A method for microbial limit test of mulberry bark formula granules

Abstract

The application provides a microbial limit test method of mulberry bark formula granules, adopts a membrane filtration method, takes pH 7.0 sodium chloride proteose peptone buffer solution as a basic diluent, adds Tween 80 and soybean lecithin, is sterilized by high pressure, is cooled, and then sterile bovine serum albumin freeze-dried powder is added before use, is shaken to prepare a diluent; and the pancreas tyrosine soybean peptone agar medium and the sand sugar glucose agar medium both are previously added with Tween 80 and soybean lecithin, are sterilized by high pressure and cooled, and then the sterile bovine serum albumin freeze-dried powder is added, so that the two kinds of culture media become two kinds of improved novel culture media with neutralizing activity. The microbial limit test method of the mulberry bark formula granules is stable, can realize qualified recovery rate of test bacteria, smooth filtration without membrane blockage, completely eliminates bacteriostatic interference, the result is accurate and reliable, and has good repeatability and practicability.

Description

Technical Field

[0001] This invention belongs to the field of quality testing technology for traditional Chinese medicine preparations, and in particular relates to a method for microbial limit testing of mulberry bark granules. Background Technology

[0002] Mulberry bark (Sangbaipi) is the dried root bark of *Morus alba* L., a plant in the Moraceae family. It is a commonly used traditional Chinese medicine with effects such as purging the lungs and relieving asthma, and promoting diuresis and reducing swelling. Its main chemical components include stilbene glycosides, isopentenyl flavonoids, polyphenols, tannins, polysaccharides, mucilage, and pectin. Among these, isopentenyl flavonoids are the core antibacterial component. The synergistic effect of other components, including mulberry bark extract, polyphenols, and tannins, provides broad-spectrum antibacterial activity. Furthermore, mulberry bark extract exhibits antibacterial effects against certain bacteria that are close to those of clinical antibiotics. These components are particularly effective against Gram-positive bacteria such as *Staphylococcus aureus* and *Bacillus subtilis*. The mechanism of action involves disrupting the integrity of the bacterial cell membrane and inhibiting cell wall and nucleic acid synthesis to produce antibacterial effects. Mulberry bark granules are granules prepared from properly processed mulberry bark slices. During testing, the antibacterial components inhibit the growth of these microorganisms, leading to low recovery rates, false negatives, and inaccurate results. Furthermore, mulberry bark granules contain a high amount of insoluble components such as starch, pectin, and mucilage, which easily clogs the filter membrane during direct filtration, making filtration difficult and affecting testing efficiency and reliability. Existing technologies struggle to simultaneously address the issues of strong antibacterial interference, membrane clogging, and substandard recovery rates. Therefore, a highly efficient and stable method specifically designed for mulberry bark granules is needed for the detection of Escherichia coli in microbial limit testing and control bacteria testing.

[0003] When conducting microbial limit tests according to the general rules of the Chinese Pharmacopoeia, the existing technology has the following shortcomings: 1. Mulberry bark has strong antibacterial properties, and the conventional plate method cannot eliminate the antibacterial effect. The recovery rate of test bacteria such as Bacillus subtilis and Staphylococcus aureus is extremely low, and false negatives are easy to occur.

[0004] 2. The neutralizing agent's ingredients or ratios are not properly used, making it impossible to completely neutralize the antibacterial components.

[0005] 3. Bovine serum albumin (BSA) is not heat-resistant and is easily inactivated when autoclaved with culture medium, thus losing its protective effect against microorganisms.

[0006] 4. Adding a neutralizing agent to the rinsing solution of the membrane filtration method can easily clog the filter membrane, causing the experiment to be unable to proceed or causing residual interference.

[0007] 5. There is a lack of novel culture media with neutralizing properties.

[0008] Therefore, establishing a microbial limit test method for mulberry bark formulation granules that are fully neutralized, non-toxic, have a stable recovery rate, and meet pharmacopoeia requirements is of great significance for ensuring the safety of clinical drug use. Summary of the Invention

[0009] The purpose of this invention is to provide a method for microbial limit testing of mulberry bark formula granules. The method has clear steps, well-defined parameters, and standardized operation. Those skilled in the art can repeat the method according to the instructions. It can reliably achieve qualified recovery rate of test bacteria, smooth filtration without membrane clogging, complete elimination of antibacterial interference, and accurate and reliable results. It has good repeatability and practicality, and can simultaneously meet the testing requirements for total aerobic bacteria, total mold and yeast count, and Escherichia coli in control bacteria testing of mulberry bark formula granules.

[0010] To achieve the above objectives, the present invention provides a method for microbial limit testing of mulberry bark formulation granules, comprising the following steps: Step 1: Preparation of diluent: Based on pH 7.0 sodium chloride peptone buffer, add Tween 80 and soybean lecithin, stir and disperse, then autoclave, cool and keep warm. Before use, add sterile bovine serum albumin lyophilized powder under aseptic conditions, shake and mix well to obtain the diluent. Step 2: Prepare the test solution: Take the mulberry bark formula granule sample, add the diluent prepared in step 1, shake to prepare a 1:10 test solution, and then use sterile nylon filters with different pore sizes for primary coarse filtration and secondary filtration in sequence. Collect the filtrate for later use. Step 3: Use membrane filtration to count the total number of aerobic bacteria, molds, and yeasts; Step 4, Control Bacterial Examination: Take the 1:10 test solution prepared in Step 2 and filter and rinse it with pH 7.0 sodium chloride peptone buffer. After rinsing, place the filter membrane in tryptic soy peptone liquid medium and incubate. Take the culture and inoculate it into MacConkey liquid medium and incubate. Then, perform separation, purification and biochemical identification according to the 2025 edition of the Chinese Pharmacopoeia Escherichia coli test method to determine whether the bacterial species are detected.

[0011] Preferably, in step 1, the high-pressure steam sterilization temperature is 121°C and the time is 15 min; then it is cooled to about 40-45°C.

[0012] Preferably, the ratio of mulberry bark granules to diluent is 1g:9mL.

[0013] The pore size is 80-120μm during primary coarse filtration and 10-20μm during secondary filtration.

[0014] Preferably, Tween 80 accounts for 0.5% (w / v) of the diluent; The proportion of soybean lecithin in the diluted solution was 0.07% (w / v); The proportion of sterile bovine serum albumin lyophilized powder in the diluent was 0.1% (w / v).

[0015] Preferably, in step 4, the culture temperature and time are the same as those for Escherichia coli in the 2025 edition of the Chinese Pharmacopoeia; The filter was rinsed using pH 7.0 sodium chloride peptone buffer, with each filter membrane being rinsed 100 mL each time, for a total rinsing volume of 400 mL.

[0016] Preferably, the membrane filtration method is used to count the total number of aerobic bacteria, molds, and yeasts, specifically including the following steps: Step 31: Take the test solution obtained in step 2, add the rinsing solution, mix well and filter; the rinsing solution is pH 7.0 sodium chloride peptone buffer. Step 32: Rinse the filter membrane several times with rinsing solution; Step 33: Remove the filter membrane and attach the front side to a modified tryptic soy peptone agar plate or a modified Sabouraud dextrose agar plate; Step 34, Total aerobic bacteria count: Incubate the modified tryptic soybean agar plates at 30-35℃ for 3-5 days; Step 35, Total count of molds and yeasts: Incubate modified Sabouraud dextrose agar plates at 23-28°C for 5-7 days.

[0017] Preferably, the preparation method of the modified tryptic soy peptone agar and modified Sabouraud dextrose agar in step 3 is as follows: Add 0.5% (w / v) Tween 80 and 0.07% (w / v) soybean lecithin to the basal culture medium, stir and disperse thoroughly, autoclave at 121℃ for 15 min, cool to 45-48℃ and keep warm. Before use, add sterile bovine serum albumin lyophilized powder to a final concentration of 0.1% (w / v) under aseptic conditions, mix well and pour into petri dishes.

[0018] Preferably, the rinsing solution described in steps 3 and 4 is a pH 7.0 sodium chloride peptone buffer solution without the addition of a neutralizing agent.

[0019] Preferably, the method further includes a bacterial suspension preparation step, wherein the bacterial suspension is prepared using Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger with a passage number not exceeding four, for use in method suitability testing.

[0020] Therefore, the present invention employs the above-mentioned method for microbial limit testing of mulberry bark formulation granules, and the technical effects are as follows: It was found that mulberry bark has a strong inhibitory effect on Bacillus subtilis and Staphylococcus aureus, and its flavonoid and phenolic acid natural antibacterial components are the root cause of the low recovery rate and false negatives in routine microbial limit tests. This study fills the gap in the specific antibacterial interference mechanism and detection adaptation technology of mulberry bark.

[0021] Breaking away from the industry's conventional practice of using only ordinary blank culture media, and no longer simply adding neutralizing agents to diluents or rinsing solutions, this invention creatively adds quantitative amounts of 0.5% (w / v) Tween 80 and 0.07% (w / v) soybean lecithin to TSA and SDA basal media. After sterilization and cooling, sterile bovine serum albumin (BSA) lyophilized powder is aseptically added to achieve a final concentration of 0.1% (w / v). This upgrades the traditional ordinary culture medium, which only has a culture function, into a novel functional culture medium that combines microbial culture, neutralizes the strong antibacterial interference of mulberry bark, and protects the growth of bacterial strains. It solves the problems of antibacterial inhibition and low bacterial counts from the root of the culture system, representing a dual original improvement in both formulation and function.

[0022] A dual neutralization system was adopted, consisting of a ternary compound neutralizing agent of 0.5% Tween 80, 0.07% soybean lecithin, and 0.1% BSA. This ternary neutralizing agent was added to both the diluent and the culture medium used for cultivation. This system targeted the antibacterial effect of mulberry bark, ensuring that the recovery rate of the test bacteria was stable and met the pharmacopoeia requirements of 50% to 200%. The neutralization was thorough and non-toxic to the strain.

[0023] To address the issue of BSA's inability to withstand high temperatures and its tendency to become inactive, an innovative process was adopted: Tween 80, lecithin, diluent, and culture medium were sterilized together under high pressure, and BSA was added later under low temperature aseptic conditions. This process maximizes the preservation of BSA's biological activity and its protective and neutralizing effects, avoiding the drawbacks of traditional methods that sterilize all components together, thus preventing inactivation.

[0024] Breaking away from the industry's conventional practice of adding neutralizing agents to rinsing solutions, this study employs a pure pH 7.0 sodium chloride peptone buffer solution without adding neutralizing agents, thus avoiding membrane clogging and filtration difficulties. It has been proven that the conventional plate method is completely unsuitable, and a solution has been established that combines diluent, ternary neutralizing agent, modified TSA / SDA culture medium, and membrane filtration as a dedicated detection method for mulberry bark. This complete method system is comprehensive, rigorous, and practical, demonstrating outstanding novelty and creativity. Detailed Implementation

[0025] The technical solution of the present invention will be further described below through embodiments.

[0026] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0027] All test strains were purchased from the National Institutes for Food and Drug Control, and the strain numbers are as follows: Pseudomonas aeruginosa (CMCC(B)10104); Staphylococcus aureus (CMCC(B)26003); Bacillus subtilis (CMCC(B)63501); Candida albicans (CMCC(F)98001); Aspergillus niger (CMCC(F)98003); Escherichia coli Escherichiacdi (CMCC(B)44102).

[0028] Electronic balance, homogenizer, constant temperature water bath, biosafety cabinet, sterile ultra-clean workbench, vertical pressure steam sterilizer, electric constant temperature incubator, mold incubator, membrane filter, sterile filter membrane (pore size 0.45μm), sterile nylon filter screen (pore size 80-120μm and 10-20μm).

[0029] Mulberry bark formula granules (produced and supplied by Wanxi Pharmaceutical Co., Ltd., and certified to meet the standards of the Chinese Pharmacopoeia), pH 7.0 sterile sodium chloride-peptone buffer, tryptic soy agar medium, Sabouraud dextrose agar medium, tryptic soy liquid medium, soybean lecithin, Tween 80, and sterile bovine serum albumin (BSA) lyophilized powder.

[0030] Example 1: Microbial limit test of conventional mulberry bark formula granules (membrane filtration method without neutralizing agent) 1. Preparation of bacterial culture 1.1 Take a small amount of fresh culture of Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis (passed no more than 4 times) and inoculate it into tryptic soy broth. Incubate at 35°C for 18-24 hours. Then, take one loopful of each culture and add it to 10 ml of pH 7.0 sterile sodium chloride-peptone buffer. Grind thoroughly and mix well. Perform a 10-fold serial dilution to prepare a 10% concentration. 3 -10 4 cfu / ml, 10 2 -10 3 CFU / ml bacterial suspension.

[0031] 1.2. Take a small amount of Candida albicans, passaged no more than 4 times, and inoculate it onto Sabouraud dextrose agar medium. Incubate at 25°C for 2 days. Take one loopful and add it to 10 ml of pH 7.0 sterile sodium chloride-peptone buffer. Grind thoroughly and mix well. Perform a 10-fold serial dilution to prepare a 10% concentration.3 -10 4 cfu / ml, 10 2 -10 3 CFU / ml bacterial suspension.

[0032] 1.3. Take a small amount of *Aspergillus niger* culture that has been passaged no more than four times and inoculate it onto Sabouraud dextrose agar medium. Incubate at 25°C for 5 days. Take this fresh *Aspergillus niger* culture and add an appropriate amount of sterile sodium chloride-peptone buffer (pH 7.0) containing 0.05% Tween 80. Elute the spores and aspirate the spore suspension into a sterile test tube. Serially dilute the suspension 10-fold with sterile sodium chloride-peptone buffer (pH 7.0) containing 0.05% Tween 80 to prepare a 10% concentration. 3 -10 4 cfu / ml, 10 2 -10 3 CFU / ml Aspergillus niger spore suspension.

[0033] 2. Preparation of the test solution Take 10g of the mulberry bark formula granules as the test sample, dilute it with pH 7.0 sodium chloride-peptone buffer (without neutralizing agent) and stir well to prepare a 1:10 test solution. Pass the 1:10 test solution through a sterile nylon filter for primary and secondary coarse filtration (first use a sterile nylon filter with a pore size of 80-120μm for primary filtration, and then use a sterile nylon filter with a pore size of 10-20μm for secondary filtration) to remove insoluble large particles such as starch. Collect the filtrate for later use.

[0034] 3. For the experimental group, take 1 ml of the prepared test solution, filter and rinse. The rinsing solution is pH 7.0 sodium chloride-peptone buffer. Rinse each filter membrane 5 times, 100 ml each time, for a total rinsing volume of 500 ml. Add 0.1 ml of a 10% solution during the last rinse. 2 -10 3 The above bacterial suspension, with a concentration of CFU / ml, included Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger. The filter membrane was then removed, bacterial side up. Aerobic bacteria were agar-agar (without neutralizing agent) and incubated at 35°C for 3 days using the following five strains: Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger. Molds were agar-agar (without neutralizing agent) and incubated at 25°C for 5 days using the following two strains: Candida albicans and Aspergillus niger. The bacterial count was determined using the membrane filtration method.

[0035] 4. For the bacterial culture control group, use sterile sodium chloride-peptone buffer at pH 7.0 instead of the 1:10 test solution and follow the same procedure as the experimental group.

[0036] 5. For the control group of the test sample, take 1 ml of the test solution at a 1:10 ratio, and follow the same procedure as the test group, without adding bacteria.

[0037] 6. Negative control group: Aseptically aspirate 1 ml of pH 7.0 sterile sodium chloride-peptone buffer solution, and perform the same procedure as the experimental group, without adding any bacteria.

[0038] 7. Recovery rate calculation: Recovery rate = (number of colonies in the test group - number of colonies in the control group) / number of colonies in the control group.

[0039] 8. The test results are shown in Table 1.

[0040] Table 1. Test results of Example 1

[0041] The results show that no colonies grew in the negative control. For the five strains tested according to the conventional membrane filtration method (without neutralizing agent) in the 2025 edition of the Chinese Pharmacopoeia, even with membrane filtration and the use of the maximum amount of rinsing solution to eliminate the antimicrobial components affecting microbial growth in the mulberry bark formulation granules, the results still indicate that the antimicrobial active components in mulberry bark still significantly inhibited the growth of Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis. Therefore, in subsequent screening, this invention improved the microbial limit detection method for mulberry bark formulation granules by targeting Staphylococcus aureus and Bacillus subtilis as the target strains.

[0042] Example 2: Screening of neutralization system for microbial limit detection of mulberry bark formula granules The experimental method was the same as in Example 1, except that the preparation of the test solution and the composition and ratio of the neutralizing agent added to the culture medium used for film application were different. Furthermore, to facilitate screening, only Staphylococcus aureus and Bacillus subtilis, which showed poor recovery rates using conventional methods, were selected as challenge microorganisms. Different groups were set up, and the composition and ratio of the neutralizing agent added to the buffer and culture medium differed for each group, as detailed below: Group 1: Sterile sodium chloride-peptone buffer at pH 7.0 containing 1% (w / v) Tween 80; tryptic soy agar (TSA) and sarcodone agar (SDA), without neutralizing agents.

[0043] Group 2: pH 7.0 sterile sodium chloride-peptone buffer containing 0.2% (w / v) soybean lecithin; tryptic soy peptone agar (TSA) and sarcodone agar (SDA), without neutralizing agents.

[0044] Group 3: Sterile sodium chloride-peptone buffer at pH 7.0 containing 0.5% (w / v) Tween 80 and 0.07% (w / v) soybean lecithin as neutralizing agents; tryptic soy peptone agar (TSA) and sarcodone agar (SDA) media, without any added neutralizing agents.

[0045] Group 4: pH 7.0 sterile sodium chloride-peptone buffer containing 0.5% (w / v) Tween 80, 0.07% (w / v) soybean lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder as neutralizing agents; tryptic soy peptone agar (TSA) and sarcodactyl dextrose agar (SDA) media, without any added neutralizing agents.

[0046] Group 5: pH 7.0 sterile sodium chloride-peptone buffer containing 0.5% (w / v) Tween 80, 0.07% (w / v) soy lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder as neutralizing agents; modified novel tryptic soy peptone agar medium (modified TSA) and modified novel Sabouraud dextrose agar medium (modified SDA) containing 0.5% (w / v) Tween 80, 0.07% (w / v) soy lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder as neutralizing agents.

[0047] The test solutions were prepared using the buffer solutions described above according to the method in Example 1, and then the tests were performed and the recovery rates were calculated. The specific results are as follows: Table 2 Recovery rate of Example 2

[0048] The results above show that simply using a neutralizing agent or just using Tween 80 and lecithin together has little effect on the recovery rate of microorganisms in mulberry bark formulation granules and cannot effectively eliminate the antibacterial activity of mulberry bark formulation granules. Only by selecting the ternary dual neutralization system of pH 7.0 sterile sodium chloride-peptone buffer containing this neutralizing agent, modified novel tryptic soybean agar medium (modified TSA) and modified novel Sabouraud dextrose agar medium (modified SDA) (this invention) containing this neutralizing agent can the antibacterial activity of mulberry bark formulation granules be eliminated. In this system, lecithin is a component of cell membranes, adsorbs cationic compounds, and can protect bacterial cell membranes from damage. Tween 80 neutralizes fat-soluble antibacterial components and also neutralizes phenolic and flavonoid components. Serum albumin (BSA) is a protein protectant, with broad-spectrum auxiliary neutralization, which can complex antibacterial substances and restore the growth of damaged microorganisms. The recovery rate data of group 5 shows that this invention can be used as a method for the microbial limit detection of mulberry bark formulation granules.

[0049] Example 3: Non-toxicity test of neutralizing agent (neutralizing agent is 0.5% (w / v) Tween 80, 0.07% (w / v) soybean lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder) 1. Neutralizing agent non-toxicity test group: The above bacterial count was taken using aseptic technique and was 10... 3 -10 4 0.1 ml of Staphylococcus aureus bacterial suspension (cfu / ml) was added to 9.9 ml of sterile pH 7.0 sodium chloride-peptone buffer (containing 0.5% (w / v) Tween 80, 0.07% (w / v) soybean lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder). After incubation for 10 minutes, 1 ml was pipetted into a sterile Petri dish. Two parallel dishes were prepared, each containing 15-20 ml of a modified novel tryptone-soybean agar medium (containing 0.5% (w / v) Tween 80, 0.07% (w / v) soybean lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder). 0.5% (w / v) soybean lecithin, 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder were used to prepare non-toxic test groups for Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger using the same method. All five strains were used for total aerobic bacterial count testing. The modified novel tryptic soy agar medium (containing 0.5% (w / v) Tween 80, 0.07% (w / v) soybean lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder) was poured into the medium and incubated at 35°C for 3 days. The counts were then observed. Candida albicans and Aspergillus niger were used for total mold and yeast count testing. The culture medium was modified Sabouraud dextrose agar (containing 0.5% (w / v) Tween 80, 0.07% (w / v) soybean lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder) and incubated at 25°C for 5 days. The counts were then observed.

[0050] 2. Bacterial suspension control group: Under aseptic conditions, 1 ml of each of the above bacterial suspensions (prepared in the same way as in Example 1, with no neutralizing agent in the diluent) was injected into a sterile Petri dish. Two dishes were prepared in parallel. The total number of aerobic bacteria was checked using tryptic soy agar medium (without neutralizing agent), and the total number of molds and yeasts was checked using sarsaparilla dextrose agar medium (without neutralizing agent).

[0051] 3. Negative control group: Using aseptic technique, 1 ml of pH 7.0 sterile sodium chloride-peptone buffer (containing 0.5% (w / v) Tween 80, 0.07% (w / v) soy lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder) was poured into sterile Petri dishes. Four dishes were prepared in parallel. Two dishes were filled with 15-20 ml of tryptic soy agar medium (containing 0.5% (w / v) Tween 80, 0.07% (w / v) soy lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder), and two dishes were filled with Sabouraud dextrose agar medium (containing 0.5% (w / v) Tween 80, 0.07% (w / v) soy lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder). The dishes were cultured under the same conditions as the non-toxic test group with neutralizing agent.

[0052] Table 3 Results of non-toxicity test

[0053] The results show that the negative control group showed no sterile growth, and the ratio of the neutralizing agent non-toxic test group to the bacterial control group was between 0.5 and 2. This indicates that there was no significant difference in the number of colonies between the neutralizing agent non-toxic test group and the bacterial control group, suggesting that the neutralization system would not cause toxicity to the microorganisms used in the experiment.

[0054] Example 4: Neutralizer effectiveness test, the method is the same as the mulberry bark formula granule method suitability test. As can be seen from the results of Example 1, it is only necessary to examine challenging Gram-positive bacteria, namely Staphylococcus aureus and Bacillus subtilis.

[0055] Table 4. Test Results

[0056] The results showed that the negative control group had no bacterial growth, and the recovery rates of each test strain were between 0.5 and 2. This indicates that the neutralization system was effective for the test, eliminating the antibacterial activity of mulberry bark and increasing the recovery rates of Staphylococcus aureus and Bacillus subtilis.

[0057] Example 5: Traditional Plate Method 1. Bacterial suspension control group test: Using aseptic technique, take a sample of the prepared solution at a concentration of 10... 3 -10 40.1 ml of each CFU / ml bacterial suspension was added to seven test tubes containing 9.9 ml of sterile pH 7.0 sodium chloride-peptone buffer (containing 0.5% (w / v) Tween 80, 0.07% (w / v) soybean lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder as neutralizing agents), and mixed well. Then, 1 ml of each buffer was injected into sterile Petri dishes. Two dishes were prepared for each test strain, each containing 15-20 ml of culture medium. All five test strains were used for total aerobic bacterial count testing, poured onto modified novel tryptic soy peptone agar, and incubated at 35°C for 3 days. Counting was then performed. Candida albicans and Aspergillus niger were used for total mold and yeast count testing, poured onto modified novel Sabouraud dextrose agar, and incubated at 25°C for 5 days. Counting was then performed.

[0058] 2. Test sample control group test: Under aseptic conditions, take 1 ml of the coarsely filtered test solution from each dilution and inject it into a sterile Petri dish. Prepare two dishes in parallel for each dilution. For the total aerobic bacteria test, pour in a modified novel tryptic soy agar medium and incubate at 35°C for 3 days. Observe and count. For the total mold and yeast test, pour in a modified novel Sabouraud dextrose agar medium and incubate at 25°C for 5 days. Observe and count.

[0059] 3. Experimental control group experiment: Under aseptic conditions, 9.9 ml of the coarsely filtered test solution of each dilution level was placed in a test tube, and a 10% concentration of [unspecified ingredient] was added. 3 -10 4 0.1 ml of the corresponding CFU / ml bacterial suspension was mixed well, and 1 ml was immediately injected into a sterile Petri dish. Two dishes were prepared for each test strain. All five test strains were used for total aerobic bacterial count testing. The medium was poured onto a modified novel tryptic soy agar and incubated at 35°C for 3 days. Counting was then observed. Candida albicans and Aspergillus niger were used for total mold and yeast count testing. The medium was poured onto a modified novel Sabouraud dextrose agar and incubated at 25°C for 5 days. Counting was then observed.

[0060] 4. Negative control group test: Aseptically, 1 ml of pH 7.0 sterile sodium chloride-peptone buffer (containing 0.2% lecithin, 0.5% Tween 80, and 0.1% BSA) was poured into a Petri dish. 15-20 ml of modified novel tryptic soy agar medium or modified novel Sabouraud dextrose agar medium was added, mixed well, and allowed to solidify. For Petri dishes containing modified novel tryptic soy agar medium, the mixture was incubated at 35°C for 3 days; for Petri dishes containing modified novel Sabouraud dextrose agar medium, the mixture was incubated at 25°C for 5 days. The results showed no colony growth in the negative control group.

[0061] Table 5 Results of 1:10 Test Solution Recovery Rate

[0062] Table 6 Results of 1:100 test solution recovery rate test

[0063] As shown in the table, even with the ternary double neutralization system of this invention, the recovery rates of the five test strains were low using the traditional plate method, regardless of whether a 1:10 or 1:100 dilution of the test solution was used. The recoveries of Bacillus subtilis and Staphylococcus aureus were both <0.5%, indicating significant antibacterial activity. Furthermore, since the mulberry bark granules are granules without raw medicinal powder, the aerobic bacteria standard is ≤10. 3 The standard for cfu / g, mold, and yeast is ≤10. 2 The cfu / g value means that the test solution can only be diluted to a maximum of 1:100. This indicates that the method is not applicable. It shows that even if the plate method uses a ternary double neutralization system and the highest dilution, the antibacterial components still remain in the system and exert antibacterial activity, resulting in a low recovery rate of each test bacterium.

[0064] Example 6: Method Suitability Test 1. Preparation of test solution: Take 10g of mulberry bark formula granules and dilute them with pH 7.0 sodium chloride-peptone buffer (containing 0.5% (w / v) Tween 80, 0.07% (w / v) soybean lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder as neutralizing agents) to prepare a 1:10 test solution. Pass the 1:10 test solution through a sterile nylon filter for primary and secondary coarse filtration to remove insoluble large particles such as starch. Collect the filtrate for later use.

[0065] 2. Experimental group (test solution with added bacteria): Take 1 ml of the above test solution, filter and rinse. The rinsing solution is pH 7.0 sodium chloride-peptone buffer. Rinse each filter membrane 4 times, 100 ml each time, for a total rinsing volume of 400 ml. Add 0.1 ml of a 10% concentration to the last rinsing solution. 2 -10 3 The above bacterial suspension, at CFU / ml, included Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger. The filter membrane was then removed with the bacterial side facing up. For aerobic bacterial count testing, the filter membrane was placed on a modified tryptophan-soybean agar medium and incubated at 35°C for 3 days using the following five strains: Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger. For mold and yeast testing, the filter membrane was placed on a modified sarsaparilla glucose agar medium and incubated at 25°C for 5 days using the following two strains: Candida albicans and Aspergillus niger. The bacterial count was determined using the membrane filtration method.

[0066] 3. For the bacterial control group, the test solution was replaced with a 1:10 solution using a pH 7.0 sterile sodium chloride-peptone buffer (containing 0.5% (w / v) Tween 80 neutralizer, 0.07% (w / v) soybean lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder) and the same procedure was performed as in the experimental group.

[0067] 4. For the control group of the test sample, take 1 ml of the test solution at a 1:10 ratio, and follow the same procedure as the test group, without adding bacteria.

[0068] 5. Negative control group: Aseptically aspirate 1 ml of pH 7.0 sterile sodium chloride-peptone buffer (containing 0.5% (w / v) Tween 80 neutralizing agent, 0.07% (w / v) soybean lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder) and perform the same procedure as the experimental group, without adding any bacteria.

[0069] Recovery rate calculation: Recovery rate = (number of colonies in the test group - number of colonies in the control group) / number of colonies in the control group.

[0070] Table 7 Experimental Results

[0071] The results show that the negative control group showed no bacterial growth, and the recovery rates of each test strain were between 0.5 and 2. This indicates that the neutralization system is effective for this test and can eliminate the antibacterial activity of mulberry bark. Therefore, this detection method is appropriate.

[0072] Example 7: Control bacteria test and verification 1. For the experimental group, take 10g of the mulberry bark formula granules as the test sample, dilute and mix them with pH 7.0 sodium chloride-peptone buffer (containing 0.5% (w / v) Tween 80, 0.07% (w / v) soybean lecithin, and 0.1% (w / v) sterile bovine serum albumin (BSA) lyophilized powder as neutralizing agents) to prepare a 1:10 test solution. After passing the solution through a sterile nylon filter for primary and secondary coarse filtration, remove insoluble large particles such as starch, collect the filtrate and set it aside.

[0073] Take 10 ml of the prepared test solution (equivalent to 1 g of test sample) and filter and rinse. The rinsing solution is pH 7.0 sodium chloride-peptone buffer. Each rinse should be 100 ml per filter membrane, for a total rinsing volume of 400 ml. Add 0.1 ml of a 10% concentration solution during the final rinse. 2 -10 3After rinsing the above Escherichia coli suspension (cfu / ml), the filter membrane was placed in 100ml of tryptic soy broth and incubated at 35°C for 24 hours. 1ml of the culture was then inoculated into 100ml of MacConkey broth and incubated at 43°C for 24 hours. Finally, a portion of the MacConkey broth was streaked onto MacConkey agar plates and incubated at 35°C for 24 hours. Colony morphology was then observed.

[0074] 2. Bacterial control group: 0.1 ml of a 10% concentration was added to 100 ml of tryptic soy peptone solution. 2 -10 3 A CFU / ml Escherichia coli suspension was mixed thoroughly and incubated at 35°C for 24 hours. 1 ml of this culture was inoculated into 100 ml of MacConkey liquid medium and incubated at 43°C for 24 hours. Finally, a MacConkey liquid culture was streaked onto a MacConkey agar plate and incubated at 35°C for 24 hours. Colony morphology was then observed.

[0075] 3. For the test sample control group, take 10 ml of a 1:10 test solution (equivalent to 1 g of test sample) and filter it through a membrane. Each membrane should be rinsed with 100 ml of solution each time, for a total rinsing volume of 400 ml. After rinsing, place the membrane in 100 ml of tryptic soy broth and incubate at 35°C for 24 hours. Finally, inoculate 1 ml of the culture into 100 ml of MacConkey broth and incubate at 43.0°C for 24 hours. Then, streak the MacConkey broth culture onto MacConkey agar plates and incubate at 35°C for 24 hours, observing the colony morphology. If colonies grow on the MacConkey agar plates, isolate and identify whether Escherichia coli is detected in the test sample. If no colonies grow on the MacConkey agar plates, or if colonies grow but the identification result is negative, the test sample control group is considered to have no Escherichia coli.

[0076] 4. Negative control group Take 100ml of tryptic soy peptone liquid culture medium from the same batch and culture it as a negative control. Perform the test according to the control group of the test sample, and culture the same conditions as above. Observe the results.

[0077] Table 8. Test Results

[0078] The results showed that the colony morphology on MacConkey agar plates of the experimental group and the bacterial suspension control group was consistent, and both were typical Escherichia coli colony morphologies. No colony growth was observed in the negative control group. The results of the method suitability test of the three batches of test samples were consistent, indicating that the method is suitable for the detection of Escherichia coli in mulberry bark formula granules.

[0079] Example 8, Repeatability Test Take batch 26030101 of mulberry bark granules as a test sample, and prepare six test solutions in parallel under aseptic conditions. Simultaneously, perform tests for total aerobic bacteria, total mold and yeast count, and control bacteria (Escherichia coli) according to the method of this invention. The repeatability test results are shown in Table 9.

[0080] Table 9 Results of Repeatability Tests

[0081] The results showed that no colonies grew in the negative controls for total aerobic bacteria count, total mold and yeast count, and Escherichia coli tests. The results of the six aerobic bacteria count and total mold and yeast count tests were consistent, with a logarithmic range of 0.19 for multiple aerobic bacteria counts and 0.22 for multiple mold and yeast counts (Lg(maximum) - Lg(minimum) ≤ 0.25). For the Escherichia coli test, the repeatability test for the control bacteria was conducted in the following groups: Group 1: six tests were performed on the test samples with bacterial suspension added according to the applicability method of this invention, each time with ≤100 cfu / ml of Escherichia coli added; Escherichia coli was detected in all six groups. Group 2: six tests were performed on the test samples without bacterial suspension; no positive bacteria were detected in any of these groups, and no colonies grew in the negative controls. This indicates that the method has good repeatability, high precision, and stable and reliable results.

[0082] Therefore, the present invention adopts the above-mentioned method for microbial limit testing of mulberry bark formula granules. The steps are clear, the parameters are well-defined, and the operation is standardized. Those skilled in the art can repeat the implementation according to the instructions. It can stably achieve qualified recovery rate of test bacteria, smooth filtration without membrane clogging, complete elimination of antibacterial interference, and accurate and reliable results. It has good repeatability and practicality, and can simultaneously meet the testing requirements of total aerobic bacteria, total mold and yeast count, and Escherichia coli in control bacteria testing of mulberry bark formula granules.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for microbial limit test of mulberry bark formula granules, characterized by, Includes the following steps: Step 1: Preparation of diluent: Based on pH 7.0 sodium chloride peptone buffer, add Tween 80 and soybean lecithin, stir and disperse, then autoclave, cool and keep warm. Before use, add sterile bovine serum albumin lyophilized powder under aseptic conditions, shake and mix well to obtain the diluent. Step 2: Prepare the test solution: Take the mulberry bark formula granule sample, add the diluent prepared in step 1, shake to prepare a 1:10 test solution, and then use sterile nylon filters with different pore sizes for primary coarse filtration and secondary filtration in sequence. Collect the filtrate for later use. Step 3: Use membrane filtration to count the total number of aerobic bacteria, molds, and yeasts; Step 4, Control Bacteria Test: Take the 1:10 test solution prepared in Step 2 and filter and rinse it with pH 7.0 sodium chloride peptone buffer. After rinsing, place the filter membrane in tryptic soy peptone liquid medium and incubate. Take the culture and inoculate it into MacConkey liquid medium and incubate. Then, isolate, purify and biochemically identify it according to the Escherichia coli control bacteria test method to determine whether the bacteria are detected.

2. The method for microbial limit test of mulberry bark formula granules according to claim 1, characterized in that, In step 1, the high-pressure steam sterilization temperature is 121℃ and the time is 15 min; then cool to 40-45℃.

3. The method for microbial limit test of mulberry bark formula granules according to claim 1, characterized in that, The ratio of mulberry bark granules to diluent was 1g:9mL; The pore size is 80-120μm during primary coarse filtration and 10-20μm during secondary filtration.

4. The method for microbial limit test of mulberry bark formula granules according to claim 1, characterized in that, The proportion of Tween 80 in the diluent is 0.5% (w / v); The proportion of soybean lecithin in the diluted solution was 0.07% (w / v); The proportion of sterile bovine serum albumin lyophilized powder in the diluent was 0.1% (w / v).

5. The method for microbial limit test of mulberry bark formula granules according to claim 1, characterized in that, In step 4, the incubation temperature and time are the same as those for Escherichia coli in the 2025 edition of the Chinese Pharmacopoeia; The filter was rinsed using pH 7.0 sodium chloride peptone buffer, with each filter membrane being rinsed 100 mL each time, for a total rinsing volume of 400 mL.

6. The method for microbial limit testing of mulberry bark granules according to claim 1, characterized in that, The membrane filtration method is used to count the total number of aerobic bacteria, molds, and yeasts, and specifically includes the following steps: Step 31: Take the test solution obtained in step 2, add an appropriate amount of rinsing solution, mix well and filter; the rinsing solution is pH 7.0 sodium chloride peptone buffer. Step 32: Rinse the filter membrane several times with rinsing solution; Step 33: Remove the filter membrane and attach the front side to a modified tryptic soy peptone agar plate or a modified Sabouraud dextrose agar plate; Step 34, Total aerobic bacteria count: Incubate the modified tryptic soybean agar plates at 30-35℃ for 3-5 days; Step 35, Total count of molds and yeasts: Incubate modified Sabouraud dextrose agar plates at 23-28°C for 5-7 days.

7. The method for microbial limit testing of mulberry bark granules according to claim 6, characterized in that, The preparation methods for the modified tryptic soy peptone agar and modified Sabouraud dextrose agar mentioned in step 3 are as follows: Add 0.5% (w / v) Tween 80 and 0.07% (w / v) soybean lecithin to the basal culture medium, stir and disperse thoroughly, autoclave at 121℃ for 15 min, cool to 45-48℃ and keep warm. Before use, add sterile bovine serum albumin lyophilized powder to a final concentration of 0.1% (w / v) under aseptic conditions, mix well and pour into petri dishes.

8. The method for microbial limit testing of mulberry bark granules according to claim 1, characterized in that, The rinsing solution described in steps 3 and 4 is a pH 7.0 sodium chloride peptone buffer solution without added neutralizing agent.

9. The method for microbial limit testing of mulberry bark granules according to claim 1, characterized in that, It also includes a bacterial suspension preparation step, wherein the bacterial suspension is prepared using Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger with no more than 4 passages, for use in method suitability testing.

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