Note: Descriptions are shown in the official language in which they were submitted.
21~17~1
BACKGROUND OF THE INVENTION
Field of the ln-ention.
The present invention relates to a safe, biodegradable disinfectant tha~ is easy to
handle and use. while exhibiting good efficacy against a variety of bacteria and fungi.
s Broadly stated, the ionone disinfectant of this invention is a composition comprising from
about l~c to about 70~c beta ionone or pseudo-ionone, from about 1% to about 70~o DL-
limonene. dipentene. citral. terpineol, or pinene, from about l ~o to about 30% surfactant. and
about l-10~O isopropyl or ethyl alcohol, all by volume. The disinfectant composition is a
substantially clear liquid and is diluted with water for use as a foot bath for animals or for
o spraying.
Description of the Prior Art.
Both the dairy industry and the veterinary profession have long recognized that foot
rot is a hard-to-treat condition that can remove cattle from production for extended periods
of time. While a variety of methods have been used to prevent foot rot, most have met with
only limited success One formulation generally acknowledged as useful for treating foot rot
and other animal hoof problems is an ionized copper solution made by SSI Corporation and
sold under the trademark "HOOFPRO+."
However. that product and other similar products have met with only limited success
It remains clear that a need for treating such hoof diseases remains, and that treatment
20 compositions must not only exhibit efficacy, but also must be economical and
environmentally-safe. Accordingly, attention has turned to other bactericides and fungicides.
21~17,Gi
Onc cla~ss of compounds considered has t~een terpene-based mixtures previously well
known for u~se as industrial cleaning agents. The use of terpene derivatives as antifungal
agents is well known and is taught in prior patent literature. For example. U. S. Patent No.
4.963.583 and U. S. Patent No. 5,001,155 both teach the use of beta-ionone derivatives as
anti-fungal agents. U. S. Patent No. 4,474,816 teaches a method for inhibiting aflatoxin
produced by strains of Aspergilllts parasiticl-s fungi by the application of beta-ionone. U. S.
Patent No. 4.814,163 discloses an antitartar mouth deodorant comprising a zinc compound.
an ionone ketone terpene derivative, and a mint flavor as the essential active ingredients. Yet
another antitartar mouth deodorant comprising a zinc compound. an ionone ketone terpene
o derivative and a flavor as the essential active ingredients is disclosed in U. S. Patent ~o.
4.8~4.164.
Thus. while the prior art teaches the use of terpenes and/or terpene derivatives as
active ingredients for controlling some bacteria and fungi, there is no teaching in the prior
art of such a disinfectant composition useful in the veterinary profession for the control of
bacteria- and fungi-related foot diseases.
21~17G 1
SU~IMARY OF THE INVENTION
The present invention relate.s to a disinfectant compo.sition containing an ionone and
another terpene which is especially efficacious against several types of bacteria and fungi.
According to a preferred formulation. the disinfectant composition comprises sbout 459~. hy
5 volume. beta-ionone or pseudo-ionone, plus about 40%, by volume. DL-limonene, plus about
109~. by volume. surfactant. plus about 5%, by volume. isopropyl alcohol. The composition
of this invention is safe, biodegradable. easy to handle and use, and creates no significant
environmental concerns.
The preferred composition de~scribed above is added to water for use as a foot bath
o for animals. or can be spraved directly on areas to be disinfected. As is set forth in greater
detail below. the disinfectant composition has been found to be effective against a broad
range of both bacteria and fun~i. there being very few non-toxic compounds that can achieve
this result.
A preferred method for preparing the disinfectant composition of this invention is also
s described below.
The invention accordingly comprises the several steps and the relation of one or more
of such steps with respect to each of the others, and the composition possessing the feature.s,
properties. and the relation of constituents which are exemplified in the following detailed
disclosure. and the scope of the invention will be indicated in the claims.
2~ 4 1 7,61
DETAILED DESCRIPTION
Through laboratory tests it was discovered that both beta-ionone and pseudo-ionone
begin IO have an effect against several types of bacteria at approximately 20 ppm. and that
greater than 100 ppm give nearly 1005'o kill of both bacteria and fungi. This is significant
becau.se it is far less than the toxic level (LD~o in mice is greater than S gAcg) and far below
a level that would normally be expected to cause eradication of the bacteria and fungi. At
this point. experiments were conducted to maximize the efficacy and to create a practical and
usable format for the ionones. At this point it should be noted that alpha-ionone was found
to have almost no efficacy.
o Because of the safety of beta-ionone and pseudo-ionone. and because of the necessity
of delivering the active ingredients in a water solution, it was next determined that a non-
ionic surfactant having an HLB range of 10 to 18 was necessary. The preferred surfactant
s polysorbate.
Further laboratory tests confirmed that the addition of another terpene would increase
efficacv when used in combination with either the beta-ionone or the pseudo-ionone. Of the
other terpenes tested. it was determined that DL-limonene, dipentene, citral, terpineol. or
pinene were all effective. However. DL-limonene was chosen because of its "generally
regarded as safe" status.
Ina.smuch as the final mixture of beta-ionone or pseudo-ionone with DL-limonene and
polysorbate forms a cloudy mixture. isopropyl alcohol was added to give a clear appearance.
The isopropyl alcohol appears to have no bearing on the activity of the two principal active
ingredients, but may contribute slightly to the disinfectant properties of the composition.
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Most oenerally stated, the di~infectant composition of this invention compri.se~ from
about 1~ to about 70qc. by volume. beta-ionone or pseudo-ionone, from about l ~o to about
70~c. by volume, other terpene (preferably DL-limonene), from about 1% to about 30~c. by
volume, non-ionic surfactant having an HLB of about 10 to about 18. and about l-1057c. by
5 volume. isopropyl or ethyl alcohol. In a preferred composition. the invention comprises about
459O. by volume. beta-ionone. about 409c, by volume, DL-limonene, about 109c. by volume.
polysorbate 80. and about 59G isopropyl alcohol.
For use as a foot bath. the disinfectant composition is added to water at about a I to
1.000 dilution. For spraying on areas to be disinfected, the disinfectant composition is diluted
o with water or organic solvents at a range of 1:1 to 1:100.
A preferred method for preparing the disinfectant composition comprises the steps of
adding the ionone to a mixing vessel maintained at about 20-25C, with mixing, next adding
about 109c. by volume. surfactant with additional mixing for about 60 minutes. then adding
the isopropyl alcohol at a rate of about 5-15 liters per minute with additional mixing for
s about 40 minutes. then slowly adding about 409c, by volume. other terpene with additional
mixing while maintaining a substantially clear mixture, and finally blending the final mixture
for about 1~2 hours to obtain the disinfectant composition.
2l~l76l
EXAMPLE I
Utilizing the procedure ou~lined above. a disinfectant composition was prepared
comprising the followin~ ingredients:
INGREDIENT VOLUME PERCENT
s beta-ionone 45
surfactant (tween 80) 10
Isopropyl alcohol 5
DL-limonene 40.
This composition. after mixing, was dispensed into bottles in 240 ml aliquots. An
o aliquot was tested for pH by placing 0.2 ml of the formula in 50 ml of deionized water. The
pH was 5.6. Ten (10) ml of material was measured. and the specific gravity was determined
as 0.901.
Standard preparations of Ca~ldida krusei were grown in tryptic soy broth at 25C.
Standard preparations of Bncillus subtilus were prepared in fluid thioglycollate at 35C.
IS Tenfold dilution blanks from 10-1 to 10-' were made in the relevant growth media. The
undilute tubes were inoculated with a final concentration of the disinfectant composition set
forth above. The undilute bacteria and fungi were subjected to the disinfectant composition
for '0 minute~s at 25C or for 20 minutes at 4C. The tubes of undilute bacteria were diluted
in the dilution blanks. The tryptic soy broth was incubated at 20-25C and the fluid
20 thioelycollate at 35C, both for four days. The tubes were then macroscopically observed.
The result.s are set forth in Table I. following.
21~1761
.
TABLE I
TEST SAMPLE TEMP OR(;ANISM MEDIA -I -2 -3 -4 -'i -6 -7 -8 -9 -10
OF
MATL
B-ionone ~0 min B. Suhlilu5 Thio
@ 7scC
Con~rol ~() min B. Suhlilll.~ Thio + + + +
@ 7s0C
3 B-ionone ~() min B. Suhlilu~ Thio
@ 4C
4 Control '0 min B. Suhlilu~ Thio + + + +
@ 4C
B-ionone 2(l min C hru.~ei SCD +
@ 7~C
6 Conlrol ~0 min C Kn~ei SCD + + + + +
t~ 'C
7 B-ionone '() min C ~ru.~er SCD
@ 1C
10 8 Control '() min C Kmsei SCD + + + + +
@ 4C
At a 1:500 dilution. the disinfectant composition of this invention was bactericidal for
B. sl~f7till~s when exposed for 20 minutes at either 4 or 25C, and was fungicidal for C.
kl l~sei when exposed for 20 minutes at either 4 or 25C. In all cases, a 4 log reduction was
observed.
21417~1
EXAMPLE II
The disinfectant composition was further subjected to other bacteria to determine its
efficacy on those organisms.
Stap11~lococcl(s aureus, Bacillus subtilus, SnlJ~ ella h'p/7imUrilO?I, Pseudom0?tas
5 aerugin~sa, and Ca~ldid~ krusei were propagated in fluid thioglycollate or tryptic soy broth
as required by the organism growth characteristics. A 1:500 dilution of the disinfectant
composition of Example I was placed with equal amounts by volume of the organisms in
individual tubes. A positive control for each organism was diluted 1:2. Accordingly the
wor}~ing solution was a 1:1000 dilution~ or about 360 ppm of beta-ionone. Both the control
o tubes and the beta-ionone-containing tubes were incubated at 4C for 20 minutes and then
diluted tenfold in a~,up,iate growth media. The dilutions were incubated at either 20-25GC
or 35CC relevant to the media and the organism. The tubes were then read for growth 48
hours after incubation with the following results:
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TABLE II
ORGANISM CONTROL TEST REDUCTION
Candida krllsei 10~ 10 99.99%/4 logs
Bacillussubtilus 10- 10- 99%/2 logs
s Staph~lococcus 10- 10- 997O/2 logs
aureus
Pseudomonas 10~ 10~ 90%/1 1g
aeruginosa
Salmonella 10~ 10~ 90%/1 log
hphimllrium
The efficacy of the disinfectant composition against these organisms is apparent.
Thus~ at a twofold dilution greater than the studv reported in Example I, the beta-ionone is
significant in reducing organism numbers even at a relatively cool 4C temperature.
21~17~1
EXAMPLE III
Inasmuch as hairy foot wan may be caused by a spirochete, efficacy of the
disinfectant composition of this invention for use in a foot bath killing spirochetes would be
a great benefit. The following test was conducted to determine the effect of the disinfectant
s composi~ion on Leptospira canicola.
Leptospira interrogans, serovar canicola (Batch No. 89/02 from ATCC) was
propagated in I X SPL leptospira growth media (301E1210) at 29C. The organism was
passed in growth media and the culture was utili~ed 10 days later. There was no agitation
of the culture during incubation. The 10 day old culture was sampled and diluted in media.
o The culture was 100% viable as demonstrated by motility as viewed by darkfield microscopy
at 400 power. The culture was quantitated in a Petroff Hausser and Helber Counting
Chamber. The resulting culture count was 3.2 x lo6 organisms per milliliter.
The disinfectant composition of Example I was diluted 1:10 in water and mixed
thoroughly. Then. 0.1 ml of the dilution was added to 10 ml of the active leptospira culture.
IS Samples were taken at I minute, 3 minutes 45 seconds. 5 minutes, and 8 minutes post
exposure. The samples were viewed and rated by darkfield microscopy as to viability with
the following results:
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TABLE III
Time of Exposure % Reduction in Viability
Test Control
I minute 50% ~
3 minutes, 45 seconds 90% 0%
5 minutes 95% ~
8 minutes 100% 0%
At 8 minutes, the remaining sample was added to 100 ml of new growth media for any
residual live Leptospira. The sample was incubated for 10 days at 29C. No growth
occurred~ indicatin~ complete inacti~ation.
21~17~i
EXAMPLE IV
An outside laboratory was provided with a 1:1.000 dilution of the disinfectant
compo~sition of Example I for testing against animal viruses to determine viricidal activity.
The disinfectant composition. at the 1:1.000 dilution, was tested against infectious Bovine
Rhinotracheitis, Bovine Viral Diarrhea, Blue Tongue Virus and Porcine Parvo Virus. In all
four instances, no viricidal activity was noted.
EXAMPLE V
The disinfectant composition wa.s prepared according to the formula of Example I,
above. and was packaged in 8 oz. polyethylene terephthalate bottles. A dairy with 800 cows
o was given somewhat more that 60 bottles of the composition to evaluate, and the product was
used for about 21/2 months. Using a 1:1.000 dilution, a foot bath was prepared, and cattle
alked through the bath twice a day. The cattle exhibited no reluctance to passing through
the bath. and there was no skin reaction or irritation within the 75-day period. The
composition mixed well and, as stated by the dairy owner. was easier to handle than other
similar products. Observations by the owner of the cattle indicated no "cure" for the clinical
disease Hairy Foot Wart. but it was noted that spread of the disea~se was significantly reduced
from cow to cow.
214176~
EXAMPLE VI
A final ex~eriment was conducted to determine the effect. if any. of prolonged
exposure to the disinfectant composition of this invention on the hooves of cattle. sheep and
horses. Hooves from three cattle. three sheep and three horses were collected, cleaned and
5 tagged as to species. sex, animal number and which hoof. The hooves were randomly placed
into five study groups. with two hooves from each species being put into each group. The
groups were identified by letters A-E and were classified by the solution used for soaking the
hooves in that group, as fo]lows:
GROUP SOLUTION
A 10 ml disinfectant solution accordin~
to Example I with 10 liters water
B 20 ml disinfectant solution according
to Example I with 10 liters water
C 3 Ibs. copper sulfate dissolved in
s 3 gallons of water
D Plain tap water
E 50 ml HOOFPRO+, as manufactured
and sold by SSI Corporation with
10 liters water.
Before treatments with the solutions identified above. samples were removed from
each hoof and tested for hardness using a PTC Instruments Durometer. After the initial
samples were taken~ the hooves were placed in plastic bags filled with the appropriate
solution and sealed with as much air removed as possible. After I hour, the solutions were
drained out and the hooves were placed on a rack to air dry. These soakings occurred 2
- 21417~:l
time~ c~ch day with a minimum of 8 hours hetween .soakings. The soakincs continued for
14 dav.~ with samples collected on day 7 and day 14.
While all hooves showed increase in hardness from day O (no treatment) to day 14
of treatment~ the hooves treated with the disinfectant composition of this invention (Groups
5 A and B) in most instances fell within the ranges defined by the tests conducted with Groups
C. D and E. In summary. the disinfectant composition of this invention exhibited no adverse
effect on hoof hardness. Also evaluated was the composition of the hooves in regards to
Nitrogen. Phoshporus, Potassium, zinc. iron, manganese, copper, calcium and magnesium on
days 0. 7 and 14 during exposure. Results indicated no significant change in the test groups
o except for the copper sulfate which resulted in dramatic increases in copper content.
It is to be remembered that the above examples are provided for illustrative purposes
only. and are not deemed to be limiting to the scopç of the present invention. Results
obtainable using pseudo-ionone rather than beta-ionone will be quite comparable to those
stated above. Also. as previously indicated. the use of terpenes other than DL-limonene such
as. for example. dipentene. citral. terpineol. and pinene exhibit similar results of increased
efficacy over the use of ionone alone, and DL-limonene is the preferred other terpene because
of its "generally regarded as safe" status. While it is contemplated that virtually any non-
ionic surfactant having an HLB range of about 10 to about 18 is acceptable, polysorbates are
preferred surfactants. It is also to be remembered that the u~se of isopropyl or ethyl alcohol
20 is for the purpose of giving the ~Inal composition a clear appearance. and this alcohol may
be omitted.
14
21~1761
lt will thus be seen that the objects set forth above, among those made apparent from
the preceding description. are efficiently attained, and, since certain changes may be made
in carrying out the above method and in the composition set fonh without departing from the
scope of the invention, it is intended that all matter contained in the above description shall
5 be interpreted as illustrative and not in a limiting sense.
It is also to be understood that the following claims are intended to cover all of the
generic and specific features of the invention herein described, and all statements of the scope
of the invention which. as a matter of language, might be said to fall therebetween.
Particularly, it is to be understood that in said claims, ingredients or compounds recited in
o the singular are intended to include compatible mixtures of such ingredients wherever the
sense permits.
Now that the invention has been described,
