JP3133336B2 - Improvements in germicidal compositions and improvements on germicidal compositions - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to bactericidal compositions (especially for surfaces), ie compositions which can destroy or inactivate microorganisms, especially microorganisms attached to surfaces.

SUMMARY OF THE INVENTION The invention, in its broader aspects, provides a surface germicidal composition comprising a dye capable of photodynamically inactivating microorganisms.

It is preferable to use a dye that generates singlet oxygen upon exposure. Due to the absorption of light energy, the dye molecule moves from its electronic ground state (S ₀ ) (electron spins are pairs and have one energy level in a magnetic field, which is a singlet state in spectroscopic terms). It is converted to a higher energy state or an excited state (S ₁ ^* ).

The excited state is short-lived and loses energy by some pathways: emission of photons as fluorescence, internal conversion as energy decay into heat, collision with molecules of different substances (quenching of fluorescence) and return to ground state. Can return.

The short-lived singlet state is also a long-lived excited state, the triplet state (high-energy-level electrons can no longer be spin-paired with lower-energy-level electrons; Because it has two energy levels, this state can be called a "triplet") and undergo a process called intrasystem crossing.

The energy is transferred to the oxygen atoms that have been advanced to the electronically excited singlet state, so that the interaction between the excited triplet state and the ground state (usually in the triplet state) oxygen molecule regenerates the dye ground state. I do. This means that in an ideal environment, a single photosensitizer molecule can generate its concentration of singlet oxygen many times.

Singlet oxygen atoms are very reactive, and photosensitized oxidation proceeding via this route is known as type II photooxidation. Type II photooxidation is independent of the photosensitizer used to generate singlet oxygen. An important feature of the sensitizer is that it must have a high triplet-forming quantum yield (ie, ideally, a triplet state must be formed for each photon absorption). Intrasystem crossing with the triplet state is facilitated by the presence of heavy atoms in the molecule.

Any biological constituent of an organism (proteins, polypeptides, amino acids, lipids having allyl hydrogen, tocophenol, sugars and cellulose) can cause cell death when subjected to photooxidation.

Currently preferred dyes include Rose Bengal (Acid
Red 94, Color Index No. 45440), erythrosin B (Acid Red 51, Color Index No. 45430) and phthalocyanine sulfonates such as aluminum phthalocyanine sulfonate (APS) and zinc phthalocyanine sulfonate (ZPS). Rose Bengal and Erythrosin B are known food colors (Rose Bengal is Food Color Red No 105, Erythrosin B is Food Color Red No 14),
Erythrosin B is on the EEC list of colorants permitted for use in cosmetics, and these two dyes are well suited for use in household compositions. Mixtures of dyes can also be used, and in some cases it may be desirable to include a visible dye in the mixture at the end of the photodynamic process.

The concentration of the dye in the composition is not critical, typically
Will be up to 0 ppm. Good results are obtained with concentrations in the range 10 ppm to 20 ppm. Concentrations as low as 1 ppm will also give reasonable results.

Because singlet oxygen is short-lived and thus has a short path length for diffusion, the photosensitizing dye that generates singlet oxygen must be close to the target material to be effective. Preferred dyes are therefore directly to (ie, capable of binding to) microorganisms, typically by binding to cellular proteins or other cellular components on the surface of the organism, such as cellular fat.

The preferred dyes described above generate singlet oxygen upon exposure, are direct to proteins, and thus are susceptible to binding to microorganisms via cellular proteins. In this way, it is possible to kill the target organism and thus to have a bactericidal action.

It is also preferred to use a dye which becomes white on exposure. By using photobleachable dyes that are direct to the microorganism, it may be possible to provide a visible target of the presence of the microorganism. As the dye becomes whiter, the photodynamic action proceeds causing death or inactivation of the microorganism. In the presence of low levels of light, bleaching and photodynamic activity are believed to proceed more slowly, while at high light intensities both processes occur more rapidly. Thus, depending on the relative rates of bleaching and photodynamic activity, the user will find that the presence of the visible dye impairs the photodynamic inactivation of all existing microorganisms.

Photodynamic inactivation of microorganisms in suspension by dyes such as Rose Bengal is known. For example, Journal o
f Applied Bacterionogy 1985, [58] 391-400
Page, Photochemistry and Photobiology 1988, [4
8] pp. 607-612 (Neckers et al.), And Shokuhin Eise
igaku Zasshi 1962, 10 (5), pages 334-347.
However, it has now been found that suitable dyes are able to photodynamically inactivate microorganisms on surfaces, and based on this the present invention was made. It is well known that microorganisms are much more susceptible to biocides in plankton or suspension. Microorganisms are much more difficult to inactivate when attached to a surface (which is its normal or favorable state). The microorganisms are usually present on the surface in the form of a "biofilm", i.e. embedded in a matrix of extracellular material. This extracellular material may be referred to in the literature as "adhesin". Thus, it is not obvious that the method of acting on microorganisms in the plankton state will act on microorganisms attached to the surface without modification. Surface-attached microorganisms represent an important and substantial source of contamination in domestic, institutional and industrial environments, and the present invention may enable targeted bactericidal action 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. In particular, the composition comprises
It is effective for the use of surface defects, joints and other relatively limited areas on surfaces where soils of possible bacteriological contamination may be present.

Since the acidic composition has been found to have a substantially enhanced effect on Gram-negative (G-) microorganisms as compared to the neutral composition, the composition is acidic, such as pH4. Preferably, it has a pH in the range of 3-5. The effect on Gram-positive (G +) microorganisms does not appear to be significantly affected by pH. The composition is a relatively mild organic acid,
It is convenient to acidify using, for example, acetic acid.

Neckers et al. (Supra) examined the contradictory evidence that penetration of the dye Rose Bengal itself through the cell wall is the nature of inactivation. They suggested that their own results supported the hypothesis of dye penetration based on the different inactivation of the first G + and G- species. The G-bacterial envelope has an additional outer membrane consisting essentially of lipopolysaccharide, and Necker et al. Considered to act as an obstacle to potentially toxic substances. Another interpretation is as follows. The amount of protein exposed on the cell wall is very different between G + and G- species due to the binding affinity for the dye, which varies with pH, with G + being higher than G-. Thus, the concentration of dye that binds to the cell wall is a function of pH. This interpretation will explain our observations of the change in kill rate and pH (however, does not account for the apparent resistance of G + Bacillus subtilis and Bacillus subtilis).

There is no difference between the endospore-forming species Bacillus subtilis and the non-spore-forming Staphylococcus aureus in sensitivity to the photodynamic effects of rose bengal in the absence of endospores. The obvious difference shown by our study is probably
Both Bacillus subtilis and Bacillus subtilis, which are significantly more resistant to singlet oxygen than the bacteria themselves, may depend on the presence of endospores. The spores survive on Rose Bengal and exposure and then germinate to form countable colonies. It is known that spores are difficult to stain and probably will not have an affinity for Rose Bengal under any conditions of use. There is little literature on the toxicity of singlet oxygen to spores. Ne
Singlet oxygen has potential in this regard from studies of conidia with the least tolerance of urospora crassa (Photochem. Photobiol., 33 , 349 (1981)).

The composition may optionally include other components, such as one or more (cleaning) surfactants and / or one or more solvents.

The surfactant is preferably alkoxylated, more preferably ethoxylated, for example in the form of an ethoxylated alcohol. The alcohol preferably has 4 to 15 carbon atoms, is in a straight or branched configuration, and
It has an HLB value (hydrophilic-lipophilic balance) of 10 to 14, for example 12.

A wide range of suitable surfactants are commercially available, one such material is the surfactant commercially available from Kolb under the trademark Imventin 91-35, which has an average of 5 moles per mole of alcohol. Nonionic C _9-11 alcohol ethoxylate with moles of ethylene oxide.

 Primary ethoxy sulfates can also be used.

 If desired, mixtures of surfactants can be used.

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 substantial amounts of dodecyl sulfate (eg Empicol L
X) is particularly preferred. Dodecyl sulfate is a known protein denaturant, suitable for removing proteins from surfaces and is biocidal.

The composition is preferably substantially free of a cationic surfactant, but may contain a minor amount of a cationic germicide.

The surfactant is preferably present in an amount of 0.05 to 2.5% by weight of the total weight of the composition, typically 0.5 to 1.5% by weight, for example 0.7% by weight of nonionic surfactant and any amount of up to 0.2% by weight of anion It is composed with an ionic surfactant.

The solvent is preferably a polar and preferably C ₂ -C ₅ alcohols, linear or branched, such as ethanol, butanol, isopropanol (propan-2-ol)
(IPN), N-butoxypropan-2-ol (propylene glycol n-butyl ether) and 2-butoxyethanol (ethylene glycol monobutyl ether). IPA is currently the preferred solvent.

Dihydric alcohols, such as ethylene glycol, and water-miscible ethers, such as dimethoxyethane, may also be used.

If desired, a mixture of solvents, such as ethanol and N-
Mixtures with butoxypropan-2-ol may also be used.

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

At least some of these solvents, such as ethanol, weaken and make the cell walls of microorganisms more permeable and more susceptible to the penetration of singlet oxygen. Such a solvent has the effect of enhancing the microbial killing effect of the dye.

The incorporation of a surfactant can reduce the photodynamic effects of the dye (possibly by solubilizing the dye and preventing adsorption to the cell wall), and the inclusion of a solvent also It has been found that photodynamic effects can be reduced (perhaps by competing for singlet oxygen). However, it has been found that when using a three-component formulation comprising a dye, a surfactant and a solvent, the reduction in the photodynamic effect of the dye is less than the expected summation effect of the surfactant and the solvent. Thus, the surfactant and the solvent have a synergistic effect, thus reducing the reduction of the photodynamic effect of the dye.

Thus, in a preferred aspect, the present invention provides a surface cleaning and disinfecting composition comprising a dye, a surfactant and a solvent capable of photodynamically inactivating microorganisms.

 The composition can include a number of optional ingredients, including:

1. A cleaning accelerator, preferably a metal chelating agent such as ethylenediaminetetraacetic acid (EDTA). Metal chelators (including EDTA) are also said to make cell walls permeable, and thus may make organisms more sensitive to biocides.

2. An electrolyte such as a buffer or salt (eg, Na ₂ SO ₄ ) helps the dye bind to the protein by promoting the movement of the dye from the aqueous phase to the protein salt. Electrolytes are generally present in various dye formulations in commercial form, but additional electrolytes may be added if desired. The total electrolyte abundance of the composition is typically between 0 and
It is 1% by weight, preferably about 0.1%.

3. fragrance.

4. Thickener.

The composition is in the form of an isotropic single-phase composition, which is particularly useful as a hard surface disinfectant (possibly also with a cleaning effect) and has a wide range of applications, domestic applications, such as bathrooms including kitchens and toilets Applications have been found for surfaces, for example in schools, hospitals and the like in facilities and for commercial buildings such as factories, offices, hotels and the like.

At least for home use, the composition is preferably formulated as a product intended for spray application, and is conveniently packaged in a suitable container, such as a container having a manual trigger spray or an aerosol propellant dispenser. The container is preferably light opaque.

In use, the composition can be applied to the surface to be treated using any convenient method, such as, for example, spraying from a suitable dispenser, smearing with a carrier such as a cloth or sponge, or pouring from a container. . Especially in industrial cleaning, this may be subsequently exposed to a light source, for example a white light source such as a quartz halogen lamp or a fluorescent "daylight" source. (This step is an alternative to using hazardous germicidal radiation, for example from a low-pressure mercury discharge lamp that produces resonant radiation at 254 nm. Such radiation is harmful to unprotected eyes.) If this is the case, this step is followed by a rinsing step, for example by drawing out with a carrier or applying a water stream.

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

The invention will be further described, by way of example, with reference to the following examples and the accompanying drawings.

FIG. 1 shows the log of the lethal effect of Rose Bengal and light on various microorganisms in suspensions at pH 4 and 7.
7 is two bar graphs of (decrease) values. FIG. 1a shows the results for Gram-positive microorganisms, and FIG. 1b shows the results for Gram-negative microorganisms and yeast.

FIG. 2 shows the log showing the biocidal effect of Rose Bengal and light as a function of pH on Staphylococcus aureus and E. coli.
5 is a pair of graphs of (decrease) versus pH. FIG. 2a shows the result after 20 minutes exposure, and FIG. 2b shows the result after 60 minutes exposure.

FIG. 3 shows a pair of graphs similar to FIG. 2 obtained using Erythrosin B instead of Rose Bengal.

Figure 4 shows Rose Bengal (RB) and Imventin C91-35
(AE), isopropanol (IPA) and Empicol LX
2 is a two bar graph of the log (decrease) showing the light hygiene effect of various combinations with (PSA). FIG. 4a shows the results obtained without exposure, and FIG. 4b shows the results obtained with exposure.

FIG. 5 is a graph showing the adsorption of Rose Bengal by Escherichia coli in terms of the amount adsorbed (nanomoles) versus the equilibrium concentration (micromoles / l). The results at pH 4 are indicated by squares and the results obtained at pH 7 are indicated by + symbols.

FIG. 6 is a pair of graphs similar to FIG. 5 showing the effect of the electrolyte. FIG. 6a shows the results at pH 4, and FIG. 6b shows the results at pH 7. No additive is indicated by a square, the result with sodium sulfate (1%) is indicated by a + sign, and sodium sulfate (5%
%) Is indicated by an asterisk.

FIG. 7 is a graph similar to FIG. 5 showing the effect of the surfactant. FIG. 7a shows the results at pH 4, and FIG. 7b shows the results at pH 7. The results without additives are indicated by squares, the results with nonionic surfactant (0.7%) (NI) are indicated by +, and PAS
(0.7%) is indicated by an asterisk (*).

FIG. 8 is a graph similar to FIG. 5 showing the effect of the solvent at pH 4. The results without additives are shown in small squares, 10
The results with% IPA are indicated by +, the results with 0.7% Imtentin are indicated by *, and the results with 10% IPA and 0.7% Imventin are indicated by large squares.

Figure 9 shows the rate of E. coli killing by Rose Bengal.
Figure 4 is a graph of the effect of pH on log (decrease) versus exposure time (minutes). The results at pH 4 are shown as squares, and the results at pH 7 are +
Shown by a mark.

Figure 10 shows the pH of Escherichia coli killing by Rose Bengal
10 is a graph similar to FIG. 9 showing the effect of the electrolyte at 7.
The results without electrolyte are indicated by squares, and the results obtained with sodium sulfate are indicated by + symbols.

Examples Experimental methods Preparation of inoculum The following microorganisms (generally the National Collection o
f Type Cultures (NCTC) or the American Type
Culture Collection (ATCC)) was used in the experiments described below.

Bacteria Staphylococcus aureus NCTC 6535 Gram positive E. coli NCTC 8196 Gram negative Pseudomonas aeruginosa NCTC 5940 Gram negative Intestinal bacteria NCTC 3281 Gram negative Klebsiella ATCC 11677 Gram negative Bacillus subtilis NCTC 6432 Gram positive Giant NCTC 7581 Gram positive Yeast Candida albicans Bacteria At 37 ° C in normal broth (28 ° C for Pseudomonas aeruginosa) and SABS broth for yeast (SABS is Sabo
Urand dextrose agar, SBAS broth is a liquid medium. The product was incubated at 28 ° C. in Oxoid Ltd) by overnight incubation. Culture
Isolated by vacuum filtration using a 0.45 um Millipore filter, washed with quarter-strength Ringer's solution and resuspended in Ringer's solution (10 ml). Serial dilution of organisms in suspension and normal agar (bacteria) or
The cells were counted by plating on SABS agar (yeast), and the total number of surviving cells (TVC) was calculated as colony forming units (cf.
It is shown as the common logarithm of the number u).

 Experiments were performed at pH 7 unless otherwise noted.

1) Agar diffusion disk method An aqueous solution containing 100 ppm of a dye was prepared. Aliquots (10 ml) of each dye solution were sterilized in glass universal screw-cap vials. Antibiotic assay disc (13m
m, BDH) was also sterilized. All organisms are normal or SA
The cells were cultured overnight in BS broth (10 ml).

For each microorganism, two normal agar plates (SABS agar for yeast) were inoculated with an overnight culture (10 ul) and confluent growth was obtained on the entire plate. Using aseptic technology
Antibiotic disks were dipped in the first dye solution and placed on the surface of the inoculated agar plate. This was repeated for the other two dye solutions to obtain three types of disks for a pair of plates.

One of the pair of plates was immediately placed in a suitable temperature incubator with minimal exposure. The remaining plates were placed on the lightbox for 3 hours. Radiation from inside the light box is a fluorescent "daylight" tube (2 x 15 watts, Ex
al X-ray Accessories Ltd, Hemal Hempstea
It was a diffuse white light with an average intensity of 4000 lux from d).
Light intensity was measured using a Megatron DA10 light meter on the surface of the diffuser. After exposure, the plates were incubated overnight to examine the area of inhibition around the disc. Using this method, the following results were obtained.

Example 1 (Disc method) A dye aqueous solution of Rose Bengal, erythrosin B and aluminum phthalocyanine sulfonate (APS) (100
Table 1 summarizes the results on agar after 180 minutes exposure at pH as described above for (ppm). In the table, the results were expressed as the difference (in mm) between the radius of the clear area of inhibition (the area without bacterial growth on the spread agar plate) and the radius of the disc. Therefore, the higher the value, the greater the number of dead bacteria.

Rose bengal was ranked as more effective than the structurally similar erythrosin B by the agar diffusion disk method. This suggests that this ranking is due to differences in quantum yield for singlet oxygen formation.
In methanol, the quantum yield of singlet oxygen formation is
Rose Bengal is 0.76 compared to 0.6 of Erythrosin B
It is. However, some other factors are also expected to contribute to the observed differences, such as differences in dye diffusion rate or dye binding to agar gel or disc material.

Characteristic of the results from the disc method is a clear difference in the sensitivity of the photodynamic action of Gram-positive (G +) and Gram-negative (G-) organisms at least at pH 7. The reasons why G-organisms can be relatively resistant are discussed elsewhere.

2) Surface test Test solution Rose bengal (20 ppm) in buffer at pH 4 and: 1. No further addition; 2. Ethanol (10% v / v) and Imventin C91-35 (0.
75); 3. propan-2-ol (10% v / v) and Imventin
C91-35 (0.7%).

A sodium hypochlorite solution (0.125%) was used as a positive control.

Evaluation of the number of organisms attached to the base of the Petri dish The microbial (eg Staphylococcus aureus) suspension (0.5 ml) was added to an aliquot (100 ml) of 4-fold diluted Ringer's solution and
The average cfu per ml was determined (TVC). Aliquots (20 ml) of these solutions were placed in sterile petri dishes and left at room temperature for 5 hours. The inoculum was pipetted into a sterile bottle and the average cfu per ml remaining in the suspension was evaluated. The number of organisms per square cm (as cfu) attached to the Petri dishes was calculated from the difference in solution concentration.

Test Method The bacterial suspension in 4-fold diluted Ringer's solution (20 ml) was placed in a sterile plastic Petri dish and left at ambient temperature for 5 hours. The inoculum was then removed and the dishes were washed once by gentle swirling with Ringer's solution and the solution was drained. 1
Plates contaminated with paired bacteria were treated with the test solution. Pour an aliquot (20 ml) of the test solution into one dish and pour the solution
Decanted after 30 seconds. The dishes were rinsed with pH 4 buffer and placed on a light box for 20 minutes. In another experiment, the solution was exposed in a second dish on a light box for 20 minutes, after which it was decanted and the dish was rinsed with pH 4 buffer. Both experiments were repeated.

After exposure, one of the plates in the set
About 5% with glucose and 0.015% Neutral Red
Covered with trypton soy agar cooled to 0 ° C. Another set of plates was stained with 0.01% acridine orange for 30 seconds, rinsed, and mounted on a microscope (100 × apochromat oil immersion objective, 10 × eyepiece and B2-A synthetic filter / 2 color mirror block and ultra high pressure mercury lamp). Having fluorescence outside (ep
Nikon "Optiphot" with ifluorescence accessory
(Microscope). The covered agar plate was incubated at 37 ° C. for 48 hours, by which time the colonies had grown out of the adherent bacteria that did not die. Colonies incorporated neutral red and were visible and counted under agar on the surface of the dish. (AMR MacKenzie and RL R
ivera-Calderon, Agar Overlay Method to Measur
e Adherence of Staphylococcus epidermidis to
Four Plastic Surfaces, Applied and Enviromen
tal Microbiology, 50, 1322 (1985)) Plates stained with acridine orange were examined and photographed. The number of stained bacteria was counted in a field of view that had been assessed by photographing on a micrometer scale (the population was counted as one).

Example 2 (Surface test) Using a direct external fluorescence microscope and no suspension
sion depletion) control experiment surface test (1
Similar values were given for the number of bacteria (Staphylococcus aureus) that could adhere to the surface of the plastic dish (of the order of 10,000 / cm ² ).

Contact with a surface positive control (sodium hypochlorite solution, 30 seconds) reduces the number of viable bacteria to zero.
The result of the photodynamically inactivated bacteria is calculated as the logarithm of the decrease in viable bacteria before and after exposure (log (reduction)), ie, l
Table 2 summarizes og (initial number) -log (final number).

Example 3 Formulation: Rose Bengal (20 ppm), nonionic surfactant (Imbentin C91-35, 0.7%), propan-2-ol (10%) (pH 4).

The experimental method is similar to that described above, except that the bacterial surface attachment required them to be in exponential growth phase. This was achieved by incubation in plastic broth (for bacteria) or SABS broth (for yeast) in plastic petri dishes for 3 hours. In Example 2, the suspension of non-proliferating bacteria in Ringer's solution was simply left for 5 hours. This worked well for Staphylococcus aureus but not for other bacteria. Table 3 shows the results.

Direct prior to using the fluorescent outside microscope experiments as long resulting fusion reproductive, this suggests that would be equivalent to an initial surface density of viable microbes of 1000000 / cm ² order I have. The surface area of the used Petri dish is about 57 cm
² , treatment in all cases reduced the level of surface contamination by at least 5 orders of magnitude (log5).

The surface test is more difficult to perform than the suspension test, and for this reason most experimental procedures demonstrating the effect of different conditions were performed in suspension.

3) Suspension test Preparation of solution The following raw material solutions were prepared by weighing (excluding those containing a solvent) and sterilizing them.

Rose Bengal in propan-2-ol (95%) (0.
2%) Nonionic surfactant (Imbentin C91-35, 14%)
(Also indicated using the abbreviation AE (alcohol ethoxylate)) Anionic surfactant (Empicol LX, 14%) (sometimes indicated as PAS) pH4 buffer (citric acid (0.1 M, 307 ml) + Dibasic sodium phosphate (0.2 M, 193 ml) pH 7 buffer (sodium dihydrogen orthophosphate (0.4
M, 468 ml) + oil disodium hydrogen phosphate dodecahydrate (0.4 M, 732 ml)) Buffers of pH 5, 6, 8 and 9 are from CRC Handbook of
Chemistry and Physics, 8 -36, the first 73 edition, CRC Pres
s (1992-1993).

The final concentration in the test solution is typically: Rose Bengal-20 ppm Ethanol-10.0% (v / v) Surfactant-0.7% (w / v) In some examples, different concentrations were used as specified. .

Test Method Test solutions were prepared in sterile plastic Petri dishes to a depth of 5 mm (30 ml). A microbial suspension (0.3 ml) was added to each solution and mixed gently. Added last to minimize exposure when in Rose Bengal test solution. Solutions were exposed on a light box, placed in the dark (reduced exposure conditions), or placed on a workbench. The average intensity of the light box diffuser surface is measured by Megatron
It was 4000 lux using a DA 10 Light meter (Megatron Ltd). After a specific exposure time, the surviving bacteria after serial dilution and plating on agar were counted as colony forming units (cfu / ml). The common log of the number of bacteria remaining (as colony forming units per ml) was determined and compared to the number before exposure as log (initial number)-log (last number). The higher the value, the higher the number of dead bacteria.

Suspension tests were performed on various organisms under various conditions to optimize the photodynamic effects of Gram negative organisms and yeast on various microorganisms. Results not mentioned are summarized in the relevant tables. In these tables, the results are expressed as the common logarithm of the ratio of the initial number of colony forming units per ml to the number remaining after exposure (lo
g (decrease)). Using this notation, the value 0 is
It means that there is no change in the number of organisms following exposure to the condition. The notation "+" before the log reduction number indicates that no microbial growth was observed (ie, all were killed).

Example 4 A suspension test was carried out as described above and showed a lethal effect on the microorganisms in the suspension of rose bengal and light, in particular the synergistic effect of low pH and ethanol solvents. Tests were conducted with Rose Bengal (20 ppm) only or ethanol (10%, (v /
v)) together with a 20 minute exposure (light box). Table 4 shows the results expressed as log (decrease) values.
Shown in

Long exposure times (60 and 100 minutes) improve performance on Gram-negative organisms, especially at pH 7.

The performance against Gram-negative organisms is much better at pH 4 than at p7H.

Example 5 Suspension tests were performed at pH 4 and pH 7 as described above, and various gram-positive and gram-negative microorganisms in suspension of rose bengal (20 ppm) and light (20 minutes exposure on light box) 1a and 1b, showing the lethal effect on yeast and yeast, expressed as log (decrease).
Is shown in the graph. FIG. 1a shows the results for gram-positive microorganisms, and FIG. 1b shows the results for gram-negative microorganisms and yeast.

Example 6 Suspension tests were performed as described above at different pH ranges, showing the lethal effect of Rose Bengal (20 ppm) and light on Staphylococcus aureus (G +) and E. coli (G-) as a function of pH. 2a (exposure time 20 minutes) and 2b (exposure time 60 minutes)
Is shown in the graph. In the figure, the + marks indicate the results for control (G-), the * marks indicate the results for E. coli, the inverted triangles indicate the results for control (G +), and the triangles indicate the results for Staphylococcus aureus. Show.

A similar suspension test was performed to show the biocidal effect of erythrosin B and light on yellow spheres and E. coli as a function of pH. The results are shown graphically in FIGS. 3a and 3b, which are otherwise similar to FIGS. 2a and 2b. They show that erythrosin B shows similarity to Rose Bengal in photobiocidal properties to pH.

Example 7 A further suspension test was performed at pH 7 as described above using the following solutions.

Nonionic surfactant Imventin C91-35 (0.7%) Imbentin C91-35 (0.7%) and rose bengal (100p
pm) Anionic surfactant Empicol LX (0.7%) Empicol LX and Rose Bengal (100 ppm) Imbentin C91-35, Empicol LX (both 0.7%)
And Rose Bengal (100 ppm) The results are shown in Table 5 below. In the table, the term "dark" indicates the condition of reduced light exposure rather than total darkness. This is because it is practically difficult to avoid exposure at all. In the tables, the term "light" indicates the average of several experiments performed over a wide range of time (20 minutes, 1 hour and 3 hours) in a statistically designed experiment.

The results similarly obtained for E. coli at pH 7 are shown as a bar graph in FIG. In this figure, PA
S is used as an abbreviation for Empicol LX, IPA is used as an abbreviation for isopropanol, and AE is used as an abbreviation for Imebentin C91-35. This figure shows the average data for 0.7% AE, 0.7% PAS, and 10% IPA.

Example 8 An additional suspension test was performed on E. coli at pH 4 using one or more of the following: Rose Bengal 40 ppm, surfactant 0.7% (Imebentin C91-35 or
Empicol LX), IPA (isopropanol) 10%. Table 6 shows the results.

The following examples are generally described above, however,
For suspension tests performed at pH 4. The concentration when using Rose Bengal is 20 ppm, but in some cases the control solution without Rose Bengal was exposed, and these results are shown in the upper column as “No Rose Bengal”.
It was shown.

In Examples 9, 10, 11 and 12, the gram-positive organism Staphylococcus aureus was used, and in Examples 13 and 14, the gram-negative organism Escherichia coli was used. Other reagents used are given in the examples. In all these examples, the samples were exposed on a lightbox for 20 minutes. The average intensity at the surface of the diffuser is measured using a Megatron DA10 light meter (Megatron Lt
d) was 4000 lux.

Example 9 Rose Bengal, ethanol and Imebentin C91-
Suspension tests were performed with Staphylococcus aureus using 35. The logarithmic log (start) of the initial concentration of Staphylococcus aureus is 6.8
Met. Table 7 shows the results.

Example 10 Rose Bengal, Dowanol PnB and Imebentin C91
Suspension tests were performed with Staphylococcus aureus using -35.
The log (start) was 6.9. Table 8 shows the results.

This example shows that Dowanol PnB has some biocidal potential.

Example 11 Rose Bengal, ethylene glycol and Imebenti
Suspension tests were performed on Staphylococcus aureus using nC91-35. The log (onset) was 6.8. Table 9 shows the results.

Example 12 A suspension test was performed on Staphylococcus aureus using Rose Bengal, IPA and Lialet 111. Lialet 111 is Eni
Trade mark for an ether sulfate formulation with an average chain length of 11 and an average degree of ethoxylation of 3 which is commercially available from chem.
The log (start) was 6.7. Table 10 shows the results.

Example 13 Rose Bengal, propan-2-ol and Imeben
Suspension tests were performed on E. coli using tin C91-35.
The log (onset) was 6.8. Table 11 shows the results.

Example 14 Rose Bengal, ethanol and Imebentin C91-
Suspension tests were performed on E. coli using 35. The log (start) was 7.1. Table 12 shows the results.

Example 15 Rose Bengal adsorption was determined from solution concentration depletion.
The concentration was determined spectrophotometrically from the absorption at the maximum absorption wavelength (ca. 549 nm) of the supernatant removed from the microorganism by centrifugation using a WPA Linton S110 spectrometer.

 The results are shown in FIGS.

The results showed that the photobiocidal effect depends on the adsorption of the dye. Dye adsorption: a) increases at low pH (FIG. 4); b) increases with neutral electrolytes (electrolytes increase the photobiocidal effect on E. coli at neutral pH) (FIG. 6); c) Decrease with surfactant (FIG. 7); d) Increase with propan-2-ol (FIG. 8).

Taking into account that the dye is believed to collect and the calculated surface of E. coli must be underestimated (not considering cilia / pilus), the amount adsorbed to E. coli by calculation is It is of the order of magnitude of a single-layer coating. Details of the calculation are shown below. The size of the Rose Bengal molecule was obtained from a scale model (Catalin L
td., London).

Escherichia coli surface area 1E7cm ² Rose Bengal area 2nm ² (plane) 0.5nm ² (side) Single layer coating 5E6 (plane) or 20E6 (side) Adsorption measured 2-8E-16 molecules / bacteria Number of bacteria 120 per bacterium 480E6 Example 16 A suspension test was performed as described above, and rose bengal (20
ppm) and electrolytes at pH 7 (sodium sulfate,
The effect of the addition of 5%) on the killing rate of E. coli was determined and the results are shown graphically in FIGS. 9 and 10, respectively.

Continued on the front page (51) Int.Cl. ⁷ Identification code FI C09B 47/04 C09B 47/04 61/00 61/00 Z (31) Priority claim number 9304732.2 (32) Priority date March 1993 9th (1993.3.9) (33) Priority country United Kingdom (GB) (56) References JP-A-58-105989 (JP, A) JP-A-63-264064 (JP, A) JP-A Sho 56-39004 (JP, A) JP-A-57-38704 (JP, A) JP-A-2-22398 (JP, A) MELVIN, I. R. , Et al. , "The Antiviral Effects of Rose Bengal and Fluorescence in" Arch. Ophthalmol. 1987, Vol. 105, No. 10, p. 1415-1417 Japan Society of Antibacterial and Fungicide Encyclopedia, Encyclopedia of Antibacterial and Antifungal Agents, Japan, Japan Society of Antifungal and Antifungal Studies, August 22, 1986, pp. 84-85, TALBET, N.M. E. FIG. , Et al. , "Sensitized Phototoxidation for Water Steward Disinfection and Detoxifica tion" Wat. Sci. Tec h. 1987, Vol. 19, No. 7, p. 1255-1258 (58) Fields investigated (Int. Cl. ⁷ , DB name) A01N 61/00 A01N 43/16 A01N 43/713 A61L 2/16 C09B 11/28 C09B 47/04 C09B 61/00 CA (STN ) REGISTRY (STN)

Claims (18)

(57) [Claims] 1. A method for killing microorganisms in a biofilm on a surface, comprising applying to the surface a composition comprising a dye capable of photodynamically inactivating the microorganisms. 2. The method of claim 1 wherein said dye generates singlet oxygen upon exposure. 3. The method according to claim 1, wherein the dye is direct to microorganisms. 4. The method according to claim 1, wherein the dye is whitened by exposure. 5. The method according to claim 1, wherein said dye is selected from the group consisting of rose bengal, erythrosine B and phthalocyanine sulfonate. 6. The method according to claim 1, wherein the dye is present in an amount of from 1 to 100 ppm. 7. The method according to claim 1, wherein the composition further comprises one or more surfactants. 8. The method according to claim 7, wherein said surfactant is alkoxylated. 9. The method according to claim 8, wherein said surfactant is ethoxylated. 10. The method according to claim 7, wherein the surfactant is at least mainly nonionic and / or anionic. 11. The method according to claim 7, wherein the surfactant is present in the composition in an amount of from 0.05 to 2.5% by weight relative to the total weight of the composition. 12. The method according to claim 1, wherein the composition comprises one or more solvents. 13. The method according to claim 12, wherein said solvent is polar. 14. The method according to claim 13, wherein the solvent is a linear or branched C2-C5 alcohol. 15. The method according to claim 12, wherein the solvent is present in the composition in an amount of from 2 to 20% by weight relative to the total weight of the composition. 16. The composition according to claim 1, wherein the composition has a pH of 3 to 5.
16. The method according to any one of claims 15 to 15. 17. The method of claim 16, wherein said composition has a pH of about 4. 18. A method for cleaning a surface comprising applying a composition comprising a dye, a surfactant and a solvent capable of photodynamically inactivating microorganisms to a surface thereof and disinfecting microorganisms in a biofilm on the surface. how to.