CN-121674385-B - Preparation method of microbial standard substance for characterizing hydrogen peroxide sterilization performance

Abstract

The invention discloses a preparation method of a microorganism standard substance for representing hydrogen peroxide sterilization performance, which belongs to the technical field of biological detection and comprises the following steps of S1, selecting raw materials, S2, preparing a composite protection liquid, namely dissolving modified chitosan in a phosphate buffer solution, adding tannic acid solution, then adding modified mesoporous nano silicon dioxide, S3, preparing a spore protection suspension, S4, inoculating and drying a carrier, and S5, packaging and storing.

Inventors

• CHEN HONGFEI
• ZHU WEN
• Hui Wenshan
• CUI XINWEN
• GAO XIANG
• NI MING
• ZHOU YUXIN
• ZHU TIANYU
• LI ZHENG

Assignees

• 南京市计量监督检测院
• 北京林电伟业电子技术有限公司

Dates

Publication Date

20260512

Application Date

20260210

Claims (5)

1. 1. A preparation method of a microorganism standard substance for representing hydrogen peroxide sterilization performance is characterized in that, the method is used for preparing a microorganism standard substance with the spore content of 1.0X10 5 to 1.0X10 6 CFU, the recovery rate of more than or equal to 95% and the relative expansion uncertainty of less than or equal to 10%, and comprises the following steps: S1, selecting raw materials, wherein the raw materials comprise an indicating microorganism, a carrier, modified chitosan, modified mesoporous nano silicon dioxide and tannic acid, wherein the modified chitosan is prepared by modifying carboxymethyl chitosan with dopamine hydrochloride, the modified mesoporous nano silicon dioxide is prepared by modifying mesoporous nano silicon dioxide with aminosilane and phenylboric acid, the indicating microorganism adopts geobacillus stearothermophilus spores, the strain number is ATCC 7953, the spore purity is more than or equal to 95%, the concentration of spore suspension is 1.0X10 8 to 1.0X10 9 CFU/mL, the carrier adopts 316L stainless steel sheet, the diameter is 6mm, the thickness is 0.5mm, and the surface roughness Ra is less than or equal to 0.8 mu m; S2, preparing a composite protection solution, namely dissolving modified chitosan in a phosphate buffer solution with the pH value of 6.5, adding a tannic acid solution according to the mass ratio of the modified chitosan to tannic acid of 10:1, stirring for 30 minutes at room temperature, adding modified mesoporous nano silicon dioxide, performing ultrasonic dispersion for 10 minutes, and continuously stirring for 2 hours to obtain the composite protection solution; S3, preparing spore protection suspension, namely mixing the spore suspension with the composite protection liquid according to the volume ratio of 1:9, and gently oscillating for 30 minutes to uniformly disperse spores in a protection system to obtain spore protection suspension; S4, inoculating and drying the carrier, namely placing the stainless steel carrier in an ultra-clean workbench, sucking 10 mu L of spore protection suspension by using a micropipette, dripping the suspension in the center of the carrier, naturally drying the suspension for 4 hours in a clean environment with the temperature of 25 ℃ and the relative humidity of 40-50%, and transferring the suspension to a dryer with the temperature of 4 ℃ and the relative humidity of less than 30% for balancing for 24 hours after the drying is finished; s5, packaging and storing, namely loading the dried standard substance into an aluminum foil bag in a single piece, vacuumizing, heat-sealing, and storing at 2-8 ℃ in a dark place; the modified chitosan is prepared by the following steps: 1, dissolving carboxymethyl chitosan in 2- (N-morpholino) ethanesulfonic acid buffer solution with the pH value of 5.5 to obtain solution A, dissolving dopamine hydrochloride in the other buffer solution to obtain dopamine hydrochloride solution, sequentially adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into the solution A, activating at room temperature, adding dopamine hydrochloride solution under the protection of nitrogen, stirring at room temperature for reaction in a dark place, dialyzing, and freeze-drying to obtain modified chitosan; the modified mesoporous nano silicon dioxide is prepared by the following method: B1, dissolving cetyl trimethyl ammonium bromide in deionized water, adding an ammonia water solution with the mass fraction of 25%, stirring to form a uniform micelle solution, adding tetraethoxysilane, continuously stirring for reaction, transferring to a hydrothermal reaction kettle, carrying out hydrothermal treatment, naturally cooling, centrifuging, washing a precipitate with deionized water and absolute ethyl alcohol in sequence, dispersing in absolute ethyl alcohol, adding concentrated hydrochloric acid with the mass fraction of 36-38%, refluxing, stirring, centrifugally collecting, washing, and vacuum drying to obtain mesoporous nano silicon dioxide; B2, dispersing mesoporous nano silicon dioxide in anhydrous toluene after vacuum drying, performing ultrasonic dispersion, adding 3-aminopropyl triethoxysilane under the conditions of nitrogen protection and magnetic stirring, heating for reflux reaction, naturally cooling, centrifuging, washing with anhydrous toluene and absolute ethanol in sequence, and performing vacuum drying to obtain an intermediate; And B3, dispersing the intermediate in N, N-dimethylformamide, performing ultrasonic dispersion to obtain a silicon dioxide suspension, dissolving 4-carboxyphenylboronic acid in other N, N-dimethylformamide, sequentially adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain an activated 4-carboxyphenylboronic acid solution, adding the activated 4-carboxyphenylboronic acid solution into the silicon dioxide suspension under the protection of nitrogen, stirring at room temperature for reaction, centrifugally collecting, sequentially washing with N, N-dimethylformamide, deionized water and absolute ethyl alcohol, and performing vacuum drying to obtain the modified mesoporous nano silicon dioxide.
2. 2. The method for preparing the microbial standard substance for characterizing the sterilization performance of hydrogen peroxide according to claim 1, wherein the particle size of the modified mesoporous nano-silica is 80-120nm, the specific surface area is 800-1000m 2 /g, and the average pore diameter is 3-4nm.
3. 3. The preparation method of the microbial standard substance for characterizing the sterilization performance of hydrogen peroxide, which is disclosed in claim 1, is characterized in that the preparation method of the tannic acid solution is that tannic acid is weighed and dissolved in phosphate buffer solution with the pH value of 6.0, stirred at room temperature and in a dark place until the tannic acid solution is completely dissolved, and the tannic acid solution with the mass concentration of 10mg/mL is obtained, is stored at the temperature of 4 ℃ and in a dark place, and the storage period is not more than 7 days.
4. 4. The method for preparing the microbial standard substance for characterizing the sterilization performance of hydrogen peroxide according to claim 1, wherein the stainless steel carrier is pretreated by ultrasonic cleaning with dilute nitric acid with the volume fraction of 5%, washing with deionized water, ultrasonic cleaning with absolute ethyl alcohol, drying, damp-heat sterilization, cooling and standby in a clean environment.
5. 5. The method for preparing the microbial standard substance for characterizing the sterilization performance of hydrogen peroxide according to claim 1, wherein the mass fraction of the composite protective solution is 1.5-2.5% of the modified chitosan, the mass fraction of the modified mesoporous nano silica is 0.8-1.2% of the modified mesoporous nano silica, and the mass fraction of the tannic acid is 0.15-0.25% of the modified mesoporous nano silica.

Description

Preparation method of microbial standard substance for characterizing hydrogen peroxide sterilization performance Technical Field The invention belongs to the technical field of biological detection, and relates to a preparation method of a microbial standard substance for characterizing hydrogen peroxide sterilization performance. Background The low-temperature hydrogen peroxide plasma sterilizer has been widely used for sterilization treatment of heat-sensitive instruments in medical institutions due to the advantages of low sterilization temperature, short period, no toxicity, no residue and the like. At present, the sterilization effect monitoring of the sterilizer mainly depends on a biological indicator, such as a rapid biological indicator for monitoring the sterilization effect and a using method thereof disclosed in the prior patent CN113398306A, and bacterial slices containing bacillus stearothermophilus spores are placed in the sterilizer, and after sterilization, the bacterial slices are cultured and observed to have spore growth, so that qualitative judgment of qualification/disqualification is made for a single sterilization process. However, the biological indicator can only verify the sterilization effect, and cannot perform quantitative measurement and traceability calibration on sterilization performance parameters such as a D value, a sterility assurance level and the like of the sterilizer. The quantitative evaluation of sterilization performance needs to use a microbial standard substance as a measurement standard, and the microbial standard substance is different from a biological indicator, and is required to have precisely known and stable spore quantity, so that viable spores can be accurately recovered and counted after treatment with different sterilization doses, and the sterilization kinetic parameters are obtained through viable curve fitting. According to the requirements of JF 1006-1994 first-class standard substance technical Specification, the standard substance needs to meet three indexes of uniformity, stability and fixed value accuracy, and the expansion uncertainty of the standard substance needs to be controlled within a reasonable range. However, the prior art has the following problems that the activity loss of spores is remarkable and the batch difference is large due to dehydration damage in the drying process of carriers when preparing microbial standard substances for evaluating the sterilization performance of hydrogen peroxide plasmas, the traditional protective agents such as trehalose, skim milk powder and the like are easy to degrade and lose efficacy in the strong oxidation environment of the hydrogen peroxide plasmas to interfere with the accuracy of spore counting, the combination state of the spores and the carriers is unstable, the elution recovery rate is low and the repeatability is poor, and the metering and tracing requirements of the standard substances are difficult to meet. Disclosure of Invention The invention aims to provide a preparation method of a microbial standard substance for characterizing the sterilization performance of hydrogen peroxide, which solves the problems of inaccurate, unstable and unreliable values of the microbial standard substance in the existing hydrogen peroxide sterilization performance evaluation. The technical scheme adopted by the invention is that the preparation method of the microbial standard substance for characterizing the sterilization performance of hydrogen peroxide is used for preparing the microbial standard substance with the spore content of 1.0 multiplied by 10 5 to 1.0 multiplied by 10 6 CFU, the recovery rate of more than or equal to 95 percent and the relative expansion uncertainty of less than or equal to 10 percent, and comprises the following steps of: S1, selecting raw materials, wherein the raw materials comprise an indicating microorganism, a carrier, modified chitosan, modified mesoporous nano silicon dioxide and tannic acid, wherein the modified chitosan is prepared by modifying carboxymethyl chitosan with dopamine hydrochloride, the modified mesoporous nano silicon dioxide is prepared by modifying mesoporous nano silicon dioxide with aminosilane and phenylboric acid, the indicating microorganism adopts geobacillus stearothermophilus spores, the strain number is ATCC 7953, the spore purity is more than or equal to 95%, the concentration of spore suspension is 1.0X10 8 to 1.0X10 9 CFU/mL, the carrier adopts 316L stainless steel sheet, the diameter is 6mm, the thickness is 0.5mm, and the surface roughness Ra is less than or equal to 0.8 mu m; S2, preparing a composite protection solution, namely dissolving modified chitosan in a phosphate buffer solution with the pH value of 6.5, adding a tannic acid solution according to the mass ratio of the modified chitosan to tannic acid of 10:1, stirring for 30 minutes at room temperature, adding modified mesoporous nano silicon dioxide, performing ultr