Method for detecting clostridium in cosmetics

Abstract

The invention relates to the technical field of microbiological detection, in particular to a method for detecting clostridium in cosmetics. The invention provides a modified reinforced clostridium culture medium, a modified Columbia agar culture medium and a method for detecting clostridium in cosmetics, and the method is specifically as follows: the modified reinforced clostridium culture medium can inhibit the activity of preservatives in the cosmetics and improve the clostridium detection rate by adding Tween 80, lecithin and sodium thiosulfate; according to the modified Columbia agar culture medium, polymyxin B and gentamicin in a specific proportion are added, so that interference of non-target bacteria can be effectively inhibited, and detection specificity is enhanced. Based on the two modified culture media, the detection method established by the invention adopts a sample preparation mode of normal temperature and heating dual-condition treatment, and the detection rate of target bacteria can be remarkably improved. The method has the advantages of rapidness, high efficiency, high specificity, high detection rate, low cost, simplicity and convenience in operation and the like, and is particularly suitable for rapid screening requirements of supervision departments and detection mechanisms.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganism detection, in particular to a method for detecting clostridium in cosmetics. BACKGROUND

[0002] Clostridium is a large group of gram-positive anaerobic or microaerophilic coarse spore-forming bacteria. The spores of the bacteria have extremely strong resistance and can survive in soil or dry environment for decades, can withstand boiling for 40-50 minutes, and have extremely strong vitality. Some pathogenic strains such as Clostridium tetani, Clostridium perfringens and botulinum bacteria can produce exotoxin and invasive enzymes, which have extremely high safety risk.

[0003] In recent years, the role and research of Clostridium in the fields of basic microbiology, drugs, food and fermentation, clinical medicine and the like have been increasingly valued. In the field of drug microorganisms, the United States Pharmacopoeia (USP) Chapter 62 stipulates the detection method of Clostridium from aspects of test solution preparation, inoculation culture, positive control, identification and the like. The 2020 edition of the Chinese Pharmacopoeia Part IV 1106 “Non-sterile product microbial limit test: control bacteria test method” stipulates the test method of Clostridium from aspects of test solution preparation and heat treatment, enrichment (i.e. enrichment culture), selection and separation culture. In the field of food, the national food safety standard GB 4789.13-2012 “Food Microbiological Examination Clostridium perfringens Examination” stipulates the detection method of Clostridium perfringens from aspects of anaerobic culture counting, selection of suspicious colonies, biochemical identification and the like.

[0004] However, the current “Cosmetic Safety Technical Specifications” 2015 edition does not clearly stipulate the test method and identification method of Clostridium. As a daily chemical industry product, cosmetics mostly contain strong preservatives, which have inhibitory effect on the growth of microorganisms, resulting in insufficient sensitivity and low detection rate when other related test methods in other fields are operated. When people use cosmetics containing such bacteria, these harmful substances may enter the human body through skin contact and the like, causing skin infection, inflammation and the like, and there is a great safety risk. In addition, the test method of Clostridium in other fields is relatively cumbersome, the detection period is long, the specificity is not strong, and it is difficult to meet the demand of the cosmetic industry for rapid and high-specificity detection. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the existing cross-field detection method in the detection of Clostridium in cosmetics, such as cumbersome operation steps, long detection period, weak specificity, low sensitivity and low detection rate, and to provide a modified and strengthened Clostridium culture medium.

[0006] Another object of the present application is to provide a modified Columbia agar culture medium.

[0007] Another object of the present application is to provide the use of the modified Clostridium culture medium or the modified Columbia agar culture medium in culturing Clostridium.

[0008] Another object of the present application is to provide the use of the modified Clostridium culture medium in improving the biomass of Clostridium.

[0009] Another object of the present application is to provide the use of the modified Columbia agar culture medium in selectively isolating Clostridium.

[0010] Another object of the present application is to provide the use of the modified Clostridium culture medium or the modified Columbia agar culture medium in detecting Clostridium.

[0011] Still another object of the present application is to provide a method for detecting Clostridium in cosmetics.

[0012] The above objects of the present application are achieved by the following technical solutions. The present application protects a modified Clostridium culture medium, which comprises the following components: soybean lecithin 0.5-1.5 g / L, Tween 80 10.0-20.0 g / L, sodium thiosulfate 7.5-10.5 g / L, beef infusion powder 9.95-10.05 g / L, peptone 9.95-10.05 g / L, yeast extract powder 2.95-3.05 g / L, starch 0.95-1.05 g / L, glucose 4.95-5.05 g / L, cysteine hydrochloride 0.46-0.54 g / L, sodium chloride 4.95-5.05 g / L, sodium acetate 2.95-3.05 g / L, and agar 0.46-0.54 g / L.

[0013] The present application adds specific proportions of Tween 80, lecithin and sodium thiosulfate as a composite neutralizing agent in the Clostridium culture medium, specifically eliminates the bacteriostatic activity of preservatives in cosmetics, fully exposes Clostridium, and significantly improves the detection efficiency. Compared with the traditional suitability verification method of increasing the volume of the culture medium or prolonging the culture time, the present application precisely controls the composite neutralizing agent, greatly reduces the cost of manpower and material resources, and significantly improves the detection efficiency and economy.

[0014] Further, the modified Clostridium culture medium comprises the following components: soybean lecithin 0.5-1.5 g / L, Tween 80 10.0-20.0 g / L, sodium thiosulfate 7.5-10.5 g / L, beef infusion powder 9.95-10.05 g / L, proteose peptone 9.95-10.05 g / L, yeast extract powder 2.95-3.05 g / L, starch 0.95-1.05 g / L, glucose 4.95-5.05 g / L, cysteine hydrochloride 0.46-0.54 g / L, sodium chloride 4.95-5.05 g / L, sodium acetate 2.95-3.05 g / L, agar 0.46-0.54 g / L and water. Wherein, the medium is a liquid medium, and a small amount of agar added in the formula can assist in constructing an anaerobic environment by limiting oxygen diffusion, thereby providing suitable conditions for the growth and reproduction of Clostridium.

[0015] Further, the preparation method of the modified Clostridium culture medium comprises the following steps: The components except sodium thiosulfate are taken according to the formula amount, sterilized at 120-122 ℃ for 14-16 min, and after cooling, the same condition sterilized sodium thiosulfate aqueous solution is added, and adjusted to pH 6.8±0.2 to obtain the modified Clostridium culture medium.

[0016] The sterile sodium thiosulfate is added alone in the present application, which aims to avoid the redox reaction of sodium thiosulfate with other components during high-temperature sterilization, thereby ensuring the effective concentration of sodium thiosulfate in the culture medium and the stability of the system.

[0017] The present application protects a modified Columbia agar culture medium, which comprises the following components: gentamicin 2-8 mg / L, polymyxin B 50000-150000 U / L, tryptone 9.95-10.05 g / L, pepsin hydrolysate 4.95-5.05 g / L, pancreatin hydrolysate 2.95-3.05 g / L, yeast extract powder 4.95-5.05 g / L, corn starch 0.95-1.05 g / L, sodium chloride 4.95-5.05 g / L, agar 14.95-15.05 g / L.

[0018] The application significantly improves the detection specificity of clostridium by adding a specific proportion of polymyxin B and gentamicin in a Columbia agar separation plate. When polymyxin B (50000-150000 U / L) or gentamicin (2-8 mg / L) is used alone, the normal growth of clostridium can be ensured, but the non-target bacteria cannot be effectively inhibited. Only through the synergistic effect of the two, the growth of common bacteria such as staphylococcus and enterobacter can be significantly inhibited, and clostridium is not inhibited. In view of the problems of weak selectivity and easy interference of sample bacteria in Columbia agar medium. The antibiotic combination is screened and verified, which can effectively solve the risk of missed detection caused by excessive proliferation of mixed bacteria, and the selectivity and specificity are verified by multiple batches of actual samples, which fully meets the strict requirements of cosmetic microbial detection.

[0019] Further, the modified Columbia agar medium comprises the following components: gentamicin 2-8 mg / L, polymyxin B 50000-150000 U / L, tryptone 9.95-10.05 g / L, pepsin hydrolysate 4.95-5.05 g / L, cardiac pancreas hydrolysate 2.95-3.05 g / L, yeast extract powder 4.95-5.05 g / L, corn starch 0.95-1.05 g / L, sodium chloride 4.95-5.05 g / L, agar 14.95-15.05 g / L and water. Among them, the purpose of adding agar is to prepare a solid medium, so as to separate the above culture by streaking operation, so as to obtain single colonies.

[0020] Further, the preparation method of the modified Columbia agar medium comprises the following steps: According to the formula amount, the components except polymyxin B and gentamicin are mixed, sterilized, cooled to 45-50 DEG C, and then polymyxin B and gentamicin are added and mixed uniformly, and then poured into a flat plate.

[0021] The application protects the application of the above-mentioned modified clostridium culture medium or the above-mentioned modified Columbia agar medium in culturing clostridium.

[0022] The application protects the application of the above-mentioned modified clostridium culture medium in improving the biomass of clostridium.

[0023] The application protects the application of the above-mentioned modified Columbia agar medium in selectively separating clostridium.

[0024] The application protects the application of the above-mentioned modified clostridium culture medium or the above-mentioned modified Columbia agar medium in detecting clostridium.

[0025] Further, the clostridium is clostridium in cosmetics.

[0026] The application protects a method for detecting Clostridium in cosmetics, comprising the following steps: S1. Two test solutions are prepared, one is placed at room temperature, and the other is cooled after incubation at 75-85 ℃ for 8-12 min; S2. Under anaerobic conditions, the two test solutions obtained in step S1 are inoculated into the aforementioned modified Clostridium culture medium for enrichment culture, and the culture obtained after enrichment culture is inoculated into the aforementioned modified Columbia agar culture medium for isolation and purification culture; S3. The single colony obtained in step S2 is picked for mass spectrometry detection to obtain a detection result.

[0027] The application provides a method for detecting Clostridium in cosmetics, which can significantly improve the detection rate of target bacteria through the sample preparation mode of normal temperature and heating. The method is suitable for various types of cosmetics such as granules and powders, and can complete the detection through enrichment culture, isolation culture and mass spectrometry identification. It has the advantages of rapid and efficient, strong specificity, high detection rate, low cost, simple operation, etc., and is especially suitable for the rapid screening needs of supervision departments and detection institutions.

[0028] Further, in step S1, the test solution is prepared according to the preparation method of the test sample in the General Principles of Microbiological Test Methods in Item 1 of Chapter 5 of the Cosmetic Safety Technical Specifications, wherein the mass-volume ratio of the sample and the solvent in the test solution is 1 g: (8-10) mL.

[0029] Further, the solvent includes one or more of physiological saline, a protein peptone aqueous solution, and a pancreas tryptone soy peptone aqueous solution.

[0030] Specifically, the test solution is prepared by the following steps: 10 g of sample is added to 90 mL of physiological saline, and the test solution is obtained after mixing.

[0031] The detection method of the application is especially suitable for detecting samples containing ≤100 CFU / mL of Clostridium.

[0032] Preferably, the incubation temperature is 80±2 ℃.

[0033] Preferably, the incubation time is 10±1 min.

[0034] Further, in step S2, the enrichment culture temperature is 36±1 ℃.

[0035] Further, in step S2, the enrichment culture time is 48±2 h.

[0036] Further, in step S2, the isolation and purification culture temperature is 36±1 ℃.

[0037] Further, in step S2, the time for separating and purifying the culture is 48-72 h.

[0038] Further, in step S3, the specific method for mass spectrometry detection comprises the following steps: The single colony obtained in step S2 is smeared on a mass spectrometer target plate, 70% formic acid is added dropwise, mixed and dried, then a matrix is added dropwise and dried, and then a full-automatic rapid mass spectrometer is used for detection.

[0039] Further, the 70% formic acid is added dropwise in a volume of 0.95-1.05 μL.

[0040] Further, the matrix is composed of α-cyano-4-hydroxycinnamic acid (solute) and a mixed solvent, wherein the mixed solvent is acetonitrile in a volume percentage of 48-52%, water in a volume percentage of 46-48%, and trifluoroacetic acid in a volume percentage of 2-3%.

[0041] Further, the mixed solvent is acetonitrile in a volume percentage of 50%, water in a volume percentage of 47.5%, and trifluoroacetic acid in a volume percentage of 2.5%.

[0042] Preferably, the volume of the matrix is 0.95-1.05 μL.

[0043] Further, in step S3, the mass spectrometry parameter conditions are as follows: scanning mode: positive ion mode; mass range: 2-20 kDa; ion source voltage 1: 20 kV; ion source voltage 2: 18.15 kV; lens voltage: 9.05 kV.

[0044] Further, in step S3, the detection result is compared with the existing bacterial standard spectrum database built in the mass spectrometer, and after similarity calculation, the possible bacterial name and corresponding confidence (for example: Clostridium perfringens, matching degree 99.9%) are given.

[0045] Compared with the prior art, the present application has the following beneficial effects: The application provides a modified Clostridium difficile culture medium, a modified Columbia agar culture medium and a method for detecting Clostridium in cosmetics, which are as follows: the modified Clostridium difficile culture medium can inhibit the activity of preservatives in cosmetics and improve the detection rate of Clostridium by adding Tween 80, lecithin and sodium thiosulfate; the modified Columbia agar culture medium can effectively inhibit the interference of non-target bacteria and enhance the detection specificity by adding a specific proportion of polymyxin B and gentamicin. Based on the above two modified culture media, the detection method established by the application uses a sample preparation method of normal temperature and heating double condition treatment, which can significantly improve the detection rate of target bacteria. The method has the advantages of rapid and efficient, strong specificity, high detection rate, low cost, simple operation and the like, and is especially suitable for the rapid screening needs of supervision departments and detection institutions. DETAILED DESCRIPTION

[0046] The application will be further described below in combination with specific examples, but the examples do not limit the application in any form. Unless otherwise specified, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the technical field.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0048] The SCDLP liquid medium is composed of the following components: casein peptone 17.0 g, soybean peptone 3.0 g, sodium chloride 5.0 g, potassium phosphate dibasic 2.5 g, glucose 2.5 g, lecithin 1.0 g, Tween 80 7.0 g and purified water 1.0 L.

[0049] The modified letheen broth is composed of the following components: beef extract powder 5.0 g, lecithin 0.7 g, protein peptone 5.0 g, sodium bisulfite 0.1 g, animal tissue digest 10.0 g, sodium chloride 5.0 g, Tween 80 5.0 g, yeast extract powder 2.0 g, protein peptone No. 3 10.0 g and purified water 1.0 L; The Eugon LT100 broth is composed of the following components: tryptone 15.0 g, sodium chloride 4.0 g, sodium sulfite 0.2 g, lecithin 1.0 g, octylphenoxypolyethoxyethanol-9 1.0 g, soybean peptone 5.0 g, L-cystine 0.7 g, glucose 5.5 g, Tween 80 5.0 g and purified water 1.0 L; The modified Eugon LT100 broth is composed of the following components: casein peptone 15.0 g, soybean peptone 5.0 g, sodium chloride 4.0 g, L-cysteine 0.7 g, sodium sulfite 0.2 g, glucose 5.5 g, lecithin 1.0 g, sodium lauryl ether sulfate 1.56 g and purified water 1.0 L.

[0050] D / E broth is composed of the following components: yeast powder 2.5 g, glucose 10.0 g, lecithin 7.0 g, bromocresol purple 0.02 g, Tween 80 5.0 g, tryptone 5.0 g, sodium bisulfite 2.5 g, sodium thiosulfate 6.0 g, sodium thio glycolate 1.0 g, and purified water 1.0 L.

[0051] The detection of clostridia in experimental examples 5-8 was obtained by directly detecting the sample, without standard addition.

[0052] Example 1 A method for detecting clostridium in cosmetics 1. Preparation of a modified enhanced clostridium culture medium Mix 1.0 g of soybean lecithin, 15.0 g of Tween 80, 10.0 g of beef infusion powder, 10.0 g of peptone, 3.0 g of yeast extract powder, 1.0 g of soluble starch, 5.0 g of glucose, 0.5 g of cysteine hydrochloride, 5.0 g of sodium chloride, 3.0 g of sodium acetate, 0.5 g of agar, and 0.95 L of purified water, heat to dissolve, sterilize by aliquot, and cool. Before use, add 50 mL of cooled 1.8 g / mL sterile sodium thiosulfate aqueous solution (i.e. the addition amount of sterile sodium thiosulfate is 9.0 g), to obtain the modified enhanced clostridium culture medium, which has a final pH of 6.8±0.2.

[0053] 2. Preparation of a modified Columbia agar culture medium Mix 10.0 g of tryptone, 5.0 g of pepsin hydrolysate, 3.0 g of cardiac trypsin hydrolysate, 5.0 g of yeast extract powder, 1.0 g of corn starch, 5.0 g of sodium chloride, 15.0 g of agar, and 1 L of purified water, heat to dissolve, and finally adjust the pH to 7.3±0.2. After sterilization, cool to 45-50°C, add polymyxin B and gentamicin to make their final concentrations 100000 U / L and 5 mg / L respectively, mix well to obtain the modified Columbia agar culture medium. Under sterile conditions, pour the un-solidified Columbia agar culture medium into a flat plate in time, and after solidification, obtain the Columbia agar culture medium plate, which is the modified Columbia agar culture medium.

[0054] 3. Method for detecting clostridium in cosmetics S1. Bacterial solution preparation: Take fresh spore clostridium culture and prepare a bacterial suspension containing ≤ about 10 2 CFU / mL with 0.9% sterile sodium chloride solution; S2. Preparation of test solution and heat treatment: Take the test product (sample to be tested), prepare the test solution according to the "Cosmetic Safety Technical Specification" Chapter 1 Item 5 General Microbiological Test Method Preparation of Test Sample, wherein the mass-volume ratio of sample and solvent in the test solution is 1 g: 9 mL; take 2 portions of 1 mL of the test solution, one portion is not treated, and the other portion is incubated at 80℃ for 10 minutes and then cooled quickly for standby; S3. Enrichment culture: under sterile operating conditions, add the two portions of test solution obtained in step S2 to 100 mL of the modified Clostridium culture medium obtained in step 1, add the bacterial suspension obtained in step S1, and incubate at 36±1℃ under anaerobic conditions for 48±2h; S4. Isolation culture: under sterile operating conditions, take the culture obtained after enrichment culture in step S3 and inoculate it on the modified Columbia agar medium obtained in step 2, and incubate at 36±1℃ under anaerobic conditions for 48~72h to obtain single colonies; S5. Full-automatic rapid mass spectrometry identification: dip the single colony grown on the modified Columbia agar medium obtained in step S4 above, evenly smear it in the sample hole position of the MALDI target plate, add 1 μL of 70% formic acid to the sample hole position, grind evenly, and air dry. Add 1 μL of matrix solution (solute (alpha-cyano-4-hydroxycinnamic acid) and mixed solvent (50% acetonitrile, 47.5% water and 2.5% trifluoroacetic acid)) to the dried sample, air dry, and then detect on the machine. The mass spectrometry parameter conditions are: scanning mode: positive ion mode; mass range: 2~20 kDa; ion source voltage 1: 20 kV; ion source voltage 2: 18.15 kV; lens voltage: 9.05 kV.

[0055] Example 2 A method for detecting Clostridium in cosmetics The difference from Example 1 is that in the preparation of the modified Clostridium culture medium, the amount of soybean lecithin is replaced from 1.0 g to 0.5 g, the amount of Tween 80 is replaced from 15.0 g to 10.0 g, and the concentration of sodium thiosulfate is replaced from 1.8 g / mL to 1.5 g / mL (i.e. the amount of sodium thiosulfate is replaced from 9.0 g to 7.5 g); in the preparation of the modified Columbia agar culture medium, the concentration of gentamicin is changed from 5 mg / L to 2 mg / L, and the concentration of polymyxin B is changed from 100000 U / L to 50000 U / L.

[0056] The other steps and conditions are the same as those in Example 1.

[0057] Example 3 A method for detecting Clostridium in cosmetics The difference from Example 1 is that in the preparation of the modified Clostridium difficile culture medium, the amount of soybean lecithin is replaced from 1.0 g to 1.5 g, the amount of Tween 80 is replaced from 15.0 g to 20.0 g, and the concentration of sodium thiosulfate is replaced from 1.8 g / mL to 2.1 g / mL (i.e. the amount of sodium thiosulfate added is replaced from 9.0 g to 10.5 g); in the preparation of the modified Columbia agar culture medium, the concentration of gentamicin is changed from 5 mg / L to 8 mg / L, and the concentration of polymyxin B is changed from 100000 U / L to 150000 U / L.

[0058] The other steps and conditions are consistent with Example 1.

[0059] Comparative Example 1 A method for detecting Clostridium sordellii in cosmetics The difference from Example 1 is that in the preparation of the modified Clostridium difficile culture medium, no soybean lecithin, Tween 80 and sodium thiosulfate are added, i.e. it is a non-modified Clostridium difficile culture medium.

[0060] The other steps and conditions are consistent with Example 1.

[0061] Comparative Example 2 A method for detecting Clostridium sordellii in cosmetics The difference from Example 1 is that in the preparation of the modified Clostridium difficile culture medium, the amount of Tween 80 is changed from 15.0 g to 7.0 g, and no sodium thiosulfate is added.

[0062] The other steps and conditions are consistent with Example 1.

[0063] Comparative Example 3 A method for detecting Clostridium sordellii in cosmetics The difference from Example 1 is that in the preparation of the modified Clostridium difficile culture medium, no sodium thiosulfate is added.

[0064] The other steps and conditions are consistent with Example 1.

[0065] Comparative Example 4 A method for detecting Clostridium sordellii in cosmetics The difference from Example 1 is that in the preparation of the modified Clostridium difficile culture medium, the concentration of sodium thiosulfate is replaced from 1.8 g / mL to 1.2 g / mL (i.e. the amount of sodium thiosulfate added is replaced from 9.0 g to 6.0 g).

[0066] The other steps and conditions are consistent with Example 1.

[0067] Experimental Example 1 Performance study of modified Clostridium difficile culture medium 1. Experimental materials and instruments (1) Experimental strain: Clostridium sporogenes CMCC (B) 64941.

[0068] (2) Enrichment medium: SCDLP liquid medium, modified letheen broth, Eugon LT100 broth, modified Eugon LT100 broth, D / E broth, modified Clostridium fortifying medium of Examples 1-3, unmodified Clostridium fortifying medium of Comparative Example 1, and modified Clostridium fortifying medium of Comparative Examples 2-4.

[0069] (3) Isolation and purification medium: modified Columbia agar medium of Example 1.

[0070] (4) Test sample: 20 cosmetic samples without Clostridium and different preservative systems, as shown in Table 2.

[0071] (5) Instrument: biochemical incubator, full-automatic rapid microbial mass spectrometry identification system.

[0072] 2. Experimental method Referring to the method for detecting Clostridium in cosmetics in Example 1, the bacterial liquid preparation, test liquid preparation and heat treatment, enrichment culture, isolation culture, and full-automatic rapid biological mass spectrometry identification experiment were sequentially performed.

[0073] 3. Experimental results Table 1 Growth of 7 samples in different enrichment media

[0074] Note: "-" in the table represents no positive growth, and "+" represents positive growth.

[0075] Table 2 Growth of 20 samples in different Clostridium fortifying media

[0076] Note: "-" in the table represents no positive growth, "+" represents positive growth, and "weak growth" represents only a few bacteria after plate culture, and the same below.

[0077] As shown in Table 1, the 7 groups of samples cannot effectively detect the target bacteria (Clostridium sporogenes) added in the control when the culture medium (SCDLP liquid medium containing neutralizing agent) for controlling bacteria in the Cosmetics Safety Technical Specification and other domestic and foreign cosmetic standards containing neutralizing agent (modified letheen broth, Eugon LT100 broth, modified Eugon LT100 broth, D / E broth) are used, indicating that the above-mentioned culture media are not suitable for the detection of Clostridium in such cosmetics. The non-modified or modified Clostridium culture medium in Comparative Examples 1 to 4 cannot completely detect the added Clostridium due to the absence of neutralizing agent or the unbalanced addition of neutralizing agent. However, by precisely controlling the addition ratio of soybean lecithin, Tween 80 and sodium thiosulfate, the target bacteria in the 7 groups of added samples are successfully detected in Examples 1 to 3.

[0078] To verify the neutralizing effect of the Clostridium culture medium in Example 1, 20 different types of cosmetic samples containing no Clostridium and having different preservative systems were selected for addition (containing Clostridium sporogenes ≤ about 10 2 CFU / mL) for testing, and the results are shown in Table 2: the non-modified Clostridium culture medium of Comparative Example 1 cannot detect the target bacteria in the 20 added samples due to the absence of neutralizing agent (soybean lecithin, Tween 80 and sodium thiosulfate); Comparative Example 3 has a nearly 0 positive detection rate due to the absence of sodium thiosulfate; Example 1 successfully detects the target bacteria in the 20 added cosmetic samples with different preservative systems by adding appropriate proportions of Tween 80, lecithin and sodium thiosulfate as a composite neutralizing agent in the non-modified Clostridium culture medium. The above results show that the neutralizing agent ratio formula developed in the present application can efficiently neutralize cosmetics of different types and different preservative systems, verifying its broad applicability.

[0079] Experimental Example 2 Selectivity test of Columbia agar culture medium 1. Experimental materials and instruments (1) Target strains: Clostridium perfringens, Clostridium beijerinckii, Clostridium bifermentans, Clostridium butyricum and Clostridium sardiniense, all from the cosmetics routinely monitored by the laboratory; Clostridium sporogenes CMCC (B) 64941 was purchased from the China Institute for Food and Drug Control.

[0080] (2) Non-target strains: Escherichia coli CMCC (B) 44102, Staphylococcus aureus CMCC (B) 26003, Pseudomonas aeruginosa CMCC (B) 10104, Bacillus subtilis CMCC (B) 63501, Salmonella CMCC (B) 50094, Shigella CMCC (B) 51572, Staphylococcus epidermidis CMCC (B) 26069, Klebsiella pneumoniae CMCC (B) 46117, all purchased from China Institute for Food and Drug Control; Bacillus megaterium ATCC 35985, Enterobacter sakazakii ATCC 51329, all purchased from Guangdong Microbial Culture Collection Center; Staphylococcus hominis CICC 23976, from the cosmetics special of China Institute for Food and Drug Control; Bacillus thuringiensis, Bacillus caldoferax, Bacillus oceanae, Bacillus lautus, Bacillus borealis, Bacillus circulans, Bacillus notoginseng, Bacillus amyloliquefaciens, Enterobacter cloacae, all from the cosmetics monitored by the laboratory.

[0081] (3) Culture medium and reagents: Columbia agar medium (10.0 g of tryptone, 5.0 g of meat peptone hydrolysate, 3.0 g of heart pancreas hydrolysate, 5.0 g of yeast extract powder, 1.0 g of corn starch, 5.0 g of sodium chloride, 15.0 g of agar, and water), gentamicin, polymyxin B.

[0082] (4) Instruments: biochemical incubator.

[0083] 2. Experimental method Referring to the method for detecting Clostridium in cosmetics in Example 1, the bacterial liquid preparation, test solution preparation and heat treatment, and enrichment culture were sequentially performed, and each bacterial culture after enrichment culture was directly inoculated into a Columbia agar medium plate containing different antibiotic groups, and placed in an anaerobic condition at 36±1 ℃ for 48-72 h, and the growth of each bacteria on the plate was observed.

[0084] 3. Experimental results Table 3 Growth of target Clostridium in different antibiotic groups of Columbia agar medium

[0085] Table 4 Growth of non-target strains in different antibiotic groups of Columbia agar medium

[0086] The detection results are shown in Tables 3-4. In the Columbia agar medium, the target Clostridium grows well when 5 mg / L of gentamicin or 100000 U / L of polymyxin B is added alone, but part of the target Clostridium grows inhibited when the concentration is increased to 10 mg / L of gentamicin or 300000 U / L of polymyxin B, that is, the growth of the target Clostridium is affected when the concentration of polymyxin B or gentamicin in the combined antibiotics is changed; although 5 mg / L of gentamicin or 100000 U / L of polymyxin B can promote the growth of the target Clostridium, the medium selectivity is poor and cannot inhibit non-target bacteria; and after 5 mg / L of gentamicin and 100000 U / L of polymyxin B are compounded, the medium selectivity is significantly improved by the synergistic effect of the two, which can ensure the normal growth of the target bacteria and effectively inhibit non-target bacteria.

[0087] Experimental Example 3: Specificity test of Clostridium in cosmetics 1. Experimental materials and instruments (1) Target strains: 4 strains of Clostridium perfringens, 4 strains of Clostridium sordellii, 4 strains of Clostridium bifermentans, 4 strains of Clostridium butyricum, 4 strains of Clostridium sardiniense, all from the cosmetics monitored by the laboratory; Clostridium sporogenes CMCC(B)64941 was purchased from China Food and Drug Inspection Institute.

[0088] (2) Non-target strains: Escherichia coli CMCC(B)44102, Staphylococcus aureus CMCC(B)26003, Pseudomonas aeruginosa CMCC(B)10104, Bacillus subtilis CMCC(B)63501, Salmonella CMCC(B)50094, Shigella CMCC(B)51572, Staphylococcus epidermidis CMCC(B)26069, Klebsiella pneumoniae CMCC(B)46117, all purchased from China Food and Drug Inspection Institute; Bacillus megaterium ATCC35985, Enterobacter sakazakii ATCC51329, all purchased from Guangdong Microbial Culture Collection Center; Staphylococcus hominis CICC23976, from the cosmetics special project of China Food and Drug Inspection Institute; Bacillus thuringiensis, Bacillus caldoferax, Bacillus okeanokoites, Bacillus lautus, Bacillus borealis, Bacillus circulans, Bacillus notoginseng, Bacillus amyloliquefaciens, Enterobacter cloacae, all from the cosmetics monitored by the laboratory.

[0089] (3) Medium: modified and enhanced Clostridium medium and modified Columbia agar medium were prepared according to the formula of Example 1.

[0090] (4) Sample: olive moisturizing toner.

[0091] (5) Instruments: biochemical incubator and full-automatic rapid microbial mass spectrometry identification system.

[0092] 2. Experimental method a. Inclusive experiment Referring to the method for detecting Clostridium cosmeticum in cosmetics in Example 1, the bacterial liquid preparation (target bacteria), test liquid preparation and heat treatment, enrichment culture, isolation culture and full-automatic rapid microbial mass spectrum identification experiment were sequentially performed.

[0093] b. Exclusive experiment Referring to the method for detecting Clostridium cosmeticum in cosmetics in Example 1, the bacterial liquid preparation (non-target bacteria), test liquid preparation and heat treatment, enrichment culture, isolation culture and full-automatic rapid microbial mass spectrum identification experiment were sequentially performed.

[0094] 3. Experimental results Table 5: Inclusive and exclusive test statistics table for detecting Clostridium cosmeticum in cosmetics

[0095] From the detection results in Table 5, 21 strains of target bacteria can be effectively detected, and 20 strains of non-target bacteria do not appear positive detection signals. This experimental data fully shows that the detection method proposed in the present application has high specificity, can accurately distinguish target bacteria from non-target bacteria, and effectively avoids the interference of non-specific detection results.

[0096] Experimental Example 4: Sensitivity test for detecting Clostridium cosmeticum in cosmetics 1. Implementation materials and instruments (1) Experimental strain: Clostridium sporogenes CMCC (B) 64941.

[0097] (2) Culture medium: modified Clostridium sporogenes culture medium and modified Columbia agar culture medium were prepared according to the formula in Example 1.

[0098] (3) Sample: 20 cosmetic samples without Clostridium sporogenes and different preservative systems, as shown in Table 6.

[0099] (4) Instruments: biochemical incubator, full-automatic rapid microbial mass spectrum identification system.

[0100] 2. Experimental method: Referring to the method for detecting Clostridium cosmeticum in cosmetics in Example 1, the above 20 kinds of cosmetics were artificially contaminated, and the bacterial concentration was 10 -1 CFU / g (mL), 10 0 CFU / g (mL), 10 1 CFU / g (mL), respectively. Each strain of different concentration needs to be repeated 3 times, test liquid preparation and heat treatment, enrichment culture, isolation culture and full-automatic rapid microbial mass spectrum identification experiment.

[0101] 3. Experimental results: Table 6 Sensitivity test results of detection method

[0102] Note: ; In the formula: - the acceptance reference value of the sample, in CFU / g (mL); - the number of confirmed positive results; m - the experimental amount, in g (mL); - the number of repeated tests; For example, taking the group corresponding to No. 1 (pure moisturizing color-blocking primer) as an example: the number "1" under "10 -1 CFU / g" in the results indicates that 1 group of Clostridium sporogenes was detected in 3 tests at a concentration of 10 -1 CFU / g; the meanings of other numbers in the results are similar (i.e., the number of groups of target bacteria detected in 3 tests at the corresponding concentration).

[0103] According to the data shown in Table 6, the 50% limit of detection (LOD 50 ) was selected as the verification standard, so the proportion of positive results in the total number of tests should be in the range of 25% to 75%. The experimental results showed that at a bacterial concentration of 10 -1 CFU / g (mL), the positive detection rate was 21.6%; at a bacterial concentration of 10 0 CFU / g (mL), the positive detection rate was 68.3%, meeting the LOD 50 requirement; at a bacterial concentration of 10 1 CFU / g (mL), the positive detection rate was 100%. Further calculation showed that at a bacterial concentration of 10 0 CFU / g (mL), the LOD 50 result was 0.61, i.e., the detection limit of the method was 0.61 CFU / g (mL).

[0104] Example 5 Method for detecting Clostridium sporogenes in seaweed granular cosmetics 1. Experimental materials and instruments (1) Experimental strain: Clostridium sporogenes CMCC (B) 6494.

[0105] (2) Culture medium: modified Clostridium sporogenes culture medium and modified Columbia agar culture medium were prepared according to the formula in Example 1.

[0106] (3) Test sample: 45 batches of commercially available seaweed granular mask cosmetic samples, as shown in Table 7.

[0107] (4) Instruments: biochemical incubator, full-automatic rapid microbial mass spectrometry identification system, and full-automatic microbial gene fingerprint identification system.

[0108] 2. Experimental method: Referring to the method for detecting Clostridium sp. in the cosmetic in Example 1, the bacterial liquid preparation, test liquid preparation, heat treatment, enrichment culture (no additional Clostridium sporosphaeroides bacterial suspension was added during the enrichment culture process to ensure that the detection result directly reflects the situation of the sample to be tested), isolation culture, full-automatic rapid biological mass spectrometry identification experiment, and 16S rDNA sequencing were sequentially performed. The method for preparing the test liquid is as follows: two parallel test liquids were prepared according to Example 1 (one was directly used at room temperature, and the other was quickly cooled after being incubated at 80 °C for 10 minutes); meanwhile, an additional 1 mL Clostridium sporosphaeroides bacterial suspension + 1 mL test liquid at room temperature was prepared. The additional test liquid was used as a positive control to verify the effectiveness of the detection method: if the target bacteria (Clostridium sporosphaeroides) can be detected, it means that the detection method can effectively detect Clostridium in the sample, and the risk of false negative is excluded; if not, it indicates that the sample to be tested may contain inhibitory components or other interference factors, resulting in detection failure, and there may be a false negative, which needs to be vigilant about the reliability of the method. Through the positive control, the two situations of “there is indeed no target Clostridium in the sample” and “the target Clostridium is not detected due to problems in the detection conditions” can be directly distinguished.

[0109] The method for 16S rDNA sequencing is as follows: 45 batches of commercially available seaweed granular mask cosmetic samples were collected to verify the present application, and the 16S rDNA sequencing and other genotypic microbial identification methods specified in the Guiding Principles for Microbial Identification in the Fourth Part of the Chinese Pharmacopoeia 2020 Edition were compared, and the test results are shown in Table 7.

[0110] 3. Experimental results Table 7 Comparison of mass spectrometry and 16S rDNA identification results of target strains in seaweed granules

[0111] Table 8 Detection of target strains in seaweed granule samples after different temperature treatments

[0112] As can be seen from Table 7, 45 batches of seaweed granular samples were taken as the detection objects in the experiment, the overall detection rate of Clostridium in the seaweed granular mask samples was as high as 48.89%, and the detection results were completely consistent with the 16S rDNA sequencing results, the relative accuracy of the method was 100%, and the false negative rate and the false positive rate were both 0%, which verified the reliability of the method. It should be noted that 16S rDNA testing often takes several days and the reagent cost is relatively high; while the mass spectrometry used in the present application can obtain results in a few minutes, which is not only efficient but also has a lower cost.

[0113] At the same time, the detection results of Clostridium in 45 batches of seaweed granular samples under normal temperature and 80 ℃ treatment are shown in Table 8. Further analysis of 22 batches of samples in which Clostridium was detected showed that the detection rate of Clostridium under normal temperature was 27.27% (6 / 22), and the detection rate of Clostridium under 80 ℃ treatment was increased to 72.73% (16 / 22). This result shows that the test solution treated at 80 ℃ for 10 minutes can not only inhibit / remove the interference bacteria in the sample, but also promote the revival of Clostridium spores, and can be used for targeted screening of high-risk spore contamination. The double-condition sample preparation of "normal temperature + 80 ℃ treatment" can significantly improve the detection rate of target bacteria and make up for the risk of missed detection under single condition.

[0114] In the experiment, highly pathogenic Clostridium perfringens (the most common pathogenic bacteria of clinical gas gangrene, which can produce gas by decomposing carbohydrates, leading to tissue emphysema, necrosis, and exacerbation of pathogenic risk due to its capsule structure) was detected. According to the detection results, it can be known that the seaweed granular mask cosmetic product is contaminated with pathogenic microorganisms, which may threaten the health of consumers. The present application can provide precise technical support for supervision and inspection, risk monitoring and other work by rapidly detecting Clostridium, and help to prevent and control the safety of cosmetic microorganisms.

[0115] Experimental Example 6: Method for detecting Clostridium in mask powder cosmetic 1. Experimental materials and instruments (1) Experimental strain: Clostridium sporogenes CMCC (B) 6494.

[0116] (2) Culture medium: modified Clostridium culture medium and modified Columbia agar culture medium were both prepared according to the formula of Example 1.

[0117] (3) Detection sample: 11 kinds of mask powder samples, as shown in Table 9.

[0118] (4) Instruments: biochemical incubator, full-automatic rapid microorganism mass spectrometry identification system, and full-automatic microorganism gene fingerprint identification system.

[0119] 2. Experimental method The experimental method for detecting Clostridium cosmeticum in cosmetics in Experimental Example 5 was referred to, and the bacterial liquid preparation, test liquid preparation and heat treatment, enrichment culture, separation culture, automatic rapid biological mass spectrum identification experiment and 16S rDNA sequencing were sequentially performed.

[0120] 3. Experimental results Table 9 Comparison table of mass spectrum and 16S rDNA identification results of target strains in mask powder

[0121] As can be seen from Table 9, 11 kinds of commercially available mask powder cosmetics were taken as objects in the experiment, and the detection rate of Clostridium cosmeticum in the above-mentioned mask powder cosmetics reached 45.45%, and the detection results were consistent with the results of the gold standard method, the relative accuracy reached 100%, and the false negative rate and the false positive rate were both 0%. This result fully verifies the reliability of the method of the application, which can accurately identify Clostridium cosmeticum in the sample and avoid the risk of missed detection and false detection.

[0122] Experimental Example 7 Method for detecting Clostridium cosmeticum in color cosmetic 1. Experimental materials and instruments (1) Experimental strain: Clostridium sporogenes CMCC (B) 6494.

[0123] (2) Culture medium: modified Clostridium sporogenes culture medium and modified Columbia agar culture medium were both prepared according to the formula of Example 1.

[0124] (3) Test sample: 50 kinds of color cosmetics, see Table 10 for details.

[0125] (4) Instruments: biochemical incubator, automatic rapid microorganism mass spectrum identification system, automatic microorganism gene fingerprint identification system.

[0126] 2. Experimental method The experimental method for detecting Clostridium cosmeticum in cosmetics in Experimental Example 5 was referred to, and the bacterial liquid preparation, test liquid preparation and heat treatment, enrichment culture, separation culture, automatic rapid biological mass spectrum identification experiment and 16S rDNA sequencing were sequentially performed.

[0127] 3. Experimental results Table 10 Comparison table of mass spectrum and 16S rDNA identification results of target strains in color cosmetic samples

[0128] The data in Table 10 show that the experiment was carried out to detect 50 kinds of commercially available cosmetic products, and the detection rate of Clostridium sphenoides in the above-mentioned cosmetic products was 16.00%, and the detection results were completely consistent with the gold standard method, the relative accuracy was 100%, and the false negative rate and the false positive rate were both 0%. This result fully verifies the reliability of the method, which can accurately identify Clostridium sphenoides in cosmetic products and effectively avoid the risk of missed detection and misjudgment.

[0129] Experimental Example 8 Method for detecting Clostridium sphenoides in foot bath powder 1. Experimental materials and instruments (1) Experimental strain: Clostridium sporogenes CMCC (B) 6494.

[0130] (2) Culture medium: Modified Strengthened Clostridium Medium and Modified Columbia Agar Medium were prepared according to the formula of Example 1.

[0131] (3) Test samples: 4 kinds of foot bath powder samples, as shown in Table 11.

[0132] (4) Instruments: biochemical incubator, full-automatic rapid microbial mass spectrometry identification system, full-automatic microbial gene fingerprint identification system.

[0133] 2. Experimental method Referring to the experimental method for detecting Clostridium sphenoides in cosmetic products in Experimental Example 5, bacterial liquid preparation, test solution preparation and heat treatment, enrichment culture, isolation culture, full-automatic rapid biological mass spectrometry identification experiment and 16S rDNA sequencing were sequentially carried out.

[0134] 3. Experimental results Table 11 Comparison of mass spectrometry and 16S rDNA identification results of target strains in foot bath powder samples

[0135] As shown in Table 11, the experiment took 4 kinds of commercially available foot bath powder samples as the detection object, and the detection rate of Clostridium sphenoides in the above-mentioned foot bath powder was 25.00%, and the detection results were completely consistent with the gold standard method, the relative accuracy was 100%, and the false negative rate and the false positive rate were both 0%. This result verifies the reliability of the method, which can accurately identify Clostridium sphenoides in foot bath powder samples and effectively avoid the risk of missed detection and misjudgment.

[0136] In summary, the modified Clostridium culture medium of the present application can inhibit the activity of preservatives in cosmetics, improve the detection rate of Clostridium by adding Tween 80, lecithin and sodium thiosulfate; the modified Columbia agar culture medium can effectively inhibit the interference of non-target bacteria and enhance the detection specificity by adding a specific proportion of polymyxin B and gentamicin. Based on the above two modified culture media, the detection method established by the present application adopts a sample preparation method of normal temperature and heating double condition treatment, which can significantly improve the detection rate of target bacteria. The method is suitable for various types of cosmetics such as granules and powders, and can be completed through enrichment culture, separation culture and mass spectrometric identification. The method has the advantages of rapid and efficient, strong specificity, high detection rate, low cost, simple operation and the like, and is especially suitable for the rapid screening needs of supervision departments and detection institutions.

[0137] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be within the scope of protection of the present application.

Claims

1. A modified Clostridium accidinifaciens culture medium, characterized in that, The modified Clostridium culture medium comprises the following components: soybean lecithin 0.5-1.5 g / L, Tween 80 10.0-20.0 g / L, sodium thiosulfate 7.5-10.5 g / L, beef infusion powder 9.95-10.05 g / L, protein peptone 9.95-10.05 g / L, yeast extract powder 2.95-3.05 g / L, starch 0.95-1.05 g / L, glucose 4.95-5.05 g / L, cysteine hydrochloride 0.46-0.54 g / L, sodium chloride 4.95-5.05 g / L, sodium acetate 2.95-3.05 g / L, agar 0.46-0.54 g / L.

2. The modified Clostridium culture medium of claim 1, wherein, The modified Clostridium culture medium comprises the following components: soybean lecithin 0.5-1.5 g / L, Tween 80 10.0-20.0 g / L, sodium thiosulfate 7.5-10.5 g / L, beef infusion powder 9.95-10.05 g / L, protein peptone 9.95-10.05 g / L, yeast extract powder 2.95-3.05 g / L, starch 0.95-1.05 g / L, glucose 4.95-5.05 g / L, cysteine hydrochloride 0.46-0.54 g / L, sodium chloride 4.95-5.05 g / L, sodium acetate 2.95-3.05 g / L, agar 0.46-0.54 g / L and water.

3. A modified Columbia Agar medium, characterized in that, The modified Columbia agar culture medium comprises the following components: gentamicin 2-8 mg / L, polymyxin B 50000-150000 U / L, tryptone 9.95-10.05 g / L, meat peptone hydrolysate 4.95-5.05 g / L, cardiac pancreas enzyme hydrolysate 2.95-3.05 g / L, yeast extract powder 4.95-5.05 g / L, corn starch 0.95-1.05 g / L, sodium chloride 4.95-5.05 g / L, agar 14.95-15.05 g / L.

4. The modified CMA medium of claim 3, wherein, The modified Columbia agar culture medium comprises the following components: gentamicin 2-8 mg / L, polymyxin B 50000-150000 U / L, tryptone 9.95-10.05 g / L, meat peptone hydrolysate 4.95-5.05 g / L, cardiac pancreas enzyme hydrolysate 2.95-3.05 g / L, yeast extract powder 4.95-5.05 g / L, corn starch 0.95-1.05 g / L, sodium chloride 4.95-5.05 g / L, agar 14.95-15.05 g / L and water.

5. The modified Clostridium culture medium of claim 1 or 2 or the modified Columbia agar culture medium of claim 3 or 4 is used for culturing Clostridium.

6. The modified Clostridium culture medium of claim 1 or 2 is used for improving the biomass of Clostridium.

7. The modified Columbia agar culture medium of claim 3 or 4 is used for selectively isolating Clostridium.

8. Use of the modified Clostridium culture medium of claim 1 or 2 or the modified Columbia agar culture medium of claim 3 or 4 in detecting Clostridium.

9. A method of detecting Clostridium spt in a cosmetic product, characterized by, The method comprises the following steps: S1. Preparing two test solutions, one of which is placed at room temperature, and the other of which is incubated at 75-85 ℃ for 8-12 min and then cooled; S2. Under anaerobic conditions, inoculating the two test solutions obtained in step S1 into the modified Clostridium culture medium of claim 1 or 2 for enrichment culture, and then inoculating the culture obtained after the enrichment culture into the modified Columbia agar culture medium of claim 3 or 4 for isolation and purification culture; S3. Picking single colonies obtained in step S2 for mass spectrometry detection to obtain a detection result.

10. The method of claim 9, wherein, In step S3, the mass spectrometry parameters are as follows: scanning mode: positive ion mode; mass range: 2-20 kDa; ion source voltage 1: 20 kV; ion source voltage 2: 18.15 kV; lens voltage: 9.05 kV.

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