Comparative Study Of Microbial Limit Test And Criteria Of TCM Decoction Pieces Among United States Pharmacopeia，European Pharmacopeia，Japanese Pharmacopeia And Chinese Pharmacopeia Ⅱ

2 Control bacteria inspection

Control bacteria inspection is an important item in the inspection of Chinese herbal medicines. The control bacteria inspection items commonly stipulated in the four pharmacopoeias include bile-resistant Gram-negative bacteria, Escherichia coli and Salmonella [10-13]. The comparison of their inspection methods is shown in Table 2. It should be pointed out that USP43 classifies bile-resistant Gram-negative bacteria in the microbial count inspection "2021". In addition, the inspection method of Staphylococcus aureus was added to USP43 and JP17; USP43 also supplemented the inspection method of Clostridium and was the first to propose the concept of unacceptable microbial risk assessment. In addition, USP43 only introduced the principle of culture medium suitability test in the control bacteria inspection method of Chinese herbal medicines, and did not clarify the specific operation method, nor did it require the conduct of method suitability test; while EP10.0, JP17 and ChP2020 all gave a relatively detailed introduction to the culture medium suitability test and method suitability test.

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2.1 Bile-resistant Gram-negative bacteria

In the pretreatment, unlike other pharmacopoeias that use TSB medium as diluent, USP43 adds pH 7.2 PBS as diluent. Although the requirements for sample recovery time are slightly different in each pharmacopoeia, they all require recovery for at least 2 h. In the bacterial enrichment stage, EP10.0 uses 4 test sample inoculation concentrations of 0.1, 0.01, 0.001, and 0.000 1 g (or mL), which are inoculated intoMEEB medium respectively, so that the number of bile-resistant Gram-negative bacteria that can be detected by this method reaches 1×104 CFU/g (or CFU/mL).
Compared with the method of other pharmacopoeias using 3 test sample inoculation concentrations of 0.1, 0.01, and 0.001 g (or mL), the detection limit of the EP10.0 method is increased by 1 order of magnitude. The methods used in selective separation of various pharmacopoeias are consistent, and VRBGA medium is used to identify suspicious colonies, but the culture temperature and culture time are slightly different.

 

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2.2 Escherichia coli

EP10.0 and JP17 respectively stipulate the qualitative and quantitative test methods for Escherichia coli, while USP43 and ChP2020 only stipulate the qualitative test methods. In the qualitative test method, the detection principles of each pharmacopoeia are consistent. The test solution is enriched with TSB medium, inoculated into MacConkey broth medium, and then separated with selective medium. Among them, USP43 uses MacConkey agar medium for separation and culture for 18 to 24 hours, and EMB medium can be used for Escherichia coli identification; the other three pharmacopoeias require the use of MacConkey agar medium for separation and culture for 18 to 72 hours, and the target bacteria are determined by appropriate identification methods. JP17 can additionally use Escherichia coli chromogenic agar medium instead of MacConkey agar medium to separate and culture samples. In the quantitative test method, both EP10.0 and JP17 use the MPN method with three test sample inoculation concentrations of 0.1, 0.01, and 0.001 g (or mL) to check Escherichia coli in samples, providing more method options for control bacteria inspection.

 

2.3 Salmonella

In terms of sampling volume, except for EP10.0, which requires 25 g (or 25 mL) of the sample to be cultured in 225 mL of buffered peptone aqueous solution, the other pharmacopoeias all stipulate that 10 g (or 10 mL) of the test sample should be added to TSB medium. In the selective bacterial enrichment stage, USP43, EP10.0 and ChP2020 all use RV medium at 30-35 °C, while JP17 raises the culture temperature to (42±0.5) °C to increase selectivity. In the separation stage, the USP43 method can select one or more of BGA, XLD or HE medium; EP10.0 and ChP2020 only require the use of XLD medium, and JP17 can also use Salmonella chromogenic agar medium. In the identification of suspicious microorganisms, USP43 screens the target bacteria through the three-sugar iron test; EP10.0 and JP17 use appropriate methods to judge; ChP2020 integrates the above two identification methods, which is more flexible and reasonable.

 

2.4 Other control bacteria

JP17 and USP43 both specify the test method for Staphylococcus aureus, while EP10.0 and ChP2020 do not specify this item. JP17 stipulates that after the test sample is cultured in TSB medium, it needs to be transferred to TSB medium containing 7.5% sodium chloride for re-culture to inhibit background bacteria; USP43 only uses TSB medium for single bacterial enrichment. When separating selective culture media, JP17 and USP43 use VJ medium and BP medium in addition to MS medium. When identifying suspicious colonies, USP43 uses plasma coagulase test, while JP17 can use appropriate methods for identification. In addition, USP43 also provides a Clostridium detection method, which is the same as the microbiological inspection method for non-sterile products under USP43 "62".

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3 Microbiological limit standards

3.1 EP10.0

EP10.0 "5.1.8" divides Chinese herbal medicine pieces and their extracts into three categories: samples for boiling water treatment (A), samples treated by other processes (B), and samples that cannot meet the requirements of B after other processes (C) [10].
For Class A samples, the limit standard for TAMC count is 1×107 CFU/g (or CFU/mL), and the limit standard for TYMC count is 1×105 CFU/g (or CFU/mL); at the same time, the maximum acceptable count results are set on this basis, expressed as a factor of 5. The most stringent standard for product counting items is Class B products, whose limit standards for TAMC count are 1×104 CFU/g (or CFU/mL) and TYMC count are 1×102 CFU/g (or CFU/mL). Except for Class A samples, which do not need to be checked for bile-resistant Gram-negative bacteria, Class B and Class C samples must be checked for TAMC, TYMC counts and bile-resistant Gram-negative bacteria, Escherichia coli and Salmonella[10], see Table 3 for details.

 

3.2 JP17

JP17 "G4" divides Chinese herbal medicine pieces and their extracts into two categories: samples treated with boiling water (Class I) and samples that are used directly without going through the extraction process (Class II)[11]. In comparison, Class I products in JP17 are equivalent to Class A products in EP10.0, and Class II in JP17 is similar to Class C in EP10.0. However, JP17 does not specify the limit standards for Class B products in EP10.0, see Table 3 for details.

 

3.3 USP43

Compared with the classification of decoction pieces in EP10.0 and JP17, USP43 has made a more detailed classification of products containing Chinese medicine (plant medicine) in the microbial limit standard "2023" for non-sterile nutrition and dietary supplements. There are 7 categories, including dried or ground plants, ground plant extracts, etc., as shown in Table 4. Among them, the requirements for dried or ground plant samples are TAMC count ≤ 1×105 CFU/g (or CFU/mL), TYMC count ≤ 1×103 CFU/g (or CFU/mL), and Escherichia coli and Salmonella must not be detected per 10 g. This requirement is higher than the requirements of EP10.0 and JP17 for products of the same classification.

 

3.4 ChP2020

Based on the 2015 edition, ChP2020 "General Rule 1108" revised the microbial limit standards for direct oral and soaked Chinese herbal medicines, and added the provisions for TAMC, TYMC counts and Escherichia coli. Except for the TYMC count requirement of 1×103 CFU/g, the rest are the same as the microbial limit standards for Class C samples in EP10.0 (see Table 3). However, ChP2020 still has not established a unified limit standard for the control bacteria inspection of Chinese herbal medicine extracts. It can be seen that the requirements of ChP2020 for the microbial limits of Chinese herbal medicines, whether it is the classification of Chinese medicines that need to control microorganisms or the microbial limit standards, are not as complete as the other three pharmacopoeias.

 

4 Discussion

The 2015 edition of the Chinese Pharmacopoeia has included some microbial limit standards for Chinese herbal medicine slices, which has played a positive role in guiding Chinese medicine manufacturers to pay attention to microbial contamination and standardize the processing of Chinese herbal medicine slices [15]. However, the overall production level and technical conditions of Chinese herbal medicine slices in my country are far behind those of developed countries. Microbial contamination is still common in products, and conditional pathogens may exist [16-18]. Although most Chinese herbal medicine slices need to be processed by decoction and other processes before use, there are still potential risks in the process of medication, and the residual pathogenic microorganisms may still cause harm to patients [3-4, 8, 17-19]. At present, although ChP2020 has revised the "Microbial Limit Standards for Chinese Herbal Medicine Extracts and Chinese Herbal Medicine Slices", compared with USP43, EP10.0 and JP17, ChP2020's regulations on sample classification, inspection items and limit standards need to be further improved.

Chinese herbal medicines are primary processed products and a special type of medicine. The applicability of their inspection methods is an important link in the process of standard setting [14]. The background contamination of Chinese herbal medicines is high, which greatly interferes with the inspection of control bacteria and has the risk of missed detection [17]. There are large differences in the bacterial enrichment and separation effects of different inspection methods. Taking the inspection of Staphylococcus aureus in JP17 as an example, the use of TSB medium containing 7.5% sodium chloride for bacterial enrichment can help inhibit the growth of background microorganisms in the sample and improve the detection rate of target bacteria. USP43 and JP17 also add the use of a variety of colorimetric culture media, which is also an effective method for distinguishing target bacteria. In addition, all four pharmacopoeias allow the use of alternative microbial inspection methods, such as electrochemical methods, gas inspection methods, chemiluminescence methods, etc. Among them, the detection technology based on molecular biology has demonstrated the advantages of high sensitivity, high specificity and rapid inspection in the fields of medical inspection and food inspection, and can play a positive role in the microbial inspection of Chinese herbal medicines and improve the detection rate of control bacteria in herbal medicines [19-20]. In terms of limit standards, USP43 has the most detailed classification of Chinese medicines and relatively strict requirements for the microbial limit standards of Chinese medicine slices. Although USP43 does not specify the types of unacceptable microorganisms, it requires manufacturers to actively evaluate the unacceptable microorganisms that may exist in the products, especially to control the microorganisms that may cause adverse effects on the products and lead to safety hazards [12]. This concept of risk assessment and risk management is worth learning from and referring to in my country. In summary, this study compared and analyzed the microbial enumeration methods, control bacteria inspection methods and microbial-related limit standards of Chinese medicine slices in four pharmacopoeias. According to the current status of microbial contamination and control of Chinese medicine slices, it is necessary to further improve the microbial inspection and limit standards of Chinese medicine-related products in the pharmacopoeia, continue to collect and organize basic microbial load data, and reasonably guide Chinese medicine manufacturers to strengthen microbial contamination control and management, and ultimately ensure the safety of Chinese medicine products. It is recommended that according to the current status of microbial contamination and control of Chinese medicine slices, the microbial inspection methods and limit standards of Chinese medicine-related products in the pharmacopoeia should be gradually improved, and the microbial limit levels of corresponding products should be reasonably refined.

 

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