Method for detecting total number of mould and saccharomycetes of amphotericin B

Abstract

The invention discloses a method for detecting the total number of mould and saccharomycetes of amphotericin B. The method comprises the following steps: (1) preparing mould and / or saccharomycetes to be detected into a bacterium suspension; (2) adding a sulfydryl-containing neutralizer into the SDA culture medium to obtain a neutralizer-containing SDA culture medium; (3) weighing an amphotericin B sample, and diluting the amphotericin B sample with a buffer solution to obtain a test solution; and (4) adding a test solution into the bacterial suspension, uniformly mixing, injecting into a neutralizer-containing SDA culture medium, culturing, and counting to obtain the total number of mould and / or saccharomycetes of amphotericin B. According to the method, a technology combining a dilution method and a neutralization method is adopted, a test sample is diluted, a sulfydryl-containing neutralizer is added into an SDA culture medium, amphotericin B molecules are effectively wrapped, and contact between the amphotericin B molecules and fungal microorganism cell membranes is reduced, so that the bacteriostatic activity of the amphotericin B molecules is eliminated, the inhibition effect of the amphotericin B on sensitive fungi is solved, the recovery rate is effectively increased, and the method is suitable for industrial production. And accurate microbial limit detection is realized.

Description

Technical Field

[0001] The present invention relates to a method for detecting the microbial limit of a drug, in particular to a method for detecting the total number of molds and yeasts of amphotericin B. Background Art

[0002] Amphotericin B (AmB), a broad-spectrum antifungal agent, presents challenges in microbial limit testing due to its inherently potent inhibitory activity against test bacteria (such as Candida albicans and Aspergillus niger). Conventional methods, including plate assays and membrane filtration, fail to effectively neutralize its activity during enumeration method suitability testing, resulting in recoveries below the pharmacopoeial requirement of 0.5-2. Currently available neutralizing agents, such as polysorbate 80 and lecithin, have limited efficacy against amphotericin B and fail to meet the validation requirements of existing methodologies. Therefore, the development of novel neutralization or inhibition strategies for the microbial testing of amphotericin B is needed. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for detecting the total number of molds and yeasts of amphotericin B with a high recovery rate.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention includes the following steps: (1) preparing the mold and / or yeast to be detected into a bacterial suspension; (2) adding a thiol-containing neutralizer to the SDA medium to obtain a neutralizer-containing SDA medium; (3) Weigh the amphotericin B sample and dilute it with buffer to obtain the test solution; (4) Add the test solution to the bacterial suspension, mix well, and then inject it into the neutralizer SDA culture medium. Culture and count to obtain the total number of amphotericin B-resistant molds and / or yeasts.

[0005] Furthermore, in step (2), the thiol neutralizing agent is selected from one or more of glutathione, thioglycolic acid, thiophenol, cysteine, sodium thioglycolate, cystine, mercaptoethanol, bovine serum albumin and homocysteine.

[0006] Furthermore, in step (2), the concentration of the thiol neutralizer in the neutralizer-containing SDA culture medium is 0.05-0.3%.

[0007] Furthermore, in step (3), the solution is diluted to 1:50 using pH 7.0 sodium chloride-peptone buffer.

[0008] Furthermore, in step (1), the mold is Aspergillus niger and the yeast is Candida albicans.

[0009] Furthermore, the Aspergillus niger is prepared into a bacterial suspension using a 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80.

[0010] Furthermore, the Candida albicans is prepared into a bacterial suspension using 0.9% sterile sodium chloride solution.

[0011] The beneficial effects of the above-mentioned technical solution are as follows: To address the problem that the strong antifungal activity of amphotericin B can lead to substandard recoveries of Candida albicans and Aspergillus niger in conventional limit tests, the present invention proposes a combined dilution and neutralization method. By adding a thiol-containing neutralizer to Sabouraud dextrose agar (SDA) medium after diluting amphotericin B, the amphotericin B molecules are effectively encapsulated through hydrophobic interactions and disulfide bonds, reducing contact with fungal cell membranes and eliminating their antibacterial activity. This addresses the inhibitory effect of amphotericin B on sensitive fungi, effectively improving the recovery rate and enabling accurate microbial limit tests. This method has been demonstrated to increase the recovery rate of Candida albicans and Aspergillus niger to 0.9, meeting the Chinese Pharmacopoeia requirement of 0.5-2. It is particularly suitable for the detection of total mold and yeast counts using amphotericin B. This method addresses the problem of false negatives in microbial limit tests for antifungal drugs and provides a reference for the testing of similar drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0013] Figure 1 1 is a flow chart of the detection method of the present invention; Figure 2 Schematic diagram of the mechanism of action of the sulfhydryl-containing neutralizer of the present invention; Figure 3 This is a photo of the Aspergillus niger counting plate described in Example 1; Figure 4 This is a photo of the Candida albicans counting plate described in Example 1. DETAILED DESCRIPTION

[0014] The method for detecting the total number of molds and yeasts of amphotericin B in the present invention comprises diluting the test sample with a buffer, adding a sulfhydryl-containing neutralizer to a Sabouraud dextrose agar medium, and counting the total number of molds and yeasts by the pouring method; Figure 1 As shown, the following steps are included: (1) Culture and preparation of bacterial strains: The yeast used is Candida albicans, and the mold used is Aspergillus niger. Both Candida albicans and Aspergillus niger are from the China Food and Drug Administration, with the Candida albicans accession number CMCC(F)98001 and the Aspergillus niger accession number CMCC(F)98003. Both Candida albicans and Aspergillus niger are cultured on SDA (Sabouraud dextrose agar) at a temperature of 20-25°C, preferably 23°C. Candida albicans is cultured for 2-3 days, preferably 2 days; Aspergillus niger is cultured for 5-7 days, preferably 7 days.

[0015] The Candida albicans is prepared into a bacterial suspension using 0.9% (g / mL) sterile sodium chloride solution, with no more than 10 4 The Aspergillus niger was prepared into a bacterial suspension using a 0.9% (g / mL) sterile sodium chloride solution containing 0.05 vol% polysorbate 80, with no more than 10 4 Aspergillus niger.

[0016] (2) Culture medium preparation: A thiol-containing neutralizer is added to the SDA culture medium, wherein the concentration of the thiol-containing neutralizer is 0.05-0.3% (g / mL), preferably 0.1%, to obtain a neutralizer-containing SDA culture medium. Figure 2 As shown, the sulfhydryl-containing neutralizing agent functions to load amphotericin B molecules through hydrophobic interactions and hydrogen bonds, thereby reducing their contact with microbial cell membranes. The sulfhydryl-containing neutralizing agent is selected from one or more of glutathione, thioglycolic acid, thiophenol, cysteine, sodium thioglycolate, cystine, mercaptoethanol, bovine serum albumin, and homocysteine.

[0017] (3) Preparation of test solution: Weigh amphotericin B and dilute it to 1:50 (g / mL) with buffer to prepare the test solution. It is best to first dilute to 1:10 with buffer and then to 1:50 with buffer. The buffer is pH 7.0 sodium chloride-peptone buffer.

[0018] (4) Solution preparation: The test solution of step (3) was added to the bacterial suspension of step (1) at a volume ratio of 9.9:0.1, and the mixture was mixed to obtain a test bacterial solution. The bacterial content of the test bacterial solution was no more than 100 cfu / mL.

[0019] In order to verify the detection effect, the test bacterial solution was used as the experimental group, and the bacterial solution control solution, the test sample control solution and the negative control solution were prepared as the control group.

[0020] The test sample control solution: take the test solution described in step (3) and add it to 0.9% sterile sodium chloride solution, test solution: sodium chloride solution = 9.9: 0.1 (volume ratio), mix well, and obtain the test sample control solution.

[0021] The bacterial suspension control solution: pH 7.0 sodium chloride-peptone buffer was added to the bacterial suspension in step (1), with the buffer: bacterial suspension ratio being 9.9:0.1 (volume ratio), and mixed to obtain a bacterial suspension control solution.

[0022] The negative control solution was prepared by adding pH 7.0 sodium chloride-peptone buffer to 0.9% sterile sodium chloride solution at a volume ratio of 9.9:0.1. The mixture was mixed to obtain a negative control solution.

[0023] (5) Recovery and cultivation of microorganisms in test samples: The test bacterial solution prepared in step (4) is placed in a sterile plate with a diameter of 90 mm, preferably a sterile plate with a diameter of 90 mm. The neutralizing SDA medium prepared in step (2) is injected at a volume ratio of 1:(15-20) of test bacterial solution to neutralizing SDA medium. The temperature of the injected neutralizing SDA medium does not exceed 45°C. The test bacterial culture medium is mixed, solidified, inverted, and cultured at 20-25°C for no more than 5 days to obtain the test bacterial culture medium. At least two plates are prepared for each test bacterial strain, and the arithmetic mean is subsequently used as the counting result.

[0024] The bacterial solution control solution, the test sample control solution and the negative control solution were used to replace the test bacterial solution respectively, and cultured according to the above process to obtain the control culture medium of each control group.

[0025] (6) Calculation of average colony count: The total colony count of Candida albicans and / or Aspergillus niger was counted on the test culture medium on the plate by the pouring method. The arithmetic mean was used as the counting result to obtain the total colony count of Candida albicans and / or Aspergillus niger in the presence of amphotericin B.

[0026] The total number of Candida albicans and / or Aspergillus niger in the control culture medium of each control group was counted by the pouring method to obtain the total number of Candida albicans and / or Aspergillus niger in each control group.

[0027] (7) Result evaluation: The recovery rate was calculated using the following method: Recovery rate = (number of colonies in the test group - number of colonies in the test sample control) / number of colonies in the bacterial solution control. The recovery rates of Candida albicans and molds described in this method can reach 0.60-0.90.

[0028] Example 1-6: The method for detecting the total number of molds and yeasts of amphotericin B in the present invention is specifically described as follows.

[0029] (1) Culture and preparation of bacterial strains: Candida albicans and Aspergillus niger were cultured separately. The culture medium used was Sabouraud dextrose agar medium and the culture temperature was 23°C. Candida albicans was cultured for 2 days and Aspergillus niger was cultured for 7 days.

[0030] After culture, the Candida albicans was prepared into bacterial suspension with 0.9% sterile sodium chloride solution. No more than 10 4 After culture, Aspergillus niger was suspended in 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80, with no more than 10 4 Aspergillus niger.

[0031] (2) Culture medium preparation: Add a thiol-containing neutralizing agent to the SDA culture medium, and then sterilize at 121°C for 15 minutes to obtain the neutralizing agent-containing SDA culture medium. The thiol-containing neutralizing agents added in each example are shown in Table 1; Table 1: The amount of sulfhydryl-containing neutralizing agent added in each embodiment (g / ml)

[0032] (3) Preparation of test solution: Weigh 10 g of amphotericin B and prepare a 1:10 (W:V) test intermediate solution with a pH 7.0 sodium chloride-peptone buffer. Then, dilute 10 mL of this 1:10 (W:V) test intermediate solution with 90 mL of buffer to make a 1:50 (W:V) test solution.

[0033] (4) Solution preparation: Prepare solutions for the test group, bacterial solution control group, test sample control group, and negative control group. For the test bacterial solution of the test group: take 9.9 mL of the test solution, add 0.1 mL of bacterial suspension, and mix well so that the bacterial count per 1 mL of the test bacterial solution is no more than 100 cfu. For the test solution control solution of the test solution control group: take the test solution and replace the bacterial suspension with 0.9% sterile sodium chloride solution, and the rest of the operation is the same as the test group. For the bacterial solution control solution of the bacterial solution control group: take pH 7.0 sodium chloride-peptone buffer solution and replace the test solution, and the rest of the operation is the same as the test group. For the negative control solution of the negative control group: take pH 7.0 sodium chloride-peptone buffer solution and replace the test solution with 0.9% sterile sodium chloride solution, and the rest of the operation is the same as the test group.

[0034] (5) Recovery and culture of microorganisms in the test sample: Take 1 ml of each solution prepared in step (4) above and place it in a sterile plate with a diameter of 90 mm. Add 20 ml of Sabouraud dextrose agar medium containing a thiol neutralizer at a temperature not exceeding 45°C, mix well, solidify, invert and culture at 23°C for no more than 5 days. Prepare two plates for each test strain.

[0035] (6) Calculate the average colony count: Figure 3 、 Figure 4 As shown, the total colony counts of Candida albicans and Aspergillus niger were performed on the test bacteria culture medium on the plate using the pouring method, and the arithmetic mean was used as the counting result to obtain the total colony counts of Candida albicans and Aspergillus niger under amphotericin B. The results are shown in Table 2; Table 2: Average colony counts for each example

[0036] As shown in Table 2, this method can effectively eliminate the antibacterial activity of amphotericin B by combining the dilution method with a neutralizer, meet the requirements of the microbial limit test of the Chinese Pharmacopoeia, and is suitable for industrial quality control.

Claims

1. A method for detecting the total number of molds and yeasts of amphotericin B, characterized in that: The method comprises the following steps: (1) preparing a bacterial suspension of the mold and / or yeast to be tested; (2) adding a thiol-containing neutralizer to the SDA medium to obtain a neutralizer-containing SDA medium; (3) Weigh the amphotericin B sample and dilute it with buffer to obtain the test solution; (4) Add the test solution to the bacterial suspension, mix well, and then inject it into the neutralizer SDA culture medium. Culture and count to obtain the total number of amphotericin B-resistant molds and / or yeasts.

2. The method for detecting the total number of molds and yeasts of amphotericin B according to claim 1, characterized in that: In the step (2), the thiol neutralizing agent is selected from one or more of glutathione, thioglycolic acid, thiophenol, cysteine, sodium thioglycolate, cystine, mercaptoethanol, bovine serum albumin and homocysteine.

3. The method for detecting the total number of molds and yeasts of amphotericin B according to claim 1, characterized in that: In the step (2), the concentration of the thiol neutralizer in the neutralizer-containing SDA culture medium is 0.05-0.3%.

4. The method for detecting the total number of molds and yeasts of amphotericin B according to claim 1, wherein: In step (3), the solution is diluted to 1:50 using pH 7.0 sodium chloride-peptone buffer.

5. The method for detecting the total number of molds and yeasts of amphotericin B according to any one of claims 1 to 4, characterized in that: In the step (1), the mold is Aspergillus niger and the yeast is Candida albicans.

6. The method for detecting the total number of molds and yeasts of amphotericin B according to claim 5, characterized in that: The Aspergillus niger is prepared into a bacterial suspension using a 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80.

7. The method for detecting the total number of molds and yeasts of amphotericin B according to claim 5, characterized in that: The Candida albicans was prepared into a bacterial suspension using 0.9% sterile sodium chloride solution.

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