A method for tracing microbial contamination in the production process of Sanhuang tablets based on whole life cycle management

Abstract

This invention relates to a method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management. It can quickly pinpoint the source of contamination when microbial contamination is detected in the product. The technical solution includes the following steps: S1: Establishing a basic microbial bank covering the entire life cycle; S2: Establishing a database linking contaminating microorganisms to their source stages; S3: Microbial tracing at the time of contamination; S4: Formulating control measures based on the tracing results. This invention can accurately locate the source of contamination, and the control measures formulated based on the tracing results effectively solve the contamination problem. Subsequent batches have not experienced similar contamination, providing a scientific basis for microbial contamination control for traditional Chinese medicine preparation manufacturers.

Description

Technical Field

[0001] This invention relates to the field of quality control technology in traditional Chinese medicine manufacturing, and in particular to a method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management. Background Technology

[0002] Currently, Chinese medicine (TCM) manufacturers primarily rely on final product testing for microbial contamination, making it difficult to quickly pinpoint the source when contamination occurs. Existing traceability methods often target single stages (such as the environment or raw materials only), lacking a systematic approach that covers the entire chain, including raw materials, excipients, packaging materials, intermediate products, formulations, production environment, facilities and equipment, personnel, and water treatment systems. Sanhuang tablets are a TCM preparation containing raw rhubarb powder (rhubarb powder accounts for approximately 50% of the prescription). Microorganisms during its production can originate from multiple sources, including raw rhubarb powder, production equipment, clean environments, and personnel operations. Once contamination occurs, companies often struggle to quickly and accurately locate the source, leading to production stoppages, losses, and quality risks.

[0003] Current research has reported methods for identifying microbial contaminants in Chinese herbal medicines or preparations (such as the literature "Identification and Source Tracing Study of Common Contaminating Bacillus in Chinese Herbal Medicines by Specific PCR and MLST Methods"). However, these methods only focus on the identification of the contaminants themselves and have not established a microbial bank covering the entire production chain, nor have they established the correlation between "strain-source link", making it impossible to quickly locate the source of contamination during the production process.

[0004] Therefore, how to provide a method for tracing the source of microbial contamination in traditional Chinese medicine preparations that covers the entire life cycle and can quickly and accurately locate the source of contamination is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] In view of the above situation and to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management, which can quickly locate the source of contamination when microbial contamination is detected in the product.

[0006] The technical solution provided by this invention includes the following steps:

[0007] S1: Establish a basic microbial bank covering the entire life cycle.

[0008] Microbial samples were collected from all stages of the Sanhuang tablet production chain, including raw materials, excipients, intermediate products, formulations, clean environments, facilities and equipment, production / inspection personnel, and backflushing waste liquid. The collected microorganisms were isolated, purified, morphologically identified, physiologically and biochemically identified, and molecularly identified. The strain number, species name, source, colony morphology, microscopic morphology, identification method, storage location, and storage conditions were recorded.

[0009] S2: Establish a database linking contaminating microorganisms to their sources.

[0010] All identified strains were classified and statistically analyzed according to their source links to establish a "strain-source link" correlation; the probability of contamination was quantitatively calculated based on the frequency of strain detection: contamination probability = number of times detected at this link ÷ total number of times detected × 100%; the probability ranking was used to form the basis for tracing and to identify the high-risk source links for each strain.

[0011] S3: Microbiological source tracing when contamination occurs

[0012] When excessive microbial levels are detected in the finished or intermediate products of Sanhuang tablets, the following steps should be performed:

[0013] 3.1 Isolate, purify, and identify the contaminating strains to obtain information on their species classification;

[0014] 3.2 Compare and match the information on contaminating bacteria with the associated database from step S2;

[0015] 3.3 Based on the comparison results, output the source of contamination and rank them by probability (e.g., Bacillus spp. → rhubarb powder source probability ≥80%, production equipment probability 15%, personnel probability 5%).

[0016] 3.4 Re-sample and verify the links with the highest probability of origin to confirm the final source of pollution;

[0017] S4: Develop prevention and control measures based on the source tracing results.

[0018] Based on the identified sources of pollution, corresponding control measures will be taken, including but not limited to: strengthening raw material drying and sterilization processes, increasing the frequency and verification of equipment cleaning and disinfection, optimizing personnel hygiene operation procedures, improving the backwashing and disinfection procedures of the water treatment system, and upgrading the clean environment monitoring level.

[0019] In step S1, the raw materials are rhubarb powder, scutellaria extract, berberine hydrochloride, and rhubarb extract.

[0020] In step S1, the excipients are corn starch, talc, magnesium stearate, and sucrose.

[0021] In step S1, the intermediate products are Sanhuang tablet intermediate product and Sanhuang tablets;

[0022] In step S1, the preparation is a coated Sanhuang tablet;

[0023] In step S1, the clean environment includes the solid production workshop (male and female changing rooms, laundry room, weighing room, pulverizing room, preparation room, tableting room, coating room), the traditional Chinese medicine extraction workshop (pulverizing room, sterilization room, spray drying room), and the microbial limit room of the quality control department.

[0024] In step S1, the facilities and equipment include a solid dosage form workshop (pulverizer, granulator, tablet press, coating machine), a traditional Chinese medicine extraction workshop (crusher, stainless steel tray, spray drying tower), and a quality control department (clean workbench, pass-through window).

[0025] In step S1, the production / inspection personnel are: ingredient mixing worker, tableting worker, coating worker, tablet picking worker, inner packaging worker, spray drying worker, and microbiological inspection worker;

[0026] In step S1, the backwash waste liquid is the backwash waste liquid from activated carbon filter, multi-media filter, softener, and quartz sand filter.

[0027] In step S2, Bacillus mainly originates from rhubarb powder, intermediate products of Sanhuang tablets, production equipment (coating machine, pulverizer), and the surface of personnel after operation; Staphylococcus mainly originates from clean environments and the surface of personnel before operation; mold mainly originates from medicinal materials, crushers, and stainless steel pans; and Copper-loving bacteria mainly originate from backflushing waste liquid.

[0028] In step S2, within the established bacterial bank: Bacillus spp. was detected 142 times, of which 113 times (79.6%) came from rhubarb powder, 21 times (14.8%) from the coating machine, and 8 times (5.6%) from personnel after the operation; Microbes oxytetracycline was detected only once, and it came from rhubarb powder, with a probability of 100%; Staphylococcus spp. was detected 48 times, of which 30 times (62.5%) came from the clean environment and 18 times (37.5%) from personnel before the operation.

[0029] The application of the microbial contamination tracing method for the production process of Sanhuang tablets based on full life cycle management described in this invention in the quality testing of traditional Chinese medicine.

[0030] It should be noted that in the method of the present invention, the samples used for constructing the basic microbial bank are completely independent of the samples of the batch to be tested for contamination, and do not include any samples from any stage of the batch to be tested, so as to ensure that the traceability determination is objective, independent and repeatable.

[0031] The beneficial technical effects of this invention are as follows:

[0032] 1. Covering the entire life cycle, a systematic microbial bank from raw materials to final products has been established for the first time (the Sanhuang tablets microbial bank currently contains 610 strains of microorganisms).

[0033] 2. A correlation between "microbial strains and processes" has been established, which can quickly locate pollution sources and shorten the pollution investigation time;

[0034] 3. It can be extended to other Chinese herbal medicines, and has wide applicability;

[0035] 4. To provide scientific basis for the prevention and control of microbial contamination for Chinese medicine preparation manufacturers;

[0036] 5. Verification through actual production batches showed that the method of this invention can accurately locate the source of pollution, and the prevention and control measures formulated based on the source tracing results effectively solved the pollution problem, with no similar pollution occurring in subsequent batches. Attached Figure Description

[0037] Figure 1 This is a distribution map showing the main bacterial strains in the bacterial library of this invention.

[0038] Figure 2 This is a schematic diagram of the pollution source tracing process of the present invention.

[0039] Figure 3 These are colony morphology photographs of some strains of the present invention on TSA.

[0040] Figure 4 These are microscope images showing the morphology of some strains of the present invention. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1

[0043] This invention validated the effectiveness of the method for tracing the source of microbial contamination in Sanhuang tablets. Following step 1 of the technical solution, microorganisms were collected and identified at each stage of the entire Sanhuang tablet production chain. A total of 610 microbial strains were identified in the microbial bank, covering samples, clean environments, facilities and equipment, personnel, and backflushing waste liquid. The source, species name, and identification method of each strain were recorded in the microbial bank. The dominant microbial species distribution at each stage was as follows: Bacillus spp. accounted for 67.5% in the samples, Staphylococcus spp. in the clean environment, Bacillus spp. in the backflushing waste liquid, Bacillus spp. in the production equipment, and Staphylococcus spp. accounted for approximately 44% before personnel operation and approximately 27% after operation. Relevant data are as follows:

[0044] (I) Microbial collection and identification at each stage of production

[0045] 1. Sample

[0046] The applicability of the method was tested on the raw materials (rhubarb powder, rhubarb extract, berberine hydrochloride, scutellaria baicalensis extract), excipients (magnesium stearate, talc, corn starch, sugar powder), Sanhuang tablet intermediate products, Sanhuang tablets, Sanhuang tablet coated tablets, and Sanhuang tablets. Microbial limit tests were performed according to the established method, and the detected microorganisms were isolated, purified, and identified.

[0047] 2. Clean environment

[0048] The following facilities were monitored for airborne bacteria, settled bacteria, and surface microorganisms in the Sanhuang tablet production workshop: male and female changing rooms, laundry room, inner packaging material storage room, weighing room 2, pulverizing room 2 and 3, preparation room 1, tablet transfer station, tablet compression room 1, coating room 3, drying room, and heat sealing room 2; the rhubarb pulverizing room and scutellaria extract pulverizing room in the traditional Chinese medicine extraction workshop; and the clean changing room and microbial limit testing room in the quality control department. The collected microorganisms were isolated, purified, and identified.

[0049] 3. Backflushing waste liquid

[0050] Waste liquids from multi-media filters, quartz sand filters, activated carbon filters, sodium ion exchangers, and softeners were collected, and the microorganisms in the waste liquids were separated, purified, and identified.

[0051] 4. Production equipment

[0052] Surface microbial samples were collected from various equipment in the Sanhuang tablet production workshop, including material shovels, pulverizers, wet granulators, stainless steel troughs, fluidized bed dryers, oscillating granulators, fixed lifting conveyors, three-dimensional motion mixers, tablet presses, coating machines, and aluminum-plastic blister packaging machines. Also collected were pulverizers, crushers, stainless steel trays in the traditional Chinese medicine extraction workshop, and clean benches and pass-through windows in the quality control department. The collected microorganisms were then isolated, purified, and identified.

[0053] 5. Personnel

[0054] Surface microbial samples were taken from the operator's five-finger gloves, wrists, elbows, and other areas that are prone to contact with materials and pose a high risk, for identification and analysis.

[0055] (II) Microbial load at each stage of sample production

[0056] The microbial load of each stage of the sample production process was investigated, including raw materials and excipients for Sanhuang tablets, cleanroom processes, buffer waste liquid, production equipment, and personnel. The results are shown in Tables 1 to 8.

[0057] Table 1. Microbial load of raw and auxiliary materials for Sanhuang tablets

[0058]

[0059] Table 2 Clean Environment of Quality Control Department

[0060]

[0061] Table 3. Airborne and Settled Bacteria in Solid Workshop

[0062]

[0063] Table 4 Microorganisms in backflushing waste liquid

[0064]

[0065] Table 5 Microorganisms in production equipment of solid production workshop

[0066]

[0067] Table 6. Surface microorganisms in the traditional Chinese medicine extraction workshop (Sabbar's glucose agar medium with antibiotics)

[0068]

[0069] Table 7. Surface microorganisms in the traditional Chinese medicine extraction workshop (tryptic soy peptone agar medium)

[0070]

[0071] Table 8 Microorganisms on Personnel Surface

[0072]

[0073] (III) Microbial identification results

[0074] 1. Sample

[0075] A total of 211 bacterial strains were identified, including 142 strains of *Bacillus*, 30 strains of *Bacillus-like*, 9 strains of *Neuralgia*, 7 strains of *Microbes*, 6 strains of *Priscilla*, 6 strains of mold, 6 strains of *Microbacterium*, 2 strains of *Enterobacter*, 1 strain of *Microbacterium*, 1 strain of *Pantheraea*, and 1 strain of *Staphylococcus*. *Bacillus* was the most abundant strain, accounting for 67.3%. Results are shown in Table 9. Figure 1 .

[0076] Table 9. Microbial identification results of samples

[0077] Rhubarb powder Curtobacterium flaccumfaciens (a type of short, wilting fungus) Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Paenibacillus lautus Rhubarb powder Priestia megaterium Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Cytobacillus horneckiae Rhubarb powder Cytobacillus horneckiae Rhubarb powder Cytobacillus horneckiae Rhubarb powder Cellulosimicrobium cellulans (cellulose-producing bacteria) Rhubarb powder Cellulosimicrobium cellulans (cellulose-producing bacteria) Rhubarb powder Bacillus circulans Rhubarb powder Cellulosimicrobium cellulans (cellulose-producing bacteria) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus subtilis / amyloliquefaciens / atrophaeus (Bacillus subtilis / amyloliquefaciens / atrophaeus) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Cellulosimicrobium cellulans (cellulose-producing bacteria) Rhubarb powder Priestia megaterium Rhubarb powder Cellulosimicrobium cellulans (cellulose-producing bacteria) Rhubarb powder Niallia circulans Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Priestia megaterium Rhubarb powder Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Paenibacillus lautus Rhubarb powder Paenibacillus cineris (a type of Bacillus cereus) Rhubarb powder Priestia megaterium Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Niallia circulans Rhubarb powder Microbacterium paraoxydans (a type of microbacterium) Rhubarb powder Microbacterium paraoxydans (a type of microbacterium) Rhubarb powder Enterobacter hormaechei Rhubarb powder Enterobacter asburiae Rhubarb powder Bacillus altitudinis / pumilus (High / Short Bacillus) Rhubarb powder Mold (Aspergillus foetidus / costaricensis / niger) Rhubarb powder Mold (Penicillium polonicum / aurantiocandidum) Rhubarb powder Mold (Neobulgaria koningiana (NCBI) / Byssochlamys sp (mycobank)) Rhubarb powder Mold (Aspergillus keveii) Rhubarb powder Mold (Aspergillus megasporus / mallochii) Rhubarb powder Mold (Aspergillus keveii) rhubarb extract Microbacterium oxydans rhubarb extract Staphlococcus cohnii ssp cohnii (Staphylococcus cohnii) rhubarb extract Paenibacillus cineris (a type of Bacillus cereus) talcum powder Bacillus altitudinis / pumilus (High / Short Bacillus) talcum powder Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Niallia circulans Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets intermediate product Niallia circulans Sanhuang tablets intermediate product Paenibacillus lautus Sanhuang tablets intermediate product Niallia circulans Sanhuang tablets intermediate product Paenibacillus lautus Sanhuang tablets intermediate product Bacillus cereus group Sanhuang tablets intermediate product Paenibacillus lautus Sanhuang tablets intermediate product Paenibacillus lautus Sanhuang tablets intermediate product Paenibacillus lautus Sanhuang tablets intermediate product Paenibacillus cineris (a type of Bacillus cereus) Sanhuang tablets intermediate product Paenibacillus cineris (a type of Bacillus cereus) Sanhuang tablets intermediate product Paenibacillus pabuli (Feed Bacillus) Sanhuang tablets intermediate product Paenibacillus lautus Sanhuang tablets intermediate product Bacillus licheniformis Sanhuang tablets intermediate product Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets intermediate product Niallia circulans Sanhuang tablets intermediate product Paenibacillus lautus Sanhuang tablets intermediate product Paenibacillus cineris (a type of Bacillus cereus) Sanhuang tablets Pantoea agglomerans (clustered pantoea) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / atrophaeus (Bacillus subtilis / amyloliquefaciens / atrophaeus) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / atrophaeus (Bacillus subtilis / amyloliquefaciens / atrophaeus) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / atrophaeus (Bacillus subtilis / amyloliquefaciens / atrophaeus) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / atrophaeus (Bacillus subtilis / amyloliquefaciens / atrophaeus) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / atrophaeus (Bacillus subtilis / amyloliquefaciens / atrophaeus) Sanhuang tablets Cellulosimicrobium cellulans (cellulose-producing bacteria) Sanhuang tablets (coated tablets) Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets (coated tablets) Paenibacillus cineris (a type of Bacillus cereus) Sanhuang tablets (coated tablets) Paenibacillus cineris (a type of Bacillus cereus) Sanhuang tablets (coated tablets) Paenibacillus lautus Sanhuang tablets (coated tablets) Niallia circulans Sanhuang tablets (coated tablets) Microbacterium paraoxydans (a type of microbacterium) Sanhuang tablets (coated tablets) Microbacterium paraoxydans (a type of microbacterium) Sanhuang tablets (coated tablets) Paenibacillus cineris (a type of Bacillus cereus) Sanhuang tablets (coated tablets) Niallia circulans Sanhuang tablets (coated tablets) Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets (coated tablets) Paenibacillus cineris (a type of Bacillus cereus) Sanhuang tablets (coated tablets) Paenibacillus cineris (a type of Bacillus cereus) Sanhuang tablets (coated tablets) Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets (coated tablets) Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets (coated tablets) Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets (coated tablets) Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets (coated tablets) Paenibacillus cineris (a type of Bacillus cereus) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Microbacterium paraoxydans (a type of microbacterium) Sanhuang tablets Paenibacillus lautus Sanhuang tablets Paenibacillus lautus Sanhuang tablets Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets Bacillus altitudinis / pumilus (High / Short Bacillus) Sanhuang tablets Bacillus altitudinis / pumilus 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subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Cellulosimicrobium cellulans (cellulose-producing bacteria) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / atrophaeus (Bacillus subtilis / amyloliquefaciens / atrophaeus) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / atrophaeus (Bacillus subtilis / amyloliquefaciens / atrophaeus) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / atrophaeus (Bacillus subtilis / amyloliquefaciens / atrophaeus) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / atrophaeus (Bacillus subtilis / amyloliquefaciens / atrophaeus) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis) Sanhuang tablets Paenibacillus lautus Sanhuang tablets Bacillus subtilis / amyloliquefaciens / atrophaeus (Bacillus subtilis / amyloliquefaciens / atrophaeus) Sanhuang tablets Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis / amyloliquefaciens / vallismortis)

[0078] Microorganisms were detected in rhubarb powder, intermediate product of Sanhuang tablets and Sanhuang tablets in the samples, while no microorganisms were detected in rhubarb extract, berberine hydrochloride and scutellaria extract, and few microorganisms were detected in excipients.

[0079] 2. Clean environment

[0080] A total of 61 bacterial strains were identified, including 31 *Staphylococcus* strains, 19 *Coccobacillus* strains, 17 *Micrococcus* strains, 4 *Bacillus* strains, 3 *Agrobacterium* strains, 2 *Escherichia* strains, 2 *Acinetobacter* strains, 2 *Bacillus* strains, 1 *Bacillus* strain, 1 *Pseudomonas* strain, and 1 *Microbacterium* strain. *Staphylococcus* accounted for the largest proportion (47.5%). Results are shown in Table 10. Figure 1 .

[0081] Table 10 Microorganisms in Clean Environments

[0082]

[0083] 3. Backflushing waste liquid

[0084] A total of 57 microorganisms were identified, including 12 strains of *Aeromonas*, 10 strains of *Aeromonas*, 8 strains of *Pseudomonas*, 4 strains of *Xanthomonas*, 5 strains of *Escherichia*, 3 strains of *Mycobacterium*, 2 strains of *Bacillus*, 2 strains of *Acidobacterium*, 2 strains of *Microbacterium*, 2 strains of *Ralstonia*, 2 strains of *Sphingosoma*, 1 strain of *Fibromonas*, 1 strain of *Trichomonas*, 1 strain of *Acinetobacter*, 1 strain of *Coxella*, and 1 strain of *Morganella*. *Aeromonas* accounted for the largest proportion, at 21.1%. The identification results are shown in Table 11 and... Figure 1 .

[0085] Table 11 Microorganisms in Backflushing Waste Liquid

[0086]

[0087] 4. Production equipment

[0088] A total of 93 bacterial strains were identified, including 32 strains of *Bacillus*, 11 strains of *Microbacterium*, 9 strains of *Staphylococcus*, 8 strains of *Stenotrophomonas*, 7 strains of *Priscilla*, 6 strains of *Ailuropoda*, 4 strains of *Micrococcus*, 4 strains of *Bacillus* spp., 2 strains of *Enterobacter*, 2 strains of *Escherichia*, 2 strains of *Cocheria*, 1 strain of *Lysinic Bacillus*, 1 strain of *Brugia*, 1 strain of *Aquaticus*, 1 strain of *Pseudomonas*, 1 strain of *Sphingospora*, and 1 strain of *Vibrio*. The results are shown in Table 12. Figure 1 .

[0089] Table 12 Microbial Identification Results of Production Equipment

[0090]

[0091] 5. Personnel

[0092] A total of 188 bacterial strains were identified, including 50 *Bacillus* strains, 48 ​​*Staphylococcus* strains, 25 *Micrococcus* strains, 13 *Escherichia* strains, 10 *Priscilla* strains, 7 *Cocheria* strains, 5 *Dermatophytes* strains, 4 *Ailuropoda* strains, 3 *Stenotrophomonas* strains, 3 *Klebsiella* strains, 3 *Bacillus* strains, 3 *Xanthomonas* strains, 2 *Fibromonas* strains, 1 *Achromobacter* strain, 1 *Bacillus brevis* strain, 1 *Brevibacter* strain, 1 *Corynebacterium* strain, 1 *Fusobacterium* strain, 1 *Squirrelococcus* strain, 1 *Microbacterium* strain, 1 *Cladosporium* strain, 1 *Pseudomonas* strain, 1 *Rhodococcus* strain, 1 *Rochetomyces* strain, and 1 *Alcaligenes* strain. *Bacillus* accounted for the largest proportion, at 26.6%. Results are shown in Table 13. Figure 1 .

[0093] Table 13 Results of Microbial Identification on Personnel Surface

[0094] Personnel 1 wrist-1 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 1 finger - 1 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 1 finger - 2 Bacillus cereus group Personnel 2 wrist-1 Microccoccus luteus Personnel 2 wrists - 2 Priestia megaterium Personnel 2 wrists - 3 Kytococcus sedentarius (Sitococcus) Personnel 2 wrists - 4 Brucella (Ochrobactrum) anthropi (human pale bacillus) Personnel 2 wrists - 5 Staphylococcus haemolyticus Personnel 2 wrists - 6 Staphylococcus haemolyticus Personnel 2 wrist-7 Staphylococcus ureilyticus Personnel 2 wrists - 8 Staphylococcus haemolyticus Personnel 2 wrist-9 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 2 wrist-10 Brucella (Ochrobactrum) anthropi (human pale bacillus) Personnel 2 wrist-11 Kytococcus sedentarius (Sitococcus) Personnel 2 wrist-12 Priestia megaterium Personnel 2 wrist-13 Staphylococcus haemolyticus Personnel 2 wrist postpartum -1 Staphylococcus haemolyticus Personnel 2 wrist postpartum -2 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 2 wrist postpartum - 3 Staphylococcus haemolyticus Personnel 2 wrist postpartum - 4 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 2 wrist postpartum - 5 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 2 wrist postpartum - 6 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 2 wrist postpartum - 7 Staphylococcus haemolyticus Personnel 2 wrist postpartum - 8 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 2 wrist postpartum -9 Microccoccus luteus Personnel 2 wrist postpartum -10 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 2 wrist postpartum - 11 Escherichia coli Personnel 2 wrist postpartum -12 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 2 wrist postpartum - 13 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 2 wrist postpartum - 14 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 2 wrist postpartum -15 Staphylococcus haemolyticus Personnel 2 wrist postpartum -16 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 2 fingers after production - 1 Brevibacillus spp. (Brief Bacillus spp.) Personnel 2 fingers after production - 2 Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis, Bacillus amyloliquefaciens) Personnel 2 fingers after production - 3 Stenotrophomonas maltophilia Personnel 2 fingers after production - 4 Escherichia coli Personnel 2 elbow postpartum -1 Klebsiella pneumoniae Personnel 2 elbow after production -2 Pectinatus frisingensis Personnel 2 elbow after production - 3 Klebsiella pneumoniae Personnel 3 wrists - 1 Staphylococcus warneri Personnel 3 wrists - 2 Staphylococcus epidermidis Personnel 3 wrists - 3 Staphylococcus warneri Personnel 3 wrists - 4 Staphylococcus warneri Personnel 3 wrists - 5 Microccoccus luteus Personnel 3 wrists - 6 Kytococcus schroeteri (Schroeteri) Personnel 3 elbows - 1 Microccoccus luteus Personnel 3 elbows - 2 Microccoccus luteus Personnel 3 elbows - 3 Microccoccus luteus Personnel 3 elbows - 4 Kocuria carniphila (Flesh-loving Coccus) Personnel 4 fingers - 1 Cellulosimicrobium cellulans (cellulose-producing bacteria) Personnel 4 elbows - 1 Cellulosimicrobium cellulans (cellulose-producing bacteria) Personnel 5 elbows - 1 Microccoccus luteus Personnel 5 elbows - 2 Staphylococcus hominis (human staphylococcus) Personnel 6 elbows - 1 Paenibacillus glucanolyticus (a type of glycolytic bacterium) Personnel 7 wrists - 1 Microccoccus luteus Personnel 7 wrists - 2 Brevibacterium sanguinis Personnel 7 fingers - 1 Staphylococcus hominis (human staphylococcus) Personnel 7 elbows - 1 Pseudoxanthomonas mexicana (Mexican Pseudomonas) Personnel 7 elbows -2 Kytococcus sedentarius (Sitococcus) Personnel 7 elbows - 3 Bacteroides cellulosilyticus Personnel 7 elbows -4 Pseudoxanthomonas mexicana (Mexican Pseudomonas) Personnel 7 elbow - 5 Pseudoxanthomonas mexicana (Mexican Pseudomonas) Personnel 8 elbows - 1 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 8 elbows - 2 Priestia fiexa (Bacillus curvaturei) Personnel 8 elbows - 3 Niallia circulans Personnel 9 wrists - 1 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 9 wrists - 2 Kocuria carniphila (Flesh-loving Coccus) Personnel 9 wrists - 3 Escherichia coli Personnel 9 wrists - 4 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 9 wrists after delivery -1 Kocuria palustris (swamp kocuria) Personnel 9 wrists after delivery -2 Kocuria palustris (swamp kocuria) Personnel 9 fingers - 1 Staphylococcus epidermidis Personnel 9 fingers - 2 Priestia fiexa (Bacillus flexuralis) Personnel 9 fingers - 3 Escherichia coli Personnel 9 fingers - 4 Kocuria palustris (swamp kocuria) Personnel 9 fingers - 5 Kytococcus sedentarius (Sitococcus) Personnel with 9 fingers after childbirth -1 Stenotrophomonas rhizophila Personnel with 9 fingers after childbirth - 2 Stenotrophomonas rhizophila Personnel 9 elbow postpartum -1 Kocuria rhizophila Personnel 9 elbows after delivery -2 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 10 fingers - 1 Brucella (Ochrobactrum) anthropi (human pale bacillus) Personnel 10 fingers - 2 Microccoccus luteus Personnel 10 fingers - 3 Staphylococcus epidermidis Personnel 10 fingers - 4 Staphylococcus ureilyticus Personnel 10 elbows - 1 Rhodococcus erythropolis Personnel 10 elbows - 2 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 10 elbows - 3 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 11 wrist-1 Priestia megaterium Personnel 11 fingers - 1 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 12 elbows - 1 Niallia circulans Personnel 12 elbows - 2 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 12 elbows - 3 Niallia circulans Personnel 13 wrist-1 Achromobacter denitrificans Personnel 13 fingers - 1 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 13 fingers - 2 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 13 elbow - 1 Mammaliicoccus sciuri (Squirrel Staphylococcus) Personnel 13 elbows -2 Escherichia coli Personnel 13 elbows - 3 Pseudomonas stutzeri Personnel 13 elbows - 4 Paenibacillus lactis (Lactobacillus) Personnel 13 elbow -5 Staphylococcus hominis (human staphylococcus) Personnel 13 elbows - 6 Staphylococcus hominis (human staphylococcus) Personnel 13 elbows -7 Paenibacillus lactis (Lactobacillus) Personnel 14 wrist-1 Staphylococcus equorum (Horse Staphylococcus) Personnel 14 fingers - 1 Klebsiella pneumonLae (Klebsiella pneumoniae) Personnel 14 fingers - 2 Staphylococcus hominis (human staphylococcus) Personnel 14 fingers after delivery -1 Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis, Bacillus amyloliquefaciens) Personnel 14 fingers after delivery - 2 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 15 wrist-1 Microccoccus luteus Personnel 15 fingers - 1 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 15 fingers - 2 Staphylococcus haemolyticus Personnel 15 fingers - 3 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 15 fingers - 4 Priestia fiexa (Bacillus curvaturei) Personnel 15 elbow - 1 Microccoccus luteus Personnel 15 elbow - 2 Fusobacterium nucleatum (a type of microbacterium) Personnel 15 elbow - 3 Microccoccus luteus Personnel 15 elbow -4 Microccoccus luteus Personnel 15 elbow - 5 Microccoccus luteus Personnel 15 elbow - 6 Microccoccus luteus Personnel 15 elbow -7 Microccoccus luteus Personnel 15 elbow - 8 Microccoccus luteus Personnel 15 elbow -9 Corynebacterium mucifaciens / ureicelerivorans (Mucolytic / Rapid Ureaplasma) Personnel 15 elbow - 10 Cytobacillus firmus (Strong Fiber Bacillus) Personnel 15 elbow - 11 Microccoccus luteus Personnel 15 elbow - 12 Cytobacillus firmus (Strong Fiber Bacillus) Personnel 15 elbow - 13 Metabacillus idriensis (Bacillus from the Institute of Pathogenic Biology) Personnel 15 elbow - 14 Rothia kristinae Personnel 15 elbow - 15 Microccoccus luteus Personnel 15 elbow - 16 Microccoccus luteus Personnel 16 wrist-1 Microccoccus luteus Personnel 16 fingers - 1 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 16 fingers - 2 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 16 fingers - 3 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 16 elbow - 1 Kocuria carniphila (Flesh-loving Coccus) Personnel 16 elbow - 2 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 16 elbow - 3 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 16 elbows -4 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 17 wrist-1 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 17 wrist-2 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 17 wrist-3 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 17 fingers - 1 Escherichia coli Personnel 17 fingers - 2 Escherichia coli Personnel 17 fingers - 3 Escherichia coli Personnel 17 fingers - 4 Escherichia coli Personnel 17 fingers - 5 Escherichia coli Personnel 17 fingers - 6 Escherichia coli Personnel 17 fingers - 7 Escherichia coli Personnel 17 elbow - 1 Staphylococcus haemolyticus Personnel 17 elbow -2 Staphylococcus haemolyticus Personnel 17 elbow -3 Staphylococcus haemolyticus Personnel 17 elbow -4 Staphylococcus haemolyticus Personnel 17 Elbow -5 Staphylococcus haemolyticus Personnel 17 elbow -6 Staphylococcus hominis (human staphylococcus) Personnel 18 wrist-1 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 18 wrists - 2 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 18 wrists - 3 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 18 wrists - 4 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 18 wrist postpartum -1 Bacillus cereus group Personnel 18 wrist postpartum - 2 Bacillus licheniformis Personnel 18 wrist postpartum - 3 Bacillus licheniformis Personnel 18 wrist postpartum - 4 Bacillus licheniformis Personnel 18 wrist postpartum - 5 Bacillus licheniformis Personnel 18 wrist postpartum - 6 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 18 wrist postpartum - 7 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 18 wrist postpartum - 8 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 18 fingers - 1 Microbcterium flavescens / laevaniformans (Pale Yellow / Fructan-producing Microbacterium) Personnel 18 fingers - 2 Escherichia coli Personnel 18 fingers after delivery -1 Microccoccus luteus Personnel 18 fingers after delivery - 2 Brucella (Ochrobactrum) anthropi (human pale bacillus) Personnel 18 elbow-1 Staphylococcus epidermidis Personnel 18 elbow - 2 Microccoccus luteus Personnel 18 elbow - 3 Staphylococcus epidermidis Personnel 18 elbows -4 Staphylococcus epidermidis Personnel 19 wrist-1 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 19 wrist-2 Priestia megaterium Personnel 19 elbow-1 Microccoccus luteus Personnel 19 elbow - 2 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 19 elbow -3 Priestia megaterium Personnel 19 elbow -4 Priestia megaterium Personnel 20 wrist-1 Priestia fiexa (Bacillus flexuralis) Personnel 20 wrist postpartum -1 Microccoccus luteus Personnel 20 wrist postpartum -2 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies) Personnel 20 wrist postpartum - 3 Staphylococcus warneri Personnel 20 fingers - 1 Bacillus subtilis / amyloliquefaciens / vallismortis (Bacillus subtilis, Bacillus amyloliquefaciens) Personnel 20 fingers after delivery - 1 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 20 fingers after delivery - 2 Bacillus altitudinis / pumilus (High / Short Bacillus) Personnel 20 elbow - 1 Staphylococcus saprophyticus Personnel 20 elbow - 2 Staphylococcus saprophyticus Personnel 20 elbow - 3 Staphylococcus cohnii ssp cohnii (Staphylococcus cohnii subspecies)

[0095] (iv) Preliminary analysis of microbial identification results

[0096] 1. Sample

[0097] Bacillus bacteria were the dominant microorganisms in the samples. Bacillus bacteria accounted for the highest proportion in rhubarb powder, intermediate products of Sanhuang tablets, coated Sanhuang tablets, and Sanhuang tablets. This contamination mainly originated from soil pollution caused by rhubarb powder.

[0098] In Sanhuang tablets, some rhubarb is used in the form of raw medicinal powder. Rhubarb is the dried root or rhizome of plants such as Rheum palmatum, Rheum tanguticum, or Rheum palmatum (medicinal variety), which are in close contact with the soil. The medicinal material itself is prone to carrying microorganisms such as Bacillus. Bacillus can produce spores under adverse environmental conditions. Spores are a dormant form of bacteria, exhibiting strong resistance to drought, high temperatures, and ultraviolet radiation. Under suitable conditions, they can revert to vegetative cells and then grow and reproduce. The microorganisms present in the fine rhubarb powder not only increase the risk of microbial contamination in Sanhuang tablet preparations but may also cause contamination of the clean environment, production equipment, and personnel during the production process.

[0099] 2. Clean environment

[0100] Staphylococcus bacteria are the dominant microorganisms in clean environments. Staphylococcus bacteria are widely present in the environment, on human skin, and on mucous membranes, and can contaminate clean environments through human operations and airflow.

[0101] 3. Production equipment

[0102] Bacillus bacteria were the dominant microorganisms in the production equipment. Analysis of the contamination sources suggests possible links to air and dust pollution, introduction of contaminants from raw materials and excipients, and introduction through personnel operations. In equipment microbial monitoring, coating machines 1, 2, and 3 in the solid product production workshop all showed a high number of microorganisms. In the traditional Chinese medicine extraction workshop, stainless steel trays, crushers, and pulverizers all showed a high number of molds. During monitoring, these equipment came into contact with the same batch of rhubarb, suggesting potential contamination between the medicinal material and the production equipment. Furthermore, the drying temperature and time after crushing the medicinal material were insufficient to kill any present molds.

[0103] 4. Backflushing waste liquid

[0104] The microbial community in backflushing wastewater reflects the ecological selection under multiple stresses (metals, organic matter, etc.) in the system. *Copper-loving bacteria* were detected in multi-media filters, activated carbon filters, and quartz sand filters, and accounted for the highest proportion (21.1%) of the backflushing wastewater microorganisms. Therefore, attention should be paid to the potential risks of pipeline corrosion, heavy metal residues, and biofilm formation caused by these bacteria. Opportunistic pathogens such as *Aeromonas* were also detected in the backflushing wastewater; these microorganisms may pose a potential risk to pharmaceutical systems.

[0105] 5. Personnel

[0106] Analysis of the types of microorganisms on personnel surfaces revealed that Staphylococcus spp. were the most prevalent before operation, while Bacillus spp. were the most prevalent after operation. Analysis of the microbial load on personnel showed a significant increase in microbial numbers after operation, indicating a potential risk of cross-contamination between materials / equipment and personnel during production. Personnel microbial monitoring data showed a high number of microorganisms on personnel's wrists and elbows before operation, suggesting that the effectiveness of ozone disinfection of current sterile workwear needs to be validated and evaluated.

[0107] (V) Analysis of the sources of microbial contamination in Sanhuang tablets

[0108] 1. Microbial contamination was detected in a certain batch of Sanhuang tablets (total aerobic bacteria count 5900 cfu / g; according to the 2025 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 1107, the total aerobic bacteria count should not exceed 10). 3 When the cfu / g was measured, the detected bacteria were isolated, purified, and identified. A total of 14 strains were identified, including 11 strains of Bacillus subtilis / Bacillus septum, 2 strains of Microbacterium oxysporum, and 1 strain of Bacillus septum.

[0109] 2. Microbial bank comparison and traceability

[0110]

[0111] The above-mentioned strains were compared with the established bacterial banks:

[0112] (1) The sources of Bacillus subtilis in the culture bank are: rhubarb powder, coating machine surface, and personnel after operation.

[0113] (2) The source distribution of oxidizing microbacteria in the library was: detected only in rhubarb powder.

[0114] (3) The source distribution of Bacillus pyriformis in the library was: it was detected only in rhubarb extract.

[0115] (4) The number of high / short Bacillus was significantly increased after the operation compared with before the operation.

[0116] According to the bacterial bank statistics, over 80% of *Bacillus subtilis* / *Bacillus pumilus* originated from rhubarb powder, approximately 15% from the coating machine, and less than 5% from personnel. *Microbacterium oxysporum* and *Bacillus sphaeroides* in the bacterial bank both originated from a single step (rhubarb powder or rhubarb extract). Therefore, based on comprehensive analysis, the most likely source of contamination is rhubarb powder (probability > 80%), followed by the coating machine, with a lower probability of human contamination.

[0117] 3. Verification Experiment

[0118] First, we conducted a re-sampling verification of the source with the highest probability of occurrence—rhubarb powder:

[0119] (1) Sampling: Three samples of rhubarb powder were collected from different locations.

[0120] (2) Detection: Microbial culture and identification were performed using the same method.

[0121] (3) Results: Using the same microbial limit test method, the total number of aerobic bacteria in the three samples was 1500 cfu / g, 80 cfu / g and 110 cfu / g, respectively. The colonies on the TSA plates were identified by VITEK MS, and Bacillus subtilis was the absolute dominant bacteria.

[0122] (4) Conclusion: The verification experiment confirmed that rhubarb powder was the main source of pollution, which is consistent with the source tracing conclusion.

[0123] Secondly, the surface of the coating machine was tested, and Bacillus subtilis was detected, confirming the existence of a secondary source of contamination.

[0124] (vi) Prevention and control measures

[0125] The above experiments revealed that: (1) Bacillus spp. were the dominant bacteria in rhubarb powder, with the highest number of bacteria. (2) Staphylococcus spp. were the dominant bacteria in clean environments, with the highest number of bacteria. (3) Bacillus spp. accounted for the highest proportion of bacteria in production equipment. (4) Staphylococcus spp. were the most prevalent bacteria before operation, while Bacillus spp. were the most prevalent bacteria after operation.

[0126] The highest proportion of *Bacillus subtilis* / *Bacillus pumilus* was found in the finished product of Sanhuang tablets. *Bacillus subtilis* / *Bacillus amyloliquefaciens* / *Bacillus oryzae*, *Bacillus scintillans*, *Bacillus moniliformis*, and *Bacillus curvatureis* were also detected. *Bacillus oryzae* and *Bacillus subtilis* / *Bacillus pumilus* were detected in the corresponding batch of rhubarb powder; *Bacillus moniliformis* was detected in rhubarb extract; *Bacillus subtilis* / *Bacillus pumilus* was detected in the excipient talc powder, but the microbial load was low; *Bacillus subtilis* / *Bacillus pumilus* was detected in the coated tablets of Sanhuang tablets. The coating machine in the production equipment had a high microbial load, and *Bacillus subtilis* / *Bacillus subtilis* / *Bacillus amyloliquefaciens* / *Bacillus moniliformis*, and *Bacillus curvatureis* were detected. The number of *Bacillus subtilis* / *Bacillus pumilus* increased significantly after personnel operation compared to before operation, indicating contamination of personnel by microorganisms in materials / equipment. Staphylococcus bacteria were predominant in the clean environment. Analysis of the types and loads of detected microorganisms indicates that the main sources of microorganisms in the production process of Sanhuang tablets are rhubarb powder and production equipment.

[0127] Based on the traceability results, strengthen the microbial control of rhubarb powder and production equipment. Specific measures are as follows:

[0128] (1) Strengthen the microbial control of rhubarb medicinal materials and rhubarb fine powder: extend the washing time of rhubarb medicinal materials (more than 30 minutes); increase the drying temperature of the medicinal materials after washing to 80-85℃, extend the drying time to 6 hours, and increase ultraviolet irradiation to kill residual microorganisms on the surface; reduce the layer thickness to less than 5cm when drying after crushing, and turn it over once every 2 hours.

[0129] (2) Strengthen the cleaning and disinfection of equipment such as coating machines: after removing detachable parts, sterilize them by soaking for 30 minutes; use high-pressure steam sterilization for crusher bags, etc.

[0130] Effect verification:

[0131] (1) Testing was conducted on a batch of Sanhuang tablets produced after the measures were taken;

[0132] (2) The microbiological test results all met the limits specified in the Chinese Pharmacopoeia (total aerobic bacteria <10 cfu / g, no bacteria growing on the plates).

[0133] The above embodiments demonstrate that the tracing method provided by the present invention can:

[0134] (1) Quickly identify the source of contamination by comparing bacterial cultures;

[0135] (2) Verification experiments were conducted to confirm the accuracy of the source tracing conclusions;

[0136] (3) The prevention and control measures formulated based on the source tracing results effectively solved the pollution problem.

[0137] Therefore, the method of the present invention has good accuracy and practicality. This embodiment uses Sanhuang tablets as an example to illustrate the traceability method of the present invention. Those skilled in the art will understand that this method is also applicable to other traditional Chinese medicine preparations; it is only necessary to establish a corresponding microbial bank for the target variety.

[0138] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any modifications or alterations made by those skilled in the art without departing from the scope of the present invention to create equivalent embodiments based on the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. A method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management, characterized in that, Includes the following steps: S1: Establish a basic microbial bank covering the entire life cycle. Microbial samples were collected from all stages of the Sanhuang tablet production chain, including raw materials, excipients, intermediate products, formulations, clean environments, facilities and equipment, production / inspection personnel, and backflushing waste liquid. The collected microorganisms were isolated, purified, morphologically identified, physiologically and biochemically identified, and molecularly identified. The strain number, species name, source, colony morphology, microscopic morphology, identification method, storage location, and storage conditions were recorded. S2: Establish a database linking contaminating microorganisms to their sources. All identified strains were classified and statistically analyzed according to their source links to establish a "strain-source link" correlation; the probability of contamination was quantitatively calculated based on the frequency of strain detection: contamination probability = number of times detected at this link ÷ total number of detections × 100%; the probability ranking was used to form the basis for tracing and to identify the high-risk source links for each strain. S3: Microbiological source tracing when contamination occurs When excessive microbial levels are detected in the finished or intermediate products of Sanhuang tablets, the following steps should be performed: 3.1 Isolate, purify, and identify the contaminating strains to obtain information on their species classification; 3.2 Compare and match the information on contaminating bacteria with the associated database from step S2; 3.3 Based on the comparison results, output the pollution source links and their probability rankings according to probability; 3.4 Re-sample and verify the links with the highest probability of origin to confirm the final source of pollution; S4: Develop prevention and control measures based on the source tracing results. Based on the identified sources of pollution, corresponding control measures will be taken, including strengthening raw material drying and sterilization processes, increasing the frequency and verification of equipment cleaning and disinfection, optimizing personnel hygiene operation procedures, and upgrading the clean environment monitoring level.

2. The method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management according to claim 1, characterized in that, In step S1, the raw materials are rhubarb powder, scutellaria extract, berberine hydrochloride, and rhubarb extract. In step S1, the excipients are corn starch, talc, magnesium stearate, and sucrose. In step S1, the intermediate products are Sanhuang tablet intermediate product and Sanhuang tablet raw tablets; In step S1, the preparation is a coated Sanhuang tablet; In step S1, the clean environment includes the solid production workshop, the traditional Chinese medicine extraction workshop, and the microbial limit room of the quality control department. In step S1, the facilities and equipment include a solid dosage form workshop, a traditional Chinese medicine extraction workshop, and a quality control department. In step S1, the production / inspection personnel are: ingredient mixing worker, tableting worker, coating worker, tablet picking worker, inner packaging worker, spray drying worker, and microbiological inspector; In step S1, the backwash waste liquid is the backwash waste liquid from activated carbon filter, multi-media filter, softener, and quartz sand filter.

3. The method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management as described in claim 2, characterized in that, The clean environment refers to the solid production workshop, which includes male and female changing rooms, a laundry room, a weighing room, a crushing room, a preparation room, a tableting room, and a coating room.

4. The method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management according to claim 2, characterized in that, The Chinese medicine extraction workshop consists of a pulverizing room, a sterilization room, and a spray drying room.

5. The method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management according to claim 2, characterized in that, The solid dosage form workshop includes a pulverizer, a granulator, a tablet press, and a coating machine.

6. The method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management according to claim 2, characterized in that, The Chinese herbal medicine extraction workshop includes a crusher, stainless steel discs, and a spray drying tower.

7. The method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management according to claim 2, characterized in that, The quality control department consists of a clean bench and a pass-through window.

8. The method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management according to claim 1, characterized in that, In step S2, Bacillus mainly originates from rhubarb powder, intermediate products of Sanhuang tablets, production equipment, and the surface of personnel after operation; Staphylococcus mainly originates from clean environments and the surface of personnel before operation; mold mainly originates from medicinal materials, crushers, and stainless steel pans; and Copper-loving bacteria mainly originate from backflushing waste liquid.

9. The method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management according to claim 1, characterized in that, In step S2, in the established bacterial bank: Bacillus spp. was detected 142 times, of which 113 times came from rhubarb powder, 21 times from the coating machine, and 8 times from personnel after the operation; Microbacterium oxysporum was detected only once, and it came from rhubarb powder, with a probability of 100%; Staphylococcus spp. was detected 48 times, of which 30 times came from the clean environment and 18 times from personnel before the operation.

10. The application of the method for tracing the source of microbial contamination in the production process of Sanhuang tablets based on full life cycle management as described in any one of claims 1-9 in the quality testing of traditional Chinese medicine.

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