Note: Descriptions are shown in the official language in which they were submitted.
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FIELD OF TEIE INVENTION
This invention relates to germicides effective against
bacteria fungi, viruses and particularly against spores.
VlJN~ OF T~E INVENTION
It has long been known that perishable foods can be
preserved by drying, by salting and by acid-producing
fermentations, and that chlorinated lime (calcium hypochlorite)
can be used to deodorize sewage and garbage (and wounds).
Sterilization denotes the use of either physical or chemical agents
to eliminate all viable microbes from a material, while
disinfection generally refers to the use of germicidal chemical
agents to destroy the potential inf ectivity of a material .
Sanitizing refers to procedures used to simply lower the bacterial
content of utensils used for food. Antisepsis refers to the
topical application of chemicals to a body surface to kill or
inhibit pathogenic microbes. Disinfectants are widely used for
skin antisepsis in preparation for surgery.
Sterilization of microbes exhibits the kinetics of a
first-order reaction, in which the logarithm of the number of
survivors decreases as a linear function of time of exposure.
Bacteria are the smallest organisms that contain all the
machinery required for growth and self-replication. A bacterium
includes a rigid cell wall surrounding the cytoplasmic membrane,
which itself encloses a single naked chromosome without a nuclear
membrane. ~he cytoplasmic membrane consists primarily of a bi-
layer of lipid molecules.
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The fundamental criterion of bactericidal action is loss
of the ability of the organism to propagate indefinitely, when
placed in a suitable environment. Bactericidal action suggests
microbe damage of various types, including the triggering of
5 irreversible damage to the cytoplasmic cell membrane or
irreversible impairment of the DNA (or viral RNA) replication.
Accordingly, sterilization is not identical with destruction of
microbes. Additionally, it is understood that damage to nucleic
acids (DNA or RNA) is not always irreversible, as it is known that
10 ultraviolet light-induced damage to viral nucleic acids can be
repaired by enzymatic and genetic mech;.ni Srnc .
Strongly acid and alkaline solutions are actively
bactericidal. Indeed, the pH range tolerated by most
microorganisms extends over 3 to 4 units, generally between pH
15 values of about 4.5 to 8; however, mycobacteria are relatively
resistant .
At sufficiently high concentrations, many chemicals are
bacteriostatic and even bactericidal. ~he term disinfectant is
restricted to chemical agents that are rapidly bactericidal at low
20 concentrations. In contrast to lethal radiations - which damage the
DNA (or viral RNA) - and to most bactericidal chemotherapeutic
agents - which interact irreversibly with various active metabolic
systems - most disinfectants act either by dissolving lipids from
the cytoplasmic membrane (detergents, lipid solvents) or by
25 damaging proteins (denaturants, oxidants, alkylating agents, and
sulfhydryl reagents). The rate of killing by disinfectants
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increases with concentration and with temperature. Different kinds
of disinfectants must be used for different purposes, due to the
very large variety of microbes.
Chlorine has been used as an antiseptic for more than a
5 century. Chlorine combines with water to form hypochlorous acid,
a strong oxidizing agent. Hypochlorite solutions are used to
sanitize clean surfaces in the food and the dairy industries and
in restaurants, and Cl2 gas i5 used to disinfeot water supplies
and swimming pools.
Alkylating agents, e.g. ethylene oxide, replace the
labile H atoms on -NH2 and -OH groups, which are abundant in
proteins and nucleic acids (DNA, RNA), and also on -COOH and -SH
groups of proteins. Indeed, ethylene oxide has proved to be the
most reliable substance available for gaseous disinfection of dry
15 surfaces. However, its use is more expensive and presents some
hazard of residual toxicity (being mutagenic to bacteria and
insects). Ethylene oxide is widely used to sterilize heat-
sensitive objects: plasticware; surgical equipment; hospital
bedding. These alkylating agents, in contrast to other
20 disinfectants, are nearly as active against spores as against
vegetative bacterial cells, because they can penetrate easily and
do not reguire water for their action.
Cationic detergents, e.g. benzalkonium chloride, are
known to be active against al l kinds of bacteria . They act by
25 disrupting the cytoplasmic membrane, causing release of metabolites
(the cytoplasmic molecules of the cell); in addition, their
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detergent action provides the advantage of dissolving lipid films
that may protect bacteria.
Fungi are similar to bacteria, yet one of their
differences is that their nucleic acid, consisting of multiple
5 chromosomes, is enveloped by a nuclear membrane. In some fungi
(as in some bacteria), the cell wall is surrounded by an external
capsular polysaccharide which, in the case of bacteria at least,
protects the pathogenic microbe from phagocytosis and thus play a
major role in determining virulence.
Spores are metabolic by-products in the life cycle of
some bacteria and fungi, and are often very resistant to physical
and chemical disinf ectant agents . Spores contain one or several
nuclei. Fungi produce a variety of exospores, including conidia,
chlamydospores (thick-walled and very resistant), and
15 sporangiospores. sacteria produce endospores, i.e. spores located
within the cytoplasm of the parental cell.
Bacterial endospores are differentiated cells formed
within a vegetative cell; they encase a genome in an insulating
dehydrated vehicle that makes the cell ametabolic and resistant to
20 various lethal agents, but permits subsequent germination in an
appropriate medium. Spores are much more resistant than the
parental (vegetative) cell to the lethal effect of heat, drying,
freezing, toxic chemicals and electromagnetic radiations. Spores
are formed by the invagination of a double layer of the cytoplasmic
25 membrane, which closes off to surround a chromosome and a small
amount of cytoplasm. A thin spore wall, and a thicker cortex with
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a much looser peptidoglycan, are synthesized between the two
layers outside the corte~ i5 a protein coat, rich in disulfide
cross-links and constituting up to 80% of the total protein of the
spore. The keratin-like impervious properties of the coat account
5 for the resistance to attack by deleterious chemicals, while the
dehydration and the presence of a large amount of Calcium and
dipicolinate contribute to the heat resistance.
A striking feature of spores is their huge content of
Catt, for which active transport units appear in the membrane of
10 the mother cell early during sporulation. Normally the Catt is
accompanied by a roughly equivalent amount of dipicolinic acid,
which can chelate Catt: dipicolinate is almost unique to bacterial
spores and may constitute as much as 15% of their weight.
Dehydration and ionic conditions are undoubtedly major factors in
15 stabilizing spore proteins. Ca dipicolinate evidently plays a
large role, by some as yet unknown mechanism, for its content
markedly influences heat resistance. Recent research results point
out to the control of calcium flow across the cytoplasmic membrane,
thanks to a ' 'calcium pump' ' assembly embedded into the bi-layer
20 lipid membrane of cells and defining a calcium selective through-
membrane channel ( ' 'The Cycling of Calcium as an Intracellular
messenger' ', Scientific American, October 1989) .
A virus consists of a single nucleic acid (either DNA or
RNA), and a protein shell or coat surrounding the nucleic acid the
25 complete viral particle is called a virion. Some viruses contain
lipids and carbohydrates. Virions lack constituents fundamental
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for growth and multiplication, they never ' 'grow' ': virions are by
themselves metabolically inert. Virions multiply (replicate) only
after cell-host invasion, and therefore are obligatory
intracellular parasites. ~ence, a virus is more than a simple
5 nucleoprotein (a chemical substance), but not quite a microbe (a
living entity); that is to say, a virus is not really ' 'alive' ' as
it is slightly short of the threshold of life as we define it.
Inactivation of virions is the permanent loss of
infectivity. The exposure of a population of virions to a chemical
10 (or physical) inactivating agent at a defined concentration for a
limited time, results in the inactivation of a proportion of the
virions; the others retain infectivity. Therefore, total
inactivation cannot be reached with certainty. Viral-inactivating
chemical agents include: lipid solvents (effective against
15 enveloped but not naked virions), alkylating agents, e.g. ethylene
oxide (effective against all virions); lipolytic en~.ymes (for some
enveloped virions).
A variety of germicides are on the market because of
patent rights. For example, Canadian patent 1,290,243 issued 8
20 October 1991 to Thomas AUCHINCLOSS, is directed to a germicide
composition comprising five ingredients: an inorganic halide
(sodium chloride), an oxidising agent (potassium persulfate triple
salt), sulfamic acid, an organic acid (malic acid), and an
anhydrous alkali metal phosphate. Enhancement of the virucidal
25 activity of the germicidal composition is claimed, due to alleged
buffering and chelating effect of the alkali metal phosphate.
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The AUCHINCLOSS patent relates to biocidal (bacteria, fungi,
et al) and virucidal compositions. However, a number of drawbacks
have been discovered by applicant with respect to such a germicide
compound:
5 (a) it is not effective against bacterial and fungi spores;
tb) because it is based on the release of chlorine in contact with
an oxidizing agent and with non reducible organic acids, it remains
of limited scope of activity:
(c) because of the presence of chlorine ions in sewage, it may
10 give rise to organochlorine derivatives (carcinogenic compounds)
and therefore, is undesirable in sewage water;
(d) the release of phosphates by the biocidal compound will
pollute sewage water, and again for this reason is undesirable in
waste water;
15 (e) sodium alkyl sulfate linear, a high foaming agent, is also
undesirable in sewage water, since it ~ill substantially reduce
the efficiency of the waste water treatment plants;
(f) the lowermost pH level obtained after use of the biocidal
composition is not acid enough to meet actual standards of sewage
20 water pH .
OBJECTS OF THE INVENTION
The gist of the invention is therefore to address the
need for a wide-spectrum disinfectant composition which will be
effective against bacteria, fungi, viruses, and particularly
25 against bacterial and fungi spores.
A more specific object of the invention is to produce a
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disinfectant composition particularly effective against bacterial
and fungi spores by disruption of the spore protein coat rich in
disulfide cross-links.
DESCRIPTION OF TElE INVENTION
Accordingly with the objects of the invention, there is
disclosed a disinfectant composition consisting of: (a) from 60
to ~90 9~ by weight of potassium monoperoxysulfate; (b) from 2 to
10% by weight of malic acid; (c) from 2 to 6% by weight of
sulfamic acid; (d) from 0.25 to 3% by weight of ethylene diamine
tetraacetic acid disodium salt (EDTA Na2); (e) from 1 to 15% by
weight of alkylated ether of polyethylene glycol; wherein a total
of 100 % by weight of the composition is obtained. The
disinfectant composition is sporicidal, bactericidal, fungicidal
and virucidal, as well as cleansing and deodorizing. That is to
say, the present disinfectant ccmposition will destroy the
potential infectivity of bacteria, fungi, bacterial (endo)spores,
and fungi (exo)spores, as well as inactivate substantially all
viroids coming in contact therewith. The molality of the
surfactant (alkylated ether of polyethylene glycol) should range
between 25 and 80.
Preferably, pota~sium monoperoxysulfate range in weight
between 77 to 88% of total composition, and most preferably,
constitutes about 80%. Similarly, malic acid preferably range~
between 3 and 89~, and most preferably constitutes about 4 % of
total composition. Similarly, sulfamic acid preferably ranges
between 3 to 6%, and most preferably constitutes 4% by weight of
~ 2~6200~
total composition. Similarly, EDTA Na2 preferably ranges between
1 and 2%, and most preferably constitutes 2% by weight of total
composition. ~imilarly, alkylated ether of polyethylene glycol
preferably ranges between 3 and 10%, and most preferably
constitutes 10% by weight of total composition.
The present disinfectant composition has a wide spectrum
of efficacity, while no phosphate is released. The di:iinfecting
system is based entirely upon decomposition of potassium
monoperoxysulfate, by irreducible organic acids, thus releasing
hydrogen peroxide and eventually oxygen. Again there is no
contamination of sewage water by phosphate ions. The use of non-
ionic detergent allows for penetration through the lipidic walls
of some micro-organisms, thus reaching the nucleic acid of the cell
(or viroid) to damage same and therefore prevent growth (or viral
replication). The further use of non-ionic detergent creates but
a smal 1 amount of foam, avoiding the inhibition of performance of
mechanical equipment used in the treatment of waste water and
permits a high degree of degradation (9ot9~). No coloring or
flavoring (polluting) agents are added.
Potassium peroxymonosul f ate wi l l oxidize hal ide ions into
halogens, ferrous ions into ferric, manganous ions into manganic,
and hydrogen peroxide into oxygen. Potassium peroxymonosulfate can
initiate the free radical polymerization of typical vinyl monomers,
e.g. vinyl acetate, ethyl acrylate, and acrylonitrile. Potassium
peroxymonosulfate is currently used as a bleaching agent in denture
cleansers, toilet-bowl cleaners, and laundry dry-bleachers.
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Potassium peroxymonosulfate is also known for use in removing
chloramines in swimming pools and as a disinfectant.
Accordingly, the active ingredient in the present
disinfectant composition is potassium peroxymonosulfate, in that
5 a byproduct of the dilution reaction is the potassium hydrogen
sulfate, which will lower the pH of the solution. Potassium
sulfate is present within the triple salt, but is not directly
involved in the above-noted reaction.
The use of organic acids gives rise to the formation of
10 hydrogen peroxide, which disinfecting properties are well known.
(H202 is also non pollutant) Mali~ acid is a fairly strong organic
acid, and a good chelating agent of di- and trivalent metal ions.
It is non toxic. Sulfamic acid is also a strong organic acid with
low toxicity.
The EDTA Na2 has been incorporated to the present
disinfectant composition in order to chelate the magnesium and
calcium ions, in view of:
(a) removing calcium ions, thus softening the water and enhancing
the detersive (disinfecting) action;
20 (b) removing levulinic acid under the form of sodium levulinate,
thus increasing likelihood of cytoplasmic membrane disruption and
therefore easing access to the nucleic acid for the disinfecting
action .
Moreover, the presence of an equal percentage of malic
25 and sulfamic acid produces a close to ideal range of acidity, to
ensure the complete release of hydrogen peroxide. Finally, the
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present disinfectant composition is freely soluble in cold, tepid
or warm water, and it can be used at tremendous ranges of
concentrations (from 1 to 20g per 100 ml of solution).
Since the oxyethylenated glycol surfactant has a high
power of wetting action and is non ionic, it will promote bacterial
and fun~us cytoplasmic wall disruption about the bi-layer lipid
component thereof, thus releasing cytoplasmic metabolites and
enabling inactivation of the growth-dependent nucleic acid.
The heart of the invention lies in the release of oxygen
from synergistic effect of the various ingredients present in this
disinfectant composition. Indeed, the organic acids ensure low pH
levels, essential for continuous and lengthy release of oxygen from
the oxidizing agent, potassium monoperoxysulfate. The chelating
agent, EDTA Na2 (ethylene diamine tetraacetic acid disodium salt)
deprives microbes from levulinic acid and calcium ions, (thus
inducing sporulation, when applicable). The low foaming surface
active agent will facilitate penetration of the cytoplasmic
membrane and will enable the active agent to reach the nucleic acid
of the microbe.
Indeed, synergism is verified by the release of oxygen
by the potassium peroxymonosulfate when in acidic medium, giving
rise to formation of hydrogen peroxide and sulfuric acid. The
malic and sulfamic acid provide at their selected concentrations
proper acidic conditions.
The pl~ of a 1% weight by volume concentration of the
composition is about 2.15 . After contact with the waste, the p31
.
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goes up to about 6 . 5, de~ending on the nature of the material being
treated, e.g. organic and body fluids, protein load. The end
products are mainly potassium sulfate, resulting from the catalysis
of potassium monoperoxysulfate, and sulfates of iron and sodium.
5 Potassium, calcium and magnesium malate are also found, as is EDTA
calcium. There are no organochlorine products present because
there are no halogenic ions in this composition. Any chlorine
compound present in the waste material would be oxidized to
hypochlorous acid and then to the halogen which would then combine
10 with sulfamic acid to form chlorosulfonic acid and also combine
with Na or K ions resulting in a chloride.
The disinfecting efficiency of the present composition
has been verified by applicant during experiments conducted over
a wide variety of microbes, to assess the disinfectant action of
15 different formulations of the present composition:
(a) viruses: herpes, adenovirus, parvovirus, coronavirus,
paramyxovirus, rhabdovirus, retrovirus.
(b) (bacterial) endospores: clostridium sporogenes.
(c) bacteria: streptococcus faecalis, staphylococcus aureus,
20 salmonella typhimurium, pseudomonas aeruginosa, salmonella
choleraesuis, escherichia coli, enterobacter spp., klebsiella
pneumoniae, serratia marcescens.
(d) fungi: Candida albicans, aspergillus flavus, trichophyton
mentagrophytes, penici11ium spp.
25 ExPeriment # 1
Various microbes were submitted to a 5% weight by volume
12
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concentration of a germicide composition consisting of the
f ol 1 owing ingredients:
potassium monoperoxysulfate: 80% by.weight
malic acid: 4 % by weight
5 sulfamic acid: 4 % by weight
EDTA Na2 sal t : 2% by weight
alkylated ether of polyethylene glycol: 10 % by weight
The growth inhibition percentage rate of the microbes
relative to defined contact time were as follows:
Serratia marcescens: 99.999999 % (10 minutes)
Escherichia coli: 100 % (30 min)
Klebsiella Pneumoniae: 100 % (30 min)
Pseudomonas aeruginosa: 100 % (30 min)
Mycobacterium phlei: 100 % (30 min)
sacteriophage MS-2: 99 . 99944 % (7 min)
Mycobacterium smegmatis: 99.98154 (10 min)
Clostridium sporogenes: 99.9999984 % (10 min)
Bacillus subtilis: 99.9583333 % (10 min)
sacillus cereus: 99.9858823 % (10 min)
Bacillus stearothermophilus: 99.2131147 % (10 min)
~accharomyces cerevisiae: 99.999840 % (10 min)
Ex~erimçnt # 2 - - -
Various microbes were submitted to a 5% weight by volume
concentration of a germicide composition consisting of the
25 ' following ingredients:
potassium monoperoxysulfate: 809~ by weight
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malic acid: 8 % by weight
sulfamic acid: 3 % by weight
EDTA Na2 salt: 0.5% by weight
alkylated ether of polyethylene glycol: 8.5 9~ by weight
The grcwth inhibition percentage rate of the microbes
relative to defined contact time were as follows:
~erratia marcescens: 100 % (10 minutes)
Mycobacterium smegmatis: 99.99769 (10 min)
Clostridium sporogenes: 99.999972 % (15 min)
lo Bacillus subtilis: 99.9750 96 (10 min)
Bacillus cereus: 99.99999 96 (10 min)
Bacillus stearothermophilus: 98.4852459 % (10 min)
8accharomyces cerevisiae: 99 . 992272 % (10 min)
The present disinfectant (powder) composition is
15 specifically for use in cleaning instruments, floors and bedding
and generally speaking for use in hospitals, bio-medical research
centers, health centers, veterinary hospitals and clinics. Contact
with the skin is not reccmmended because of the high pH of the
composition; however, it is not corrosive. It is freely soluble in
20 cold water.
Directions for use can be summarized as follows:
(a) routine cleaning and disinfection: prepare and wash with a
0,5% by weight solution of the present composition (e.g., 25g in
5 liters of water).
25 (b) terminal disinfection of various areas: wash carefully with
a 1% solution of the present ccmpositicn (e.g., 50g in 5 liters of
~ 2062006
wat er ) .
(c) disinfection of laboratory ware: if heavily soiled, soak in
a 1% solution for 10 minutes, then rinse with running water. If
lightly soiled, use a 1% solution of the present composition.
5 (d) disinfection of ambient air: a mechanical or manual sprayer
may be used (there is increased risk of infection caused by a high
degree of humidity) to vaporize a 0 . 2% by weight solution of the
present composition (e.g., 10g in 5 liters of water).
With spores or other highly resistant microbes, or where important
10 organic loads (feces, blood, urine) are present, the concentration
of the present disinfectant composition could be increased to 5%
weight by volume, and the contact time, increased. Also, a 0.2%
weight by volume solution of the present composition could be
applied in the form of spray (manually or mechanically).
It is understcod that a 1$ solution of the present
disinfecting solution is not irritating to the skin; however,
contact with eyes and mucous membranes should be avoided. The
present composition should be stored in a cool, dry place separate
from other chemicals. A 1% solution of this disinfectant will lose
20 20% of its potency after ten days. It is best tc use the solution
within iw~ d y5 irom d lution ~ h~ ~owd r ~om~osition.
