A specific culture medium for detecting propionibacterium acnes in cosmetics and a detection method thereof

Abstract

The present application relates to a kind of specific culture medium for detecting propionibacterium acnes in cosmetic and its detection method, belong to microorganism detection technical field.The specific culture medium of the present application contains 5-25g nitrogen source per 1000mL culture medium, 5-10g carbon source, 0.02-16g inhibitor, 0.01-0.02g color indicator, 0.1-13g regulator, and the rest is solvent;The inhibition includes Tween 80, tea tree oil extract, bile salt, sodium laurate and hydroxyproline.The specific culture medium provided by the present application is a kind of antibiotic-free, high-selectivity, color-sensitive and low-cost propionibacterium acnes selective culture medium.By optimizing the composition of culture medium, adding selective inhibitor and color indicator, the growth of other facultative anaerobes such as lactic acid bacteria can be effectively inhibited, the specificity of propionibacterium acnes is cultured and color detection is carried out, and the accuracy and reliability of detection are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganism detection, and in particular to a specific culture medium for detecting Cutibacterium acnes in cosmetics and a detection method thereof. BACKGROUND

[0002] Cutibacterium acnes (C. acnes for short) is a gram-positive facultative anaerobic bacterium closely related to the pathogenesis of acne. It is a normal flora on the surface of human skin and mainly exists in the sebaceous glands of hair follicles. Under normal physiological conditions, it belongs to the symbiotic bacteria of the human body. However, when the secretion of sebaceous glands increases or other conditions change, C. acnes will multiply in large numbers and cause skin inflammatory reactions, leading to skin problems such as acne. In the laboratory, in order to detect and identify C. acnes, a selective culture medium is usually required. The contamination of C. acnes in cosmetics can adversely affect human skin health. Therefore, it is necessary to detect the content of C. acnes in cosmetics to ensure the safety of cosmetics and prevent adverse effects on human skin health.

[0003] To solve the problem of detecting C. acnes in cosmetics, some patents have been developed to solve these problems. For example:

[0004] Patent document CN118028139A discloses a lactic acid bacterium with skin cell repair ability and its application in cosmetics. The lactic acid bacterium has the characteristics of fast growth rate and short fermentation period, and its fermentation supernatant has strong inhibitory effect on C. acnes and Staphylococcus epidermidis. However, this patent still has the problem of further optimizing the culture conditions of Lactobacillus plantarum to improve its growth rate and fermentation period.

[0005] Patent document CN118773084A discloses a strain of C. acnes and its application. The fermentation supernatant of the C. acnes has the effect of promoting the growth of Malassezia globosa, inhibiting the growth of Staphylococcus capitis and limiting the growth of Malassezia restricta, which is beneficial to the health of the scalp. However, this patent still has the problem of optimizing the fermentation conditions of the C. acnes to improve the function of its fermentation supernatant in regulating the scalp flora.

[0006] The existing technology has the following disadvantages:

[0007] 1. The existing C. acnes culture medium lacks specific inhibitors and cannot effectively inhibit the growth of gram-positive bacteria such as lactic acid bacteria. The growth and reproduction of other facultative anaerobic bacteria such as lactic acid bacteria in the culture medium will interfere with the growth of C. acnes, leading to interference in the detection of C. acnes and poor accuracy of the detection results.

[0008] 2. Insensitive color reaction: The existing color development system has low sensitivity, making it difficult to quickly and accurately identify P. acnes.

[0009] 3. Antibiotic-dependent inhibitor system: Existing media usually use antibiotics such as chloramphenicol and gentamicin as inhibitors to inhibit the growth of other microorganisms. However, the use of antibiotics not only increases the complexity of the medium configuration, but also may lead to antibiotic residues, affecting the accuracy of experimental results, and even causing drug resistance problems.

[0010] Therefore, it is necessary to optimize the specific medium for P. acnes, which not only inhibits the growth of other facultative anaerobes such as lactic acid bacteria, but also ensures that P. acnes can specifically grow in the medium, thereby improving the accuracy of detection. SUMMARY

[0011] The present application aims to overcome the shortcomings of the prior art and provide a specific medium for detecting P. acnes in cosmetics and a detection method thereof. The specific medium provided by the present application is an antibiotic-free, highly selective, sensitive color development, and low-cost P. acnes selective medium. The detection method of the present application effectively inhibits the growth of other non-target facultative anaerobes and aerobes such as lactic acid bacteria by optimizing the composition of the medium, adding selective inhibitors and color indicators, and combining anaerobic culture methods, thereby promoting the specific growth and color detection of P. acnes, and improving the accuracy and reliability of P. acnes detection.

[0012] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0013] In a first aspect, the present application provides a specific medium for detecting P. acnes in cosmetics, containing 5-25 g of nitrogen source, 5-10 g of carbon source, 0.02-16 g of inhibitor, 0.01-0.02 g of color indicator, 0.1-13 g of adjusting agent, and the rest of the solvent per 1000 mL of medium; wherein the inhibitor includes Tween 80, tea tree oil extract, bile salt, sodium laurate and hydroxyproline.

[0014] The present application uses non-antibiotic inhibitors: Tween 80, tea tree oil extract, bile salt, sodium laurate and hydroxyproline in combination, effectively inhibiting the growth of other facultative anaerobes and aerobes such as lactic acid bacteria, avoiding the use of antibiotics and potential drug resistance problems, while basically not affecting the growth of P. acnes. By adding a color indicator to the medium, the growth of P. acnes can be quickly and sensitively detected. When P. acnes grows, the color of the medium changes, and the color reaction is sensitive and highly specific.

[0015] The present application combines Tween 80, tea tree oil extract, sodium laurate and hydroxyproline to obtain a composite bacteriostatic agent, which can selectively inhibit miscellaneous bacteria (including lactic acid bacteria, yeast, Staphylococcus aureus, Streptococcus thermophilus, Bacillus subtilis, etc.), but basically does not affect the growth of target bacteria (Propionibacterium acnes), and has specific synergistic selective inhibition characteristics for microorganisms.

[0016] As a preferred embodiment of the first aspect, the nitrogen source comprises peptone and yeast extract; the content of peptone is 10-15 g per 1000 mL of culture medium, and the content of yeast extract is 5-10 g.

[0017] As a preferred embodiment of the first aspect, the carbon source comprises glucose.

[0018] As a preferred embodiment of the first aspect, the content of Tween 80 is 1-10 g, the content of tea tree oil extract is 0.02-0.2 g, the content of bile salt is 0.1-2 g, the content of hydroxyproline is 0.2-2 g, and the content of sodium laurate is 0.02-0.4 g per 1000 mL of culture medium.

[0019] The content of Tween 80 can be any value or value range in 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g or 10 g; the content of tea tree oil extract can be any value or value range in 0.02 g, 0.04 g, 0.06 g, 0.08 g, 0.1 g or 0.2 g; the content of bile salt can be any value or value range in 0.1 g, 0.3 g, 0.5 g, 0.7 g, 0.9 g, 1 g, 1.2 g, 1.4 g, 1.6 g, 1.8 g or 2 g; the content of hydroxyproline can be any value or value range in 0.2 g, 0.3 g, 0.5 g, 0.7 g, 0.9 g, 1 g, 1.2 g, 1.4 g, 1.6 g, 1.8 g or 2 g; the content of sodium laurate can be any value or value range in 0.02 g, 0.04 g, 0.06 g, 0.08 g, 0.1 g, 0.2 g, 0.3 g or 0.4 g.

[0020] Sodium laurate is a surfactant that can destroy the structure of bacterial cell membranes, especially has a strong inhibitory effect on gram-positive bacteria. By adding an appropriate concentration (0.02-0.4 g) of sodium laurate, the growth of miscellaneous bacteria can be effectively inhibited while the growth of target bacteria (Propionibacterium acnes) is not significantly affected, thereby improving the selective inhibition of the culture medium. Therefore, sodium laurate can selectively inhibit the growth and pollution of other unnecessary microorganisms, ensuring the controllability of the culture process and the dominance of the target strain.

[0021] The experiment proves that Tween 80 and hydroxyproline have no effect on the growth of Propionibacterium acnes, and can promote the growth of Propionibacterium acnes to a certain extent, but have a certain inhibitory effect on miscellaneous bacteria, such as lactic acid bacteria, yeast, Staphylococcus aureus, Streptococcus thermophilus and Bacillus subtilis. Tea tree oil extract and sodium cholate also have a certain inhibitory effect on Propionibacterium acnes, but have little effect at a lower concentration, so the concentration thereof should not be too high, and the concentration thereof should be limited in the range of the present application, which has no inhibitory effect on Propionibacterium acnes and can also inhibit other miscellaneous bacteria.

[0022] The present application can effectively inhibit the growth of lactic acid bacteria and other facultative anaerobes and other aerobic bacteria by optimizing the components of the inhibitor and the ratio thereof, and can effectively inhibit the growth of lactic acid bacteria and other facultative anaerobes and other aerobic bacteria, while not excessively affecting the normal growth of the target bacteria Propionibacterium acnes.

[0023] As a preferred embodiment of the first aspect, the color developing indicator is bromocresol purple.

[0024] Propionibacterium acnes is a facultative anaerobe, which consumes carbon sources (such as glucose) and produces acidic metabolites such as lactic acid during growth and metabolism in the culture medium. The accumulation of lactic acid can cause the pH value of the culture medium to gradually decrease from the initial neutral or slightly alkaline (pH 7.2-7.4) to the acidic range (pH < 6.0).

[0025] Bromocresol purple (BCP) is an acid-base indicator, the color of which changes with the pH value of the solution: under neutral or alkaline conditions (pH > 7.2), bromocresol purple is purple; under neutral and slightly acidic conditions (pH 6.0-7.2), bromocresol purple is green; under acidic conditions (pH < 6.0), bromocresol purple turns yellow.

[0026] At the same time, in order to ensure the specificity of the color developing reaction, a suitable concentration of selective inhibitors (Tween 80, tea tree oil extract, hydroxyproline, sodium laurate and cholate) is added to the culture medium to inhibit the growth of other facultative anaerobes and some aerobic bacteria (such as lactic acid bacteria). Therefore, only Propionibacterium acnes can specifically grow in the culture medium of the present application and produce enough acidic metabolites to trigger the color developing reaction. Other bacteria or microorganisms (such as lactic acid bacteria, yeast, etc.) cannot grow or metabolize in the culture medium due to the inhibition of the selective inhibitors, and therefore cannot produce enough acidic metabolites to trigger the color developing reaction. Therefore, the culture medium of the present application combined with bromocresol purple and selective inhibitors can effectively detect the contamination of Propionibacterium acnes in cosmetics. When Propionibacterium acnes grows, the color of the culture medium changes from purple to yellow, and the color developing reaction is sensitive and specific.

[0027] As a preferred embodiment of the first aspect, the regulator comprises dipotassium hydrogen phosphate, sodium chloride and L-cysteine hydrochloride; the content of dipotassium hydrogen phosphate is 0.5-1 g per 1000 mL of culture medium, the content of sodium chloride is 5-10 g, and the content of L-cysteine hydrochloride is 0.1-1 g.

[0028] The present application adds L-cysteine hydrochloride as one of the regulators in the culture medium, which aims to provide a reducing environment to meet the growth needs of anaerobic bacteria Propionibacterium acnes. Specifically, the functions of L-cysteine hydrochloride include:

[0029] As a reducing agent: L-cysteine hydrochloride contains sulfhydryl (-SH) and has reducing property, which can help maintain the reducing state of the culture medium of the present application and prevent the toxic effect of oxygen on anaerobic bacteria, thereby promoting the growth of bacterial cells. Provide nitrogen and sulfur sources: L-cysteine hydrochloride provides essential nitrogen and sulfur sources for microorganisms, supporting their metabolic activities and protein synthesis.

[0030] In a second aspect, the present application provides a method for detecting Propionibacterium acnes in cosmetics, comprising the following steps:

[0031] S1, taking a cosmetic sample for enrichment culture to obtain an enrichment culture solution;

[0032] S2, inoculating the enrichment culture solution into the specific culture medium of the first aspect for anaerobic culture;

[0033] S3, determining whether the sample is contaminated with Propionibacterium acnes by whether the anaerobic culture of step S2 changes color, if it changes color, it is determined to be contaminated, if it does not change color, it is determined to be uncontaminated.

[0034] As a preferred embodiment of the second aspect, the culture medium used for enrichment culture in step S1 is Reinforced Clostridial Medium (RCM, Reinforced Clostridial Medium), which is used for culturing Clostridium and the like.

[0035] As a preferred embodiment of the second aspect, the enrichment culture in step S1 adopts the following method:

[0036] (1) Take the emulsion sample, add sterile physiological saline, and homogenize it as an inoculum;

[0037] (2) Take the above inoculum and add it to liquid Reinforced Clostridial Medium (RCM), and place it in a 36℃±1℃ incubator for anaerobic culture for 24-48h to obtain an enrichment solution.

[0038] As a preferred embodiment of the second aspect, the culture method of anaerobic culture in step S2 is:

[0039] (1) Preparation of specific culture medium plate;

[0040] (2) Take the enrichment liquid and coat or streak on the culture medium plate, and perform anaerobic culture for 24-48h.

[0041] As a preferred embodiment of the second aspect, the color change in step S3 is that the culture medium changes from purple to yellow.

[0042] Compared with the prior art, the present application has the following beneficial effects:

[0043] 1. The culture medium of the present application has a significant selective inhibition effect: it can effectively inhibit the growth of other facultative anaerobic bacteria such as lactic acid bacteria and other aerobic bacteria, while basically not affecting the normal growth of the target bacteria Propionibacterium acnes. Avoid their interference with the detection of Propionibacterium acnes, and improve the accuracy of the detection results;

[0044] 2. By adding bromocresol purple specific color indicator in the culture medium of the present application, the growth of Propionibacterium acnes and other bacteria can be accurately distinguished, further improving the specificity and accuracy of the detection.

[0045] 3. Environmental protection and safety are improved. The culture medium of the present application completely avoids the use of antibiotics, reduces the potential contribution to antibiotic resistance, meets the current requirements of green chemistry and environmental protection. At the same time, the use of antibiotics also reduces the toxicity risk of the culture medium, and is safer to use. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The color reaction test results of the Propionibacterium acnes culture plate and the lactic acid bacteria culture plate in Test Example 1 are shown in the schematic diagram (the Propionibacterium acnes culture plate changes from purple to yellow, and the colony morphology is clear, and the lactic acid bacteria culture plate does not change color and is still purple);

[0047] Figure 2 The color reaction sensitivity test results of the Propionibacterium acnes culture plate with an initial concentration of 1x10 4 -9x10 4 (CFU / mL) changing from purple to yellow are shown in the schematic diagram (the yellow plate is the Propionibacterium acnes culture plate, and the purple plate is the blank control). DETAILED DESCRIPTION

[0048] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific embodiments.

[0049] The tea tree oil extract is purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.; product name: tea tree oil, pinene-4-ol content: ≥35% 100g; product number: T819562.

[0050] Reinforced Clostridial Medium(RCM) was purchased from Guangzhou Haibo Technology Co., Ltd., product name: Reinforced Clostridial Medium(RCM), product number: HB0316 。

[0051] The preparation method of sodium laurate is as follows:

[0052] Lauric acid(C 12 H 24 O2), sodium hydroxide(NaOH, Guang test reagent), distilled water; lauric acid and sodium hydroxide were mixed in a molar ratio of 1:1 and dissolved in water, and a saponification reaction occurred to obtain sodium laurate.

[0053] Bovine bile salt: Shanghai Macklin Biochemical Technology Co., Ltd., product number B875069;

[0054] L-hydroxyproline: Shanghai Macklin Biochemical Technology Co., Ltd., product number L811195, CAS number: 51-35-4;

[0055] L-cysteine hydrochloride: Shanghai Macklin Biochemical Technology Co., Ltd., product number: C6183, L-cysteine hydrochloride (anhydrous) (biotechnology grade / 25g), CAS number: 52-89-1;

[0056] Oleic acid: oleic acid Guang test reagent technology Co., Ltd. CP 500ml, CAS number: 112-80-1;

[0057] Peppermint oil: Aradin, product number: C304394, peppermint oil ≥99%, CAS number: 68917-18-0;

[0058] Sodium deoxycholate: Shanghai Macklin Biochemical Technology Co., Ltd., product number: D6128, sodium deoxycholate(10g), CAS number: 302-95-4;

[0059] Glycine: Shanghai Macklin Biochemical Technology Co., Ltd., product number: G800880, glycine, 99.5-100.5%(500g), CAS number: 56-40-6;

[0060] L-methionine: S20060-100g L-methionine BR, 99%, source leaf, CAS number: 63-68-3;

[0061] The specific culture medium for detecting P. acnes in cosmetics in Examples 1-3 and Comparative Examples 1-14 is shown in the following table:

[0062] Table 1:

[0063]

[0064]

[0065] The preparation method of the medium of Example 1 is as follows:

[0066] According to the content in Table 1 of Example 1, respectively take proteose peptone, yeast extract, glucose, dipotassium hydrogen phosphate, sodium chloride, adjust the pH to 7.2, high temperature and high pressure sterilization (121℃, 15 minutes), wait for the medium to cool to room temperature (about 50℃ or less); then according to the content in Table 1 of Example 1, add Tween 80, tea tree oil extract, butylated hydroxytoluene, hydroxyproline, bromocresol purple, L-cysteine hydrochloride, sodium laurate, stir well to dissolve, prepare the specific medium for detecting propionibacterium acnes in cosmetics.

[0067] The preparation of the medium of Examples 2-3 and Comparative Examples 1-14 is the same as that of Example 1.

[0068] Test Example 1: Selective inhibition and color reaction test of the medium of the application

[0069] 1. Strain culture:

[0070] Propionibacterium acnes (ATCC 11827), Lactobacillus casei, Bacillus subtilis, yeast (CICC 1965), Staphylococcus aureus (CICC 10384), Streptococcus thermophilus were inoculated into nutrient agar slant, MRS medium, PDA medium slant or RCM medium slant, respectively, and cultured at 36℃±1℃ for 24-48h. Among them, Propionibacterium acnes, Lactobacillus casei and Streptococcus thermophilus were cultured anaerobically, and Bacillus subtilis, yeast and Staphylococcus aureus were cultured aerobically.

[0071] The culture medium of Propionibacterium acnes (ATCC 11827) was RCM;

[0072] The culture medium of Lactobacillus casei was MRS medium;

[0073] The culture medium of Bacillus subtilis was nutrient agar medium;

[0074] The culture medium of yeast (CICC 1965) was PDA medium;

[0075] The culture medium of Staphylococcus aureus (CICC 10384) was nutrient agar medium;

[0076] The culture medium of Streptococcus thermophilus is MRS culture medium.

[0077] 2. Preparation of bacterial suspension

[0078] The P. acnes bacterial solution, the lactic acid bacterial solution, the yeast bacterial solution, the S. aureus bacterial solution, the Streptococcus thermophilus bacterial solution and the B. subtilis bacterial solution are respectively prepared with sterile normal saline, and the concentration is 1.0×10 6 CFU / mL ~ 9.0×10 7 CFU / mL.

[0079] 3. Dilution and inoculation

[0080] The bacterial suspension is diluted to a suitable concentration (for example, 1:10, 1:100, 1:1000). 100 μL of the dilution is respectively coated or streaked on the selective medium of Example 1, and cultured at 37°C for 24-48h, wherein the P. acnes, the lactic acid bacteria and the Streptococcus thermophilus are cultured in an anaerobic manner, and the B. subtilis, the yeast and the S. aureus are cultured in an aerobic manner, so that each kind of bacteria grows in the most suitable condition.

[0081] 4. Observation of results

[0082] The P. acnes forms a grayish white colony (1-2mm in diameter) on the culture medium, and the color of the culture medium changes from purple to yellow. Other interfering bacteria do not grow or only grow in a small amount, and the culture medium remains purple and does not change color. The results are shown in Figure 1 , Table 2:

[0083] Table 2:

[0084]

[0085] As can be seen from Table 2, the culture medium of the present application has a selective inhibition, and the inhibition effect on the target bacteria is not obvious, but the inhibition on other bacteria is significant.

[0086] Test Example 2: Selectivity of culture method

[0087] 1. Culture of bacteria

[0088] The P. acnes (ATCC 11827), Lactobacillus casei, Bacillus subtilis, yeast (CICC 1965), Staphylococcus aureus (CICC 10384) and Streptococcus thermophilus are inoculated on nutrient agar slant, MRS medium, PDA medium slant or RCM medium slant respectively, and cultured at 36℃±1℃ for 24-48h, wherein the P. acnes, Lactobacillus casei and Streptococcus thermophilus are cultured in an anaerobic way, and the Bacillus subtilis, yeast and Staphylococcus aureus are cultured in an aerobic way.

[0089] The culture medium of the P. acnes (ATCC 11827) is RCM;

[0090] The culture medium of the Lactobacillus casei is MRS medium

[0091] The culture medium of the Bacillus subtilis is nutrient agar medium

[0092] The culture medium of the yeast (CICC 1965) is PDA medium

[0093] The culture medium of the Staphylococcus aureus (CICC 10384) is nutrient agar medium

[0094] The culture medium of the Streptococcus thermophilus is MRS medium

[0095] 2. Preparation of bacterial suspension:

[0096] The P. acnes, Lactobacillus casei, yeast, Staphylococcus aureus, Streptococcus thermophilus and Bacillus subtilis are prepared into bacterial liquid with sterile normal saline respectively, and the concentration is 1.0×10 6 CFU / mL-9.0×10 7 CFU / mL.

[0097] 3. Dilution and inoculation:

[0098] The above bacterial suspension is diluted to a suitable concentration (such as 1:10, 1:100, 1:1000). 100μL of the diluent is spread or streaked on the culture medium, and cultured at 37℃ anaerobically for 24-48h; wherein,

[0099] The culture medium of the P. acnes (ATCC 11827) is RCM;

[0100] The culture medium of the Lactobacillus casei is MRS medium

[0101] The culture medium of Bacillus subtilis is nutrient agar medium

[0102] The culture medium of Saccharomyces is PDA medium

[0103] The culture medium of Staphylococcus aureus (CICC 10384) is nutrient agar medium

[0104] The culture medium of Streptococcus thermophilus is MRS medium

[0105] 4. Result observation:

[0106] Propionibacterium acnes forms grayish white colonies (1-2 mm in diameter) on the culture medium, and the color of the culture medium changes from purple to yellow. Other interfering bacteria do not grow or only grow in small amounts. The results are shown in Table 3:

[0107] Table 3:

[0108]

[0109]

[0110] As can be seen from Table 3, Propionibacterium acnes can grow well under anaerobic culture conditions, while other aerobic interfering bacteria or part of facultative anaerobes are difficult to grow or grow slowly under anaerobic culture conditions. Therefore, the present application selects the performance effective inhibition of other aerobic interfering bacteria by means of anaerobic culture.

[0111] Test Example 3: Combination of selective culture medium and anaerobic culture method

[0112] 1. Strain culture:

[0113] Propionibacterium acnes (ATCC 11827), Lactobacillus casei, Bacillus subtilis, Saccharomyces (CICC 1965), Staphylococcus aureus (CICC 10384), Streptococcus thermophilus strains are inoculated into nutrient agar slant, MRS medium, PDA medium slant or RCM medium slant, respectively, and cultured at 36℃±1℃ for 24-48h, wherein Propionibacterium acnes, Lactobacillus, Streptococcus thermophilus are cultured by anaerobic culture method, and Bacillus subtilis, Saccharomyces, Staphylococcus aureus are cultured by aerobic culture method.

[0114] The culture medium of Propionibacterium acnes (ATCC 11827) is RCM;

[0115] The culture medium of Lactobacillus casei is MRS culture medium

[0116] The culture medium of Bacillus subtilis is nutrient agar culture medium

[0117] The culture medium of yeast (CICC 1965) is PDA culture medium

[0118] The culture medium of Staphylococcus aureus (CICC 10384) is nutrient agar culture medium

[0119] The culture medium of Streptococcus thermophilus is MRS culture medium

[0120] 2. Preparation of bacterial suspension:

[0121] The P. acnes bacterial liquid, the Lactobacillus bacterial liquid, the yeast bacterial liquid, the Staphylococcus aureus bacterial liquid, the Streptococcus thermophilus bacterial liquid and the Bacillus subtilis bacterial liquid are respectively prepared by using sterile normal saline, and the concentration is 1.0×10 6 CFU / mL-9.0×10 7 CFU / mL.

[0122] 3. Dilution and inoculation:

[0123] The above bacterial suspension is diluted to a suitable concentration (for example, 1:10, 1:100, 1:1000). 100 μL of the diluent is respectively coated or streaked on the selective medium of Example 1, and all are cultured at 37°C anaerobically for 24-48h.

[0124] The P. acnes forms a grayish white colony (1-2mm in diameter) on the culture medium, and the color of the culture medium changes from purple to yellow, and other interfering bacteria do not grow. The results are shown in Table 4:

[0125] Table 4:

[0126]

[0127]

[0128] In summary, the method of the present application combines the selective inhibitory culture medium and the anaerobic culture method to purposefully grow P. acnes and inhibit the growth of other miscellaneous bacteria, so as to achieve the purpose of detecting P. acnes in the sample.

[0129] Test Example 4: Sensitivity test of color reaction

[0130] 1. Culture of bacterial strains:

[0131] P. acnes (ATCC 11827) was inoculated on RCM medium slant and incubated anaerobically at 36°C±1°C for 24-48h.

[0132] 2. Preparation of bacterial suspension:

[0133] The bacterial suspension of P. acnes was prepared with sterile normal saline, with a concentration of 1.0x10 6 CFU / mL-9.0x10 7 CFU / mL.

[0134] 3. Dilution and inoculation:

[0135] The P. acnes bacterial solution was diluted to different concentration ranges of 1x10 3 -9x10 3 CFU / mL, 1x10 4 -9x10 4 CFU / mL, 1x10 5 -9x10 5 CFU / mL, respectively. 100 μL of the dilutions were spread or streaked on the selective medium of Example 1, respectively, to obtain different initial concentrations of P. acnes ranging from 1x10 2 -9x10 2 CFU / mL, 1x10 3 -9x10 3 CFU / mL, 1x10 4 -9x10 4 CFU / mL. The culture was incubated anaerobically at 37°C for 24-48h.

[0136] 4. Observation of results:

[0137] The results are shown in Figure 2 , Table 5. P. acnes formed grayish white colonies (1-2 mm in diameter) on the medium, and the color of the medium changed from purple to yellow. The sensitivity of the color reaction of P. acnes at different concentrations was different, and the time of color development also varied. As shown in Table 5, the medium can detect P. acnes as low as 10 2 CFU / mL, and the color development time is 48h.

[0138] Table 5:

[0139]

[0140]

[0141] Test Example 5: Detection of emulsion samples

[0142] 1. Sample pretreatment:

[0143] Take 10g of different emulsion samples, labeled A, B, C, D, and E, and add 90mL of sterile physiological saline to each sample, then homogenize to prepare the inoculum. Emulsion samples A, B, and C were respectively inoculated with a solution containing 6.5 × 10⁻⁶ mg / L sterile saline. 2 Each of the following samples contained 1 ml of Propionibacterium acnes: the emulsion sample D contained 1 ml of mixed bacteria (including Lactobacillus casei, Bacillus subtilis, yeast (CICC 1965), Staphylococcus aureus (CICC 10384), and Streptococcus thermophilus) at the same concentration; the emulsion sample E contained no Propionibacterium acnes and served as a blank control.

[0144] 2. Enrichment culture:

[0145] Add 10 mL of the above inoculum to 90 mL of liquid-enhanced Clostridium tumefaciens liquid culture medium (RCM), and anaerobic culture in an incubator at 36℃±1℃ for 24-48 h to obtain enrichment broth.

[0146] 3. Detection and culture:

[0147] 100 μL of the above enrichment broth was spread or streaked onto the culture medium plates of Example 1 and incubated anaerobically at 37°C for 48 h. The results are shown in the table below:

[0148] Table 6

[0149] Sample name Color change of medium Number of colonies (CFU / g) Emulsion A Purple to yellow 1.5 x 10 5 ]]> Emulsion B Purple to yellow 2.2 x 10 5 ]]> Emulsion C Purple to yellow 1.9 x 10 5 ]]> Emulsion D Purple Not detected Emulsion E Purple Not detected

[0150] As shown in Table 6, the culture medium prepared by this invention can detect Propionibacterium acnes in cosmetics. In lotion AE, Propionibacterium acnes was detected in lotions A, B, and C, indicating that lotions A, B, and C were contaminated with Propionibacterium acnes. However, the culture medium for lotion D did not change color, indicating that Propionibacterium acnes was not detected, and other bacteria failed to grow, therefore the culture medium did not change color. Similarly, the culture medium for lotion E did not change color, indicating that Propionibacterium acnes was not detected, and lotion E was not contaminated by this bacterium.

[0151] Test Example 6: Growth and reaction of Propionibacterium acnes in the culture media prepared in Examples 1-3 and Comparative Examples 1-14.

[0152] Test method:

[0153] 1. Preparation of mixed bacterial culture:

[0154] 1.1 Microbial culture:

[0155] The P. acnes (ATCC 11827), Lactobacillus casei, Bacillus subtilis, yeast (CICC 1965), Staphylococcus aureus (CICC 10384), Streptococcus thermophilus were inoculated on nutrient agar slant, PDA medium slant or RCM medium slant respectively, and cultured at 36℃±1℃ for 24-48h.

[0156] 1.2 Preparation of bacterial suspension:

[0157] 1.2.1 Preparation of P. acnes bacterial suspension: The P. acnes bacterial solution was prepared with sterile normal saline at a concentration of 1.0×10 6 CFU / mL-9.0×10 6 CFU / mL.

[0158] 1.2.2 Preparation of mixed bacterial suspension without P. acnes: The Lactobacillus casei bacterial solution, yeast bacterial solution, Staphylococcus aureus bacterial solution, Streptococcus thermophilus bacterial solution, and Bacillus subtilis bacterial solution were prepared with sterile normal saline respectively at a concentration of 1.0×10 6 CFU / mL-9.0×10 6 CFU / mL. The Lactobacillus casei bacterial solution, yeast bacterial solution, Staphylococcus aureus bacterial solution, Streptococcus thermophilus bacterial solution, and Bacillus subtilis bacterial solution were mixed in equal volumes to prepare the mixed bacterial suspension.

[0159] The mixed bacterial suspension without P. acnes was used to detect the selective culture performance of the culture medium of Examples 1-3 and Comparative Examples 1-14. If the culture medium inoculated with the mixed bacterial suspension without P. acnes showed a color reaction, it indicated that other miscellaneous bacteria grew on the culture medium, and the culture medium had a lower inhibitory ability to other miscellaneous bacteria, indicating that the culture medium had a lower selectivity.

[0160] 3. Inoculation:

[0161] 100μL of the bacterial suspension was respectively spread or streaked on the selective culture medium prepared in Examples 1-3 and Comparative Examples 1-14, and cultured at 37℃ for 24-48h.

[0162] Among them, P. acnes, Lactobacillus casei, and Streptococcus thermophilus were cultured anaerobically, and Bacillus subtilis, yeast, and Staphylococcus aureus were cultured aerobically, so that each bacterium grew in the most suitable conditions, and only the culture medium was different.

[0163] 4. The observation results are shown in the following table:

[0164] Table 7:

[0165]

[0166] Note: The meaning of the symbols in the table: "++++" indicates very obvious effect; "+++" indicates obvious effect; "++" indicates weaker effect; "+" indicates no obvious effect; "-" indicates no effect. In addition, since lactic acid bacteria and Streptococcus thermophilus can also produce acid, when the selective medium has low selective inhibition, lactic acid bacteria and Streptococcus thermophilus grow and accumulate acid, causing the selective medium to change color.

[0167] As can be seen from Table 7, the effect of Examples 1-3 is better than that of Comparative Examples 1-14.

[0168] The absence of sodium laurate, Tween 80, tea tree oil extract, bovine bile salt, and hydroxyproline in Comparative Examples 1-5, respectively, destroys the synergistic effect of specific inhibition of the complex inhibitor, resulting in a decrease in the selective antibacterial effect of the medium, and the growth of both the target bacteria and the interfering bacteria, all of which appear to be colored. Sodium laurate is an important bacteriostatic agent that can effectively inhibit the growth of lactic acid bacteria and other bacteria. After the absence of sodium laurate in Comparative Example 1, the growth of interfering bacteria increased significantly, and the interference color reaction was observed.

[0169] Comparative Example 6 replaces bromocresol purple with phenol red, and the color reaction of phenol red is less sensitive than that of bromocresol purple, resulting in a decrease in color effect.

[0170] Comparative Example 7 lacks L-cysteine hydrochloride: L-cysteine hydrochloride provides a sulfur source or regulates the redox state, and after its absence, the inhibitory effect of the interfering bacteria and the growth of P. acnes decrease.

[0171] Comparative Example 8 replaces sodium laurate with oleic acid: the antibacterial effect of oleic acid is weaker than that of sodium laurate, resulting in a decrease in the inhibitory effect of the interfering bacteria.

[0172] Comparative Example 9 replaces tea tree oil extract with peppermint oil extract, which may have a different selective antibacterial effect than tea tree oil, resulting in a decrease in the color reaction of the target bacteria and a decrease in the inhibitory effect of the interfering bacteria.

[0173] Comparative Example 10 replaces sodium deoxycholate with bile salt, and the selective antibacterial effect of sodium deoxycholate is not as good as that of bovine bile salt, resulting in a decrease in the color reaction of the target bacteria and a decrease in the inhibitory effect of the interfering bacteria.

[0174] Comparative Example 11 replaces hydroxyproline with glycine, which cannot effectively regulate metabolism, resulting in a decrease in the color reaction.

[0175] Comparative Example 12 replaces L-cysteine hydrochloride with L-methionine, although L-methionine provides a certain sulfur source, the intensity and sensitivity of the color reaction are significantly reduced, and the redox state regulation effect is not good, indicating the unique role of L-cysteine hydrochloride in providing a sulfur source and regulating the redox state.

[0176] Comparative Examples 13-14 destroyed the synergistic selective bacteriostatic effect of the composite inhibitor because the content ratio of Tween 80, tea tree oil extract, sodium laurate, bile salt and hydroxyproline was not within the specific range defined in the application, resulting in a weaker inhibitory effect.

[0177] Example 4

[0178] The present example provides a method for detecting P. acnes in cosmetics, comprising the following steps:

[0179] S1, taking a cosmetic sample for enrichment culture to obtain an enrichment culture solution;

[0180] S1-1, taking 10 g of emulsion sample, adding 90 mL of sterile normal saline, homogenizing, as inoculum;

[0181] S1-2, taking 10 mL of the above inoculum and adding it to 90 mL of liquid Clostridium difficile liquid medium (RCM), and placing it in a 36℃±1℃ incubator for anaerobic culture for 24-48h to obtain an enrichment solution;

[0182] S2, inoculating the enrichment culture solution into the specific medium of Example 1 for anaerobic culture;

[0183] S2-1, weighing each component according to the proportion of the medium formula of Example 1, and adding 1.5% agar, then sterilizing by high-pressure steam to prepare the specific medium plate;

[0184] S2-2, taking the above enrichment solution and spreading or streaking it on the medium plate of Example 1, and incubating it at 37℃ for 48h under anaerobic conditions.

[0185] S3, determining whether the sample is contaminated with P. acnes by whether the medium plate of step S2 changes color, if the color changes to yellow and the colony morphology is consistent with P. acnes (gram-positive bacilli under a microscope, no spores), it is determined to be contaminated, if the color changes to purple and no colonies grow, it is determined to be uncontaminated.

[0186] In summary, the medium of the present application can specifically detect P. acnes in cosmetics, effectively inhibit the growth of other facultative anaerobes such as lactic acid bacteria and other aerobic bacteria, and at the same time, does not affect the normal growth of the target bacteria P. acnes. By adding specific color developing indicators such as bromocresol purple, the growth of P. acnes and other bacteria can be accurately distinguished. The medium of the present application completely avoids the use of antibiotics, reduces the potential contribution to antibiotic resistance, and meets the current requirements of green chemistry and environmental protection. At the same time, the use of antibiotics is also reduced, which reduces the risk of toxicity of the medium and makes it safer to use.

[0187] Finally, it should be noted that the above examples are merely intended to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A specific medium for detecting Propionibacterium acnes in a cosmetic, characterized by, Only contains: nitrogen source, carbon source, inhibitor, color developing indicator and regulator, the solvent is deionized water; The nitrogen source is composed of peptone and yeast extract; The carbon source is glucose; The inhibitor is composed of Tween 80, tea tree oil extract, bile salt, sodium laurate and hydroxyproline; The color developing indicator is bromocresol purple; The regulator is composed of dipotassium hydrogen phosphate, sodium chloride and L-cysteine hydrochloride; Moreover, the content of glucose is 5-25 g per 1000 mL of the medium; The content of peptone is 10-15 g, and the content of yeast extract is 5-10 g; The content of Tween 80 is 1-10 g, the content of tea tree oil extract is 0.02-0.2 g, the content of bile salt is 0.1-2 g, the content of sodium laurate is 0.02-0.4 g, and the content of hydroxyproline is 0.2-2 g; The content of bromocresol purple is 0.01-0.02 g; The content of dipotassium hydrogen phosphate is 0.5-1 g, the content of sodium chloride is 5-10 g, and the content of L-cysteine hydrochloride is 0.1-1 g.

2. A method for detecting P. acnes in a cosmetic product, characterized in that, The method comprises the following steps: S1, taking a cosmetic sample for enrichment culture to obtain an enrichment culture solution; S2, inoculating the enrichment culture solution into the specific medium of claim 1 for anaerobic culture; S3, determining whether the sample is contaminated with Propionibacterium acnes by whether the anaerobic culture of step S2 changes color, if it changes color, it is determined to be contaminated, if it does not change color, it is determined to be not contaminated.

3. The detection method of claim 2, wherein, The medium used for enrichment culture in step S1 is a reinforced Clostridium liquid medium.

4. The detection method as described in claim 2, characterized in that, The culture method for anaerobic culture in step S2 is: (1) preparing a specific medium plate; (2) taking the enrichment culture solution and coating or streaking it on the medium plate for anaerobic culture for 24-48 h.

5. The detection method as described in claim 2, characterized in that, The color change in step S3 is that the medium changes from purple to yellow.

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