JP6288752B2 - Method for evaluating the efficacy of preventing secondary contamination of non-aqueous skin external products - Google Patents

Description

  The present invention relates to a method for evaluating the effect of preventing secondary contamination by microorganisms in a non-aqueous external skin product.

  It is known that skin external products such as cosmetics are contaminated by microorganisms from the external environment during the production or use of the product. In products contaminated with microorganisms, the products may decay or deteriorate due to the effects of the grown microorganisms and their metabolites, resulting in off-flavors (hereinafter also referred to as “odour” in this specification), discoloration, quality deterioration, etc. There is. Non-aqueous skin external products using non-water-soluble bases such as oil-based bases are considered to have a relatively low risk of contamination by microorganisms because the products do not contain sufficient moisture for microbial growth. However, in reality, it has been reported that a strange odor is generated during use (Patent Document 1).

Microbial contamination is broadly divided into primary contamination that occurs during the manufacturing stage of products and secondary contamination that occurs during the consumer use phase. In order to prevent secondary contamination, the products are usually subjected to antiseptic and antifungal treatments.
As a confirmation test for the efficacy of preventing secondary contamination in products for external use on skin, referring to the Japanese Pharmacopoeia preservation efficacy test (Non-patent document 1) and the microbiology guidelines of CTFA (Non-patent document 2), there are methods based on this. Conceived and implemented. In Non-Patent Document 1, the target product is forcibly inoculated with microorganisms, mixed uniformly, and the number of surviving microorganisms in the product is measured over time to examine the death behavior of the microorganisms. A method for assessing the storage efficacy of a product against microorganisms is described. Non-Patent Document 2 describes the preservation of a product against microorganisms by applying microorganisms to the surface of the target product, and measuring the number of remaining microorganisms in the product over time, and examining the killing behavior of the microorganisms. A method for assessing efficacy is described.

JP 2005-247776 A

16th revision Japanese Pharmacopoeia Reference Information G4. Microorganism-related preservation efficacy test method, p. 2044-2046 CTFA (Cosmetic Toiletry and Fragrance Association) Microbiology Guideline Section 20, 21, 22, 23, 24, 2007

When the present inventors examined the methods described in the above-mentioned documents, these methods are suitable for the evaluation of water-based skin external products using a water-soluble base, but non-water-based skin using a water-insoluble base. It was not necessarily suitable for evaluation of external products. When a non-aqueous external skin product was tested by the above method, the results changed greatly due to changes in the test environment, the reproducibility was poor, and stable evaluation results could not be obtained. Now, when using the method for actual product evaluation, to ensure safety for consumers, confirm that the test environment does not affect the results. In addition to confirming the stability of the product, human use tests (tests in which humans actually use the target product for a certain period to confirm the occurrence of off-flavors, etc.) are also performed to confirm the validity of the results. It is necessary to conduct a test. That is, in the above method, it is difficult to easily and quickly evaluate a non-aqueous skin external product, and the time and cost burden is large.
Furthermore, the above method evaluates only the killing behavior of microorganisms, and it has been difficult to evaluate the physical property change of the product such as odor and discoloration.

  It is an object of the present invention to provide a laboratory evaluation method capable of evaluating a risk of occurrence of a strange odor at the time of use that occurs in a non-aqueous external skin product by a simple method quickly and with good reproducibility.

  In view of the above problems, the present inventors first elucidated the cause mechanism of the generation of a strange odor when using a non-aqueous external skin product. Next, the construction of a laboratory evaluation system using the odor generation model clarified above was examined. As a result, the nutrient source (substrate) necessary for the generation of off-flavor is identified, the microorganisms and the substrate are attached to the surface of the non-aqueous product, and this is placed in a high humidity environment where the microorganisms can grow. It was found that the risk of off-flavor generation during use can be evaluated. Furthermore, it has been found that by using an auxiliary substrate together with the above substrate, the growth of bacteria and the accompanying odor generation are accelerated, and the evaluation result can be obtained quickly. The present invention has been completed based on these findings.

That is, this invention relates to the evaluation method of the secondary contamination prevention effect of the nonaqueous skin external product containing the process of following (1)-(3).
(1) The surface of the nonaqueous skin external product is selected from the group consisting of microorganisms, at least one substrate selected from the group consisting of keratinocytes, proteins and peptides, and amino acids, sugars, vitamins, metal salts and lipids. Contacting with at least one auxiliary substrate;
(2) A step of maintaining the non-aqueous skin external product of (1) for a certain period of time under a humidity condition in which microorganisms can grow, and (3) Proliferation of odors or microorganisms generated in the non-aqueous skin external product of (2) Process for evaluating sex

  According to the method of the present invention, the efficacy of preventing secondary contamination by microorganisms in a non-aqueous skin external product can be evaluated quickly and with good reproducibility by a simple technique. By using the method of the present invention, it is possible to appropriately evaluate and confirm the risk of off-flavor generation when using a non-aqueous skin external product.

The non-aqueous external skin product to be evaluated by the method of the present invention uses a water-insoluble base such as an oil base and does not substantially contain moisture necessary for the growth of microorganisms. Specifically, it is a skin external product having a water activity of 0.75 or less.
Water activity is an index used to evaluate the ability of microorganisms to grow in foods, cosmetics, pharmaceutical preparations, etc., and is a unit representing the ratio of free water in a product. The water activity (Water Activity: Aw) is defined by the ratio of the water vapor pressure (P) in the sealed container containing the product to the vapor pressure (Po) of pure water at that temperature: P / Po. The water activity can be measured by, for example, a rotronic water activity measurement system (manufactured by GSI Creos).
Moisture contained in cosmetics and the like is classified into bound water in a state where it is combined with other components and free water that is easy to move. As mentioned above, spoilage and mold generation in cosmetics and preparations are caused by microbial contamination, but it is essential that a certain amount of free water is present for the microorganisms to propagate in these products. Each microorganism has a range of water activity that can be grown, and cannot grow under water activity less than that. The minimum water activity necessary for growth is referred to as minimum growth water activity, which is about Aw = 0.90 or more for general bacteria and about Aw = 0.75 or more for general mold / yeast.

For non-aqueous skin external products with low water activity, microorganisms cannot grow in the product, so it is considered that the risk of contamination is low even when microorganisms are mixed during use. Was not established. In fact, it has been reported that even non-aqueous products generate off-flavors due to use, but the cause of off-flavor generation has not been elucidated.
Therefore, the present inventors examined the cause of off-flavor generation in non-aqueous skin external products, and when using the product, microorganisms such as skin resident bacteria, moisture such as human saliva and sweat, exfoliated keratin and sebum The components derived from the body surface, etc. adhere to the surface of the product and are kept in a sealed state in the container while they are adhered to form a high humidity environment due to the adhered moisture, and the microorganisms nourish the components derived from the body surface It has been found that it proliferates as a source and generates off-flavors. In other words, it was found that the generation of off-flavor was caused by secondary contamination by microorganisms, and was caused by a combination of factors such as adhesion of microorganisms to the product surface, supply of external nutrients, and high humidity environment. Furthermore, it has been specified that a nitrogen source is particularly necessary as a nutrient source that causes the generation of off-flavors.

Next, by recreating the above three factors for the generation of off-flavors, the adhesion of microorganisms to the product surface, the supply of external nutrients, and the formation of a high humidity environment, the effectiveness of secondary contamination prevention for non-aqueous skin products can be improved. A laboratory evaluation system was constructed. Furthermore, when the nutrient sources (substrates and auxiliary substrates) to be used were examined, it is possible to easily and quickly evaluate and obtain stable test results by using proteins as substrates and amino acids as auxiliary substrates. I understood it.
That is, the method of the present invention is a method for evaluating the effectiveness of preventing secondary contamination by microorganisms in a non-aqueous skin external product, and includes at least the following steps (1) to (3) in this order.
(1) The surface of the nonaqueous skin external product is selected from the group consisting of microorganisms, at least one substrate selected from the group consisting of keratinocytes, proteins and peptides, and amino acids, sugars, vitamins, metal salts and lipids. Contacting with at least one auxiliary substrate;
(2) A step of maintaining the non-aqueous skin external product of (1) for a certain period of time under a humidity condition where microorganisms can grow (proliferate), and a non-aqueous skin external product of (3) (2) Steps for evaluating the odor generated or the growth of microorganisms Steps (1) to (3) are described in detail below.

In the present invention, the external skin product is a product that is used by directly contacting the product with the body surface (including the surface of the skin, oral cavity, mucous membrane, nail, body hair (such as hair)). Specific examples of the external skin product include external skin preparations including cosmetics and pharmaceuticals. The cosmetics include those belonging to both cosmetics and quasi-drugs under the Japanese Pharmaceutical Affairs Law.
Especially, the cosmetics are preferable for the skin external product to be evaluated in the present invention. Cosmetics include lip cosmetics (for example, lip gloss, lipstick (both liquid and solid), lip balm, etc.), skin cosmetics (for example, foundation, concealer, beauty oil, sunscreen, etc.) ), Cosmetics for eye makeup (such as mascara), cosmetics for cleaning (such as oil cleansing), cosmetics for hair (such as hair styling and tic agents), and the like. The evaluation method of the present invention is particularly preferably used for lip cosmetics.

  In the present invention, “secondary contamination” refers to contamination by microorganisms at the stage where a consumer uses a product. The concept for this is "primary contamination", which is the stage from product production to the consumer's hands, raw materials (raw materials, containers, etc.), manufacturing environment (manufacturing equipment, workers, working environment, etc.) It means that microorganisms get mixed in through the above.

The microorganism used in the method of the present invention is not particularly limited as long as it may adhere to the product during use. Such microorganisms include both bacteria and fungi. Moreover, in the method of this invention, the microorganisms used may be 1 type or multiple types.
The microorganism is preferably a human resident bacteria, more preferably a skin and oral cavity resident bacteria. Specifically, Staphylococcus (Staphylococcus) genus Actinomyces (Actinomyces) genus Propionibacterium (Propionibacterium) genus Streptococcus (Streptococcus) genus Acinetobacter (Acinetobacter) genus Bacillus (Bacillus) genus, Cochlear ( Kocuria ) and bacteria or fungi belonging to the genus Candida are preferred.
Examples of Staphylococcus bacteria include Staphylococcus epidermidis , Staphylococcus aureus , Staphylococcus warneri , Staphylococcus saprophyticus subsp. Saprophyticus , Staphylococcus hominis, and the like.
Examples of Actinomyces bacteria include Actinomyces naeslundii , Actinomyces viscosus and the like.
Examples of Propionibacterium bacteria include Propionibacterium acnes .
Examples of Streptococcus bacteria include Streptococcus pseudopneumoniae , Streptococcus mitis , Streptococcus cristatus , Streptococcus sanguinis, and the like.
Examples of Acinetobacter bacteria include Acinetobacter calcoaceticus .
Examples of Bacillus bacteria include Bacillus subtilis .
Examples of Candida fungi include Candida albicans .
Among them, the bacterium belonging to the genus Staphylococcus is preferable as the microorganism used in the method of the present invention, Staphylococcus epidermidis or Staphylococcus aureus is more preferable, and Staphylococcus epidermidis or Staphylococcus epidermidis. Staphylococcus Oureusu Anerobiusu (Staphylococcus aureus subsp. anaerobius) is more preferable.

As a nutrient substrate, at least one selected from keratinocytes, proteins and peptides is used.
Examples of the protein include keratin, elastin, albumin, glycoprotein (for example, mucin, proline-rich protein, lactoferrin, fibronectin, staserine, acidic proline-rich protein, histidine-rich protein), milk protein (for example, skim milk) and the like. Considering the ease of use in the evaluation system, the protein is preferably keratin (more preferably, water-soluble keratin (oligokeratin)), elastin, albumin (more preferably serum albumin, more preferably bobbin serum albumin ( BSA)), more preferably keratin, and still more preferably water-soluble keratin.
In consideration of ease of use in an evaluation system, the peptide is preferably a peptone (eg, bactopeptone, polypeptone, tryptone, casein peptone, soy peptone).
The substrate is preferably at least one selected from keratinocytes, keratin, elastin, and albumin, more preferably at least one selected from keratinocytes and keratin, more preferably keratin, and even more preferably water-soluble keratin.

The auxiliary substrate used together with the substrate includes at least one selected from amino acids, sugars, vitamins, metal salts, and lipids. By using an auxiliary substrate together with the substrate, the propagation of microorganisms can be promoted to shorten the culture period, and the time required for evaluation can be shortened.
The amino acids are leucine, casamino acid, histidine, methionine, cysteine, proline, glycine, glutamic acid, serine, aspartic acid, isoleucine, alanine, arginine, lysine, tryptophan, valine, threonine, serine, tyrosine, phenylalanine, glutamine, and asparagine. It is preferably at least one selected from
The saccharide is preferably at least one selected from monosaccharides, disaccharides and oligosaccharides, more preferably at least one selected from monosaccharides, and even more preferably glucose.
The vitamins are preferably at least one selected from water-soluble vitamins and fat-soluble vitamins, and at least selected from vitamins contained in peptones and extracts (eg, yeast extract, brain extract, heart extract). One type is more preferable.
The metal salt is preferably at least one selected from phosphates such as sodium, potassium, magnesium, calcium, copper, zinc and iron, chlor salt, sulfate or nitrate, and sodium, potassium or magnesium phosphorus Acid salts are more preferred.
The lipid is preferably at least one selected from simple lipids, complex lipids, derived lipids, etc., and includes stratum corneum (containing ceramide, fatty acid, cholesterol, cholesteryl ester as a main lipid), peptone, extract (for example, More preferably, it is at least one selected from lipids contained in yeast extract, brain extract, heart extract).
It is preferable to use at least one kind of amino acid as an auxiliary substrate from the point that the evaluation result can be obtained quickly by generating odor, and selected from leucine, casamino acid, glycine, glutamic acid, aspartic acid, methionine, cysteine, and proline. It is more preferable to use at least one selected from the group consisting of leucine and casamino acid, and it is particularly preferable to use leucine and casamino acid.

  In the step (1), the method of bringing the microorganism, the substrate, and the auxiliary substrate into contact with the surface of the nonaqueous skin external product is not particularly limited. For example, a method in which a microorganism, a substrate, and an auxiliary substrate are directly applied to the surface of a test product, a method in which a microorganism is smeared on an agar medium containing the substrate and the auxiliary substrate, and a test product is laminated thereon, a filter Examples include a method in which a microorganism, a substrate, and an auxiliary substrate are placed on top and this filter is overlaid on a test product.

The amount of the microorganism, substrate, and auxiliary substrate to be brought into contact with the surface of the test product is not particularly limited, and can be selected according to the type of the test product. An example of each usage amount per unit surface area of the test product is shown below.
-Surface area of the product to be tested: 12.5 mm x 12.5 mm x 3.14 = 490 mm ²
-Amount of microorganisms When using Staphylococcus epidermidis : 10 ² to 10 ⁸ cfu, more preferably 10 ⁴ to 10 ⁸ cfu
Staphylococcus Oureusu Anerobiusu (. Staphylococcus aureus subsp anaerobius) For use: 10 to 10 ⁷ cfu, more preferably ¹⁰ 3 ^{to 10} 7 cfu
Base mass When using keratinocytes: 10 ² to 10 ⁵ cells, more preferably 10 ³ to 10 ⁵ cells
When water-soluble keratin is used: 0.001 mg or more and 10 mg or less, more preferably 0.01 mg or more and 10 mg or less / Auxiliary group mass When amino acid is used: 0.02 mg or less, more preferably 0.0025 mg or more and 0.02 mg or less

In the step (2), the test product that is in contact with the microorganism, the substrate and the auxiliary substrate in the step (1) is held for a certain period of time under humidity conditions where the microorganism can grow, and the microorganism is cultured. Note that “microorganisms can grow” means that microorganisms can grow.
The humidity during cultivation (relative humidity) may be higher than the humidity at which microorganisms can grow, and varies depending on the type of microorganism used, but is preferably more than 75%, more preferably 86% or more, and 88% More preferably, the above is used. The upper limit of humidity can be 100%.
The method of keeping the test product in the above humidity environment is not particularly limited. For example, the test product may be cultured in a sealed container or in a room where the humidity can be controlled so as to keep the humidity of the test product. The humidity in the sealed container can be adjusted using water or a salt solution such as a saturated ammonium dihydrogen phosphate solution. The container used for the culture is not particularly limited as long as it does not affect the test product, the substrate, and the growth of microorganisms, and a normal glass bottle, glass tube, centrifuge tube, plastic container, or the like can be used.
The culture temperature may be appropriately selected depending on the type of microorganism used, but it is preferably performed at a temperature of about 30 ° C., which is a general microorganism growth condition.
Since the growth rate of microorganisms varies depending on the type of microorganism, substrate, and auxiliary substrate used, the culture period is appropriately selected in consideration of these factors. Usually, it can be about 1 day or more and about 1 month, preferably about 1 week.

For example, Staphylococcus epidermidis as inoculum microorganisms (Staphylococcus epidermidis) or Staphylococcus Oureusu Anerobiusu the (Staphylococcus aureus subsp. Anaerobius), if the keratinocytes or water-soluble keratin as a substrate, using an amino acid as a co-substrate, humidity conditions of 100%, The culture temperature is preferably around 30 ° C., and the culture is preferably performed for about one week.

In step (3), the odor generated or the growth of microorganisms is evaluated for the test product that has undergone the culture step of (2) above. Secondary contamination by microorganisms can be evaluated by both the growth of the inoculated microorganisms and the off-flavor generated as a result of the microorganism growth.
The method of the present invention evaluates the preventive effect of a product against the secondary contamination of microorganisms, thereby knowing the risk of off-flavor generation when using the product. As shown in the examples described later, the generation of off-flavor and the growth of microorganisms are correlated, and the risk of the generation of odor can be appropriately evaluated using the growth of microorganisms as an index. From the viewpoint of simplicity of evaluation, it is preferable to evaluate based on the growth of microorganisms.

As an odor evaluation method, an evaluation method by odor component analysis such as sensory evaluation, SPME (Solid phase micro extraction) -GC / MS analysis, HPLC analysis or the like can be used.
The type of odor to be detected may vary depending on the microorganism used and the type of substrate, but as a result of studies by the present inventors, in the sensory evaluation of products after actual human use, isovaleric acid odor, butyric acid odor, sulfur Odor, stuffy odor, miso odor, methional odor, etc. were reported. In the SPME-GC analysis of the product after use, isovaleric acid, isovaleraldehyde, acetic acid, butyric acid, acetoin and the like were detected as off-flavor components. Note that isovaleraldehyde is a precursor compound of isovaleric acid. From these findings, when evaluating odor by sensory evaluation in the method of the present invention, isovaleric acid odor, isovaleraldehyde odor, acetic acid odor, butyric acid odor, sulfur odor, stuffy odor, miso odor, methional odor are detected. In the case of odor component analysis such as SPME-GC, it is preferable to detect isovaleric acid, isovaleraldehyde, acetic acid, butyric acid, and acetoin. Especially, it is preferable to make evaluation object the odor of isovaleric aldehyde which is an isovaleric acid or its precursor, or these odor components which are often seen in the claim of the generation of off-flavor.
In sensory evaluation, it can be determined that there is a risk of occurrence of a bad odor when the occurrence of the above-mentioned type of odor is detected from the product to be tested that has undergone the steps (1) and (2).
In the evaluation by odor component analysis such as SPME-GC, a test product that has undergone the above steps (1) and (2) and a comparative product that has not undergone the above step (1) (ie, microorganisms, substrates and auxiliary substrates) The detected amount of the odorous component is compared with the product not in contact), and when the detected amount of the above-mentioned type of odorous component in the test product is increased, it can be determined that there is a risk of occurrence of a bad odor.

The growth of microorganisms can be evaluated by measuring the number of microorganisms in the test product after culturing. As a method for measuring the number of microorganisms, methods such as measurement of the number of bacteria by smearing on a plate, absorbance measurement, calorimetry, ATP measurement, impedance measurement and the like can be used.
As a result of the measurement, if the number of microorganisms has not decreased with respect to the initial inoculation amount, it can be determined that there is a risk of occurrence of a foul odor because microorganisms having metabolic activity may exist.

  In relation to the above-described embodiment, the present invention further discloses the following method.

<1> A method for evaluating the efficacy of preventing secondary contamination of a nonaqueous skin external product, including the following steps (1) to (3).
(1) The surface of the nonaqueous skin external product is selected from the group consisting of microorganisms, at least one substrate selected from the group consisting of keratinocytes, proteins and peptides, and amino acids, sugars, vitamins, metal salts and lipids. Contacting with at least one auxiliary substrate;
(2) A step of maintaining the non-aqueous skin external product of (1) for a certain period of time under a humidity condition in which microorganisms can grow, and (3) Proliferation of odors or microorganisms generated in the non-aqueous skin external product of (2) Process for evaluating sex

<2> The evaluation method according to <1>, wherein the water activity of the non-aqueous external skin product is 0.75 or less.
<3> The evaluation method according to <1> or <2>, wherein the substrate is at least one selected from the group consisting of keratinocytes, keratin, elastin, and albumin.
<4> The evaluation method according to any one of <1> to <3>, wherein the auxiliary substrate is an amino acid.
<5> The auxiliary substrate is casamino acid, leucine, histidine, methionine, cysteine, proline, glycine, glutamic acid, serine, aspartic acid, isoleucine, alanine, arginine, lysine, tryptophan, valine, threonine, serine, tyrosine, phenylalanine, The evaluation method according to any one of <1> to <4>, which is at least one selected from the group consisting of glutamine and asparagine.
<6> The microorganism is at least one selected from Staphylococcus epidermidis (Staphylococcus epidermidis), and Staphylococcus Oureusu Anerobiusu (Staphylococcus aureus subsp. Anaerobius), <1> ~ <5> either claim 1 Evaluation method according to item.
<7> The evaluation method according to any one of <1> to <6>, wherein in the step (3), the odor component to be evaluated is at least one selected from isovaleric acid and isovaleraldehyde.
<8> The evaluation method according to any one of <1> to <7>, wherein the external skin product is a cosmetic.
<9> The evaluation method according to any one of <1> to <8>, wherein the skin external product is a lip cosmetic.
<10> The evaluation method according to any one of <1> to <9>, wherein the humidity condition under which the microorganism can grow is adjusted using water or a saturated ammonium dihydrogen phosphate solution.
<11> The evaluation method according to any one of <1> to <10>, wherein at least leucine or casamino acid is used as the auxiliary substrate.
<12> The evaluation method according to any one of <1> to <11>, wherein leucine and casamino acid are used as the auxiliary substrate.
<13> The evaluation method according to any one of <1> to <12>, wherein the humidity is 100% in the step (2).

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

The SPME-GC / MS analysis used for the analysis of odor components was performed under the following conditions.
[SPME]
Fiber: Carboxen / Polydimethyl Siloxane (manufactured by Supelco)
Temperature: 40 ° C
Extraction time: 35min
[GC]
Equipment: HP6890 (Agilent)
Column: DB-WAX / 60m × 0.25mm
Film 0.25μm
Oven temperature: 40 ° C (4min hold) -6 ° C / min-70 ° C-3 ° C / min-240 ° C
[MS]
Equipment: HP 5973 MSD (Agilent)

[Culture medium]
The following was used as a medium for confirming the growth of microorganisms.
・ BHI (Brain Heart Infusion) Agar (BD): Calf Brains, Infusion from 7.7g, Beef Heart, Infusion from 9.8g, Proteose Peptone 10g, Dextrose 2g, Sodium Chloride 5g, Disodium Phosphate 2.5g (pH7. 4 ± 0.2)
・ GP (glucose peptone) agar (manufactured by Nippon Pharmaceutical): Glucose 20g, Casein Peptone 5g, Yeast Extract 2g, MgSO ₄ 0.5g, KH ₂ PO ₄ 1g, Agar 15g (pH 5.7 ± 0.1)

I. Reference Example Human Use Test 1. Test Gloss The following three types of lip glosses A, B, and C were used as non-aqueous skin external products. The gloss containers used were dome-type containers that were applied directly to the lips.
[Lip gloss]
・ Lip gloss A
Oil agent, coloring agent, no preservative added Water activity: 0.561
・ Lip gloss B
Oil agent, coloring agent, preservative (phenoxyethanol)
Water activity: 0.476
・ Lip gloss C
Oil agent, coloring agent, preservative (phenoxyethanol, paraben)
Water activity: 0.604

2. Human actual use test About 40 female subjects, using the above-mentioned lip gloss A, B, C for about 1 month, 2 to 3 times a day, and carrying it during the test period . There are no restrictions on activities during life. Moreover, the test was stopped immediately when a strange odor was felt, and a sample was collected. The collected sample after the test was immediately examined for microorganisms at the tip of the container, and the odor at the tip of the container was confirmed by sensory evaluation.
The sensory evaluation evaluated the strength of the odor in three levels: strong, weak, and none. The ratio of samples in which a weak odor was generated in all samples was defined as a weak odor rate, and the ratio of samples in which a strong odor was generated was defined as a strong odor rate.

3. Results of actual human use test (1) Sensory evaluation As a result of sensory evaluation, gross A is 16% as weak odor rate, 22% as strong odor rate, gloss B is 11% as weak odor rate, strong odor rate 6%, gloss C was 9% as the weak odor rate, and 0% as the strong odor rate (Table 1-1). From these results, the risk of occurrence of odor change in each gloss was in the order of A>B> C, and it was confirmed that the preventive effect of each gloss against odor change was A <B <C.

(2) Microbial analysis As a result of the above sensory evaluation, a sample of gross A in which a strange odor was confirmed was used to examine the relationship between the odor intensity, the detection of bacteria, and the number of cell fragments attached to the container surface.
[Bacteria detection]
Immediately after collecting the gloss sample, the tip of the container was scraped with a 1 μL loop and applied to a BHI agar medium and a GP agar medium. The BHI agar medium after application was cultured at 32-37 ° C., and the GP agar medium after application was cultured at 25 ° C. for 2-7 days. After culturing, the number of colonies that appeared on the plate was visually observed, and the number of bacteria was evaluated in three stages: small, medium, and large.
[Number of cell pieces attached to the container surface]
The tip of the container of the gross sample was placed on a slide glass and observed with an optical microscope, and the number of cell pieces was evaluated in three stages: small, medium and large.

Table 1-2 shows the relationship between the odor intensity (two steps of strong and weak) of each sample shown in the sensory evaluation, the number of detected bacteria evaluated above, and the number of cell fragments attached to the surface of the container. The numbers in parentheses in the table represent the number of samples.
As a result, it was observed that a strong odor was generated in the case where the number of detected bacteria was large and the number of attached cell pieces was large.

[Segregation of strains from odor samples]
Furthermore, the same test as the above-mentioned actual use was performed, and the strain was isolated from the gross sample in which a strange odor was confirmed. Of the isolated strains, the following 5 genera and 7 species were isolated as strains capable of stable growth.

(3) Deodorized component analysis Next, the deodorized component was analyzed by SPME-GC / MS for two samples of gloss A in which the deodorized odor was confirmed and an unused product. In the sample, the cap part and the application part were cut, and the total of the detected component abundance was calculated.
As a result, 16 components were identified as components involved in the deodorization, and components such as lower aldehydes, lower fatty acids and ketones were confirmed (Table 1-4). From the off-flavor generation sample, isovaleraldehyde and acetoin which were not detected in the control (unused product) were detected, and odorous components such as acetic acid, butyric acid and isovaleric acid were greatly increased.

II. Development of a model for generating a foul odor We examined a model for generating a foul odor that can be used in place of human use.
A filter was used for the odor generation model, and models A and B in which a lip-derived component was attached thereto, and a model C in which microorganisms, a substrate, and an auxiliary substrate imitating the lip-derived component were attached to the filter were prepared. Using these models, the relationship between the generation of odor and the growth of microorganisms, the culture conditions, the type and amount of microorganisms used, the amount of substrate used, the effect of adding an auxiliary substrate, etc. were examined.

1. Odor generation model A
The filter used was 0.2 μm / 25 mm (made of polycarbonate, manufactured by Millipore).
The filter was rubbed directly onto the lips and placed on the side of a 20 ml glass sample bottle that had been conditioned. Various humidity adjusting liquids (humidity 100%: sterilized water, humidity 75%: saturated sodium chloride solution, humidity 43%: saturated potassium carbonate solution, humidity 0%: silica gel) were added to the bottom of the glass bottle and sealed. After culturing at 30 ° C. for 24 hours, the presence or absence of a strange odor was confirmed by sensory evaluation.
The sensory evaluation was performed based on an index of two steps of odor change (Yes: change odor is felt, No: change odor is not felt).

2. Odor generation model B
The lips were rubbed several times with a sterile cotton swab moistened with physiological saline, and the cotton swab used in 0.5 mL physiological saline was washed. After 200 μL of this cleaning solution was filtered through a filter having a pore size of 11 μm or 0.2 μm (both made of polycarbonate, manufactured by Millipore, 25 mm), the surface of the filter was washed with 2 mL of physiological saline, and the side of a 30 mL glass sample bottle This filter was installed in the section. 500 μL of sterilized water was added to the bottom of the glass bottle and sealed (humidity 100%). After culturing at 30 ° C. for 1 to 2 days, the presence or absence of a strange odor was confirmed by sensory evaluation and SPME-GC / MS analysis, and the growth of microorganisms was confirmed.
The sample using a pore size 11 μm filter is a system mainly targeting lip corneum cells as a substrate and microorganisms present in the lip horn layer as a microorganism species, and the sample using a pore size 0.2 μm filter is saliva as a substrate. It mimics a system mainly composed of components, horny keratinocytes, and all microorganisms present in the lips and oral cavity as microbial species.

[sensory evaluation]
Sensory evaluation evaluated the bad smell by the following six-stage indexes.
A: A strong odor is felt B: A strange odor is felt C: A weak odor is felt D: A slightly weak odor is felt E: A rather weak odor is felt F: No odor is felt

  To confirm the growth of microorganisms, physiological saline was added to the sample bottle and mixed well, and a certain amount of the solution was smeared on a BHI agar medium. After smearing the smeared agar medium at 30 ° C. for 1-2 days, the number of remaining bacteria was confirmed.

3. Odor generation model C
20 μL of a predetermined concentration of bacterial solution and 20 μL of various auxiliary substrates adjusted to a predetermined concentration were added to the filter with the substrate attached. A filter attached with a substrate, fungus, and auxiliary substrate was placed on the side of a 30 mL glass sample bottle. 500 μL of sterilized water was added to the bottom of the glass bottle and sealed (humidity 100%). After culturing at 30 ° C. for a predetermined time, the presence or absence of a strange odor was confirmed by sensory evaluation or SPME-GC / MS, and the growth of microorganisms was confirmed.

[filter]
0.2 μm / 25 mm (made of polycarbonate, manufactured by Millipore) was used.

[Test microorganism]
I. above. In an actual human use test, a strain isolated from a sample exhibiting a strange odor was used. Various strains were purely isolated, suspended in a 10% glycerol solution before use, and stored frozen at -80 ° C.
The cryopreserved test microorganisms were thawed before the test and inoculated on a BHI agar medium. After culturing at 30 ° C. for 24 to 48 hours, the suspension was suspended in physiological saline, adjusted to a predetermined number of bacteria, and used for the experiment.

[Substrate]
As a substrate, keratinocytes (manufactured by KURABO, normal human cells) or 5% aqueous solution of water-soluble keratin (manufactured by TCI) were mainly used.
Keratinocytes are cultured with EpiLife-KG2 (manufactured by KURABO), then the cultured cells are attached to a filter with a pore size of 0.2 μm (manufactured by Millipore, 25 mm) to a predetermined number of cells, and the culture solution is removed by suction filtration. did. The cultured cells were washed with a phosphate buffer to prepare a keratinocyte-attached filter.
A 5% keratin aqueous solution was sterilized in an autoclave and then attached on a filter having a pore diameter of 0.2 μm so that a predetermined amount of keratin was obtained to prepare a keratin-attached filter.

[Test auxiliary substrate]
As auxiliary substrates, a 0.1% leucine solution and a 0.1% casamino acid solution were mainly used.

[sensory evaluation]
Sensory evaluation evaluated the bad smell by the following six-stage indexes.
A: A strong odor is felt B: A strange odor is felt C: A weak odor is felt D: A slightly weak odor is felt E: A rather weak odor is felt F: No odor is felt

  To confirm the growth of microorganisms, physiological saline was added to the sample bottle and mixed well, and a certain amount of the solution was smeared on a BHI agar medium. After smearing the smeared agar medium at 30 ° C. for a predetermined period, the number of remaining bacteria was confirmed.

Verification 1. Examination of culture humidity conditions using the odor generation model A Using the odor generation model A, the humidity condition is changed to 0 to 100% by changing the type of the humidity control liquid in the container. The relationship was verified. The presence or absence of odor generation was determined by sensory evaluation.
As a result, when the humidity was 75% or less, no odor was confirmed (Table 2-1). From this result, it was found that the odor was generated in a high humidity environment exceeding 75%.

Verification 2. Analysis of Odor Generation and Microorganism Proliferation by Odor Generation Model B, and Change Odor Component About both filter samples of pore size 11μm and 0.2μm of Odor Generation Model B, sensory evaluation, microorganism growth, SPME- GC / MS analysis was performed.
For sensory evaluation, a sample after one day of culture was used. As a result, both samples showed a strange odor (Table 3-1).

  Microbial growth was performed on samples after 2 days of culture. As a result, it was confirmed that in both filter samples having a pore diameter of 11 μm and 0.2 μm, the microorganisms grew after 2 days of culture compared to the time of the start of culture (Table 3-2).

  Samples after 2 days of culture were used for SPME-GC / MS analysis. As a result, in both filter samples having a pore diameter of 11 μm and 0.2 μm, a component causing the odor was detected (Table 3-3).

  From these results, it was confirmed that even when the substrate and the microorganism species adhering to the filter are different using a filter having a different pore size, the microorganism grows in a high humidity environment, and a strange odor is generated accordingly. That is, it was found that the model system can be used to reproduce odors in a high humidity environment, and that there is a relationship between the growth of microorganisms and the generation of odor-related components.

Verification 3. Examination of each condition by the odor generating model C Using the odor generating model C, the conditions of the substrate, microorganisms and auxiliary substrate to be attached to the filter were verified.

3.1 Substrate: Keratinocyte 3.1.1 Examination of amount of keratinocyte Samples were prepared and evaluated by changing the number of keratinocyte cells attached to the filter.

Microorganisms and inoculum quantity ・Staphylococcus epidermidis Ae 7-1 strain Inoculum quantity 1 × 10 ⁸ cfu
・Staphylococcus aureus subsp. Anaerobius Ae 44-2 Inoculum Amount 1 × 10 ³ cfu
Auxiliary substrate: casamino acid 0.02mg, leucine 0.02mg
Culture conditions: humidity 100%, 30 ° C., 2-15 days culture evaluation: sensory evaluation

As a result, in the sample inoculated with Staphylococcus epidermidis Ae 7-1 strain, a strange odor was confirmed when the number of keratinocyte cells was 10 ² to 10 ⁵ cells (Table 4-1). In the sample inoculated with Staphylococcus aureus subsp. Anaerobius Ae 44-2 strain, a strange odor was confirmed when the number of keratinocyte cells was 10 ² to 10 ⁵ cells (Table 4-2).

3.1.2 Examination of inoculum amount Samples were prepared by changing the amount of inoculum on the filter and evaluated.

Microbes, Staphylococcus epidermidis Ae 7-1 strain, Staphylococcus aureus subsp. Anaerobius Ae 44-2 strain Keratinocyte addition amount (cell count): 6 × 10 ⁴ cell (inoculated sample of Staphylococcus epidermidis Ae 7-1 strain), 4 × 10 ⁵ cell ( Staphylococcus aureus subsp. Anaerobius Ae 44-2 inoculated sample)
Auxiliary substrate: casamino acid 0.02mg, leucine 0.02mg
Culture conditions: humidity 100%, 30 ° C., 2 days culture evaluation: sensory evaluation

As a result, in Staphylococcus epidermidis Ae 7-1 strain, a strange odor was confirmed when the inoculum was 10 ² to 10 ⁸ cfu (Table 4-3). Moreover, in Staphylococcus aureus subsp. Anaerobius Ae 44-2 strain, a strange odor was confirmed when the inoculum was 10 ² to 10 ⁶ cfu (Table 4-4).

3.1.3 Verification of supplementary substrate addition effect (1) Presence / absence of supplementary substrate Samples with and without supplementary substrate were prepared and evaluated.

Microorganisms and inoculum
Mixture of 3 strains of Staphylococcus epidermidis Ae 7-1, Staphylococcus epidermidis An 7-1, and Propionibacterium acnes An 7-2 (inoculated with 1 × 10 ⁸ cfu of each strain)
Keratinocyte addition amount (number of cells): 3 × 10 ⁵ cells
Auxiliary substrate: casamino acid 0.02 mg, various amino acid mixtures (histidine (His), methionine (Met), cysteine (Cys), proline (Pro), leucine (Leu), 0.02 mg each)
Culture conditions: humidity 100%, 30 ° C., 2 days culture evaluation: sensory evaluation

  As a result, the samples with and without the auxiliary substrate were confirmed to have a bad odor, but it was confirmed that the strength of the odor was particularly strong in the sample to which the auxiliary substrate was added (Table 4-5).

(2) Types of auxiliary substrates Samples were prepared and evaluated by changing the types of auxiliary substrates added to the samples as shown in Table 4-6.

Microorganisms and inoculum
Three strains of Staphylococcus epidermidis Ae 7-1, Staphylococcus epidermidis An 7-1, and Propionibacterium acnes An 7-2 (mixed with 1 × 10 ⁶ cfu of each strain)
Keratinocyte addition amount (number of cells): 2 × 10 ⁵ cells
Auxiliary substrate: casamino acid 0.02 mg, various amino acid mixtures (histidine (His), methionine (Met), cysteine (Cys), proline (Pro), leucine (Leu), 0.02 mg each)
Culture conditions: humidity 100%, 30 ° C., 2 days culture evaluation: sensory evaluation

  As a result, it was confirmed that when casamino acid and leucine were added, the odor was stronger (Table 4-6).

(3) Type of auxiliary substrate Samples were prepared and evaluated by changing the type of auxiliary substrate added to the sample as shown in Table 4-7.

Microorganisms and inoculum
Staphylococcus epidermidis Ae 7-1 strain, inoculum amount 1 × 10 ⁸ cfu
Keratinocyte addition amount (cell count): 4 × 10 ⁵ cells
Auxiliary substrate: casamino acid 0.02 mg, various amino acids (glycine (Gly), glutamic acid (Glu), serine (Ser), aspartic acid (Asp), leucine (Leu), 0.02 mg each)
Culture conditions: humidity 100%, 30 ° C., 2 days culture evaluation: sensory evaluation

  As a result, although it was possible to confirm odor change with other amino acids, it was confirmed again that the effect of adding leucine was particularly high (Table 4-7).

3.1.4 Optimization of bacterial species and strains Samples were prepared and evaluated by changing the type of microorganisms inoculated on the filter. The microorganism is the above-mentioned I. Each of the bacterial species and strains isolated from the samples that showed a strange odor in the actual human use test were used.

Microorganisms (see Tables 4-8 and 4-9 for strains and inoculum amounts)
・Staphylococcus epidermidis
・Staphylococcus aureus subsp. Anaerobius
・Candida albicans
・Staphylococcus warneri
・Staphylococcus saprophyticus subsp.saprophyticus
・Actinomyces naeslundii
Keratinocyte addition amount (cell number): 6 × 10 ⁴ cells (Table 4-8), 4 × 10 ⁵ cells (Table 4-9)
Auxiliary substrate: casamino acid 0.02mg, leucine 0.02mg
Culture conditions: humidity 100%, 30 ° C., 2 days culture evaluation: sensory evaluation

As a result, Staphylococcus epidermidis, Staphylococcus aureus subsp. Anaerobius, Candida albicans, Staphylococcus warneri, Staphylococcus saprophyticus subsp. Saprophyticus, confirmed the strange odor in the Actinomyces naeslundii. In particular, Staphylococcus epidermidis, Staphylococcus aureus subsp. Difference of degree even if the strain is different in anaerobius is there, but it was confirmed that strange odor (Table 4-8,4-9).

3.1.5 Deodorized component analysis Samples were prepared and evaluated with different combinations of substrate and auxiliary substrate.

Microorganisms and inoculum
Staphylococcus aureus subsp. Anaerobius Ae 44-2 strain Inoculum 2 × 10 ⁴ cfu
Keratinocyte addition amount (cell count): 1 × 10 ⁴ cell
Auxiliary substrate: 0.02 mg leucine, 0.02 mg casamino acid
Culture conditions: humidity 100%, 30 ° C., 2 days culture evaluation: SPME-GC / MS analysis

  As a result, when keratinocytes were added, isovaleraldehyde, a deodorizing component, was detected. Furthermore, by adding casamino acid and leucine, isovaleric acid components were also detected, and it was confirmed that these odor components tend to increase (Table 4-10).

3.2 Substrate: Protein, Peptide Samples were prepared and evaluated using various proteins or peptides shown in Table 4-11 as substrates.

Microorganisms and inoculum
Staphylococcus epidermidis Ae 7-1 strain Inoculum amount 1 × 10 ⁸ cfu
Substrate / Bactopeptone (BD) ^{* 1)}
・ Polypeptone (Nippon Pharmaceutical Co., Ltd.) ^{* 1)}
・ Skim milk (made by BD)
・ Tryptone (made by BD)
・ Bovin serum albumin (BSA, manufactured by SIGMA)
・ Wool-derived keratin (insoluble, manufactured by TCI) suspension solution ^{* 1)}
・ Elastin (manufactured by SIGMA) ^{* 2)}
・ Water-soluble keratin (using 5% aqueous solution, manufactured by TCI) <sup>* 3)
* 1) A</sup> 0.1% suspension was autoclaved, pulverized by sonication, diluted 10-fold with sterilized water, and used.
^{* 2) A} 5% solution was prepared, autoclaved, sonicated and used after pulverization.
^{* 3) A} commercially available product was directly sterilized in an autoclave and then diluted 10-fold with sterilized water.
Substrates other than the above were used after adjusting 0.5% aqueous solution.
Base mass, except wool keratin: 1mg
・ Wool-derived keratin: 0.02mg
Auxiliary substrate: casamino acid 0.02mg, leucine 0.02mg
Culture conditions: humidity 100%, 30 ° C., 6 days culture evaluation: sensory evaluation

  As a result, odors different from those initially added to the filter were confirmed in all substrates (Table 4-11). Especially, since water-soluble keratin was easy to determine the degree of odor, it was further investigated whether it could be used as an optimal substrate.

3.3 Substrate: Water-soluble keratin 3.3.1 Examination of amount of keratin Samples were prepared and evaluated by changing the amount of water-soluble keratin attached to the filter.

Microorganisms and inoculum
Staphylococcus aureus subsp. Anaerobius Ae 44-2 strain Inoculum 6 × 10 ⁵ cfu
Auxiliary substrate: casamino acid 0.02mg, leucine 0.02mg
Culture conditions: humidity 100%, 30 ° C., 2 days and 5 days Culture evaluation: sensory evaluation

  As a result, when 0.001 mg or more of water-soluble keratin was used, a strange odor was confirmed (Table 4-12).

3.3.2 Examination of inoculum amount and culture time Samples were prepared and evaluated by changing the inoculum amount and culture time.

Microbe
Staphylococcus aureus subsp. Anaerobius Ae 44-2 strain Inoculum 1 × 10 ¹ 〜1 × 10 ⁷ cfu
Base mass: Water-soluble keratin 1mg
Auxiliary substrate: casamino acid 0.02mg, leucine 0.02mg
Culture conditions: humidity 100%, 30 ° C., 9 hours to 2 days Culture evaluation: sensory evaluation

As a result, in the sample after 2 days of culture, the amount of inoculum was 1 × 10 ¹ cfu or more, and the odor was confirmed (Tables 4-13 and 4-14).

3.3.3 Verification of supplementary substrate addition effect Samples with and without supplemental substrate were prepared and evaluated.

Microorganisms and inoculum
Staphylococcus aureus subsp. Anaerobius Ae 44-2 strain Inoculum 2 × 10 ⁴ cfu
Base mass: Water-soluble keratin 1mg
Auxiliary substrate: casamino acid 0.02mg, leucine 0.02mg
Culture conditions: humidity 100%, 30 ° C., 2 days to 21 days Culture evaluation: sensory evaluation

  As a result, it was confirmed that, by culturing for 2 days or more, it was possible to confirm the odor change even when the auxiliary substrate was not added, and that the intensity of the odor was particularly increased in the sample to which the auxiliary substrate was added (Table 4-15).

3.3.4 Deodorized component analysis The deodorized component when water-soluble keratin was used as a substrate was analyzed by sensory evaluation and SPME-GC / MS analysis.

Microorganisms and inoculum
Staphylococcus aureus subsp. Anaerobius Ae 44-2 strain Inoculum 2 × 10 ⁷ cfu
Base mass: Water-soluble keratin 1mg
Auxiliary substrate: casamino acid 0.02mg, leucine 0.02mg
Culture conditions: humidity 100%, 30 ° C., 7 days culture evaluation: sensory evaluation, SPME-GC / MS analysis

  As a result, a strange odor was confirmed by sensory evaluation. Moreover, as a result of SPME-GC / MS analysis, it was confirmed that isovaleraldehyde and isovaleric acid were produced (Table 4-16).

III. Evaluation of non-aqueous skin external products using a model for generating strange odor II. The following evaluation model was created based on the conditions obtained by using the above-mentioned odor generation model, and the evaluation of the generation of off-flavor in non-aqueous skin external products was performed.

1. Test Product As a non-aqueous external skin product, The same three types of lip glosses A, B and C used in the human actual use test were used.

2. Evaluation model for non-aqueous products (lip gloss) (1) Filter method A predetermined amount of gloss is added to the bottom of a 100 mL glass sample bottle, and the surface of the filter with substrate, microorganisms, and auxiliary substrate attached on the added gloss is glossed. It installed so that it might contact. As a substrate, 0.125 mg of water-soluble keratin was attached to the filter. As the inoculated microorganism, the above-mentioned I. Staphylococcus aureus subsp. Anaerobius Ae 44-2 strain, which is an isolated bacterium of the human actual use test, was attached to the filter with 2 × 10 ⁴ cfu. Furthermore, 0.0025 mg of leucine and 0.0025 mg of casamino acid were attached to the filter as auxiliary substrates, respectively. 0.2 g of each gloss (A, B, C) was added to the bottom of the sample bottle, and the attached filter was brought into contact with the gloss surface.
A small-capacity glass bottle to which 500 μL of sterilized water was added was placed inside the 100-mL sample bottle, and then the 100-mL sample bottle was sealed to create a high humidity environment (humidity 100%). In addition, as a comparison object, a sample prepared in the same manner as above except that no gloss was added (control sample), and prepared in the same manner as above except that a filter with a substrate, bacteria, or auxiliary substrate was not contacted with the added gloss. Samples (gross-only samples) were prepared respectively. These samples were cultured at 30 ° C., and after 1 day and 2 days of culture, the presence or absence of a strange odor was confirmed by sensory evaluation, and the growth of microorganisms was confirmed. Moreover, about the sample 2 days after culture | cultivation, the smell change component analysis by SPME-GC was performed.

Sensory evaluation evaluated the bad smell by the following six-stage indexes.
A: A strong odor is felt B: A strange odor is felt C: A weak odor is felt D: A slightly weak odor is felt E: A rather weak odor is felt F: No odor is felt

  To confirm the growth of microorganisms, add a lecithin polysorbate solution (LP diluted solution, manufactured by Nippon Pharmaceutical Co., Ltd.) to a glass sample bottle of 100 mL so that the amount is 10 times the amount of gloss, and then mix well. A certain amount of was smeared on a BHI agar medium. After smearing the smeared agar medium at 30 ° C. for 1-2 days, the number of remaining bacteria was confirmed.

(2) Multilayer Method A predetermined amount of gloss was added to the bottom of a 30 mL glass sample bottle, and a substrate, microorganisms, and auxiliary substrate were directly added to the top of the added gloss and layered. A phosphate buffer solution (pH 6.4) containing 2.25 mg of water-soluble keratin as a substrate and 0.009 mg of leucine and 0.009 mg of casamino acid as auxiliary substrates was used as a substrate / auxiliary substrate solution. As the inoculum, I. Staphylococcus aureus subsp. Anaerobius Ae 44-2, which is an isolated bacterium of the human real-use test, was used. 1 g of each gloss (A, B, C) was added to the bottom of the sample bottle, and 0.45 mL of substrate / auxiliary substrate solution and 0.05 mL of bacterial solution (approximately 10 ⁴ cfu) were added to the gloss surface.
The sample bottle was placed in a box whose humidity was adjusted to 100%, and the entire box was sealed to make a high humidity environment (humidity 100%). For comparison, a sample prepared in the same manner as above except that no gloss was added (control sample), and a sample prepared in the same manner as above except that no substrate, fungus, or auxiliary substrate was added on the added gloss (gross). Only samples) were prepared respectively. These samples were cultured at 30 ° C., and the growth of microorganisms was confirmed on the 5th and 7th days.

  To confirm the growth of microorganisms, add a lecithin polysorbate solution (LP diluted solution, manufactured by Nihon Pharmaceutical Co., Ltd.) to a 30 mL sample bottle so that it is 10 times the amount of gloss, and then mix well. A certain amount was smeared on BHI agar medium. After smearing the smeared agar medium at 30 ° C. for 1-2 days, the number of remaining bacteria was confirmed.

3. Evaluation result As a result of sensory evaluation by the filter method, the odor was confirmed in the gloss A, but the odor was not confirmed in the gloss B and C (Table 5-1).

  As a result of confirming the number of surviving bacteria in the sample by the filter method, in Gross A, the tendency of the inoculated bacteria to grow was confirmed. On the other hand, the tendency to decrease was confirmed in gross B and C (Table 5-2).

  In the result of confirming the number of surviving bacteria in the sample by the multi-layer method, the tendency of the inoculated bacteria to grow was confirmed in gross A, and the tendency to decrease in gross B and C was confirmed (Table 5-3).

  From the results of SPME-GC / MS analysis by the filter method, a large amount of isovaleraldehyde, which is a deodorizing component, was detected from the gross A sample. Although isovaleraldehyde was detected from the gloss B sample having higher antiseptic properties than the gloss A, the amount thereof was less than that of the gloss A. Furthermore, no isovaleraldehyde was detected in the gross C sample (Table 5-4).

  As is clear from the above results, sensory evaluation results, SPME-GC / MS analysis results, and microbial growth are correlated with each other in the human evaluation test results and the laboratory evaluation system using the odor generation model. It was. Using the evaluation system of the odor change model built in the present invention, it was confirmed that the risk of off-flavor generation by microorganisms of non-aqueous products, that is, the effectiveness of preventing secondary contamination of non-aqueous products can be appropriately evaluated in actual use conditions. .

IV. Comparative Example: Japanese Pharmacopoeia Preservative Efficacy Test Japanese Pharmacopoeia Preservative Efficacy Test Method (16th Revised Japanese Pharmacopoeia Reference Information G4. Microorganism-related Preservative Efficacy Test Method, p.2044-2046) Then, the effect of preventing secondary contamination of lip gloss was evaluated.

As the inoculated microorganism, several kinds of microorganisms including the following strains described in the preservation efficacy test method were used.
・Escherichia coli NBRC3972
・Pseudomonas aeruginosa NBRC13275
・Staphylococcus aureus NBRC13276
Lip gloss is the The same lip glosses A, B, and C used in the human actual use test were used.

The microorganism is suspended in physiological saline so as to be 1 × 10 ⁸ cfu / mL, and 1% by mass of this suspension is inoculated with respect to the lip gloss sample (10 ⁶ cfu / g) and mixed thoroughly. . After the inoculated sample was stored at 30 ° C. for a predetermined period, a certain amount was sampled, inactivated with LP diluent, and then smeared on the SCDLP agar medium. After culturing at 30 ° C. for a predetermined period, the number of viable bacteria was measured.
The number of days until the number of viable bacteria became 0.01% or less was measured with the number of inoculated bacteria as 100%.
The above test was performed twice on different days. The first and second results are shown in Table 6-1.

As shown in Table 6-1, in the gross A, B, and C, the number of days until the viable cell count became 0.01% or less was greatly different between the first test and the second test. That is, in the said test method, it turned out that the reduction rate of a bacteria count is not stable and the reproducibility of a result is bad. Furthermore, in the second test, the rate of decrease in the number of bacteria of gross A, B, and C was all the same, and did not correlate with the results of the human use test.
From these results, it was considered that it was difficult for this test method to quickly and appropriately evaluate the secondary contamination prevention effect of non-aqueous products.

Claims (11)

The evaluation method of the secondary contamination prevention effect of the nonaqueous skin external product containing the process of following (1)-(3).
(1) on the surface of the non-aqueous skin external products, Staphylococcus (Staphylococcus) genus Actinomyces (Actinomyces) genus Propionibacterium (Propionibacterium) genus Streptococcus (Streptococcus) genus Acinetobacter (Acinetobacter) genus Bacillus (Bacillus) genus, and microorganisms that are indigenous bacteria Cochlear (Kocuria) genus and Candida (Candida) at least one human skin and oral cavity selected from the group consisting of bacteria or fungi belonging to the genus, keratinocytes, keratin and Contacting at least one substrate selected from the group consisting of peptone with at least one auxiliary substrate selected from the group consisting of leucine and casamino acid ,
(2) step for a certain period of time retaining the non-aqueous skin external products in the microorganism can grow humidity conditions (1), and (3) in a non-aqueous skin external products maintained for a predetermined period of (2), the microorganism A process to evaluate the preventive effect of non-aqueous external skin products on secondary contamination of microorganisms by evaluating the odor generated by secondary contamination or the growth of microorganisms.   The evaluation method according to claim 1, wherein the water activity of the non-aqueous skin external product is 0.75 or less. Wherein the substrate is at least one selected from keratinocytes and Kerachi down or Ranaru group, according to claim 1 or 2 evaluation method according. The evaluation method according to claim 1, wherein the substrate is water-soluble keratin. The evaluation method according to claim 1 , wherein the auxiliary substrate contains at least leucine and casamino acid . Wherein the microorganism, Staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus Oureusu (Staphylococcus aureus), Staphylococcus warneri (Staphylococcus warneri), Staphylococcus saprophyticus (Staphylococcus saprophyticus subsp. Saprophyticus), Propionibacterium acnes (Propionibacterium acnes), and is at least one selected from the group consisting of Candida albicans (Candida albicans), the evaluation method of any one of claims 1-5. Wherein the microorganism is at least one selected from Staphylococcus epidermidis (Staphylococcus epidermidis), and Staphylococcus Oureusu-Anerobiusu (Staphylococcus aureus subsp. Anaerobius), according to any one of claims 1 to 6 Evaluation method. Wherein in the step (3) is at least one odor component to evaluate is selected from isovaleric acid and isovaleraldehyde claim 1-7 Evaluation method according to any one of. The skin external products are cosmetic, evaluation method of any one of claims 1-8. The skin external products, a lip cosmetic, evaluation method of any one of claims 1-9. The evaluation method according to any one of claims 1 to 10 , wherein the humidity condition under which the microorganism can grow is conditioned using water or a saturated ammonium dihydrogen phosphate solution.