Microbial limit detection method of iron sulfide nano-enzyme

Abstract

The invention relates to the technical field of microbial limit detection, in particular to a microbial limit detection method of iron sulfide nano-enzyme. The microbial limit detection method comprises the following steps: step 1, dispersing an iron sulfide nano-enzyme sample into a buffer solution containing a compound neutralizer I to obtain a test solution; step 2, using a culture medium containing a compound neutralizer II, and detecting the total number of aerobic bacteria in the test solution by adopting a plate method; and step 3, using a culture medium containing a compound neutralizer III, and detecting the total number of mould and saccharomycetes in the test solution by adopting a plate method. On the basis of a traditional plate method, the accuracy of the microbial limit detection method of the iron sulfide nano-enzyme is higher by improving experimental operation steps, optimizing experimental parameters and adding the neutralizer and the like. The method can be used for controlling the quality of the iron sulfide nano-enzyme and protecting the safety of an iron sulfide nano-enzyme product.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial limit detection, and in particular to a method for detecting the microbial limit of iron sulfide nanozyme. Background Art

[0002] Prior art has discovered that nano-iron sulfide is a nanozyme with PQD-like and CAT-like activity. It can catalyze the oxidation of H₂O₂ and accelerate the release of polysulfide hydrogen, demonstrating high antibacterial efficacy. Considering that the main components of nano-iron sulfide are iron and sulfur, both essential elements for the human body, its use in biological antibacterial treatment is more biosafety-friendly than other metal sulfide nanozymes. As a pharmaceutical, iron sulfide nanozymes have shown promising potential for wound disinfection, treatment of oral ulcers, enteritis, myositis, lung infections, and bacterial keratitis. Given that iron sulfide nanozymes have a strong antibacterial effect, when the adaptability test of the counting culture medium or technical method was checked using the method described in the "2020 Edition of the Chinese Pharmacopoeia" - Microbial Limit Test for Non-sterile Products: Microbial Counting Method (General Chapter 1105), when polysorbate 80 and egg yolk lecithin were used as neutralizers, it was found that Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Escherichia coli all grew well, while Bacillus subtilis and Staphylococcus aureus could not grow, resulting in the recovery of the two test bacteria Bacillus subtilis and Staphylococcus aureus not meeting the requirements. The above also shows that the antibacterial activity of iron sulfide nanozymes in the test solution prepared by selecting a conventional neutralizer and following the conventional test solution preparation steps cannot be effectively eliminated. In the prior art, there are few reports on the microbial limit detection method of iron sulfide nanozymes. Because iron sulfide nano has broad application prospects as a new type of high-efficiency antibacterial material, it is very necessary to study the microbial limit detection method of iron sulfide nanozymes. Summary of the Invention

[0003] In order to address the defect that the recovery of Bacillus subtilis and Staphylococcus aureus does not meet the acceptance criteria in the applicability test using conventional microbial limit tests, the present invention aims to provide a method for microbial limit detection of iron sulfide nanozymes. Based on the traditional plate method, the microbial limit test of iron sulfide nanozymes is made to comply with relevant regulations by improving the experimental operation steps, optimizing the experimental parameters and combining the addition of neutralizing agents.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] The present invention provides a method for detecting the microbial limit of iron sulfide nanozymes, comprising the following steps:

[0006] Step 1: Disperse the iron sulfide nanozyme sample in a buffer solution containing a composite neutralizer I and dilute it to a dilution of 1:10 to 600 as a test solution;

[0007] Step 2: using a culture medium containing compound neutralizer II, and detecting the total number of aerobic bacteria in the test solution by plate method;

[0008] Step 3: Using a culture medium containing the compound neutralizer III, the total number of molds and yeasts in the test solution is detected by a plate method.

[0009] Preferably, the compound neutralizer I, compound neutralizer II or compound neutralizer III all include polysorbate 80 and lecithin.

[0010] Preferably, in the buffer solution containing the composite neutralizer I in step 1, the volume concentration of polysorbate 80 is 2% to 5%, and the mass concentration of lecithin is 0.2% to 0.5%; and / or

[0011] In the culture medium containing the composite neutralizer II in step 2, the volume concentration of polysorbate 80 is 2% to 5%, and the mass concentration of lecithin is 0.2% to 0.5%; and / or

[0012] In the culture medium containing the composite neutralizer III in step 3, the volume concentration of polysorbate 80 is 2% to 5%, and the mass concentration of lecithin is 0.2% to 0.5%.

[0013] Preferably, when the plate method is used for detection in step 2, the diameter of the plate selected is 140 to 160 mm. For example, in the present invention, a commercially available plate with a diameter of 150 mm is selected;

[0014] When the plate method is used for detection as described in step 3, the diameter of the plate selected is 90 mm.

[0015] Preferably, the buffer solution in step 1 is a sterile sodium chloride-peptone buffer solution with a pH of 6.8 to 7.2; illustratively, the sterile sodium chloride-peptone buffer solution selected in the present invention is a sterile sodium chloride-peptone buffer solution with a pH of 7.0; and / or

[0016] The culture medium in step 2 is trypticase soy agar medium; and / or

[0017] The culture medium described in step 3 is Sabouraud dextrose agar medium.

[0018] Preferably, when the total aerobic bacteria count in the test solution is detected by the plate method in step 2, the dilution of the test solution is 1:400-600; and / or

[0019] When the total number of molds and yeasts in the test solution is detected by the plate method as described in step 3, the dilution of the test solution is 1:80-120.

[0020] Preferably, the iron sulfide nanozyme is heptaferron octasulfide nanozyme.

[0021] The microbial limit detection method of iron sulfide nanozyme provided by the present invention comprises the following specific steps:

[0022] Step 1: Weigh 10 g of heptasulfide nanozyme sample and add 2-5% polysorbate 80 and 0.2-0.5% egg yolk lecithin in a pH 7.0 sterile sodium chloride-peptone buffer to 100 ml as a 1:10 test solution;

[0023] The 1:10 test solution was sequentially diluted with the aforementioned sterile sodium chloride-peptone buffer at pH 7.0 containing 2-5% polysorbate 80 and 0.2-0.5% egg yolk lecithin to prepare test solutions of 1:80, 1:100, 1:120, 1:400, 1:500, and 1:600;

[0024] Step 2: Take two portions of the 1:400-600 test solution (illustratively, a 1:500 test solution is selected in the embodiment of the present invention), 1 ml each, and inject them into sterile plates with a diameter of 140-160 mm. In each plate, inject 46-55 ml of tryptic soy agar medium containing 2-5% polysorbate 80 and 0.2-0.5% egg yolk lecithin at a temperature not exceeding 45°C. Mix well, invert and culture at 30°C to 35°C after solidification for 3-5 days, count the number of colonies on each plate, and use the arithmetic mean as the counting result;

[0025] Replace the test solution with sterile sodium chloride-peptone buffer (pH 7.0) containing 2-5% polysorbate 80 and 0.2-0.5% egg yolk lecithin. Prepare two plates as negative controls in the same manner. The negative controls should show no bacterial growth.

[0026] Step 3: Take two portions of the 1:80-120 test solution (illustratively, a 1:100 test solution is selected in the present embodiment), 1 ml each, and inject them into sterile plates with a diameter of 90 mm. In each plate, inject 15-20 ml of Sabouraud dextrose agar medium containing 2-5% polysorbate 80 and 0.2-0.5% egg yolk lecithin at a temperature not exceeding 45°C. Mix thoroughly, wait for solidification, and invert the plate to incubate at 20°C-25°C for 5-7 days. Count the number of colonies on each plate, and use the arithmetic mean as the count result.

[0027] Replace the test solution with sterile sodium chloride-peptone buffer at pH 7.0 containing 2-5% polysorbate 80 and 0.2-0.5% egg yolk lecithin. Prepare two plates as negative controls in the same manner. The negative controls should show sterile growth.

[0028] More preferably, the detection method further comprises a suitability check of the aerobic bacterial count method and a suitability check of the aerobic bacterial count culture medium.

[0029] Preferably, the suitability check of the total aerobic bacterial count method includes:

[0030] S1. Test group: Add the test bacterial solution to the test solution to obtain a bacteria-containing test solution; culture the bacteria-containing test solution in a trypticase soy agar medium containing polysorbate 80 and lecithin, and count the bacteria.

[0031] S2. Test control group: 0.9% sterile sodium chloride solution was added to the test solution to obtain a test control solution; the test control solution was placed in a tryptic soy agar medium containing polysorbate 80 and lecithin and cultured, and counted;

[0032] S3. Bacteria control group: Sterile sodium chloride pH 7.0 - peptone buffer was added to the test bacteria to obtain a bacteria control solution; the bacteria control solution was placed in a tryptic soy peptone agar medium containing polysorbate 80 and lecithin and cultured, and counted;

[0033] S4 negative control group: pH7.0 sterile sodium chloride - peptone buffer was added 0.9% sterile sodium chloride solution to obtain a negative control solution; the negative control solution was placed in trypticase soy peptone agar medium containing polysorbate 80 and lecithin and cultured, and counted;

[0034] S5. Neutralizer control group: sterile sodium chloride-peptone buffer containing polysorbate 80 and lecithin was taken, and the test bacteria solution was added to obtain a neutralizer-containing bacteria control solution; the neutralizer-containing bacteria control solution was placed in a trypticase soy agar medium containing polysorbate 80 and lecithin and cultured, and counted;

[0035] S6. Calculate the recovery test ratio of each test bacteria.

[0036] Preferably, the test bacterial solution includes a bacterial suspension containing Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Candida albicans, and also includes a spore suspension containing Aspergillus niger spores.

[0037] Due to the adoption of the above technical solution, the present invention has the following technical advancements compared with the prior art:

[0038] ① Based on the conventional microbial limit test, the present invention makes the microbial limit test of iron sulfide nanozyme more accurate by optimizing the neutralizing agent, optimizing the experimental steps, and selecting an appropriate buffer. This is of great significance for improving the quality standard construction of iron sulfide nanozyme and fills the gap in the microbial limit detection method of iron sulfide nanozyme;

[0039] ② In the microbial limit detection method of iron sulfide nanozyme provided by the present invention, the selected compound neutralizer is composed of polysorbate 80 and lecithin, and the optimized experimental steps are also easy to master for those skilled in the art. The detection method is simple and practical, and is easy to promote and apply;

[0040] ③ The microbial limit detection method of iron sulfide nanozyme provided by the present invention lays the foundation for the promotion and use of the new broad-spectrum antibacterial material - iron sulfide nanozyme.

[0041] The microbial limit detection method of iron sulfide nanozyme provided by the present invention can be used to control the quality of iron sulfide nanozyme and ensure the safety of iron sulfide nanozyme products. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0043] The iron sulfide nanozyme in the present invention is a heptaferrosulfide nanozyme sample prepared by a hydrothermal method, and the content of heptaferrosulfide in the sample is 90% to 95%.

[0044] Example 1

[0045] The present invention provides a method for detecting the microbial limit of iron sulfide nanozymes, comprising the following steps:

[0046] Step 1: Weigh 10 g of heptasulfide nanozyme sample and add 3% polysorbate 80 and 0.3% egg yolk lecithin in a pH 7.0 sterile sodium chloride-peptone buffer to 100 ml as a 1:10 test solution;

[0047] The 1:10 test solution was diluted sequentially with the sterile sodium chloride-peptone buffer (pH 7.0) containing 3% polysorbate 80 and 0.3% egg yolk lecithin to prepare test solutions of 1:20, 1:50, 1:100, and 1:500.

[0048] Step 2: Take two 1 ml portions of the 1:500 test solution and inject them into sterile 150 mm diameter plates. Add 50 ml of trypticase soy agar containing 3% polysorbate 80 and 0.3% egg yolk lecithin, kept at a temperature not exceeding 45°C, to each plate. Mix thoroughly, allow to solidify, and incubate at 30°C for 4 days. Count the number of colonies on each plate, and take the arithmetic mean as the count result. The total aerobic bacterial count in the heptasulfide nanozyme sample was calculated to be <500 cfu / g.

[0049] Step 3: Take two 1 ml portions of the 1:100 test solution and inject them into sterile 90 mm diameter plates. Add 20 ml of Sabouraud dextrose agar containing 3% polysorbate 80 and 0.3% egg yolk lecithin, maintained at a temperature not exceeding 45°C, to each plate. Mix thoroughly, allow to solidify, and invert the plate to incubate at 25°C for 6 days. Count the number of colonies on each plate, and use the arithmetic mean as the count result. The total number of mold and yeast colonies in the heptasulfide nanozyme sample was calculated to be <100 cfu / g.

[0050] It should be noted that the percentage of polysorbate 80 in this embodiment is the volume concentration; the percentages before egg yolk lecithin are all mass concentrations;

[0051] The applicability of the aerobic bacteria count method in the above-mentioned microbial limit test method is checked as follows:

[0052] S1. Test group: Add the test bacterial solution to the 1:500 test solution to obtain a test solution containing no more than 100 cfu / ml of bacteria;

[0053] The test bacterial solution includes a bacterial suspension containing Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Candida albicans, and also includes a spore suspension containing Aspergillus niger spores. The specific preparation method of the test bacterial solution is prepared with reference to the corresponding method recorded in the "2020 Edition of the Chinese Pharmacopoeia" - Microbial Limit Test for Non-sterile Products: Microbial Count Method (General Chapter 1105);

[0054] After shaking the bacterial test solution, draw 1 ml from the bacterial test solution and inject it into a sterile plate with a diameter of 150 mm. Pour 50 ml of trypticase soy peptone agar medium containing 3% polysorbate 80 and 0.3% egg yolk lecithin at a temperature not exceeding 45°C. Prepare two parallel plates for each test bacteria strain, culture, and count.

[0055] S2. Test sample control group: Add the test bacterial solution to 0.9% sterile sodium chloride solution to obtain a bacterial control solution containing no more than 100 cfu / ml;

[0056] After the test sample control solution is shaken, 1 ml of the test sample control solution is drawn from the test sample control solution and injected into a sterile plate with a diameter of 150 mm. Pour the plate into a tryptic soy agar medium containing 3% polysorbate 80 and 0.3% egg yolk lecithin at a temperature not exceeding 45°C. Prepare two parallel plates for each test bacteria strain, culture, and count.

[0057] S3. Bacterial control group: Add 0.1 ml of the test bacterial solution to 9.9 ml of sterile sodium chloride-peptone buffer at pH 7.0 to obtain a bacterial control solution with a concentration of no more than 100 cfu / ml. After shaking, inject 1 ml of the bacterial control solution into a sterile plate and pour over a tryptic soy agar medium containing 3% polysorbate 80 and 0.3% egg yolk lecithin at a temperature not exceeding 45°C. Prepare two parallel plates for each test bacterial strain, culture, and count;

[0058] S4 negative control group: pH7.0 sterile sodium chloride - peptone buffer 9.9ml was added 0.9% sterile sodium chloride solution 0.1ml to obtain a negative control solution; the negative control solution was placed in trypticase soy peptone agar medium containing polysorbate 80 and lecithin and cultured and counted;

[0059] S5. Neutralizer control group: The test bacteria were added to a sterile sodium chloride-peptone buffer containing 3% polysorbate 80 and 0.3% egg yolk lecithin at pH 7.0 to obtain a bacterial neutralizer control solution containing no more than 100 cfu / ml;

[0060] After shaking the bacterial neutralizer control solution, draw 1 ml and inject it into a sterile plate with a diameter of 150 mm. Pour 50 ml of tryptic soy agar medium containing 3% polysorbate 80 and 0.3% egg yolk lecithin at a temperature not exceeding 45°C. Prepare 2 plates in parallel for each test bacteria, culture, and count.

[0061] S6. Calculate the recovery test ratio of each test bacteria.

[0062] After calculation, the recovery test ratios of each test bacteria in the test group were all in the range of 0.5 to 2. The recovery test ratios of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were 0.94, 1.04, 0.94, 0.92 and 0.90, respectively.

[0063] The recovery test ratios of each test bacteria in the neutralizer control group were also in the range of 0.5 to 2. The recovery test ratios of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were 0.91, 0.88, 0.90, 0.87 and 0.94 respectively.

[0064] The cultures in steps S1-S5 are all inverted and cultured at 30-35°C for 3 days. In this embodiment, the culture temperature is set at 32°C. The counting is to count the number of colonies on each plate, and the arithmetic mean is used as the counting result.

[0065] Example 2

[0066] The present invention provides a method for detecting the microbial limit of iron sulfide nanozymes, comprising the following steps:

[0067] Step 1: Weigh 10 g of heptasulfide nanozyme sample and add 2% polysorbate 80 and 0.2% egg yolk lecithin in a pH 7.0 sterile sodium chloride-peptone buffer to 100 ml as a 1:10 test solution;

[0068] The 1:10 test solution was diluted sequentially with the sterile sodium chloride-peptone buffer (pH 7.0) containing 2% polysorbate 80 and 0.2% egg yolk lecithin to prepare test solutions of 1:20, 1:50, 1:100, and 1:500.

[0069] Step 2: Take two 1 ml portions of the 1:500 test solution and inject them into sterile 150 mm diameter plates. Add 46 ml of trypticase soy agar containing 3% polysorbate 80 and 0.5% egg yolk lecithin, kept at a temperature not exceeding 45°C, to each plate. Mix thoroughly, allow to solidify, and incubate at 35°C for 5 days. Count the number of colonies on each plate, and take the arithmetic mean as the count result. The total aerobic bacterial count in the heptasulfide nanozyme sample was calculated to be <500 cfu / g.

[0070] Step 3: Take two 1 ml portions of the 1:100 test solution and inject them into sterile 90 mm diameter plates. Add 20 ml of Sabouraud dextrose agar containing 4% polysorbate 80 and 0.2% egg yolk lecithin, maintained at a temperature not exceeding 45°C, to each plate. Mix thoroughly, allow to solidify, and invert the plate to incubate at 20°C for 7 days. Count the number of colonies on each plate, and use the arithmetic mean as the count result. The total number of mold and yeast in the heptasulfide nanozyme sample was calculated to be <100 cfu / g.

[0071] It should be noted that the percentage of polysorbate 80 in this embodiment is the volume concentration; the percentages before egg yolk lecithin are all mass concentrations;

[0072] The applicability of the aerobic bacteria count method in the above-mentioned microbial limit test method is checked as follows:

[0073] S1. Test group: Add the test bacterial solution to the 1:500 test solution to obtain a test solution containing no more than 100 cfu / ml of bacteria;

[0074] The test bacterial solution is the same as that in Example 1;

[0075] After shaking the test solution containing bacteria, draw 1 ml from the test solution and inject it into a sterile plate with a diameter of 150 mm. Pour tryptic soy agar medium containing 2% polysorbate 80 and 0.2% egg yolk lecithin at a temperature not exceeding 45°C. Prepare 2 parallel plates for each test bacteria strain, culture, and count.

[0076] S2. Test sample control group: Add the test bacterial solution to 0.9% sterile sodium chloride solution to obtain a bacterial test sample control solution containing no more than 100 cfu / ml;

[0077] After shaking the test sample control solution, draw 1 ml from the test sample control solution and inject it into a sterile plate with a diameter of 150 mm. Pour the plate into a tryptic soy agar medium containing 2% polysorbate 80 and 0.2% egg yolk lecithin at a temperature not exceeding 45°C. Prepare two parallel plates for each test bacteria strain, culture them, and count them.

[0078] S3. Bacterial control: Add 0.1 ml of the test bacterial solution to 9.9 ml of sterile sodium chloride-peptone buffer at pH 7.0 to obtain a bacterial control solution with a count of no more than 100 cfu / ml. After shaking, inject 1 ml of the bacterial control solution into a sterile plate and pour over a tryptic soy agar medium containing 2% polysorbate 80 and 0.2% egg yolk lecithin at a temperature not exceeding 45°C. Prepare two parallel plates for each test bacterial strain, culture, and count;

[0079] S4 negative control group: pH7.0 sterile sodium chloride - peptone buffer 9.9ml was added 0.9% sterile sodium chloride solution 0.1ml to obtain a negative control solution; the negative control solution was placed in trypticase soy peptone agar medium containing polysorbate 80 and lecithin and cultured and counted;

[0080] S5. Neutralizer control group: The test bacteria were added to a sterile sodium chloride-peptone buffer containing 2% polysorbate 80 and 0.2% egg yolk lecithin at pH 7.0 to obtain a bacterial neutralizer control solution containing no more than 100 cfu / ml;

[0081] After shaking the neutralizer control solution containing bacteria, 1 ml was drawn and injected into a sterile plate with a diameter of 150 mm. 50 ml of trypticase soy agar medium containing 2% polysorbate 80 and 0.2% egg yolk lecithin at a temperature not exceeding 45° C. was poured in. Two plates were prepared in parallel for each test bacteria strain, and the culture and counting were performed.

[0082] S6. Calculate the recovery test ratio of each test bacteria.

[0083] After calculation, the recovery test ratios of each test bacteria in the test group were all in the range of 0.5 to 2. The recovery test ratios of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were 0.98, 1.10, 0.90, 1.00 and 1.01, respectively.

[0084] The recovery test ratios of each test bacteria in the neutralizer control group were also in the range of 0.5 to 2. The recovery test ratios of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were 0.97, 1.07, 0.97, 0.97 and 0.98 respectively.

[0085] The culture conditions and counting methods in steps S1-S5 are the same as those described in Example 1.

[0086] Example 3

[0087] The present invention provides a method for detecting the microbial limit of iron sulfide nanozymes, comprising the following steps:

[0088] Step 1: Weigh 10 g of heptasulfide nanozyme sample and add 3% polysorbate 80 and 0.3% egg yolk lecithin in a pH 7.0 sterile sodium chloride-peptone buffer to 100 ml as a 1:10 test solution;

[0089] The 1:10 test solution was diluted sequentially with the sterile sodium chloride-peptone buffer (pH 7.0) containing 3% polysorbate 80 and 0.3% egg yolk lecithin to prepare test solutions of 1:20, 1:50, 1:100, and 1:500.

[0090] Step 2: Take two 1:500 test solution aliquots (1 ml each) and dispense them into sterile 150 mm diameter plates. Add 55 ml of trypticase soy agar containing 5% polysorbate 80 and 0.2% egg yolk lecithin, kept at a temperature no higher than 45°C, to each plate. Mix thoroughly, allow to solidify, and incubate at 35°C for three days. Count the number of colonies on each plate, and use the arithmetic mean as the count result. The total aerobic bacterial count in the heptasulfide nanozyme sample was calculated to be <500 cfu / g.

[0091] Step 3: Take two 1:100 test solution aliquots (1 ml each) and inject them into sterile 90 mm diameter plates. Add 18 ml of Sabouraud dextrose agar containing 2% polysorbate 80 and 0.5% egg yolk lecithin, kept at a temperature no higher than 45°C, to each plate. Mix thoroughly, allow to solidify, and invert the plate to incubate at 25°C for 5 days. Count the number of colonies on each plate, and use the arithmetic mean as the count result. The total number of mold and yeast colonies in the heptasulfide nanozyme sample was calculated to be <100 cfu / g.

[0092] It should be noted that the percentage of polysorbate 80 in this embodiment is the volume concentration; the percentage before egg yolk lecithin is the mass concentration;

[0093] The applicability of the aerobic bacteria count method in the above-mentioned microbial limit test method is checked as follows:

[0094] S1. Test group: Add the test bacterial solution to the 1:500 test solution to obtain a test solution containing no more than 100 cfu / ml of bacteria;

[0095] The test bacterial solution is the same as that in Example 1;

[0096] After shaking the test solution containing bacteria, draw 1 ml from the test solution and inject it into a sterile plate with a diameter of 150 mm. Pour in trypticase soy peptone agar medium containing 5% polysorbate 80 and 0.5% egg yolk lecithin at a temperature not exceeding 45°C. Prepare 2 parallel plates for each test bacteria strain, culture them, and count them.

[0097] S2. Test sample control group: Add the test bacterial solution to 0.9% sterile sodium chloride solution to obtain a bacterial control solution containing no more than 100 cfu / ml;

[0098] After shaking the test sample control solution, draw 1 ml from the test sample control solution and inject it into a sterile plate with a diameter of 150 mm. Pour the plate into a tryptic soy peptone agar medium containing 5% polysorbate 80 and 0.5% egg yolk lecithin at a temperature not exceeding 45°C. Prepare 2 parallel plates for each test bacteria strain, culture them, and count them.

[0099] S3. Bacterial control: Add 0.1 ml of the test bacterial solution to 9.9 ml of sterile sodium chloride-peptone buffer at pH 7.0 to obtain a bacterial control solution with a concentration of no more than 100 cfu / ml. After shaking, inject 1 ml of the bacterial control solution into a sterile plate and pour over a tryptic soy agar medium containing 5% polysorbate 80 and 0.5% egg yolk lecithin at a temperature not exceeding 45°C. Prepare two parallel plates for each test bacterial strain, culture, and count;

[0100] S4 negative control group: pH7.0 sterile sodium chloride - peptone buffer 9.9ml was added 0.9% sterile sodium chloride solution 0.1ml to obtain a negative control solution; the negative control solution was placed in trypticase soy peptone agar medium containing polysorbate 80 and lecithin and cultured and counted;

[0101] S5. Neutralizer control group: The test bacteria were added to a sterile sodium chloride-peptone buffer containing 5% polysorbate 80 and 0.5% egg yolk lecithin at pH 7.0 to obtain a bacterial neutralizer control solution containing no more than 100 cfu / ml;

[0102] After shaking the neutralizer control solution containing bacteria, 1 ml was drawn and injected into a sterile plate with a diameter of 150 mm. 50 ml of trypticase soy agar medium containing 5% polysorbate 80 and 0.5% egg yolk lecithin at a temperature not exceeding 45° C. was poured in. Two plates were prepared in parallel for each test bacteria strain, and the culture and counting were performed.

[0103] S6. Calculate the recovery test ratio of each test bacteria.

[0104] After calculation, the recovery test ratios of each test bacteria in the test group were all in the range of 0.5 to 2. The recovery test ratios of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were 0.92, 1.13, 0.93, 0.95 and 0.99, respectively.

[0105] The recovery test ratios of each test bacteria in the neutralizer control group were also in the range of 0.5 to 2. The recovery test ratios of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were 0.95, 1.04, 0.93, 0.92 and 0.89, respectively.

[0106] The culture conditions and counting methods in steps S1-S5 are the same as those described in Example 1.

[0107] Example 4

[0108] An embodiment of the present invention provides a microbial limit detection method for iron sulfide nanozyme, which is basically the same as Example 1, except that the volume concentration of polysorbate 80 involved in the above microbial limit detection method is changed from 3% to 4%, and the mass concentration of egg yolk lecithin involved is changed from 0.3% to 0.4%. The other steps and parameters remain unchanged.

[0109] When the applicability of the total aerobic bacterial count method was checked, the recovery test ratios of each test bacteria in the test group were all within the range of 0.5 to 2. The recovery test ratios of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were 0.95, 1.01, 0.92, 0.95 and 0.94, respectively.

[0110] The recovery test ratios of each test bacteria in the neutralizer control group were also within the range of 0.5 to 2. The recovery test ratios of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were 0.95, 0.93, 0.87, 0.87 and 0.91, respectively, which were in line with the regulations.

[0111] Comparative Example 1

[0112] Comparative Example 1 of the present invention provides a microbial limit detection method for iron sulfide nanozymes, which is basically the same as Example 1, except that: (1) the pH 7.0 sterile sodium chloride-peptone buffer used in this comparative example does not contain polysorbate 80 and egg yolk lecithin; (2) the tryptic soy agar medium used in this comparative example does not contain polysorbate 80 and egg yolk lecithin; (3) when the aerobic bacterial count method is used for applicability testing, the plate diameter is 90 mm; (4) when the aerobic bacterial count method is used for applicability testing, in step S1, "1:500 test solution" is replaced with "1:20, 1:50, and 1:100 test solution are drawn respectively", and 15 to 20 ml of tryptic soy agar medium is poured; (5) when the aerobic bacterial count method is used for applicability testing, step S5 is not performed. Except for the above differences, the remaining steps and parameters of Comparative Example 1 are the same as those of Example 1.

[0113] During the suitability test of the total aerobic bacterial count method, the recovery test ratio for Staphylococcus aureus was 0.03 at a test solution dilution of 1:20; 0.14 at a test solution dilution of 1:50; and 0.19 at a test solution dilution of 1:100. None of these ratios were within the range of 0.5 to 2, indicating that the heptasulfide nanozyme has strong antibacterial activity against Staphylococcus aureus. The recovery test ratio for Bacillus subtilis at test solution dilutions of 1:20, 1:50, and 1:100 was 0, indicating that the heptasulfide nanozyme has strong antibacterial activity against Bacillus subtilis. Therefore, the above test solution preparation method and the 1:20, 1:50, and 1:100 plate method could not be used for method suitability testing.

[0114] Comparative Example 2

[0115] Comparative Example 2 of the present invention provides a microbial limit detection method for iron sulfide nanozymes. This detection method is basically the same as Comparative Example 1, except that: in step 1, the pH 7.0 sterile sodium chloride-peptone buffer used in this comparative example contains 0.5% polysorbate 80 and 0.1% egg yolk lecithin to prepare the test solution; and in the suitability check of the total aerobic bacterial count method - step S1, the test solution prepared by the above method is used. Except for the above differences, the remaining steps and parameters of Comparative Example 2 are the same as those of Comparative Example 1.

[0116] During the applicability check of the total aerobic bacterial count method, it was found that when the test solution dilution was 1:100, the recovery test ratio of Staphylococcus aureus was 0.17; when the test solution dilution was 1:200, the recovery test ratio was 0.29; when the test solution dilution was 1:500, the recovery test ratio was 0.41, all of which were not within the range of 0.5-2, indicating that the heptasulfide iron nanozyme has strong antibacterial activity against Staphylococcus aureus, and when the test solution dilution was 1:500, it still could not eliminate the antibacterial activity against Staphylococcus aureus. When the dilution ratios of the test solution for Bacillus subtilis were 1:100, 1:200, and 1:500, the recovery test ratios were all 0, indicating that the heptasulfide iron nanozyme had strong antibacterial activity against Bacillus subtilis. Similarly, when the dilution ratio of the test solution was 1:500, the antibacterial activity against Bacillus subtilis could not be eliminated. Therefore, this test solution preparation method and the plate method with dilution ratios of 1:100, 1:200, and 1:500 could not be selected for the method applicability test.

[0117] Comparative Example 3

[0118] The microbial limit detection method of the iron sulfide nanozyme provided in Comparative Example 3 of the present invention is basically the same as that in Comparative Example 2, except that the tryptic soy agar medium used in this comparative example contains 3% polysorbate 80 and 0.3% egg yolk lecithin, that is, the tryptic soy agar medium used in Comparative Example 3 is the same as that in Example 1, both containing 3% polysorbate 80 and 0.3% egg yolk lecithin. Except for the above differences, the remaining steps and parameters of Comparative Example 3 and Comparative Example 2 are the same.

[0119] During the applicability check of the total aerobic bacterial count method in this comparative example, it was found that when the dilution of the test solution was 1:100, the recovery test ratio of Staphylococcus aureus was 0.39; when the dilution of the test solution was 1:200, the recovery test ratio was 0.68; when the dilution of the test solution was 1:500, the recovery test ratio was 0.77. The recovery rate of heptasulfide iron nanozyme for Staphylococcus aureus was in the range of 0.5 to 2 when the dilution of the test solution was 1:200, which has met the requirements of the 2020 edition of the Chinese Pharmacopoeia and complies with the regulations. However, when the dilution of the test solution was 1:200, the recovery test ratio of Bacillus subtilis was only 0.08; when the dilution of the test solution was 1:500, the recovery test ratio was 0.45. The recovery rate of the heptasulfide iron nanozyme for Bacillus subtilis still did not reach the range of 0.5-2 when the dilution of the test solution was 1:500. According to the 2020 edition of the Chinese Pharmacopoeia, it did not comply with the regulations. Therefore, the above test solution preparation method and the plate method with dilutions of 1:100, 1:200, and 1:500 could not be selected for method applicability testing.

[0120] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting the microbial limit of iron sulfide nanozymes, characterized in that: The following steps are involved: Step 1: Disperse the iron sulfide nanozyme sample in a buffer solution containing a composite neutralizer I and dilute it to a dilution of 1:10 to 600 as a test solution; Step 2: using a culture medium containing compound neutralizer II, and detecting the total number of aerobic bacteria in the test solution by plate method; Step 3: Using a culture medium containing the compound neutralizer III, the total number of molds and yeasts in the test solution is detected by a plate method.

2. The microbial limit detection method of iron sulfide nanozyme according to claim 1, characterized in that: The compound neutralizer I, compound neutralizer II or compound neutralizer III all include polysorbate 80 and lecithin.

3. The microbial limit detection method of iron sulfide nanozyme according to claim 2, characterized in that: In the buffer solution containing the composite neutralizer I in step 1, the volume concentration of polysorbate 80 is 2% to 5%, and the mass concentration of lecithin is 0.2% to 0.5%; and / or In the culture medium containing the composite neutralizer II in step 2, the volume concentration of polysorbate 80 is 2% to 5%, and the mass concentration of lecithin is 0.2% to 0.5%; and / or In the culture medium containing the compound neutralizer III in step 3, the volume concentration of polysorbate 80 is 2% to 5%, and the mass concentration of lecithin is 0.2% to 0.5%.

4. The microbial limit detection method of iron sulfide nanozyme according to claim 1, characterized in that: When the plate method is used for detection as described in step 2, the diameter of the plate selected is 140 to 160 mm.

5. The microbial limit detection method of iron sulfide nanozyme according to claim 1, characterized in that: The buffer described in step 1 is sterile sodium chloride-peptone buffer; and / or The culture medium in step 2 is trypticase soy agar medium; and / or The culture medium described in step 3 is Sabouraud dextrose agar medium.

6. The microbial limit detection method of iron sulfide nanozyme according to claim 1, characterized in that: When the total aerobic bacteria count in the test solution is detected by the plate method in step 2, the dilution of the test solution is 1:400-600; and / or When the total number of molds and yeasts in the test solution is detected by the plate method as described in step 3, the dilution of the test solution is 1:80-120.

7. The microbial limit detection method of iron sulfide nanozyme according to claim 1, characterized in that: The iron sulfide nanozyme is heptaferron octasulfide nanozyme.

8. The method for detecting the microbial limit of iron sulfide nanozymes according to any one of claims 1 to 7, characterized in that: The detection method also includes a suitability check of the total aerobic bacterial count method and a suitability check of the culture medium for the total aerobic bacterial count.

9. The method for detecting the microbial limit of iron sulfide nanozyme according to claim 8, wherein: The suitability check of the total aerobic bacteria count method includes: S1. The test solution was added to the test solution to obtain a test solution containing bacteria; the test solution containing bacteria was placed in a tryptic soy peptone agar medium containing polysorbate 80 and lecithin and cultured, and counted; S2. 0.9% sterile sodium chloride solution was added to the test solution to obtain a test sample control solution; the test sample control solution was placed in a tryptic soy peptone agar medium containing polysorbate 80 and lecithin and cultured, and counted; S3. Take sterile sodium chloride - peptone buffer solution was added to the test bacteria to obtain a bacterial solution control solution; the bacterial solution control solution was placed in a tryptic soy peptone agar medium containing polysorbate 80 and lecithin and cultured and counted; S4. Take sterile sodium chloride - peptone buffer was added 0.9% sterile sodium chloride solution to obtain a negative control solution; the negative control solution was placed in trypticase soy peptone agar medium containing polysorbate 80 and lecithin and cultured and counted; S5. Take sterile sodium chloride containing polysorbate 80 and lecithin - peptone buffer, add the test bacteria solution to obtain a neutralizer-containing bacteria control solution; the neutralizer-containing bacteria control solution was placed in a tryptic soy peptone agar medium containing polysorbate 80 and lecithin and cultured and counted; S6. Calculate the recovery test ratio of each test bacteria.

10. The microbial limit detection method of iron sulfide nanozyme according to claim 9, characterized in that: The test bacterial solution includes a bacterial suspension containing Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Candida albicans and a spore suspension containing Aspergillus niger spores.