Method for testing preservation efficacy of cosmetic

Abstract

The present invention involves: inoculating a test target cosmetic with a microorganism mixture including six species consisting of a yeast that is Candida albicans and five species of bacteria which are Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacian, and Enterobacter cloacae, and storing the same; inoculating, with a sample collected from the test target cosmetic during the storage period, an agar medium having tetrazolium blue added thereto, and culturing the same; and using, as an evaluation standard for preservation efficacy, the reduction in number of live cells, which corresponds to the total number of colony formation units measured in a state of being cultured for a short period of time, by a predetermined number or more with respect to the theoretical number of cells inoculated on the agar medium.

Description

Testing method for preservative effectiveness of cosmetics

[0001] The present invention relates to a method for testing the preservative effectiveness of cosmetics, which allows for simple and efficient preliminary screening of preservative effect confirmation tests based on various standards for microbiologically evaluating the preservative effectiveness of cosmetics.

[0002] Generally, preservative effectiveness testing is a method for microbiologically evaluating the effectiveness of pharmaceutical and cosmetic formulations or the preservatives contained therein, and is known to include tests to confirm the preservative effectiveness of cosmetics based on the International Organization for Standardization (ISO) and the Japanese Pharmacopoeia (JP).

[0003] The 19th Edition of the Japanese Pharmacopoeia describes the preservative effectiveness test method as reference information, explaining that it is a test method in which "the test strain to be tested is forcibly inoculated and mixed into the preparation, and the change in the test bacteria is tracked over time to evaluate the preservative effectiveness," and specifies the specified test strain and culture conditions as shown in Table 1 below.

[0004]

[0005] A similar preservative effectiveness test is also specified in the ISO 11930 standard of the International Organization for Standardization (ISO), which requires the incubation period of the test sample to be at least four weeks, the same as in the Japanese Pharmacopoeia, and in reality it takes four to five weeks to obtain the test results.

[0006] Since the prescribed preservative effectiveness tests take a long time, in order to test many prototypes in the cosmetics manufacturing business, it is necessary to carry out efficient product development in a short period of time while keeping costs as low as possible, and predictions of preservative properties are carried out as a preliminary screening.

[0007] As an example of such screening, Patent Document 1 describes a method in which the concentration ratio of ingredients that affect the antiseptic properties of cosmetics, such as preservatives, to be blended at the minimum inhibitory concentration for a specific bacterium is defined as an antibacterial index, and the antiseptic properties are predicted by calculating the antibacterial index when formulating a preparation.

[0008] JP 2009-203165 A

[0009] However, when developing cosmetics, there are cases where products are developed that do not contain any specific preservatives, or where formulations are developed that combine multiple ingredients that can replace specific preservatives.When such complex formulations are required, safer formulation designs are required.

[0010] Therefore, it is necessary to not only predict the effectiveness of preservatives based on their antibacterial index, but also to perform preliminary screening of preservative effectiveness tests simply and efficiently.

[0011] As mentioned above, since a standard preservative effectiveness test usually requires 5 to 6 weeks, it was necessary to carry out preliminary screening that would enable a reliable judgment of the preservative effect in as short a test period as possible.

[0012] Therefore, an object of the present invention is to solve the above-mentioned problems and provide a novel preservative effectiveness testing method that enables preliminary determination comparable to the International Organization for Standardization (ISO) and the Japanese Pharmacopoeia (JP) in a shorter period than the conventional required testing period, for example, 2 to 3 weeks (or 14 to 21 days) or less.

[0013] In order to solve the above-mentioned problems, the present invention provides a method for producing a microbial colony containing five types of bacteria, namely, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacia, and Enterobacter cloacae, and a yeast, Candida albicans. The cosmetic product to be tested was inoculated with a six-species mixed microorganism consisting of 6 species of microorganisms and 6 species of microbial organisms (CFU) and stored at 20-25°C. During this storage period, samples collected from the cosmetic product to be tested were inoculated onto an agar medium containing the color-developing reagent tetrazolium blue and cultured. The total number of colony-forming units (CFU) per unit mass (1 g) of the cosmetic product to be tested was then measured for the six species of mixed microorganisms. The effectiveness of the cosmetic product to be tested was evaluated based on whether the viable cell count A corresponding to the measured total number of colony-forming units was reduced by a predetermined number or more relative to the theoretical cell count B of the six species of mixed microorganisms inoculated onto the agar medium.

[0014] The six types of mixed microorganisms used in this invention do not include microorganisms including molds specified in the preservative effectiveness test method in the reference information of the Japanese Pharmacopoeia or in the ISO 11930 standard, and consist of bacteria and yeasts that have a greater impact than molds on the preservative properties of the test cosmetics, which are general cosmetics. In this invention, the six types of mixed microorganisms are used to screen for contamination risks in a short period of time, and a preliminary judgment is made on the preservative effectiveness of the test cosmetics.

[0015] In the present invention, the uniquely employed Burkholderia cepacia and Enterobacter cloacae have the properties of being able to assimilate a wide variety of organic substances and having a wide range of habitats, and all of the six types of mixed microorganisms consisting of bacteria and yeast increase in number and form colonies in a relatively short period of time within the specified temperature range of 20 to 25°C during the storage period.

[0016] Burkholderia cepacia is a soil bacterium known as an opportunistic pathogen that causes respiratory diseases and other illnesses. Like fungi, it is a bacterium that can utilize a variety of organic substances.

[0017] Enterobacter cloacae is an intestinal bacterium that can cause pneumonia and cystitis. Although it rarely infects healthy individuals, it is a bacterium that, like mold, is ubiquitous in terrestrial and aquatic environments.

[0018] The pathogenic bacteria Burkholderia cepacia and Enterobacter cloacae that can adhere to the skin in this way do not produce spores like molds, so their numbers increase and decrease rapidly, just like the other three types of bacteria and one type of yeast (fungus).

[0019] Furthermore, Burkholderia cepacia and Enterobacter cloacae have properties similar to those of specific fungi designated in the Japanese Pharmacopoeia or ISO 11930 standard in terms of their ability to assimilate a variety of organic substances and their wide range of habitats, and are therefore suitable microorganisms for use as indicators in the present invention.

[0020] The six types of mixed microorganisms, including these two types of bacteria, are inoculated into a test cosmetic and stored at 20-25°C. The storage period is preferably 14 days or longer. During this storage period, a sample taken from the test cosmetic is inoculated onto an agar medium containing the color-developing reagent tetrazolium blue and cultured.

[0021] During the storage, sampling is performed, for example, on the 7th and 14th days, and the effectiveness is evaluated by whether the viable cell count A corresponding to the measured total number of colony-forming units is reduced by a predetermined number or more relative to the theoretical cell count B of the six-type mixed microorganism inoculated on the agar medium.

[0022] As evaluation criteria comparable to ISO 11930 standard and the Japanese Pharmacopoeia (JP), it is preferable that the decrease in the viable cell count A of a sample collected on the 7th day of the storage period relative to the theoretical cell count B is 3.0 or more in common logarithm, and that the decrease in the viable cell count A of a sample collected on the 14th day of the storage period relative to the theoretical cell count B is 3.5 or more in common logarithm.

[0023] Furthermore, in order to enable sufficiently accurate counting of the total number of colony forming units (CFU) distinguishable by bacterial species or by species, it is preferable that the agar medium on which the sample is cultured is an SCDLP agar medium (a lecithin-polysorbate 80-added soybean-casein digest agar medium) to which the coloring reagent tetrazolium blue has been added, and that the culture conditions are a culture temperature of 30 to 35°C and a culture period of 3 days or longer.

[0024] The colonies formed by each of the six types of mixed microorganisms are stained with tetrazolium blue in a specific blue color compared to other microorganisms that are accidentally mixed in, and each species forms colonies of different sizes or shapes on the agar medium, making it extremely easy to identify and count colonies by species.

[0025] Furthermore, SCDLP agar medium cultivates microorganisms while weakening the effects of preservatives contained in the test cosmetic, improving the appearance and formation of colonies, and enabling more accurate measurement of the number of viable bacteria contained in the test cosmetic after storage for a specified period of time.

[0026] In addition to testing the preservative effectiveness of cosmetics, it is also preferable to check whether specific microorganisms, such as the six mixed microorganisms, are viable in samples contained in the test cosmetics after storage for a predetermined period of time, in order to strictly conduct hygiene inspections and the like required at the time of shipping the cosmetics.

[0027] To do this, a sample of the cosmetic to be tested is inoculated into an agar medium and the same sample is inoculated into a liquid medium and cultured in both media simultaneously.If no colony-forming units are detected in the agar medium that has been stored for a specified period, the presence or absence of the six mixed microorganisms in a culture collected from a liquid medium that has been stored for the same period is confirmed by the streak culture method.

[0028] In particular, a sample of the cosmetic to be tested is inoculated onto an SCDLP agar medium to which the color-developing reagent tetrazolium blue has been added, and the same sample is also inoculated into a liquid medium and cultured in both media simultaneously.If no colony-forming units are detected in the SCDLP agar medium that has been stored for a specified period of time, the presence or absence of the six mixed microorganisms in a culture collected from a liquid medium that has been stored for the same specified period of time can be confirmed by a streak culture method, thereby improving the visibility of colony morphology, etc., and further improving the accuracy of viable cell count measurement.

[0029] This invention provides a novel preservative effectiveness testing method that can provide a preliminary determination comparable to the International Organization for Standardization (ISO) and the Japanese Pharmacopoeia (JP) in a shorter period than the test period required for conventional preservative effectiveness tests, for example, 2 to 3 weeks (or 14 to 21 days) or less, because Burkholderia cepacia and Enterobacter cloacae are bacteria capable of assimilating a wide variety of organic substances and having a wide range of habitats. The method involves culturing a specific six-species mixture of microorganisms including these bacteria, staining the colonies formed with a specific color-developing reagent for identification, and quantifying the number of colony-forming units (CFUs) as the total number of viable bacteria in a relatively short period of time.

[0030] Photograph substituting a drawing showing the culture state of Example 1 on SCDLP agar medium supplemented with tetrazolium blue. Photograph substituting a drawing showing the typical colony morphology of Pseudomonas aeruginosa in the culture state of Example 1. Photograph substituting a drawing showing the typical colony morphology of Burkholderia cepacia in the culture state of Example 1. Photograph substituting a drawing showing the typical colony morphology of Staphylococcus aureus in the culture state of Example 1. Photograph substituting a drawing showing the typical colony morphology of Escherichia coli in the culture state of Example 1. Photograph substituting a drawing showing the typical colony morphology of Enterobacter cloacae in the culture state of Example 1. Photograph substituting a drawing showing the typical colony morphology of Candida albicans in the culture state of Example 1.

[0031] As an embodiment of the present invention, a method for testing the preservative effectiveness of cosmetics will be described in more detail and specifically, in which the above-mentioned six specific mixed microorganisms are inoculated into a test cosmetic and stored at 20 to 25°C, samples collected from the test cosmetic during this storage period are inoculated onto an agar medium to which the color-developing reagent tetrazolium blue has been added and cultured, and the total number of colony-forming units (CFU) per unit mass (1 g) of the test cosmetic is then measured for the six mixed microorganisms, and the effectiveness is evaluated based on whether the viable cell count A corresponding to the measured total number of colony-forming units is reduced by a predetermined number or more relative to the theoretical cell count B of the six mixed microorganisms inoculated onto the agar medium.

[0032] This invention is not particularly limited to the type of cosmetic product to be tested, and can be applied to cosmetics containing water or oil, and includes, for example, basic cosmetics such as lotion, essence, emulsion, cream, pack, sunscreen, etc.; makeup cosmetics such as liquid foundation, mascara, eyeliner, eye shadow, lipstick, concealer, blush, nail color, etc.; hair cosmetics such as shampoo, conditioner, hair styling agent, etc.; body cleansers such as body shampoo, etc.; makeup removers, facial cleansers, etc.; and can also be applied to the same topical compositions that are quasi-drugs.

[0033] Such test cosmetics usually contain preservatives (i.e., preservatives) to prevent the growth of microorganisms such as bacteria and mold, but the "method for testing the preservative effectiveness of cosmetics" of this invention can be used regardless of whether or not the cosmetics contain preservatives.

[0034] Well-known preservatives used in cosmetics and quasi-drugs include, for example, paraoxybenzoic acid esters (parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben); phenoxyethanol; alkanediols; alkyl glyceryl ethers; glycerin fatty acid esters; salicylic acid; sodium benzoate; isothiazolinone derivatives; imidazolinium urea; dehydroacetic acid and its salts; phenols; halogenated bisphenols, acid amides, quaternary ammonium salts; trichlorocarbanide, zinc pyrithione, benzalkonium chloride, benzethonium chloride, sorbic acid, chlorhexidine, chlorhexidine gluconate, halocarban, hexachlorophene, hinokitiol; phenylethyl alcohol, photosensitizers, antibacterial zeolites, and silver ions.

[0035] In order to weaken the effect of the preservatives contained in the cosmetic product to reduce the viable bacterial count and to evaluate the storage conditions of the cosmetic product under stricter conditions, it is preferable to use a sample that is diluted, for example, 10 times, to be inoculated onto the agar medium, and to conduct the preservative effectiveness test under conditions in which the effect of the preservatives contained in the cosmetic product is reduced to 1 / 10.

[0036] The mixed microorganisms used in this invention are used to inoculate the test cosmetics, and they consist of five types of bacteria, namely, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacia, and Enterobacter cloacae, and one type of yeast, namely, Candida albicans.

[0037] Of the six microorganisms mentioned above, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Candida albicans are also used in the preservative effectiveness testing method described as reference information in the 19th Edition of the Japanese Pharmacopoeia, and ATCC strains or NBRC strains as shown in Table 1 can be used as test strains.

[0038] Furthermore, Burkholderia cepacia and Enterobacter cloacae are Gram-negative, non-spore-forming, aerobic, polar-flagellated rod-shaped bacteria that are the type species of the Burkholderiales order and the Burkholderiaceae family of eubacteria. They are soil bacteria that are normally present in the natural environment and can act as opportunistic pathogens, causing respiratory or bloodstream infections.

[0039] Enterobacter cloacae is a Gram-negative, facultatively anaerobic, peritrichous, periflagellated rod-shaped bacterium in the Enterobacteriaceae family of the eubacteria order Enterobacteriaceae. It is mesophilic and can grow in a wide range of pH from 4 to 10, and is ubiquitous in terrestrial and aquatic environments (water, mud, soil, food, and hot water treatment tanks). This bacterium is a symbiotic microorganism found in the intestinal flora of humans and animals in general, and although infection with it is unlikely to cause disease, it can also cause respiratory tract infections in immunodeficient individuals.

[0040] The various microorganisms used in the microbial mixture of the present invention can be NBRC strains such as Pseudomonas aeruginosa (NBRC 13275), Staphylococcus aureus (NBRC 13276), Escherichia coli (NBRC 3972), Candida albicans (NBRC 1594), Burkholderia cepacia (NBRC 14595), and Enterobacter cloacae (NBRC 13535) shown below as test strains.

[0041] These mixed microorganisms are inoculated into the test cosmetic product, mixed uniformly, and stored at 20 to 25° C. The storage temperature is within a range that allows the growth of the mixed microorganisms used in this invention and that also assumes the normal storage and use conditions of cosmetics.

[0042] The storage period may be selected appropriately depending on the type of cosmetic product to be tested, but is usually 14 days or more, and at most 28 days as a guideline. For example, if samples are collected on the 2nd, 7th, 14th, and 28th days after storage, comparison with the prescribed preservative effectiveness test (the preservative effectiveness test method in the reference information of the Japanese Pharmacopoeia or the ISO 11930 standard) can be easily performed.

[0043] The agar medium used to culture the collected sample is an agar medium containing tetrazolium blue, and may be an agar plate medium or an agar slant medium.

[0044] Furthermore, in order to test the above-mentioned cosmetics to be tested under storage conditions that are as favorable as possible for microbial growth, it is preferable to use SCDLP agar medium, and the lecithin-polysorbate-80-added soybean casein digest agar medium (SCDLP agar medium) listed in the Microbiological Testing Methods for Pharmaceuticals can be used. SCDLP agar medium is a medium that intentionally weakens the effectiveness of preservatives by incorporating lecithin and polysorbate 80 into the basic SCD agar medium (soybean casein digest agar), making it possible to check for the presence or absence of viable bacteria.

[0045] Tetrazolium blue is a type of reducing color-developing reagent, and is a compound represented by the structural formula of Chemical Formula 1. Tetrazolium blue has excellent staining properties for the colonies formed by the six types of mixed microorganisms used in this invention, allowing the colonies to be clearly identified without affecting colony development, as confirmed in the Reference Examples described below.

[0046]

[0047] The above-mentioned culture may be carried out by smearing the sample on an agar plate medium in a conventional manner, or by forming a mixture of the agar medium and the sample into a plate by the agar plate pour method, and then culturing the mixture.

[0048] Cultivation on the agar medium at 30 to 35° C. for at least 3 days allows the six types of mixed microorganisms to grow sufficiently, allowing the number of colony forming units (CFU) to be measured accurately.

[0049] In this measurement, the total number of colony-forming units (CFU) per unit mass of the test cosmetic for the six types of mixed microorganisms is measured using a commercially available automatic bacteria counting device, etc., and it is confirmed that the viable cell count corresponding to the measured total number of colony-forming units is reduced by a specified number or more relative to the theoretical cell count of the six types of mixed microorganisms inoculated onto the agar medium. The theoretical cell count of the mixed microorganisms can be calculated by conventional methods from the number of colonies formed in the petri dish, the volume of the inoculated liquid, and the dilution ratio, and the viable cell count per unit volume of the sample.

[0050] Furthermore, when a hygiene test is to be conducted by checking for the presence or absence of specific microorganisms by the streak culture method simultaneously with the preservative effectiveness test, a sample of the cosmetic to be tested is inoculated onto an agar medium, and an appropriate amount of the same sample is also inoculated into a liquid medium, and the sample is simultaneously cultured in both media. When simultaneously culturing on an agar medium and a liquid medium, appropriate culture can be achieved by inoculating, for example, 1 / 100 of a 100-volume sample of the cosmetic to be tested onto an agar medium and simultaneously inoculating 99 / 100 into a liquid medium.

[0051] The liquid medium may be any medium suitable for the growth of the mixed microorganisms, and may be, for example, a soybean casein digest medium or a Sabouraud glucose liquid medium, both of which are described in the reference information of the Japanese Pharmacopoeia and contain the same components as the agar medium described above.

[0052] The liquid culture is performed under the same temperature and period as the agar culture conditions, for example, at 30 to 35°C for at least three days to allow the six types of mixed microorganisms to grow sufficiently. Then, the presence or absence of the six types of mixed microorganisms in the culture sample collected from the liquid medium is confirmed by the streak culture method.

[0053] In the so-called enrichment test using streak culture, a streaked agar medium similar to that described above is cultured at 30-35°C for at least 18 hours, and the presence or absence of independently formed colonies is confirmed. If bacteria are detected in a sample taken on or before the seventh day of liquid culture, a retest is conducted. If bacteria are not detected in a sample taken after the seventh day, for example, on the eighth or 14th day, the preservative effectiveness of the cosmetic product is deemed confirmed and the test is terminated. Furthermore, if a preservative effectiveness test for fungi is required in addition to the preservative effectiveness test of the present invention, an additional test for fungi is conducted using conventional methods.

[0054] Example 1 As shown in Table 2, a mixed microorganism of six types, namely, Pseudomonas aeruginosa (NBRC 13275), Staphylococcus aureus (NBRC 13276), Escherichia coli (NBRC 3972), Burkholderia cepacia (NBRC 14595), Enterobacter cloacae (NBRC 13535), and Candida albicans (NBRC 1594), was used to test the preservative effectiveness of cosmetics (in Table 2, these are referred to as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, two specific types of bacteria, and yeast).

[0055] That is, each microorganism was appropriately cultured by a conventional method, and then a bacterial suspension was prepared. Equal amounts of all the bacterial suspensions were mixed together to prepare a six-type mixed bacterial suspension (mixed microorganism solution).

[0056] In addition, 20 g of commercially available lotion A was prepared as a sample of the cosmetic to be tested, weighed out and placed in a sterilized container, and this sample was inoculated with 0.2 ml of the above mixed microbial liquid, stirred to disperse uniformly, diluted 10-fold, and stored for 14 days at 22.5° C. The cosmetic to be tested was commercially available lotion A, which had previously met the pass criteria in the test results according to ISO 11930.

[0057] Sampling was carried out on the 2nd, 7th and 14th days of the storage period, and each sample was subjected to a microbial test in which the samples were simultaneously cultured by agar culture and liquid culture.

[0058] In the agar culture, the samples were cultured at 32°C for 3 days using SCDLP agar medium supplemented with 30 ppm of tetrazolium blue (Dojindo Laboratories) as a reducing color-developing reagent, and the number of remaining viable bacteria was measured for each sample stored for 2, 7, and 14 days. The decrease in the number of remaining viable bacteria A cultured from the sample stored for each period relative to the theoretical number of bacteria B of the six-type mixed microorganism inoculated on the agar medium by a predetermined number or more was expressed as a common logarithm, and the results are shown in Table 2.

[0059] The state of culture on the SCDLP agar medium on the 14th day is shown in a photograph in FIG. 1, and comparative diagrams of typical examples of each colony that appeared on the surface of the medium are shown in FIGS. 2A to 2F.

[0060]

[0061] Comparative Example 1 Using the same sample of lotion A as used in Example 1, the preservative effectiveness was examined according to a general preservative effectiveness (antiseptic) test method (JP). The test conditions and test results are shown in Table 2.

[0062] Comparative Example 2 Using the same sample of lotion A as used in Example 1, a preservative effectiveness test was carried out according to ISO 11930. The test conditions and test results are shown in Table 2.

[0063] As is clear from the results shown in Table 2, when the correlation between the method for testing the preservative effectiveness of cosmetics using the mixed microorganisms consisting of bacteria and yeast of the present invention and ISO 11930 was analyzed, it was shown that the preservative effectiveness could be evaluated within 14 days when representative commercially available cosmetics and the formulation of Example 1 were subjected to the mixed bacteria and yeast inoculation method.

[0064] The test cosmetics that were judged to be in compliance (criterion A) in the ISO 11930 test results also met the predetermined evaluation standard for effectiveness, which is the reduction in the amount of CFU (B) relative to A (common logarithm value) of 3.0 or more, in the preservative effectiveness test method of Example 1.

[0065] It should be noted that all of the cosmetics that were judged to be in compliance (criterion B) and not in compliance with ISO 11930 (examples omitted) failed the preservative effectiveness test of the present invention.

[0066] Furthermore, as is clear from FIG. 1, in the SCDLP agar medium to which tetrazolium blue was added at about 30 ppm (e.g., 28 to 32 ppm), the colonies of the six specified mixed microorganisms were stained so as to be clearly identifiable, and it was possible to measure the total number of colony forming units (CFU).

[0067] The SCDLP agar medium to which tetrazolium blue was added was capable of culturing the six specified mixed microorganisms in the same manner as the SCDLP agar medium to which tetrazolium blue was not added, and each colony could be produced. As shown in Figures 2A to 2F, the morphology of each colony that appeared on the surface of the medium indicated that it was possible to distinguish the strains (bacterial species) of the six specified mixed microorganisms from the degree of staining and the morphology of the colonies.

[0068] Thus, Example 1, which is a mixed bacterial and yeast inoculation method, was recognized to have a preservative effectiveness equivalent to or greater than that of Criteria A of ISO 11930, and was confirmed to be a novel preservative effectiveness testing method that allows preliminary judgment (screening) comparable to the International Organization for Standardization (ISO) and the Japanese Pharmacopoeia (JP).

[0069] Reference Example 1 A four-type mixed microorganism mixture consisting of Pseudomonas aeruginosa (NBRC 13275), Staphylococcus aureus (NBRC 13276), Escherichia coli (NBRC 3972), and Candida albicans (NBRC 1594) was appropriately cultured by a conventional method, and then equal amounts of all the bacterial suspensions were mixed to prepare a four-type mixed bacterial suspension.

[0070] The bacterial suspension was inoculated onto an SCDLP agar medium containing 30 ppm of tetrazolium blue (TB) by the agar plate smear method, and after culturing at 32°C for 3 days, the stainability of the colonies and the ability to form or express colonies were evaluated. The evaluation was made on a three-level scale: good (◯), somewhat poor (△), and poor (×), and the results are shown in Table 3.

[0071] [Reference Example 2] In Reference Example 1, the staining ability of colonies and the ability to form or express colonies were evaluated in exactly the same manner as in Reference Example 1, except that nitroblue tetrazolium chloride (Nitro-TB) was used instead of tetrazolium blue (TB) as the reductive color-developing reagent. The results are also shown in Table 3.

[0072] [Reference Example 3] The staining properties of colonies and the formation or expression of colonies were evaluated in exactly the same manner as in Reference Example 1, except that triphenyltetrazolium chloride (TTC) was used instead of tetrazolium blue (TB) as the reductive color-developing reagent. The results are also shown in Table 3.

[0073]

[0074] Comparative Example 3 Storage was carried out in exactly the same manner as in Example 1, except that instead of the six types of mixed microorganisms, Burkholderia cepacia (NBRC 14595) and Enterobacter cloacae (NBRC 13535) were not used, and instead Aspergillus brasiliensis (NBRC 9455) was used as a representative example of mold. Samples on the 14th and 28th days of storage were examined for visibility by colony staining, and it was found that a storage period of at least 28 days was required to determine the preservative effectiveness of reducing the number of mold cells by 1.5 or more common logarithms relative to the theoretical cell count at the time of inoculation.

[0075] Examples 2-6 As shown in Table 4, the same procedures as in Example 1 were repeated except that commercially available lotion B (Example 2), a commercially available all-in-one gel (Example 3), a commercially available cosmetic cream (Example 4), a commercially available shampoo (Example 5), and a commercially available liquid foundation (Example 6) were used as the test cosmetic samples instead of commercially available lotion A. The remaining viable cell counts were measured for each sample stored for 2, 7, 14, and 28 days, and the decrease in the remaining viable cell count cultured from the sample for each storage period relative to the theoretical cell count of the six-species mixed microorganism inoculated onto the agar medium by a specified number or more was expressed as a common logarithm, and the results are shown in Table 4. Note that if the viable cell count decreased by a common logarithm of 6.0 or more after 7 days of storage, it was clear that there would be no further decrease, and the test was terminated at that point.

[0076]

[0077] As is clear from the results shown in Table 4, Examples 2 to 6 showed suitable test results for preservative effectiveness test on various cosmetics characterized by water solubility, gel, emulsion, surfactant, oiliness, etc., by the preservative effectiveness test method of this invention, at the latest on the 14th day.

[0078] For the test cosmetics whose preservative effectiveness was confirmed in Examples 2-6, a preservative effectiveness test according to ISO 11930 was conducted to confirm the results of the preservative effectiveness test.

[0079] According to this method, the reduction in the number of remaining viable bacteria relative to the theoretical number of bacteria for each of the four test bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) inoculated onto four agar media was examined on the 7th, 14th, and 28th days of storage, and the results are shown in Table 5 as common logarithmic values.

[0080]

[0081] As is clear from the results shown in Table 5, it was confirmed that all of the test cosmetics whose preservative effectiveness was confirmed in Examples 2-6 were capable of obtaining results comparable to Criteria A in the preservative effectiveness test according to the International Organization for Standardization (ISO) 11930. This confirmed that the preservative effectiveness test in Examples 2-6 is a preservative effectiveness test method capable of making a preliminary judgment comparable to the test specified by the International Organization for Standardization (ISO) and the like.

[0082] It can be used for screening as a preliminary test for tests prescribed by laws and regulations or industries to microbiologically evaluate the effectiveness of pharmaceutical and cosmetic formulations or the preservatives contained therein, as well as for hygiene inspections and inspections of cosmetics at the time of shipment.

Claims

1. Five types of bacteria, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacian, and Enterobacter cloacae, and the yeast Candida albicans A cosmetic preservative effectiveness test method in which a cosmetic test is inoculated with a six-type mixed microorganism consisting of 6 types of microorganisms and 6 types of microbial organisms (Cas9 and Ca2+ / Cas9) and stored at 20-25°C, samples taken from the cosmetic test during the storage period are inoculated onto an agar medium containing a color-developing reagent, tetrazolium blue, and cultured, and the total number of colony-forming units (CFU) per unit mass of the cosmetic test is measured for the six types of mixed microorganisms, and the effectiveness is evaluated based on whether the viable cell count A corresponding to the measured total number of colony-forming units is reduced by a predetermined number or more with respect to the theoretical cell count B of the six types of mixed microorganisms inoculated onto the agar medium.

2. The method for testing the preservative effectiveness of cosmetics according to claim 1, wherein the storage period is 14 days or more, the reduction in the viable cell count A of the sample taken on the 7th day relative to the theoretical cell count B is 3.0 or more in common logarithm, and the reduction in the viable cell count A of the sample taken on the 14th day of the storage period relative to the theoretical cell count B is 3.5 or more in common logarithm.

3. The method for testing the preservative effectiveness of cosmetics according to claim 1 or 2, wherein the agar medium is an SCDLP agar medium, and the culture is carried out at a culture temperature of 30 to 35°C for a culture period of 3 days or more.

4. A method for testing the preservative effectiveness of cosmetics according to claim 1 or 2, comprising inoculating a sample of the cosmetic to be tested into an agar medium and a liquid medium, culturing the sample in both media simultaneously, and if no colony-forming units are detected in the agar medium stored for a specified period of time, confirming the presence or absence of the six mixed microorganisms in a culture taken from a liquid medium stored for the same period of time by a streak culture method.

5. A method for testing the preservative effectiveness of cosmetics as described in claim 3, in which a sample of the cosmetic to be tested is inoculated onto an SCDLP agar medium containing tetrazolium blue as a color-developing reagent, and the same sample is also inoculated into a liquid medium and cultured in both media simultaneously, and if no colony-forming units are detected in the SCDLP agar medium stored for a specified period of time, the presence or absence of the six mixed microorganisms in a culture taken from a liquid medium stored for the same period of time is confirmed by a streak culture method.

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