Method for testing storage efficacy of cosmetics

Abstract

The present invention relates to a method for preparing a cosmetic sample, which is characterized in that: six kinds of mixed microorganisms comprising five kinds of bacteria, i.e., Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacian, and Enterobacter cloacae, and Candida albicans yeast, are inoculated into a cosmetic sample to be tested, and are stored; a sample collected from the cosmetic to be tested during the storage period is inoculated into an agar culture medium to which tetrazole blue is added and cultured; the number of viable cells corresponding to the total number of colony formation units measured in a short-time culture state is reduced by a predetermined number or more with respect to the theoretical number of cells inoculated in the agar culture medium.

Description

Technical Field

[0001] The present invention relates to a method for testing the preservation efficacy of cosmetics, which is used for simply and effectively conducting preliminary screening of various standard preservative effect confirmation tests for evaluating the preservation efficacy of cosmetics based on microbiology. Background Art

[0002] Generally, as a method for microbiologically evaluating the effectiveness of pharmaceuticals, cosmetics, or preservatives contained therein, a cosmetic preservative effect confirmation test based on the International Organization for Standardization (ISO) or the Japanese Pharmacopoeia (JP) is known as a preservative effectiveness test.

[0003] The 19th revised edition of the Japanese Pharmacopoeia records the preservation efficacy test method as reference information, and explains that it is a test method that "evaluates the preservation efficacy by forcibly inoculating and mixing the test subject bacteria in the preparation and tracking the growth and decline of the test bacteria over time." As shown in Table 1 below, the specified test strains and culture conditions are specified.

[0004] [Table 1]

[0005]

[0006] The same preservation efficacy test is also stipulated in the ISO11930 standard of the International Organization for Standardization (ISO). The incubation time of the test sample is the same as that in accordance with the Japanese Pharmacopoeia, which is at least 4 weeks. In fact, it takes 4 to 5 weeks to obtain the test results.

[0007] As described above, the prescribed preservation effectiveness test requires a long time, and therefore, in the cosmetics manufacturing business, in order to test a large number of trial products, it is necessary to minimize costs and conduct efficient product development in a short period of time, and to perform a prediction of the preservation properties as a preliminary screening.

[0008] As such a screening, Patent Document 1 describes that for cosmetics, etc., the concentration ratio of preservatives and other raw materials that affect the antiseptic properties at the minimum inhibitory concentration against specific bacteria is defined as the antibacterial index, and the antibacterial index is calculated in the formulation of the preparation to predict the antiseptic properties.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application Publication No. 2009-203165 Summary of the invention

[0012] However, in the development of cosmetics, there are cases where a formulation is developed without any specific preservatives or where a formulation is developed by combining a plurality of raw materials that replace specific preservatives. When such a complex formulation is required, safer formulation design is required.

[0013] Therefore, there is a need not only for the prediction of the antimicrobial index based on preservatives, but also for simple and efficient preliminary screening based on preservation efficacy tests.

[0014] As mentioned above, a given preservation efficacy test usually takes 5 to 6 weeks, so a preliminary screening that can reliably determine the preservation effect within the shortest possible test period is required.

[0015] Therefore, the object of the present invention is to solve the above-mentioned problems and to provide a novel preservation efficacy test method which can perform a preliminary judgment comparable to the International Organization for Standardization (ISO) or the Japanese Pharmacopoeia (JP) in a period shorter than the conventional required test period, for example, 2 to 3 weeks (or 14 to 21 days) or less.

[0016] In order to solve the above-mentioned problems, the present invention adopts a method for testing the preservation effectiveness of cosmetics, wherein a six-type mixed microorganism consisting of five types of bacteria, namely, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacian and Enterobacter cloacae, and a yeast, namely, Candida albicans, is inoculated into a test cosmetic and stored at 20 to 25° C. A sample collected from the test cosmetic during the storage period is inoculated into an agar medium supplemented with a color developing reagent, tetrazolium blue, and cultured. The total number of colony forming units (CFU) per unit mass (1 g) of the test cosmetic is measured for the six types of mixed microorganisms, and a reduction of a predetermined number or more in the number of viable cells A corresponding to the measured total number of colony forming units relative to a theoretical number B of the six types of mixed microorganisms inoculated into the agar medium is used as a criterion for evaluating the effectiveness.

[0017] The six mixed microorganisms used in the present invention do not include microorganisms including molds specified in the preservation efficacy test method of the reference information of the Japanese Pharmacopoeia or the ISO11930 standard, and are composed of bacteria and yeasts that have a greater impact on the preservation of the test cosmetics as general cosmetics than molds. In the present invention, the above six mixed microorganisms are used to screen the contamination risk in a short time and perform a preliminary judgment on the preservation efficacy of the test cosmetics.

[0018] In the present invention, the Burkholderia cepacia and Enterobacter cloacae independently used have properties such as the assimilation of various organic substances and the wide range of habitats. All six mixed microorganisms consisting of bacteria and yeast increase the bacterial count in a relatively short period of time within the specified temperature range of 20 to 25°C during the above-mentioned storage period to form colonies.

[0019] Burkholderia cepacia is a soil bacterium known as an opportunistic pathogen that causes respiratory diseases and the like, and is a bacterium that can assimilate a variety of organic matter like fungi.

[0020] In addition, Enterobacter cloacae is an intestinal bacterium that can also cause pneumonia and cystitis. Although it rarely infects healthy people, it is a common bacterium in terrestrial and aquatic environments like fungi.

[0021] Since Burkholderia cepacia and Enterobacter cloacae, pathogenic bacteria that may adhere to the skin, do not produce spores like fungi, they increase and decrease rapidly like the other three bacteria and one yeast (fungus).

[0022] In addition, Burkholderia cepacia and Enterobacter cloacae have the same properties as those of specific fungi specified in the Japanese Pharmacopoeia or ISO 11930 in terms of assimilation of various organic substances and wide range of habitats, and are suitable microorganisms for use as indicators in the present invention.

[0023] The above-mentioned six kinds of mixed microorganisms including these two kinds of bacteria are inoculated into the test cosmetics and stored at 20-25° C. The storage period is preferably 14 days or more. During the storage period, the sample collected from the test cosmetics is inoculated into an agar medium added with a color developing reagent, tetrazolium blue, and cultured.

[0024] Then, during the above storage, sampling is performed, for example, on the 7th and 14th days, and the effectiveness is evaluated by reducing the viable bacterial count A corresponding to the total number of colony forming units measured by more than a specified number relative to the theoretical bacterial count B of the above 6 mixed microorganisms inoculated on the above agar medium.

[0025] The reduction in the number of viable bacteria A of the sample collected on the 7th day during the above-mentioned storage period relative to the above-mentioned theoretical number of bacteria B is 3.0 or more in terms of a common logarithm, and the reduction in the number of viable bacteria A of the sample collected on the 14th day during the above-mentioned storage period relative to the above-mentioned theoretical number of bacteria B is 3.5 or more in terms of a common logarithm, which is preferably used as an evaluation standard comparable to the ISO11930 standard or the Japanese Pharmacopoeia (JP).

[0026] In addition, in order to be able to count the colony forming units (CFU) identifiable by bacterial species sufficiently accurately in total or by type, it is preferred that the above-mentioned agar medium for culturing the sample is SCDLP agar medium (soybean casein digest agar medium containing lecithin and polysorbate 80) supplemented with a color developing reagent tetrazolium blue, and the culture conditions are a culture temperature of 30 to 35°C and a culture time of more than 3 days.

[0027] The colonies formed by each of the above 6 mixed microorganisms can be stained with tetrazolium blue to become a specific blue color compared with other accidentally mixed microorganisms, and colonies of different sizes or shapes are formed on the agar medium according to the species, so it is extremely easy to identify and count colonies according to the species.

[0028] In addition, since the SCDLP agar medium cultured microorganisms with reduced influence of preservatives contained in the test cosmetics, the colony presentation and formation properties were improved, and the number of viable bacteria contained in the test cosmetics after storage for a predetermined period could be measured more accurately.

[0029] Furthermore, in addition to the preservation effectiveness test of cosmetics, in order to further strictly perform the hygienic inspection required when cosmetics are shipped, it is also preferred to investigate whether specific microorganisms such as the above-mentioned 6 mixed microorganisms are alive in the samples contained in the test cosmetics after being stored for a prescribed period.

[0030] For this purpose, the sample of the test cosmetics is inoculated into an agar medium and the sample is inoculated into a liquid medium, and cultured in both culture media simultaneously. If no colony forming units are detected in the agar medium stored for a predetermined period, the presence of the six mixed microorganisms is confirmed by the streak culture method in a culture collected from the liquid medium stored for the same period as the predetermined period.

[0031] In particular, by inoculating the sample of the above-mentioned cosmetics to be tested into an SCDLP agar medium supplemented with a color developing reagent, blue tetrazolium, and also inoculating the sample of the above-mentioned cosmetics to be tested into a liquid culture medium, and culturing them simultaneously in the two culture media, when no colony forming units are detected in the above-mentioned SCDLP agar medium stored for a specified period, the presence or absence of the above-mentioned six mixed microorganisms in the culture collected from the liquid culture medium stored for the same period as the above-mentioned specified period is confirmed by the streak culture method, thereby improving the visibility of the morphology of the colonies, etc., and further improving the accuracy of the determination of the number of viable bacteria.

[0032] In the present invention, Burkholderia cepacia and Enterobacter cloacae are bacteria that have the ability to assimilate a variety of organic substances and have a wide range of habitats. The colonies formed by culturing specific 6 mixed microorganisms containing them are stained with a specific color developing reagent for identification, and the number of colony forming units (CFU) as the total number of viable bacteria can be quantified in a relatively short period of time. Therefore, it has become a new preservation effectiveness test method that can perform preliminary judgments comparable to the International Organization for Standardization (ISO) or the Japanese Pharmacopoeia (JP) in a period shorter than the test period required for existing preservation effectiveness tests, for example, 2 to 3 weeks (or 14 to 21 days) or less. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a photograph as a drawing showing the culture state of Example 1 in SCDLP agar medium supplemented with tetrazolium blue.

[0034] Figure 2A This is a photograph as an alternative to a drawing showing a typical morphology of a Pseudomonas aeruginosa colony in a culture state in Example 1.

[0035] Figure 2B This is a photograph as an alternative to a drawing showing a typical morphology of a colony of Burkholderia cepacia in the culture state of Example 1.

[0036] Figure 2C This is a photograph as an alternative to a drawing showing a typical morphology of a colony of Staphylococcus aureus in a culture state in Example 1.

[0037] Figure 2D This is a photograph as an alternative to a drawing showing a typical morphology of a colony of Escherichia coli in a culture state in Example 1.

[0038] Figure 2E This is a photograph as an alternative to a drawing showing a typical morphology of a colony of Enterobacter cloacae in a culture state in Example 1.

[0039] Figure 2F This is a photograph as an alternative to a drawing showing a typical morphology of a Candida albicans colony in a culture state in Example 1. DETAILED DESCRIPTION

[0040] As an embodiment of the present invention, a method for testing the preservation effectiveness of cosmetics is described in more detail and specifically. The method for testing the preservation effectiveness of cosmetics comprises inoculating the above-mentioned specific 6 mixed microorganisms into the test cosmetics and storing them at 20-25°C. After inoculating a sample collected from the test cosmetics during the storage period into an agar medium supplemented with a color developing reagent, tetrazolium blue, and culturing the sample, the total number of colony forming units (CFU) per unit mass (1g) of the test cosmetics is measured for the above-mentioned 6 mixed microorganisms, and a reduction of a specified number or more in the number of live bacteria A corresponding to the measured total number of colony forming units relative to the theoretical number B of the above-mentioned 6 mixed microorganisms inoculated into the above-mentioned agar medium is used as an evaluation criterion for effectiveness.

[0041] In the present invention, the types of cosmetics to be tested are not particularly limited, and can be applied to cosmetics containing water or oil, for example, including basic cosmetic materials such as lotions, essences, emulsions, creams, facial masks, and sunscreens, color cosmetic materials such as foundation, mascara, eyeliner, eye shadow, lipstick, concealer, blush, and nail polish, hair cosmetic materials such as shampoo, conditioner, and hair fixative, body cleansers such as shower gels, and external compositions such as makeup removers and facial cleansers, and can also be applied to external compositions that are quasi-drugs.

[0042] Such test cosmetics usually contain preservatives (i.e., preservatives) in order to prevent the proliferation of microorganisms such as bacteria and molds, but the "cosmetic preservation effectiveness test method" of the present invention can be used regardless of whether or not the preservatives are contained.

[0043] Examples of known preservatives used in cosmetic materials or quasi-drugs include parabens (parabens such as methyl paraben, ethyl paraben, propyl paraben, and butyl paraben); phenoxyethanol; alkyl glycols; alkyl glyceryl ethers; glyceryl fatty acid esters; salicylic acid; sodium benzoate; isothiazolinone derivatives; imidazoline Urea; dehydroacetic acid and its salts; phenols; halogenated bisphenols, acid amides, quaternary ammonium salts; triclocarban, zinc pyrithione, benzalkonium chloride, benzethonium chloride, sorbic acid, chlorhexidine, chlorhexidine gluconate, halocarban, hexachlorophene, cypressol; phenylethyl alcohol, photosensitizers, antibacterial zeolite, silver ions, etc.

[0044] In order to weaken the effect of reducing the number of viable bacteria caused by the preservatives contained in the tested cosmetics and to determine the storage conditions of the cosmetics under more stringent conditions, it is preferred that the sample inoculated on the agar medium is diluted 10 times, for example, and the preservation effectiveness test is carried out in a state where the influence of the preservatives contained in the cosmetics is suppressed to 1 / 10.

[0045] In addition, the mixed microorganisms used in the present invention are used for inoculation into the test cosmetics, which are five types of bacteria consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacian and Enterobacter cloacae, and one type of yeast consisting of Candida albicans.

[0046] Among the above-mentioned 6 microorganisms, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Candida albicans are also used in the preservation efficacy test method recorded as reference information in the 19th revised edition of the Japanese Pharmacopoeia, and ATCC strains or NBRC strains as recorded in Table 1 can also be used as test strains.

[0047] In addition, Burkholderia cepacian and Enterobacter cloacae are Gram-negative, non-spore-forming, aerobic, polar flagellated rods, typical species of the Burkholderiales and Burkholderiaceae families, which are true bacteria. They are common soil bacteria in the natural environment and are microorganisms that sometimes cause infection through the respiratory system or the bloodstream as opportunistic pathogens.

[0048] In addition, Enterobacter cloacae is a Gram-negative, facultative anaerobic, peritrichous, and periflagellated bacillus of the Enterobacteriaceae order and the Enterobacteriaceae family of true bacteria. It is mesophilic and can grow in a wide range of pH 4 to 10, and is commonly found in terrestrial and aquatic environments (water, mud, soil, food, and warm water treatment tanks). This bacterium is a symbiotic microorganism found in the general intestinal flora of humans and animals, and has a low probability of causing disease through infection. It is a microorganism that sometimes causes respiratory tract infections even in the case of immunodeficiency.

[0049] For the various microorganisms used in the mixed microorganism of the present invention, as test strains, 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) can be used as shown below.

[0050] These mixed microorganisms are inoculated into the test cosmetics, mixed uniformly, and stored at 20 to 25° C. The storage temperature is a temperature range at which the mixed microorganisms used in the present invention can grow and assumes normal storage and use conditions of the cosmetics.

[0051] The storage period can be appropriately selected according to the type of cosmetic to be tested, but is generally 14 days or more, with a maximum of about 28 days being the standard. For example, if samples are collected on the 2nd, 7th, 14th, and 28th days after storage, it is easy to compare with an established preservation effectiveness test (preservation effectiveness test method of reference information of the Japanese Pharmacopoeia or ISO 11930 standard).

[0052] The agar medium for culturing the collected sample uses an agar medium supplemented with tetrazolium blue, and the agar medium may be an agar plate medium or an agar slant medium.

[0053] In addition, in order to make the storage state of the above-mentioned test cosmetics as easy as possible for microorganisms to proliferate, it is preferred to use SCDLP agar medium, and soybean casein digest agar medium containing lecithin and polysorbate 80 (SCDLP agar medium) included in the microbiological test method for pharmaceuticals can be used.

[0054] SCDLP agar medium is a medium in which the effectiveness of the preservative is intentionally weakened by adding lecithin and polysorbate 80 to SCD agar medium (soybean casein digest agar) as a base medium, thereby enabling confirmation of the presence of live bacteria.

[0055] Tetrazolium blue is a kind of reducing color developing reagent, and is a compound represented by the following structural formula: Tetrazolium blue has excellent staining property for the colonies formed by the six mixed microorganisms used in the present invention, can clearly identify the colonies, and does not affect the colony presentation, which has been confirmed by the reference examples described later.

[0056]

[0057] The above-mentioned culture can be carried out by smearing the sample on an agar plate medium by a conventional method, or by forming a mixture of an agar plate medium and a sample into a plate form by an agar plate mixing method.

[0058] The culture using the agar medium is carried out at 30 to 35° C. for at least 3 days, thereby allowing the six types of mixed microorganisms to sufficiently proliferate and the number of colony forming units (CFU) to be measured sufficiently accurately.

[0059] In this measurement, the total number of colony forming units (CFU) per unit mass of the cosmetics to be tested is measured for the above-mentioned 6 mixed microorganisms using a commercially available automatic colony count measuring device, and it is confirmed that the number of viable bacteria corresponding to the measured total number of colony forming units is reduced by more than a specified number relative to the theoretical number of bacteria of the above-mentioned 6 mixed microorganisms inoculated on the above-mentioned agar medium.

[0060] The theoretical bacterial count of the mixed microorganisms can be obtained by conventionally determining the number of viable bacteria per unit volume of the specimen from the number of colonies formed on the culture dish, the volume of the inoculated liquid, and the dilution ratio.

[0061] In addition, at the same time as the preservation efficacy test, the presence of specific microorganisms is confirmed by the streak culture method. When conducting a hygienic inspection, the sample of the above-mentioned test cosmetics is inoculated into an agar medium, and an appropriate amount of the above-mentioned test cosmetics sample is also inoculated into a liquid medium, and cultured in both culture media at the same time.

[0062] When culturing is performed simultaneously in an agar medium and a liquid medium, appropriate culturing can be performed if, for example, 1 / 100 of 100 parts by volume of the test cosmetic sample is inoculated in the agar medium and 99 / 100 is inoculated in the liquid medium.

[0063] The liquid medium may be any medium suitable for the growth of the mixed microorganisms, and a soybean casein digest medium or a Sabouraud dextrose liquid medium described in the reference information of the Japanese Pharmacopoeia, which is composed of the same components as the above-mentioned agar medium, may be used.

[0064] The liquid culture conditions are carried out at the same temperature and period as the agar culture conditions, for example, by culturing at 30 to 35° C. for at least 3 days, the six mixed microorganisms can be fully proliferated. Then, the presence of the six mixed microorganisms in the culture collected from the liquid culture medium is confirmed by the streak culture method.

[0065] The so-called bacterial enrichment test by streak culture is to culture the same agar medium as above at 30-35°C for at least 18 hours to confirm the presence or absence of independent colonies. If bacteria are detected in samples collected on the 7th day or before the liquid culture, the test is repeated, and if bacteria are not detected in samples collected after the 7th day, such as the 8th day or the 14th day, the preservation effectiveness of the cosmetic can be confirmed and the test is terminated.

[0066] Furthermore, when a preservation effectiveness test on molds is required in addition to the preservation effectiveness test of the present invention, an additional test on molds can be performed by a conventional method.

[0067] Example

[0068] [Example 1]

[0069] As shown in Table 2, as a cosmetic preservation effectiveness test, a six-type mixed microorganism consisting of Pseudomonas aeruginosa (NBRC 13275), Staphylococcus aureus (NBRC 13276), Escherichia coli (NBRC 3972), Burkholderia cepacian (NBRC 14595), Enterobacter cloacae (NBRC 13535) and Candida albicans (NBRC 1594) was used (in Table 2, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, two specific bacteria, and yeast) .

[0070] That is, after each microorganism is appropriately cultured by a conventional method, a bacterial suspension is prepared, and all bacterial suspensions are mixed in equal amounts to prepare six kinds of mixed bacterial suspensions (mixed microorganism liquids).

[0071] In addition, as a sample of the cosmetics to be tested, 20 g of commercially available lotion A was weighed in a sterilized container, 0.2 ml of the above-mentioned mixed microbial solution was inoculated into the sample, stirred to make it uniformly dispersed, diluted 10 times, and stored at 22.5° C. for 14 days. It should be noted that as the cosmetics to be tested, commercially available lotion A that had previously met the pass standard based on the test results of ISO11930 was used.

[0072] Sampling was performed on the 2nd, 7th and 14th days of the above storage period, and each sample was simultaneously cultured by an agar culture method and a liquid culture method for a microbial test.

[0073] In agar culture, SCDLP agar medium to which 30 ppm of tetrazolium blue (Dojin Chemical Research Institute) as a reducing color developing reagent was added was used for culture at 32°C for 3 days. The number of remaining viable bacteria was measured for samples on the 2nd, 7th and 14th days of storage, and the number of remaining viable bacteria A cultured from samples at each storage period was reduced by more than a specified number relative to the theoretical number B of the above-mentioned 6 mixed microorganisms inoculated on the above-mentioned agar medium as the common logarithm. The results are shown in Table 2.

[0074] In addition, Figure 1 The photograph shows the culture state of SCDLP agar medium on the 14th day. Figure 2A to Figure 2F A comparison diagram showing typical examples of various colonies appearing on the surface of the above-mentioned culture medium is shown in FIG.

[0075] [Table 2]

[0076]

[0077] (*) Preservation effectiveness test method (JP) (**) ISO11930

[0078] [Comparative Example 1]

[0079] The same sample of lotion A as that used in Example 1 was used to examine the preservation efficacy by a general preservation efficacy (preservative property) test method (JP). The test conditions and test results are shown in Table 2.

[0080] [Comparative Example 2]

[0081] Using the same sample of lotion A as that used in Example 1, a preservation effectiveness test was conducted in accordance with ISO11930. The test conditions and test results are shown in Table 2.

[0082] As shown in the results in Table 2, when the correlation between the preservation efficacy test method of the cosmetics using the mixed microorganisms composed of bacteria and yeast of the present invention and ISO11930 was analyzed, representative commercially available cosmetics and the formulation of Example 1 were used for the bacteria and yeast mixed bacteria inoculation method, and the results showed that the preservation efficacy could be evaluated until the 14th day.

[0083] The cosmetics tested that were judged to be suitable (standard A) in the ISO11930 test results also met the prescribed effectiveness evaluation criteria in the preservation efficacy test method of Example 1, that is, the common logarithm of the reduction (CFU) of A relative to B was 3.0 or more.

[0084] In addition, cosmetics (not shown) that are judged as suitable (standard B) and outside the standard in ISO11930 are not suitable in the preservation effectiveness test of the present invention.

[0085] In addition, by Figure 1 It was found that in the SCDLP agar medium to which about 30 ppm (eg, 28 to 32 ppm) of tetrazolium blue was added, colonies of the above-specified six mixed microorganisms were stained so as to be clearly identifiable, and the total number of colony forming units (CFU) could be determined.

[0086] The SCDLP agar medium added with tetrazolium blue can culture the above-mentioned 6 kinds of mixed microorganisms equally with the SCDLP agar medium without tetrazolium blue, and each colony can be formed. The morphology of each colony appearing on the surface of the culture medium is as follows: Figure 2A to Figure 2F As shown, it was determined that the strains (bacterial species) could be identified based on the staining degree and colony morphology of the above-specified 6 mixed microorganisms.

[0087] Thus, Example 1, which is a mixed bacterial yeast inoculation method, is considered to have a preservation efficacy equal to or better than Standard A of ISO11930, and is confirmed to be a new preservation efficacy test method capable of performing preliminary judgment (screening) comparable to the International Organization for Standardization (ISO) or the Japanese Pharmacopoeia (JP).

[0088] [Reference Example 1]

[0089] Four mixed microorganisms consisting of Pseudomonas aeruginosa (NBRC 13275), Staphylococcus aureus (NBRC 13276), Escherichia coli (NBRC 3972) and Candida albicans (NBRC 1594) (NBRC 1594) were appropriately cultured by conventional methods, and all bacterial suspensions were mixed in equal amounts to prepare four mixed bacterial suspensions.

[0090] The bacterial suspension was inoculated into SCDLP agar medium supplemented with 30 ppm of tetrazolium blue (TB) by the agar plate smear method, and cultured at 32°C for 3 days, and then the colony staining and colony formation or presentation were evaluated. The evaluation was set to three levels: good (○), slightly poor (△), and poor (×), and the results are shown in Table 3.

[0091] [Reference Example 2]

[0092] In Reference Example 1, except that nitro blue tetrazolium chloride (Nitro-TB) was used instead of tetrazolium blue (TB) as a reducing color developing reagent, the same procedure was followed to evaluate the colony staining property and the colony formation or presentation property. The results are recorded in Table 3.

[0093] [Reference Example 3]

[0094] In Reference Example 1, except that triphenyltetrazolium chloride (TTC) was used instead of tetrazolium blue (TB) as a reducing color developing reagent, the same procedures were followed to evaluate the colony staining property and the colony formation or presentation property. The results are shown in Table 3.

[0095] [Table 3]

[0096]

[0097] [Comparative Example 3]

[0098] In Example 1, instead of using Burkholderia cepacian (NBRC 14595) and Enterobacter cloacae (NBRC 13535), Aspergillus brasiliensis (NBRC 9455), which is a representative example of molds, was used instead of the above-mentioned 6 mixed microorganisms. Except for this, the storage was exactly the same. For the samples on the 14th and 28th days during the storage period, the visibility was confirmed by colony staining. As a result, a storage period of at least 28 days was required to determine the preservation efficacy of the mold, which was a reduction of 1.5 or more in common logarithm relative to the initial theoretical bacterial count at the time of inoculation.

[0099] [Example 2-6]

[0100] As shown in Table 4, in Example 1, as samples of the cosmetics to be tested, commercially available lotion B (Example 2), commercially available all-in-one gel (Example 3), commercially available cosmetic cream (Example 4), commercially available shampoo (Example 5), and commercially available liquid foundation (Example 6) were used instead of commercially available lotion A. The same procedure was followed except that for the samples whose storage periods were the 2nd day, the 7th day, the 14th day, and the 28th day, the number of remaining viable bacteria was measured respectively, and the number of remaining viable bacteria cultured from the samples at each storage period was reduced by a specified number or more relative to the theoretical number of the above-mentioned 6 mixed microorganisms inoculated on the above-mentioned agar medium as a common logarithm, and the results are shown in Table 4.

[0101] In addition, when the storage period was 7 days or more and there was a decrease in the number of viable cells to a common logarithm value of 6.0 or more, it was clear that there was no further decrease, and therefore the test was terminated at that point.

[0102] [Table 4]

[0103]

[0104] As shown in the results in Table 4, in Examples 2 to 6, for various cosmetics having characteristics such as water solubility, gel, emulsion, surface activity, and oiliness, the preservation effectiveness test method of the present invention obtained results that were appropriate as test results of the preservation effectiveness test at the latest on the 14th day.

[0105] For the test cosmetics confirmed to have preservative efficacy in Examples 2 to 6, a preservative efficacy test was conducted in accordance with ISO11930 in order to confirm the results of the preservative efficacy test.

[0106] According to this method, the reduction number was investigated based on the number of residual viable bacteria on the 7th, 14th and 28th days of storage relative to the theoretical bacterial count of four test bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans) inoculated respectively in four agar culture media, and the results were set as common logarithms and shown in Table 5.

[0107] [Table 5]

[0108]

[0109] As shown in the results in Table 5, it was found that all the test cosmetics confirmed to have preservative efficacy in Examples 2 to 6 obtained results comparable to those of Standard A in the preservative efficacy test based on the International Organization for Standardization (ISO) 11930.

[0110] Therefore, it was confirmed that the preservation effectiveness test of Example 2-6 is a preservation effectiveness test method capable of performing a preliminary judgment comparable to the test prescribed by the International Organization for Standardization (ISO) and the like.

[0111] Industrial Applicability

[0112] It can be used in screening as a preliminary test for microbiological evaluation of the efficacy of preparations such as pharmaceuticals or cosmetics or preservatives contained therein, hygiene inspections, and inspections of cosmetics before shipment.

Claims

1. A method for testing the preservation effectiveness of cosmetics, comprising inoculating a test cosmetic with a mixture of six microorganisms consisting of five bacteria, namely, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacian and Enterobacter cloacae, and Candida albicans yeast, and storing the cosmetics at 20 to 25°C; inoculating a sample collected from the test cosmetics during the storage period in an agar medium supplemented with a color developing reagent, tetrazolium blue, and culturing the sample; and measuring the total number of colony forming units (CFU) per unit mass of the test cosmetics for the six mixed microorganisms; and using a reduction in the number of viable cells A corresponding to the measured total number of colony forming units relative to the theoretical number B of the six mixed microorganisms inoculated on the agar medium by a predetermined number or more as a criterion for evaluating effectiveness.

2. The method for testing the preservation effectiveness of cosmetics according to claim 1, wherein: The storage period is more than 14 days, the reduction in the number of viable bacteria A of the sample collected on the 7th day relative to the theoretical number of bacteria B is greater than 3.0 in terms of common logarithm, and the reduction in the number of viable bacteria A of the sample collected on the 14th day during the storage period relative to the theoretical number of bacteria B is greater than 3.5 in terms of common logarithm.

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

4. The method for testing the preservation effectiveness of cosmetics according to claim 1 or 2, wherein: The sample of the test cosmetics is inoculated into an agar medium and in a liquid medium, and the two culture media are simultaneously cultured. When no colony forming unit is detected in the agar medium stored for a predetermined period, the presence or absence of the six mixed microorganisms in a culture collected from the liquid medium stored for the same period as the predetermined period is confirmed by a streak culture method.

5. The method for testing the preservation effectiveness of cosmetics according to claim 3, wherein: The sample of the cosmetic to be tested is inoculated into an SCDLP agar medium supplemented with a color developing reagent, blue tetrazolium, and is also inoculated into a liquid medium, and cultured simultaneously in the two culture media. When no colony forming units are detected in the SCDLP agar medium stored for a specified period, the presence or absence of the six mixed microorganisms in a culture collected from the liquid culture medium stored for the same period as the specified period is confirmed by a streak culture method.

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