Applicability,of,Microbial,Limit,Test,Method,for,Qingyan,Zhisou,Powder

Changping TAI, Yuanfeng YANG, Yongmin CHEN, Yuling LUO, Keqin MA, Renhui YANG

Abstract ［Objectives］ This study was conducted to establish a microbial limit test method for Qingyan Zhisou Powder.

［Methods］ Applicability tests were carried out according to Microbial Limit Tests of Non-sterile Products， General Rules 1105-1107 of Part IV of Chinese Pharmacopoeia， 2020.

［Results］ Qingyan Zhisou Powder had a strong bacteriostatic effect on Pseudomonas aeruginosa. After increasing the dilution ratio （1：40）， the bacteriostatic effect was obviously eliminated. When the total number of aerobic microbes was determined by the test liquid dilution method （1：40）， the recovery values were in the range of 50%-200%; and when using the test liquid （1：10） to determine the total number of mold and yeasts， the recovery ranged from 50% to 200%. Escherichia coli， Salmonella and bile-tolerant Gram-negative bacteria were detected by both the routine method and dilution method in experimental groups.

［Conclusions］ The microbial limit test of Qingyan Zhisou Powder adopted the test liquid dilution method， and the routine method could be used for the test of Escherichia coli， Salmonella and bile-tolerant Gram-negative bacteria. The applicability test of the method is feasible， with scientific and accurate results， and the method can effectively control the quality of the preparation and is recommended for popularization.

Key words Qingyan Zhisou Powder; Microbial limit test; Method applicability

Received：
November 12， 2022  Accepted：
January 15， 2023

Supported by Traditional Chinese Medicine and Ethnic Minority Medicine Technology Research Subject of Guizhou Administrative Bureau （QZYY-2018-095）.

Changping TAI （1980-）， male， P. R. China， pharmacist-in-charge， devoted to research about drug dispensing， drug evaluation， and development of hospital preparations.

*Corresponding author. E-mail：
645818689@qq.com.

Qingyan Zhisou Powder is a traditional Chinese medicine prescription prepared by the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. The prescription is derived from the proved prescription of Professor Wang Yulin， a famous provincial Chinese medicine professor， used to treat wind-heat cold， tonsillitis， acute and chronic pharyngitis and other diseases due to yin deficiency and lung dryness. The symptoms include sore throat， itchy throat， hoarse voice， qi stagnation and coagulated phlegm in throat， and persistent cough.  Its main ingredients are composed of 12 traditional Chinese medicines， such as Folium Mori， Herba Menthae， Radix Scrophulariae， Radix Ophiopogonis， Radix Glycyrrhizae， etc. In order to effectively control the quality of the preparation， according to the provisions of Chinese Pharmacopoeia （General Rule of Part IV， 2020 edition）， the applicability tests of the microbial limit test methods were carried out for the preparation， and the microbial limit test method of the preparation was established. Formulating effective quality control standards for preparations can ensure the accuracy and effectiveness of test results and improve the safety of clinical medication.

Materials and Methods

Instruments and materials

Test product

Qingyan Zhisou Powder， specification：
6 g/bag， 12 bags per box， batch number：
20200701， 20200702， 20200703， all provided by the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine.

Instrument

Electronic balance （model TP-320C， Xiangyi Balance Instrument Equipment）; electrothermal thermostatic water bath （model BSG-24， Shanghai Yiheng Scientific Instrument Co.， Ltd.）; electrothermal thermostatic incubator （model DHP-9162， Shanghai Yiheng Scientific Instrument Co.， Ltd.）; biosafety cabinet （model HR30-II A2， Qingdao Haier Special Electrical Appliance Co.， Ltd.）; microbe tester （model JPX-2010， Shanghai Bingyue Electronic Instrument Co.， Ltd.）.

Media and reagents

Trypticase soy broth （TSB， batch number 1075741）， trypticase soy agar （TSA， batch number 1075861）， sabouraud dextrose broth （SDB， batch number 1076161）， sabouraud dextrose agar （SDA， batch number 1075841）， MacConkey broth medium （batch number 1111831）， MacConkey agar medium （batch number 1076291）， RV Salmonella enrichment broth （batch number 1106591）， xylose lysine desoxycholate agar （batch number 1104921）， enterobacteria enrichment broth mossel  （batch number 1070971）， and violet red bile glucose agar （batch number 1087471）， all produced by Guangdong Huankai Biology Co.， Ltd.）.

Strains

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 ［CMCC（B）44102］， and Salmonella paratyphi B （CMCC（B）50094），  all of which were derived strains from National Institutes for Food and Drug Control.

Experimental methods

Preparation of microbial liquids

The microbial liquids were prepared according to the requirements of Part IV of Chinese Pharmacopoeia （2020 edition）， with reference to the method reported by Liu et al.［2-5］.  S. aureus and P. aeruginosa were cultured on trypticase soy broth medium and prepared into bacterial suspensions with bacterial concentrations not higher than 10 000 and 1 000 cfu/ml， respectively. Each of B. subtilis and C. albicans was prepared into a bacterial suspension with a bacterial concentration not higher than 10 000 cfu/ml. Each of Salmonella paratyphi B and Escherichia coli was prepared into a bacterial suspension with a bacterial concentration not higher than 1 000 cfu/ml. A. niger was prepared into a fungal suspension with a spore concentration not higher than 10 000 cfu/ml.

Preparation of test liquids

A certain amount of the tested sample （10 g） was added with TSB to 100 ml， forming a 1：10 test liquid after shaking well. In order to screen an effective method for eliminating the antimicrobial activity， the 1：10 test liquid was also diluted to 1：20 and 1：40 test liquids.

Strain recovery rate

Recovery （%）=（Average number of colonies in the experimental group-Average number of colonies in the control group of test liquid）/Average number of colonies in the control group of microbial liquid

A recovery value in the range of 50%-200% indicated that the method was feasible［6］.

Applicability of microbial counting method

（i） Experimental groups

Seven portions （10 ml each） of test liquids 1：10， 1：20 and 1：40 were added into sterile glass tubes， respectively. Among them， five portions were added with 0.1 ml of bacterial suspensions under the item "Preparation of microbial liquids" （five strains with a concentration no higher than 10 000 cfu/ml corresponding to aerobic microbe counting）， respectively， and the remaining two portions were added with 0.1 ml of C. albicans and A. niger corresponding to mold and yeast counting， respectively. The obtained liquids were shaken well to ensure the final concentrations of microbes equal to or lower than 100 cfu/ml. Next， 1 ml of the seven test liquids were， respectively， added into 90 mm sterile culture dishes， into each of which 20 ml of culture medium （TSA for aerobic microbe counting plates， SDA for mold and yeast counting plates） thawed at about 45 ℃ was added， and the obtained liquids were quickly mixed， cooled and solidified.

（ii） Control group of microbial liquid

The test liquids were replaced with TSB， and the operation accorded to the above method under "Experimental groups".

（iii） Control group of test liquid

The microbial liquids were replaced with TSB， and the operation accorded to the above method under "Experimental groups".

（iv） Negative group

The test liquids were replaced with TSB， and no microbial liquids were added， and the operation accorded to the above method under "Experimental groups".

Results and Analysis

Pre-experiment of test liquid dilution counting method

The recovery data of the test liquid dilution method （one decimal place） are shown in Table 1. The results of the pre-experiment showed that when the dilution ratio was 1：40， the recovery of the seven test groups involving five test microbes ranged from 50% to 200%. The results showed that when the dilution ratios were 1：10 and 1：20， the product had strong inhibition on P. aeruginosa， and its recovery was lower than the lower limit of 50%. When the dilution ratio was 1：40， the antimicrobial activity of the preparation could be significantly eliminated， and the recovery values of the total aerobic microbes， mold and yeasts were all in the range of 50%-200%.

The results showed that the recovery of P. aeruginosa was less than 50% when the aerobic microbe counting of the product was checked by the routine method （1：10 test liquid）， so the routine method was not suitable for aerobic microbe counting of the product. The dilution method （1：20， 1：40） was adopted for verification. When the dilution ratio was 1：40， the recovery values of the total number of the five aerobic microbes were in the range of 50%-200%. When taking the 1：10， 1：20 and 1：40 test liquids of the product to count and check the total number of mold and yeasts of the product， the recovery values of C. albicans and A. niger were both within the range of 50%-200%， so the routine method was selected for counting and checking the total number of mold and yeasts of the product.

Counting experiment on three batches of samples

The recovery values of total aerobic microbes， mold and yeasts obtained by the test liquid dilution method （1：40） and the routine method （1：10） in the seven experimental groups were all in the range of 50%-200%， so the antimicrobial activity of the preparation against P. aeruginosa could be eliminated by the test liquid dilution method. The results are shown in Table 2.

The results showed that the aerobic microbe counting of the product was checked by the test liquid dilution method （1：40 test liquid）， and the total number of mold and yeasts of the product was checked by the routine method （1：10 test liquid）. The recovery values of the three batches of samples were all in the range of 50%-200%.

Applicability of control bacteria examination methods

E. coli

（i） Experimental groups：
First， 10 ml of 1：10 test liquid prepared under the item "Preparation of test liquids" was inoculated into 100， 150 and 200 ml of trypticase soy broth， respectively， and 0.1 ml of prepared bacterial suspensions of E. coli and S. aureus were added， respectively. After mixing well， the strains were cultured at 34 ℃ for 18-24 h. Next， 1 ml of each above culture was inoculated into 100 ml of MacConkey liquid medium， and cultured at 44 ℃ for 48 h. The MacConkey liquid cultures were streak-inoculated on MacConkey agar medium plates， and cultured at 34 ℃ for 24-72 h.

（ii） Control groups of test liquid：
The test bacteria were replaced with TSB， and the operation accorded to the above method under "Experimental groups".

（iii） Negative control：
The test liquid was replaced with TSB， and the operation accorded to the above method under "Experimental groups".

（iv） Verification results：
The experimental groups of the above method were positive， and the colonies on the plates were isolated， cultured and subjected to Gram-staining microscopy， which confirmed that the strain was E. coli. Both the control groups of test liquid and the negative control group were tested to be negative. Therefore， the control bacteria examination of the product could be operated by the routine method. The results are shown in Table 3.

Changping TAI et al. Applicability of Microbial Limit Test Method for Qingyan Zhisou Powder

Bile-tolerant gram-negative bacteria

（i） Experimental groups：
The test sample was prepared with trypticase soy broth liquid medium as the diluent according to the item "Preparation of test liquids" into a 1：10 test liquid， which was incubated at 20-25 ℃ for about 2 h. Next， the incubated liquid was diluted with trypticase soy broth liquid medium into 1：100 and 1：1 000 test liquids. Next， 10 ml of enterobacteria enrichment borth medium was added with 1 ml of 1：10， 1：100， 1：1 000 test liquids and 0.1 ml of prepared E. coli， P. aeruginosa and S. aureus， respectively （within 100 cfu）， and the obtained liquids were mixed well， and cultured at 34 ℃ for 48 h. Each of the above cultures was streak-inoculated on violet red bile glucose agar medium plates， and cultured at 34 ℃ for 24 h.

（ii） Test liquid groups：
Into 10， 15 and 20 ml of enterobacteria enrichment borth medium， 1 ml of 1：10， 1：100 and 1：1 000 test liquids were added， respectively. The obtained liquids were mixed well and cultured according to culture conditions and examination method the same as those of the experimental groups.

（iii） Negative control groups：
Into 10， 15 and 20 ml of enterobacteria enrichment borth medium， 1 ml of trypticase soy broth liquid medium， respectively. The obtained liquids were mixed well and cultured according to culture conditions and examination method the same as those of the experimental groups.

（iv） Verification results：
The experimental groups were tested to be positive， and the test liquid groups and negative control groups were tested to be negative. Therefore， the quantitative examination of the control bacteria， i.e.， bile-tolerant gram-negative bacteria， could be operated by the routine method. The results are shown in Table 3.

Salmonella

（i） Experimental groups：
Into 100， 150 and 200 ml of enterobacteria enrichment borth medium was added 10 g of test sample， respectively， and obtained mixtures were heated in water baths at 45 ℃ to dissolve the test sample and mix well. Next， 0.1 ml of prepared bacterial suspensions of S. paratyphi B and S. aureus were added， respectively， and the obtained liquids were mixed well， and cultured in an incubator at 34 ℃ for 24 h. Next， 0.1 ml of each culture was inoculated into 10 ml of RV Salmonella enrichment broth， and the obtained liquids were mixed well， and cultured in an incubator at 34 ℃ for 24 h. A small amount of each culture was streak-inoculated on xylose lysine desoxycholate agar plates， and cultured at 34 ℃ for 48 h.

（ii） Test liquid groups：
Into 100， 150 and 200 ml of trypticase soy broth liquid medium， was added 10 g of test sample， respectively， and the obtained mixtures were heated in water baths at 45 ℃ to dissolve the test sample and mix well. The culture conditions and examination method were the same as those of the experimental groups.

（iii） Negative control groups：
Into 100， 150 and 200 ml of trypticase soy broth liquid medium， was added 10 ml of trypticase soy broth liquid medium， respectively， and the culture conditions and examination method were the same as those of the experimental groups.

（iv） Verification results：
The experimental groups were tested to be positive， and the test liquid groups and negative control groups were tested to be negative. Therefore， it was determined that the trypticase soy broth liquid medium used for detection of Salmonella was 100 ml， and the routine method could be used for detection of Salmonella. The results are shown in Table 3.

Conclusions and Discussion

According to the method applicability tests， the inhibitory effects of antimicrobial ingredients in Qingyan Zhisou Powder to total aerobic microbes could be eliminated by the test liquid dilution method. When the dilution ratio of test liquid was 1：40， the recovery values of aerobic strains were all in the range of 50%-200%. When the total number of mold and yeasts was tested with the 1：10 test liquid， the recovery values were in the range of 50%-200%. In control bacteria examination， the test bacteria could be detected in the experimental groups by both the routine method and the culture medium dilution method， so the routine method was selected to examine control bacteria. The TSB volume for both E. coli and Salmonella examination was 100 ml， and the volume of enterobacteria enrichment borth medium for examination of bile-tolerant Gram-negative bacteria was 10 ml. Through the verification on three batches of Qingyan Zhisou Powder samples， the conclusions obtained were consistent with the pre-experiments. During the study on the applicability of the microbial examination methods for Qingyan Zhisou Powder， on the one hand， it was due to multiple bacteriostatic ingredients in the preparation， such as Folium Mori， Flos Chrysanthemi， Flos Lonicerae， Herba Menthae， etc.［7-11］. After powdering and bagging of the prescription combination， the powder finally showed obvious bacteriostasis to the recovery of P. aeruginosa in the method applicability test. When the dilution ratio of test liquid was 1：10， the recovery of P. aeruginosa was only 20.8%， which was far less than 50%. When the test liquid was diluted to 1：20， the recovery of P. aeruginosa was only 39.0%， which was still lower than the minimum of 50%. When the dilution ratio was 1：40， the results of the pre-experiment showed that the recovery of P. aeruginosa was 80.5%， which was in the range of 50%-200%. It could be seen that the test liquid dilution method could effectively eliminate the antibacterial activity in this study， and could truly reflect the contamination of the preparation by external microorganisms during production， use and storage. On the other hand， according to the provisions of Chinese Pharmacopoeia （2020 edition） on non-sterile solid oral preparation of traditional Chinese medicine containing raw powder， the total number of aerobic microbes is required to be ≤104 cfu/g， and the total number of mold and yeasts is ≤102 cfu/g. The ranges are relatively wide. Therefore， the culture medium dilution plate method could be adopted to gradually dilute the effects of bacteriostatic ingredients on tests. The formulated methods can ensure effective formulation of control standards and microbial safety quality control.

According to Part IV of Chinese Pharmacopoeia （2020 edition）， when the recovery values of experimental groups are less than 50%， it is necessary to eliminate the bacteriostatic activity of test products. The methods for reducing the bacteriostatic activity include：
adding diluent or increasing the volume of the culture medium， adding neutralizer or inactivator in an appropriate amount， using membrane filtration method or using the combination of the above three methods. When there are bacteriostatic ingredients in a preparation， it may lead to bacteriostatic efficiency in the verification process of microbial limit detection method when establishing the quality standard of the preparation， resulting in the recovery of some strains less than 50%. Meanwhile， when the medium dilution method or adding neutralizer and inactivator is adopted for sterilization， the bacteriostatic efficiency of different concentrations of neutralizer and inactivation on bacteriostatic ingredients is different， and it is more reasonable to test with the pilot products produced with different batches of medicinal materials. For this reason， the bacteriostatic efficiency of preparations produced from raw medicinal materials in different periods and production batches may not be consistent. Therefore， the test method was only applicable to the microbial limit test of the three batches of Qingyan Zhisou Powder， which is a controversial topic among microbiological researchers in recent years.

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