Biological challenge device with combined adjustable resistance for evaluating sterilization process effect

Abstract

The invention discloses a biological challenge device with combined adjustable resistance for evaluating the effect of a sterilization process, which comprises a shell, the shell comprises a plastic pipe and an inner pipe cap detachably connected with the inner wall of the plastic pipe, the inner pipe cap is provided with a breathable window and covered by a breathable film, and a bacterial contamination carrier for bearing indicator biological spores is attached to the inner bottom of the plastic pipe. An openable container containing a liquid culture medium is arranged in the plastic pipe cavity; the outer side of the inner pipe cap is sleeved with an outer pipe cap detachably connected with the outer wall of the plastic pipe, and the outer pipe cap is provided with a flow blocking structure used for adjusting the speed of the sterilizing agent entering the area of the inner pipe cap; the indicator biological spores are pretreated by a stable protective agent containing one or more of polyethylene glycol, trehalose, sorbitol, polyvinylpyrrolidone and glyceryl stearate. The speed of a sterilizing agent entering the device can be limited, the spores are dry in a suspension added with a stable protective agent, the capability of resisting general environmental changes is very good, and the performance of the device can be maintained for a long time.

Description

Technical Field

[0001] This invention belongs to the field of sterilization indicator technology, and specifically relates to a biochallenge device with combined adjustable resistance for evaluating the effectiveness of the sterilization process. Background Technology

[0002] In clinical practice, sterilization failure of medical devices can lead to serious nosocomial infections and even endanger lives. Therefore, sterilization monitoring is one of the core tasks of CSSD (Clinical Sterilization Management) quality control. Traditional sterilization methods such as steam sterilization and ethylene oxide sterilization rely on physical or chemical means to kill microorganisms, but their effectiveness needs to be verified by biological indicators (BI). BI, as the "gold standard" of sterilization effectiveness, reflects the reliability of the sterilization process through the survival or death status of live microorganisms and is a key technology for ensuring the sterility level of medical devices.

[0003] Steam sterilization is a physical method of sterilizing heat-resistant items using high-temperature saturated steam (typically in the range of 121°C to 135°C). Its core principle is based on the latent heat and penetrability of steam, achieving sterilization by destroying the protein and enzyme systems of microorganisms. Sterilization effectiveness is closely related to temperature and time: at 121°C, instruments need to be exposed for at least 30 minutes to ensure sterilization, while at 132°C, this can be reduced to 3-4 minutes. However, sterilization effectiveness is affected by various factors, including temperature fluctuations in the sterilizer, steam penetration, and the way instruments are loaded. Although steam sterilization is highly efficient, proper operating procedures and continuous monitoring remain crucial to ensuring sterilization quality.

[0004] Biological indicators use *Bacillus stearothermophilus* as the strain, whose resistance characteristics make it the "gold standard" for sterilization effectiveness. Traditional bioindicators (BI) use pH indicators to detect growth, requiring up to 24 hours for the nutrient composition to come into contact with the indicator organism and for a detectable number of microorganisms to be produced, which cannot meet the need for real-time feedback for rapid sterilization results.

[0005] Rapid detection technology using the related enzyme activities generated by this technology can reduce the time required to detect viable indicator organisms. Compared to traditional pH detection technology, the rapid detection technology developed based on spore enzyme activity detection can shorten the time to test for detectable numbers of indicator organisms to less than 3 hours.

[0006] The hospital primarily uses biochallenge test kits prepared from indicators, as they have stronger resistance than the bioindicators themselves and can monitor longer sterilization cycles to ensure the effectiveness of the sterilization process. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a biochallenge device with adjustable combined resistance for evaluating the effectiveness of sterilization processes. The device includes a detachable double-cap system with a specially designed structure, indicator spores pretreated with a stabilizing agent, and a supporting device for rapidly detecting the effectiveness of the sterilization process using these spores. This device is simple in structure and easy to use. Switching between the indicator and the challenge device is achieved by installing and removing the outer cap, effectively shortening the detection time for the bioindicator. The detection results show good consistency with the spore growth results.

[0008] The technical solution provided by this invention is as follows:

[0009] This invention provides a biochallenge device with adjustable combined resistance, comprising a housing, which includes a plastic tube and an inner cap detachably connected to the inner wall of the plastic tube. The inner cap has a venting window covered by a venting membrane. A contaminating carrier for indicator spores is attached to the bottom of the plastic tube. An openable container containing liquid culture medium is provided inside the plastic tube. An outer cap detachably connected to the outer wall of the plastic tube is fitted around the inner cap. The outer cap has a flow-blocking structure for adjusting the rate at which a sterilizing agent enters the area of ​​the inner cap. The indicator spores are pretreated with a stabilizer containing one or more of polyethylene glycol, trehalose, sorbitol, polyvinylpyrrolidone, and glyceryl stearate.

[0010] Furthermore, the concentration of the stabilizer used is 5~30 mg / ml.

[0011] Furthermore, the flow-blocking structure includes one or more vents formed on the outer cap, the number, size, and / or shape of which are selected to provide predetermined device resistance.

[0012] Furthermore, the inner side of the vent is provided with a baffle to increase the complexity of the sterilizing agent's entry path.

[0013] Furthermore, the pore size of the breathable membrane is no greater than 0.5 μm, and the material of the breathable membrane is selected from filter paper, Tyvek paper, or dialysis paper.

[0014] Furthermore, the openable container is an ampoule with both ends sealed by fusion.

[0015] Furthermore, the detachable connection between the plastic tube and the inner cap, and between the plastic tube and the outer cap, is achieved through a slot or thread.

[0016] Furthermore, the liquid culture medium comprises bacteriological peptone, sodium chloride, bromocresol purple, enzyme substrate, distilled water, and amino acids.

[0017] Furthermore, the enzyme substrate is 4-methylumbelliferone-aD-glucopyranoside, and the amino acid is selected from one or more of alanine, valine, and methionine.

[0018] Furthermore, the indicator microbial spore is a thermophilic strobilus spore.

[0019] Beneficial effects

[0020] The biochallenge device of this invention uses indicator spores pretreated with stabilizing agents, which exhibit longer stability and increased resistance to sterilizing agents compared to untreated raw spores. Some of the stabilizers, such as trehalose, provide stability to membranes and other macromolecular structures essential for cell survival under extreme environmental conditions; others, such as polyvinylpyrrolidone, can create an artificial membrane around the spores, enhancing their resistance to environmental changes. Furthermore, this invention uses a double-cap design, with the outer cap featuring flow-blocking structures to regulate the rate at which sterilizing agents enter the inner cap region. These structures include smaller vents, fewer vents, and baffles inside the vents, increasing the difficulty for sterilizing gases to enter the device and significantly improving its resistance.

[0021] The components of this invention exist independently and will not interfere with each other before formal use, exhibiting excellent and reliable performance. The breathable membrane ensures that the bacterial carrier and the openable container containing liquid culture medium will not be contaminated by other microorganisms before use.

[0022] During use, steam enters the interlayer between the two caps through the vent of the outer cap, then passes through the vent membrane between the inner cap and the plastic tube to enter the inner chamber of the plastic tube and make full contact with the bacterial substrate. This biochallenge device can effectively participate in the steam sterilization process, and after sterilization, detection results can be obtained within 3 hours using a fluorescence detection instrument. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the inner and outer cap structures of the present invention;

[0024] Figure 2 This is a schematic diagram of the structure of the biological indicator of the present invention;

[0025] Figure 3 This is a schematic diagram of the biochallenge device of the present invention;

[0026] Figure 4 This is an exploded view of the biochallenge device of the present invention.

[0027] Explanation of reference numerals in the attached diagram: 1. Plastic tube; 2. Infecting carrier; 3. Openable container; 4. Breathable membrane; 5. Inner cap; 6. Outer cap. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0029] like Figure 2 and Figure 3 As shown, this embodiment of the invention provides a biochallenge device with adjustable combined resistance, including a shell, which includes a plastic tube 1 and an inner cap 5 detachably connected to the inner wall of the plastic tube 1. The inner cap 5 has a venting window and is covered by a venting membrane 4. A contamination carrier 2 carrying indicator spores is attached to the bottom of the plastic tube 1. An openable container 3 containing liquid culture medium is provided inside the cavity of the plastic tube 1. An outer cap 6 detachably connected to the outer wall of the plastic tube 1 is fitted outside the inner cap 5. The outer cap 6 has a flow-blocking structure for adjusting the rate at which the sterilizing agent enters the area of ​​the inner cap 5. Both the inner cap 5 and the outer cap 6 are opaque, high-temperature resistant cylindrical plastic caps with one open end. The indicator spores are pretreated with a stabilizer containing one or more of polyethylene glycol, trehalose, sorbitol, polyvinylpyrrolidone, and glyceryl stearate. This stabilizer can form a barrier layer between the spores and the external environment to maintain the long-term stability of the spores or dilute the sterilizing agent to protect the spores.

[0030] Specifically, the plastic tube 1 is a semi-transparent, frosted, cylindrical, high-temperature resistant plastic tube open at one end. The inner cap 5 is a high-temperature resistant cylindrical plastic cap open at one end with a ventilation window on the side. The inner cap 5 is tightly bonded to the inside of the plastic tube. A breathable membrane 4 is provided between the plastic tube 1 and the inner cap 5. This breathable membrane 4 allows air to pass through while preventing bacteria from passing through; that is, the membrane material with a pore size not exceeding approximately 0.5 micrometers is used. This membrane material can be filter paper, Tyvek paper, dialysis paper, etc. The outer cap 6 is a high-temperature resistant cylindrical plastic cap open at one end, designed with a special structure to control the rate at which sterilizing gas enters the device. The outer cap 6 is tightly bonded to the outside of the plastic tube 1. When the outer cap 6 is removed, the device functions as a biological indicator. After the outer cap 6 is installed... The device is a biochallenge device; the openable container containing liquid culture medium is placed inside the plastic tube 1, below the inner cap 5; the plastic tube 1 and the inner and outer caps 6 can be made of polymers such as polycarbonate (PC), polypropylene (PP), polyphenylene ether (PPE), polyethylene (PE), polystyrene (PS), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA or acrylic resin), acrylonitrile-butadiene-styrene copolymer (ABS), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polysulfone (PSU), polyethersulfone (PES), polyetherimide (PEI), polybutylene terephthalate (PBT), etc., or combinations thereof.

[0031] In this embodiment, the stabilizing agent is added to the spore suspension beforehand, and the concentration of the stabilizing agent used is 5-30 mg / ml. Preferably, the concentration used is 10-25 mg / ml.

[0032] In this embodiment, the flow-blocking structure includes one or more vents formed on the outer cap 6, the number, size, and / or shape of which are selected to provide predetermined device resistance. Figure 1 and 4 ).

[0033] In this embodiment, a baffle is provided on the inner side of the vent to increase the complexity of the sterilizing agent's entry path.

[0034] Specifically, the vents on the outer cap 6 can be located on the side or top. By designing the number, size, and shape of the vents, the speed at which the sterilizing agent enters the interlayer between the two caps through the outer cap 6 can be adjusted, as can the speed at which the sterilizing agent enters the inner cap 5, the speed at which the contaminated carrier 2 contacts the sterilizing agent, and the resistance of the biochallenge device. For example, smaller vents and fewer vents can improve the resistance of the biochallenge device. A baffle is also designed on the inner side of the vent of the outer cap 6, so that the sterilizing agent impacts the baffle after entering the vent. This design can also slow down the speed at which the sterilizing agent enters the inner cap 5. It should be understood that the baffle is one implementation example of this design, and its form can be any design that increases the difficulty for the sterilizing agent to enter the inner cap 5 from the vent of the outer cap 6, such as an increased tortuous path.

[0035] In this embodiment, the pore size of the breathable membrane 4 is no greater than 0.5 μm, and the material of the breathable membrane 4 is selected from filter paper, Tyvek paper, or dialysis paper. A breathable membrane 4 is provided between the plastic tube 1 and the inner cap 5. This breathable membrane 4 allows air to pass through while preventing bacteria from passing through; that is, the membrane material with a pore size not greater than approximately 0.5 μm can be filter paper, Tyvek paper, dialysis paper, etc.

[0036] In this embodiment, the openable container 3 is an ampoule with both ends sealed by welding. The length after sealing is suitable for placement inside the biological indicator device. Before use, one end is sealed, and an appropriate amount of liquid culture medium is added in a sterile environment. The capacity of the openable container 3 can be 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0.7ml, etc., preferably 0.5ml, 0.6ml, or 0.7ml. After adding the liquid culture medium, the other end is also sealed. The liquid culture medium is stored in a sealed environment, and the entire openable container 3 is sterilized. It should be understood that "openable" in this invention refers to breaking the openable container 3 by external force, including but not limited to squeezing, to allow the liquid culture medium to come into contact with the indicator organism.

[0037] In this embodiment, the detachable connection between the plastic tube 1 and the inner cap 5, and between the plastic tube 1 and the outer cap 6, is achieved through a slot or thread. The housing used in this invention includes a plastic tube 1, an outer cap 6, and an inner cap 5. The inner cap 5 can be tightly connected to the inside of the plastic tube 1, and the outer cap 6 can be tightly connected to the outside of the plastic tube. The tight connection methods include, but are not limited to, slots, threads, etc.

[0038] In this embodiment, the liquid culture medium includes bacteriological peptone, sodium chloride, bromocresol purple, enzyme substrate, distilled water, and amino acids.

[0039] In this embodiment, the enzyme substrate is 4-methylumbelliferone-aD-glucopyranoside, and the amino acid is selected from one or more of alanine, valine, and methionine.

[0040] Specifically, the liquid culture medium used in this invention consists of bacteriological peptone, sodium chloride, bromocresol purple, enzyme substrate, distilled water, amino acids, etc. The pH of the liquid culture medium can be 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, etc., with preferred pH values ​​of 7.2, 7.3, 7.4, and 7.5. Although a higher pH value is not conducive to spore recovery, it is beneficial for the detection of detectable signals generated by the enzyme substrate. The liquid culture medium is normally sealed in a container and does not come into contact with the indicator organism until the container is opened. The enzyme substrate used in this invention is 4-methylumbelliferone-aD-glucopyranoside, which can react with α-glucosidase to produce a detectable fluorescent signal. The amino acid is one or more of alanine, valine, and methionine.

[0041] In this embodiment, the indicator spore is *Bacillus stearothermophilus* spore, and the predetermined quantity is 1 × 10⁻⁶. 5 ~1×10 6 CFU. The *Bacillus stearothermophilus* spores are derived from ATCC 7953. The spore activity-related enzyme is α-glucosidase, which reacts with its substrate to produce a detectable fluorescent signal. *Bacillus stearothermophilus* spores exhibit suitable resistance to moist heat sterilization; compared to vegetative cells, spores show stronger resistance to moist heat sterilization. To obtain suitable resistance to moist heat sterilization, the number of indicator organisms added to the contaminated carrier 2 should be at least 1 × 10⁻⁶. 5 .

[0042] In this embodiment, the bacterial carrier 2 used can be made of fibrous material or polypropylene material, cut into a suitable size, such as a square piece of 0.5×2.5cm. After the indicator organism is dripped on, it is placed at the bottom of the plastic tube 1. The placement method includes, but is not limited to, curling it into an arc shape or placing it flat at the bottom.

[0043] In this embodiment, the environment used includes, but is not limited to, pressure steam sterilization processes, as the indicator organism can also be used to assess the effectiveness of hydrogen peroxide sterilization processes. Taking pressure steam sterilization processes as an example, sterilization cycles at temperatures such as 121°C, 132°C, 134°C, and 135°C, including but not limited to bottom exhaust, pre-vacuum, and pulsed vacuum sterilization cycles, can all utilize this invention. During sterilization, steam passes through two caps and a breathable membrane into the biological indicator, ensuring good contact between the indicator organism and the steam during sterilization. It should be understood that the above sterilization temperatures and processes are not intended to limit the invention, but are merely examples to aid understanding by those skilled in the art.

[0044] The present invention will be further illustrated by specific embodiments below, which will help those skilled in the art to further understand the present invention.

[0045] Example 1: Culture of *Bacillus stearothermophilus* spores

[0046] Amplification medium. The indicator organism was amplified using 40 ml of tryptone soybean broth medium and cultured at 60°C for 18 h.

[0047] Spore formation plates. The preparation method for spore formation plates is as follows: 5g beef extract powder, 3g tryptone, 3g yeast powder, 0.1g manganese sulfate, and 20g agar are dissolved in 1L of pure water, sterilized at 121℃, and then poured into sterile petri dishes to cool.

[0048] Spore incubation and collection. In a biosafety cabinet, amplified indicator organisms were inoculated onto spore-forming plates and incubated at 60°C for 3 days. Spores were scraped from the plates and suspended in sterile distilled water. The suspension was centrifuged at 7000 rpm and 4°C for 20 minutes to separate the spores from other debris. The supernatant was discarded, and the spores were resuspended in sterile distilled water. This cleaning process was repeated several times to obtain clean spores.

[0049] Example 2: Openable container configuration

[0050] Liquid culture medium. The liquid culture medium contains bacteriological peptone, sodium chloride, bromocresol purple, enzyme substrate, distilled water, and amino acids. A preferred liquid culture medium formulation contains 15g of bacteriological peptone, 9g of sodium chloride, 0.02g of bromocresol purple, 0.01g of 4-methylumbelliferone-aD-glucopyranoside, 0.03g of alanine, 0.03g of valine, and 0.03g of methionine per 1L of liquid culture medium, adjusted to pH 7.3 with sodium hydroxide. In a clean environment, 0.5ml of the prepared liquid culture medium is injected into an ampoule sealed at one end, sealed, sterilized, and stored. When needed, the ampoule is placed inside a biological indicator to indicate the rapid recovery of organisms.

[0051] Example 3 Preparation of biological indicators

[0052] Preparation of the inoculum carrier. Spores of the indicator organism were harvested according to Example 1. The spores required heat activation treatment before use to prolong their viability. Based on the counting results, 1×10⁻⁶ spores were... 5 ~1×10 6A quantity of spores is dropped onto a contamination carrier in a biosafety cabinet. The contamination carrier can be made of materials such as Whatman filter paper, glass fiber filter paper, or polypropylene. Taking Whatman filter paper as an example, it is cut to fit the inner wall of a plastic tube, coated with a certain quantity of indicator spores, and then left to air dry overnight.

[0053] Biological indicator assembly. The biological indicator assembly process is as follows. It should be understood that this process is only to help those skilled in the art understand this embodiment and not to limit the invention:

[0054] (1) Bend the bacterial carrier and place it on the bottom of the inner wall of the plastic tube.

[0055] (2) The openable ampoule containing liquid culture medium prepared according to Example 2 is placed inside the plastic tube, with the bottom of the ampoule passing through the channel formed by the bending of the bacterial carrier and contacting the bottom of the plastic tube.

[0056] (3) The breathable membrane is attached to the breathable window of the inner tube cap to form the inside and outside of the biological indicator, blocking bacteria from entering.

[0057] (4) Tightly connect the inner tube cap to the plastic tube.

[0058] Example 4: Preparation of biological indicators with added stabilizers

[0059] Similar to Example 3, except that the spore suspension was supplemented with polyvinylpyrrolidone and trehalose at a final concentration of 25 mg / ml before being added dropwise.

[0060] Example 5: Preparation of the Biochallenge Device

[0061] Similar to Example 3, except that the outer cap is tightly bonded to the plastic tube, building upon Example 3.

[0062] Example 6: Incomplete sterilization cycle of biological indicators by pressurized steam sterilization

[0063] The biological indicator prepared according to Example 3 was exposed to different sublethal sterilization cycles at 121°C, and the resulting detectable signals were captured and analyzed by an independently developed fluorescence reader. The reader displayed the detection results after 3 hours of incubation of the biological indicator and compared them with the results of 24 hours of isothermal incubation of spore growth. The test results are shown in the table below, and the results show that the 3-hour test results are in good agreement with the 24-hour test results.

[0064] Table 1: Fluorescence test results

[0065]

[0066] Table 2: Spore growth results

[0067]

[0068] Example 7: A biological indicator with added stabilizer was subjected to a cyclic sterilization process involving incomplete pressure steam sterilization.

[0069] Similar to Example 6, except that the biological indicator was prepared according to Example 4, the test results showed that the biological indicator with added stabilizing agent had higher resistance and the 3h test results were in good agreement with the 24h test results.

[0070] Table 3: Fluorescence test results

[0071]

[0072] Table 4: Spore growth results

[0073]

[0074] Example 8: Biochallenge device undergoes incomplete pressure steam sterilization cycle sterilization process

[0075] Similar to Example 6, except that the biological indicator was replaced with the biological challenge device prepared according to Example 5. The results showed that the biological challenge device had higher resistance than the biological indicator.

[0076] Table 5: Spore growth results

[0077]

[0078] Example 9: The Influence of the Number and Diameter of Holes in the Outer Cap on the Resistance of the Biochallenge Device

[0079] In this embodiment, circular holes with diameters of 1 mm and 2 mm were used, both located on the side of the outer cap. The number of holes was one and two, respectively. Five biochallenge devices were prepared according to the method in Example 5. These devices were exposed at 121°C for 14 minutes and then incubated at a constant temperature for 24 hours before observing spore growth. The results show that smaller ventilation windows and fewer ventilation windows can improve the resistance of the biochallenge device.

[0080] Table 6: Spore growth results

[0081]

[0082] Example 10: A biochallenge device with added stabilizer undergoes a cyclic sterilization process involving incomplete pressure steam sterilization.

[0083] Biochallenge devices with added stabilizers were prepared according to Examples 4 and 5. The prepared bioindicators were exposed to different sublethal sterilization cycles at 121°C, and the resulting detectable signals were captured and analyzed by an independently developed fluorescence reader. The reader displayed the detection results after 3 hours of incubation of the bioindicators and compared them with the results of 24 hours of isothermal incubation of spore growth. The test results are shown in the table below. The results show that the biochallenge device prepared with added stabilizers has the highest resistance under the same sterilization conditions, and the 3-hour test results are in good agreement with the 24-hour test results.

[0084] Table 7: Fluorescence test results

[0085]

[0086] Table 8: Spore growth results

[0087]

[0088] In this invention, the amount of stabilizing agent added, the number of holes in the outer cap, the hole diameter, the shape of the holes (not shown in the examples), and the position of the holes are combined to realize a combined adjustable resistance biochallenge device. The simple switching between the indicator and the challenge device is realized by installing and removing the outer cap.

[0089] The specific embodiments of the present invention have been described in detail above, but the scope of the present invention is not limited to the above embodiments. For those skilled in the art, various adjustments, modifications, or substitutions can be made to the embodiments without departing from the core principles and spirit of the present invention, but these modifications still fall within the protection scope of the present invention.

Claims

1. A biochallenge device with combined adjustable resistance for evaluating the effectiveness of a sterilization process, characterized in that, The device includes a shell comprising a plastic tube and an inner cap detachably connected to the inner wall of the plastic tube. The inner cap has a venting window covered by a venting membrane. A contaminating carrier for indicator spores is attached to the bottom of the plastic tube. An openable container containing liquid culture medium is located inside the plastic tube. An outer cap detachably connected to the outer wall of the plastic tube is fitted around the inner cap. The outer cap has a flow-blocking structure for regulating the rate at which the sterilizing agent enters the area of ​​the inner cap. The indicator spores are pretreated with a stabilizer containing one or more of polyethylene glycol, trehalose, sorbitol, polyvinylpyrrolidone, and glyceryl stearate.

2. The biochallenge device with combined adjustable resistance for evaluating the effectiveness of a sterilization process according to claim 1, characterized in that, The concentration of the stabilizer is 5-30 mg / ml.

3. The biochallenge device with combined adjustable resistance for evaluating the effectiveness of a sterilization process according to claim 1, characterized in that, The flow-blocking structure includes one or more vents formed on the outer cap, the number, size and / or shape of which are selected to provide predetermined device resistance.

4. The biochallenge device with combined adjustable resistance for evaluating the effectiveness of a sterilization process according to claim 3, characterized in that, The inside of the vent is provided with a baffle to increase the complexity of the sterilizing agent's entry path.

5. The biochallenge device with combined adjustable resistance for evaluating the effectiveness of a sterilization process according to claim 1, characterized in that, The pore size of the breathable membrane is no greater than 0.5 μm, and the material of the breathable membrane is selected from filter paper, Tyvek paper, or dialysis paper.

6. The biochallenge device with combined adjustable resistance for evaluating the effectiveness of a sterilization process according to claim 1, characterized in that, The openable container is an ampoule with both ends sealed by fusion.

7. The biochallenge device with combined adjustable resistance for evaluating the effectiveness of a sterilization process according to claim 1, characterized in that, The detachable connection between the plastic tube and the inner cap, and between the plastic tube and the outer cap, is achieved through a slot or thread.

8. The biochallenge device with combined adjustable resistance for evaluating the effectiveness of a sterilization process according to claim 1, characterized in that, The liquid culture medium includes bacteriological peptone, sodium chloride, bromocresol purple, enzyme substrate, distilled water, and amino acids.

9. The biochallenge device with combined adjustable resistance for evaluating the effectiveness of a sterilization process according to claim 8, characterized in that, The enzyme substrate is 4-methylumbelliferone-aD-glucopyranoside, and the amino acid is selected from one or more of alanine, valine, and methionine.

10. The biochallenge device with combined adjustable resistance for evaluating the effectiveness of a sterilization process according to claim 1, characterized in that, The indicator spore is a thermophilic strobilus spore.

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