Microbial limit test method of adapalene benzoyl peroxide gel
By using sterile calcium chloride solution as a neutralizing agent and a dilution-combination counting method, the problem of antimicrobial interference in the microbial limit test of adapalene peroxybenzoyl gel was solved, ensuring the accuracy and compliance of the test results.
Patent Information
- Application Number
- CN202511698454.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-19
- Publication Date
- 2026-01-13
AI Technical Summary
Existing methods for testing the microbial limits of adapalene peroxybenzoyl gel suffer from antimicrobial interference and sample homogeneity issues, leading to false negative results and inaccurate detection. There is a lack of suitable methods for testing the microbial limits.
A sterile calcium chloride solution of 9.6%–10.4% was used as a neutralizing agent. The antibacterial activity of benzoyl peroxide gel was eliminated by dilution and multi-plate counting methods, thus ensuring the accuracy of microbial detection.
The results of the microbial limit test of adapalene peroxybenzoyl gel were made more accurate, met the pharmacopoeia requirements, eliminated interference with antibacterial activity, and improved the microbial recovery rate.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of microbial limit test, and particularly relates to a microbial limit test method for adapalene and peroxi benzoyl gel. BACKGROUND
[0002] The pharmacopoeias at home and abroad (such as Chinese Pharmacopoeia (ChP), USP, EP) all stipulate that the microbial limit test method needs to be subjected to method suitability test to prove: culture medium applicability; counting method effectiveness (such as whether the recovery rate of the plate method meets the standard); control bacteria checking specificity (such as whether Pseudomonas aeruginosa and Staphylococcus aureus can be accurately detected); since the drug has antibacterial activity, the conventional microbial limit test method cannot be realized, and the antibacterial activity should be eliminated first, and whether the antibacterial activity is completely removed is verified through experiment. The microbial limit test is a key link of drug quality control, which is used for evaluating the aerobic bacteria, mold and yeast, and the microbial index level of Staphylococcus aureus and Pseudomonas aeruginosa in non-sterile preparations, and ensuring that the drug meets the safety standard.
[0003] Adapalene is a retinoid compound, which is proved to have anti-inflammatory properties in in vivo and in vitro inflammation models, and the product is a gel, which may interfere with microbial detection, so a reliable microbial limit test method needs to be established to ensure the accuracy of the test results.
[0004] The detection of the gel preparation in the prior art has the following difficulties: (1) bacteriostatic interference: peroxi benzoyl has strong oxidizing property, which may inhibit microbial growth, leading to false negative results. (2) sample uniformity: the gel matrix may affect the dispersion and recovery of microorganisms. (3) anti-inflammatory interference: the preparation may inhibit microbial growth and interfere with the effect of neutralizing agent, and high osmotic pressure, extreme pH, organic solvents, etc. may directly damage microbial cells. There is no microbial limit test method suitable for the adapalene and peroxi benzoyl gel drug in the prior art. Since the drug has its unique properties, the microbial limit test method of other gel drugs is not suitable for this product, so it is urgent to establish a reliable microbial limit test method for adapalene and peroxi benzoyl gel to ensure the microbial safety of the gel preparation, meet the requirements of GMP and pharmacopoeia, and provide a scientific basis for drug quality control. SUMMARY
[0005] In view of the defects in the prior art, in order to solve the problem that there is no reliable microbial limit test method for adapalene and peroxides gel, the purpose of the present application is to design a microbial limit test method for adapalene and peroxides gel. The present application eliminates the bacteriostatic activity of interfering substances by using an improved dilution level, adding 9.6% to 10.4% calcium chloride solution as a neutralizing agent for microbial limit test, and using a multi-dish combined counting method, to obtain a microbial limit test method that meets the requirements of the pharmacopoeia.
[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:
[0007] On the one hand, the present application provides a microbial limit test method for adapalene and peroxides gel, comprising the following steps:
[0008] (1) Preparation of test solution: Take adapalene and peroxides gel, add physiological saline to make it completely dissolved, add 9.6% to 10.4% sterile calcium chloride solution, mix well, stand for a while, take the supernatant, dilute and use as the test solution;
[0009] Preferably, the concentration of the sterile calcium chloride solution is 10%.
[0010] (2) Take several portions of the above test solution, group them, and place them in different sterile plates, pour the culture medium into the plates, and then culture after cooling, and calculate the total number of aerobic bacteria, total number of mold and / or yeast after combined counting after the end of the culture;
[0011] (3) Take the supernatant and inoculate it into sterile plates containing culture medium for enrichment culture, then streak inoculate it into agar medium after the end of the culture, and then perform selective culture, and finally count the typical colonies and determine whether the growth control bacteria are present.
[0012] The microbial limit test method for adapalene and peroxides gel, the mass of the adapalene and peroxides gel is 9.6 to 10.4 g;
[0013] The physiological saline is 0.9% sterile sodium chloride solution;
[0014] The volume ratio of the physiological saline to the sterile calcium chloride solution is 9:1.
[0015] The microbial limit test method for adapalene and peroxides gel, the mass of the adapalene and peroxides gel in the test solution is 1:50 g / mL compared to the sum of the volumes of the physiological saline and the sterile calcium chloride solution.
[0016] The standing time is 5-10 min.
[0017] The microbial limit test method of the adapalene peroxide benzoyl gel, the several parts are 10 parts, and are divided into two groups, 5 parts in each group.
[0018] The microbial limit test method of the adapalene peroxide benzoyl gel, the aerobic bacteria include Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans or Aspergillus niger;
[0019] The mold and / or yeast are Candida albicans and Aspergillus niger;
[0020] The control bacteria are Staphylococcus aureus and Pseudomonas aeruginosa.
[0021] The microbial limit test method of the adapalene peroxide benzoyl gel, in step (2), the culture medium is a melted culture medium with a temperature of not more than 45 DEG C, and the volume ratio of the culture medium to the test liquid is (15-20):1.
[0022] The microbial limit test method of the adapalene peroxide benzoyl gel, in step (2), when the microorganism is aerobic bacteria, the culture medium is tryptose soy broth agar medium, and the culture condition is a temperature of 30-35 DEG C and a culture time of 3-5 days.
[0023] When the microorganism is mold and / or yeast, the culture medium is sabouraud dextrose agar medium, and the culture condition is a temperature of 20-25 DEG C and a culture time of 5-7 days.
[0024] The microbial limit test method of the adapalene peroxide benzoyl gel, in step (3), when the control bacteria are Staphylococcus aureus, the culture medium is tryptose soy broth liquid medium, the volume ratio of the supernatant to the culture medium is 1:10, the enrichment culture condition is a temperature of 30-35 DEG C and a culture time of 18-24 hours, the agar medium is mannitol sodium chloride agar medium, and the selection culture condition is a temperature of 30-35 DEG C and a culture time of 18-72 hours.
[0025] When the control bacteria are Pseudomonas aeruginosa, the culture medium is tryptose soy broth liquid medium, the volume ratio of the supernatant to the culture medium is 1:10, the enrichment culture condition is a temperature of 30-35 DEG C and a culture time of 18-24 hours, the agar medium is cetyltrimethylammonium bromide agar medium, and the selection culture condition is a temperature of 30-35 DEG C and a culture time of 18-72 hours.
[0026] The microbial limit test method of the adapalene peroxide benzoyl gel, the calculation method of the combined count is that the count results of each petri dish in each group after culture are added, and the count results of the adapalene peroxide benzoyl gel are obtained.
[0027] Secondly, the present invention provides the application of any of the microbial limit testing methods described in the invention in detecting the number of microorganisms in adapalene peroxybenzoyl gel.
[0028] This invention uses a sterile calcium chloride solution of approximately 10% as a neutralizing agent. Its working principle is as follows: the oxidizing power of benzoyl peroxide depends on the peroxide bond (-OO-) in the molecule. This bond is unstable and easily decomposes in aqueous solution or upon contact with microorganisms, releasing reactive oxygen species (such as ·OH free radicals, O2). 2- These reactive oxygen species (ROS), etc., disrupt the structure of microorganisms. Calcium chloride (CaCl2) is a strong electrolyte and completely dissociates into Ca in aqueous solution. 2+ and Cl - Ca 2+ It is a divalent cation with strong coordination ability, capable of forming coordinate bonds with oxygen atoms near the peroxide bond in the benzoyl peroxide molecule, effectively stabilizing the peroxide bond structure and reducing its decomposition rate; simultaneously, Ca... 2+ The positive charge of benzoyl peroxide attracts the negatively charged region in the benzoyl peroxide molecule, altering its molecular conformation and further inhibiting its binding ability to microbial cell membranes and proteins, thereby reducing the release of reactive oxygen species and weakening its oxidizing properties. - It possesses a certain reducing property, enabling it to undergo a redox reaction with the small amount of residual reactive oxygen species produced by the decomposition of benzoyl peroxide. This step effectively removes any remaining reactive oxygen species, preventing residual oxidizing substances from further inhibiting microbial growth and providing a stable growth environment for microorganisms (such as positive control bacteria inoculated during testing, or microorganisms that may be present in the sample). This solves the problem of neutralizing the antibacterial effect of antimicrobial agents. The final results demonstrate that calcium ions in 10% sterile calcium chloride can neutralize the antibacterial effect of adapalene-benzoyl peroxide gel, improving the recovery rate of the test bacteria and ensuring that the test results meet the requirements of the Chinese Pharmacopoeia.
[0029] On the other hand, when excessively high concentrations of sterile calcium chloride are used as a neutralizing agent, high concentrations of Ca... 2+ and Cl - This can lead to excessively high osmotic pressure in the system, causing microbial cells to dehydrate and shrink, thus inhibiting growth and resulting in inaccurate results. Too low a concentration of sterile calcium chloride, Ca... 2+ and Cl - If the amount of calcium chloride solution is insufficient, it cannot completely neutralize the oxidizing properties of benzoyl peroxide, and will still have residual antibacterial effect, thus failing to achieve the neutralization and antibacterial effect. Therefore, it is necessary to strictly follow the concentration limit of sterile calcium chloride solution during the experimental testing process.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] The microbial limit test of adapalene peroxybenzoyl gel using the method of this invention can eliminate the antibacterial activity of interfering substances. The total number of aerobic bacteria, total number of molds and yeasts, as well as Staphylococcus aureus and Pseudomonas aeruginosa were tested according to law (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapters 1105 and 1106), and the results met the requirements. Detailed Implementation
[0032] The present invention will be further illustrated by the following examples.
[0033] Example 1:
[0034] I. Test samples, culture media and experimental instruments
[0035] 1. Test sample
[0036] Adapalene peroxybenzoyl, batch number: S4240301, was provided by Hangzhou Minsheng Pharmaceutical Co., Ltd.
[0037] 2. Culture medium and reagents
[0038] Tryptic soybean agar medium (batch number: 221227; supplier: Beijing Sanyao Technology Co., Ltd.).
[0039] Tryptic soybean peptone liquid culture medium (batch number: 221104; supplier: Beijing Sanyao Technology Co., Ltd.).
[0040] Mannitol sodium chloride agar medium (batch number: 230221; supplier: Beijing Sanyao Technology Co., Ltd.).
[0041] Hexadecanetrimethylamine bromide agar medium (batch number: 1091355; supplier: Guangdong Huankai).
[0042] Sabouraud dextrose agar medium (batch number: 1107925; supplier: Guangdong Huankai Microbial Technology Co., Ltd.).
[0043] 0.9% physiological sodium chloride solution (batch number: 1B23102305; supplier: Shandong Qidu Pharmaceutical Co., Ltd.).
[0044] Anhydrous calcium chloride (batch number: 20220719; supplier: Sinopharm Chemical Reagent Co., Ltd.).
[0045] All culture media were prepared according to the instructions for use.
[0046] 3. Experimental instruments
[0047] ME 2002E Electronic Balance (Item No.: H-1-106, H-1-110; Supplier: Mettler Toledo Instruments (Shanghai) Co., Ltd.).
[0048] MLS-3781 High-Pressure Steam Sterilizer (Item No.: Y-10-53; Supplier: Sanyo Corporation, Japan).
[0049] HF safe-1200 biosafety cabinet (part numbers: Y-40-14, Y-40-10; supplier: Shanghai Lishen Scientific Instruments Co., Ltd.).
[0050] SF 450 sterilization box (Item No.: X-5-72; Supplier: Memert GmbH, Germany).
[0051] ICP 55 Low Temperature Incubator (Item No.: X-5-81; Supplier: Memert GmbH, Germany).
[0052] IF 450 constant temperature incubator (Item No.: X-5-96; Supplier: Memert GmbH, Germany).
[0053] IF 450 constant temperature incubator (Item No.: X-5-94; Supplier: Memert GmbH, Germany).
[0054] II. Preparation of bacterial culture
[0055] 1. Staphylococcus aureus [CMCC(B)26003]
[0056] Take a Staphylococcus aureus cryopreservation tube, inoculate it into 10 mL of tryptic soy broth, incubate at 33°C for 18 hours, and then dilute it with 0.9% sterile sodium chloride solution to prepare a bacterial suspension of appropriate concentration.
[0057] 2. Pseudomonas aeruginosa [CMCC(B)10104]
[0058] Take a cryopreservation tube of Pseudomonas aeruginosa, inoculate it into 10 mL of tryptic soy peptone liquid medium, incubate at 33°C for 18 hours, and then dilute it with 0.9% sterile sodium chloride solution to prepare a bacterial suspension of appropriate concentration.
[0059] 3. Bacillus subtilis [CMCC(B)63501]
[0060] Take a cryopreserved tube of Bacillus subtilis, inoculate it onto a tryptic soy agar slant, and incubate at 33°C for 24 hours. Then wash off the bacterial growth with a sterile 0.9% sodium chloride solution, and then dilute the bacterial suspension with a sterile 0.9% sodium chloride solution to a suitable concentration.
[0061] 4. Candida albicans [CMCC(F)98001]
[0062] Take a cryopreserved tube of Candida albicans, inoculate it into 10 mL of Sabouraud dextrose liquid medium, incubate at 23°C for 2 days, and dilute with 0.9% sterile sodium chloride solution to prepare a bacterial suspension of appropriate concentration.
[0063] 5. Aspergillus niger [CMCC(F)98003]
[0064] Take a cryopreserved tube of Aspergillus niger, inoculate it onto Sabouraud dextrose agar slant, and incubate at 23°C for 7 days. Then, wash the spores with 5 mL of 0.9% sterile sodium chloride solution containing 0.05% (mL / mL) polysorbate 80. Aspirate the spore suspension into a sterile test tube using a sterile pipette, and dilute it with 0.9% sterile sodium chloride solution containing 0.05% (mL / mL) polysorbate 80 to a suitable concentration of bacterial suspension.
[0065] All the above test strains were provided by the National Institutes for Food and Drug Control, and bacterial suspensions of appropriate concentrations were prepared using the methods provided in the 2020 edition of the Chinese Pharmacopoeia for later use.
[0066] III. Methods and Results
[0067] (I) Suitability test of counting method
[0068] In the following method, a 10% calcium chloride solution was used as a demulsifying and neutralizing agent. Since a precipitate forms between the 10% calcium chloride solution and the pH 7.0 sodium chloride-peptone buffer, a 0.9% sterile sodium chloride solution was selected as the diluent. The suitability of the counting method was tested by increasing the dilution level and combining multiple dishes for counting to eliminate the antibacterial activity of interfering substances.
[0069] 1. Preparation of test solution: Take 10 g of sample, add 0.9% sterile sodium chloride solution to 90 mL, shake to dissolve, then add 10 mL of 10% sterile calcium chloride solution, shake well, let stand for 5 to 10 minutes to allow precipitation, the resulting supernatant is the 1:10 test solution, further dilute to 1:50 test solution.
[0070] 2. Experimental Group: ① Total aerobic bacteria count: Take 1 mL of the 1:50 test solution, pour it into a Petri dish, add 1 mL of the prepared bacterial solution, and prepare 5 Petri dishes using the same method. Pour the solution into tryptic soy agar medium. Prepare two replicates for each test strain. The test strains are Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger. ② Total mold and yeast count: Take 50 mL of the 1:50 test solution, add 0.5 mL of the prepared bacterial solution, and mix well. Take 1 mL of the test solution containing bacteria, pour it into a Petri dish, and prepare 5 Petri dishes using the same method. Pour the solution into Sabouraud dextrose agar medium. Prepare two replicates for each test strain. The test strains are Candida albicans and Aspergillus niger.
[0071] 3. Control group of test sample: Take the prepared test solution, without adding bacterial solution, and use diluent instead of bacterial solution in the same way as the test group.
[0072] 4. Control group of bacterial suspension: Replace the test solution with 0.9% sterile sodium chloride solution, add the test bacterial suspension according to the operation of the test group, and determine the bacterial count according to the counting method of the test group.
[0073] 5. Neutralizing agent control group: Replace the test solution with 10% calcium chloride solution, add the test bacterial solution according to the operation of the test group, and determine the bacterial count according to the counting method of the test group.
[0074] 6. Negative control group: Use 0.9% sterile sodium chloride solution instead of the test sample (without adding bacterial solution), and follow the test solution preparation method and counting method.
[0075] 7. The test results are shown in Table 1-8 below:
[0076] Table 1. Total aerobic bacteria count (CFU) results in the experimental group.
[0077] Table 2. Total count of molds and yeasts in the experimental group (CFU)
[0078] Table 3. Count results (CFU) of the control group for the test sample.
[0079] Table 4. Total aerobic bacteria count (CFU) in the bacterial culture control group.
[0080] Table 5. Total count of molds and yeasts (CFU) in the control group of the bacterial culture.
[0081] Table 6. Total aerobic bacteria count (CFU) results in the neutralizing agent control group.
[0082] Table 7. Total count of molds and yeasts (CFU) in the neutralizing agent control group.
[0083] Table 8. Count results (CFU) of the negative control group.
[0084] Based on the experimental data obtained above, recovery tests were conducted on the experimental group and the neutralizing agent control group. The results are shown in Tables 9 and 10 below.
[0085] Table 9 Results of the recovery test in the experimental group
[0086] Table 10 Results of the recovery test of the neutralizing agent control group
[0087] The above results indicate that, according to regulations, the recovery ratio is calculated as the ratio of the number of colonies in the experimental group minus the number of colonies in the control group to the number of colonies in the bacterial solution control group. Using the counting method of this invention, the recovery ratios of all five strains were within the range of 0.5 to 2 specified in the pharmacopoeia. The recovery ratio of the neutralizing agent control group was also within the range specified in the pharmacopoeia, demonstrating that the neutralizing agent of this invention can effectively neutralize the residual activity of disinfectants and has no inhibitory or growth-promoting effect on microorganisms.
[0088] (II) Suitability test of control methods
[0089] 1. Staphylococcus aureus
[0090] 1.1 Preparation of test solution: Same as the suitability test of the counting method.
[0091] 1.2 Suitability Test: Take 10 mL of 1:10 supernatant and inoculate it directly into 100 mL of tryptic soy broth, then add 1 mL of Staphylococcus aureus culture and incubate at 33°C for 18 hours. Streak the above culture onto mannitol-sodium chloride agar plates and incubate at 33°C for 18 hours.
[0092] 1.3 Test sample group: Take 10 mL of 1:10 supernatant, without adding bacterial solution, treat according to the method of suitability test group, and culture according to law.
[0093] 1.4 Bacterial count group: Staphylococcus aureus bacterial suspension was taken and its bacterial count was determined by the plate method. Tryptic soy agar medium was used as the culture medium.
[0094] 1.5 Negative control group: Use 0.9% sterile sodium chloride solution instead of the test sample, and determine the bacterial count according to the test solution preparation method and suitability test counting method, and culture according to the law.
[0095] 1.6 Test Results:
[0096] Table 11 Results of the suitability test for the control bacteria method
[0097] The above results indicate that the control bacteria test for Staphylococcus aureus using this product was conducted using the conventional method with a culture medium volume of 100 mL.
[0098] 2. Pseudomonas aeruginosa
[0099] 2.1 Preparation of test solution: Same as the suitability test of the counting method.
[0100] 2.2 Suitability Test: Take 10 mL of 1:10 supernatant and inoculate it directly into 100 mL of tryptic soy broth, then add 1 mL of Pseudomonas aeruginosa culture and incubate at 33°C for 18 hours. Streak the above culture onto hexadecanetrimethylammonium bromide agar plates and incubate at 33°C for 18 hours.
[0101] 2.3 Test sample group: Take 10 mL of 1:10 test solution, without adding bacterial solution, treat according to the method of suitability test group, and culture according to law.
[0102] 2.4 Bacterial count group: Pseudomonas aeruginosa bacterial suspension was taken and its bacterial count was determined by the plate method. Tryptic soy agar medium was used as the culture medium.
[0103] 2.5 Negative control group: Use 0.9% sterile sodium chloride solution instead of the test sample, and determine the bacterial count according to the test solution preparation method and suitability test counting method, and culture according to the law.
[0104] 2.6 Test Results:
[0105] Table 12 Results of the suitability test for the control bacteria method
[0106] The above results indicate that the control bacteria test for Pseudomonas aeruginosa using this product was conducted using the conventional method with a culture medium volume of 100 mL.
[0107] Based on the above verification results, the microbial limit test method for adapalene peroxybenzoyl gel is as follows:
[0108] Take 10 g of adapalene peroxybenzoyl gel, add 0.9% sterile sodium chloride solution to 90 mL, shake to dissolve, then add 10 mL of 10% sterile calcium chloride solution, shake well, let stand for 5 to 10 minutes to precipitate, the resulting supernatant is the 1:10 test solution, further dilute to 1:50 test solution.
[0109] Total aerobic bacteria count, total mold and yeast count: Take 1 mL of 1:50 test solution, place it in a sterile petri dish, prepare 5 portions in the same way, combine and count them, and test according to the law (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 1105, Petri dish method). It should meet the requirements.
[0110] Staphylococcus aureus and Pseudomonas aeruginosa: Take 10 mL of 1:10 supernatant and inoculate it into 100 mL of tryptic soy peptone liquid medium. Test according to the law (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 1106), and it meets the requirements.
Claims
1. A method for microbial limit testing of adapalene-peroxybenzoyl gel, characterized in that, Includes the following steps: (1) Preparation of test solution: Take adapalene peroxybenzoyl gel, add it to physiological saline to dissolve it completely, add 9.6%~10.4% sterile calcium chloride solution, mix well, let stand, take the supernatant, dilute it and use it as test solution; (2) Take several portions of the above test solution, group them, place them in different sterile petri dishes, pour in culture medium, and incubate after cooling. After incubation, calculate the total number of aerobic bacteria, mold and / or yeast by merging and counting. (3) Take the supernatant and inoculate it into a sterile Petri dish containing culture medium for enrichment culture. After the culture is completed, streak it into agar medium and carry out selective culture. After the culture is completed, count the typical colonies to determine whether it is a growth control bacterium.
2. The method for microbial limit testing of adapalene peroxybenzoyl gel as described in claim 1, characterized in that, The saline solution is a 0.9% sterile sodium chloride solution; The volume ratio of the physiological saline to the sterile calcium chloride solution is 9:
1.
3. The method for microbial limit testing of adapalene peroxybenzoyl gel as described in claim 1, characterized in that, The ratio of the mass of adapalene peroxybenzoyl gel in the test solution to the sum of the volumes of physiological saline and sterile calcium chloride solution is 1:50 g / mL. The settling time is 5 to 10 minutes.
4. The method for microbial limit testing of adapalene peroxybenzoyl gel as described in claim 1, characterized in that, The number of portions is 10, divided into two groups of 5 portions each.
5. The method for microbial limit testing of adapalene peroxybenzoyl gel as described in claim 1, characterized in that, The aerobic bacteria include Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, or Aspergillus niger. The mold and / or yeast are Candida albicans and Aspergillus niger; The controlled bacteria are Staphylococcus aureus and Pseudomonas aeruginosa.
6. The method for microbial limit testing of adapalene peroxybenzoyl gel as described in claim 1, characterized in that, The culture medium mentioned in step (2) is a melted culture medium at a temperature not exceeding 45°C, and the volume ratio of the culture medium to the test solution is (15~20):
1.
7. The method for microbial limit testing of adapalene peroxybenzoyl gel as described in claim 1, characterized in that, In step (2), when the microorganism is an aerobic bacterium, the culture medium is tryptic soy agar medium, and the culture conditions are a temperature of 30-35°C and a culture time of 3-5 days. When the microorganism is a mold and / or yeast, the culture medium is Sabouraud dextrose agar medium, and the culture conditions are a temperature of 20-25°C and a culture time of 5-7 days.
8. The method for microbial limit testing of adapalene peroxybenzoyl gel as described in claim 1, characterized in that, In step (3), when the control bacterium is Staphylococcus aureus, the culture medium is tryptic soy broth, the volume ratio of the supernatant to the culture medium is 1:10, the enrichment culture conditions are a temperature of 30-35℃ and a culture time of 18-24 hours; the agar medium is mannitol sodium chloride agar medium, and the selective culture conditions are a temperature of 30-35℃ and a culture time of 18-72 hours. When the control bacterium is *Pseudomonas aeruginosa*, the culture medium is tryptic soy peptone liquid medium, and the volume ratio of the supernatant to the culture medium is 1:10; the enrichment culture conditions are a temperature of 30-35℃ and a culture time of 18-24 hours; the agar medium is cetyltrimethylammonium bromide agar medium, and the selective culture conditions are a temperature of 30-35℃ and a culture time of 18-72 hours.
9. The method for microbial limit testing of adapalene peroxybenzoyl gel as described in claim 1, characterized in that, The method for calculating the combined count is as follows: the sum of the count results of each plate after incubation in each group is the microbial count result of adapalene peroxyformyl gel.
10. The application of the microbial limit test method according to any one of claims 1-9 in the detection of microbial counts in adapalene peroxybenzoyl gel.