A method for counting microorganisms of sirolimus gel
By using a combination of siromus gel, Tween 80 and calcium chloride in the microbial counting detection of sirolimus gel, the problems of gel bacteriostatic activity and filtration obstruction were solved, and the detection efficiency and accuracy were improved.
Patent Information
- Application Number
- CN202411995245.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-01
AI Technical Summary
Sirolimus gel has the problem of antibacterial activity in microbial counting detection, and the gel structure hinders filtration and uneven sample distribution affects detection efficiency and accuracy.
Add Sipan and Tween 80 to the test solution to prepare a 1:10 test solution, and add 1%-2% calcium chloride to the rinse solution, filter through a membrane and add Tween and β-cyclodextrin to the culture medium for microbial counting.
Effectively reduce the surface tension of the test solution, ensure that the test solution passes through the membrane smoothly, improves detection efficiency, and improves the microbial recovery ratio by optimizing the use of neutralizing agents to ensure the accuracy of the detection results.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to the field of inspection and detection of sirolimus gel, and in particular to a microbial counting method of sirolimus gel. Background Art
[0002] Sirolimus gel, the main ingredient of which is sirolimus, is indicated for the treatment of facial angiofibromas associated with tuberous sclerosis complex in adults and children aged 6 years and above.
[0003] Sirolimus is a macrolide compound. As an immunosuppressant, it has strong antibacterial activity against many fungi and also has a certain inhibitory effect on the growth of some bacteria. This characteristic poses a severe challenge in the process of microbial limit testing, which may lead to the problem of missed microbial detection, thus affecting the accuracy of the test results.
[0004] In addition, in the process of establishing the microbial limit test method and applicability test of gel (gel preparation containing antibacterial ingredients), it was found that the antibacterial property must be eliminated by membrane filtration, and the gel brought new challenges to the membrane filtration method:
[0005] (1) Gel structure hinders filtration: Due to its high molecular weight polymer network structure, the gel can easily clog the filter membrane during the filtration process, resulting in the test solution being unable to pass smoothly, thus affecting the efficiency and accuracy of the test.
[0006] (2) Problem of uneven sample distribution: The physical properties of the gel may lead to uneven distribution of the test solution components, further affecting the recovery ratio of the microorganisms and the reliability of the test results.
[0007] It has been reported in the literature that calcium chloride can effectively chelate carbomer in gelling agents. Most methods involve adding calcium chloride during the preparation of the test solution. However, low concentrations of calcium chloride may not be able to fully neutralize the carbomer in the gelling agent, and the test solution may still have unbroken gel, making it difficult for the test solution to pass through the membrane filter smoothly. High concentrations of calcium chloride may have a direct toxic effect on microorganisms and damage microbial cells.
[0008] Some methods try to lay a layer of diatomaceous earth before filtering. Although the addition of diatomaceous earth does help the test solution to pass through the membrane smoothly, the diatomaceous earth particles remain on the filter membrane to form a thick coating, which greatly increases the difficulty of accurately counting microorganisms and greatly reduces the credibility of the test results.
[0009] Another method is to deal with it through membrane separation. However, this method not only consumes a huge amount of filter membranes and reagents, but also is cumbersome to operate and significantly increases the workload. This has led to a sharp increase in inspection costs and seriously affected work efficiency, making the entire inspection process lengthy and inefficient. Summary of the invention
[0010] The technical problem to be solved by the present invention is to provide a method for counting microorganisms of sirolimus gel, thereby improving the detection efficiency and the detection results.
[0011] The present invention is achieved in that:
[0012] A method for counting microorganisms of sirolimus gel comprises adding Span and Tween 80 to a test solution to prepare a 1:10 test solution, and adding 1%-2% calcium chloride to a flushing solution.
[0013] Furthermore, the preparation method of the test solution is as follows:
[0014] Take 10g of the test sample and add it to a conical flask containing a mixture of 5g Span and 10g Tween 80 with a melting temperature not exceeding 45℃, add glass beads, shake to mix, and slowly add pH 7.0 sterile sodium chloride-peptone buffer with a temperature not exceeding 45℃ to 100ml, mixing while adding to fully emulsify the test sample, and use it as a 1:10 test solution.
[0015] Furthermore, the flushing solution is a 1%-2% calcium chloride solution containing 1% Tween 80.
[0016] Furthermore, during flushing, 1 ml of the test solution was taken at a ratio of 1:10, 100 ml of the flushing solution was added, and the membrane was filtered, and 300 ml / membrane was flushed with the corresponding flushing solution.
[0017] Furthermore, when counting microorganisms, the filter membrane is taken out and placed with the bacterial surface facing upward in a culture medium containing 0.5% Tween 80 and 1%-2% β-cyclodextrin for culture.
[0018] Furthermore, the culture medium is TSA culture medium.
[0019] Furthermore, the microorganisms include Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger.
[0020] The present invention has the following advantages:
[0021] (1) Highly effective surfactant / calcium chloride combination
[0022] The preparation method of the present invention uses two surfactants, Span and Tween 80, to prepare the test solution, which can effectively reduce the surface tension of the test sample and make the test solution easier to dilute and emulsify. After being treated with the surfactant of the present invention, adding low-concentration CaCl2 to the flushing liquid can effectively complex the carbomer component, thereby ensuring that the test solution can be smoothly filmed and improving the detection efficiency.
[0023] (2) Optimization of neutralizer usage
[0024] The present invention does not need to directly add β-cyclodextrin to the test solution, but adds Tween and a certain proportion of β-cyclodextrin to the culture medium to achieve a good antibacterial neutralization effect. This design simplifies the preparation process of the test solution and improves the recovery value of each test bacteria. DETAILED DESCRIPTION
[0025] The technical scheme of the present invention will be described clearly and completely below in conjunction with specific embodiments. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.
[0026] The microbial counting method of sirolimus gel of the present invention has the following specific experimental process: 1. Instruments and materials
[0027] 1.1 Main instruments and equipment
[0028] TX2202L electronic balance (Shimadzu, Japan), HVA-110 high-pressure steam sterilizer (Hirayama, Japan), MSCAdvantage1.8 biological safety cabinet (Thermo Fisher Scientific), HTY-306A filtration system (Zhejiang Tailin) and IQ822C incubator (Yamato, Japan), etc.
[0029] 1.2 Bacterial strains
[0030] Staphylococcus aureus [CMCC(B)26003], Bacillus subtilis [CMCC(B)63501], Pseudomonas aeruginosa [CMCC(B)10104], Candida albicans [CMCC(F)98001], Aspergillus niger
[0031] [CMCC(F)98003] were purchased from China Food and Drug Inspection Institutes and were passaged for the third time.
[0032] 1.3 Culture medium and reagents
[0033] Tryptic soy bean broth (TSA), Sabouraud dextrose agar (SDA) and pH 7.0 sterile sodium chloride-peptone buffer were purchased from Beijing Sanyao Technology Co., Ltd.
[0034] Tween 80, Span, anhydrous calcium chloride and β-cyclodextrin were purchased from Sinopharm Group.
[0035] 1.5 Test sample
[0036] Sirolimus gel.
[0037] 1.6 Testing Basis
[0038] Pharmacopoeia of the People's Republic of China 2020 Edition, Part 4.
[0039] 2 Experimental methods
[0040] 2.1 Effect of calcium chloride diluent on gel breaking of the test product
[0041] Preparation of test solution: Take 10g of the test sample, add different concentrations of calcium chloride solution (5% CaCl2 solution, 10% CaCl2 solution and 20% CaCl2 solution) to 100ml, add glass beads, shake and mix, and use as 1:10 test solution. Take 1ml of 1:10 test solution, add 100ml of flushing solution (pH7.0 sterile sodium chloride-peptone buffer, 0.9% sterile sodium chloride, 0.5% CaCl2 solution, 1% CaCl2 solution and 2% CaCl2 solution), filter through the membrane, flush 300ml / membrane with the corresponding flushing solution, and observe the residual amount of gel on the filter membrane and the filtration speed.
[0042] 2.2 Effect of calcium chloride diluent / calcium chloride rinse on microbial growth
[0043] The membrane filtration method was used to verify the effect of the diluent / rinsing solution on the growth of microorganisms in the experiment. Based on the experimental results of 2.1, the calcium chloride concentration with better gel breaking effect and the rinsing solution with faster filtration speed were selected to further verify the effect of the system on the growth of microorganisms.
[0044] Test group: Take 1 ml of 20% CaCl2 solution, add 100 ml of 1% CaCl2 and 2% CaCl2 respectively, and add 1 ml of bacterial suspension with a bacterial content of no more than 100 cfu, filter with membrane, rinse 300 ml / membrane with corresponding CaCl2 flushing solution, and filter dry. Take out the filter membrane, stick it on TSA with the bacterial surface facing up, and culture it at 30-35℃. The culture time of Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis shall not exceed 3 days, and the culture time of Candida albicans and Aspergillus niger shall not exceed 5 days, and observe the counting results.
[0045] Bacterial solution control group: pH 7.0 sterile sodium chloride-peptone buffer was used instead of calcium chloride diluent and flushing solution. The test bacterial suspension was added in the same manner as the test group and a microbial recovery test was performed.
[0046] Recovery ratio = average colony count of the test group / average colony count of the bacterial solution control group (the recovery ratio of the test bacteria should be between 0.5 and 2).
[0047] 2.3 Effect of Span-Tween-pH7.0 Sterile Sodium Chloride-Peptone Buffer Diluent / Calcium Chloride Rinse on the Gel Breaking of the Test Product
[0048] Preparation of test solution: Take 10g of the test sample and add it to a conical flask containing a mixture of 5g Span and 10g Tween 80 dissolved, add glass beads, shake to mix, slowly add pH 7.0 sterile sodium chloride-peptone buffer at a temperature not exceeding 45°C to 100ml, shake while adding to fully emulsify the test sample, and use it as a 1:10 test solution.
[0049] Take 1 ml of the test solution at a ratio of 1:10, add 100 ml of flushing solution (pH 7.0 sterile sodium chloride-peptone buffer, 0.9% sterile sodium chloride, 1% CaCl2 solution, 2% CaCl2 solution, 3% CaCl2 solution, 1% Tween 80 + 1% CaCl2 solution, 1% Tween 80 + 2% CaCl2 solution and Tween plus 3% calcium chloride), filter through the membrane, rinse 300 ml / membrane with the corresponding flushing solution, and examine the residual amount of gel on the filter membrane and the filtration speed.
[0050] 2.4 Effect of Span-Tween-pH 7.0 Sterile Sodium Chloride-Peptone Buffer Diluent / Calcium Chloride Rinse on Microbial Growth
[0051] Test group: Add glass beads to the conical flask of the dissolved 5g Span and 10g Tween 80 mixture, shake and mix, slowly add pH7.0 sterile sodium chloride-peptone buffer at a temperature not exceeding 45°C to 100ml, shake and set aside. Take 1ml of the above Span-Tween-pH7.0 sterile sodium chloride-peptone buffer dilution mixture, add 100ml 3% CaCl2 solution and 1% Tween 80+2% CaCl2 solution respectively, and add 1ml of bacterial suspension with a bacterial content of no more than 100cfu, filter with a membrane, rinse 300ml / membrane with the corresponding flushing solution, and filter dry. Take out the filter membrane and stick it on TSA or SDA culture medium with the bacterial surface facing up. Stick the filter membrane containing Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis on the TSA plate, culture at 30-35°C, and the culture time shall not exceed 3 days, and observe the counting results. The filter membrane containing Candida albicans and Aspergillus niger was attached to TSA and SDA plates, where TSA plates were cultured at 30-35°C and SDA plates were cultured at 20-25°C. The culture time was no more than 5 days, and the counting results were observed. At the same time, a test sample control group and a bacterial solution control group were prepared, the counting results were observed, and the recovery ratio was calculated.
[0052] Test sample control group: Use pH 7.0 sterile sodium chloride-peptone buffer instead of bacterial solution and operate in the same way as the test group. Incubate at the specified temperature and time, and observe the counting results.
[0053] Bacterial solution control group: pH 7.0 sterile sodium chloride-peptone buffer was used instead of diluent and flushing solution, and the test bacterial suspension was added in the same manner as the test group, and a microbial recovery test was performed.
[0054] Recovery ratio = (average colony count of the test group - average colony count of the test sample control group) / average colony count of the bacterial solution control group (the recovery ratio of the test bacteria should be between 0.5 and 2).
[0055] 2.5 Study on the applicability of the sirolimus gel microbial enumeration method
[0056] Prepare 1:10 test solution of sirolimus gel as described in 2.3. Take 1 ml of the test solution, add 100 ml of 1% Tween 80 + 2% CaCl2 solution, filter through a membrane, rinse 300 ml / membrane with 1% Tween 80 + 2% CaCl2 solution, add 1 ml of bacterial suspension with a bacterial content of no more than 100 cfu to the last rinse, and filter dry. Take out the filter membrane and stick it on different culture medium plates with the bacterial surface facing up. The filter membrane containing Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger was stuck on TSA, TSA containing 0.5% β-cyclodextrin and 0.5% Tween 80, TSA containing 1% β-cyclodextrin and 0.5% Tween 80, and TSA containing 2% β-cyclodextrin and 0.5% Tween 80. The filter membrane containing Candida albicans and Aspergillus niger was attached to SDA, SDA containing 0.5% β-cyclodextrin and 0.5% Tween 80, SDA containing 1% β-cyclodextrin and 0.5% Tween 80, and SDA containing 2% β-cyclodextrin and 0.5% Tween 80. At the same time, a test sample control group and a bacterial solution control group were prepared, the counting results were observed, and the recovery ratio was calculated.
[0057] 3 Results
[0058] 3.1 Effect of calcium chloride diluent on gel breaking of the test product
[0059] As shown in Table 1, the CaCl2 solution can neutralize the carbomer in this sample. However, when the calcium chloride concentration is 5% and 10%, gel blocks still remain on the filter membrane, indicating that the carbomer in the sample is not completely neutralized, which not only affects the filtration speed of the film, but also inhibits the microorganisms due to the antibacterial drugs wrapped in the gel. When using 20% CaCl2 solution, the carbomer in the sample can be neutralized well, and the test solution is prepared evenly. However, when using conventional pH 7.0 sterile sodium chloride-peptone buffer or 0.9% sterile sodium chloride as a flushing solution, the filter membrane is blocked, making it difficult for the test solution to pass through the filter membrane, and thus the test cannot be carried out smoothly. When 0.5% CaCl2 solution is used as a flushing solution, the test solution still has difficulty passing through the filter membrane; when the concentration of CaCl2 solution is increased to 1%, the test solution can pass through the filter membrane smoothly; when the concentration of CaCl2 is increased to 2%, the filtration speed is greatly accelerated.
[0060] Table 1 Effect of calcium chloride diluent on sirolimus gel treatment
[0061]
[0062]
[0063] Note: "-" indicates slow filtration speed, with a flow rate of less than 100 ml in 20 minutes, so the gel residue on the filter membrane could not be observed; "+" indicates filtration speed, the more "+" the faster the speed. The less gel residue on the filter membrane, the better the gel breaking effect.
[0064] 3.2 Effect of calcium chloride diluent / calcium chloride rinse on microbial growth
[0065] The test was conducted using 20% CaCl2 solution as the test diluent, 1% CaCl2 solution and 2% CaCl2 solution as the flushing solution. The results are shown in Table 2. The recovery ratio of Staphylococcus aureus in the five test bacteria was less than 0.5, which did not meet the requirements of the pharmacopoeia. Therefore, this product cannot be tested for microbial count using this diluent and flushing solution system.
[0066] Table 2 Calcium chloride diluent / calcium chloride flushing solution suitability test results
[0067]
[0068] 3.3 Effect of Span-Tween-pH7.0 Sterile Sodium Chloride-Peptone Buffer Diluent / Calcium Chloride Rinse on the Gel Breaking of the Test Product
[0069] Although 20% CaCl2 solution can well neutralize the carbomer in the test sample, it will inhibit the growth of microorganisms. Therefore, the present invention intends to use a combination of Span-Tween-pH7.0 sterile sodium chloride-peptone buffer diluent, and according to the addition method described in 2.4, the sample can be well dispersed and emulsified, and the test solution is uniform and stable.
[0070] When conventional pH7.0 sterile sodium chloride-peptone buffer or 0.9% sterile sodium chloride solution was used as the flushing liquid, the film still had clogging phenomenon. When 1% CaCl2 solution, 2% CaCl2 solution or 3% CaCl2 solution was used as the flushing liquid, the test solution could pass through the film smoothly, indicating that after the gel was treated with a surfactant, adding a low concentration of CaCl2 in the flushing liquid could effectively complex carbomer, thereby promoting the test solution to pass through the film smoothly, especially when 3% CaCl2 solution was used as the flushing liquid, the filtration speed was the fastest. In addition, when 1% Tween 80 was added to the corresponding flushing liquid, the filtration speed was significantly improved, but when 1% Tween 80 was added to the 3% CaCl2 solution, the filtration speed no longer increased, as shown in Table 3.
[0071] Table 3 Effects of different flushing solutions on sirolimus gel treatment
[0072]
[0073] Note: "-" indicates slow filtration speed, with the flow rate less than 100 ml in 20 minutes, so the gel residue on the filter membrane could not be observed; "+" indicates filtration speed, the more "+" the faster the speed.
[0074] 3.4 Effect of Span-Tween-pH 7.0 Sterile Sodium Chloride-Peptone Buffer Diluent / Calcium Chloride Rinse on Microbial Growth
[0075] The diluent of the test sample was a combination of Span-Tween-pH7.0 sterile sodium chloride-peptone buffer solution, and 1% Tween 80+2% CaCl2 and 3% CaCl2 were used as flushing solutions. The results are shown in Table 4. When 3% CaCl2 solution was used as the flushing solution, the recovery ratio of Staphylococcus aureus was less than 0.7. In the process of developing the microbial counting method, we preferred the condition with a recovery ratio greater than 0.7. Therefore, 1% Tween 80+2% CaCl2 was finally selected as the flushing solution. The recovery ratios of the five test bacteria were all between 0.8 and 1.2, which met the requirements of the pharmacopoeia, indicating that the diluent / flushing solution system had no effect on microbial growth.
[0076] Table 4 Test results of suitability of Span-Tween-pH7.0 sterile sodium chloride-peptone buffer diluent / calcium chloride rinse solution
[0077]
[0078]
[0079] 3.5 Study on the applicability of the sirolimus gel microbial enumeration method
[0080] As shown in Table 5, the test strains used in the experiment, including Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Aspergillus niger, had recovery ratios between 0.85 and 1.1 in the provided culture medium, which met the requirements of the pharmacopoeia. In contrast, Candida albicans showed higher sensitivity in evaluating the removal effect of the test system on the antibacterial activity of sirolimus. In standard TSA and SDA culture media, the recovery ratio of Candida albicans was 0, indicating that it was difficult to survive without neutralizing the antibacterial activity of sirolimus. However, when Tween and β-cyclodextrin were added to the culture medium as neutralizers, the recovery ratio was significantly improved. Under the conditions of TSA and SDA with the addition of 0.5% β-cyclodextrin and 0.5% Tween 80, the recovery ratios of Candida albicans were 0.49 and 0.45, respectively. Although improved, they still did not meet the requirements of the pharmacopoeia.
[0081] To further optimize the culture conditions, the present invention keeps the addition amount of 0.5% Tween 80 unchanged and increases the concentration of β-cyclodextrin to 1% and 2% respectively. This optimization strategy significantly improves the recovery ratio of Candida albicans to a range of 0.88-0.96, which is fully in line with the provisions of the pharmacopoeia. This optimization provides a strong scientific basis and technical support for β-cyclodextrin as an antibacterial neutralizer.
[0082] Table 5 Results of recovery ratio of test bacteria in different culture media
[0083]
[0084]
[0085] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.
Claims
1. A method for enumerating microorganisms of sirolimus gel, characterized in that: Add Span and Tween 80 to the test solution to prepare a 1:10 test solution, and add 1%-2% calcium chloride to the rinse solution.
2. The method for microbial counting of sirolimus gel according to claim 1, characterized in that: The preparation method of the test solution is as follows: Take 10g of the test sample and add it to a conical flask containing a mixture of 5g Span and 10g Tween 80 with a melting temperature not exceeding 45℃, add glass beads, shake to mix, and slowly add pH 7.0 sterile sodium chloride-peptone buffer with a temperature not exceeding 45℃ to 100ml, mixing while adding to fully emulsify the test sample, and use it as a 1:10 test solution.
3. The method for microbial counting of sirolimus gel according to claim 1, characterized in that: The flushing solution is a 1%-2% calcium chloride solution containing 1% Tween 80.
4. The method for microbial counting of sirolimus gel according to claim 1, characterized in that: When flushing, take 1 ml of the test solution at a ratio of 1:10, add 100 ml of the flushing solution, filter through the membrane, and flush 300 ml / membrane with the corresponding flushing solution.
5. The method for microbial counting of sirolimus gel according to claim 1, characterized in that: When counting microorganisms, remove the filter membrane and place it with the bacterial surface facing up in a culture medium containing 0.5% Tween 80 and 1%-2% β-cyclodextrin for culture.
6. The method for microbial counting of sirolimus gel according to claim 1, characterized in that: The culture medium is TSA culture medium.
7. The method for microbial counting of sirolimus gel according to claim 1, characterized in that: The microorganisms include Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger.