Preservative effectiveness test method for cosmetics

Abstract

To provide a novel preservative effectiveness test method that allows preliminary determination comparable to the International Organization for Standardization (ISO) and the Japanese Pharmacopoeia (JP) in a short period of time, preferably 2 to 3 weeks or less, more preferably about 1 week or less.SOLUTION: Six types of mixed microorganisms consisting of yeast Candida albicans and five types of bacteria of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacia, and Enterobacter cloacae, are inoculated into a cosmetic to be tested and are stored at 20 to 25°C. During this storage period, a sample taken from the cosmetic to be tested is inoculated into an agar medium in which tetrazolium blue as a coloring reagent is added and is cultured. An evaluation standard for preservative effectiveness is such that the number of live bacteria corresponding to the total number of colony-forming units measured in a relatively short culture period is reduced by a specified number or more compared to the number of theoretical bacteria inoculated into the agar medium.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for testing the preservative effectiveness of cosmetics, for simply and efficiently conducting preliminary screening for preservative effect confirmation tests based on various standards for microbiologically evaluating the preservative effectiveness of cosmetics. [Background technology]

[0002] Generally, preservative effectiveness testing is a method for microbiologically evaluating the effectiveness of pharmaceutical and cosmetic preparations 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 lists the preservative effectiveness test method as reference information, explaining that it is a test method in which "the test species to be tested is forcibly inoculated and mixed into the preparation, and the preservative effectiveness is evaluated by tracking the change in the test bacteria over time," and specifies the specified test strains and culture conditions as shown in Table 1 below.

[0004] [Table 1]

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

[0006] Because 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 down as much as possible. Predictions of preservative effectiveness are carried out as a preliminary screening method.

[0007] As an example of such screening, Patent Document 1 describes a method of predicting the preservative properties of cosmetics and the like by calculating the antibacterial index, which is the concentration ratio of ingredients that affect the preservative properties, such as preservatives, at the minimum inhibitory concentration for a specific bacterium, and calculating the antibacterial index when formulating a preparation. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2009-203165 A Summary of the Invention [Problem to be solved by the invention]

[0009] However, in the development of cosmetics, there are cases where products are developed that do not contain any specific preservatives at all, or where a formulation is developed by combining multiple ingredients that replace specific preservatives. When such complex formulations are required, a safer formulation design is 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 conduct a preliminary screening that would enable a reliable judgment on the preservative effect in as short a test period as possible.

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

[0013] In order to achieve the above object, the present invention provides a method for the preparation of a microbial colony-stimulating agent comprising the steps of: (1) detecting and / or treating a bacterial infection caused by ... The cosmetic product to be tested was inoculated with six types of mixed microorganisms consisting of Pseudomonas aeruginosa and Pseudomonas albicans, and stored at 20 to 25°C. During this storage period, samples taken 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 (1g) of the cosmetic product to be tested was then measured for the six types 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 specified number or more relative to the theoretical cell count B of the six types 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 the ISO 11930 standard, and consist of bacteria and yeasts that have a greater impact than molds on the preservative effectiveness of the test cosmetic, which is a general cosmetic. 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 cosmetic.

[0015] The unique Burkholderia cepacia and Enterobacter cloacae employed in the present invention have the properties of being able to assimilate a wide variety of organic matter and having a wide range of habitats, and all of the six types of mixed microorganisms consisting of bacteria and yeasts 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 conditions, and like molds, it is a bacterium that can utilize a variety of organic matter.

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

[0019] In addition, Burkholderia cepacia and Enterobacter cloacae have similar properties to certain molds specified in the Japanese Pharmacopoeia or ISO 11930 standard in terms of their ability to assimilate a wide variety of organic matter and their wide range of habitats, and are therefore suitable microorganisms to be used as indicators in this invention.

[0020] The six types of mixed microorganisms including these two types of bacteria are inoculated into a test cosmetic and stored at 20 to 25° C. The storage period is preferably 14 days or longer. During this storage period, a sample taken from the test cosmetic is inoculated into 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 specified 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 ISO11930 standard and Japanese Pharmacopoeia (JP), it is preferable that the reduction 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 reduction 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] In order to allow sufficiently accurate counting of the total number or species-specific number of colony forming units (CFU) that can be distinguished by species, it is preferable that the agar medium on which the sample is cultured is SCDLP agar medium (lecithin-polysorbate 80-added soyabean-casein digest agar medium) to which the color-developing 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 mixed microorganisms are stained a specific blue color by tetrazolium blue compared to other microorganisms that are accidentally mixed in, and each species forms colonies of different sizes and shapes on the agar medium, making it extremely easy to identify and count the colonies by species.

[0025] In addition, SCDLP agar medium cultures microorganisms by weakening the effects of preservatives contained in the cosmetic product under test, improving the expression and formation of colonies, and enabling more accurate measurement of the number of viable bacteria contained in the cosmetic product under test after storage for a specified period of time.

[0026] In addition to testing the preservative effectiveness of cosmetics, in order to strictly carry out hygiene inspections, etc., required at the time of shipping cosmetics, it is also preferable to check whether specific microorganisms, such as the six mixed microorganisms, are viable in samples contained in the tested cosmetics after a specified period of storage.

[0027] To do this, a sample of the cosmetic to be tested is inoculated into an agar medium and also into a liquid medium, and the sample is cultured in both media simultaneously. If no colony-forming units are detected in the 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 streak culture method.

[0028] In particular, a sample of the cosmetic to be tested is inoculated onto an SCDLP agar medium containing the color-developing reagent tetrazolium blue, 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 stored for a specified period of time, the presence or absence of the six mixed microorganisms in the culture taken from the liquid medium 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. Effect of the Invention

[0029] This invention has the advantage that Burkholderia cepacia and Enterobacter cloacae are bacteria with the ability to assimilate a wide variety of organic matter and have a wide range of habitats, and that the colonies formed when a specific six-species mixed microorganism including these are cultured can be identified by staining them with a specific color-developing reagent, 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, making it a novel preservative effectiveness testing method that can make a preliminary determination comparable to the International Organization for Standardization (ISO) and the Japanese Pharmacopoeia (JP) in a shorter test period than that required for conventional preservative effectiveness tests, for example, 2 to 3 weeks (or 14 to 21 days) or less. [Brief description of the drawings]

[0030] [Figure 1] A photograph showing the culture state of Example 1 on SCDLP agar medium containing tetrazolium blue. [Diagram 2]Photographs substituting drawings showing typical morphologies of colonies of (a) Pseudomonas aeruginosa, (b) Burkholderia cepacia, (c) Staphylococcus aureus, (d) Escherichia coli, (e) Enterobacter cloacae, and (f) Candida albicans under the culture conditions of Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] As an embodiment of the present invention, a method for testing the preservative effectiveness of cosmetics is 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, and a sample taken from the test cosmetic during this storage period is inoculated onto an agar medium to which the color-developing reagent tetrazolium blue has been added and cultured, after which the total number of colony-forming units (CFU) per unit mass (1 g) of the test cosmetic is 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 specified 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 in type to the cosmetic product to be tested, and can be applied to cosmetics that contain water or oil, and includes, for example, basic cosmetics such as lotion, essence, milky lotion, 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, makeup remover, facial cleanser, 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; glycerol 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 to be tested in reducing the number of viable bacteria and to evaluate the storage conditions of the cosmetic under more severe conditions, it is preferable to use a sample that is inoculated onto the agar medium that is diluted, for example, 10 times, and to conduct the preservative effectiveness test under conditions in which the effect of the preservatives contained in the cosmetic is reduced to 1 / 10.

[0036] The mixed microorganisms used in this invention are used to inoculate the test cosmetic product, and they 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.

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

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

[0039] Enterobacter cloacae is a gram-negative, facultatively anaerobic, peritrichous, periflagellated rod-shaped bacterium in the order Enterobacteriaceae and family 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 it is unlikely to develop as a result of infection, it can cause respiratory tract infections in cases of immunodeficiency.

[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), and Candida albicans (NBRC 1594), Burkholderia cepacia (NBRC 14595), and Enterobacter cloacae (NBRC 13535) shown below as test strains.

[0041] The test cosmetic is inoculated with these mixed microorganisms and mixed uniformly, and then 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 assumes normal storage and use conditions for cosmetics.

[0042] The storage period may be selected appropriately depending on the type of cosmetic product to be tested, but is usually at least 14 days, and as a guideline, at most 28 days. For example, samples taken on the 2nd, 7th, 14th, and 28th days after storage will facilitate 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).

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

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

[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, and therefore the colonies can be clearly identified, and it has been confirmed in the Reference Examples described below that the colony formation is not affected.

[0046] [ka]

[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 pouring method, and culturing the mixture.

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

[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, and it is confirmed that the viable bacteria count corresponding to the measured total number of colony forming units is reduced by a specified number or more compared to the theoretical bacteria count of the six types of mixed microorganisms inoculated onto the agar medium. The theoretical number of bacteria in a mixed microorganism can be calculated in a standard manner by calculating the number of colonies formed in a petri dish, the amount of inoculated liquid, and the dilution ratio, and thus the number of viable bacteria per unit volume of the sample.

[0050] In addition, when a hygiene test is to be conducted by checking for the presence or absence of specific microorganisms by the streak culture method at the same time as 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 cultured in both media simultaneously. When culturing simultaneously in agar medium and liquid medium, appropriate culturing can be achieved by inoculating, for example, 1 / 100 of a 100 volume part sample of the cosmetic to be tested into the agar medium and simultaneously inoculating 99 / 100 into the liquid medium.

[0051] The liquid medium may be any medium suitable for the growth of the mixed microorganisms, and may be, for example, Soyabean-Casein-Digest Medium or 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, by culturing for at least 3 days at 30 to 35° C. to sufficiently grow the six types of mixed microorganisms. Then, the presence or absence of the six types of mixed microorganisms in the culture sample collected from the liquid medium is confirmed by a streak culture method.

[0053] In the so-called enrichment test using streak culture, a streak of the same agar medium as above is cultured at 30-35°C for at least 18 hours, and the presence or absence of colonies formed independently is confirmed. If bacteria are detected in a sample taken on or before the 7th day of liquid culture, a retest is conducted, and if bacteria are not detected in a sample taken after the 7th day, for example on the 8th or 14th day, the preservative effectiveness of the cosmetic product is confirmed and the test is terminated. In addition to the preservative effectiveness test of the present invention, if a preservative effectiveness test for molds is required, an additional test for molds is carried out by a conventional method. EXAMPLES

[0054] [Example 1] As shown in Table 2, a mixture of six types of microorganisms 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 for the preservative effectiveness test of cosmetics (Table 2 lists 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 microbial solution).

[0056] In addition, 20 g of commercially available lotion A was weighed out and placed in a sterilized container as a sample of the cosmetic to be tested, and 0.2 ml of the mixed microbial liquid described above was inoculated into this sample, stirred to disperse it evenly, and then diluted 10-fold and stored for 14 days at 22.5° C. The cosmetic to be tested was commercially available lotion A, which had already passed the ISO 11930 test criteria.

[0057] Sampling was performed on the 2nd, 7th, and 14th days of the above storage period, and microbial tests were performed by simultaneously culturing each sample using the agar culture method and liquid culture method.

[0058] For the agar culture, SCDLP agar medium containing 30 ppm of tetrazolium blue (Dojindo Laboratories) as a reducing color-developing reagent was used to culture the samples at 32°C for 3 days, and the number of remaining viable bacteria was measured for each sample stored on the 2nd, 7th, and 14th days. The reduction in the number of remaining viable bacteria A cultured from the sample at each storage period compared to the theoretical number of bacteria B of the six types of mixed microorganisms inoculated onto the agar medium by a specified number or more was expressed as a common logarithm, and the results are shown in Table 2.

[0059] FIG. 1 shows a photograph of the culture state on the SCDLP agar medium on the 14th day, and FIG. 2 shows a comparison diagram of typical examples (a) to (f) of the colonies that appeared on the surface of the medium.

[0060] [Table 2]

[0061] [Comparative Example 1] Using the same sample of lotion A as the sample 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] A preservative effectiveness test according to ISO11930 was carried out using the same sample of lotion A as the sample used in Example 1, and 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 cosmetic preservative effectiveness test method using the mixed microorganisms consisting of bacteria and yeast of the present invention and ISO11930 was analyzed, it was shown that the preservative effectiveness could be evaluated within 14 days when the bacteria and yeast mixed inoculation method was used with representative commercially available cosmetics and the formulation of Example 1.

[0064] The test cosmetics that were judged to be in compliance (criterion A) in the ISO11930 test results also met the prescribed effectiveness evaluation standard in the preservative effectiveness test method of Example 1, which is that the reduction in the amount of B relative to A (CFU) is a common logarithm value of 3.0 or more.

[0065] In addition, all of the cosmetics that were judged to be in compliance (criterion B) or outside the standard in ISO11930 (examples omitted) were found to be non-compliant in the preservative effectiveness test of the present invention.

[0066] Furthermore, as is clear from FIG. 1, in 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, making it possible to measure the total number of colony forming units (CFUs).

[0067] The SCDLP agar medium to which tetrazolium blue had been added was capable of culturing the six specified mixed microorganisms in the same manner as the SCDLP agar medium to which tetrazolium blue had not been added, and was capable of causing each colony to appear. It was determined that the morphology of each colony that appeared on the surface of the medium was such that the strains (bacterial species) of the six specified mixed microorganisms could be identified from the degree of staining and the morphology of the colonies, as shown in Figure 2 (a) to (f).

[0068] Thus, Example 1, which is a mixed bacterial and yeast inoculation method, was recognized to have a preservative effectiveness equal to or greater than that of Criteria A of ISO11930, and was confirmed to be a novel preservative effectiveness testing method capable of 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 suspension consisting of Pseudomonas aeruginosa (NBRC 13275), Staphylococcus aureus (NBRC 13276), Escherichia coli (NBRC 3972), and Candida albicans (NBRC 1594) was appropriately cultured by standard methods, and then equal amounts of all the bacterial suspensions were mixed to prepare a four-type mixed bacterial suspension.

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

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

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

[0073] [Table 3]

[0074] [Comparative Example 3] Storage was performed in exactly the same manner as in Example 1, except that instead of the six types of mixed microorganisms, Burkholderia cepacian (NBRC 14595) and Enterobacter cloacae (NBRC 13535) were not used, and instead, Aspergillus brasiliensis (NBRC 9455) was used as a representative example of mold. When the visibility of the samples on the 14th and 28th days of storage was confirmed by colony staining, a storage period of at least 28 days was required to determine the preservative effectiveness of reducing the theoretical number of mold bacteria at the time of inoculation by 1.5 or more in common logarithm.

[0075] [Example 2-6] As shown in Table 4, in Example 1, 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 bacterial counts were measured for samples stored for 2, 7, 14, and 28 days in exactly the same manner, and the reduction in the remaining viable bacterial count cultured from the samples for each storage period by a specified number or more relative to the theoretical bacterial count of the six mixed microorganisms inoculated onto the agar medium was expressed as a common logarithm, and the results are shown in Table 4. If the storage period was 7 days or longer and there was a decrease in the viable cell count of 6.0 or more logarithms, it was clear that there would be no further decrease, and the test was terminated at that point.

[0076] [Table 4]

[0077] As is clear from the results shown in Table 4, the preservative effectiveness test results of Examples 2-6 were found to be appropriate for various cosmetics characterized by water solubility, gel, emulsion, surface activity, oiliness, etc., by the preservative effectiveness test method of this invention, no later than the 14th day.

[0078] For the test cosmetics whose preservative effectiveness was confirmed in Examples 2-6, a preservative effectiveness test according to ISO11930 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 on the 7th, 14th, and 28th days of storage was investigated for each of the four test bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) inoculated onto four agar media, relative to the theoretical number of bacteria. The results are shown in Table 5 as common logarithm values.

[0080] [Table 5]

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

[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 preparations or the preservatives contained therein, as well as for hygiene inspections and inspections of cosmetics at the time of shipment.

Claims

1. Five species of bacteria, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacian, and Enterobacter cloacae, and the yeast Candida albicans were included. 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. 2. The method for testing the preservative effectiveness of a cosmetic product according to claim 1, wherein the storage period is 14 days or more, the reduction in the viable cell count A of the 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 the reduction in the viable cell count A of the 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.

3. 3. The method for testing the preservative effectiveness of a cosmetic product according to claim 1, 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. 3. The method for testing the preservative effectiveness of a cosmetic according to claim 1 or 2, further comprising inoculating an agar medium with a sample of the cosmetic to be tested and a liquid medium with the same sample, and culturing the sample in both media simultaneously; if no colony-forming units are detected in the agar medium stored for a specified period of time, the presence or absence of the six kinds of mixed microorganisms in a culture taken from a liquid medium stored for the same period of time is confirmed by a streak culture method.

5. 4. The method for testing the preservative effectiveness of cosmetics according to claim 3, further comprising the steps of inoculating a sample of the cosmetic to be tested onto an SCDLP agar medium containing tetrazolium blue as a color-developing reagent, and simultaneously inoculating the same sample onto a liquid medium and culturing the sample in both media at the same time, and if no colony-forming units are detected in the SCDLP 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.

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