Specific culture medium for detecting propionibacterium acnes in cosmetics and detection method thereof

Abstract

The invention relates to a specific culture medium for detecting propionibacterium acnes in cosmetics and a detection method thereof, and belongs to the technical field of microbiological detection. Every 1000 mL of the specific culture medium contains 5-25 g of a nitrogen source, 5-10 g of a carbon source, 0.02-16 g of an inhibitor, 0.01-0.02 g of a color indicator, 0.1-13 g of a conditioning agent and the balance of a solvent. The inhibitor comprises Tween 80, a tea tree oil extract, cholate, sodium laurate and hydroxyproline. The specific culture medium provided by the invention is a propionibacterium acnes selective culture medium which is free of antibiotics, high in selectivity, sensitive in color development and low in cost. By optimizing the components of the culture medium and adding the selective inhibitor and the color indicator, the growth of other facultative anaerobic bacteria such as lactic acid bacteria can be effectively inhibited, specific culture and color development detection of propionibacterium acnes are carried out, and the accuracy and reliability of detection are improved.

Description

Technical Field

[0001] The present invention relates to the field of microbial detection, and in particular to a specific culture medium for detecting Propionibacterium acnes in cosmetics and a detection method thereof. Background Art

[0002] Propionibacterium 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 is mainly found in the sebaceous glands of hair follicles. Under normal physiological conditions, it belongs to the human commensal bacteria. However, when the secretion of sebaceous glands increases or other conditions change, Propionibacterium acnes will multiply in large quantities and cause skin inflammation, leading to skin problems such as acne. In the laboratory, in order to detect and identify Propionibacterium acnes, selective culture media are usually required. Cosmetics may be contaminated with Propionibacterium acnes, which has an adverse effect on human skin health. Therefore, it is necessary to detect the content of Propionibacterium acnes in cosmetics to ensure the safety of cosmetics and prevent them from having an adverse effect on human skin health.

[0003] Regarding the problem of detecting Propionibacterium acnes in cosmetics, there are currently some patents that address these issues. For example:

[0004] Patent document CN118028139A discloses a lactic acid bacterium with skin cell repair capabilities and its use in cosmetics. This lactic acid bacterium exhibits rapid growth and a short fermentation cycle, and its fermentation supernatant exhibits a strong inhibitory effect against Propionibacterium acnes and Staphylococcus epidermidis. However, this patent still faces the challenge of further optimizing the culture conditions of Lactobacillus plantarum to increase its growth rate and fermentation cycle.

[0005] Patent document CN118773084A discloses a strain of Propionibacterium acnes and its uses. The fermentation supernatant of this strain promotes the growth of Malassezia globosa while inhibiting the growth of Staphylococcus capitis and Malassezia restricta, thus promoting scalp health. However, this patent still faces the challenge of optimizing the fermentation conditions of this strain to enhance the fermentation supernatant's ability to regulate scalp flora.

[0006] The existing technology currently has the following shortcomings:

[0007] 1. The existing Propionibacterium acnes culture medium lacks targeted 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 Propionibacterium acnes, resulting in interference with Propionibacterium acnes detection and poor accuracy of test results.

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

[0009] 3. Antibiotic-Reliant Inhibitor Systems: Existing culture media often 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 culture medium configuration but can also lead to antibiotic residues, affecting the accuracy of experimental results and even causing drug resistance.

[0010] Therefore, it is necessary to optimize the specific culture medium for Propionibacterium acnes, which can not only inhibit the growth of other facultative anaerobic bacteria such as lactic acid bacteria, but also ensure that Propionibacterium acnes can grow specifically in the culture medium, thereby improving the accuracy of detection. Summary of the Invention

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

[0012] To achieve the above object, the technical solution adopted by the present invention is:

[0013] In a first aspect, the present invention provides a specific culture medium for detecting Propionibacterium acnes in cosmetics, wherein each 1000 mL of culture medium contains: 5-25 g of a nitrogen source, 5-10 g of a carbon source, 0.02-16 g of an inhibitor, 0.01-0.02 g of a color indicator, 0.1-13 g of a regulator, and the remainder is solvent; wherein the inhibitor includes Tween 80, tea tree oil extract, bile salts, sodium laurate, and hydroxyproline.

[0014] This invention combines the non-antibiotic inhibitors Tween 80, tea tree oil extract, bile salts, sodium laurate, and hydroxyproline to effectively inhibit the growth of other facultative anaerobes and aerobic bacteria, such as lactic acid bacteria, avoiding the use of antibiotics and their potential resistance, while also having little effect on the growth of Propionibacterium acnes. By adding a colorimetric indicator to the culture medium, this invention enables rapid and sensitive detection of P. acnes growth. When P. acnes grows, the culture medium changes color, resulting in a sensitive and highly specific color reaction.

[0015] The present invention combines Tween 80, tea tree oil extract, ox bile salt, sodium laurate and hydroxyproline to obtain a composite antibacterial agent. The antibacterial agent 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 properties against microorganisms.

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

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

[0018] As a preferred embodiment of the first aspect, the content of Tween 80 in each 1000 mL of culture medium 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.

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

[0020] Sodium laurate is a surfactant that disrupts the structure of bacterial cell membranes, with a particularly strong inhibitory effect against Gram-positive bacteria. By adding an appropriate concentration (0.02-0.4g) of sodium laurate, the growth of miscellaneous bacteria can be effectively inhibited while not significantly affecting the growth of the target bacteria (Propionibacterium acnes), thereby enhancing the selective inhibitory properties of the culture medium. Therefore, sodium laurate can selectively inhibit the growth and contamination of other unwanted microorganisms, ensuring controllable culture processes and the dominance of the target strain.

[0021] Experiments conducted in the present invention demonstrate that Tween 80 and hydroxyproline have no effect on the growth of Propionibacterium acnes and, to a certain extent, promote its growth. However, they exhibit a limited inhibitory effect on other bacteria, such as lactic acid bacteria, yeast, Staphylococcus aureus, Streptococcus thermophilus, and Bacillus subtilis. Tea tree oil extract and ox bile salts also exhibit a limited antibacterial effect on Propionibacterium acnes, but at relatively low concentrations, the effect is minimal. Therefore, their concentrations should not be too high, and within the scope of the present invention, they have no inhibitory effect on Propionibacterium acnes while still inhibiting other bacteria.

[0022] The present invention optimizes the inhibitor components and the ratios therebetween, so that the culture medium has a significant selective inhibitory effect, which can effectively inhibit the growth of other facultative anaerobic bacteria such as lactic acid bacteria 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 indicator is bromocresol purple.

[0024] Propionibacterium acnes is a facultative anaerobic bacterium that consumes carbon sources (such as glucose) and produces acidic metabolites such as lactic acid during its growth and metabolism in the culture medium. The accumulation of lactic acid will 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 whose color changes with the pH value of the solution: under neutral or alkaline conditions (pH>7.2), bromocresol purple is purple; under neutral to 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 development reaction, appropriate concentrations of selective inhibitors (Tween 80, tea tree oil extract, hydroxyproline, sodium laurate and bile salts) are added to the culture medium to inhibit the growth of other facultative anaerobic bacteria and some aerobic bacteria (such as lactic acid bacteria). Therefore, the culture medium of the present invention only has Propionibacterium acnes that can specifically grow and produce enough acidic metabolites to trigger a color development reaction. Other bacteria or microorganisms (such as lactic acid bacteria, yeast, etc.) are inhibited by the selective inhibitor and cannot grow or metabolize in the culture medium, so they will not produce enough acidic metabolites and will not cause a color development reaction. Therefore, the culture medium of the present invention, 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 development reaction is sensitive and highly specific.

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

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

[0029] As a reducing agent: L-cysteine hydrochloride contains a sulfhydryl group (-SH), which has reducing properties and can help maintain the reduced state of the culture medium of this invention, preventing the toxic effects of oxygen on anaerobic bacteria, thereby promoting bacterial growth. Providing a nitrogen and sulfur source: 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 invention provides a method for detecting Propionibacterium acnes in cosmetics, comprising the following steps:

[0031] S1. Take a cosmetic sample and perform bacterial culture to obtain a bacterial culture solution;

[0032] S2. Inoculating the enriched culture solution into the specific culture medium described in the first aspect for anaerobic culture;

[0033] S3. Determine whether the sample is contaminated with Propionibacterium acnes by checking whether the anaerobic culture in step S2 changes color. If the sample changes color, it is determined to be contaminated; if not, it is determined to be uncontaminated.

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

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

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

[0037] (2) Take the above inoculum and add it to liquid reinforced Clostridium liquid medium (RCM), place it in an incubator at 36℃±1℃ for anaerobic culture for 24-48 hours to obtain enrichment liquid.

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

[0039] (1) Prepare specific culture medium plates;

[0040] (2) Spread or streak the enrichment solution on a culture medium plate and perform anaerobic culture for 24-48 hours.

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

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

[0043] 1. The culture medium of the present invention has a significant selective inhibitory effect: it can effectively inhibit the growth of other facultative anaerobic bacteria such as lactic acid bacteria and other aerobic bacteria, while essentially not affecting the normal growth of the target bacteria Propionibacterium acnes. This prevents these bacteria from interfering with P. acnes detection and improves the accuracy of test results.

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

[0045] 3. Improved environmental friendliness and safety: The culture medium of this invention completely avoids the use of antibiotics, reducing the potential contribution to antibiotic resistance and meeting current green chemistry and environmental requirements. Furthermore, avoiding the use of antibiotics also reduces the risk of toxicity, making the culture medium safer to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the color reaction results of the Propionibacterium acnes culture plate and the Lactobacillus culture plate in Test Example 1 (the Propionibacterium acnes culture plate changed from purple to yellow with clear colony morphology, while the Lactobacillus culture plate did not change color and remained purple);

[0047] Figure 2 For the color reaction sensitivity test of Test Example 4, the initial concentration of Propionibacterium acnes was 1×10 4 -9×10 4 Schematic diagram of the results of the culture plate changing from purple to yellow (the yellow plate is the culture plate for Propionibacterium acnes, and the purple plate is the blank control for inoculation). DETAILED DESCRIPTION

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

[0049] Tea tree oil extract was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; product name: tea tree oil, terpinen-4-ol content: ≥35% 100g; item number: T819562.

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

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

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

[0053] Ox bile salts: Shanghai MacLean Biochemical Technology Co., Ltd., catalog number B875069;

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

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

[0056] Oleic acid: Oleic acid Guangshi Reagent Technology Co., Ltd. CP 500ml, CAS number: 112-80-1;

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

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

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

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

[0061] The components and content ratios of the specific culture medium used for detecting Propionibacterium acnes in cosmetics in Examples 1-3 and Comparative Examples 1-14 are shown in the table below:

[0062] Table 1:

[0063]

[0064]

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

[0066] Peptone, yeast extract, glucose, dipotassium hydrogen phosphate, and sodium chloride were dissolved in distilled water according to the contents in Table 1, Example 1, and the pH was adjusted to 7.2. The mixture was sterilized under high temperature and high pressure (121° C., 15 minutes). The culture medium was cooled to room temperature (about 50° C. or less). Tween 80, tea tree oil extract, ox bile salt, hydroxyproline, bromocresol purple, L-cysteine hydrochloride, and sodium laurate were then added according to the contents in Table 1, Example 1, and stirred thoroughly until dissolved to prepare a specific culture medium for detecting Propionibacterium acnes in cosmetics.

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

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

[0069] 1. Bacteria culture:

[0070] Propionibacterium acnes (ATCC 11827), lactic acid bacteria (Lactobacillus casei), Bacillus subtilis, yeast (CICC 1965), Staphylococcus aureus (CICC 10384), and Streptococcus thermophilus were inoculated onto nutrient agar slant, MRS medium, PDA medium slant, or RCM medium slant, and cultured at 36°C±1°C for 24-48 hours. Propionibacterium acnes, lactic acid bacteria, and Streptococcus thermophilus were cultured anaerobically, while Bacillus subtilis, yeast, and Staphylococcus aureus were cultured aerobically.

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

[0072] The culture medium for lactic acid bacteria ( Lactobacillus casei ) is MRS medium;

[0073] The culture medium for Bacillus subtilis is nutrient agar;

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

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

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

[0077] 2. Preparation of bacterial suspension:

[0078] Propionibacterium acnes, lactic acid bacteria, yeast, Staphylococcus aureus, Streptococcus thermophilus and Bacillus subtilis were prepared with sterile saline at a concentration of 1.0×10 6 CFU / mL~9.0×10 7 CFU / mL.

[0079] 3. Dilution and inoculation:

[0080] Dilute the bacterial suspension to an appropriate concentration (e.g., 1:10, 1:100, 1:1000). Apply or streak 100 μL of the dilution onto the selective culture medium of Example 1 and incubate at 37°C for 24-48 hours. For Propionibacterium acnes, Lactobacillus, and Streptococcus thermophilus, use anaerobically, while for Bacillus subtilis, yeast, and Staphylococcus aureus, use aerobic culture to ensure optimal growth of each bacterium.

[0081] 4. Observation of results:

[0082] Propionibacterium acnes forms off-white colonies (1-2 mm in diameter) on the culture medium, and the culture medium changes from purple to yellow. Other interfering bacteria do not grow or only grow in trace amounts, and the culture medium remains purple without changing color. Figure 1 Table 2:

[0083] Table 2:

[0084]

[0085] As can be seen from Table 2, the culture medium of the present invention has selective inhibition, and has little inhibitory effect on the target bacteria, but has significant inhibitory effect on other bacteria.

[0086] Test Example 2: Selectivity of culture method

[0087] 1. Bacteria culture:

[0088] Propionibacterium acnes (ATCC 11827), lactic acid bacteria (Lactobacillus casei), Bacillus subtilis, yeast (CICC 1965), Staphylococcus aureus (CICC 10384), and Streptococcus thermophilus were inoculated onto nutrient agar slant, MRS medium, PDA medium slant, or RCM medium slant, and cultured at 36°C±1°C for 24-48 hours. Propionibacterium acnes, lactic acid bacteria, and Streptococcus thermophilus were cultured anaerobically, while Bacillus subtilis, yeast, and Staphylococcus aureus were cultured aerobically.

[0089] The culture medium for Propionibacterium acnes (ATCC 11827) was RCM;

[0090] The culture medium for Lactobacillus casei is MRS medium

[0091] The culture medium for Bacillus subtilis is nutrient agar medium

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

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

[0094] The culture medium for Streptococcus thermophilus is MRS medium

[0095] 2. Preparation of bacterial suspension:

[0096] Propionibacterium acnes, lactic acid bacteria, yeast, Staphylococcus aureus, Streptococcus thermophilus and Bacillus subtilis were prepared with sterile saline at a concentration of 1.0×10 6 CFU / mL~9.0×10 7 CFU / mL.

[0097] 3. Dilution and inoculation:

[0098] Dilute the above bacterial suspension to an appropriate concentration (e.g. 1:10, 1:100, 1:1000). Take 100 μL of the dilution and spread or streak on the culture medium, and incubate anaerobically at 37°C for 24-48 hours.

[0099] The culture medium for Propionibacterium acnes (ATCC 11827) was RCM;

[0100] The culture medium for Lactobacillus casei is MRS medium

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

[0102] The culture medium for yeast (CICC 1965) is PDA medium

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

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

[0105] 4. Observation of results:

[0106] Propionibacterium acnes formed off-white colonies (1-2 mm in diameter) on the culture medium, and the culture medium color changed from purple to yellow. Other interfering bacteria did not grow or only grew in trace 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 bacteria or some facultative anaerobes have difficulty growing or grow slowly under anaerobic culture conditions. Therefore, the present invention can effectively inhibit other aerobic bacteria by selectively cultivating them through anaerobic culture.

[0111] Test Example 3: Combination of Selective Culture Medium and Anaerobic Culture

[0112] 1. Bacteria culture:

[0113] Propionibacterium acnes (ATCC 11827), lactic acid bacteria (Lactobacillus casei), Bacillus subtilis, yeast (CICC 1965), Staphylococcus aureus (CICC 10384), and Streptococcus thermophilus were inoculated onto nutrient agar slant, MRS medium, PDA medium slant, or RCM medium slant, and cultured at 36°C±1°C for 24-48 hours. Propionibacterium acnes, lactic acid bacteria, and Streptococcus thermophilus were cultured anaerobically, while Bacillus subtilis, yeast, and Staphylococcus aureus were cultured aerobically.

[0114] The culture medium for Propionibacterium acnes (ATCC 11827) was RCM;

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

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

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

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

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

[0120] 2. Preparation of bacterial suspension:

[0121] Propionibacterium acnes, lactic acid bacteria, yeast, Staphylococcus aureus, Streptococcus thermophilus and Bacillus subtilis were prepared with sterile saline at a concentration of 1.0×10 6 CFU / mL~9.0×10 7 CFU / mL.

[0122] 3. Dilution and inoculation:

[0123] Dilute the bacterial suspension to an appropriate concentration (e.g., 1:10, 1:100, 1:1000). Take 100 μL of the dilution and spread or streak onto the selective culture medium of Example 1. Incubate anaerobically at 37°C for 24-48 hours.

[0124] Propionibacterium acnes formed off-white colonies (1-2 mm in diameter) on the culture medium, and the culture medium color changed from purple to yellow. Other interfering bacteria did not grow. The results are shown in Table 4:

[0125] Table 4:

[0126]

[0127]

[0128] In summary, the present invention selectively selects an inhibitory culture medium combined with an anaerobic culture method to purposefully promote the growth of Propionibacterium acnes while inhibiting the growth of other bacteria, thereby achieving the purpose of detecting Propionibacterium acnes in a sample.

[0129] Test Example 4: Color reaction sensitivity test

[0130] 1. Bacteria culture:

[0131] Propionibacterium acnes (ATCC 11827) was inoculated onto a slant of RCM culture medium and cultured anaerobically at 36°C ± 1°C for 24-48 hours.

[0132] 2. Preparation of bacterial suspension:

[0133] A bacterial suspension of Propionibacterium acnes was prepared in sterile saline at a concentration of 1.0 × 10 6 CFU / mL~9.0×10 7 CFU / mL.

[0134] 3. Dilution and inoculation:

[0135] The Propionibacterium acnes bacterial solution was diluted to different concentrations ranging from 1×10 3 -9×10 3 CFU / mL, 1×10 4 -9×10 4 CFU / mL, 1×10 5 -9×10 5 CFU / mL. 100 μL of the dilution was applied or streaked onto the selective culture medium of Example 1, and different initial concentrations of P. acnes were obtained, ranging from 1×10 2 -9×10 2 CFU / mL, 1×10 3 -9×10 3 CFU / mL, 1×10 4 -9×10 4 CFU / mL. Incubate anaerobically at 37°C for 24-48 hours.

[0136] 4. Observation of results:

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

[0138] Table 5:

[0139]

[0140]

[0141] Test Example 5: Emulsion Sample Testing

[0142] 1. Sample pretreatment:

[0143] Take 10g of different emulsion samples, numbered A, B, C, D, E, add 90mL of sterile saline, and homogenize as inoculum. The emulsion samples numbered A, B, and C were added with a concentration of 6.5×10 2 acnes were added to the emulsion sample numbered D, with 1 ml of mixed bacteria (including lactic acid bacteria (Lactobacillus casei), Bacillus subtilis, yeast (CICC 1965), Staphylococcus aureus (CICC 10384), and Streptococcus thermophilus) added at the same concentration; the emulsion sample numbered E was not added with P. acnes and served as a blank control.

[0144] 2. Bacterial enrichment culture:

[0145] Take 10 mL of the above inoculum and add it to 90 mL of liquid reinforced Clostridium liquid medium (RCM), place it in an incubator at 36℃±1℃ for anaerobic culture for 24-48 hours to obtain the enrichment solution.

[0146] 3. Detection and cultivation:

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

[0148] Table: 6

[0149] Sample name Culture medium color change Colony count (CFU / g) Lotion A Purple turns yellow <![CDATA[1.5×10 5 ]]> Lotion B Purple turns yellow <![CDATA[2.2×10 5 ]]> Lotion C Purple turns yellow <![CDATA[1.9×10 5 ]]> Lotion D Purple Not detected Lotion E Purple Not detected

[0150] As shown in Table 6, the culture medium prepared by the present invention can detect Propionibacterium acnes in cosmetics. In emulsions AE, P. acnes was detected in emulsions A, B, and C, indicating that emulsions A, B, and C were contaminated with P. acnes. However, the culture medium in emulsion D did not change color, indicating that P. acnes was not detected. Furthermore, other bacteria were unable to grow, resulting in no discoloration. The culture medium in emulsion E did not change color, indicating that P. acnes was not detected and that emulsion E was not contaminated by this bacteria.

[0151] Test Example 6: Testing the Growth and Display 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 solution:

[0154] 1.1 Bacteria culture:

[0155] Propionibacterium acnes (ATCC 11827), lactic acid bacteria (Lactobacillus casei), Bacillus subtilis, yeast (CICC 1965), Staphylococcus aureus (CICC 10384), and Streptococcus thermophilus were inoculated onto nutrient agar slant, PDA medium slant, or RCM medium slant, and cultured at 36°C ± 1°C for 24-48 hours.

[0156] 1.2 Preparation of bacterial suspension:

[0157] 1.2.1 Preparation of Propionibacterium acnes suspension: Use sterile saline to prepare a Propionibacterium acnes suspension with 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 Propionibacterium acnes: Prepare lactic acid bacteria suspension, yeast suspension, Staphylococcus aureus suspension, Streptococcus thermophilus suspension, and Bacillus subtilis suspension respectively with sterile saline, with a concentration of 1.0×10 6 CFU / mL~9.0×10 6 CFU / mL. Equal volumes of lactic acid bacteria liquid, yeast liquid, Staphylococcus aureus liquid, Streptococcus thermophilus liquid, and Bacillus subtilis liquid were mixed to prepare a mixed bacterial suspension.

[0159] A mixed bacterial suspension that does not contain Propionibacterium acnes was used to test the selective culture performance of the culture media of Examples 1-3 and Comparative Examples 1-14. If a color reaction occurs in the culture inoculated with the mixed bacterial suspension that does not contain Propionibacterium acnes, it indicates that other bacteria are growing on the culture medium, and the culture medium has a low inhibitory ability against other bacteria, indicating that the culture medium has low selectivity.

[0160] 3. Vaccination:

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

[0162] Among them, Propionibacterium acnes, lactic acid bacteria, and Streptococcus thermophilus are cultured anaerobicly, while Bacillus subtilis, yeast, and Staphylococcus aureus are cultured aerobicly, so that each bacterium grows under the most suitable conditions, with only the culture medium being different.

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

[0164] Table 7:

[0165]

[0166] Note: Symbols in the table mean: "++++" indicates a very significant effect; "+++" indicates a significant effect; "++" indicates a weak effect; "+" indicates a moderate effect; and "——" indicates no effect. Furthermore, since lactic acid bacteria and thermophilic Streptococcus also produce acid, when the selective medium has a low selectivity, the cumulative acid production from the growth of lactic acid bacteria and thermophilic Streptococcus may cause the selective medium to change color.

[0167] It can be seen from Table 7 that, compared with Comparative Examples 1-14, the effects of Examples 1-3 are better than those of Comparative Examples.

[0168] Comparative Examples 1-5, lacking sodium laurate, Tween 80, tea tree oil extract, ox bile salts, and hydroxyproline, respectively, disrupted the synergistic effect of the combined inhibitor's specific inhibition, resulting in reduced selective antibacterial activity in the culture medium. This resulted in the growth of target bacteria alongside the growth of other bacteria, all of which exhibited a color reaction. Sodium laurate is an important antibacterial agent, effectively inhibiting the growth of lactic acid bacteria and other bacteria. The absence of sodium laurate in Comparative Example 1 significantly increased the growth of interfering bacteria, disrupting the color reaction.

[0169] In Comparative Example 6, bromocresol purple was replaced by phenol red. The color development reaction of phenol red was not as sensitive as that of bromocresol purple, and the color development effect was weakened.

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

[0171] Comparative Example 8: Sodium laurate is replaced by oleic acid: The antibacterial effect of oleic acid is weaker than that of sodium laurate, so the inhibitory effect on interfering bacteria is weakened.

[0172] In Comparative Example 9, the tea tree oil extract was replaced by peppermint oil extract. The selective antibacterial effect of peppermint oil extract may be different from that of tea tree oil, resulting in a weakened color reaction of the target bacteria and a weakened inhibitory effect on interfering bacteria.

[0173] In Comparative Example 10, bile salt was replaced by sodium deoxycholate. The selective antibacterial effect of sodium deoxycholate was not as good as that of ox bile salt, resulting in a weakened color reaction of the target bacteria and a weakened inhibitory effect on the interfering bacteria.

[0174] In Comparative Example 11, hydroxyproline was replaced by glycine. However, glycine could not effectively regulate metabolism, and the color reaction was weakened.

[0175] In Comparative Example 12, L-cysteine hydrochloride was replaced by L-methionine. Although L-methionine provided a certain sulfur source, the intensity and sensitivity of the color development reaction were significantly reduced, and the redox state regulation effect was poor, indicating the unique role of L-cysteine hydrochloride in providing a sulfur source and regulating the redox state.

[0176] In Comparative Examples 13-14, since the content ratio of Tween 80, tea tree oil extract, sodium laurate, bile salt and hydroxyproline is not within the specific range defined by the present invention, the synergistic selective antibacterial effect of the composite inhibitor is destroyed, resulting in a weak inhibitory effect.

[0177] Example 4

[0178] This embodiment provides a method for detecting Propionibacterium acnes in cosmetics, comprising the following steps:

[0179] S1. Take a cosmetic sample and perform bacterial culture to obtain a bacterial culture solution;

[0180] S1-1 Take 10 g of the emulsion sample, add 90 mL of sterile saline, and homogenize to use as the inoculum;

[0181] S1-2 Take 10 mL of the above inoculum and add it to 90 mL of liquid reinforced Clostridium liquid medium (RCM), place it in an incubator at 36℃±1℃ for anaerobic culture for 24-48 hours to obtain enrichment solution;

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

[0183] S2-1 Weigh each component according to the medium formula ratio of Example 1, add 1.5% agar, and sterilize with high-pressure steam to prepare a specific medium plate;

[0184] S2-2: Spread or streak the above enrichment solution on the culture medium plate of Example 1 and culture anaerobically at 37°C for 48 hours.

[0185] S3. Determine whether the sample is contaminated with Propionibacterium acnes by checking whether the culture medium plate in step S2 changes color. If the color changes to yellow and the colony morphology is consistent with that of Propionibacterium acnes (microscopic examination shows Gram-positive rods without spores), the sample is determined to be contaminated. If the color changes to purple and no colonies grow, the sample is determined to be uncontaminated.

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

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

Claims

1. A specific culture medium for detecting Propionibacterium acnes in cosmetics, characterized in that: Each 1000 mL of culture medium contains: 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 regulator, and the remainder of solvent; wherein the inhibitor includes Tween 80, tea tree oil extract, bile salt, sodium laurate and hydroxyproline.

2. The specific culture medium according to claim 1, wherein The nitrogen source includes peptone and yeast extract; the content of peptone in each 1000 mL of culture medium is 10-15 g, and the content of yeast extract is 5-10 g.

3. The specific culture medium according to claim 1, wherein The carbon source is glucose.

4. The specific culture medium according to claim 1, wherein In every 1000 mL of the culture medium, 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.

5. The specific culture medium according to claim 1, wherein The color indicator is bromocresol purple.

6. The specific culture medium according to claim 1, wherein The regulator comprises dipotassium hydrogen phosphate, sodium chloride and L-cysteine hydrochloride; in each 1000 mL of culture medium, 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.

7. A method for detecting Propionibacterium acnes in cosmetics, characterized in that: The steps include: S1. Take a cosmetic sample and perform bacterial culture to obtain a bacterial culture solution; S2. inoculating the enrichment culture solution into the specific culture medium according to any one of claims 1 to 6 for anaerobic culture; S3. Determine whether the sample is contaminated with Propionibacterium acnes by checking whether the anaerobic culture in step S2 changes color. If the sample changes color, it is determined to be contaminated; if not, it is determined to be uncontaminated.

8. The detection method according to claim 7, wherein The culture medium used for the bacterial enrichment culture in step S1 is a fortified Clostridium liquid culture medium.

9. The detection method according to claim 7, wherein The anaerobic culture method in step S2 is: (1) Prepare specific culture medium plates; (2) Spread or streak the enrichment solution on a culture medium plate and perform anaerobic culture for 24-48 hours.

10. The detection method according to claim 7, wherein The color change in step S3 specifically refers to the culture medium changing from purple to yellow.

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