Neutralizing agent for detecting Burkholderia cepacia flora in elution cosmetics and application of neutralizing agent

Abstract

The invention discloses a neutralizing agent for detecting Burkholderia cepacia flora in elution cosmetics and application of the neutralizing agent. The neutralizing agent contains lecithin, Tween-80, sodium thioglycolate, sodium thiosulfate, sodium hydrogen sulfite, histidine, glycine, L-cysteine hydrochloride, a nitrogen source component, a carbon source component and the like, and can effectively neutralize a complex preservative system in the leaching cosmetics. When the Burkholderia cepacia detection kit is applied to the detection of Burkholderia cepacia in washing cosmetics, the influence of preservatives in the washing cosmetics on Burkholderia cepacia flora detection can be effectively eliminated, and the problems that the washing cosmetics have relatively strong antibacterial activity on Burkholderia cepacia flora due to complex preservative systems; the method is beneficial for protecting the safety and health of consumers, and the problem that the detection rate of the positive control of the burkholderia cepacia flora is relatively low is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and in particular relates to a neutralizer for detecting Burkholderia cepacia in rinse-off cosmetics and application thereof. Background Art

[0002] Burkholderia cepacia Complex (Bcc) is a conditional pathogen that easily infects people with low immune function. The presence of Bcc in cosmetics may cause an imbalance in the skin microecology of people who use cosmetics, resulting in dermatitis, wound infection, abscess, conjunctivitis and other diseases. There have been many pathogenic events caused by Bcc contamination at home and abroad, such as urinary tract infection, respiratory system infection, wound infection, blood infection, etc. caused by Bcc contamination of hand sanitizer, mouthwash, liquid soap, etc.; urinary tract infection and respiratory tract infection caused by Bcc contamination of cosmetic moisturizing body lotion; there are also reports of sepsis and acute renal failure (ARF) complicated by skin Bcc. The US FDA and the EU Rapid Alert System for Non-Food Consumer Products have repeatedly reported recalls of cosmetics contaminated with Bcc (samples include disposable cleansing foam, mouthwash, body cream, spray, etc.). At present, Bcc has been identified by the FDA as an unacceptable microorganism.

[0003] At present, the Bcc test method is not included in my country's "Technical Specifications for Safety of Cosmetics", nor is there a relevant method suitability test standard. At present, the main Bcc test methods for cosmetics at home and abroad are: United States Pharmacopoeia USP 43-NF38 <60> (1) SN / T 4684-2016 “Test Method for Burkholderia cepacia in Imported Cosmetics”; (2) SN / T 4684-2016 “Test Method for Burkholderia cepacia in Imported and Exported Oral Hygiene Products”; (3) SN / T 4485-2016 “Test Method for Burkholderia cepacia in Imported and Exported Oral Hygiene Products”. These three methods do not specify the need to eliminate antibacterial ingredients in cosmetics during the preparation of sample test solutions and bacterial culture, so the detection rate of Bcc positive controls in cosmetics is relatively low.

[0004] The existing neutralizers used in microbial detection in the prior art are mainly for conventional control bacteria such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. There is no neutralizer for Bcc detection of rinse-off cosmetics, and the existing neutralizers are not compounded for the preservative system of cosmetics. The preservative system of rinse-off cosmetics is very complex and often contains a variety of preservatives, such as isothiazolinones, benzoic acid and its derivatives, alcohols, phenols, organic acids and their salts, formaldehyde sustained-release bodies, and other organic compounds. Conventional, single neutralizers cannot effectively neutralize the effect of preservatives on Bcc, such as conventional D / E neutralizing broth and the neutralizer disclosed in patent CN103642893A, a neutralizer for cosmetic microbial detection and its preparation method. The research group of the inventor of this application found in the previous study that the preservative system in rinse-off cosmetics has a strong inhibitory effect on Bcc. At present, there is a lack of neutralizers that can effectively neutralize the preservative system in rinse-off cosmetics. The existing neutralizers have a low detection rate for Bcc positive controls, which poses a safety and health risk to the user population. Therefore, it is particularly important to develop a neutralizer that can be used to eliminate the influence of preservatives in rinse-off cosmetics on Bcc testing during sample processing and can effectively neutralize the preservative system in cosmetics. Summary of the invention

[0005] The object of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and to provide a neutralizing agent for microbial testing of rinse-off cosmetics.

[0006] The second object of the present invention is to provide the use of the neutralizer for microbial testing of rinse-off cosmetics.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The invention firstly provides a neutralizer for testing Burkholderia cepacia Complex (Bcc) in rinse-off cosmetics, which contains the following components in mass fractions: 2-5% lecithin, 2-5% Tween-80, 0.5-3% sodium thioglycolate, 0.5-3% sodium thiosulfate, 0.5-3% sodium bisulfite, 0.5-3% histidine, 0.5-3% glycine, 0.5-3% L-cysteine ​​hydrochloride, 0.7-2% nitrogen source component, 0.5-3% carbon source component, and the balance is water.

[0009] The present invention screens the components of the neutralizer according to the complex preservative system of rinse-off cosmetics: lecithin, Tween-80, sodium bisulfite, sodium thiosulfate, sodium thioglycolate can neutralize isothiazolinones, benzoic acid and its derivatives, alcohols, phenols, organic acids and their salts in rinse-off samples; histidine and glycine are used to neutralize aldehyde preservatives; L-cysteine ​​hydrochloride is used to neutralize metal and organic mercury preservatives. Nitrogen source components and carbon source components are used to provide nutrients necessary for microbial growth. The above components are compounded to form a neutralizer, which has a good neutralizing effect on preservatives in rinse-off cosmetics, can eliminate the influence of preservatives on Burkholderia cepacia Complex (Bcc), effectively detect Burkholderia cepacia Complex in samples, and is non-toxic and safe.

[0010] Furthermore, the cepacia group is selected from one or more of Burkholderia cepacia, Burkholderia cenocepacia, and Burkholderia aenigmatica.

[0011] Preferably, the cepacia Burkholderia flora is selected from Burkholderia cepacia.

[0012] Furthermore, the nitrogen source components are trypsin and yeast powder, and the carbon source component is glucose.

[0013] Preferably, the mass ratio of tryptone to yeast powder is (0.5-1):(0.2-1).

[0014] Preferably, the neutralizer contains the following components in mass fractions: 3-5% lecithin, 3-5% Tween-80, 1-3% sodium thioglycolate, 1-3% sodium thiosulfate, 1-3% sodium bisulfite, 1-3% histidine, 1-3% glycine, 1-3% L-cysteine ​​hydrochloride, 0.75-2% nitrogen source component, 1-3% carbon source component, and the balance is water.

[0015] Preferably, the neutralizer contains the following components in mass fractions: 3% lecithin, 3% Tween-80, 1% sodium thioglycolate, 1% sodium thiosulfate, 1% sodium bisulfite, 1% histidine, 1% glycine, 1% L-cysteine ​​hydrochloride, 0.75% nitrogen source component, 1% carbon source component, and the balance is water.

[0016] Furthermore, the preparation method of the neutralizer comprises dissolving the above-mentioned corresponding mass fractions of lecithin and Tween-80 in a small amount of water, adding corresponding mass fractions of sodium thioglycolate, sodium thiosulfate, sodium bisulfite, histidine, glycine, L-cysteine ​​hydrochloride, nitrogen source components, and carbon source components, adding the remaining mass fraction of water, mixing, adjusting the pH to 7.5-8, and sterilizing to obtain the product.

[0017] Preferably, the pH is adjusted to pH 7.6.

[0018] Preferably, the sterilization is high pressure sterilization at 121° C. for 15 min.

[0019] The present invention provides the use of any of the above-mentioned neutralizing agents in the microbiological testing of rinse-off cosmetics.

[0020] Furthermore, the microbiological testing method may be a conventional microbiological testing method in the art or a developed standard testing method.

[0021] Furthermore, the microbial test is to test the Burkholderia cepacia flora.

[0022] Preferably, the cepacia group is selected from one or more of Burkholderia cepacia, Burkholderia cenocepacia, and Burkholderia aenigmatica.

[0023] The present invention also provides a method for testing Burkholderia cepacia in rinse-off cosmetics, comprising the following steps:

[0024] S1. dilute the elution sample 10 to 50 times using any of the above-mentioned neutralizing agents to prepare a test solution;

[0025] S2. The test solution is cultured to obtain a sample test solution;

[0026] S3. Isolate and culture the sample test fluid for microbial identification to check whether it contains cepacia bacteria.

[0027] Furthermore, step S1 is to dilute the elution sample by 20 to 50 times using any of the above-mentioned neutralizing agents to prepare a test solution.

[0028] Furthermore, the bacterial enrichment culture in step S2 is performed using a SCDLP bacterial enrichment solution containing polymyxin B for proliferation culture.

[0029] Furthermore, the separation and culture in step S3 is carried out using BCSA agar containing polymyxin B, gentamicin and vancomycin.

[0030] Preferably, the typical bacterial colonies isolated and cultured in step S3 are grayish white, grayish pink or earthy yellow (brown) in color, and the surrounding culture medium turns orange-red or rose-red, and the typical bacterial colonies are picked for microbial identification.

[0031] Preferably, the microbial identification includes but is not limited to one or more of physiological and biochemical identification, morphological identification, molecular biology identification, and microbial mass spectrometry identification.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention provides a neutralizer for testing Burkholderia cepacia complex (Bcc) in rinse-off cosmetics. The neutralizer contains components such as lecithin, Tween-80, sodium thioglycolate, sodium thiosulfate, sodium bisulfite, histidine, glycine, L-cysteine ​​hydrochloride, nitrogen source components, carbon source components, acid-base indicators, etc., and can effectively neutralize the complex preservative system in rinse-off cosmetics. The neutralizer is applied to the test of Burkholderia cepacia complex in rinse-off cosmetics, which can effectively eliminate the influence of preservatives in rinse-off cosmetics on Bcc testing, solves the problem that rinse-off cosmetics have strong antibacterial properties against Bcc due to their complex preservative system, resulting in a low detection rate of Bcc positive controls, and is beneficial to protecting the safety and health of consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a test method for Burkholderia cepacia in rinse-off cosmetics.

[0035] Figure 2 The growth conditions of 10 batches of samples in BCSA agar in the positive control experiment and the identification results of the growing strains are shown. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] D / E neutralization broth was purchased from Beijing Luqiao Technology Co., Ltd., batch number: 221226.

[0039] Example 1 Preparation of Neutralizer (Self-Made Neutralizer Broth) for Testing Burkholderia cepacia in Rinse-off Cosmetics

[0040] The preservative systems in rinse-off cosmetics mainly include: isothiazolinones, benzoic acid and its derivatives, alcohols, phenols, organic acids and their salts, formaldehyde sustained-release agents and other organic compound preservatives. The preservative systems in rinse-off cosmetics are classified with reference to the classification standards for preservatives, as shown in Table 1.

[0041] Table 1 Preservative systems in rinse-off cosmetics

[0042]

[0043] According to the types of preservatives commonly used in rinse-off cosmetics, the development ideas of the neutralizer for testing Burkholderia cepacia in rinse-off cosmetics (also called "self-made neutralizer broth") are as follows: lecithin, Tween, sodium bisulfite, sodium thiosulfate, sodium thioglycolate can neutralize isothiazolinones, benzoic acid and its derivatives, alcohols, phenols, organic acids and their salts in rinse-off samples; histidine and glycine are used to neutralize aldehyde preservatives; L-cysteine ​​hydrochloride is used to neutralize metal and organic mercury preservatives. The composition of the neutralizer for testing Burkholderia cepacia in rinse-off cosmetics prepared by the present invention (self-made neutralizer broth) is shown in Table 2.

[0044] Table 2 Neutralizer (homemade neutralizer broth) formula for testing Burkholderia cepacia in rinse-off cosmetics

[0045]

[0046]

[0047] Preparation method: Dissolve the weighed lecithin and Tween-80 by heating with a small amount of distilled water, then add the above ingredients to 1000 mL of distilled water, mix well, adjust the pH to 7.6 after cooling, sterilize at 121°C for 15 minutes, and divide into portions for use.

[0048] Example 2 Preparation of Neutralizer (Self-Made Neutralizer Broth) for Testing Burkholderia cepacia in Rinse-off Cosmetics

[0049] The formula of the self-made neutralizer broth is: 2% lecithin, 2% Tween-80, 0.5% sodium thioglycolate, 0.5% sodium thiosulfate, 0.5% sodium bisulfite, 0.5% histidine, 0.5% glycine, 0.5% L-cysteine ​​hydrochloride, 0.7% nitrogen source component, and 0.5% carbon source component.

[0050] The nitrogen source component is 0.5% tryptone and 0.2% yeast powder, and the carbon source component is 0.5% glucose.

[0051] The preparation method is the same as that of Example 1.

[0052] Example 3 Preparation of Neutralizer (Self-Made Neutralizer Broth) for Testing Burkholderia cepacia in Rinse-off Cosmetics

[0053] The formula of the self-made neutralizer broth is: 5% lecithin, 5% Tween-80, 3% sodium thioglycolate, 3% sodium thiosulfate, 3% sodium bisulfite, 3% histidine, 3% glycine, 3% L-cysteine ​​hydrochloride, 2% nitrogen source component, and 3% carbon source component.

[0054] The nitrogen source component is 1% tryptone and 1% yeast powder, and the carbon source component is 3% glucose.

[0055] The preparation method is the same as that of Example 1.

[0056] Example 4 Toxicity test of neutralizer to microorganisms

[0057] The tested bacteria include the following bacteria and fungi: (1) Burkholderia cepacia standard strains: ATCC 25416 (Burkholderia cepacia), ATCC BAA-245 (Burkholderia cenocepacia) and ATCC BAA-247 (Burkholderia multivorans), all purchased from the American Type Culture Collection; Pseudomonas aeruginosa [CMCC (B) 10104], Staphylococcus aureus [CMCC (B) 26003], Bacillus subtilis [CMCC (B) 63501], all purchased from the China National Center for the Administration of Microbiological Collection of Medical Sciences. (2) Candida albicans [CMCC (F) 98001], Aspergillus niger [CMCC (F) 98003], all purchased from the China National Center for the Administration of Microbiological Collection of Medical Sciences.

[0058] Experimental method: (1) Preparation of experimental bacterial suspension: The above bacterial strains were streaked onto TSA plates and incubated at 30-35°C for 18-24 h. Fresh TSA culture was prepared into a bacterial suspension of appropriate McFarland concentration and diluted 10% with 0.9% sterile saline. 3 ~10 4 CFU / mL bacterial suspension for later use. Take the above-mentioned fungus Candida albicans and streak it onto the SDA plate and culture it at 20-25℃ for 2-3 days. Take the fresh SDA culture to prepare a bacterial suspension with appropriate McFarland concentration; take Aspergillus niger and inoculate it onto the SDA slant, culture it at 20-25℃ for 5-7 days, add 3-5mL of pH7.0 sterile sodium chloride-peptone buffer containing 0.05% polysorbate-80, wash off the mold spores, and prepare a spore suspension. Take the Candida albicans suspension and the Aspergillus niger spore suspension, dilute them step by step to 10 with pH7.0 sterile sodium chloride-peptone buffer. 3 ~10 4 CFU / mL, for future use.

[0059] (2) Experimental groups:

[0060] Neutralizer group: Take 10 portions (9.9 mL each) of the homemade neutralizer broth prepared in Example 1 and add 10 portions (9.9 mL each) of the prepared 3 ~10 4 Mix 0.1 mL of each CFU bacterial suspension, let it stand for 30 min, take 1 mL of the mixed bacterial suspension and pour it onto a TSA agar plate, incubate it at 30-35°C for 3-5 days, and prepare 2 plates in parallel.

[0061] Bacterial solution control group: except using 9.9 mL of 0.9% saline instead of the neutralizer, the rest of the steps were the same as those of the neutralizer group.

[0062] (3) Experimental results:

[0063] Refer to USP <60> The three standard strains of Burkholderia cepacia in the method and the five standard strains in the enumeration method suitability test under the microbial limit test item 1105 of the Chinese Pharmacopoeia were subjected to a microbial recovery test to evaluate the toxicity of the homemade neutralizer broth of Example 1 to microorganisms. The results are shown in Table 3, and the recovery rates of all the test strains were greater than 0.8; the homemade neutralizer broth prepared using Examples 2 and 3 also had a high recovery rate for the test bacteria, indicating that the neutralizer for testing the Burkholderia cepacia group in rinse-off cosmetics provided by the present invention is non-toxic to the test bacteria and can be used for the preparation of the test solution in the Bcc test.

[0064] Table 3 Neutralizer bacterial solution recovery rate determination results

[0065] Test strains Neutralizer Group Bacterial liquid control group Recovery rate (%) Burkholderia cepacia 72 82 0.9 Burkholderia cenocepacia 68 85 0.8 Burkholderia cepacia 78 89 0.9 Staphylococcus aureus 65 66 1.0 Pseudomonas aeruginosa 74 70 1.1 Bacillus subtilis 63 71 0.9 Candida albicans 65 67 1.0 Aspergillus niger 54 50 1.1

[0066] Example 5: Neutralization effect verification of homemade neutralizer broth USP <60> method

[0067] Experimental method: 28 batches of elution samples were used according to USP <60> Method: (1) 0.9% saline (1:10); (2) D / E neutralizing broth (1:10); (3) homemade neutralizing broth of Example 1 (1:10); (4) homemade neutralizing broth of Example 1 (1:20); (5) homemade neutralizing broth of Example 1 (1:50) were used to prepare test solutions to verify the neutralization effect.

[0068] Experimental results: As shown in Table 4, (1) 0.9% saline was used to prepare the test solution at a ratio of 1:10: none of the positive controls of 28 batches of samples were detected; (2) D / E neutralizing broth was used to prepare the test solution at a ratio of 1:10: none of the positive controls of 18 batches of samples were detected, with a risk of missed detection of 64.3%; (3) Neutralizer broth was used to prepare the test solution at a ratio of 1:10: none of the positive controls of 6 batches of samples were detected, with a risk of missed detection of 21.4%; (4) Neutralizer broth was used to prepare the test solution at a ratio of 1:20: only two batches of samples were not detected, with a risk of missed detection of 7.1%; (5) Neutralizer broth was used to prepare the test solution at a ratio of 1:50: all samples were tested for Burkholderia cepacia. The above results show that the positive control detection rate of the self-made neutralizer broth in Example 1 is significantly increased compared with the positive control detection rate of the D / E neutralization broth pretreatment method, indicating that the neutralizer for testing Burkholderia cepacia in rinse-off cosmetics of the present invention has a good neutralization effect, and the sample can be basically eliminated after being diluted by 1:20 and 1:50, indicating that the neutralizer for testing Burkholderia cepacia in rinse-off cosmetics of the present invention can be used for USP <60> Methods for the detection of Burkholderia cepacia.

[0069] Table 4USP <60> Method verification of the neutralization effect test results of homemade neutralizer broth

[0070]

[0071]

[0072] Note: “+” means that Burkholderia cepacia was detected, “-” means that Burkholderia cepacia was not detected.

[0073] Example 6 Neutralizing effect verification of neutralizing agent broth SN / T 4684-2016 method

[0074] Experimental method: 28 batches of elution samples were used according to the SN / T 4684-2016 method, using (1) 0.9% saline (1:10); (2) D / E neutralizing broth (1:10); (3) homemade neutralizer broth in Example 1 (1:10); (4) homemade neutralizer broth in Example 1 (1:20); (5) homemade neutralizer broth in Example 1 (1:50) to prepare test solutions to verify the neutralization effect.

[0075] Experimental results: The results are shown in Table 5. (1) 0.9% saline was used to prepare a 1:10 test solution: 18 batches of samples were not detected in the positive control; (2) D / E neutralization broth was used to prepare a 1:10 test solution: 16 batches of samples were not detected in the positive control, and the risk of missed detection was 57.1%; (3) Example 1 homemade neutralizer broth was used to prepare a 1:10 test solution: 6 batches of samples were not detected in the positive control, and the risk of missed detection was 21.4%; (4) Example 1 homemade neutralizer broth was used to prepare a 1:20 and 1:50 test solution: Burkholderia cepacia was detected in all samples. The positive control detection rate of the homemade neutralizer broth was significantly increased compared with the D / E neutralization broth pretreatment method, indicating that the homemade neutralizer broth of Example 1 has a good neutralization effect, and the sample can eliminate the influence of the preservative after being diluted at 1:20 and 1:50. It is shown that the neutralizer for testing Burkholderia cepacia in rinse-off cosmetics of the present invention can be used in the SN / T4684-2016 Burkholderia cepacia testing method.

[0076] Table 5 SN / T 4684-2016 Verification of the neutralization effect test results of homemade neutralizer broth

[0077]

[0078]

[0079] Note: “+” indicates that Burkholderia cepacia was detected, and “-” indicates that Burkholderia cepacia was not detected.

[0080] Example 7 Application of homemade neutralizer broth in the detection of Burkholderia cepacia in cosmetics

[0081] Experimental method: (according to Figure 1 Experimental process (carry out the experiment)

[0082] (1) Prepare a homemade neutralizer broth according to the mass fraction in Example 1, sterilize at 121°C for 15 min and set aside. Select 10 batches of rinse-off cosmetics, weigh 10 g (10 mL) of sample or use a sterile pipette to draw 10 mL of sample and add it to 90 mL of neutralizer broth (containing glass beads), mix well, and let stand for 30 min to prepare a 1:10 test solution. Use a sterile pipette to draw 10 mL of the 1:10 test solution and add it to 90 mL of SCDLP liquid culture medium containing polymyxin B, and culture it at 36°C±1°C for 48h~72h. Use a sterile inoculation loop to take a loop of SCDLP enrichment solution (containing 250,000 U / L polymyxin B), streak it on a BCSA agar plate (containing 600,000 U / L polymyxin B, 10.0 mg / L gentamicin and 2.5 mg / L vancomycin), and culture it at 36°C±1°C for 48h~72h. At the same time, a negative control was made. Observe the growth of the colonies on each plate and the color change of the culture medium around the colonies. Typical colonies on BCSA agar (containing 600,000 U / L polymyxin B, 10.0 mg / L gentamicin and 2.5 mg / L vancomycin) plates are grayish white, grayish pink or earthy yellow (brown), and the surrounding culture medium turns orange or rose red.

[0083] (2) Positive control (sample suitability verification)

[0084] Preparation of bacterial suspension: Streak Burkholderia cepacia (CMCC(B)23005), Burkholderia cenocepacia CMCC(B)23006, Burkholderia mysticum CMCC(B)23010 or equivalent strains onto TSA plates, incubate at 30-35°C for 18-24h, prepare bacterial suspension with appropriate McFarland concentration from fresh TSA culture, and dilute to 10% with 0.9% sterile saline. 3 ~10 4 CFU / mL bacterial suspension for later use.

[0085] Take 9.7 mL of the prepared 1:10 test solution and add the bacterial suspensions of Burkholderia cepacia (CMCC(B)23005), Burkholderia cenocepacia CMCC(B)23006, and Burkholderia mystica CMCC(B)23010 (10 3 ~10 4CFU / mL) were added to 0.1 mL each, and after being fully mixed, they were transferred to 90 mL of SCDLP enrichment solution (containing 250,000 U / L polymyxin B), and cultured at 36°C ± 1°C for 48 h to 72 h. The subsequent separation and culture was performed in the same manner as step (1) of this embodiment. The growth of the colonies on each plate and the color change of the culture medium around the colonies were observed. The typical colonies on the BCSA agar (containing 600,000 U / L polymyxin B, 10.0 mg / L gentamicin and 2.5 mg / L vancomycin) culture plate were grayish white, grayish pink or khaki (brown) in color, and the surrounding culture medium turned orange or rose red.

[0086] (3) Identification: Pick suspicious colonies on the BCSA agar plate, streak inoculate on the TSA agar plate, and culture at 36℃±1℃ for 18-24h (if the colony grows slowly, the culture can be extended to 48h), observe the colony morphology, pick the isolated and purified colonies for Gram staining, and observe the colony morphology under a microscope. And identify the suspicious colonies according to the instructions for use of the microbial identification mass spectrometry system.

[0087] Experimental results: Using the self-established method, the positive control groups of 10 batches of elution samples all had typical colony morphology on BCSA agar containing polymyxin B, gentamicin and vancomycin. The colonies were grayish white, grayish pink or khaki (brown), and the surrounding culture medium turned orange-red or rose-red. This showed that the positive controls of all 10 batches of samples grew. The colonies on the plates were picked and identified using the microbial mass spectrometry identification system, and the identification results were all Burkholderia cepacia Complex; that is, Burkholderia cepacia was detected on the positive control agar plates of all 10 batches of samples, indicating that the self-established method - the test method for Burkholderia cepacia in cosmetics can be used, and the Burkholderia cepacia in the samples can be effectively detected. The results are shown in Table 6. Figure 2 The homemade neutralizer broth prepared by Example 2 and Example 3 can also effectively detect the cepacia Burkholderia in the sample, indicating that the homemade neutralizer broth provided by the present invention can effectively eliminate the influence of preservatives in rinse-off cosmetics on the detection of cepacia Burkholderia.

[0088] Table 6 Growth of 10 batches of samples in BCSA agar and identification results of growing strains

[0089]

[0090]

[0091] Note: “+” means that Burkholderia cepacia was detected, “-” means that Burkholderia cepacia was not detected.

[0092] Comparative Example

[0093] Using D / E neutralization broth (control reagent 1), the neutralizer for cosmetic microbial detection and the neutralizer in the preparation method thereof in patent CN103642893A (control reagent 2) as control reagents, and the neutralizer prepared in Example 1 of the present invention, 5 batches of rinse-off cosmetics with strong antibacterial properties were selected for neutralization treatment and then tested for Burkholderia cepacia.

[0094] The results are shown in Table 7. The detection rate of Burkholderia cepacia was 0 when D / E neutralizing broth was diluted 1:10, the detection rate of Burkholderia cepacia was 0 when control reagent 2 was diluted 1:10, and the detection rate of the neutralizer of the present invention was 100% when diluted 1:10. In summary, the neutralizer for microbial testing of rinse-off cosmetics provided by the present invention can effectively eliminate the influence of preservatives in rinse-off cosmetics on Bcc testing, and is suitable for testing Burkholderia cepacia in preservatives in rinse-off cosmetics.

[0095] Table 7 Bcc test results of 1:10 dilutions of three neutralizers

[0096]

[0097] Note: “+” means that Burkholderia cepacia was detected, “-” means that Burkholderia cepacia was not detected.

Claims

1. A neutralizing agent for testing Burkholderia cepacia in rinse-off cosmetics, characterized in that: The invention contains the following components in mass fractions: 2-5% lecithin, 2-5% Tween-80, 0.5-3% sodium thioglycolate, 0.5-3% sodium thiosulfate, 0.5-3% sodium bisulfite, 0.5-3% histidine, 0.5-3% glycine, 0.5-3% L-cysteine ​​hydrochloride, 0.7-2% nitrogen source component, 0.5-3% carbon source component, and the balance is water.

2. The neutralizing agent according to claim 1, characterized in that The invention contains the following components in mass fractions: 3-5% lecithin, 3-5% Tween-80, 1-3% sodium thioglycolate, 1-3% sodium thiosulfate, 1-3% sodium bisulfite, 1-3% histidine, 1-3% glycine, 1-3% L-cysteine ​​hydrochloride, 0.75-2% nitrogen source component, 1-3% carbon source component, and the balance is water.

3. The neutralizing agent according to claim 1, characterized in that The nitrogen source components are tryptone and yeast powder.

4. The neutralizing agent according to claim 3, characterized in that The mass ratio of the tryptone to the yeast powder is 0.5-1:0.2-1.

5. The neutralizing agent according to claim 1, characterized in that The carbon source component is glucose.

6. Use of the neutralizer according to any one of claims 1 to 5 in the detection of Burkholderia cepacia in rinse-off cosmetics.

7. A method for testing cepacia Burkholderia in rinse-off cosmetics, characterized in that: The following steps are involved: S1. Dilute the elution sample 10 to 50 times using the neutralizer according to any one of claims 1 to 5 to prepare a test solution; S2. The test solution is cultured to obtain a sample test solution; S3. Isolate and culture the sample test fluid for microbial identification.

8. The method according to claim 7, characterized in that: Step S1 is to dilute the elution sample by 20 to 50 times using the neutralizer described in any one of claims 1 to 5 to prepare a test solution.

9. The method according to claim 7, characterized in that: The bacterial enrichment culture in step S2 is performed by using a SCDLP bacterial enrichment solution containing polymyxin B for proliferation culture.

10. The method according to claim 7, characterized in that: The separation and culture in step S3 is carried out using BCSA agar containing polymyxin B, gentamicin and vancomycin.

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