SK6495A3 - Method of microorganism killing - Google Patents

Abstract

A surface germicidal composition comprises a dyestuff which is capable of photo-dynamic inactivation of micro-organisms. The dyestuff preferably generates singlet oxygen on exposure to light and is substantive to microorganisms, conveniently being selected from the group comprising Rose Bengal, Erythrosin B and phthalocyanin sulphonates. The composition may optionally include other ingredients such as one or more surfactants (for cleaning purposes) and/or one or more solvents.

Description

Technical field

The present invention relates to germicidal compositions, in particular for topical use, i. preparations capable of disrupting or inactivating microorganisms, in particular surface-bound microorganisms.

In a broader sense, the invention provides a surface germicidal dye-containing preparation capable of photodynamically inactivating microorganisms.

BACKGROUND AND SUMMARY OF THE INVENTION

It is recommended to use a dye that develops singlet oxygen when exposed to light. The absorption of light energy a dye molecule is converted into energy or exited state (S ^ ') from its electronic ground state (S _o). Electron dirt is paired and so they are in the singlet stage in terms of spectroscopy, containing a simple energy stage in the magnetic field.

The aroused stage is short-lived and can lose energy when returning to the initial stage in a number of ways: by emission of quantum light as fluorescence; by internal conversion, since energy is degraded to heat; collision with a different substance molecule (quenching fluorescence).

The short term singlet stage may also undergo a process called intersystem crossover to a longer lasting aroused stage, the triplet stage. The stage is termed triplet because the electron at the higher energy stage is no longer spin-paired with the electron at the lower stage and the aroused stage has three energy levels in the magnetic field.

The interaction of the aroused triplet stage with molecular oxygen at the starting stage, which normally exists at the triplet stage, regenerates the initial dye state when energy is transferred to oxygen, which is promoted to the electronically aroused singlet stage. This means that, under ideal conditions, a single photosensitizer molecule can repeatedly recover its own concentration of singlet oxygen.

Singlet oxygen is highly reactive and photosensitive, the oxidation process in this way is known as photooxidation type

II. Type II photooxidation is independent of the photosensitizer used to recover singlet oxygen. An important feature of the photosensitizer is that it has a high quantum yield of the triplet formation, which means that ideally, the triplet stage should be formed for each photon absorbed. Intersystem cross-linking to the triplet stage is facilitated in the presence of heavy atoms in the molecule.

Photooxidation of any vital component of the body can result in cell death, protein, polypeptide, amino acids, allylic hydrogen lipids, tocopherols, sugars, and cellulose.

Currently preferred dyes include Bengal pink (red acid 94, Color Index No. 45440), Erythrosine B (red acid 51, Color Index No. 45430)

and phthalocyanine sulfonates such as aluminum phthalocyanine sulfonate (APS), zinc phthalosulfonate (ZPS).

- Bengal pink and Erythrosine B are known dyes in the food industry (Bengal pink is food red No. 105 and Erythosin B is food red No. 14), and Erythrosine B is included in the EEC dyes list for use in cosmetic products, so the two the colorants are well suited for use in household preparations. Mixtures of dyes may be used and in some cases it may be desirable to have a dye in the mixture that remains visible at the end of the photodynamic process.

The dye concentration in the formulation is not limited and typically up to 100 ppm, good results were obtained with concentrations ranging from 10ppm to 20ppm. Lower concentrations up to 1ppm should also show significant results.

Singlet oxygen has a short lifetime and therefore a short scattering path length, so for efficiency the photosensitive dye developing singlet oxygen must be close to the target substrate. Preferred dyes are therefore direct dyes for microorganisms, i. capable of binding, typically by binding to a cell protein on the surface of an organism or other cellular components, such as cell fats.

The preferred dyes mentioned above develop singlet oxygen upon exposure to light and are direct to the protein and thus are capable of binding to microorganisms via the cellular protein. In this way, targeted killing of organisms and hence germicidal effect is possible.

It is also advantageous to use a dye which has been bleached upon exposure to light. By using a photo-bleaching dye, which is direct to microorganisms, it can be visibly concluded that microorganisms are present. As the dye is whitened, the photo-dynamic effect causes the killing or inactivation of the microorganisms. In the presence of low levels of light, both bleaching and photodynamic activity are slower, while at higher light intensities, both processes act faster. Thus, depending on the relative rates of bleaching and photo-dynamic activity, the presence of a visible dye indicates to the user that photo-dynamic inactivation of any microorganisms present is incomplete.

Photodynamic inactivation of microorganisms in suspension by dyes such as Bengal pink is known. See, for example, Journal of Applied Bacteriology 1985, (58), p. 391-400, Photochemistry and Photobiology 1988, (48), p. 607-612 (Necker et al.), And Shokuhin Eiseigaku Zasshi 1962, 10 (5), p.

344-347. However, it has now been found that suitable dyes are capable of photo-dynamic inactivation of microorganisms on surfaces and this is the basis of the present invention. It is well known that microorganisms are much more sensitive to biocides in their plankton or suspended state: they are much more difficult to inactivate when attached to surfaces, representing their usual or preferred state. Microorganisms are normally on surfaces in the form of biofilms that are incorporated into a matrix of extracellular material. This extracellular material can sometimes be referred to in the literature as adhesin. Therefore, it is not usual for a process that acts on the microorganisms at their plankton stage to act on surface-bound organisms without modification. Surface-bound microorganisms represent an important and essential source of contamination in households, institutional and industrial environments, and the present invention may allow a targeted germicidal effect on such microorganisms.

The compositions of the present invention are particularly suitable for use on hard surfaces in the home and industry, such as glass, plastic, ceramic and metal surfaces. The compositions are particularly effective for use on surfaces that can retain soil that is potentially contaminated with bacteria on surface irregularities, joints, and other relatively delimited areas.

The composition is preferably of an acidic nature, i. it has a pH in the range of 3 to 5, i. a pH of about 4, since it has been found that the acidic preparations have a substantially improved anti-potency activity

Gram-negative (G-) microorganism compared to neutral preparations. The efficacy against Gram-positive (6+) microorganisms does not appear to be significantly affected by pH. The formulations can be easily acidified using relatively weak organic acids, such as acetic acid.

Necker et al. (above) provide an overview that contradicts the claim that penetration of Bengal pink itself through the cell wall is the basis for inactivation. They report that their own results support the hypothesis of dye penetration primarily based on different G + and Gdruh inactivation. The G-bacterial sheath has another outer membrane composed essentially of a lipopolysaccharide by Necker et al. as a barrier to potentially toxic substances. A possible explanation is that the amount of protein exposed in cell membranes is very different in G + and G- species, greater in G + than in G-, with dye binding affinity that varies according to pH. The concentration of dye bound to the cell wall could therefore be a function of pH. This interpretation could explain our findings in differences in pH killing rates (but not the apparent G + resistance of B. subtilis and B. megaterium species).

There is no difference between endospore-forming B. subtilis and S. aureus, which does not form endospores in terms of their sensitivity to the bengal pink photodynamic effect in the absence of endospores. The apparent differences shown by our studies are believed to be due to the presence of endospores in B.subtilis and B. megaterium. which are clearly more resistant to singlet oxygen than native bacteria. The spores survive exposure to Bengal pink and light and then germinate to form numerous colonies. It is known that spores are difficult to stain and probably do not show affinity for Bengal pink under any of the conditions used. There is little data on spore toxicity of singlet oxygen in the literature. From studies of possibly less resistant Neurospora crassa conidia (Photochem Photobiol., 33, 349 (1981)), singlet oxygen is effective in this aspect.

The composition may optionally contain other ingredients, such as one or more surfactants, for cleaning purposes and / or one or more solvents.

The surfactant is preferably alkoxylated, more preferably ethoxylated, for example in the form of ethoxylated alcohols. The alcohol preferably has 4 to 15 carbon atoms, is in a straight or branched configuration, and its HLB value, hydrophilic-ipofi Other equilibrium, is in the range of 10 to 14, for example 12.

A wide variety of suitable surfactants are commercially available, one of which is known under the trade name Imbentin 91-35, from Kolb, a nonionic alcohol ethoxylate having an average of 5 moles of ethylene oxide per mole of alcohol.

Primary ethoxy sulfates may also be used.

Mixtures of surfactants may also be used if desired.

The surfactant is preferably nonionic or anionic, or a mixture of both types.

Preferred anionic surfactants for this purpose include primary alkyl sulfates (PAS), preferably sodium dodecyl sulfate (SDS). Commercial mixtures containing a substantial proportion of dodecyl sulfate, for example Empicol LX, are particularly preferred. Dodecyl sulfate is a known protein denaturant, is suitable for removing proteins from surfaces and is biocidal.

The formulation is preferably substantially free of cationic surfactant, but may contain minor amounts of cationic germicide.

The surfactant is an amount in the range of 0.05 to 2.5% based on the total weight of the formulation, typically 0.5% to 1.5%, for example 0.7% based on the weight of the nonionic surfactant, with any amount up to 0, 2% based on the weight of anionic surfactant.

The solvent is preferably polar and straight or branched chain C2 to C5 alcohol such as ethanol, butanol, isopropanol (propan-2-ol) (IPA), N-butoxypropan-2-ol (propylene glycol n-butyl ether), 2-butoxyethanol ( ethylene glycol monobutyl ether). Currently, the preferred solvent is IPA.

A dihydric alcohol such as ethylene glycol, water miscible ethers such as dimethoxyethane may also be used.

Mixtures of solvents may be used, if appropriate, for example mixtures of ethanol and N-butoxypropan-2-ol.

The solvent is preferably present in an amount of 2 to 20% by weight. of the total weight of the preparation.

At least some of these solvents, such as ethanol, weaken the cell walls of the microorganisms, increasing their premeability, making the microorganisms more sensitive to the penetration of singlet oxygen. This represents an increase in the killing effect of the dye on microorganisms.

It has been found that the incorporation of a surfactant can reduce the photo-dynamic effect of dyes, presumably by solubilizing the dye and preventing adsorption to the cell wall, and the use of a solvent can also reduce the photo-dynamic effect of dyes, possibly by singlet oxygen competition. However, it has been found that in the three-component formulations containing the colorant, surfactant and solvent, the reduction in the photo-dynamic effect on the colorants is less than would be expected from the combined effects of the surfactant and the solvent. Thus, the surfactant and the solvent together have a synergistic effect resulting in a reduction in the reduction of the photodynamic effect of the dye.

Thus, in a preferred embodiment, the present invention provides a surface-cleaning and germicidal composition comprising a colorant capable of photo-dynamically inactivating microorganisms further comprising a surfactant and a solvent.

The composition may contain a number of any ingredients including:

Detergent activators, preferably metal chelating agents, such as ethylenediaminotetraacetic acid (EDTA). Chelates (including EDTA) are required to permeabilize cell walls, i.e., to increase the sensitivity of organisms to biocides.

2. An electrolyte as a buffer or salt, for example Na 2 SO 4, which participates in the binding of the dye to the protein by promoting the movement of the dye from the aqueous phase to the protein salt. The electrolyte is co-present in various manufactured dye preparations commercially available, although additional electrolyte may be added if necessary. The total amount of electrolyte in the formulation should generally be in the range of 0 to 1% by weight, preferably about 0.1%.

3. Fragrances.

4. Thickeners.

The preparation is in the form of an isotropic, single-phase preparation and is mainly used as a germicide, also capable of utilizing cleaning capabilities, on hard surfaces, with a wide range of applications, including households such as kitchen and bathroom surfaces, including toilet bowl, in institutions such as schools, hospitals, etc., in the premises of factories, factories, offices, hotels, etc.

For domestic use, the formulation is preferably adapted for spray application, suitably adjusted in a container, for example with a manually operated valve or aerosol dispenser. Preferably, the container is inaccessible to light transmission.

In use, the composition is applied to a surface to be treated in a conventional manner, for example by spraying from a suitable dispenser, wiping with a carrier such as a cloth or sponge, or spilling from a container, etc. This method, especially in industrial cleaning, could be followed by exposure to a light source, for example a white light source such as a silicon halogen lamp or a fluorescent daylight source. The method could represent an alternative to the use of hazardous germicidal radiation, for example from a low pressure mercury lamp emitting resonant radiation at 254 nm. Such radiation is dangerous to exposed eyes. This is generally followed by a rinsing step, for example by wiping with a carrier, applying a flow of running water, and the like.

Thus, in another aspect, the invention provides a method of killing bacteria on a surface comprising applying the composition to a surface according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described, by way of illustration, with the following examples of Ladies and with reference to the figures in which:

Fig. 1 shows two band diagrams of log (reduction) values showing the lethal effect of Bengal pink and light on various microorganisms in suspension at pH 4 and pH 7; FIG. 1a shows the results for Gram-positive microorganisms; and FIG. 1b results for G-negative organisms and for yeast;

Figure 2 presents a few log (reduction) versus pH graphs expressing the biocidal effect of Bengal pink and light on

S. aureus and E. coli as a function of pH; Fig. 2a shows the results after 20 minutes of light exposure; 2b results after 60 min exposure;

Figure 3 shows a few graphs similar to those of Figure 3. 2, except that the Bengal pink is replaced by Erythrosine B;

Figure 4 is two band diagrams of logs (reduction) showing the photohygienic effect of different combinations of Bengal pink (RB), Imbentin C91-35 (AE), isopropanol (IPRA) and Empicol LX (PAS); 4a shows the results obtained without light exposure, and FIG. 4b results obtained with light exposure;

Figure 5 is a graphical representation of Bengal pink adsorption in E. coli with adsorbed amount (nanomoles) versus equilibrium concentration (micromoles / L), with results at pH 4 shown by squares and with results at pH 7 shown by crosses;

Figure 6 shows a pair of graphs similar to those of Figure 1. 5, which show the effect of an electrolyte, and FIG. 6a shows the results at pH 4 and FIG. 6b results at pH 7, no additive results in squares, sodium sulfate (1%) results in crosses, and sodium sulfate (5%) results in double crosses;

Figure 7 shows a pair of graphs similar to those of Figure 1. 5 showing the effect of a surfactant, and FIG. 7a shows the results at pH 4 and FIG. 7b results at pH 7, no additive results show squares, results with non-ionic surfactant (0.7%) (NI) represent squares, and results with PAS (0.7%) represent double squares;

Figure 8 shows a pair of graphs similar to those of Figure 1. 5 showing solvent effect at pH 4, no additive results in small squares, 10% IPA results in crosses, 0.7% Imbentin results in double crosses and 10% IPA and 0.7% Imbentin results in large squares;

Figure 9 graphically depicts log (reduction) versus exposure time (min) and expresses the effect of pH on the killing rate E, bengal coli, results at pH 4 are indicated by squares and results at pH 7 by crosses; and

Figure 10 is a graph similar to that of Figure 10. 9 and expresses the effect of the electrolyte at pH 7 on the bengal pink E. coli killing rate, the results without the electrolyte show squares and the results with sodium sulfate indicate crosses.

DETAILED DESCRIPTION OF THE INVENTION

Experimental procedures

Preparation of inoculum

The following microorganisms (generally each of the National Collection of Standardized Cultures (TCTC) or the American Collection of Standardized Cultures) were used for the experiments described:

bacteria:

Staphvlococcus aureus NCTC 6538 (G +) Escherichia coli NCTC 8196 (G) Pseudomonas aeruginosa NCTC 5940 (G) Enterobacter sp. NCTC 3281 (G) Klebsiella sd. ATCC 11677 (G) Bacillus subtilis NCTC 6432 (G +) Bacillus megaterium NCTC 7581 (G +)

yeast:

Candida albicans

Organisms were grown by incubation in bacteria nutrient broth overnight at 37 ° C (28 ° C for Ps. Aeruginosa) or in SABS broth (SABS is Sabourand's dextrose agar, with liquid medium in the case of SABS broth, from Oxoid Ltd.) at 28 ° C. C for yeast. The cultures were isolated by vacuum filtration using a 0.45 gm Millipore filter and were washed with a quarter concentration of Ringer's solution before resuspending in Ringer's solution (10 mL). Organisms in suspension were calculated by serial dilution and plated on nutrient agar (bacteria) or SABS agar (yeast) and total viable count (TVC) was expressed as the decimal logarithm of the number of colony forming units (CFU) in ml. The experiments were performed at pH 7 unless otherwise specified.

1) Agar diffusion disk method

Aqueous solutions containing 100 ppm dye were prepared.

• Aliquots (10ml) of each dye solution were sterilized in threaded glass universal ampoules.

* Antibiotic assay discs (13 mm from BDH) were also sterilized. All organisms were grown overnight in nutritional or SABS broth (10 ml).

For each microorganism, two nutrient agar plates (SABS for yeast) were inoculated with overnight culture (10μ1) to achieve whole plate growth. Using aseptic technique, the antibiotic disc was immersed in the first dye solution and placed on the surface of an inoculated agar plate. This was repeated with two additional dye solutions to obtain three discs on duplicate plates.

from each pair of duplicate plates was immediately placed in an incubator at a suitable temperature with minimal light. The remaining plates were placed in light boxes for 3 hours. Lighting from the light box was diffuse white light with an average intensity of 4000 lux from fluorescent daylight tubes (2x 15 watts, Exal X-ray Accessories Ltd, Hemel Hempstead).

Light intensities were measured on the diffuser surface using a Megatron DA10 headlamp. After exposure, the plates were incubated overnight and then evaluated for the inhibition zones around the disc.

Using this procedure, the following results were obtained:

Example 1 (Disk method)

Results for aqueous solutions of Bengal pink dye, Erythrosine B and aluminum phthalocyanine sulfonate (APS) (100 ppm) per

One placed by exposure to the upper part of the agar at pH after light exposure for 180 minutes as described above is summarized in Tab. 1. The results in the table are expressed as the difference (in mm) between the radius of the clear inhibition zone (area without bacterial growth on the ground agar plate) and the radius of the disc. Thus, the higher the value, the more effective the killing of bacteria.

The agar disk diffusion method has identified Bengal pink as more effective than the structurally similar Erythrosine B. It is convincing to attribute this order of difference in quantum yield to the formation of singlet oxygen. In methanol, the quantum yield for singlet oxygen formation is 0.76 for Bengal pink compared to 0.6 for Erythrosine B. However, many other factors can be expected to contribute to the observed differences, such as diffusion dye velocity or dye bond differences for agar gel or disc material.

A particular feature of the results obtained by the disk method is a clear distinction in the sensitivity of Gram-positive (G +) and Gram-negative (G-) organisms to the photodynamic effect, at least at pH 7. Possible causes of the relative resistance of G-organisms are discussed elsewhere.

2) Surface Test Test solutions

Bengal pink (20 ppm) in pH 4 buffer and:

1. No additive

2. Ethanol (10% v / v) and Imbentin C91-35 (0.7%).

3. Propan-2-ol (10% v / v) and Imbentin C91-35 (0.7%).

Sodium hypochlorite solution (0.125%) was used as a control.

Determination of the number of organisms adhering to the substrate in a Petri dish

A microorganism such as S. aureus. a suspension (0.5 ml) was added to a solution (100 ml) of a quarter strength Ringer's solution and the cfu per ml (TVC) was determined. Aliquots of these solutions (20 ml) were pipetted into sterile Petri dishes and left for 5 hours. at room temperature overnight. Then, the inoculum was pipetted into a sterile bottle and the diameter of cfu per ml remaining in the suspension was determined. The number of organisms (as cfu) per cm adhered to the Petri dish was calculated from the difference in solution concentrations.

Test method

Bacterial suspensions in a quarter strength of Ringer's solution (20ml) were pipetted into sterile plastic Petri dishes, which were allowed to stand at room temperature for 5 hours. The inoculum was then removed, the dishes were washed once with Ringer's solution by gently swirling the hands and the solution was discarded. Paired bacterially contaminated plates were treated with test solutions. An aliquot of the test solution (20 ml) was poured into one dish and the solution was decanted for 30 seconds. The plate was washed with buffer at pH 4, placed in a light box for 20 minutes. In a separate experiment, the solution in the second dish was exposed to the light box 20 minutes before it was decanted and the dish was washed with pH 4 buffer. Both experiments were repeated.

After exposure to light, one of the paired plates was overlaid with tryptone-soy agar containing 1% glucose with 0.015% neutral red cooled to about 50 ° C. Additional paired plates were stained with 0.01% acridine orange for 30 sec, washed and evaluated microscopically (Nikon Optiphot microscope equipped with 100x apochromatic immersion objective, 10x eyepiece and fluorescent attachment with B2-A combined filter / dichroic mirror and super high pressure mercury The stacked plates were incubated at 37 ° C for 48 hours until colonies of adherent non-killed bacteria had grown up, collecting neutral red and visible and counted in agar on the plate surface (AMR MacKenzie and RL Rivera-Calderon, Agar). Layering method for measuring adherence of Staphvlococcus epidermidis to 4 plastic surfaces, Appl. Environ. Mic14 robiol., 50, 1322 (1985).

Plates stained with acridine orange were evaluated and photographed. The number of stained bacteria was counted in the field of view (clusters counted as 1), which was previously determined by taking a micrometer scale photograph.

Example 2 (Surface Test)

Surface tests of control experiments using epifluorescence microscopy as described above and suspension depletion yielded similar values for the number of bacteria (S. aureus) adhered to the surface of the plastic dish (of the order of 1 million per cm).

In contrast, the positive control surface (sodium hypochlorite solution, 30 seconds) reduced the number of surviving bacteria to zero. The results for photodynamically inactivated bacteria are summarized in Tab. 2 as the decimal logarithm of the reduction (log reduction) of surviving bacteria before and after exposure, i. log (default count) - log (final count).

Example 3

Ingredients: Bengal pink (20ppm), nonionic surfactant (Imbentin C91-35, 0.7%), propan-2-ol (10%) (pH 4).

The experimental procedure corresponds to that previously described, except that the surface attachment of the bacteria required them to be in the exponential phase of growth. This was achieved by incubation in nutrient broth (for bacteria) or SABS broth (for yeast) in a plastic Petri dish for 3 hours. In Example 2, a suspension of non-growing bacteria in Ringer's solution was allowed to stand for 5 hours simply. This has worked well for S. aureus, but not for other bacteria. The results are shown in Tab. Third

Previous experience using epifluorescence microscopy suggests that where confluent growth has been obtained, this can be considered equivalent to an initial surface density of viable microbes of the order of 1 million / cm 2. The surface area of the Petri dishes used was about 57 cm 2, so that the treatment in all cases reduced the surface contamination level by at least 5 rows of amplitude (log 5).

Surface tests are more difficult to perform than suspension tests and for this reason most experiments were carried out in suspension to demonstrate the effect under varying conditions.

3) Suspension test

Preparation of solutions

Stock solutions were prepared by weighing and sterilized (except for those containing solvent):

Bengal pink (0.2%) in propan-2-ol (95%)

Nonionic surfactant (Imbentin C91-35, 14%) (sometimes abbreviated as AE, for alcohol ethoxylate) Anionic surfactant (Empicol LX, 14%) (sometimes referred to as PAS) «buffer pH 4 (citric acid (0,0, 1 M, 307 ml) + sodium dihydrogen phosphate (0.2 M, 193 ml) • buffer pH 7 (sodium ortho-phosphate (0.4 M, 468 ml) + disodium orthophosphate dodecahydrate (0.4M, 732 ml)

Buffers pH 5, 6, 8, 9 were prepared as reported by the CRC Handbook of Chemistry and Physics, 8-36, 73rd Ed., CRC Press (1992-1993)

Final concentrations in the test solutions were typically:

Bengal pink - 20 ppm ethanol - 10.0% v / v total surfactant - 0.7% w / v total volume

In some cases, different concentrations were used as specified.

Test method

Test solutions were made up in sterile Petri dishes to a depth of 5 mm (30 ml). A microorganism suspension (0.3 ml) was added to each solution and gently mixed. If Bengal Pink was to be included in the test solution, it was added only at the end to minimize light exposure. The solutions were or exposed to a light box placed in the dark (reduced light exposure conditions) or left on a laboratory table. The average intensity on the surface of the light box diffuser was 4000 lux and was measured with a Megatron DA 10 (from Megatron Ltd.). After the specified exposure times, surviving bacteria were counted as colony forming units (cfu / ml) followed by incubation after a series of dilutions and inoculated on agar. The decimal logarithm of the number of bacteria remaining as cfu / ml was determined and compared to the pre-exposure count as log (baseline) - log (finite count). The higher the value, the greater the killing of the bacteria.

Suspension tests were performed against a variety of organisms under various conditions to optimize the photodynamic effect against microorganisms, including Gram-negative organisms and yeast. The output results are summarized in the corresponding tables. In these tables, the results are expressed as the decimal logarithm of the ratio of the initial number of colony-forming units to their number after exposure, log (reduction). Using this designation, a value of 0 indicates that there were no changes in the number of organisms after exposure. The designation + before the log reduction number indicates that no growth of microorganisms (i.e. total killing) was detected.

Example 4

Suspension tests were performed as described above to demonstrate the lethal effect of Bengal pink and light on the microorganisms in suspension; in particular the synergism of low pH and ethanol solvent. Tests were performed with Bengal pink (20 ppm) alone or with ethanol (10% v / v volume, with light exposure of 20 minutes (light box). Results expressed as log values (reduction) are shown in Table 4.

Longer exposure times (60 and 100 minutes) increase efficacy against Gram negative organisms, especially at pH 7.

It will be appreciated that the efficacy against Gram negative organisms is significantly increased at pH 4 compared to pH 7.

Example 5

Suspension tests were performed as above at pH 4 and pH 7 to demonstrate the lethal effect of Bengal pink (20 ppm) and light (20 minute light box exposure) in various Gram-positive and Gram-negative microorganisms and yeasts. in suspension, and the results expressed as log (reduction) are shown in FIG. 1a and 1b, FIG. 1a shows the results for Gram-positive microorganisms and FIG. 1b results for Gram-negative microorganisms and yeast.

Example 6

Suspension tests were performed as above in the range of different pH to demonstrate the biocidal effect of Bengal pink (20 ppm) and light on S. aureus (G +) and E. coli (G-) as a function of pH and results expressed as log (reduction) are shown graphically in FIG. 2a (exposure time 20 minutes) and 2b (exposure time 60 minutes). In the figures, crosses show results for control (G-), double crosses for E. coli. inverted triangles show results for S. aureus.

Similar suspension tests were performed to demonstrate the biocidal effect of Erythrosin B and light on S. aureus and E. coli as a function of pH, and the results are graphically depicted in FIG. 3a and 3b, which are otherwise identical to FIGS. 2a and 2b. These show that Erythrosine B works similarly to Bengal pink in terms of its photobiocidal profile with pH.

Example 7

Additional suspension tests were performed as above at pH 7 using the following solutions: Non-ionic surfactant Imbentin C91-35 (0.7%) Imbentin C91-35 (0 to 7%) with Bengal pink (100 ppm) Anionic surface-active Empicol LX (0.7%) Empicol LX with Bengal pink (100 ppm)

Imbentin C91-35, Empicol LX (0.7%) and Bengal pink (100 ppm).

The results are explained in Table 5. the term darkness refers to light exposure reduction conditions better than total darkness, due to the practical problems of avoiding any light exposure.

In the table, the term light indicates the results that are the average of some experiments performed over time (20 minutes, 1 hour, 3 hours) in a statistically designed experiment.

Results obtained similarly for E. coli at pH 7 are graphically shown in the bar graphs of FIG. 4. In this figure, PAS is an abbreviation for Empicol LX, IPA is an abbreviation for isopropanol, and AE is an abbreviation for Imbentin C91-35. This figure represents mean data for AE 0.7%, PAS 0.7%, IPA 10%.

Example 8

Additional suspension tests were performed at pH 4 for E. coli using one or more of the following: pink rose 40 ppm, surfactant 0.7% (Imbentin C91-35 or Empicol LX), IPA (isopropanol) 10%. The results are shown in Tab. 6th

The following examples include suspension tests performed generally as described above, but at pH 4. Bengal pink, if used, was at a concentration of 20 ppm, although in some cases control solutions without Bengal pink were exposed to light and these results are shown in column labeled No Bengal Pink.

Examples 9, 10, 11 and 12 used the Gram positive organism of S. aureus, and Examples 13 and 14 the Gram negative organism of E. coli.

Other reagents used are exemplified. In all these examples, samples were exposed for 20 minutes in a light box. The average intensity at the diffuser surface was 4000 lux as measured by the Megatron DA 10 (from Megatron Ltd.).

Example 9

Suspension tests were performed using Bengal pink, ethanol and Imbentin C91-35, with S. aureus. The decimal logarithm of the initial concentration, the log (start) for S. aureus was 6.8. The results are shown in Tab. 7th

Example 10

Suspension tests were performed using Bengal Pink, Dowanol PnB and Imbentin C91-35, with S. aureus. The log was 6.9. The results are shown in Tab. 8th

This example shows that Dowanol PnB exhibits certain biocidal properties.

Example 11

Suspension tests were performed using Bengal pink, ethylene glycol and Imbentin C91-35, with S. aureus. The log was 6.8. The results are shown in Tab. 9th

Example 12

Suspension tests were performed using Bengal Pink, IPA and Lialet 111, with S. aureus. Lialet 111 is the trade name of a sulfate ether preparation, commercially available from Enichem, with an average chain length of 11, with an average degree of ethoxylation of 3. The log (start) was 6.7. The results are shown in Tab. 10th

Example 13

Suspension tests were performed using Bengal pink, propan-2-ol and Imbentin C91-35, with E, coli. The log was 6.8.

The results are shown in Table 11.

Example 14

Suspension tests were performed using Bengal pink, ethanol and Imbentin C91-35, with E. coli. The log was 7.1. The results are shown in Tab. 12th

Example 15

Bengal pink adsorption was determined from solution depletion concentration. Concentrations were obtained spectroscopically from absorbances measured at the maximum absorbance wavelength (ca. 549 nm) using a VPA Linton S110 spectrophotometer on supernatant liquids devoid of microbes by centrifugation.

The results are shown in Figures 5 to 8.

The results show that the photobiocidal effect depends on the dye adsorption. Dye adsorption is:

a) increased at low pH (Fig. 4);

b) increased with neutral electrolyte (which also increases the photobiocidal effect at neutral pH on E. coli (Fig. 6);

c) lowered by a surfactant (Fig. 7);

d) increased propan-2-ol (Fig. 8).

The calculations show that the amount absorbed on E. coli is in the correct order of monolayer coverage, showing that the dyes aggregate and the recalculated surface of E. coli must be evaluated lower (fimbrios and piles not considered). Brief details of the calculations are given below. Molecular dimensions of Bengal pink were taken from scale model (Catalin E. coli surface area Bengal pink area

Monolayer coverage

Measured adsorption number of molecules

Example 16

Ltd., London).

1E7 nm ²

O nm (straight)

0.5 nm ² (lateral)

5E6 (straight) or 2OE6 (side)

2-8 E-16 moles / bacteria

120 - 480 E6 per bacteria

Suspension tests were performed as above to determine the effect on the killing rate of E. coli Bengal pink (20ppm) on pH and the addition of electrolyte (sodium sulfate, 5%) at pH 7, and the results are graphically shown in FIG. 9 and 10.

Table 1 (Example 1)

The photobiocidal effect of said agents

Inhibition zones around disk microorganisms gram Type Bengal pink Erythrosine B APS WITH. aureus + 4 1 2 B. subtilis + 3 1 1 B. megaterium + 3 1 1.5 E. coli - 0 0 0 K. pneumoniae - 1.5 0 3 ps aeruginosa - 0 0 0 Enterobacter sp . 0 0 0 C. albicans 0 0 0

Table 2 (Example 2)

Lethal effect of Bengal pink and light on Staphylococcus aureus attached to plastic surface

solution Log (ratio) Bengal pink 4.7 Bengal pink + Imbentin C91-35 + ethanol 6.0

Table 3 (Example 3)

Microor- Number Results of their growth in the ganisms of experiments in agar technique

Control After exposure

WITH. aureus 1 blend into growth no growth 2 blend in growth no growth E. coli 1 big growth 45 cfu 2 blend in growth no growth K. pneumoniae 1 blend into growth no growth 2 blend in growth no growth P. aeruginosa 1 big growth 5 cfu 2 blend in growth no growth C. albicans 1 blend in growth 5 cfu 2 blend in growth 4 cfu

Table 4 (Example 4)

microorganisms Gram typ Exposure conditions without solvent with ethanol pH 7 pH 4 pH 7 pH 4 S. aureus + 7.0 7.1 7.1 6.9 B. subtilis + 0.6 2.1 3.1 0.8 B. megaterium + 1.3 0.3 1.1 0.3 E. coli - 0.2 5.3 0.1 6.9 K. pneumoniae - 0.9 5.6 0 7.0 Ps. aeruginosa - 0 7.0 0 6.9 Enterobacter sp. - 1.1 7.3 0 7.4 C. albicans 0 5.7 0 5.7 Controls (without Bengali pink) E. coli - 0.1 0.8

Table 5 (Example 7)

Microorganisms s Imbentin Imbentin / RB EMPICOL EMPICOL / RB greater dark the light dark the light dark the light dark St. aureus 4.5 4.0 8.0 3.5 4.5 4.5 5.5 5.0 E. coli 9.0 9.5 10.5 10,0 5.5 5.0 6.5 6.0 Entero. 0 0 0 0 0 0 0 0 KlebsieU a 7.0 6.0 6.0 7.0 6.0 6.0 6.0 6.0 Ps. aeruginosa. 0 0 0 0 0 0 0 0 C. albicans 0 0 1.0 1.0 2.5 1.5 3.0 0

Table 5 (Example 7) continued

Microor IMBENTIN / EMPICOL Imbentin / EMPICOL / RB BENGÁL.RUŽOVÁ -organisms the light dark the light dark the light dark St. aureus 3.0 4.0 2.0 0 4.0 0 E. coli 4.0 4.0 4.0 4.5 0 0 Entero. 0 0 0 0 0 0 Klebsiella 3.5 3.5 4.0 2.5 0 0 Ps.aerug. 0 0 0 0 0 0 C. albicans 0 0 0 0 0 0

Table 6 (Example 8)

EXPOSURE TIME 1 HOUR 2 HOURS 3 HOURS the light dark the light dark the light dark Bengal pink 3.8 0.5 7.2 0.4 7.2 0.7 RB / Imbentin C91-35 0.2 0.2 1.0 0.3 3.8 0.5 RB / Empicol LX 1.7 0.4 3.9 0.7 7.2 0.7 RB / IPA 5.4 3.6 4.7 5.0 7.2 7.2 Imbentin C91-35 0.1 0.3 0.9 Empicol LX 0.1 0.6 1.0 IPA 0.3 1.7 4.4

Table 7 (Example 9)

ethanol % Imbentin C91-35% After light « Log (reduction) without exposure of Bengal pink 5 0.2 + 6.8 5 0.6 4.6 10 0.6 + 6.8 15 0.6 + 6.8 5 - + 6.8 0.1 10 - + 6.8 - 0,4 15 - + 6.8 0.0 - 0.2 4.6 2.5 - 0.6 3.5 2.3 - - + 6.8 2.3 table 8 (Example 10)

Log (reduction)

Dowanol Imbentin After without % C91-35% light exposure Bengal pink 3 0.7 + 6,9 3 - + 6,9 4.0 - 0.7 5.2 3.4 - - + 6,9

Table 9 (Example 11)

Log (reduction)

IN Ethylene glycol% Imbentin C91-35% After light exposure without Bengal pink 10 0.7 + 6.8 10 - + 6,9 - 0,2 - 0.7 + 6.8 3.4 - - + 6.8

Table 10 (Example 12)

propane 2-ol% Lialet 111 % After light Log (reduction) without exposure of Bengal pink 15 0.5 + 6.7 15 - + 6.7 4.9 - 0.5 + 6.7 + 6.7 - - + 6.7

Table 11 (Example 13)

propane 2-ol% Imbentin C91-35% After light Log (reduction) without exposure of Bengal pink 5 0.1 2.3 10 0.1 + 6.8 10 0.5 + 6.8 10 0.7 + 6.8 - - 4.8 5 - 5.5 0.3 10 - 3.0 1.9 - 0.1 1.2 1.3 - 0.5 1.0 1.2 - 0.7 1.1 1.1

Table 12 (Example 14)

Ethanol Imbentin% C91-35%

Log (reduction)

After No Light Exposure And Bengal Pink

5 0.2 4.1 15 0.6 + 7.1 5 - 5.0 0.2 10 - + 7.1 0.2 15 - + 7.1 2.2 - 0.2 3.3 2.5 - 0.6 3.4 2.6 - - 3.9

P /

Claims (18)

PATENT CLAIMS CLAIMS 1. A method for killing microorganisms adhered to a surface, comprising forging a dye-containing composition onto a surface, wherein the dye has the ability to photodynamically inactivate microorganisms. Method according to claim 1, characterized in that the dye generates singlet oxygen upon exposure to light. Method according to claim 1 or 2, characterized in that the dye is direct for the microorganisms. Method according to claim 1, 2 or 3, characterized in that the dye fades after exposure to light. The method according to any one of the preceding claims, wherein the colorant is selected from the group consisting of Bengal pink, Erythrosine B and phthalocyanine sulfonates. The process according to any one of the preceding claims, wherein the colorant is present in an amount in the range of 1 to 100 ppm. The method according to any one of the preceding claims, characterized in that the formulation further comprises one or more surfactants. 8. The method of claim 7, wherein the surfactant is alkoxylated. The method of claim 8, wherein the surfactant is ethoxylated. Method according to claim 7, 8 or 9, characterized in that the surfactant is at least predominantly non-ionic and / or anionic. The method of any one of claims 7 to 10, wherein the surfactant is present in the composition in an amount ranging from 0.05 to 2.5% by weight. to the total weight of the preparation. A method according to any one of the preceding claims, characterized in that the composition comprises one or more solvents. Process according to claim 12, characterized in that the solvent is polar. 14. The method of claim 13 wherein the solvent is a linear or branched C2 to C5 alcohol. Process according to claim 12, 13 or 14, characterized in that the solvent is present in an amount in the range of 2 to 20% by weight. to the total weight of the preparation. Process according to any one of the preceding claims, characterized in that the pH of the preparation is in the range of 3 to 5. 17. The method of claim 16, wherein the pH of the formulation is about 4. 18. A method of killing micro-organisms adhered to a surface, comprising topically applying a dye-containing composition capable of photodynamically inactivating micro-organisms and containing a surfactant and a solvent.