WO 2010/133001 PCT/CA2010/000789
Barrel 4 110
Barrel 5-6 110
Barrel?-8 41
Barrel 9-10 38
Wheat starch, Whetstar 4TM (ADMTM) was used. Exfoliated bentonite 0.5%, WT-325
mesh National Premium (BPN) was also used. The water "solvent" for the
bentonite was
20.8% of the total weight. The chromogenic solution was the same as that of
Example 3
and was introduced at barrel 9.
The product that was obtained was much more reactive. However, it has a
certain
heterogeneity due to the short mixing time in barrels 9 and 10 prior to the
extruder outlet.
An intense blue-green develops rapidly when in contact with blood, which is in
sharp
contrast with its initial light amber color prior to reaction.
Example 5
Following the extrusion installation of Example 4, a mono-screw extrruder was
introduced (AmutTM, model BrebaTM). The screw diameter was 48 mm, UD = 20/1 to
increase the mixing and enable improved homogenisation of the product.
The temperature profile in the extruder was the following:
Extruder temperature profile
T C
Barrel 2-3 35
Barrel 4 110
Barrel 5-6 110
Barrel 7-8 41
Barrel 9-10 38
Wheat starch, Whetstar 4TM (ADMTM) was used. The aqueous solution was the same
as
that used in Example 2. Improvement in homogenisation and dispersion of the
components was achieved.
Example 6
Another example was done where the reactive components were introduced
separately
into the polysaccharide mixture. At the inlet of the extruder, for each 1 kg
of starch, a 200
g aqueous solution containing 0.46 g of TMB-HC, 0.24 g lepidine and 0.5 g
bentonite,
were used. Near the outlet of the extruder, another solution (60 g) was
introduced
containing 0.12 g EDTA, 5 g citric acid and 0.36 g cumene hydroperoxide. The
same
temperature profile was used as for the previous example.
24
CA 02737489 2011-07-21
Example 7
For the purpose of comparison, some solution compositions, which may function
as
desired when impregnated into paper substrates, provided less effective
activity when
combined with the starch-bentonite mixture by extrusion:
Solution A Solution B Solution C Product %
0,35 0,35 0,54 TMB
0,27 0,27 0,42 C HP
0,18 0,18 0,18 Lepidine
3,8 3,8 3,8 Citric acid
1 1,27 1,27 NaOH
The concentrations and relative proportions used in the other examples enabled
composite materials with superior processability and color indication
properties than
those prepared using solutions A-C.
