Microbial Limit Test of Zhideke Granules

Yunjuan PANG, Yushan ZHOU, Jie LIANG,3,5*, Hui XU, Jinyu WEI, Dongfang HUANG, Zhengyi SUN

1. Guangxi University of Chinese Medicine, Nanning 530200, China; 2. Yulin Institute for Food and Drug Control, Yulin 537000, China; 3. Guangxi Zhuang Yao Pharmaceutical Engineering Technology Research Center, Nanning 530200, China; 4. Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning 530011, China; 5. Guangxi Superior Chinese Patent Medicine and National Medicine Development Engineering Technology Center, Nanning 530200, China

Abstract [Objectives] To test the microbial limit of Zhideke granules. [Methods] According to the relevant regulations on the testing of granules in Chinese Pharmacopoeia in 2015, the applicability of the methods for counting the total number of aerobic bacteria, molds and yeasts and objectionable organisms in Zhideke granules was studied. [Results] The total number of aerobic bacteria in Zhideke granules was determined by dilution plate method (1∶20, 1 mL per dish), the total number of molds and yeasts was determined by conventional plate method (1∶10, 1 mL per dish), and E. coli was detected by conventional method of TTB (1∶10 test solution, 10-100 mL TSB). [Conclusions] The method was suitable for the microbial limit test of Zhideke granules.

Key words Zhideke granule, Microbial limit, Methodological verification, Aerobic bacteria, Mold, Yeast, Plate method, Conventional method, E. coli

1 Introduction

Zhideke granule is a hospital preparation of Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine. It is an oral solid preparation made of 10 kinds of traditional Chinese medicine includingS.baicalensis, blackberry lily,R.Bupleuri, andP.grandiflorum. It has been used clinically for more than five years.S.baicalensishas bacteriostatic, anti-inflammatory, hypoglycemic effect, and blackberry lily can treat sore throat arthralgia[1-2]. Zhideke granule has the effect of clearing lung and resolving phlegm, anti-inflammation and relieving cough. It is used to treat cold, fever, sneeze, headache, pneumonia, cough, laryngitis, acute and chronic tracheitis[3]. In order to better control the product quality, the microbial limit test of Zhideke granules was carried out according to the relevant regulations of microbiological examination of traditional Chinese medicine granules in the 2015 edition ofChinesePharmacopoeia. The microbial limit test of traditional Chinese medicine granules is a compulsory item, and the pollution control of granular microorganisms is an important index of drug quality control[4]. In the 2015 edition ofChinesePharmacopoeia, great changes have been made to the microbial limit verification method, culture system and operating environment under microbiological examination, stipulating that as long as it is a preparation that needs to be tested for microbial limit, the relevant verification should be carried out again in accordance with the requirements of the current version of theChinesePharmacopoeia[5]. The total number of aerobic bacteria in Zhideke granules should be less than 1×103cfu/g, and the total number of molds and yeasts should be less than 100 cfu/g, andE.colishould not be found in 1 g of samples[6].

2 Materials

Type 371 incubator (Bio-Rad, USA); MJ-70-I mold incubator (Shanghai Yuejin Medical Equipment Co., Ltd.); BSC-1004 II A2 biosafety cabinet (Suzhou Antai Air Technology Co., Ltd.); Tryptic (Trypticase) Soy Agar (TSA), Tryptic (Trypticase) Soy Broth(TSB), Sabouraud Dextrose Agar (SDA), Sabouraud Dextrose Broth (SDB), Maconkey Agar (MAC), pH 7.0 Buffered Sodium Chloride-Peptone Solution (SPB), 0.9% aseptic sodium chloride solution (Beijing Luqiao Technology Co., Ltd.); Zhideke granule (Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, specification: 15 g); B. subtilis [CMCC(B)63501],S.aureus[CMCC(B)26003],P.aeruginosa[CMCC(B)10104],E.coli[CMCC(B)44102],C.albicans[CMCC(F)98001],A.niger[CMCC(F)98003] (China Institute for Food and Drug Control).

3 Methods and results

3.1 Preparation of bacterial solution and test solutionS.aureus,P.aeruginosaandB.subtiliswere inoculated into TSB and cultured respectively at 30-35 ℃ for 18-24 h as fresh culture; fresh cultures were taken and 0.9% sterile sodium chloride solution was used to prepare bacterial suspension containing less than 1×104cfu/mL bacteria;C.albicanswas inoculated into SDB and cultured at 20-25 ℃ for 2 to 3 d as fresh culture; the fresh culture was taken and 0.9% sterile sodium chloride solution was used to prepare bacterial suspension containing less than 1×104cfu/mL bacteria;A.nigerwas inoculated into SDB, cultured at 20-25 ℃ for 5-7 d, and the spores were washed with 5 mL of 0.9% sterile sodium chloride solution, the spore suspension was sucked into a sterile test tube, and the spore suspension containing less than 1×104cfu/mL spores was prepared with 0.9% sterile sodium chloride solution. the spore suspension was sucked into a sterile test tube, and the spore suspension containing less than 1×104cfu/mL spores was prepared with 0.9% sterile sodium chloride solution;E.coliwas inoculated into TSB and cultured at 30-35 ℃ for 18-24 h as fresh culture; the fresh culture was taken and made into a bacterial suspension with a bacterial content less than 100 cfu/mL with 0.9% sterile sodium chloride solution; 10 g of Zhideke granule was mixed with pH 7.0 SPB to 100 mL to make 1∶10 test solution, and pH 7.0 SPB was used as diluent to prepare 1∶20 and 1∶50 test solution respectively.

3.2 Applicability test of microbial counting methodIn the experimental group, 10 mL of test solution was put into sterilized test tube, and 0.1 mL of bacterial suspension was added to tube. After shaking well, it was injected into dishes, 1 mL per dish, and 2 dishes were prepared in parallel in the test group of each culture medium, and then injected into the corresponding medium immediately. In the bacterial liquid group, 10 mL of TSB was put into the sterilized test tube, and the other steps were the same as those in the experimental group. Sample control group: the test solution was taken and injected into dishes, 1 mL per dish, and was immediately injected into the corresponding medium. The total number of aerobic bacteria was counted by TSA, and the total number of molds and yeasts was counted by SDA. After the culture medium was solidified, TSA was cultured at the temperature of 30-35 ℃. The culture time of bacteria was less than 3 d, the culture time ofC.albicansandA.nigerwas less than 5 d, and the culture time of SDA was not more than 5 d at 20-25 ℃. The recovery ratio=(the number of colonies in the test group-the number of colonies in the control group)/the number of colonies in the control group, the range was 0.5-2.0.S.aureusandC.albicanswere selected as sensitive strains for pre-test. It can be seen from Table 1 that in the group of 1∶20 test solution (1 mL per dish), the bacteria recovery ratio ofS.aureuswas 0.80, which was in the range of 0.5-2.0, indicating that it was feasible to count the total number of aerobic bacteria by dilution plate method of test solution (1∶20 test solution, 1 mL per dish).

Table 1 Pre-test results of microbial counting method suitability test

In the group of 1∶10 test solution (1 mL per dish), the recovery ratio ofC.albicanswas 1.12, which was in the range of 0.5-2.0, indicating that Zhideke granule had no inhibitory effect on fungi, and it was feasible to count the total number of molds and yeasts by conventional plate method (1∶10 test solution, 1 mL per dish). According to the results of the pre-test, the dilution plate method for the test solution (1∶20 test solution, 1 mL per dish) was determined as the method for counting the total number of aerobic bacteria in Zhideke granules. The conventional plate method (1∶10 test solution, 1 mL per dish) was the method for counting the total number of molds and yeasts. Three batches of Zhideke granule samples were collected and the recovery test was conducted on the total number of aerobic bacteria according to the dilution plate method for the test solution (1∶20 test solution, 1 mL per dish) and the total number of molds and yeasts according to the conventional plate method (1∶10 test solution, 1 mL per dish). The results are shown in Table 2.

3.3 Applicability test of objectionable organisms test method

Using the conventional enrichment method, 10 mL of 1∶10 test solution was inoculated into 100 mL TSB. At the same time,E.coli

Table 2 Recovery ratio results of three batches of Zhideke granules

(no more than 100 cfu) was added and cultured at 30-35 ℃ for 18-24 h as a culture. 1 mL of culture was inoculated into 100 mL MAC and cultured at 42-44 ℃ for 24-48 h. The liquid culture of MAC was taken and inoculated on MAC plate and cultured at 30-35 ℃ for 18-72 h as the experimental group, and the colony morphology was observed.S.aureuswas taken as the negative control test bacteria and cultured by the same method as the negative control group. After being separated and cultured on MAC plate, theE.coliin the experimental group was pink, round, neat at edges, smooth and moist; there was no colony growth forS.aureusin the negative control group. It can be seen that the conventional method of TTB (1∶10 test solution, 10-100 mL TSB) was feasible to detectE.coliin Zhideke granules. Three batches of Zhideke granule samples were taken, and the recovery test was conducted onE.coliaccording to the conventional method of TTB, and then separated and cultured on MAC plate. After that, the colony was observed, and the result was the same as above. The results showed that using the conventional method of TTB (1∶10 test solution, 10-100 mL TSB) for the determination ofE.coliin Zhideke granules was in line with the relevant provisions ofChinesePharmacopoeiain 2015 (microbial limit test for non-aseptic products: objectionable organisms test), and the method was feasible.

3.4 Test of microbial limits of samplesThe total number of aerobic bacteria was determined by dilution method (1∶20 test solution), the total number of molds and yeasts was determined by conventional method (1∶10 test solution), and the objectionable organisms were detected by conventional method of TTB (1∶10 test solution, 10-100 mL TSB). Three batches of samples were tested. It can be seen from Table 3 that the microbial limit test method of Zhideke granules was as follows: dilution method (1∶20) for the total number of aerobic bacteria, conventional method (1∶10) for the total number of molds and yeasts, and conventional method of TTB (1∶10 test solution, 10-100 mL TSB) for objectionable organisms. The established method was in line with the regulations and could be used for the detection of microbial limits in Zhideke granules.

Table 3 Microbial limit test results of three batches of Zhideke granules

4 Discussion

After the verification of the pre-experimental method, the microbial limit test of three batches of Zhideke granules showed that noE.coliwas detected, the total number of aerobic bacteria was less than 20 cfu/g, and the total number of molds and yeasts was less than 15 cfu/g. After verification, the microbial limit test method of Zhideke granules established in this paper can be used for the microbiological examination of the product. The microbial limit test of three batches of Zhideke granules are in line with the provisions of the 2015 edition ofChinesePharmacopoeia. Oral solid preparations of traditional Chinese medicine are generally composed of a variety of medicinal materials, and if the medicinal materials contain bacteriostatic agents, it may affect the accuracy of microbial limit test methods. Therefore, theChinesePharmacopoeiastipulates that methodological applicability tests should be carried out before test to ensure the accuracy, reliability and completeness of the results. In the study on the applicability of microbiological detection methodology of samples, when the 1∶10 test solution (1 mL per dish) was used, the inhibitory activity againstS.aureuswas obvious, and the recovery ratio was 0.43, which was lower than the limit set in the 2015 edition ofChinesePharmacopoeia(0.5-2.0), indicating that the test solution still had antibacterial activity when the dilution ratio was 1∶10. When the sample concentration was diluted to 1∶20 in the test solution (1 mL per dish), the recovery ratio ofS.aureuswas 0.80, which was in the range. This showed that it was feasible to count the total number of aerobic bacteria by dilution plate method (1∶20 test solution, 1 mL per dish). Oral solid preparation of traditional Chinese medicine has a certain bacteriostatic effect, which will interfere with the detection of microorganisms to a certain extent. By means of methodological applicability test, appropriate methods can be selected to eliminate the influence of bacteriostatic components on microbial detection[7-9].E.colican be detected in three batches of Zhideke granules in the test group, but not in the negative control group, indicating that the conventional bacteriostatic solution can be used to detect the objectionable organisms in Zhideke granules. Some compound preparations of traditional Chinese medicine have many flavors and large amounts, and their pharmacological activities will be changed after preparation. Therefore, it is necessary to establish and verify the microbial limit test method of traditional Chinese medicine compound preparation, so as to minimize the influence of its own antibacterial activity on the pollution degree of drug preparation, simplify the test steps and improve the test efficiency.