JPH04275202A - Non-toxic enzymatic sterilizing agent - Google Patents

Abstract

PURPOSE: To provide a method for sterilizing all the microorganisms including spores, Mycobacterium tuberculosis, fungi, dehydrating bacteria and viruses. CONSTITUTION: This method for sterilizing pathogens including spores, Mycobacterium tuberculosis, fungi, dehydrating bacteria and viruses in the presence of water comprises steps for selecting four components of a peroxide source, a peroxidase, a buffer agent and an iodide salt, storing the selected four components under unreacted conditions and bringing the stored four components into contact with an aqueous medium and initiating a catalytic reaction for producing a sterilizing agent for sterilizing the pathogens from the iodide salt.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION This invention relates to a method for sterilizing pathogens in the presence of non-toxic chemical disinfectants and water.

0002

BACKGROUND OF THE INVENTION Microorganisms are adapted to a wide range of environments. Sterilization is defined as the ability to inactivate all active pathogens (bacteria, viruses, fungi, algae, and spores) compared to just bacteria (bactericides) or viruses (virucides). There is. Sterilizing agents inactivate all pathogens in contrast to disinfectants, which have a limited range of activity.

Sterilization can be accomplished using heat, chemicals, irradiation, and filtration. Traditional chemical sterilants and sterilization formulations include formaldehyde, glutaraldehyde, peroxoacetic acid/heat, ultraviolet radiation, chlorine and chlorine derivatives. All known chemical sterilants that are either toxic or corrosive will pose environmental safety problems if not treated with care and proper care. Furthermore, most of these chemicals cause human irritation to the eyes, skin and olfactory nerves. Non-toxic chemical sterilants that kill all pathogens and are environmentally safe would have significant utility.

Besides limiting the activity of drugs against pathogens, toxicological properties are also important properties of chemical sterilants. The prior art described in US Pat. Nos. 4,476,231 and 4,473,550 uses organic compounds as donor molecules that, when oxidized by peroxidases, can bind to toxic and unpleasant drugs. Essentially,
These drugs 1) do not irritate the eyes or skin, 2) do not cause allergic reactions, 3) can be safely taken incidentally, 4) do not have an unpleasant odor, and 5) are mutagenic. It is important that it is not sexual. It has proven very difficult, if not impossible, to find drugs that are rapidly toxic to all pathogens and completely non-toxic and non-irritating to humans. The present invention is suitable for all types of pathogens (spores, viruses, Mycobacterium tuberculosis).
uberculosis), dehydrated pathogens, fungi). In addition, it 1) maintains a nascent sterile environment for long periods of time; 2) provides ideal toxicological properties for cold chemical sterilizers; and 3) has a significant organic load.
4) exhibits activity in the presence of viscosities of 1.5 centipoise or higher; 5) modulates hue formation and duration in peroxidase-based disinfection systems; 6) Methods and conditions to maximize activation rate are described.

Peroxidase is defined as an enzyme that reduces hydrogen peroxide to water. Peroxidases perform the reduction of hydrogen peroxide by removing electrons from donor molecules and transferring these reducing equivalents to hydrogen peroxide. Two reducing equivalents are required per hydrogen peroxide molecule. The reaction cycle consists of: 1) binding hydrogen peroxide to peroxidase; 2) transferring one electron from the first donor molecule to hydrogen peroxide; and 3) transferring one electron from the second donor molecule.
It involves transferring electrons to hydrogen peroxide. Each time an electron is removed from a donor molecule, a corresponding free radical is generated.

[0006] In US Pat. No. 4,476,231, a mechanism for the antimicrobial action of selected peroxidase-initiated reactions, which is based on the formation of free radicals, is described. The mechanism attributes the antibacterial action to free radicals or free radical by-products.

[0007] It is unexpected that bactericides will inactivate viruses, yeasts and spores. U.S. Patent No. 44850
No. 29 states that ``surprisingly and unexpectedly'' a known bactericidal formulation of glycerol monolaurate and para-hydroxybenzoic acid was used to kill the yeast Candida albicans.
and Aspergillus fumigatis (Aspergi
llus fumigatis) is inactivated.

[0008]

[Means for Solving the Problems] The present invention provides that in the presence of water,
The formulation of peroxidase, peroxide producing compounds and iodide is taught to inactivate all pathogens including spores, Mycobacterium tuberculosis, fungi, dehydrated bacteria and viruses. The compositions of the invention can remain active for several weeks and exhibit no harmful toxicological properties.

When an environment is sterilized, it is often required to maintain the activity of the sterile environment for a limited period of time. One way to actively maintain a sterile environment when using a peroxidase, peroxide, iodide system is to increase the concentration of peroxide to a concentration that acts as an inhibitor to bacterial growth. The problem with this solution is that high concentrations of peroxide can inhibit the ability of the peroxidase, peroxide and iodide system of the present invention. At high peroxide concentrations, the bactericidal ability of peroxide contributes to its bactericidal activity rather than as an enzyme-catalyzed reaction. According to the formulation of the present invention, the peroxide concentration can have a lower limit of 0.0003% by weight and an upper limit of 0.3% by weight based on the volume.

The present invention describes a method for logarithmically increasing the rate at which the sterilizing properties of peroxidase-based iodide-containing systems operate. The time required to perform a sterilization process limits applicability and affects user acceptance of the process. It has therefore been observed that by carefully adjusting the conditions and selection of donor molecules, it is possible to logarithmically increase the sterilization rate using peroxidase-based enzymatic chemistries. Increased sterilization speed
It is obtained by adjusting the iodide concentration and pH of the environment in which the peroxidase-based sterilizer acts. The rate at which peroxidase-based sterilants inactivate pathogens increases significantly when the environmental pH is adjusted between pH 4 and 6.5.

The preferred peroxidase of the present invention is horseradish peroxidase (horseradish peroxidase).
adish peroxidase) has been identified by the International Union of Biochemistry and the International Union of Pure and Applied Chemistry, Enzyme Commission with the identification number E. C. 1.11.1.
It was identified as 7. During use, the peroxidase component in the sterile composition of the present invention may be 0.00005 to 1.0 m
g/ml.

Iodide is surprisingly unique and critical to the compositions of the present invention as a source of donor molecules. Other donor molecules may be effective in producing bactericidal action, but other donor molecules do not result in sterilization. Other donor molecules may 1) increase the rate of pathogen killing at pH ranges between 4 and 6.5; 2) inactivate pathogens in the presence of significant organic load; 3) 1.5 centipoise or higher or 4) does not exhibit inactivation of dehydrated bacteria. It is not understood why iodide has uniquely beneficial properties.

Suitable iodine sources for the present invention include sodium iodide and potassium iodide, although many other iodide salts are also suitable. After dissolving in aqueous basic solvent,
Any compound that produces iodine ions is potentially suitable for use. Simple salts of iodide have the advantage of being cheap and stable. The concentration of iodide in solution is critical to the invention, with a minimum concentration of 1.0 mmol (
0.15 mg sodium iodide/ml), preferably at least 3.32 mmol (0.5 mg sodium iodide/ml).

The oxidizing agent of the present invention is hydrogen peroxide or a compound that produces hydrogen peroxide after being dissolved in an aqueous basic carrier. Also, methyl peroxide can be formulated in the product. Materials suitable for this use include hydrogen peroxide, persulfates, perphosphates, peroxyesters, urea peroxides, peroxyacids, alkyl peroxides and perborates. Furthermore, mixtures of two or more of these substances can also be used.

The components absolutely necessary to complete the present invention are 1) a peroxide source; 2) a pH of 4.0 and 6.5;
3) peroxidase; and 4) iodide salts. After mixing with aqueous medium,
A pH buffer adjusts the pH of the medium between pH 4.0 and 6.5. Suitable buffers include citrate, phosphate, phthalate and N.E.Go.
Analytical Biochemistry (
Analytical Biochemistry) (
1980), Vol. 104, p. 300,
Includes buffers referred to as "Good buffers."

[0016] The formulation must be stored in such a way that enzymatic reactions do not begin until sterile properties are required. To achieve this objective, it is acceptable to combine any two components from the group consisting of peroxide source, iodide or peroxidase and to separate these two components from the third component. Furthermore, it is possible to store the components of this system in powder or pill form such that enzymatic reactions are prevented until the time the powder or pill is mixed in a suitable carrier. If it is desired to maintain a sterile environment active for a limited period of time by operating the reaction at high peroxide concentrations, it is preferred to add the peroxide component of the reaction last. for example,
High concentrations of iodide and peroxidase can be combined in a suitable carrier and peroxide added to the carrier as a final step. If it is desired to use a powder or pill, it is also possible to prepare a formulation in which the iodide and enzyme dissolve quickly and the peroxide is released slowly over time.

[0017] Accordingly, the present invention provides a peroxide source, E. C. peroxidase selected from the group comprising 1.11.1.7 and iodide.
Selecting the species components, storing the three components in an unreacted state, introducing the three components into an aqueous medium, and producing a sterilizing agent from the iodide salt for sterilizing the medium. Broadly covers methods of sterilizing pathogens that involve catalyzing reactions that eliminate virtually all pathogens.

Additional control of sterilization is achieved by adding a buffer as a fourth component to adjust the pH of the liquid carrier between pH 4.0 and pH 6.0.
5, optimally between pH 5.0 and 6.5.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the concentration of Aspergillus fumigatis (As) active in a viscous liquid formulation after 5 minutes as a function of pH.
pergillus fumigatis).

PREFERRED EMBODIMENTS OF THE INVENTION The conditions necessary to store either formulation are such that the three main components are depleted and thus prevented from combining under the conditions in which the catalytic reaction begins. That's true. Interacting the enzyme components before intended for sterilization results in the inevitable depletion of the enzyme's substrate molecules (peroxide and iodide), thereby weakening the effectiveness of the formulation. It is acceptable to combine any two of the enzyme components of this system (peroxide source, iodide and peroxidase) and separate them from the third component to achieve this goal.

Furthermore, it is possible to store the four components of this system in powder or pill form such that enzymatic reactions are prevented until the time the powder or pill is mixed in a suitable carrier. be. If powder or pill use is desired in applications where a sterile environment is required to remain active for a limited period of time, a formulation in which the iodide and enzymes dissolve rapidly and the peroxides are released slowly over time is recommended. It is possible to use

The preferred pH range for operating the sterile enzymatic chemicals of this invention is between pH 6.5 and 4.0. Sterilization rate increases as the pH decreases from pH 6.5. Sterilization occurs more rapidly at pH 5.5 than at pH 6.0. If the pH is too low, the enzymes will not function quickly and in that environment there is a greater likelihood that human tissues that come into contact with either the sterile chemicals or the means that come into contact with the sterile chemicals will cause inflammation.

The concentration of iodide in solution is critical to the present invention because it determines the extent of pathogenic activity and the rate of its activity. The iodide concentration range is from 1.0 mmol (sodium iodide 0.15 mg/ml) to 33
．． It is 4 mmol. The preferred range of iodide is 3.3
4 mmol to 33.4 mmol. Upper amounts are preferred.

The sterilizing compositions of the present invention provide sterilization of any aqueous medium even if it has a viscosity of 1.5 centipoise or greater. In the latter case, the sterilizing composition may be formulated to form a soap formulation when mixed with water and used as an epidermal cleaner and sterilizing agent.

Next, the present invention will be explained in more detail with reference to Examples. All parts and percentages are by weight unless otherwise noted.

[0026]

[Example] Example 1 Horseradish peroxidase 10 mg, sodium iodide 1 g, sodium perborate 0.158 g
and 300 mg of sodium phosphate were dissolved in 1 liter of tap water to obtain a final pH of less than 6.5. Clinically positive specimens of M. tuberculosis were collected in Lowenstein-Jensen medium slants (
Lowenstein-Jensen medium
slants) (BBL#20909), Middle Broke 7H-9 Broth (BBL#97151) 5m
It was cultured using l. After culturing for one week, the culture was 0.
010 ml was successively diluted 10 times in saline water, cultured, and the number of colony forming units (cfu) was measured. Approximately 1
~2,000,000 cfu was added to 2 ml of the above peroxidase-containing solution and to the saline control. The peroxidase-containing samples were incubated at room temperature. sample(
0.05 ml) were collected at 30, 60, 120 and 240 minutes and cultured. These samples were incubated on Lowenstein-Jensen medium for 3 weeks at 36°C and tested for growth on a weekly basis. After 3 weeks of incubation, the number of typical "rough" colonies of M. tuberculosis was counted.

[0027]
Results Mycobacterium tuberculosis versus time
Number of active colonies per ml
time(
minute) sample
30 60
120 240 saline control
106 106
106 106 Peroxidase Chemical 5.0×105 5.0×105
10 2

The saline control did not show any inactivation of M. tuberculosis as expected. Mycobacterium tuberculosis samples treated with peroxidase-based sterilization chemicals showed that the number of active microorganisms decreased to 10 in 0 minutes.
The number decreased from 6 cells/ml to 2 cells/ml in 240 minutes.

Example 2 Pseudomonas aeruginosa
s aeruginosa) (ATCC #15442)
, Staphylococcus aureus (Staphylo
coccus aureus) (ATCC #6538)
and Salmonella cholerae suis
ATCC #107
08) were grown overnight in Anatone nutrient broth (AOAC p.65,4001a) supplemented with 5% calf serum. stainless steel pen cylinder
icylinder) were soaked overnight in 1N sodium hydroxide, washed in water, autoclaved in 0.1% asparagine, and used as bacterial carriers. The stainless steel carrier was exposed to one type of microorganism as follows. Add 60 sterile stainless steel penicylinder per 20 ml of culture.
Soaked for 5 minutes and dried on filter paper in a sterile Patri dish for 40 minutes at 35°C. To determine the number of colony forming units (cfu) on each penicylinder, the carrier was stirred in saline for 3 minutes to remove attached microorganisms and the number of cfu per penicylinder was determined.

Horseradish peroxidase 1
A powder containing 0 mg, 1 g of sodium iodide, 0.158 g of sodium perborate, and 0.30 g of sodium phosphate is dissolved in 1 liter of tap water and has a p of 6.5 or less.
I got H. Each contaminated and dried carrier cylinder was treated with sterilizer 1 in a temperature controlled water bath for 10 minutes at 20°C.
into a separate tube containing 0 ml. Each treated carrier was then transferred to 10 ml of Letheen broth containing lecithin and Tween-80 using a grappling needle in 30 second staggered intervals. These samples were incubated at 35° C. for 48 hours and the survival of microorganisms was determined. If no active microorganisms are detected, the medium is
or less cfu of challenge microorganisms and demonstrated neutralization. Repeat this protocol three times, totaling 18
0 carriers were tested with each microorganism (Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella cholerae suis).

Using the same experimental procedure, the ability of the organic donor molecules phenol and para-cresol to inactivate dehydrated bacteria was tested. In each test, instead of using 60 stainless steel penicylinder, 8 cylinders were tested. Three cylinders were used for each bacterium and the number of active bacteria per cylinder was determined by stirring the cylinders in saline water and measuring the number of colony forming units transferred into the solution. The disinfectant formulation was as described above (10 mg of horseradish peroxidase in 1 liter of tap water, 0 sodium perborate) except that the donor molecules used were phenol and para-cresol at a concentration of 0.10 mmolar.
．． 158 g and 0.30 g of sodium phosphate, a pH of below 6.5 was obtained).

Organic donor molecules (phenol and para-
Cresol) did not inactivate any of the dehydrated bacteria on either stainless steel penicylinder. This is consistent with previous observations using organic donor molecules showing that at high concentrations of dehydrated bacteria, the organic donor molecules act more as inhibitors than as bactericides. In contrast, no active microorganisms were found on any of the carriers after treatment with iodide as donor. This clearly indicates that iodide has the ability to serve as an effective donor molecule in peroxidase-based inactivation of microorganisms dried on solid surfaces.

Test results for iodide as a donor Microorganisms
initial number of microorganisms
Bacteria active after treatment
/carrier
Career 1. Pseudomonas
2.0×105
0/60 Aeruginosa Staphylococcus 2.0×107
0/60 Salmonella aureus 3.6
×104 0/60
Cholera Swiss 2. Pseudomonas・2.2×105
0/60 Aeruginosa Staphylococcus 3.0×105
0/60 Salmonella aureus 2.0
×104 0/60
Cholera Swiss 3. Pseudomonas・2.1×105
0/60 Aeruginosa Staphylococcus・2.8×105
0/60 Salmonella aureus 1.9
×105 0/60
Cholerae suis When phenol is used as a donor 4. Pseudomonas
4.6×105
5/5 Aeruginosa Staphylococcus・1.8×105
5/5 Salmonella aureus 6.0
×104 5/5
Cholera Switzerland as a donor
When using cresol 5. Pseudomonas
4.6×105
5/5 Aeruginosa Staphylococcus・1.8×105
5/5 Salmonella aureus 6.0
×104 5/5
cholera suis

Example 3 A peroxidase-based sterilization/disinfection chemical was
00,000 microorganisms/ml Bacillus pamilus
tested against Clorox bleach spores.
) (NaOCl) and sporocidin (Sporoci
din) Lot #LO853. The reagents used are shown below: A- 150 mg of sodium iodide was dissolved in 100 ml of phosphate buffered saline (PBS). B- 1 part 30% hydrogen peroxide at 100% in PBS
00 parts. C-Clorox was 0.85% in PBS. D- horseradish peroxidase at 0.
5 and 0.05 mg/ml. E- Clorox was diluted to 0.5% in oil and 0.01% in water. F-sporocidin was used undiluted, diluted to 1/16 according to the manufacturer's instructions. G- subjecting the microorganism to centrifugation and removing the culture medium;
Diluted in PBS to obtain 1 million microorganisms/ml.

The reagents were mixed in the indicated amounts in the following order:

ml phosphate buffered saline
1.75
bacillus pamilus
0.05 Sodium iodide
1.75 Hydrogen peroxide
0.50 Horseradish peroxidase 0.0
5

Bacillus with 30 million spores
A 0.50 ml volume of Pamilus was added to 4.5 ml of various dilutions of sterile chemicals based on Clorox, sporocidin and peroxidase of the invention. Samples were taken at 10, 20 and 60 minutes and at 24 hours and 2%
Diluted in trypticase soy broth with Tween-20 and 0.5% lecithin. For Clorox treated samples, 0.1% sodium sulfate was added to the trypticase soy broth mixture. Increase the number of active colonies to 48
Measurements were taken at each time point after hours of incubation to determine the time required to reduce active Bacillus pamilus by a factor of 10.

[0037]
Test results Chemical substances
1
It takes to reduce the amount by 1/0.

peroxidase (0.050mg
/ml) 9.8 minutes peroxidase (0.0050mg/ml)
12.8 minutes Clorox (0.5%)
1
．． 8 minute Clorox (0.1%)
1.8 minutes Clorox (0.01%)
1.8 min sporocidin (undiluted)
2.78 min sporocidin (1/16 dilution) 3
1.8 minutes

These results demonstrate that peroxidase-based sterilizing chemicals have the ability to inactivate spores in a time comparable to currently used sporicides.

Example 4 Bacteriophage T2 was isolated from Escherichia coli (Es
cherichia coli) strain B, 1
1000 plaque forming units (pfu) per ml, assayed by double agar layer technique in 00 mm Petri dishes.
The titer was obtained. Potassium iodide (0.010 m
l), hydrogen peroxide (0.010 ml) at 0.03% and phosphate buffer (100 mM, pH 5.0 and 7
．． 0) was added to T2 (0.8 ml). 10 μl of horseradish peroxidase was added to the sample; minimum amount of peroxidase per ml of final mixture was 0.
．． A stock solution of peroxidase (5 mg/ml in water) was diluted to be 0.0007 mg/ml. The suspension was incubated for 5 minutes. A portion of the suspension was taken out and added to a suspension of warm agar containing Escherichia coli strain B. The agar mixture was poured onto a plate and spread evenly over its surface. After solidification at room temperature, the plate was incubated at 37°C. The plates were checked for plaque formation.

At pH 5.0, no plaques were formed; at pH 7.0, plaque formation was observed on some plates at a peroxidase concentration of 0.070 mg/ml.

Example 5 Autoclaving of two matched scalpels and forceseps and shaking of a glass container containing a suspension of T2 virus in Escherichia coli strain B at 10,000 pfu/ml placed on the platform. Take out two instruments, 1
50 ml of 00 mM sodium phosphate (pH 5.5),
Placed in a mixture containing 0.50 ml (40 mg/ml) potassium iodide and 0.50 ml (0.3%) hydrogen peroxide. Horseradish peroxidase 0.
50 ml (5 mg/ml) was added and the solution was gently shaken. Controls were treated similarly except that peroxidase was omitted.

The reaction was allowed to proceed for 10 minutes and then 20 ml of sodium fluoride was added (2.3 g/l). removing the Scapels and Forceps from the solution;
It was placed on soft agar containing Escherichia coli strain B.

The agar was allowed to solidify at room temperature, incubated at 37° C. in a stationary incubator, and checked for plaque formation. The control showed plaque formation, while the sample treated with horseradish peroxidase did not form any plaques.

Example 6 Using Pseudomonas aeruginosa, pH value 4.0,
The relative rates of bacterial inactivation at 5.0, 6.0 and 7.0 were determined. An overnight culture of Pseudomonas aeruginosa was grown in 0.010 ml of potassium iodide (4
0mg/ml), hydrogen peroxide 0.010ml (0.03
%) at the indicated pH and suspended in a solution containing 0.010 ml (0.5 mg/ml) of peroxidase. Aliquots were removed from the solution at various times, serially diluted 10 times, and cultured in culture medium for 1
The number of active colonies per ml was determined.

[0045] Pseudomonas aeruginosa concentration to 10
The time required to reduce the amount to 1/2 was measured. Time (seconds)/log reduction was calculated by dividing the incubation time by the log value of the initial number of microorganisms minus the log value of the number of surviving microorganisms at each time. The inactivation rate of Pseudomonas aeruginosa was 4.5% compared to the inactivation rate at pH 7.0.
It was greater at pH values between 0 and 6.5.

Pseudomonas aeruginosa Time required to achieve a 10-fold reduction in bacteria (seconds) Sampling time pH 4.0 pH 5.0
pH6.0 pH7.0
45 <7.7
27.8 40 7
0.3 90 <7.7
28.4 37
68.7 180
<7.7 -
34 65

Example 7 Sodium iodide, hydrogen peroxide and sodium phosphate were mixed as follows: Sodium iodide, 10.0 mg/ml
0.40ml hydrogen peroxide, 0.003%
0.10ml Sodium phosphate, pH5.0, 0.10M 0.40
ml

[0048] The mixture was mixed from 0.5 mg/ml to 0.
．． 0.10 ml of horseradish peroxidase at concentrations varying up to 00005 mg/ml was added to several tubes containing 0.10 ml of horseradish peroxidase. After incubating for 5 minutes,
The spectrophotometric activity of the resulting mixture was determined for Bacillus in solution.
The activity of these mixtures was evaluated by comparison with the known sporicidal activity of the reactants, previously determined using Pamilus spores.

Sporicidal activity was greatest at concentrations between 0.05 and 0.0005 mg/ml. concentration is 0.00
The activity did not decrease significantly until it was reduced to 0.0005 mg/ml.

Example 8 The oral toxicity of a mixture of sodium iodide, peroxide, hydrogen peroxide and sodium phosphate was determined in male and female live albino Sprague Dawley.
- Dawley strain of rats by administering a dose level of 5 g/kg body weight. Mixture of sodium iodide (600 parts), sodium perborate (63 parts), horseradish peroxidase (1 part) and monobasic sodium phosphate (120 parts) 3
．． 5 g was added to 1000 ml of tap water, dissolved, and the pH was adjusted to 6.5 or less. Five female and five male rats were deprived of food for 24 hours and were administered 0.97 ml of the test solution (specific gravity 1.03) per 200 g of body weight.

Animals were observed for signs of disease and mortality for a total of 14 days. All animals are alive and 1
On the 4th, he was killed by CO2 asphyxiation and an autopsy was performed.

It was evident that all animals were not affected by oral administration of the test substance throughout the test period. All animals gained weight during the study period. The average weight gain during the study period in male rats was 113.2 g. The average weight gain during the study period in female rats was 39.2 g. Necropsy of the 10 rats used in this experiment was negative for each rat.

This composition imparts potent bactericidal, fungicidal, virucidal and sporicidal activity, and the formulation
It was surprising and unexpected that it was non-toxic orally at a dose of 5 g per day.

Example 9 The ocular toxicity of a mixture of sodium iodide, peroxide, hydrogen peroxide and monobasic sodium phosphate was tested in six different 3 month old male and female New Zealand White Rabbits. white
rabbit) (body weight 2-3 kg). Sodium iodide (600 parts), sodium perborate (63 parts), horseradish peroxidase (
1 part) and monobasic sodium phosphate (120 parts) were added to 1000 ml of tap water and dissolved to obtain a pH below 6.5. This solution was used to irrigate each rabbit's eye. Using a 1 cc syringe, 0.10 ml of the test solution was injected into the lower conjunctival sac of the left eye of each rabbit.

After instillation of the test solution, each rabbit closed its eyelids for 1 second. The contralateral eye of each rabbit was left untreated and served as a control.

[0056] Prior to instillation of the test material, all test and control eyes were judged to be free of significant ocular inflammation. To detect or confirm pre-existing corneal damage, the eyes were treated with fluorescent dye and observed under ultraviolet light in the dark.

One hour after instillation of the test substance, and even one day later,
The eyes were tested for ocular reactions on days 2, 3, 4, 7, 14 and 21. Fluorescent staining procedures were performed during each interval.

The composition imparts potent bactericidal, bactericidal, virucidal and sporicidal activity and the formulation did not cause any corneal opacification or iritis in any of the animals during the test period. This was surprising and unexpected. Minimal transient conjunctivitis was observed in 2 out of 6 animals within 24 hours after treatment. This symptom disappeared within 2 days. No accessory orbital growth or inflammation to surrounding ocular structures was observed in any of the animals. That is, under the test conditions, the sterilizing chemicals of the invention were not irritants to ocular tissue in non-irrigated eyes.

Example 10 The skin irritation intensity of a mixture of sodium iodide, peroxide, hydrogen peroxide and monobasic sodium phosphate on intact and abraded skin was determined in six different 3-month-old male and female animals. Measurements were made using New Zealand White rabbits (body weight 2-3 kg). Before applying the samples, the left flank of each rabbit was clipped free of fur with electric clippers. Sodium iodide (600 parts)
, sodium perborate (63 parts), horseradish peroxidase (1 part) and sodium monobasic phosphate (120 parts) in 100 g of tap water.
0 ml and dissolved to obtain a pH below 6.5. This solution was used to test for skin irritation.

A 0.5 ml sample of the peroxidase-based sterile test solution was applied to the skin site (1 site/rabbit) by inserting it under a 1 x 1 inch two-layer gauze patch. The patch was covered with non-inflammatory tape and the entire test site was wrapped with air permeable orthopedic stockinette. After 4 hours of exposure, the stockinette and test solution were removed. The test site was wiped with tap water to remove any remaining test solution. Observe for erythema and edema at 4 and 2 of application.
Tests were carried out after 4, 48 and 72 hours.

It is surprising that this composition imparts potent bactericidal, fungicidal, virucidal and sporicidal activity and that the formulation did not cause any irritation in any of the animals during the test period. It was both unexpected and unexpected. That is, the peroxidase-based sterilizing chemicals of the invention are non-irritating to rabbit skin.

Example 11 The intensity of allergic reactions due to peroxidase-based sterilization chemicals of the present invention was determined in experimental groups of 5 males and 5 males.
Two female Hartley guinea pigs were measured with three male and three female guinea pigs as controls. The method used for this measurement utilized local inflammatory effects at the sensitized site. Administration of adjuvants and repeated applications of peroxidase-based sterile chemicals were performed within one week to enhance hypersensitivity acquisition.

These measurements show that the peroxidase-based sterilizing chemicals of the invention do not cause local allergic reactions.

Sodium iodide (600 parts), sodium perborate (63 parts), horseradish peroxidase (1 part) and monobasic sodium phosphate (
120 parts) was added to 1000 ml of tap water and dissolved to obtain a pH of below 6.5. This solution was used for all measurements. Control animals were used to measure the skin irritation intensity of peroxidase-based sterilization chemicals. One 0.5 ml dose of peroxidase-based germicidal chemical at four concentrations (100%, 50%, 25%, 10%)
It was applied to the right and left skin sites of each of six guinea pigs by inserting it under an inch square gauze patch. Four skin sites per animal were occlusively covered with elastoplast and masking tape.

After 24 hours, the patch was removed and the skin sites were evaluated for erythema immediately and again at 48 and 72 hours. The undiluted formulation was used for hypersensitivity attacks, as neither developed skin irritation.

Allergic sensitivity to the peroxidase-based sterilization chemicals of the invention was induced as follows. Hair from 10 different laboratory animals was clipped with an Oster Animal Clipper using a #40 blade. Undiluted peroxidase-based sterilization chemistry (4
x 0.5 ml) was applied under a 1 inch square two-layer gauze patch that was applied topically to the sanded shoulder skin area. The skin was scraped again before each administration of peroxidase-based sterilizing chemicals. By exfoliating the surface, the keratin was liberated and removed, leaving the surface red-hot without bleeding. The application site was then covered with an occlusive wrap of elastoplast and masking tape. Induction administration was performed on days 0, 2, 4 and 7.

At 24, 48 and 72 hours after treatment, the animals were tested for allergic contact dermatitis. Each animal was given a score ranging from 0 for unresponsiveness to 3 for deep red erythema with vesicle formation.

This composition conferred potent bactericidal, fungicidal, virucidal, and sporicidal activity, with no response in 5 male or 5 female guinea pigs to a test regimen using this formulation. It is surprising and unexpected that they did not.

In addition, 6 animals were similarly sensitized with 25% isoeugenol/petroleum and served as a positive control group. All these animals exhibited either mild or moderate allergic contact dermatitis.

Example 12 E. coli was grown from a starter culture of brain heart infusion (BHI). Cells were harvested by centrifugation and washed twice with standard saline. The cells were suspended in 3 ml of standard saline, and a standard plate count (SPC) was performed using a BHI agar plate. This suspension was diluted in standard saline and approximately 2.38 x 107 colony forming units (cfu)/m
1 concentration was obtained. Immediately before the experiment, dilute the suspension 1/1 with sterile water.
It was diluted to 0.00 and used as a stock solution for the experiment.

Reaction buffers with pH values of 5.5, 6.0, 6.5, 6.8 and 7.0 were prepared using sodium phosphate and sodium hydroxide. Control reactions containing all reactants except peroxidase were run at each pH value and the number of microorganisms surviving at each pH was determined. p
The relative effect that H has on the rate of peroxidase-catalyzed killing was calculated by determining the percent active microorganisms based on each pH control.

The reaction conditions were as follows: 100mM reaction buffer 0.
70ml, donor (2.21mM tyrosine or 1mM
g/ml sodium iodide) 0.10ml, 0.3%
0.10ml hydrogen peroxide, 0.050ml bacteria and 0
．． Consists of 0.10 ml of horseradish peroxidase solution at a concentration of 50 mg/ml. Control reactions added 0.10 ml of distilled water and did not contain peroxidase. At each pH value, the bacteria and buffer were mixed and then 0.8 ml of this suspension was aliquoted into two separate tubes. One of these tubes served as a control, and peroxidase was added to the other.

The reaction was started and incubated for 15 minutes at 37°C. Samples were taken from each tube and diluted (0.25 mol) in a solution of sodium fluoride to generate SPC for each reaction. The number of active microorganisms in the tyrosine control reaction did not change by more than 16%. All bacteria were inactivated when sodium iodide was used as a donor molecule with peroxidase. Like the effect of pH when using tyrosine as a donor molecule,
The number of active microorganisms did not change significantly. The percentage of microorganisms surviving at each pH for tyrosine and sodium iodide is shown below.

Percentage of active microorganisms
Tyrosine pH 5.5
6.0 6.5 6.8
7.0 E. Collie 70
67 75 71
73
Sodium iodine pH
5.5
6.0 6.5 6.8
7.0 E. Collie 0
0 0 0
0

Example 13 Determining the activity of iodide, phenol and o-cresol served as donor molecules in a peroxidase-based bactericidal reaction as a function of viscosity in the range of 1 and 2.6 centipoise (cp) Measured by viscosity.

[0076] Staphylococcus aureus (ATC
C 6538) was grown overnight from a starter culture in brain heart infusion (BHI) and collected by centrifugation. Cells were washed twice in standard saline and resuspended in 5 ml of standard saline. Perform a standard plate count (SPC)
The number of colony forming units (cfu)/ml was determined and the stock was prepared using 0.1 molar sodium phosphate,
Diluted to 8.20 x 10E7 cfu/ml at pH 7.0. Phenol (10.6mM), o-cresol (92mM).
4mM) and sodium iodide (1.5mg/ml)
A stock solution of was prepared for use as a donor molecule.

Glucose, glycerol and fructose were used to increase viscosity. Sterilize the reaction with various levels of thickener (glucose, fructose or glycerol) such that the final viscosity is between 1 and 2.6 cp.
The operation was carried out in a solution containing . At each concentration of thickener, control reactions were run without peroxidase. The number of microorganisms in the control was compared to the number of microorganisms in the reaction to determine the relative effectiveness at a given viscosity.

[0078] For the bactericidal reaction, 0.5 ml of the preserved bacterial suspension,
0.1 ml of 0.05 mg/ml peroxidase, 0.1 ml of donor molecule, 0.1 ml of 0.03% hydrogen peroxide.
and 0.8 ml of thickener at various concentrations. The microorganism concentration in the initial reaction is 5.
It was determined to be 12×10E6/ml. The reaction was incubated at 37°C for 15 minutes. Take out a portion, dilute 1/10 in sodium fluoride solution, and SP
C to determine the concentration of active microorganisms.

At each viscosity level, iodide inactivated all microorganisms in the solution. When using phenol or o-cresol as the donor, increasing the viscosity above 1 cp decreased the percentage of microorganisms inactivated. Both phenol and o-cresol had no effect when increasing the viscosity above 2.4 centipoise.

Glucose as a thickener
Percentage of microorganisms remaining
iodide
Phenol o-cresol viscosity (
cp) 1.0 0
80.8
84.6 1.194 0
99.3
94.8 1.459
0 103
108 2.453
0 97.3
101

Fructose as a thickener
Percentage of microorganisms remaining
iodide
Phenol o-cresol viscosity (
cp) 1.0 0
86.0
86.3 1.196 0
93.0
97.4 1.480
0 96.5
95.9 2.557
0 101
104

Glycerol as a thickener
Percentage of microorganisms remaining
iodide
Phenol o-cresol viscosity (
cp) 1.0 0
91.5
86.3 1.218 0
98.6
97.4 1.530
0 95.2
95.9 2.632
0 99.4
104

[0083]

According to the present invention, it is possible to obtain a non-toxic sterilizing agent that sterilizes all pathogens including spores, Mycobacterium tuberculosis, fungi, dehydrated bacteria and viruses in the presence of water. can.

[Brief explanation of the drawing]

FIG. 1 shows the reduction of active Aspergillus fumigatis in a viscous liquid formulation after 5 minutes as a function of pH.

Claims (16)

[Claims] 1. A method for sterilizing pathogens, including spores and Mycobacterium tuberculosis, in the presence of water, characterized in that it consists of continuously generating iodine anions. 2. The method of claim 1, further comprising adding a buffer to adjust the pH of the aqueous medium to a preferred pH range of 4.0 to 6.5. Claim 3: (a) Four components: peroxide source;
E. C. (b) selecting a peroxidase selected from the group comprising 1.11.1.7, a pH buffer that maintains the pH below 6.5, and an iodide salt with a minimum concentration of 1.67 mmolar; storing the four components in an unreacted state; and (
c) contacting said four components with an aqueous medium to cause a catalytic reaction that produces a sterilizing agent for sterilizing pathogens from said iodide salt, in the presence of water. and methods for sterilizing pathogens including Mycobacterium tuberculosis. 4. The method for sterilizing pathogens in the presence of water according to claim 3, wherein the concentration of iodide salt relative to peroxide is 0.5 to 5.0 mg/ml. 5. Peroxidase is present in the active composition.
．． 00005 to 1.0 mg/ml, and the peroxide is present in an amount of 0.0003% to 0.3% by weight relative to the volume of the active composition.
Sterilization method as described. 6. The method of claim 3, further comprising adding a buffer to adjust the pH of the aqueous medium to a preferred pH range of 4.0 to 6.5. 7. The method according to claim 6, wherein the pH is adjusted between pH 5.0 and pH 6.5. 8. The method of any one of claims 3 to 7, wherein the components are combined in an aqueous basic environment with a viscosity of 1.5 centipoise or greater. 9. The method of claim 8, wherein the ingredients are combined in an aqueous basic environment containing one or more surfactants, foaming agents, thickeners, wetting agents, and foam stabilizers. 10. A composition according to claim 3, wherein the iodide salt is sodium iodide or potassium iodide and the molar ratio of iodide to peroxide is between 0.5 and 5.0. [Claim 11] The iodide salt has a final concentration of 1.5 mg.
/ml sodium iodide, sodium perborate 0
．． 157 mg/ml, horseradish peroxidase 0.005 mg/ml, and monobasic sodium phosphate 0.30 mg/ml. 12. The method of any one of claims 3 to 7, wherein the resulting composition is non-toxic to mammalian cells and tissues. 13. The method according to claim 3, wherein the composition obtained is non-toxic after oral administration to mammals. 14. The method of any one of claims 3 to 7, wherein the resulting composition is non-inflammatory to unirrigated mammalian eye tissue. 15. A method according to claim 3, wherein the composition obtained is non-inflammatory to mammalian epidermal tissue. 16. The method according to claim 3, wherein the composition obtained is non-allergenic to mammals.