Method for detecting bacteriostatic effect of shampoo
A method for culturing Malassezia furfur under specific conditions addresses the lack of suitable culture methods in existing technologies, allowing for efficient and cost-effective verification of antibacterial shampoo efficacy on Malassezia.
Patent Information
- Application Number
- CN202411506961.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-25
- Publication Date
- 2025-01-03
AI Technical Summary
Existing methods for evaluating the antibacterial effect of shampoos on Malassezia, which cause dandruff and other skin infections, require human efficacy testing and lack suitable culture conditions, leading to long test cycles and high costs.
A method involving culturing Malassezia furfur under specific conditions (29-31°C, MEA plus Tween 40 culture medium, with a sterile saline diluent) to create a bacterial suspension above 1×10^6 cfu/mL, followed by measuring the antibacterial effect using this suspension, including dilution, mixing, and colony counting.
This method ensures Malassezia activity while reducing test costs by providing a suitable culture method, enabling efficient verification of antibacterial shampoo efficacy on Malassezia.
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Figure CN119242761A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of detection and analysis technology, and in particular to a method for detecting the antibacterial effect of shampoo. Background Art
[0002] Malassezia is a lipophilic yeast, one of the normal flora on the skin of humans and warm-blooded animals. As a conditional pathogen, under the influence of certain susceptibility factors, Malassezia often invades the stratum corneum of the skin and causes superficial fungal infections, such as pityriasis versicolor, Malassezia folliculitis, seborrheic dermatitis, atopic dermatitis, psoriasis, onychomycosis and neonatal impetigo. Malassezia infection can easily cause pathological dandruff, which is generally manifested as excessive dandruff and clogged pores, resulting in a weakened hair state, easy bacterial proliferation, and irritation of the skin to cause itchy scalp.
[0003] Hair products that claim to remove dandruff generally need to undergo relevant efficacy evaluation tests and human efficacy tests. In particular, the efficacy of removing dandruff requires the recruitment of special subjects. At the same time, the testing cycle is long and the cost is high.
[0004] Moreover, the culture conditions of Malassezia are more special than those of general bacterial species, and it is impossible to directly refer to the culture methods of other antibacterial and antibacterial products. If only a simple reference culture condition is used, the culture of Malassezia will be affected, and it cannot be normally subcultured, preserved, and used in normal antibacterial tests. Summary of the invention
[0005] In view of the above problems, the present application is proposed to provide a method for detecting the antibacterial effect of shampoo that overcomes the above problems or at least partially solves the above problems, comprising:
[0006] A method for detecting the antibacterial effect of shampoo, comprising the steps of:
[0007] Malassezia furfur is cultured according to preset conditions to obtain a test colony; wherein the preset conditions are 29-31° C., MEA plus Tween 40 culture medium, and the diluent is a sterile saline solution containing Tween 40;
[0008] The bacterial suspension is prepared by the test colony; wherein the concentration of the bacterial suspension is higher than 1×10 6 cfu / mL;
[0009] The bacterial suspension is used to measure the object to be tested to determine the antibacterial effect of the object to be tested.
[0010] Furthermore, the step of measuring the object to be tested by the bacterial suspension to determine the antibacterial effect of the object to be tested includes:
[0011] Diluting the analyte according to preset conditions to obtain a test sample solution;
[0012] Adding the first volume of bacterial suspension to the test sample solution to obtain a test mixed solution, and placing the solution at a temperature of 29-31° C. for 50-70 seconds;
[0013] The test mixture is sampled, and a neutralizer is added to obtain a culture solution; wherein the volume ratio of the test mixture to the neutralizer is 1:9;
[0014] The culture solution is cultured to obtain the number of colonies, and the antibacterial effect of the test object is determined based on the number of colonies.
[0015] Furthermore, the step of culturing the culture medium comprises:
[0016] The culture solution is placed in an environment of 29-31° C. for 290-310 seconds;
[0017] Dividing the culture solution into several portions, and diluting them according to a preset dilution gradient to obtain several diluted culture solutions;
[0018] The diluted culture solution was sampled into a sterile plate, 10-15 mL of 44-46° C. MEA medium containing Tween 40 was added, and cultured at 29-31° C. for 48-72 hours.
[0019] Furthermore, it also includes:
[0020] Diluting the bacterial suspension to a concentration of 300-1000 cfu / mL to obtain a verification bacterial solution;
[0021] The assay conditions were verified using the verification bacterial solution.
[0022] Furthermore, the step of verifying the assay conditions using the verification bacterial solution includes:
[0023] The neutralization effect of the neutralizer is determined by the verification bacterial solution. Specifically, 9 ml of the neutralizer is added to 1 ml of the test object, heated to 29-31° C. and maintained for 50-70 seconds, 1 ml of the verification bacterial solution is added, and placed in an environment of 29-31° C. for 290-310 seconds, sampling is calculated to calculate the number of colonies, and the neutralization effect is determined based on the number of colonies.
[0024] Furthermore, the step of verifying the assay conditions using the verification bacterial solution includes:
[0025] The toxic effect of the neutralizer is determined by the verification bacterial solution; specifically, 9 ml of the neutralizer is added to 1 ml of the buffer solution, heated to 29-31° C. and maintained for 50-70 seconds, 1 ml of the verification bacterial solution is added, and placed in an environment of 29-31° C. for 290-310 seconds, sampling is calculated to calculate the number of colonies, and the toxic effect of the neutralizer is determined based on the number of colonies.
[0026] Furthermore, the step of verifying the assay conditions using the verification bacterial solution includes:
[0027] The bacterial activity is determined by the verification bacterial solution; specifically, 1 ml of the verification bacterial solution is added to 10 ml of the buffer solution, placed in an environment of 29-31° C. for 290-310 seconds, sampling is performed to calculate the number of colonies, and the bacterial activity is determined based on the number of colonies.
[0028] Furthermore, the step of verifying the assay conditions using the verification bacterial solution includes:
[0029] The environmental effect is determined by the verification bacterial solution; specifically, 1 ml of the verification bacterial solution is added to 10 ml of the test object, placed in an environment of 29-31° C. for 290-310 seconds, samples are taken to calculate the number of colonies, and the bacterial activity is determined based on the number of colonies.
[0030] Furthermore, the bacterial activity of the test colony is greater than 6, and the colony is white and raised.
[0031] Furthermore, the step of culturing Malassezia furfur to obtain a test colony according to preset conditions comprises:
[0032] Inoculate the first generation culture by streaking on a rough slant of MEA plus Tween 40 medium;
[0033] The inoculated culture medium was placed in an environment of 29-31°C and cultured for 48-72 hours;
[0034] The colonies in the culture medium were eluted using a mixture of sterile saline and Tween 40 to obtain test colonies.
[0035] This application has the following advantages:
[0036] In the embodiments of the present application, the detection schemes for the anti-dandruff effect of shampoo products in the prior art all require human efficacy testing and do not have a suitable culture method for Malassezia, the test cycle is long, and the cost is high. The present application provides a method for detecting the antibacterial effect of shampoo, including the steps of: culturing Malassezia furfur to obtain a test colony according to preset conditions; wherein the preset conditions are 29-31°C, MEA plus Tween 40 culture medium, and the diluent is a sterile saline containing Tween 40; configuring a bacterial suspension through the test colony; wherein the concentration of the bacterial suspension is higher than 1×106cfu / mL; and determining the antibacterial effect of the test object through the bacterial suspension. Malassezia is cultured under preset conditions to ensure the activity of Malassezia while reducing the test cost, thereby verifying the antibacterial effect of anti-dandruff shampoo on Malassezia. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the description of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.
[0038] Figure 1 This is a flow chart of the steps of a method for detecting the antibacterial effect of shampoo provided in one embodiment of the present application;
[0039] Figure 2 This is the culture status of Malassezia furfur under different culture conditions in a method for detecting the antibacterial effect of a shampoo provided in one embodiment of the present application. DETAILED DESCRIPTION
[0040] In order to make the objects, features and advantages of the present application more obvious and understandable, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.
[0041] The inventors have found through analysis of the prior art that the prior art does not have the most suitable culture conditions for the production of Malassezia and cannot be normally propagated, so when evaluating anti-dandruff hair products, subjects need to spend a lot of time on evaluation and comparison, which results in a long cycle and high cost.
[0042] Reference Figure 1 , showing a flow chart of the steps of a method for detecting the antibacterial effect of shampoo provided in an embodiment of the present application:
[0043] The method comprises:
[0044] S110, culturing Malassezia furfur according to preset conditions to obtain test colonies; wherein the preset conditions are 29-31° C., MEA plus Tween 40 culture medium, and the diluent is sterile saline containing Tween 40;
[0045] S120, preparing a bacterial suspension using the test colonies; wherein the concentration of the bacterial suspension is higher than 1×106 cfu / mL;
[0046] S130, measuring the object to be tested by using the bacterial suspension to determine the antibacterial effect of the object to be tested.
[0047] In the embodiments of the present application, the detection schemes for the anti-dandruff effect of shampoo products in the prior art all require human efficacy testing and do not have a suitable culture method for Malassezia, the test cycle is long, and the cost is high. The present application provides a method for detecting the antibacterial effect of shampoo, including the steps of: culturing Malassezia furfur to obtain a test colony according to preset conditions; wherein the preset conditions are 29-31°C, MEA plus Tween 40 culture medium, and the diluent is a sterile saline containing Tween 40; configuring a bacterial suspension through the test colony; wherein the concentration of the bacterial suspension is higher than 1×106cfu / mL; and determining the antibacterial effect of the test object through the bacterial suspension. Malassezia is cultured under preset conditions to ensure the activity of Malassezia while reducing the test cost, thereby verifying the antibacterial effect of anti-dandruff shampoo on Malassezia.
[0048] Next, a method for detecting the antibacterial effect of shampoo in this exemplary embodiment will be further described.
[0049] As described in step S110, Malassezia furfur is cultured according to preset conditions to obtain test colonies; wherein the preset conditions are 29-31°C, MEA plus Tween 40 culture medium, and the diluent is sterile saline containing Tween 40.
[0050] It should be noted that Malassezia is a yeast. By comparing the most suitable culture temperature for yeast, that is, within the range of 25℃-35℃, the culture conditions of Malassezia at various temperature gradients were found. Malassezia is a lipid species, and the general fungal culture medium cannot meet the growth conditions of Malassezia, so fat needs to be added.
[0051] like Figure 2As shown in the figure, the culture conditions of Malassezia furfur under different culture media at different temperatures were recorded. The growth rate of the target bacteria required G≥6. In the test results in the following table, the test conditions of G≥6 were: at a culture temperature of 28℃±1℃, MEA+Tween 40 and MEA+Tween 20 were used; at a culture temperature of 30℃±1℃, MEA+Tween 80, MEA+Tween 40 and MEA+Tween 20 were used; at a culture temperature of 32.5℃±2.5℃, MEA+Tween 80, MEA+Tween 40 and MEA+Tween 20 were used; among them, MEA+Tween 40 had G=10 under the conditions of 30℃±1℃ and 32.5℃±2.5℃. However, when cultured under the conditions of 32.5℃±2.5℃, the colony morphology was dry, which may lead to insufficient activity of the strain. In summary, combined with the growth rate, morphology, activity and other conditions of the strain, Malassezia furfur (AATCC 44344) Under the experimental conditions of this laboratory, the optimal growth and culture conditions are: the culture medium is MEA + Tween 40 medium, the diluent is sterile saline containing Tween 40, and the culture temperature is 30℃±1℃.
[0052] In one embodiment of the present invention, the specific process of "culturing Malassezia furfur to obtain test colonies according to preset conditions" in step S110 can be further explained in combination with the following description.
[0053] Inoculate the first generation culture by streaking on a rough slant of MEA plus Tween 40 medium as described in the following steps;
[0054] Incubate the inoculated culture medium at 29-31°C for 48-72 hours as described in the following steps;
[0055] As described in the following steps, the colonies in the culture medium were eluted with a mixture of sterile saline and Tween 40 to obtain test colonies.
[0056] As an example, use an inoculation loop to streak the first generation culture on the rough slope of MEA + Tween 40 medium, and place the inoculated medium at 30℃±1℃ for 48-72h. After each culture, observe whether the culture and surface bacterial morphology are abnormal. The morphology of Malassezia furfur (ATCC 44344) should be white, small, and scurfy. Use 10mL of sterile saline + Tween 40 to elute the surface colonies of the MEA slope containing Tween 40. Use a glass rod or other suitable tools to gently scrape the agar surface to better elute the colonies.
[0057] As described in step S120, a bacterial suspension is prepared by using the test colonies; wherein the concentration of the bacterial suspension is higher than 1×10 6 cfu / mL.
[0058] As described in step S130, the bacterial suspension is used to measure the object to be tested to determine the antibacterial effect of the object to be tested.
[0059] In one embodiment of the present invention, the specific process of "determining the antibacterial effect of the object to be tested by using the bacterial suspension" in step S130 can be further explained in combination with the following description.
[0060] As described in the following steps, the test object is diluted according to preset conditions to obtain a test sample solution;
[0061] As described in the following steps, a first volume of bacterial suspension is added to the test sample solution to obtain a test mixture, and the mixture is placed at a temperature of 29-31° C. for 50-70 seconds;
[0062] As described in the following steps, the test mixture is sampled, a neutralizer is added, and a culture solution is obtained; wherein the volume ratio of the test mixture to the neutralizer is 1:9;
[0063] As described in the following steps, the culture solution is cultured to obtain the number of colonies, and the antibacterial effect of the test object is determined based on the number of colonies.
[0064] In one embodiment of the present invention, the following description can be combined to further illustrate the specific process of culturing the culture solution to obtain the number of colonies and determining the antibacterial effect of the test object based on the number of colonies.
[0065] As described in the following steps, the culture medium is placed in an environment of 29-31° C. for 290-310 seconds;
[0066] As described in the following steps, the culture solution is divided into several portions, and diluted according to a preset dilution gradient to obtain several diluted culture solutions;
[0067] As described in the following steps, the diluted culture solution is sampled into a sterile plate, 10-15 mL of 44-46° C. MEA medium containing Tween 40 is added, and the culture is carried out at 29-31° C. for 48-72 hours.
[0068] In a specific implementation, take 9.5 mL of sample solution into a sterile empty tube, add 0.5 mL of working bacterial solution, and mix thoroughly. Place in a water bath at the corresponding temperature for 2 min ± 10 s. At the end of the timing, take 1.0 mL of the above mixture and transfer it to a test tube containing 9.0 mL of neutralizer. After mixing, place the test tube in a water bath at the corresponding temperature for 5 min ± 10 s, and then select the appropriate dilution level for dilution. Take 1.0 mL of each dilution level into a sterile plate, pour 10-15 mL of 45 ± 1 ° C Tween-containing MEA medium, and culture in a 30 ± 1 ° C incubator for 48-72 hours.
[0069] Counting scheme: Select plates with 30-300 colonies for counting, and count the colonies under a 10x magnifying glass.
[0070] Evaluation standard of sample antibacterial effect: Calculate the logarithmic value of the reduction in the number of colonies of the test sample. If the reduction value is ≥4.0, the shampoo antibacterial effect is qualified.
[0071] Calculate the percentage of reduction in the number of colonies in the test sample. If the reduction value is ≥99.99%, the shampoo's antibacterial effect is qualified.
[0072] In one embodiment of the present invention, the method further comprises diluting the bacterial suspension to a concentration of 300-1000 cfu / mL to obtain a verification bacterial solution;
[0073] The assay conditions were verified using the verification bacterial solution.
[0074] It should be noted that the assay conditions need to be verified by the verification bacterial solution, and the assay conditions include the neutralization effect of the neutralizer, the toxic effect of the neutralizer, bacterial activity and other environmental conditions.
[0075] In an embodiment of the present invention, the specific process of verifying the assay conditions by using the verification bacterial solution can be further described in combination with the following description.
[0076] As described in the following steps, the neutralization effect of the neutralizer is determined by the verification bacterial solution. Specifically, 9 ml of the neutralizer is added to 1 ml of the test object, heated to 29-31° C. and maintained for 50-70 seconds, 1 ml of the verification bacterial solution is added, and placed in an environment of 29-31° C. for 290-310 seconds, sampling is calculated to calculate the number of colonies, and the neutralization effect is determined based on the number of colonies.
[0077] As described in the following steps, the toxic effect of the neutralizer is determined by the verification bacterial solution; specifically, 9 ml of the neutralizer is added to 1 ml of the buffer solution, heated to 29-31° C. and maintained for 50-70 seconds, 1 ml of the verification bacterial solution is added, and placed in an environment of 29-31° C. for 290-310 seconds, sampling is calculated to calculate the number of colonies, and the toxic effect of the neutralizer is determined based on the number of colonies.
[0078] As described in the following steps, the bacterial activity is determined by the verification bacterial solution; specifically, 1 ml of the verification bacterial solution is added to 10 ml of the buffer solution, placed in an environment of 29-31° C. for 290-310 seconds, sampling is performed to calculate the number of colonies, and the bacterial activity is determined based on the number of colonies.
[0079] As described in the following steps, the environmental effect is determined by the verification bacterial solution; specifically, 1 ml of the verification bacterial solution is added to 10 ml of the test object, placed in an environment of 29-31° C. for 290-310 seconds, samples are taken to calculate the number of colonies, and the bacterial activity is determined based on the number of colonies.
[0080] The requirements for the test colonies in any embodiment of the present invention require that the bacterial activity is greater than 6 and the appearance is white raised colonies.
[0081] Although the preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present application.
[0082] Finally, it should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or terminal device including the elements.
[0083] The above is a detailed introduction to the detection method for the antibacterial effect of shampoo provided by the present application. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea; at the same time, for general technicians in this field, according to the idea of the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.
Claims
1. A method for detecting the antibacterial effect of shampoo, characterized in that: Includes steps: Malassezia furfur is cultured according to preset conditions to obtain a test colony; wherein the preset conditions are 29-31° C., MEA plus Tween 40 culture medium, and the diluent is a sterile saline solution containing Tween 40; The bacterial suspension is prepared by the test colony; wherein the concentration of the bacterial suspension is higher than 1× 10 6 cfu / mL; The bacterial suspension is used to measure the object to be tested to determine the antibacterial effect of the object to be tested.
2. The detection method according to claim 1, characterized in that: The step of measuring the object to be tested by the bacterial suspension to determine the antibacterial effect of the object to be tested comprises: Diluting the analyte according to preset conditions to obtain a test sample solution; Adding the first volume of bacterial suspension to the test sample solution to obtain a test mixed solution, and placing the solution at a temperature of 29-31° C. for 50-70 seconds; The test mixture is sampled, and a neutralizer is added to obtain a culture solution; wherein the volume ratio of the test mixture to the neutralizer is 1:9; The culture solution is cultured to obtain the number of colonies, and the antibacterial effect of the test object is determined based on the number of colonies.
3. The detection method according to claim 2, characterized in that: The step of culturing the culture fluid comprises: The culture solution is placed in an environment of 29-31° C. for 290-310 seconds; Dividing the culture solution into several portions, and diluting them according to a preset dilution gradient to obtain several diluted culture solutions; The diluted culture solution was sampled into a sterile plate, 10-15 mL of 44-46° C. MEA medium containing Tween 40 was added, and cultured at 29-31° C. for 48-72 hours.
4. The detection method according to claim 1, characterized in that: Also includes: Diluting the bacterial suspension to a concentration of 300-1000 cfu / mL to obtain a verification bacterial solution; The assay conditions were verified using the verification bacterial solution.
5. The detection method according to claim 4, characterized in that: The step of verifying the assay conditions by using the verification bacterial solution comprises: The neutralization effect of the neutralizer is determined by the verification bacterial solution. Specifically, 9 ml of the neutralizer is added to 1 ml of the test object, heated to 29-31° C. and maintained for 50-70 seconds, 1 ml of the verification bacterial solution is added, and placed in an environment of 29-31° C. for 290-310 seconds, sampling is calculated to calculate the number of colonies, and the neutralization effect is determined based on the number of colonies.
6. The detection method according to claim 4, characterized in that: The step of verifying the assay conditions by using the verification bacterial solution comprises: The toxic effect of the neutralizer is determined by the verification bacterial solution; specifically, 9 ml of the neutralizer is added to 1 ml of the buffer solution, heated to 29-31° C. and maintained for 50-70 seconds, 1 ml of the verification bacterial solution is added, and placed in an environment of 29-31° C. for 290-310 seconds, sampling is calculated to calculate the number of colonies, and the toxic effect of the neutralizer is determined based on the number of colonies.
7. The detection method according to claim 4, characterized in that: The step of verifying the assay conditions by using the verification bacterial solution comprises: The bacterial activity is determined by the verification bacterial solution; specifically, 1 ml of the verification bacterial solution is added to 10 ml of the buffer solution, placed in an environment of 29-31° C. for 290-310 seconds, sampling is performed to calculate the number of colonies, and the bacterial activity is determined based on the number of colonies.
8. The detection method according to claim 4, characterized in that: The step of verifying the assay conditions by using the verification bacterial solution comprises: The environmental effect is determined by the verification bacterial solution; specifically, 1 ml of the verification bacterial solution is added to 10 ml of the test object, placed in an environment of 29-31° C. for 290-310 seconds, samples are taken to calculate the number of colonies, and the bacterial activity is determined based on the number of colonies.
9. The detection method according to claim 1, characterized in that: The bacterial activity of the test colonies is greater than 6, and the colonies are white convex colonies.
10. The detection method according to claim 1, characterized in that: The step of culturing Malassezia furfur to obtain a test colony according to preset conditions comprises: Inoculate the first generation culture by streaking on a rough slant of MEA plus Tween 40 medium; The inoculated culture medium was placed in an environment of 29-31°C and cultured for 48-72 hours; The colonies in the culture medium were eluted using a mixture of sterile saline and Tween 40 to obtain test colonies.