Preparation method of microbial standard substance for characterizing hydrogen peroxide sterilization performance

Abstract

The application discloses a kind of microbial standard substance preparation methods of characterizing hydrogen peroxide sterilization performance, belong to biological detection technical field, comprising the following steps: S1, raw material selection: including indicating microorganism, carrier, modified chitosan, modified mesoporous nanometer silicon dioxide and tannic acid;S2, preparation composite protective solution: modified chitosan is dissolved in phosphate buffer solution, add tannic acid solution;Again add modified mesoporous nanometer silicon dioxide;S3, preparation spore protection suspension;S4, the inoculation and drying of carrier;S5, packaging and storage;The application solves the problem that microbial standard substance quality value is not accurate, unstable, unreliable in the existing hydrogen peroxide sterilization performance evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology and relates to a method for preparing microbial standard substances that characterize the sterilization performance of hydrogen peroxide. Background Technology

[0002] Hydrogen peroxide low-temperature plasma sterilizers are widely used in the sterilization of heat-sensitive medical devices in medical institutions due to their advantages such as low sterilization temperature, short cycle time, and no toxic residue. Currently, monitoring the sterilization effect of this type of sterilizer mainly relies on biological indicators, such as the rapid biological indicator and its usage method disclosed in existing patent CN113398306A for monitoring sterilization effect. This involves placing a bacterial tablet containing thermophilic stearobacter spores inside the sterilizer, culturing it after sterilization, and observing whether spore growth occurs, thereby making a qualitative judgment of "qualified / unqualified" for a single sterilization process. However, biological indicators can only verify the sterilization effect and cannot quantitatively measure or trace the sterilization performance parameters of the sterilizer, such as the D-value and aseptic assurance level.

[0003] Quantitative evaluation of sterilization performance requires the use of microbial reference materials as a metrological standard. Unlike biological indicators, microbial reference materials require a precisely known and stable number of spores, enabling accurate recovery and counting of surviving spores after treatment with different sterilization doses, and obtaining sterilization kinetic parameters through survival curve fitting. According to JJF 1006-1994 "Technical Specifications for Primary Reference Materials," reference materials must meet three indicators: homogeneity, stability, and accuracy of value assignment, and their expanded uncertainty should be controlled within a reasonable range.

[0004] However, existing technologies face the following challenges in preparing microbial reference materials for evaluating the sterilization performance of hydrogen peroxide plasma: First, spores suffer significant activity loss and batch-to-batch variations due to dehydration damage during carrier drying; second, traditional protective agents such as trehalose and skim milk powder are easily degraded and rendered ineffective under the strong oxidizing environment of hydrogen peroxide plasma, interfering with the accuracy of spore counting; and third, the binding state between spores and the carrier is unstable, resulting in low elution recovery rates and poor repeatability, making it difficult to meet the metrological traceability requirements of reference materials. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing microbial standard substances that characterize the sterilization performance of hydrogen peroxide, thereby solving the problems of inaccurate, unstable, and unreliable quality values ​​of microbial standard substances in the existing hydrogen peroxide sterilization performance evaluation.

[0006] The technical solution adopted in this invention is as follows: a method for preparing microbial standard substances characterizing the sterilization performance of hydrogen peroxide, wherein each tablet contains 1.0 × 10⁻⁶ spores. 5 Up to 1.0×10 6Microbial reference materials with CFU, recovery rate ≥95%, and relative expanded uncertainty ≤10%, including the following steps:

[0007] S1. Raw material selection: including indicator microorganisms, carrier, modified chitosan, modified mesoporous nano-silica, and tannic acid; wherein, the modified chitosan is prepared by modifying carboxymethyl chitosan with dopamine hydrochloride; the modified mesoporous nano-silica is prepared by modifying mesoporous nano-silica with aminosilane and phenylboronic acid; the indicator microorganism is Bacillus stearothermophilus spores, strain number ATCC 7953, spore purity ≥95%, and spore suspension concentration 1.0×10⁻⁶. 8 Up to 1.0×10 9 CFU / mL; the carrier is made of 316L stainless steel sheet with a diameter of 6 mm, a thickness of 0.5 mm, and a surface roughness Ra≤0.8 μm;

[0008] S2. Preparation of composite protective solution: Dissolve modified chitosan in phosphate buffer solution with pH 6.5, add tannic acid solution at a mass ratio of modified chitosan to tannic acid of 10:1, stir at room temperature for 30 minutes; then add modified mesoporous nano silica, ultrasonically disperse for 10 minutes, and continue stirring for 2 hours to obtain composite protective solution;

[0009] S3. Preparation of spore protection suspension: Mix the spore suspension and the composite protection solution at a volume ratio of 1:9, and gently shake for 30 minutes to ensure that the spores are evenly dispersed in the protection system to obtain the spore protection suspension;

[0010] S4. Inoculation and drying of the carrier: Place the stainless steel carrier in a clean bench, use a micropipette to add 10 μL of spore protection suspension to the center of the carrier, and place it in a clean environment at 25℃ and 40-50% relative humidity to dry naturally for 4 hours. After drying, transfer it to a desiccator at 4℃ and less than 30% relative humidity for equilibration for 24 hours.

[0011] S5. Packaging and Storage: Pack the dried standard material flakes into aluminum foil bags, vacuum seal them, and store them at 2-8℃ away from light.

[0012] Furthermore, the modified chitosan is prepared by the following method:

[0013] A1. Dissolve carboxymethyl chitosan in a 2-(N-morpholino)ethanesulfonic acid buffer solution with a pH of 5.5 to obtain solution A. Dissolve dopamine hydrochloride in another of the above buffer solutions to obtain dopamine hydrochloride solution. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to solution A in sequence. Activate at room temperature. Add dopamine hydrochloride solution under nitrogen protection. Stir the reaction at room temperature in the dark. Dialyze and freeze-dry to obtain modified chitosan.

[0014] Furthermore, the modified mesoporous nano-silica is prepared by the following method:

[0015] B1. Dissolve hexadecyltrimethylammonium bromide in deionized water, add 25% ammonia solution, stir to form a homogeneous micelle solution, add tetraethyl orthosilicate, continue stirring to react, transfer to a hydrothermal reactor, hydrothermally treat, cool naturally and centrifuge, wash the precipitate with deionized water and anhydrous ethanol in sequence, disperse in anhydrous ethanol, add 36-38% concentrated hydrochloric acid, reflux and stir, collect by centrifugation, wash, and vacuum dry to obtain mesoporous nano silica;

[0016] B2. After vacuum drying, mesoporous nano-silica is dispersed in anhydrous toluene and ultrasonically dispersed. Under nitrogen protection and magnetic stirring, 3-aminopropyltriethoxysilane is added, and the mixture is heated to reflux. After natural cooling, it is centrifuged, washed successively with anhydrous toluene and anhydrous ethanol, and vacuum dried to obtain the intermediate.

[0017] B3. The intermediate was dispersed in N,N-dimethylformamide and ultrasonically dispersed to obtain a silica suspension. 4-Carboxyphenylboronic acid was dissolved in another N,N-dimethylformamide, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added sequentially to obtain an activated 4-carboxyphenylboronic acid solution. Under nitrogen protection, the activated 4-carboxyphenylboronic acid solution was added to the silica suspension, and the reaction was stirred at room temperature. The mixture was collected by centrifugation and washed sequentially with N,N-dimethylformamide, deionized water and anhydrous ethanol. The mixture was then vacuum dried to obtain modified mesoporous nano-silica.

[0018] Furthermore, the modified mesoporous nano-silica has a particle size of 80-120 nm, a specific surface area of ​​800-1000 m² / g, and an average pore size of 3-4 nm.

[0019] Furthermore, the tannic acid solution is prepared as follows: weigh tannic acid and dissolve it in a phosphate buffer solution with a pH of 6.0, stir at room temperature in the dark until completely dissolved, to obtain a tannic acid solution with a mass concentration of 10 mg / mL, and store it at 4°C in the dark for no more than 7 days.

[0020] Furthermore, the stainless steel carrier undergoes the following pretreatment before use: ultrasonic cleaning with 5% dilute nitric acid, rinsing with deionized water, ultrasonic cleaning with anhydrous ethanol, drying and then wet heat sterilization, and cooling before use in a clean environment.

[0021] Furthermore, the composite protective liquid contains, by total mass, 1.5-2.5% modified chitosan, 0.8-1.2% modified mesoporous nano silica, and 0.15-0.25% tannic acid.

[0022] Furthermore, the method for recovering the standard substance is as follows: the standard substance is placed in a centrifuge tube containing 10 mL of citrate-disodium hydrogen phosphate buffer with a pH of 4.5, and incubated at 37°C with shaking for 30 minutes, followed by vortexing for 2 minutes and sonication for 5 minutes.

[0023] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0024] 1. This invention improves the survival rate and batch stability of spores during the drying process through a composite protection system of modified chitosan and modified mesoporous nano-silica. Modified chitosan forms a protective film on the spore surface, effectively slowing down the dehydration rate, while modified mesoporous nano-silica maintains the local microenvironment humidity through its nanoporous structure. The synergistic effect of the two reduces the activity loss of spores after drying, and controls the coefficient of variation of spore number between batches to within 8%, ensuring the accuracy and consistency of the standard substance quality value.

[0025] 2. The composite protection system constructed in this invention exhibits excellent anti-degradation performance and spore protection ability under the strong oxidizing environment of hydrogen peroxide plasma. The phenolic hydroxyl structures rich in modified chitosan and tannic acid can efficiently scavenge hydroxyl free radicals and protect spores from oxidative damage. Meanwhile, the modified mesoporous nano-silica stabilizes the spores through electrostatic interaction, preventing them from falling off or dispersing under plasma impact. At the same time, the protection system can achieve complete dissociation under specific pH conditions, so that the spore recovery rate is stable at over 95% and the relative expansion uncertainty is controlled within 10%. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort, wherein:

[0027] Figure 1 This is a flowchart of a method for preparing microbial standard substances to characterize the sterilization performance of hydrogen peroxide, according to Embodiment 1 of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments. The components of the embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0029] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0030] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0031] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0032] Example 1: This example provides a method for preparing microbial standard substances characterizing the sterilization performance of hydrogen peroxide. This method is used to prepare a sample with a spore content of 1.0 × 10⁻⁶ per tablet. 5 Up to 1.0×10 6 Microbial reference materials with CFU, recovery rate ≥95%, and relative expanded uncertainty ≤10%, including the following steps:

[0033] S1. Raw material selection: including indicator microorganisms, carrier, modified chitosan, modified mesoporous nano-silica, and tannic acid; wherein, the modified chitosan is prepared by modifying carboxymethyl chitosan with dopamine hydrochloride; the modified mesoporous nano-silica is prepared by modifying mesoporous nano-silica with aminosilane and phenylboronic acid; the indicator microorganism is Bacillus stearothermophilus spores, strain number ATCC 7953, with a spore purity of 96% and a spore suspension concentration of 5.0 × 10⁻⁶. 8CFU / mL; the carrier is made of 316L stainless steel sheet with a diameter of 6 mm, a thickness of 0.5 mm, and a surface roughness Ra of 0.6 μm;

[0034] S2. Preparation of composite protective solution: Dissolve modified chitosan in phosphate buffer solution with pH 6.5, add tannic acid solution at a mass ratio of modified chitosan to tannic acid of 10:1, stir at room temperature for 30 minutes; then add modified mesoporous nano silica, ultrasonically disperse for 10 minutes, and continue stirring for 2 hours to obtain composite protective solution;

[0035] S3. Preparation of spore protection suspension: Mix the spore suspension and the composite protection solution at a volume ratio of 1:9, and gently shake for 30 minutes to ensure that the spores are evenly dispersed in the protection system to obtain the spore protection suspension;

[0036] S4. Inoculation and drying of the carrier: Place the stainless steel carrier in a clean bench, use a micropipette to add 10 μL of spore protection suspension to the center of the carrier, and place it in a clean environment at 25°C and 45% relative humidity to dry naturally for 4 hours. After drying, transfer it to a desiccator at 4°C and less than 30% relative humidity for equilibration for 24 hours.

[0037] S5. Packaging and Storage: Pack the dried standard material flakes into aluminum foil bags, vacuum seal them, and store them at 2-8℃ away from light.

[0038] The modified chitosan was prepared by the following method:

[0039] A1. Dissolve 5g of carboxymethyl chitosan in 250mL of 2-(N-morpholino)ethanesulfonic acid buffer solution with pH 5.5 to obtain solution A. Separately, dissolve 3.65g of dopamine hydrochloride in 50mL of the above buffer solution to obtain dopamine hydrochloride solution. Add 2.87g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.73g of N-hydroxysuccinimide to solution A sequentially. Activate at room temperature for 30 minutes. Under nitrogen protection, add dopamine hydrochloride solution dropwise over 30 minutes. Stir and react at room temperature in the dark for 24 hours. Dialyze the reaction solution through a dialysis bag with a molecular weight cutoff of 8000 to 14000 Daltons. First, dialyze with hydrochloric acid solution with pH 4.0 for 24 hours, changing the dialysate every 6 hours. Then, dialyze with deionized water for 48 hours, changing the dialysate every 8 hours. Freeze-dry to obtain modified chitosan and store at -20℃ in a sealed container in the dark.

[0040] The modified mesoporous nano-silica was prepared by the following method:

[0041] B1. Dissolve 2g of hexadecyltrimethylammonium bromide in 960mL of deionized water, add 7mL of 25% ammonia solution, and stir at 35℃ for 30 minutes to form a uniform micelle solution. Under stirring conditions of 800 to 1000 rpm, add 10mL of tetraethyl orthosilicate dropwise at a rate of 1mL / min, and continue stirring for 2 hours. Transfer the reaction solution to a hydrothermal reactor lined with polytetrafluoroethylene, and hydrothermally treat at 100℃ for 24 hours. After natural cooling, centrifuge at 10000 rpm for 15 minutes. Wash the precipitate three times each with deionized water and anhydrous ethanol. Disperse the precipitate in 200mL of anhydrous ethanol, add 20mL of 36-38% concentrated hydrochloric acid, reflux and stir at 80℃ for 24 hours, collect the product by centrifugation, wash with anhydrous ethanol until the pH of the supernatant is neutral, and vacuum dry at 60℃ for 12 hours to obtain mesoporous nano silica.

[0042] B2. After activating 2g of mesoporous nano-silica by vacuum drying at 120℃ for 4 hours, it was dispersed in 100mL of anhydrous toluene and ultrasonically dispersed for 30 minutes. Under nitrogen protection and magnetic stirring, 4mL of 3-aminopropyltriethoxysilane was added dropwise. The mixture was heated to 110℃ and refluxed for 24 hours. After natural cooling, it was centrifuged at 8000 rpm for 10 minutes and washed three times each with anhydrous toluene and anhydrous ethanol. The mixture was then vacuum dried at 60℃ for 12 hours to obtain the intermediate.

[0043] B3. Disperse 1.5g of the intermediate in 75mL of N,N-dimethylformamide and sonicate for 20 minutes to obtain a silica suspension. Separately, dissolve 0.75g of 4-carboxyphenylboronic acid in 25mL of N,N-dimethylformamide, then add 0.86g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.52g of N-hydroxysuccinimide. Activate at room temperature for 30 minutes to obtain an activated 4-carboxyphenylboronic acid solution. Under nitrogen protection, add the activated 4-carboxyphenylboronic acid solution dropwise to the silica suspension and stir at room temperature for 24 hours. Collect the product by centrifugation at 10,000 rpm for 15 minutes. Wash three times each with N,N-dimethylformamide, deionized water, and anhydrous ethanol. Dry under vacuum at 50℃ for 12 hours to obtain modified mesoporous nano-silica. Store in a sealed container at room temperature under dry and light-protected conditions.

[0044] The modified mesoporous nano-silica has a particle size of 100 nm and a specific surface area of ​​900 m². 2 / g, with an average pore size of 3.5nm.

[0045] The tannic acid solution is prepared as follows: Weigh 1g of tannic acid and dissolve it in 100mL of phosphate buffer solution with a pH of 6.0. Stir at room temperature in the dark until completely dissolved to obtain a tannic acid solution with a mass concentration of 10mg / mL. Store at 4℃ in the dark for no more than 7 days.

[0046] The stainless steel carrier undergoes the following pretreatment before use: ultrasonic cleaning with 5% dilute nitric acid for 10 minutes, rinsing with deionized water 3 times, ultrasonic cleaning with anhydrous ethanol for 10 minutes, drying at 60°C, and then wet heat sterilization at 121°C and 0.1 MPa for 20 minutes. After cooling, it is stored in a clean environment for later use.

[0047] The composite protective liquid contains, by total mass, 2.0% modified chitosan, 1.0% modified mesoporous nano-silica, and 0.20% tannic acid.

[0048] The standard substance was placed in a centrifuge tube containing 10 mL of citrate-disodium hydrogen phosphate buffer at pH 4.5, and incubated at 37°C with shaking for 30 minutes. Then, it was vortexed for 2 minutes and sonicated for 5 minutes to completely release the spores from the protection system. The number of spores was determined by plate counting.

[0049] Example 2: This example is based on Example 1, but differs in that the concentration of the spore suspension in this example is 1.0 × 10⁻⁶. 8 The CFU / mL concentration, based on the total mass of the composite protective solution, is 1.5% for modified chitosan, 0.8% for modified mesoporous nano silica, and 0.15% for tannic acid. The remaining steps and parameters are the same as in Example 1.

[0050] Example 3: This example is based on Example 1, but differs in that the concentration of the spore suspension in this example is 1.0 × 10⁻⁶. 9 The CFU / mL concentration, based on the total mass of the composite protective solution, is 2.5% modified chitosan, 1.2% modified mesoporous nano silica, and 0.25% tannic acid. The remaining steps and parameters are the same as in Example 1.

[0051] Example 4: This example differs from Example 1 in that the modified mesoporous nano-silica has a particle size of 80 nm and a specific surface area of ​​1000 m². 2 / g, with an average pore size of 3nm, and the remaining steps and parameters are the same as in Example 1.

[0052] Example 5: This example differs from Example 1 in that the modified mesoporous nano-silica has a particle size of 120 nm and a specific surface area of ​​800 m². 2 / g, with an average pore size of 4nm, and the remaining steps and parameters are the same as in Example 1.

[0053] Comparative Example 1: This comparative example is based on Example 1, but differs from Example 1 in that unmodified carboxymethyl chitosan is used instead of modified chitosan to prepare the composite protective solution. That is, the dopamine hydrochloride modification step is not performed, and the carboxymethyl chitosan is directly dissolved in phosphate buffer with a pH of 6.5 for use. The remaining steps and parameters are the same as in Example 1.

[0054] Comparative Example 2: This comparative example is based on Example 1, but differs from Example 1 in that unmodified mesoporous nano-silica is used instead of modified mesoporous nano-silica to prepare the composite protective liquid. That is, only step B1 of the modified mesoporous nano-silica preparation method in Example 1 is completed, and steps B2 and B3 are not performed. The remaining steps and parameters are the same as in Example 1.

[0055] Comparative Example 3: This comparative example is based on Example 1, but differs from Example 1 in that the preparation of modified mesoporous nano silica in this comparative example only completes steps B1 and B2, without performing step B3 phenylboronic acid modification. That is, an intermediate is used to replace the modified mesoporous nano silica in the preparation of the composite protective liquid. The remaining steps and parameters are the same as in Example 1.

[0056] Comparative Example 4: This comparative example is based on Example 1, but differs from Example 1 in that the composite protective liquid in this comparative example does not include modified chitosan. The remaining steps and parameters are the same as in Example 1.

[0057] Comparative Example 5: This comparative example is based on Example 1, but differs from Example 1 in that the composite protective liquid in this comparative example does not include modified mesoporous nano silica. The remaining steps and parameters are the same as in Example 1.

[0058] Comparative Example 6: This comparative example is based on Example 1, but differs from Example 1 in that the composite protective solution in this comparative example does not contain tannic acid. The remaining steps and parameters are the same as in Example 1.

[0059] Comparative Example 7: This comparative example is based on Example 1, but differs from Example 1 in that a 10% trehalose aqueous solution is used as the protective solution instead of the composite protective solution. That is, the spore suspension and the trehalose solution are mixed at a volume ratio of 1:9 and then directly added to the carrier. The remaining steps and parameters are the same as in Example 1.

[0060] Comparative Example 8: This comparative example differs from Example 1 in that no protective agent is used. Instead, 10 μL of the spore suspension is diluted with phosphate buffer (pH 6.5) at a volume ratio of 1:9 and added directly to the center of the carrier. The remaining steps and parameters are the same as in Example 1.

[0061] Experiment 1: To verify the effect of the synergistic effect of modified chitosan, modified mesoporous nano silica and tannic acid in this invention on the spore drying survival rate, antioxidant protection ability and recycling performance. The results are shown in Table 1.

[0062] Test samples: Examples 1-5 and Comparative Examples 1-8, with 10 standard samples prepared in parallel for each group.

[0063] The detection indicators and methods are as follows:

[0064] Spore survival rate determination: The prepared standard substance was placed in a centrifuge tube containing 10 mL of citrate-disodium hydrogen phosphate buffer with a pH of 4.5, and cultured at 37°C with shaking for 30 minutes. Then, it was vortexed for 2 minutes and sonicated for 5 minutes to release spores. The number of recovered spores was determined by plate counting method. The survival rate (%) was calculated by comparing it with the theoretical inoculation amount of spore suspension before inoculation. The result was calculated as (number of recovered spores / theoretical number of inoculated spores) × 100%. Each group was measured 10 times in parallel.

[0065] Determination of spore survival rate after hydrogen peroxide plasma treatment: The standard material was placed in a hydrogen peroxide low-temperature plasma sterilizer and treated with a sublethal dose (58% hydrogen peroxide concentration, 45°C, treatment time 3 minutes). Spores were released and counted according to the recovery method described above. The survival rate after plasma treatment was calculated by comparing with the untreated group. Each group was measured in parallel 5 times.

[0066] Spore recovery rate determination: Take the spore suspension from the same batch as the prepared standard substance, dilute it with phosphate buffer (pH 6.5) to the same concentration as the spore protection suspension (i.e., 1 / 10 of the original spore suspension concentration), and accurately pipette 10 μL directly into a centrifuge tube containing 10 mL of citrate-disodium hydrogen phosphate buffer (pH 4.5) as a spiked control group. At the same time, take the prepared standard substance and release spores according to the standard recovery method. The number of spores in both groups is determined by plate counting method. Calculate the recovery rate (%) = (number of spores recovered from the standard substance / number of spores in the spiked control group) × 100%. Each group is measured 10 times in parallel.

[0067] Determination of the coefficient of variation between batches: Three batches of samples were prepared consecutively for each group of samples, with 10 tablets in each batch. The number of spores was determined using the standard recovery method, and the relative standard deviation (RSD%) of the number of spores between batches was calculated as the coefficient of variation index.

[0068] Table 1. Synergistic effect of the three substances on spores:

[0069]

[0070] As shown in Table 1, the present invention can effectively improve the spore drying survival rate, antioxidant protection capacity and recycling performance through the synergistic effect of modified chitosan, modified mesoporous nano silica and tannic acid.

[0071] Experiment 2: Verify the storage stability, accuracy of value determination, and sterilization dose response characteristics of the microbial standard material prepared in this invention. The results are shown in Table 2.

[0072] Test samples: Examples 1-5 and Comparative Examples 7 and 8. 60 standard samples were prepared for each group for stability testing and 30 samples were prepared for dose response testing.

[0073] The detection indicators and methods are as follows:

[0074] Accelerated stability test: The standard substances were stored at a constant temperature of 37℃ and samples were taken on days 0, 7, 14, 21 and 28. The number of spores was determined according to the standard recovery method. The retention rate of spores at each time point relative to the initial value was calculated and stability curves were plotted. Five samples were measured in parallel for each group at each time point. Based on the results of the accelerated test, the shelf life under 2-8℃ conditions was extrapolated according to the Arrhenius equation.

[0075] Long-term stability test: The standard substance was stored at 2-8℃ in the dark. The number of spores was measured on days 0, 30, 60, 90 and 180 to investigate the trend of spore number during the storage period. Linear regression analysis was used to evaluate stability. Five samples were measured in parallel at each time point for each group.

[0076] Relative expanded uncertainty assessment: In accordance with the requirements of JJF 1006-1994 "Technical Specification for Primary Standard Materials", the uncertainty components introduced by homogeneity, stability, and determination method are comprehensively considered to calculate the combined standard uncertainty. The coverage factor k=2 is taken to calculate the relative expanded uncertainty (%).

[0077] Sterilization dose-response curve determination: The standard substance was placed in a hydrogen peroxide low-temperature plasma sterilizer, and five different treatment time gradients (1, 2, 3, 4, 5 minutes) were set. Other parameters were fixed (58% hydrogen peroxide concentration, 45℃). After treatment, surviving spores were recovered and counted. A survival curve was plotted with treatment time as the x-axis and the logarithm of the number of surviving spores as the y-axis. The D-value (time required for the number of spores to decrease by one logarithmic order) and the regression correlation coefficient R were calculated using linear regression. 2 Five tablets were measured in parallel at each dose point in each group.

[0078] Table 2. Performance of the prepared microbial standard substances:

[0079]

[0080] It should be noted that the D-value measured by the microbial standard material prepared in this invention includes the apparent D-value, which is influenced by the protective system. Because the composite protective system provides a certain degree of antioxidant protection to the spores during sterilization, this apparent D-value is slightly higher than the inherent D-value of naked spores, but it does not affect the quantitative evaluation of the sterilizer performance by the standard material. In practical metrological traceability applications, the standard material's value is determined based on the measured apparent D-value, ensuring the traceability and consistency of the measurement results. Furthermore, the data in Table 2 show that the correlation coefficient R² of the survival curves of the examples is greater than 0.99, significantly better than the comparative examples, indicating that the composite protective system significantly improves the linearity of the spore response under different sterilization doses, making the D-value determination more reliable and repeatable. For microbial standard materials used for metrological traceability, the repeatability of the D-value determination and the linearity of the survival curve are more critical quality indicators than the absolute value of the D-value.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a microbial standard substance characterizing the sterilization performance of hydrogen peroxide, characterized in that, This method is used to prepare spores with a content of 1.0 × 10⁻⁶ per piece. 5 Up to 1.0×10 6 Microbial reference materials with CFU, recovery rate ≥95%, and relative expanded uncertainty ≤10%, including the following steps: S1. Raw material selection: including indicator microorganisms, carrier, modified chitosan, modified mesoporous nano-silica, and tannic acid; wherein, the modified chitosan is prepared by modifying carboxymethyl chitosan with dopamine hydrochloride; the modified mesoporous nano-silica is prepared by modifying mesoporous nano-silica with aminosilane and phenylboronic acid; the indicator microorganism is Bacillus stearothermophilus spores, strain number ATCC 7953, spore purity ≥95%, and spore suspension concentration 1.0×10⁻⁶. 8 Up to 1.0×10 9 CFU / mL; the carrier is made of 316L stainless steel sheet with a diameter of 6 mm, a thickness of 0.5 mm, and a surface roughness Ra≤0.8 μm; S2. Preparation of composite protective solution: Dissolve modified chitosan in phosphate buffer solution with pH 6.5, add tannic acid solution at a mass ratio of modified chitosan to tannic acid of 10:1, stir at room temperature for 30 minutes; then add modified mesoporous nano silica, ultrasonically disperse for 10 minutes, and continue stirring for 2 hours to obtain composite protective solution; S3. Preparation of spore protection suspension: Mix the spore suspension and the composite protection solution at a volume ratio of 1:9, and gently shake for 30 minutes to ensure that the spores are evenly dispersed in the protection system to obtain the spore protection suspension; S4. Inoculation and drying of the carrier: Place the stainless steel carrier in a clean bench, use a micropipette to add 10 μL of spore protection suspension to the center of the carrier, and place it in a clean environment at 25℃ and 40-50% relative humidity to dry naturally for 4 hours. After drying, transfer it to a desiccator at 4℃ and less than 30% relative humidity for equilibration for 24 hours. S5. Packaging and storage: Pack the dried standard material flakes into aluminum foil bags, vacuum seal them, and store them at 2-8℃ away from light. The modified chitosan was prepared by the following method: A1. Dissolve carboxymethyl chitosan in a 2-(N-morpholino)ethanesulfonic acid buffer solution with a pH of 5.5 to obtain solution A. Dissolve dopamine hydrochloride in another of the above buffer solutions to obtain dopamine hydrochloride solution. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to solution A in sequence. Activate at room temperature. Add dopamine hydrochloride solution under nitrogen protection. Stir the reaction at room temperature in the dark. Dialyze and freeze-dry to obtain modified chitosan. The modified mesoporous nano-silica was prepared by the following method: B1. Dissolve hexadecyltrimethylammonium bromide in deionized water, add 25% ammonia solution, stir to form a homogeneous micelle solution, add tetraethyl orthosilicate, continue stirring to react, transfer to a hydrothermal reactor, hydrothermally treat, cool naturally and centrifuge, wash the precipitate with deionized water and anhydrous ethanol in sequence, disperse in anhydrous ethanol, add 36-38% concentrated hydrochloric acid, reflux and stir, collect by centrifugation, wash, and vacuum dry to obtain mesoporous nano silica; B2. After vacuum drying, mesoporous nano-silica is dispersed in anhydrous toluene and ultrasonically dispersed. Under nitrogen protection and magnetic stirring, 3-aminopropyltriethoxysilane is added, and the mixture is heated to reflux. After natural cooling, it is centrifuged, washed successively with anhydrous toluene and anhydrous ethanol, and vacuum dried to obtain the intermediate. B3. The intermediate was dispersed in N,N-dimethylformamide and ultrasonically dispersed to obtain a silica suspension. 4-Carboxyphenylboronic acid was dissolved in another N,N-dimethylformamide, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added sequentially to obtain an activated 4-carboxyphenylboronic acid solution. Under nitrogen protection, the activated 4-carboxyphenylboronic acid solution was added to the silica suspension, and the reaction was stirred at room temperature. The mixture was collected by centrifugation and washed sequentially with N,N-dimethylformamide, deionized water and anhydrous ethanol. The mixture was then vacuum dried to obtain modified mesoporous nano-silica.

2. The method for preparing a microbial standard substance for characterizing the sterilization performance of hydrogen peroxide according to claim 1, characterized in that, The modified mesoporous nano-silica has a particle size of 80-120 nm and a specific surface area of ​​800-1000 m². 2 / g, with an average pore size of 3-4nm.

3. The method for preparing a microbial standard substance for characterizing the sterilization performance of hydrogen peroxide according to claim 1, characterized in that, The tannic acid solution is prepared as follows: weigh tannic acid and dissolve it in a phosphate buffer solution with a pH of 6.

0. Stir at room temperature in the dark until completely dissolved to obtain a tannic acid solution with a mass concentration of 10 mg / mL. Store at 4°C in the dark for no more than 7 days.

4. The method for preparing a microbial standard substance for characterizing the sterilization performance of hydrogen peroxide according to claim 1, characterized in that, The stainless steel carrier undergoes the following pretreatment before use: ultrasonic cleaning with 5% dilute nitric acid, rinsing with deionized water, ultrasonic cleaning with anhydrous ethanol, drying, moist heat sterilization, and cooling before use in a clean environment.

5. The method for preparing a microbial standard substance for characterizing the sterilization performance of hydrogen peroxide according to claim 1, characterized in that, The composite protective liquid contains, by total mass, 1.5-2.5% modified chitosan, 0.8-1.2% modified mesoporous nano-silica, and 0.15-0.25% tannic acid.

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