Preservative efficacy test method for cosmetic

Abstract

Six kinds of mixed microorganisms consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacian, and Enterobacter cloacae, and Candida albicans yeast are inoculated into the cosmetic to be tested for storage, and a sample collected from the cosmetic to be tested during the storage is inoculated into an agar medium to which tetrazolium blue is added and cultured, and the number of viable bacteria corresponding to the total number of colony forming units determined in a short time is reduced by a predetermined amount or more from the theoretical number of bacteria inoculated into the agar medium, and this is used as an evaluation criterion for preservation efficacy.

Description

TECHNICAL FIELD

[0001] The present application relates to a preservation efficacy test method for cosmetics, for simply and efficiently performing preliminary screening of various standard preservative effect confirmation tests for evaluating the preservation efficacy of cosmetics based on microbiology. BACKGROUND

[0002] Generally, for preservation efficacy tests, as a method for evaluating the efficacy of a preservative or the like contained in a preparation or the like, or a cosmetic, a preservative effect confirmation test based on the International Organization for Standardization (ISO) or the Japanese Pharmacopoeia (JP) is known.

[0003] In the nineteenth revised edition of the Japanese Pharmacopoeia, as reference information, a preservation efficacy test method is described, and it is explained that it is a test method in which "by forcibly inoculating and mixing test bacteria into a preparation, and tracing the growth and decline of the test bacteria over time, the preservation efficacy is evaluated", and as shown in Table 1 below, a specified test bacterial strain and culture conditions are prescribed.

[0004] [Table 1]

[0005]

[0006] The same preservation efficacy test is also prescribed in the ISO 11930 standard of the International Organization for Standardization (ISO), and the culture time of the sample as a test object is at least 4 weeks, and in practice, 4 to 5 weeks are required until the test result is obtained, as in the case of the Japanese Pharmacopoeia.

[0007] Thus, the prescribed preservation efficacy test requires a long time, and therefore, as a cosmetic manufacturing business, in order to test a large number of trial products, it is necessary to suppress expenses as much as possible and to perform efficient product development in a short period of time, and as preliminary screening, a prediction of preservative properties is performed.

[0008] As such screening, in Patent Literature 1, for cosmetics and the like, an antibacterial index defined as the concentration ratio at which a specific bacterium is inhibited at the minimum inhibitory concentration in a raw material that affects the preservative properties of a preservative and the like is defined, and the antibacterial index is calculated in the formulation of a preparation to predict the preservative properties.

[0009] PRIOR ART DOCUMENTS

[0010] PATENT LITERATURE

[0011] Patent Literature 1: Japanese Patent Application Publication No. 2009-203165 SUMMARY

[0012] However, in the development of cosmetics, there are cases where a formulation is developed that does not at all match a specific preservative and cases where a formulation is developed by combining a plurality of raw materials that replace a specific preservative, and when such a complex formulation is required, a safer formulation design is required.

[0013] Therefore, not only prediction based on the antibacterial index of a preservative is required, but also a preliminary screening that is simple and effective for a preservation efficacy test is required.

[0014] As described above, the established preservation efficacy test generally requires 5 to 6 weeks, and thus a preliminary screening that is highly reliable for judging the preservation effect within the shortest possible test period is required.

[0015] Therefore, the present application aims to solve the above-described problems and provide a novel preservation efficacy test method that enables a preliminary determination comparable to the International Organization for Standardization (ISO) or the Japanese Pharmacopoeia (JP) in a shorter period than the required test period of the prior art, for example, 2 to 3 weeks (or 14 to 21 days) or less.

[0016] To solve the above-described problems, the present application employs a preservation efficacy test method for cosmetics, in which six kinds of mixed microorganisms consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacian, and Enterobacter cloacae, and Candida albicans yeast are inoculated into a cosmetic to be tested and stored at 20 to 25°C, a sample collected from the cosmetic to be tested during the storage period is inoculated into an agar medium to which a chromogenic reagent tetrazolium blue is added and cultured, and then the total number of colony forming units (CFU) per unit mass (1 g) of the cosmetic to be tested is determined for the six kinds of mixed microorganisms, and the number of viable bacteria A corresponding to the total number of colony forming units determined is reduced by a predetermined amount or more relative to the theoretical number B of the six kinds of mixed microorganisms inoculated into the agar medium, and the reduction is used as an evaluation criterion for effectiveness.

[0017] The six kinds of mixed microorganisms used in the present application do not include microorganisms containing mold species specified in the preservation efficacy test method of the reference information of the Japanese Pharmacopoeia or the ISO 11930 standard, and are composed of bacteria and yeast that have a greater impact on the preservability of a cosmetic to be tested as a general cosmetic than mold species. In the present application, the contamination risk is screened in a short period of time using the above-described six kinds of mixed microorganisms, and a preliminary determination of the preservation efficacy of the cosmetic to be tested is performed.

[0018] In the present application, Burkholderia cepacia and Enterobacter cloacae, which are used alone, have properties of assimilability of various organic matters and wide habitat, and six kinds of mixed microorganisms composed of bacteria and yeast all increase the number of bacteria in a short time to form colonies in a prescribed temperature range of 20 to 25°C during the above storage period.

[0019] Burkholderia cepacia is a soil bacterium known as an opportunistic pathogen causing respiratory diseases and the like, and is a bacterium capable of assimilating various organic matters like molds.

[0020] In addition, Enterobacter cloacae is an intestinal bacterium that also causes pneumonia and cystitis, and although it rarely infects healthy people, it is a bacterium that is ubiquitous in terrestrial and aquatic environments like molds.

[0021] Thus, the pathogenic bacteria, Burkholderia cepacia and Enterobacter cloacae, which can adhere to the skin, do not produce spores like molds, and thus increase and decrease rapidly like the other three kinds of bacteria and one kind of yeast (fungus).

[0022] In addition, Burkholderia cepacia and Enterobacter cloacae have properties of assimilability of various organic matters and wide habitat, which are the same as specific molds specified in the Japanese Pharmacopoeia or the ISO 11930 standard, and are microorganisms suitable for use as indicators in the present application.

[0023] The above six kinds of mixed microorganisms including the two kinds of bacteria are inoculated into the cosmetic to be tested and stored at 20 to 25°C. The storage period at this time is preferably 14 days or more. A sample collected from the above cosmetic to be tested during the storage period is inoculated into an agar medium to which a chromogenic reagent, tetrazolium blue, is added and cultured.

[0024] Then, sampling is performed at, for example, the 7th day and the 14th day during the above storage, and the number of viable bacteria A corresponding to the total number of colony-forming units determined is reduced by a prescribed number or more relative to the theoretical number B of the above six kinds of mixed microorganisms inoculated into the above agar medium, and this is used as an evaluation criterion for effectiveness.

[0025] The number of reduction of the number of viable bacteria A of the sample collected on the 7th day during the above storage period relative to the above theoretical number B is 3.0 or more in terms of a common logarithmic value, and the number of reduction of the number of viable bacteria A of the sample collected on the 14th day during the above storage period relative to the above theoretical number B is 3.5 or more in terms of a common logarithmic value, and this is preferably used as an evaluation criterion that matches the ISO 11930 standard or the Japanese Pharmacopoeia (JP).

[0026] In addition, in order to accurately count the colony-forming units (CFU) that can be identified by species, either by total number or by type, the preferred agar medium for culturing the sample is SCDLP agar medium (soy casein digest agar medium containing lecithin and polysorbate 80) with added tetrazolium blue as a colorimetric reagent, and the culture conditions are a culture temperature of 30-35°C and a culture time of more than 3 days.

[0027] The colonies formed by the above six mixed microorganisms can be stained with tetrazolium blue in a blue color that is specific to them compared to other microorganisms that are accidentally mixed together. Furthermore, they form colonies of different sizes or shapes on agar medium according to their species, making it extremely easy to identify and count the colonies according to their species.

[0028] In addition, SCDLP agar medium reduces the influence of preservatives contained in the tested cosmetics while culturing microorganisms, thus improving colony presentation and formation, and enabling more accurate determination of the number of viable bacteria contained in the tested cosmetics after the specified storage period.

[0029] In addition to the preservation efficacy test of cosmetics, in order to further strictly conduct the hygiene inspections required when cosmetics leave the factory, it is also preferable to investigate whether there are any specific microorganisms, such as the above-mentioned six mixed microorganisms, in the samples contained in the tested cosmetics after the specified storage period.

[0030] Therefore, the sample of the cosmetic product to be tested was inoculated into agar medium and also into liquid medium. The sample was cultured simultaneously in both media. If no colony-forming units were detected in the agar medium after being stored for the specified period, the presence of the above six mixed microorganisms was confirmed by streak plating method in the culture collected from the liquid medium after being stored for the same specified period.

[0031] In particular, by inoculating the sample of the aforementioned cosmetic product onto SCDLP agar medium containing the colorimetric reagent tetrazolium blue, and also inoculating the sample of the aforementioned cosmetic product onto liquid medium, and simultaneously culturing it on both media, if no colony-forming units are detected in the aforementioned SCDLP agar medium that has been stored for the specified period, the presence of the aforementioned six mixed microorganisms can be confirmed by streak plating method in the culture collected from the liquid medium that has been stored for the same specified period as above. This improves the visibility of colony morphology and other characteristics, and further improves the accuracy of viable count determination.

[0032] In this invention, Burkholderia cepacia and Enterobacter cloacae are bacteria with the ability to assimilate a variety of organic substances and a wide range of habitats. Colonies formed by culturing a specific mixture of six microorganisms containing them are identified by staining with a specific chromogenic reagent. The number of colony-forming units (CFU), which is the total number of viable bacteria, can be quantified in a shorter period of time. Therefore, this invention provides a novel preservation efficacy test method that can make preliminary determinations comparable to those of the International Organization for Standardization (ISO) or the Japanese Pharmacopoeia (JP) in a shorter period of time than that required for existing preservation efficacy tests, such as 2 to 3 weeks (or 14 to 21 days) or less. Attached Figure Description

[0033] Figure 1 The accompanying photograph is a substitute photograph showing the culture status of Example 1 in SCDLP agar medium supplemented with tetrazolium blue.

[0034] Figure 2A The accompanying photograph is a substitute for the typical morphology of Pseudomonas aeruginosa colonies in the culture state of Example 1.

[0035] Figure 2B The accompanying photograph is a substitute photograph showing the typical morphology of Burkholderia cepacia colonies under the culture conditions of Example 1.

[0036] Figure 2C The accompanying photograph is a substitute for the typical morphology of Staphylococcus aureus colonies in the culture state of Example 1.

[0037] Figure 2D The accompanying photograph is a substitute for the typical morphology of Escherichia coli colonies in the culture state of Example 1.

[0038] Figure 2E The accompanying photograph is a substitute photograph showing the typical morphology of Enterobacter cloacae colonies under culture conditions in Example 1.

[0039] Figure 2F The accompanying photograph is a substitute for the typical morphology of Candida albicans colonies in the culture state of Example 1. Detailed Implementation

[0040] As an embodiment of the present invention, the method for testing the preservation efficacy of cosmetics will be described in more detail and specifically. The method for testing the preservation efficacy of cosmetics involves inoculating the cosmetics tested with the aforementioned six specific mixed microorganisms and storing them at 20-25°C. During the storage period, samples collected from the cosmetics tested are inoculated into an agar medium containing the colorimetric reagent tetrazolium blue and cultured. The total number of colony-forming units (CFU) per unit mass (1g) of the cosmetics tested is determined. The viable count A corresponding to the total number of colony-forming units measured is reduced by a predetermined number or more relative to the theoretical count B of the six mixed microorganisms inoculated into the agar medium as the evaluation criterion for effectiveness.

[0041] In this invention, the type of cosmetic product being tested is not particularly limited. It can be applied to cosmetics containing water or oil, such as basic cosmetic materials including toner, serum, lotion, cream, mask, sunscreen, etc.; color cosmetic materials including foundation, mascara, eyeliner, eyeshadow, lipstick, concealer, blush, nail polish, etc.; hair cosmetic materials including shampoo, conditioner, hair styling agent, etc.; body cleansing agents such as shower gel; and external compositions such as makeup remover oil and facial cleanser. It can also be applied to the external composition as a quasi-pharmaceutical.

[0042] In such tested cosmetics, preservatives (i.e., preservatives) are usually contained to prevent the proliferation of microorganisms such as bacteria and mold. However, regardless of whether preservatives are contained, the "Test Method for Preservation Efficacy of Cosmetics" of this invention can be used.

[0043] Well-known preservatives used in cosmetic materials or quasi-pharmaceuticals include, for example, parabens (methylparaben, ethylparaben, propylparaben, butylparaben, etc.); phenoxyethanol; alkyl glycols; alkyl glycerol ethers; glycerol fatty acid esters; salicylic acid; sodium benzoate; isothiazolinone derivatives; and imidazolines. Urea; dehydroacetic acid and its salts; phenols; halogenated bisphenols, acid amides, quaternary ammonium salts; triclocarban, zinc pyrithione, benzalkonium chloride, benzyl chloride, sorbic acid, chlorhexidine, chlorhexidine gluconate, halocarban, hexachlorophenol, physalisol; phenethyl alcohol, photosensitizers, antibacterial zeolites, silver ions, etc.

[0044] In order to mitigate the reduction in viable bacteria caused by preservatives in the tested cosmetics and to determine the preservation conditions of cosmetics under more stringent conditions, it is preferable to use samples inoculated on agar medium diluted, for example, 10 times, and to conduct preservation efficacy tests under conditions where the effect of preservatives in cosmetics is suppressed to 1 / 10.

[0045] In addition, the mixed microorganisms used in this invention are used to inoculate the cosmetic product being tested. They consist of five types of bacteria, including Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacia, and Enterobacter cloacae, as well as one type of yeast, Candida albicans.

[0046] Of the six microorganisms mentioned above, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Candida albicans were also used as reference information in the preservation efficacy test method in the 19th revised edition of the Japanese Pharmacopoeia. The ATCC strain or NBRC strain listed in Table 1 can also be used as the test strain.

[0047] In addition, Burkholderia cepacian and Enterobacter cloacae are typical species of Gram-negative, non-spore-forming, aerobic, polar flagellated bacilli belonging to the order Burkholderia and family Burkholderiaceae. They are common soil bacteria in the natural environment and are sometimes opportunistic pathogens that cause infection through the respiratory tract or bloodstream.

[0048] In addition, *Enterobacter cloacae* is a Gram-negative, facultative anaerobic, peritrichous, flagellated bacillus belonging to the order Enterobacteriaceae, family Enterobacteriaceae. It is mesophilic and can grow in a wide pH range of 4–10, and is widely found in terrestrial and aquatic environments (water, mud, soil, food, warm water treatment tanks). This bacterium is a symbiotic microorganism found in the general gut microbiota of humans and animals. While the likelihood of disease development through infection is low, it can sometimes cause respiratory infections in immunocompromised individuals.

[0049] For the various microorganisms used in the mixed microorganisms 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.

[0050] These mixed microorganisms were inoculated into the cosmetic product being tested and mixed evenly, then stored at 20–25°C. The storage temperature is the range within which the mixed microorganisms used in this invention can grow and assumes the normal storage and use conditions of the cosmetic product.

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

[0052] The agar medium used to culture the collected samples is agar medium supplemented with tetrazolium blue. This agar medium can be agar plate medium or agar slant medium.

[0053] In addition, in order to conduct the test on the above-mentioned cosmetic products in a state that is as conducive to the proliferation of microorganisms as possible, it is preferable to use SCDLP agar medium, which is a soybean casein digest agar medium containing lecithin and polysorbate 80 (SCDLP agar medium) included in the microbial test methods for pharmaceuticals.

[0054] SCDLP agar medium is a medium that uses lecithin and polysorbate 80 added to SCD agar medium (soy casein digest agar) as a base medium to intentionally reduce the effectiveness of preservatives and to confirm the presence or absence of live bacteria.

[0055] Tetrazolium blue is a type of reduction-based chromogenic reagent and is a compound represented by the following structural formula. Tetrazolium blue exhibits excellent staining properties for colonies formed by the six mixed microorganisms used in this invention, clearly identifying the colonies without affecting colony presentation, as confirmed by the reference examples described later.

[0056]

[0057] The above-mentioned culture can be carried out by spreading the sample on an agar plate using conventional methods. Alternatively, the agar plate mixing method can be used to form a mixture of agar medium and sample into a plate for culture.

[0058] The culture using the above-mentioned agar medium, which is incubated at 30–35°C for at least 3 days, allows for the full proliferation of the six mixed microorganisms and enables the accurate determination of colony forming units (CFU).

[0059] In this test, a commercially available automated bacterial count measuring device was used to measure the total number of colony-forming units (CFU) per unit mass of the tested cosmetic product containing the above six mixed microorganisms. The test confirmed that the number of viable bacteria corresponding to the total number of colony-forming units was reduced by a specified number or more compared to the theoretical number of the above six mixed microorganisms inoculated on the above agar medium.

[0060] The theoretical number of mixed microorganisms can be calculated using conventional methods from the number of colonies formed on the petri dish, the volume of inoculated liquid, and the dilution ratio, to determine the number of viable bacteria per unit volume of the sample.

[0061] In addition, while conducting the preservation efficacy test, the presence or absence of specific microorganisms is confirmed by the streak plating method. During the hygiene inspection, the sample of the above-mentioned cosmetic product is inoculated into an agar medium, and an appropriate amount of the above-mentioned cosmetic product sample is also inoculated into a liquid medium and cultured simultaneously in both media.

[0062] When culturing simultaneously in agar medium and liquid medium, appropriate culturing can be achieved by, for example, inoculating 1 / 100 of the sample of the tested cosmetic in agar medium and simultaneously inoculating 99 / 100 in liquid medium out of 100 volume portions of the sample.

[0063] The liquid culture medium can be any medium suitable for the growth of mixed microorganisms. Soybean casein digest medium or Sabouraud dextrose liquid medium, which are composed of the same components as the agar medium mentioned above, can be used as described in the reference information of the Japanese Pharmacopoeia.

[0064] Liquid culture was conducted under the same temperature and duration as agar culture, for example, by incubating at 30–35°C for at least 3 days, which allowed the six mixed microorganisms to proliferate sufficiently. Then, the presence of the six mixed microorganisms in the cultures collected from the liquid culture medium was confirmed by streak plating.

[0065] The so-called enrichment test, performed by streak plating, involves incubating the same agar medium as described above at 30–35°C for at least 18 hours to confirm the presence or absence of isolated colonies. For samples collected on or before day 7 of liquid culture, if bacteria are detected, the test is repeated. For samples collected after day 7, such as day 8 or day 14, if no bacteria are detected, the test is considered to confirm the preservation effectiveness of the cosmetic and is terminated.

[0066] In addition, if a preservation efficacy test for molds is required besides the preservation efficacy test of the present invention, additional tests for molds can be conducted using conventional methods.

[0067] Example

[0068] [Example 1]

[0069] As shown in Table 2, for the preservation efficacy test of cosmetics, a mixture of six microorganisms was used, consisting of 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). (In Table 2, these are listed as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, two specific bacteria, and yeast.)

[0070] That is, after culturing each microorganism appropriately using conventional methods, a bacterial suspension is prepared, and all the bacterial suspensions are mixed in equal amounts to make six mixed bacterial suspensions (mixed microbial liquids).

[0071] In addition, as a sample of the cosmetic product being tested, 20g of commercially available toner A was weighed into a sterile container. 0.2ml of the aforementioned mixed microbial solution was inoculated into this sample, stirred to ensure uniform dispersion, diluted 10 times, and stored at 22.5°C for 14 days. It should be noted that the commercially available toner A used as the cosmetic product being tested had previously met the acceptance criteria according to ISO 11930 test results.

[0072] Samples were taken on the 2nd, 7th, and 14th days of the above storage period to conduct microbial tests by simultaneously culturing each sample using both agar and liquid culture methods.

[0073] In agar culture, SCDLP agar medium containing 30 ppm of tetrazolium blue (Dongjin Chemical Research Institute) as a reducing chromogenic reagent was used and cultured at 32°C for 3 days. For samples stored on day 2, day 7, and day 14, the number of surviving bacteria was determined. The number of surviving bacteria A from the samples stored for each period that was less than or equal to the theoretical number B of the above-mentioned 6 mixed microorganisms inoculated on the above-mentioned agar medium was set as the common logarithm. The results are shown in Table 2.

[0074] In addition, Figure 1 The photo shows the culture status of SCDLP agar medium on day 14, and further, in Figures 2A-2F The diagram shows a comparison of typical examples of colonies that appeared on the surface of the culture medium described above.

[0075] [Table 2]

[0076]

[0077] (*) Preservative efficacy test method (JP) (**) ISO 11930

[0078] [Comparative Example 1]

[0079] Using the same sample of lotion A as that used in Example 1, the preservation efficacy was investigated by the general preservation efficacy (preservative) test method (JP), and the test conditions and test results are recorded in Table 2.

[0080] [Comparative Example 2]

[0081] Using the same lotion A sample as used in Example 1, a preservation efficacy test was conducted according to ISO 11930. The test conditions and results are recorded in Table 2.

[0082] As shown in Table 2, when analyzing the correlation between the test method for the preservation efficacy of cosmetics using a mixture of bacteria and yeast of the present invention and ISO 11930, representative commercially available cosmetics and the formulation of Example 1 were used for the bacterial-yeast mixed inoculation method. The results showed that the preservation efficacy could be evaluated up to the 14th day.

[0083] The tested cosmetic product that is deemed suitable (Standard A) in the ISO 11930 test results also meets the prescribed effectiveness evaluation criteria in the preservation efficacy test method of Example 1, namely, the commonly used logarithmic value of the reduction in amount (CFU) of A relative to B is 3.0 or higher.

[0084] It should be noted that cosmetics deemed suitable (Standard B) and outside the standard in ISO 11930 (examples omitted) are not suitable in the preservation efficacy test of this invention.

[0085] In addition, by Figure 1 It is known that in SCDLP agar medium containing approximately 30 ppm (e.g., 28–32 ppm) of tetrazolium blue, the colonies of the six mixed microorganisms specified above are stained in a way that allows for clear identification and determination of the total number of colony forming units (CFU).

[0086] SCDLP agar medium supplemented with tetrazolium blue is equally capable of culturing the six mixed microorganisms specified above as SCDLP agar medium without tetrazolium blue, and can display the morphology of each colony on the surface of the medium as follows: Figures 2A-2F As shown, it is determined that the bacterial strains (species) can be identified based on the staining degree and colony morphology of the six mixed microorganisms specified above.

[0087] Thus, Example 1, as a mixed bacterial and yeast inoculation method, is considered to have preservation efficacy equivalent to or higher than Standard A of ISO 11930, confirming that it is a novel preservation efficacy test method capable of conducting preliminary judgments (screening) comparable to those of 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), were cultured separately using conventional methods. Then, all the bacterial suspensions were mixed in equal volumes to prepare four mixed bacterial suspensions.

[0090] The bacterial suspension was inoculated onto SCDLP agar medium supplemented with 30 ppm tetrazolium blue (TB) using the agar plate spread method. After incubation at 32°C for 3 days, the staining properties and colony formation or presentation were evaluated. The evaluation was graded as follows: good (〇), slightly poor (△), and poor (×). The results are shown in Table 3.

[0091] [Reference Example 2]

[0092] In Reference Example 1, Nitro-TB was used instead of Tetraazole Blue (TB) as the reducing colorimetric reagent. Otherwise, the staining and colony formation or presentation were evaluated in exactly the same manner, and the results are recorded in Table 3.

[0093] [Reference Example 3]

[0094] In Reference Example 1, triphenyltetrazolium chloride (TTC) was used instead of tetrazolium blue (TB) as a reducing colorimetric reagent. Otherwise, the staining and colony formation or presentation were evaluated in exactly the same manner, and the results are recorded in Table 3.

[0095] [Table 3]

[0096]

[0097] [Comparative Example 3]

[0098] In Example 1, Burkholderia cepacian (NBRC 14595) and Enterobacter cloacae (NBRC 13535) were not used. Instead, Aspergillus brasiliensis (NBRC 9455), a representative example of mold, was used instead of the above six mixed microorganisms. Otherwise, the preservation was carried out in exactly the same manner. For samples on the 14th and 28th days of preservation, the visibility was confirmed by colony staining. The preservation efficacy was determined to be at least 28 days of preservation if the mold count decreased by more than 1.5 relative to the initial theoretical count of molds by the commonly used logarithmic value.

[0099] [Examples 2-6]

[0100] As shown in Table 4, in Example 1, as samples of the cosmetics to be tested, commercially available toner B (Example 2), commercially available all-in-one gel (Example 3), commercially available cream (Example 4), commercially available shampoo (Example 5), and commercially available foundation (Example 6) were used instead of commercially available toner A. Otherwise, the same procedure was performed. For samples stored for 2 days, 7 days, 14 days, and 28 days, the number of viable bacteria remaining was measured. The number of viable bacteria remaining from the samples cultured for each storage period that was less than or equal to the theoretical number of the above six mixed microorganisms inoculated on the above agar medium was set as the common logarithm. The results are shown in Table 4.

[0101] It should be noted that if the storage period is more than 7 days and there is a decrease in the number of viable bacteria with a commonly used logarithmic value of 6.0 or higher, and it is clear that there is no further decrease, then the experiment is terminated at that point.

[0102] [Table 4]

[0103]

[0104] As shown in Table 4, in Examples 2-6, for various cosmetics with characteristics such as water solubility, gel, emulsion, surface activity, and oiliness, the preservation efficacy test method of the present invention yielded appropriate test results as preservation efficacy test results no later than the 14th day.

[0105] For the tested cosmetic products that were confirmed to have preservation efficacy in Examples 2-6, preservation efficacy tests were conducted in accordance with ISO 11930 to confirm the results of the preservation efficacy test.

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

[0107] [Table 5]

[0108]

[0109] As shown in Table 5, the tested cosmetics that were confirmed to have preservation efficacy in Examples 2-6 all achieved results comparable to Standard A through preservation efficacy testing based on International Organization for Standardization (ISO) 11930.

[0110] Therefore, it can be confirmed that the preservation efficacy test of Examples 2-6 is a preservation efficacy test method that can make a preliminary judgment comparable to the tests specified by the International Organization for Standardization (ISO) and other organizations.

[0111] Industrial availability

[0112] It can be used in screening for preliminary tests, hygiene inspections, and cosmetic product inspections as prescribed by laws or industry regulations for the microbiological evaluation of the efficacy of pharmaceutical or cosmetic preparations or the preservatives contained therein.

Claims

1. A method for testing the shelf-life efficacy of a cosmetic product, comprising a mixture of six microorganisms—five bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Burkholderia cepacia, and Enterobacter cloacae) and Candida albicans yeast—inoculated into the cosmetic product being tested and stored at 20–25°C. During this storage period, samples collected from the cosmetic product are inoculated onto agar medium supplemented with tetrazolium blue for culturing. The total number of colony-forming units (CFU) per unit mass of the cosmetic product is determined. The viable count (A) corresponding to the determined total number of CFUs is considered to be less than the theoretical count (B) of the six mixed microorganisms inoculated onto the agar medium by a predetermined amount, serving as the evaluation criterion for effectiveness. The storage period is 14 days or more, and 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 3.0 or more in terms of commonly used logarithmic value, and the reduction in the number of viable bacteria A of the sample collected on the 14th day of the storage period relative to the theoretical number of bacteria B is 3.5 or more in terms of commonly used logarithmic value.

2. The method for testing the preservation efficacy of cosmetics according to claim 1, wherein, The agar medium is SCDLP agar medium, and the culture is carried out at a temperature of 30-35℃ for a period of more than 3 days.

3. The method for testing the preservation efficacy of cosmetics according to claim 1, wherein, The sample of the cosmetic product to be tested was inoculated into an agar medium and also into a liquid medium. The samples were cultured simultaneously in both media. If no colony-forming units were detected in the agar medium after a specified period of storage, the presence of the six mixed microorganisms was confirmed by streak plating from the liquid medium collected after a specified period of storage.

4. The method for testing the preservation efficacy of cosmetics according to claim 2, wherein, The sample of the cosmetic product being tested was inoculated onto SCDLP agar medium containing tetrazolium blue as a colorimetric reagent, and the sample of the cosmetic product being tested was also inoculated onto liquid medium. The samples were cultured simultaneously on both media. If no colony-forming units were detected in the SCDLP agar medium after a specified period of storage, the presence of the six mixed microorganisms was confirmed by streak plating from the culture collected from the liquid medium after a specified period of storage.

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