阅读说明：本技术 黄连配方颗粒的微生物限度检查方法 (Microbial limit inspection method for coptis chinensis formula granules ) 是由 胡梦莹 王志斌 张连中 高春 牛策 秦柳 李娜娜 于 2020-04-30 设计创作，主要内容包括：本发明公开了一种黄连配方颗粒的微生物限度检查方法,包括以下步骤：S1分别配制金黄色葡萄球菌、铜绿假单胞菌、枯草芽孢杆菌、白色念珠菌和黑曲霉的菌悬液；S2供试液的制备：配制含有3%组氨酸的供试液；S3需氧菌总数计数方法适用性试验；S4霉菌和酵母菌总数计数方法适用性试验；S5大肠埃希菌检查方法适用性试验；其中限度检测标准为：1 g供试品中,需氧菌总数≤10<Sup>3</Sup> cfu,霉菌和酵母菌总数≤10<Sup>2</Sup> cfu,大肠埃希菌不得检出。本发明的方法可用于黄连配方颗粒中微生物限度检查,具有有效地消除样品的抑菌性、简化检查过程、减少试剂用量、高效检测出黄连配方颗粒中微生物的优点。(The invention discloses a microbial limit inspection method of coptis chinensis formula granules, which comprises the following steps: s1 respectively preparing bacterial suspensions of staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, candida albicans and aspergillus niger; s2 preparation of test solution: preparing a test solution containing 3% histidine; s3 test of applicability of total aerobic bacteria count method; s4 mould and yeastCarrying out a total bacteria count method applicability test; S5E, performing an Escherichia coli inspection method applicability test; wherein the limit detection criteria are: 1g of the sample contains aerobic bacteria with a total number of less than or equal to 10 3 cfu, total number of mould and microzyme less than or equal to 10 2 cfu, Escherichia coli could not be detected. The method can be used for checking the microbial limit in the coptis chinensis formula particles, and has the advantages of effectively eliminating the bacteriostatic activity of a sample, simplifying the checking process, reducing the reagent dosage and efficiently detecting the microbes in the coptis chinensis formula particles.)

1. A method for checking the microbial limit of coptis chinensis formula granules is characterized by comprising the following steps:

preparing a suspension of S1 bacteria: respectively preparing bacterial suspensions of staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, candida albicans and aspergillus niger;

s2 preparation of test solution:

s2-1, taking 10g of coptis chinensis formula particles, adding sterile sodium chloride-peptone buffer solution with pH7.0 and 3% histidine to 100ml, and shaking until a test sample is uniformly dispersed to serve as a test solution with a ratio of 1: 10;

s2-2, taking 20ml of 1:10 test solution, adding 80ml of sterile sodium chloride-peptone buffer solution with pH7.0 and 3% histidine, and obtaining the 1:50 test solution;

s2-3, taking 10ml of 1:10 test solution, adding 90ml of sterile sodium chloride-peptone buffer solution with pH7.0 and 3% histidine, and obtaining 1:100 test solution;

s3 total aerobic count method applicability test: respectively setting a test group, a test article group, a bacteria liquid control group and a diluent control group, and respectively counting the total number of aerobic bacteria in each group;

s4 test for applicability of the method for counting the total number of molds and yeasts: respectively setting a test group, a test article group, a bacteria liquid control group and a diluent control group, and respectively counting the total number of moulds and yeasts in each group;

s5 Escherichia coli test method applicability test: the test group, the test article group and the negative control group are respectively provided with cultures, then the cultures are inoculated in corresponding culture media, and the results are observed.

2. The method as claimed in claim 1, wherein the step S3 comprises the steps of:

s3-1 test group: taking five parts of 1:100 test solutions, wherein the volume of each part of test solution is 1ml, adding the five parts of test solutions into 100ml of sterile sodium chloride-peptone buffer solution with the pH value of 7.0, washing the test solutions for multiple times by using the sterile sodium chloride-peptone buffer solution with the pH value of 7.0 after filtering by a membrane filtration method, adding 1ml of test bacteria solution with the bacteria content of 10-100cfu into the five parts of test solutions in the last washing solution, taking down a filter membrane, attaching the filter membrane to a trypticase soy agar culture medium, and culturing for 3 days at the temperature of 33 ℃; wherein, the test bacteria added into the five test solutions are respectively as follows: staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, candida albicans and aspergillus niger;

s3-2 test article group: 1ml of 1:100 test solution is taken, sterile sodium chloride-peptone buffer solution with the pH value of 7.0 is used for replacing bacterial solution to operate the same test group;

s3-3 bacterial liquid control group: replacing the test solution with 1:100 sterile sodium chloride-peptone buffer solution at pH7.0 to operate with the test group;

s3-4 diluent control group: the test group was operated with a corresponding amount of sterile NaCl-peptone buffer pH7.0 containing 3% histidine, instead of the test solution.

3. The method as claimed in claim 1, wherein the step S4 comprises the steps of:

s4-1 test group: the difference from the step S3-1 is that the test solution is 1:50, the test bacteria solution is Candida albicans test bacteria solution and Aspergillus niger test bacteria solution, the filter membrane is taken down and then is pasted on a Sabouraud' S dextrose agar culture medium, and the culture is carried out for 5 days at the temperature of 20-25 ℃;

s4-2 test article group: taking 1ml of 1:50 test solution, and replacing the bacterial liquid with the diluent to operate the test group in the step S4-1;

s4-3 bacterial liquid control group: replacing the test solution with sterile sodium chloride-peptone buffer solution with pH7.0 at a ratio of 1:50, and performing the same operation with the test group;

s4-4 diluent control group: the test group was operated with a corresponding amount of sterile NaCl-peptone buffer pH7.0 containing 3% histidine, instead of the test solution.

4. The method as claimed in claim 1, wherein the step S5 comprises the following steps:

preparing S5-1 bacterial liquid: preparing a bacterial suspension of Escherichia coli;

s5-2 test group: taking 10ml of test solution with the ratio of 1:10, adding the test solution into 500ml of tryptic soy peptone liquid medium containing 3% of polysorbate 80 and 0.3% of soy lecithin, taking 1ml of escherichia coli liquid with the bacterial content of 10-100cfu, injecting the escherichia coli liquid into the 500ml of tryptic soy peptone liquid medium containing 3% of polysorbate 80 and 0.3% of soy lecithin, mixing uniformly, culturing at 33 ℃ for 18-24h, and observing;

s5-3 test article group: taking 10ml of test solution with the ratio of 1:10, adding the test solution into 500ml of tryptone soy peptone liquid medium containing 3% polysorbate 80 and 0.3% soybean lecithin, taking 1ml of diluent to replace bacterial liquid, operating the test group, and observing;

s5-4 negative control group: replacing the test solution with a corresponding amount of sterile sodium chloride-peptone buffer solution with pH7.0 containing 3% histidine;

s5-5, taking 1ml to 100ml of the culture respectively, culturing for 24-48h at 43 ℃, streaking and inoculating the Mackanka liquid culture on a Mackanka agar plate, culturing for 18-72h at 33 ℃, and observing the result.

5. The method of claim 1, wherein the preparation of the Staphylococcus aureus bacterial suspension comprises the following steps: inoculating Staphylococcus aureus to 10ml trypticase soy peptone liquid medium, culturing at 30-35 deg.C for 18-24h, and making into bacterial suspension containing 10-100cfu bacteria per 1ml with pH7.0 sterile sodium chloride-peptone buffer solution.

6. The method of claim 1, wherein the preparation of the Pseudomonas aeruginosa suspension comprises the following steps: inoculating pseudomonas aeruginosa into 10ml trypticase soy peptone liquid medium, culturing for 18-24h at 30-35 ℃, and preparing bacterial suspension containing 10-100cfu of bacteria per 1ml by using sterile sodium chloride-peptone buffer solution with pH 7.0.

7. The method of claim 1, wherein the preparation of the bacillus subtilis suspension comprises the following steps: inoculating Bacillus subtilis into 10ml trypticase soy peptone liquid medium, culturing at 30-35 deg.C for 18-24 hr, and making into bacterial suspension containing 10-100cfu bacteria per 1ml with pH7.0 sterile sodium chloride-peptone buffer solution.

8. The method of claim 1, wherein the preparation of the Candida albicans suspension comprises the steps of: inoculating Candida albicans into 10ml of Sabouraud's dextrose liquid culture medium, culturing at 20-25 ℃ for 18-24h, and preparing bacterial suspension containing 10-100cfu bacteria per 1ml of sterile sodium chloride-peptone buffer solution with pH 7.0.

9. The method of claim 1, wherein the Aspergillus niger suspension is prepared by the following steps: collecting fresh culture of Aspergillus niger cultured at 20-25 deg.C for 5-7 days, adding 3-5ml of pH7.0 sterile sodium chloride-peptone buffer solution containing 0.05% polysorbate 80 to elute spore, sucking spore suspension into sterile test tube, and preparing spore suspension containing 10-100cfu spore number per 1ml with pH7.0 sterile sodium chloride-peptone buffer solution.

Technical Field

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for checking microbial limit of coptis chinensis formula granules.

Background

Coptis chinensis has the effects of clearing heat and drying dampness, purging fire and removing toxicity, and is commonly used for treating damp-heat distention and fullness, vomiting and acid regurgitation, dysentery, jaundice, high fever and coma, hyperactivity of heart-fire, vexation and insomnia, blood heat and hematemesis and epistaxis, conjunctival congestion, toothache, thirst, carbuncle swelling and furuncle and other symptoms, so the Coptis chinensis is prepared into a traditional Chinese medicine preparation for treating related diseases.

The coptis chinensis is usually made into granules, and before entering the market for circulation, the coptis chinensis needs to be checked for the microbial limit according to the relevant requirements of the pharmacopoeia of the people's republic of China published by the State food and drug administration. Currently, many studies have been made on the examination of the limit of microorganisms in drugs.

The invention patent with application number 201810077354.4 discloses a microbial limit inspection method of bisoprolol fumarate capsules, which comprises the following steps: (1) preparing a bacterial liquid; (2) preparing a test solution: taking 10g of bisoprolol fumarate capsule sample, placing the bisoprolol fumarate capsule sample in a sterile homogenizing bag, adding 100ml of 45 ℃ sterile sodium chloride-peptone buffer solution with the pH value of 7.0, beating for 1min by using a homogenizer, shaking up to obtain a mixture as 1:10, a test solution; (3) determination of total number of aerobic bacteria, mold and yeast; (4) examination of Escherichia coli.

The above is a conventional method for examining the microbial limit in a medicament, but a method for examining the microbial limit in coptis chinensis has not been disclosed yet. According to the microbial limit inspection method in traditional Chinese medicine recorded in the pharmacopoeia of the people's republic of China, an effective neutralizer needs to be added in the detection process, and the effective neutralizer can be combined with interferents in the coptis chinensis to eliminate the bacteriostatic activity of the coptis chinensis sample, so that the microbial limit in the coptis chinensis can be effectively detected, but the pharmacopoeia of the people's republic of China does not record a neutralizer or an inactivation method capable of eliminating the interferents in the coptis chinensis, and the interferents can directly influence the limit inspection result of the microbes in the coptis chinensis. Therefore, the microbial limit in coptis chinensis cannot be accurately detected according to the relevant records of the pharmacopoeia of the people's republic of China.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method for detecting the microbial limit of coptis chinensis formula particles, which adopts a neutralizing agent and has the advantages of effectively eliminating the bacteriostatic property of a sample, simplifying the process of detecting the microbial limit in coptis chinensis, reducing the dosage of reagents and efficiently detecting the microbes in the coptis chinensis formula particles.

In order to achieve the purpose, the invention provides the following technical scheme: a method for checking microorganism limit of Coptidis rhizoma granule comprises the following steps:

preparing a suspension of S1 bacteria: respectively preparing bacterial suspensions of staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, candida albicans and aspergillus niger;

s2 preparation of test solution:

s2-1, taking 10g of coptis chinensis formula particles, adding sterile sodium chloride-peptone buffer solution with pH7.0 and 3% histidine to 100ml, and shaking until a test sample is uniformly dispersed to serve as a test solution with a ratio of 1: 10;

s2-2, taking 20ml of 1:10 test solution, adding 80ml of sterile sodium chloride-peptone buffer solution with pH7.0 and 3% histidine, and obtaining the 1:50 test solution;

s2-3, taking 10ml of 1:10 test solution, adding 90ml of sterile sodium chloride-peptone buffer solution with pH7.0 and 3% histidine, and obtaining 1:100 test solution;

s3 total aerobic count method applicability test: respectively setting a test group, a test article group, a bacteria liquid control group and a diluent control group, and respectively counting the total number of aerobic bacteria in each group;

s4 test for applicability of the method for counting the total number of molds and yeasts: respectively setting a test group, a test article group, a bacteria liquid control group and a diluent control group, and respectively counting the total number of aerobic bacteria in each group;

s5 Escherichia coli test method applicability test: the test group, the test article group and the negative control group are respectively provided with cultures, then the cultures are inoculated in corresponding culture media, and the results are observed.

By adopting the technical scheme, as the effective neutralizer histidine is added into the test solution and is combined with the effective components in the coptis, the influence of interferents in the coptis formula granules on the inspection result in the microbial limit inspection of the coptis formula granules is eliminated, so that the microbes existing in the coptis formula granules can be more accurately inspected according to the method, and the quality of the product is ensured.

Meanwhile, in the microbial limit inspection of the coptis chinensis formula particles, the microbial inspection requirement is met only by selecting one neutralizing agent (histidine), the complexity of using a plurality of neutralizing agents is reduced, the washing frequency of membrane filtration is reduced, the culture medium and the reagent used in the test are reduced, and the inspection process is simpler.

Further, the step S3 includes the following steps:

s3-1 test group: taking five parts of 1:100 test solutions, wherein the volume of each part of test solution is 1ml, adding the five parts of test solutions into 100ml of sterile sodium chloride-peptone buffer solution with the pH value of 7.0, washing the test solutions for multiple times by using the sterile sodium chloride-peptone buffer solution with the pH value of 7.0 after filtering by a membrane filtration method, adding 1ml of test bacteria solution with the bacteria content of 10-100cfu into the five parts of test solutions in the last washing solution, taking down a filter membrane, attaching the filter membrane to a trypticase soy agar culture medium, and culturing for 3 days at the temperature of 33 ℃; wherein, the test bacteria added into the five test solutions are respectively as follows: staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, candida albicans and aspergillus niger; s3-2 test article group: 1ml of 1:100 test solution is taken, sterile sodium chloride-peptone buffer solution with the pH value of 7.0 is used for replacing bacterial solution to operate the same test group;

s3-3 bacterial liquid control group: replacing the test solution with 1:100 sterile sodium chloride-peptone buffer solution at pH7.0 to operate with the test group;

s3-4 diluent control group: the test group was operated with a corresponding amount of sterile NaCl-peptone buffer pH7.0 containing 3% histidine, instead of the test solution.

By adopting the technical scheme, the applicability test of the aerobic bacteria total number counting method is carried out in simple and efficient steps, an accurate and effective test result is obtained, and the applicability of the method is improved.

Further, the step S4 includes the following steps:

s4-1 test group: the difference from the step S3-1 is that the test solution is 1:50, the test bacteria solution is Candida albicans test bacteria solution and Aspergillus niger test bacteria solution, the filter membrane is taken down and then is pasted on a Sabouraud' S dextrose agar culture medium, and the culture is carried out for 5 days at the temperature of 20-25 ℃;

s4-2 test article group: taking 1ml of 1:50 test solution, and replacing the bacterial liquid with a diluent to operate the test group in the step S4-1;

s4-3 bacterial liquid control group: replacing the test solution with sterile sodium chloride-peptone buffer solution with pH7.0 at a ratio of 1:50, and performing the same operation with the test group;

s4-4 diluent control group: the test group was operated with a corresponding amount of sterile NaCl-peptone buffer pH7.0 containing 3% histidine, instead of the test solution.

By adopting the technical scheme, the applicability test of the mould and yeast total number counting method is carried out in simple and efficient steps, accurate and effective test results are obtained, and the applicability of the method is improved.

Further, the step S5 specifically includes the following steps:

preparing S5-1 bacterial liquid: preparing a bacterial suspension of Escherichia coli;

s5-2 test group: taking 10ml of test solution with the ratio of 1:10, adding the test solution into 500ml of tryptic soy peptone liquid medium containing 3% of polysorbate 80 and 0.3% of soy lecithin, taking 1ml of escherichia coli liquid with the bacterial content of 10-100cfu, injecting the escherichia coli liquid into the 500ml of tryptic soy peptone liquid medium containing 3% of polysorbate 80 and 0.3% of soy lecithin, mixing uniformly, culturing at 33 ℃ for 18-24h, and observing;

s5-3 test article group: taking 10ml of test solution with the ratio of 1:10, adding the test solution into 500ml of tryptone soy peptone liquid medium containing 3% polysorbate 80 and 0.3% soybean lecithin, taking 1ml of diluent to replace bacterial liquid, operating the test group, and observing;

s5-4 negative control group: replacing the test solution with a corresponding amount of sterile sodium chloride-peptone buffer solution with pH7.0 containing 3% histidine;

s5-5, taking 1ml to 100ml of the culture respectively, culturing for 24-48h at 43 ℃, streaking and inoculating the Mackanka liquid culture on a Mackanka agar plate, culturing for 18-72h at 33 ℃, and observing the result.

By adopting the technical scheme, the applicability test of the escherichia coli inspection method is carried out by simple and efficient steps, accurate and effective test results are obtained, and the applicability of the method is improved.

Further, the preparation of the staphylococcus aureus bacterial suspension comprises the following steps: inoculating Staphylococcus aureus to 10ml trypticase soy peptone liquid medium, culturing at 30-35 deg.C for 18-24h, and making into bacterial suspension containing 10-100cfu bacteria per 1ml with pH7.0 sterile sodium chloride-peptone buffer solution.

By adopting the technical scheme, the influence of interference substances generated in the growth process of staphylococcus aureus on the detection result is eliminated by adopting the 3% neutralizing agent, so that the limit check on the staphylococcus aureus in the coptis formula particles is not influenced.

Further, the preparation of the pseudomonas aeruginosa suspension comprises the following steps: inoculating fresh culture of Pseudomonas aeruginosa, culturing in 10ml trypticase soy peptone liquid medium at 30-35 deg.C for 18-24 hr, and making into bacterial suspension containing 10-100cfu per 1ml with pH7.0 sterile sodium chloride-peptone buffer solution.

By adopting the technical scheme, 3 percent of neutralizer is adopted, so that the influence of interference substances generated by the pseudomonas aeruginosa in the growth process on the detection result is eliminated, and the limit inspection of the pseudomonas aeruginosa in the coptis chinensis formula particles is not influenced.

Further, the preparation of the bacillus subtilis bacterial suspension comprises the following steps: inoculating fresh Bacillus subtilis culture, culturing at 30-35 deg.C for 18-24 hr in trypticase soy peptone liquid medium, and making into bacterial suspension containing 10-100cfu per 1ml with pH7.0 sterile sodium chloride-peptone buffer solution.

By adopting the technical scheme, the 3 percent of neutralizer is adopted, so that the influence of interfering substances generated in the growth process of the bacillus subtilis on the detection result is eliminated, and the limit inspection of the bacillus subtilis in the coptis formula particles is not influenced.

Further, the preparation of the candida albicans suspension comprises the following steps: inoculating Candida albicans fresh culture into 10ml of Sabouraud's dextrose liquid culture medium, culturing for 18-24h at 20-25 ℃, and preparing bacterial suspension containing 10-100cfu bacteria per 1ml of sterile sodium chloride-peptone buffer solution with pH7.0.

By adopting the technical scheme, the influence of the interference substances generated by the candida albicans in the growth process on the detection result is eliminated by adopting the 3 percent neutralizing agent, so that the limit check of the candida albicans in the coptis formula granules is not influenced.

Further, the preparation of the aspergillus niger suspension comprises the following steps: collecting fresh culture of Aspergillus niger cultured at 20-25 deg.C for 5-7 days, adding 3-5ml of pH7.0 sterile sodium chloride-peptone buffer solution containing 0.05% polysorbate 80 to elute spore, sucking spore suspension into sterile test tube, and preparing spore suspension containing 10-100cfu spore number per 1ml with pH7.0 sterile sodium chloride-peptone buffer solution.

By adopting the technical scheme, the influence of an interference substance generated in the growth process of the aspergillus niger on the detection result is eliminated due to the adoption of the 3 percent neutralizing agent, so that the limit inspection of the aspergillus niger in the coptis chinensis formula particles is not influenced.

In conclusion, the invention has the following beneficial effects:

firstly, the method of the invention adds effective neutralizer histidine into the test solution, effectively eliminates the influence of interferents in the coptis formula granules on the microbial limit inspection result, enables the method to more accurately inspect the microbes in the coptis formula granules, and ensures the product quality; meanwhile, the requirement of microbial limit inspection of the coptis chinensis formula particles is met under the condition that only one neutralizing agent is selected, so that the test process is simplified, the use of reagents is reduced, and the inspection process is simple.

Secondly, in the invention, the pH7.0 sterile sodium chloride-peptone buffer solution with 3% histidine added in the prepared test solution is preferably adopted, so that the influence of interferents generated in the microbial growth process on the microbial limit inspection result is effectively eliminated, the content of the microbes in the coptis formula particles can be more accurately inspected according to the method, and the product quality is ensured.

Detailed Description

The present invention will be described in further detail with reference to examples.

The strains adopted in the invention are purchased from China medical bacteria collection and management center, wherein the strain number of Escherichia coli (Escherichia coli) is [ CMCC (B)44102], the strain number of Bacillus subtilis is [ CMCC (B)63501], the strain number of Candida albicans (Monilia albicans or Candida albicans) is [ CMCC (F)98001], the strain number of Pseudomonas aeruginosa (Pseudomonas aeruginosa) is [ CMCC (B)10104], the strain number of Staphylococcus aureus (Staphylococcus aureus) is [ CMCC (B)26003], and the strain number of Aspergillus niger (CMCC (F)98003 ].

Tryptone soy peptone liquid medium was purchased from Beijing three pharmaceutical technology development company; the Sa's glucose liquid culture medium is purchased from the development company of the three pharmaceutical sciences of Beijing, and the Sa's glucose agar culture medium is purchased from the development company of the three pharmaceutical sciences of Beijing; histidine was purchased from national drug group chemical agents, ltd; the Mackanka liquid medium was purchased from the development company of the three pharmaceutical sciences of Beijing, and the Mackanka agar medium was purchased from the development company of the three pharmaceutical sciences of Beijing.