Screening method and application of cosmetic preservative

Abstract

The invention relates to a screening method and application of cosmetic preservatives, and belongs to the technical field of application of preservatives. The invention relates to a method for screening cosmetic preservatives, in particular to a method for screening cosmetic preservatives which can inhibit pathogenic bacteria and are friendly to skin bacteria by taking SVS values and / or CVS values as screening indexes. Wherein the SVS value is equal to the MIC value of the staphylococcus epidermidis / the MIC value of the staphylococcus aureus, and the CVS value is equal to the MIC value of the propionibacterium acnes / the MIC value of the staphylococcus aureus. The preservative composition constructed by the method has a good preservative effect, and has certain micro-ecological friendliness to the growth of resident skin flora (namely skin bacteria).

Description

Technical Field

[0001] The invention relates to a method for screening a cosmetic preservative and application thereof, belonging to the technical field of preservative application. Background Art

[0002] The growth and reproduction of microorganisms in cosmetics may cause product deterioration and cause infection to consumers. Therefore, cosmetics widely use preservatives to ensure product safety. Chinese patent document CN110634540A (application number 201911094696.8) proposes a cosmetic preservative application evaluation system and method, which includes: establishing a database of minimum inhibitory concentrations of each preservative; calculating the antibacterial efficiency of each preservative on the inhibited bacteria; when the total antibacterial efficiency of each preservative on the inhibited bacteria meets the preset antibacterial efficiency, the cosmetic preservative ratio is judged to be qualified, otherwise, it is unqualified. The types of inhibitory bacteria that need to be evaluated for cosmetics include Pseudomonas aeruginosa-Gram-negative, Escherichia coli-Gram-negative, Staphylococcus aureus-Gram-positive, Aspergillus niger-mold, and Candida albicans-yeast.

[0003] Nowadays, while people are paying attention to the preservative effect of preservatives, they are also paying more and more attention to their safety. There are safety disputes about some preservatives, such as methylisothiazolinone causing contact allergies, and parabens (parabens) interfering with endocrine. In addition, while cosmetic preservatives inhibit and kill pathogenic bacteria, they also interfere with and damage the resident flora of the skin. Studies have shown that the short-term use of 0.5% to 5% methylparaben in cosmetics has a significant inhibitory effect on Propionibacterium acnes and Staphylococcus epidermidis. The purpose of using cosmetics is to be beautiful and healthy. Its short-term or long-term use should not kill or significantly inhibit the resident flora of the skin, which poses new challenges to the use of preservatives in cosmetics.

[0004] Skin microecology is a balance achieved by the continuous interaction between different microbial communities on the skin and between skin microorganisms and the host. Maintaining this balance is very important for skin health. Staphylococcus epidermidis and Propionibacterium acnes are the main abundant bacteria resident on the skin, also known as sentinel bacteria. They are very important for maintaining the balance of skin microecology. Changes in their abundance and diversity can lead to many skin problems, such as atopic dermatitis, dandruff and acne.

[0005] Currently, the recommended types and dosages of preservatives used in cosmetics are often based on toxicological safety data of cells, animals and humans, without considering the impact on resident microorganisms on human skin. The relationship between the use of preservatives and the health of skin microbial communities is still unclear. The development of preservative systems in cosmetics needs to include the impact of skin flora in the scope of risk assessment research.

[0006] Preservatives are widely used in cosmetics, which interfere with and damage the resident bacteria on the skin, thus affecting the skin's microecological environment and easily causing skin problems or even skin diseases. In order to reduce the damage of preservatives to resident bacteria on the skin, it is necessary to explore the types and dosages of preservatives used in cosmetics to fill the gaps in existing technologies. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a method for screening cosmetic preservatives and applications thereof. The present invention obtains the minimum inhibitory concentration (MIC) of preservatives commonly used in the cosmetics catalog against skin bacteria and common pathogenic bacteria (cosmetic preservative challenge test strains), and uses SVS value and CVS value as indicators to discover preservatives that are friendly to skin flora. The preservative combination constructed by this method has a good preservative effect and has a certain microecological friendliness to the growth of resident skin flora (i.e., skin bacteria).

[0008] The technical solution of the present invention is as follows:

[0009] A method for screening cosmetic preservatives, using SVS value and / or CVS value as screening indexes, to screen cosmetic preservatives that can inhibit pathogenic bacteria and are friendly to skin bacteria; wherein:

[0010] SVS value = MIC value of Staphylococcus epidermidis / MIC value of Staphylococcus aureus,

[0011] CVS value = MIC value of Propionibacterium acnes / MIC value of Staphylococcus aureus.

[0012] The present invention uses the CVS value as a standard for measuring the friendliness to Propionibacterium acnes to eliminate the influence of the difference in the amount of different preservatives used. A value ≥1 indicates that the MIC value for inhibiting Propionibacterium acnes is greater than or equal to the MIC value of Staphylococcus aureus, and is friendly to Propionibacterium acnes, and the larger the value, the friendlier; a value <1 indicates poor friendliness to Propionibacterium acnes. Similarly, the SVS value is used as a standard for measuring the friendliness to Staphylococcus epidermidis. A value ≥1 indicates that the MIC value for inhibiting Staphylococcus epidermidis is greater than or equal to the MIC value of Staphylococcus aureus, and is friendly to Staphylococcus epidermidis, and the larger the value, the friendlier; a value <1 indicates poor friendliness to Staphylococcus epidermidis.

[0013] Preferably according to the present invention, the Staphylococcus epidermidis is Staphylococcus epidermidis CCSM 0287, Staphylococcus epidermidis ATCC 12228 or Staphylococcus epidermidis ATCC 35984; more preferably, it is Staphylococcus epidermidis CCSM 0287.

[0014] Preferably according to the present invention, the Propionibacterium acnes is Propionibacterium acnes CCSM 0331 or Propionibacterium acnes ATCC 6919; further preferably, it is Propionibacterium acnes CCSM 0331.

[0015] Preferably according to the present invention, the Staphylococcus aureus is Staphylococcus aureus ATCC 6538.

[0016] Application of the above screening method in the screening of cosmetic preservatives.

[0017] A cosmetic preservative screened by the screening method comprises at least one of 1,2-hexanediol, phenoxyethanol and ethylhexylglycerin.

[0018] Preferably according to the present invention, the cosmetic preservative is a combination of 1,2-hexanediol and phenoxyethanol.

[0019] More preferably, the concentration of 1,2-hexanediol is 0.5%-1%, and the concentration of phenoxyethanol is 0.25%-0.5%, both of which are mass percentages.

[0020] Preferably according to the present invention, the cosmetic preservative is a combination of 1,2-hexanediol and ethylhexylglycerin.

[0021] More preferably, the concentration of 1,2-hexanediol is 0.5%-1%, and the concentration of ethylhexylglycerol is 0.125%-0.25%, both of which are mass percentages.

[0022] Beneficial effects:

[0023] The present invention provides a method for screening microecologically friendly cosmetic preservatives. On the basis of obtaining the minimum inhibitory concentration (MIC) of each preservative against skin bacteria and common pathogenic bacteria (strains of cosmetic preservative challenge test), the preservatives that can inhibit common pathogenic bacteria and be friendly to skin bacteria are screened with SVS value and CVS value as indicators. The new preservative combination screened by the present invention can pass the preservative challenge test and is friendly to skin bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is a bar graph showing the MIC values ​​of 16 common preservatives against Staphylococcus epidermidis CCSM 0287.

[0025] Figure 2 The figure is a bar graph showing the MIC values ​​of 16 common preservatives against Staphylococcus epidermidis ATCC12228.

[0026] Figure 3 The figure is a bar graph showing the MIC values ​​of 16 common preservatives against Staphylococcus epidermidis ATCC 35984.

[0027] Figure 4 The figure is a bar graph showing the MIC values ​​of 16 common preservatives against Staphylococcus aureus CCSM 0349.

[0028] Figure 5 The figure is a bar graph showing the MIC values ​​of 16 common preservatives against Propionibacterium acnes CCSM 0331.

[0029] Figure 6 The figure is a bar graph showing the MIC values ​​of 16 common preservatives against Propionibacterium acnes ATCC 6919.

[0030] Figure 7 The figure is a bar graph showing the MIC values ​​of 16 common preservatives against Staphylococcus aureus ATCC 6538.

[0031] Figure 8 The figure is a bar graph showing the MIC values ​​of 16 common preservatives against Escherichia coli ATCC 8739.

[0032] Fig. 9 The figure is a bar graph showing the MIC values ​​of 16 common preservatives against Pseudomonas aeruginosa ATCC 9027.

[0033] Fig.10 The figure is a bar chart of SVS values ​​of 16 common preservatives.

[0034] Fig.11 The following is a bar chart showing the CVS values ​​of 16 common preservatives.

[0035] Fig.12 The results of the chessboard test of the combination of 1,2-hexanediol and phenoxyethanol on four bacteria; among them, (A) is Pseudomonas aeruginosa ATCC 9027; (B) is Escherichia coli ATCC 8739; (C) is Staphylococcus aureus ATCC6538; (D) is Staphylococcus epidermidis CCSM 0287; dark color means inhibition rate <20%, medium color means inhibition rate between 25%-50%, light color means inhibition rate between 50%-90%, and colorless means inhibition rate >90%.

[0036] Fig.13 The results of the chessboard test of the combination of 1,2-hexanediol and ethylhexylglycerol on four bacteria; among them, (A) is Pseudomonas aeruginosa ATCC 9027; (B) is Escherichia coli ATCC 8739; (C) is Staphylococcus aureus ATCC 6538; (D) is Staphylococcus epidermidis CCSM 0287; dark color means inhibition rate <20%, medium color means inhibition rate between 25%-50%, light color means inhibition rate between 50%-90%, and colorless means inhibition rate >90%.

[0037] Fig.14This is a bar graph showing the inhibition rates of five preservative combinations against four types of bacteria. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0039] The preservatives and experimental strains involved in this embodiment are as follows:

[0040] Preservatives: 1,2-butylene glycol, 1,2-pentanediol, 1,2-hexanediol, sodium salicylate, sodium benzoate, benzyl alcohol, phenoxyethanol, benzalkonium bromide, p-hydroxyacetophenone, ethylhexylglycerin, chlorphenesin, imidazolidinyl urea, potassium sorbate, caprylhydroxamic acid, DMDM-hydantoin, bis(hydroxymethyl)imidazolidinyl urea, and methylparaben were all purchased from Aladdin Biochemical Technology Co., Ltd.

[0041] Experimental strain: Propionibacterium acnes CCSM 0331: isolated from healthy human cheek skin, deposited in China Center for Type Culture Collection, deposit number CCTCC No: M 2022781, which has been disclosed in the patent document with application number 202210704625.0. This patent does not involve microbial preservation;

[0042] Propionibacterium acnes ATCC 6919: purchased from Guangdong Microbiological Culture Collection Center, the strain number on the platform is GDMCC NO.1.243;

[0043] Staphylococcus capitis CCSM 0349: isolated from healthy human cheek skin, deposited in China Center for Type Culture Collection, number CCTCC NO: M2022772; this strain is an existing strain that has been disclosed, and this patent does not involve microbial preservation;

[0044] Staphylococcus epidermidis CCSM 0287: isolated from healthy human cheek skin, deposited in China Center for Type Culture Collection, number CCTCC NO: M2022779, has been disclosed in the patent document with application number 202210704624.6. This patent does not involve microbial preservation;

[0045] Staphylococcus epidermidis ATCC 12228: purchased from the Beina bacterial strain resource library, and the strain number on the platform is BNCC330867;

[0046] Staphylococcus epidermidis ATCC 35984: purchased from the American Type Culture Collection;

[0047] Staphylococcus aureus ATCC 6538: purchased from China General Microbiological Culture Collection Center (CGMCC), the strain number on the platform is CGMCC NO.1.2465;

[0048] Escherichia coli ATCC 8739: purchased from China General Microbiological Culture Collection Center (CGMCC), the strain number on the platform is CGMCC NO.1.2463;

[0049] Pseudomonas aeruginosa ATCC 9027: purchased from China General Microbiological Culture Collection Center (CGMCC), and the strain number on the platform is CGMCC NO.1.2421.

[0050] Example 1: Determination of MIC values ​​of common preservatives against skin bacteria

[0051] The skin bacteria selected in this embodiment include: Staphylococcus epidermidis CCSM 0287, Staphylococcus epidermidis ATCC12228, Staphylococcus epidermidis ATCC 35984, Staphylococcus capitis CCSM 0349, Propionibacterium acnes CCSM 0331, Propionibacterium acnes ATCC 6919. The MIC value determination method of common preservatives for the above skin bacteria is as follows:

[0052] 1) Cultivate skin bacteria using conventional methods and measure the OD of the culture solution 600 , substitute into the standard curve to obtain the bacterial concentration, and dilute the bacterial concentration to 1.0×10 8 CFU / mL;

[0053] 2) Continue to dilute the bacterial concentration to 1.0×10 6 CFU / mL (used up within half an hour);

[0054] 3) In a 96-well plate, 100 μL of bacterial solution and 100 μL of preservatives of different concentrations were added to each well of the experimental group, 100 μL of TSB medium and 100 μL of preservatives of different concentrations were added to each well of the blank group, 200 μL of TSB medium was added to each well of the sterile control group, and 100 μL of TSB medium and 100 μL of bacterial solution were added to each well of the growth control group. Four replicate wells were made for each group;

[0055] 4) Culture at 37°C for 24 hours (anaerobic culture of Propionibacterium acnes for 72 hours);

[0056] 5) OD measurement 600 The lowest concentration of preservative at which the number of colonies remains unchanged is the MIC value of the preservative for the strain, that is, no optical turbidity is produced.

[0057] The MIC values ​​of common preservatives against Staphylococcus epidermidis CCSM 0287 are as follows: Figure 1 As shown, the results showed that the MIC values ​​of 16 common preservatives against Staphylococcus epidermidis CCSM 0287 were 200-125000 mg / L;

[0058] The MIC values ​​of common preservatives against Staphylococcus epidermidis ATCC12228 are as follows: Figure 2 As shown, the results showed that the MIC values ​​of 16 common preservatives against Staphylococcus epidermidis ATCC12228 were 200-125000 mg / L;

[0059] The MIC values ​​of common preservatives against Staphylococcus epidermidis ATCC 35984 are as follows: Figure 3 As shown, the results showed that the MIC values ​​of 16 common preservatives against Staphylococcus epidermidis ATCC 35984 were 200-125000 mg / L.

[0060] The MIC values ​​of common preservatives against Staphylococcus aureus CCSM 0349 are as follows: Figure 4 As shown, the results showed that the MIC values ​​of 16 common preservatives against Staphylococcus aureus CCSM 0349 were 100-125000 mg / L.

[0061] The MIC values ​​of common preservatives against Propionibacterium acnes CCSM 0331 are as follows: Figure 5 As shown in the figure, the results showed that the MIC values ​​of 16 common preservatives against Propionibacterium acnes CCSM 0331 were 100-62500 mg / L.

[0062] The MIC values ​​of common preservatives against Propionibacterium acnes ATCC 6919 are as follows: Figure 6 As shown, the results showed that the MIC values ​​of 16 common preservatives against Propionibacterium acnes ATCC 6919 were 200-62500 mg / L.

[0063] Example 2: Determination of MIC values ​​of common preservatives against pathogenic bacteria

[0064] The pathogenic bacteria selected in this embodiment include: Staphylococcus aureus ATCC 6538, Escherichia coli ATCC8739, Pseudomonas aeruginosa ATCC 9027, and the MIC value determination method of common preservatives for the above pathogenic bacteria is as follows:

[0065] 1) Cultivate pathogenic bacteria using conventional methods and measure the OD of the culture solution 600 , substitute into the standard curve to obtain the bacterial concentration, and dilute the bacterial concentration to 1.0×10 8 CFU / mL;

[0066] 2) Continue to dilute the bacterial concentration to 1.0×106 CFU / mL (used up within half an hour);

[0067] 3) In a 96-well plate, 100 μL of bacterial solution and 100 μL of preservatives of different concentrations were added to each well of the experimental group, 100 μL of TSB medium and 100 μL of preservatives of different concentrations were added to each well of the blank group, 200 μL of TSB medium was added to each well of the sterile control group, and 100 μL of TSB medium and 100 μL of bacterial solution were added to each well of the growth control group. Four replicate wells were made for each group;

[0068] 4) Incubate at 37°C for 24 hours;

[0069] 5) OD measurement 600 The lowest concentration of preservative at which the number of colonies remains unchanged is the MIC value of the preservative for the strain, that is, no optical turbidity is produced.

[0070] The MIC values ​​of common preservatives against Staphylococcus aureus ATCC 6538 are as follows: Figure 7 As shown, the results showed that the MIC values ​​of 16 common preservatives against Staphylococcus aureus ATCC 6538 were 200-125000 mg / L;

[0071] The MIC values ​​of common preservatives against Escherichia coli ATCC 8739 are as follows: Figure 8 As shown, the results showed that the MIC values ​​of 16 common preservatives against Escherichia coli ATCC 8739 were 200-62500 mg / L;

[0072] The MIC values ​​of common preservatives against Pseudomonas aeruginosa ATCC 9027 are as follows: Fig. 9 As shown, the results showed that the MIC values ​​of 16 common preservatives against Pseudomonas aeruginosa ATCC 9027 were 200-31250 mg / L.

[0073] Example 3: Screening method for microecologically friendly cosmetic preservatives

[0074] Propionibacterium acnes and Staphylococcus epidermidis are the main abundant bacteria on the skin and are also sentinel bacteria. The stability of Propionibacterium acnes and Staphylococcus epidermidis is crucial to maintaining the balance of skin microecology and skin health. The MIC value is only the minimum concentration at which the preservative can completely inhibit bacterial growth. The friendliness of the preservative to skin bacteria cannot be directly judged based on the MIC value.

[0075] The purpose of building a skin microecological friendly preservative system is to minimize the impact on skin resident bacteria at a concentration that completely inhibits pathogenic bacteria. The results of MIC value determination show that most preservatives can completely inhibit Escherichia coli and Pseudomonas aeruginosa at a concentration that can completely inhibit Staphylococcus aureus. Therefore, we chose the MIC value of Staphylococcus aureus as the standard control, introduced SVS value and CVS value, and judged the friendliness of preservatives to Staphylococcus epidermidis and Propionibacterium acnes. Among them:

[0076] SVS value = MIC value of Staphylococcus epidermidis CCSM 0287 / MIC value of Staphylococcus aureus ATCC 6538

[0077] CVS value = MIC value of Propionibacterium acnes CCSM 0331 / MIC value of Staphylococcus aureus ATCC 6538

[0078] The CVS value is used as a standard to measure the friendliness to Propionibacterium acnes to eliminate the influence of the different amounts of preservatives used. A value ≥ 1 indicates that the MIC value of inhibiting Propionibacterium acnes is greater than or equal to the MIC value of Staphylococcus aureus, which is friendly to Propionibacterium acnes, and the larger the value, the friendlier it is; a value < 1 indicates poor friendliness to Propionibacterium acnes. Similarly, the SVS value is used as a standard to measure the friendliness to Staphylococcus epidermidis to eliminate the influence of the different amounts of preservatives used.

[0079] The SVS values ​​of 16 common preservatives are shown in Table 1 and Fig.10 As shown in the figure, from the SVS values, except for methylparaben, the SVS values ​​of other preservatives are all ≥1, which means that the MIC values ​​of most preservatives to Staphylococcus epidermidis CCSM 0287 are greater than or equal to the MIC values ​​to Staphylococcus aureus ATCC 6538, and they are relatively friendly to Staphylococcus epidermidis CCSM 0287. The preservatives that are friendly to Staphylococcus epidermidis are 1,2-hexanediol, ethylhexylglycerol, imidazolidinyl urea, and DMDM-hydantoin.

[0080] Table 1. SVS values ​​of 16 common preservatives

[0081]

[0082] The CVS values ​​of 16 common preservatives are shown in Table 2 and Fig.11 As shown in the figure, from the CVS value, the CVS values ​​of most preservatives are less than 1, which indicates that the preservatives are less friendly to Propionibacterium acnes CCSM 0331. Preservatives that are relatively friendly to Propionibacterium acnes include sodium salicylate, sodium benzoate, ethylhexylglycerin, caprylhydroxamic acid, DMDM-hydantoin, imidazolidinyl urea, and chlorphenesin.

[0083] Table 2. CVS values ​​of 16 common preservatives

[0084]

[0085] Judging from the SVS and CVS values, almost all preservatives have high SVS values ​​and are more friendly to Staphylococcus epidermidis, while their CVS values ​​are low and are less friendly to Propionibacterium acnes.

[0086] Example 4: Construction of preservative combinations

[0087] According to the SVS value and CVS value of the preservative in Example 3, combined with the application of common preservatives in the industry, in order to increase the potential for widespread application of preservatives in the future, two combinations of 1,2-hexanediol and phenoxyethanol, and 1,2-hexanediol and ethylhexylglycerol were selected to conduct the following experiment of constructing a preservative combination, wherein 1,2-hexanediol can be used as both a moisturizer and a preservative. Currently, 1,2-hexanediol is widely used in the industry as an ingredient outside the preservative list. The specific steps are as follows:

[0088] 1) preparing a preservative stock solution, for a combination of 1,2-hexanediol and phenoxyethanol, 1,2-hexanediol is recorded as preservative X, the stock solution mass concentration is 5%, phenoxyethanol is recorded as preservative Y, the stock solution mass concentration is 5%; for a combination of 1,2-hexanediol and ethylhexylglycerol, 1,2-hexanediol is recorded as preservative X, the stock solution mass concentration is 5%, ethylhexylglycerol is recorded as preservative Y, the stock solution mass concentration is 1.25%;

[0089] 2) For preservative X, two-fold dilution was performed to obtain 7 solutions of preservative X with different concentrations, and the same operation was performed for preservative Y;

[0090] 3) In a 96-well plate, add 40 μL of serially diluted preservative X solution to each well in columns 1-7, and add 40 μL of TSB medium to column 8;

[0091] 4) Add 40 μL of serially diluted preservative Y solution to each well in rows 1-7, and add 40 μL of TSB medium to row 8;

[0092] 5) Add 80 μL TSB medium to the 11th column as a growth control well, and add 100 μL TSB medium to the 12th column as a sterility control well;

[0093] 6) Add 20 μL of diluted bacterial solution to each well in columns 1-8 and 11 to ensure that the bacterial concentration in each well is 5×10 5 CFU / mL;

[0094] 7) Incubate at 37°C for 20 h and measure OD 600value, and according to OD 600 The value is used to calculate the inhibition rate of preservatives on the strain;

[0095] 8) Calculate the PVOA value. A PVOA value > 0 indicates that the two preservatives have a synergistic effect, whereas a value below 0 indicates an inhibitory effect.

[0096] PVOA(%)=(PA-OA) / PA×100%

[0097] PA: predicted indifference area, which refers to the area of ​​the rectangular area defined by the indifference lines of preservatives X and Y (each grid square is regarded as a unit area), that is, the number of wells contained in the rectangular area defined by the minimum inhibitory concentrations of preservatives X and Y;

[0098] OA: actual interaction area, that is, the number of holes where bacteria actually grow.

[0099] Fig.12 The results of the chessboard test of the combination of 1,2-hexanediol and phenoxyethanol on four bacteria. The data displayed in the chessboard is the strain inhibition rate data. The highest concentrations of 1,2-hexanediol and phenoxyethanol are both 2%, that is, preservative X is 2% 1,2-hexanediol, and preservative Y is 2% phenoxyethanol. The PVOA values ​​of Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC8739, Staphylococcus aureus ATCC 6538, and Staphylococcus epidermidis CCSM 0287 are calculated as follows:

[0100] PVOA against Pseudomonas aeruginosa = 100% × (5 × 5-21) / (5 × 5) = 16%

[0101] PVOA against Escherichia coli = 100% × (5 × 6-28) / (5 × 6) = 6.67%

[0102] PVOA against Staphylococcus aureus = 100% × (6 × 7-40) / (6 × 7) = 4.76%

[0103] PVOA against Staphylococcus epidermidis = 100% x (6 x 7-47) / (6 x 7) = -11.9%

[0104] In summary, the combination of 1,2-hexanediol and phenoxyethanol has a synergistic effect on Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8739, and Staphylococcus aureus ATCC 6538, and an antagonistic effect on Staphylococcus epidermidis CCSM 0287. The combination of 1% 1,2-hexanediol and 0.5% or 0.25% phenoxyethanol was screened out, which was friendly to Staphylococcus epidermidis CCSM 0287 while inhibiting the three pathogens.

[0105] Fig.13 The results of the chessboard test of the combination of 1,2-hexanediol and ethylhexylglycerol on four bacteria. The data displayed in the chessboard is the strain inhibition rate data. The maximum concentration of 1,2-hexanediol is 2%, and the maximum concentration of ethylhexylglycerol is 0.5%, that is, preservative X is 2% 1,2-hexanediol, and preservative Y is 0.5% ethylhexylglycerol. Calculate the PVOA values ​​of Pseudomonas aeruginosa ATCC9027, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, and Staphylococcus epidermidis CCSM 0287. The following is the calculation of the PVOA values ​​of the combination of 1,2-hexanediol and ethylhexylglycerol on four bacteria:

[0106] PVOA against Pseudomonas aeruginosa = 100% × (5 × 7-37) / (5 × 7) = -5.71%

[0107] PVOA against Escherichia coli = 100% × (6 × 6-39) / (6 × 6) = -8.33%

[0108] PVOA against Staphylococcus aureus = 100% × (7 × 7-44) / (7 × 7) = 10.20%

[0109] For Staphylococcus epidermidis CCSM 028 PVOA = 100% × (7 × 7-49) / (7 × 7) = 0%

[0110] In summary, the combination of 1,2-hexanediol and ethylhexylglycerol has an antagonistic effect on Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 8739, and a synergistic effect on Staphylococcus aureus ATCC 6538. The POVA value cannot be used to determine whether it is a synergistic or antagonistic effect on Staphylococcus epidermidis CCSM 0287, and the inhibition rate is used to determine that it is a synergistic effect. The combination of 1% or 0.5% 1,2-hexanediol and 0.25% or 0.125% ethylhexylglycerol was screened out to be more friendly to Staphylococcus epidermidis CCSM0287.

[0111] Example 5: Preservative Combination Friendliness

[0112] According to the preservative combination in Table 3, a 2-fold concentration combined preservative solution was prepared.

[0113] Table 3. Preservative combinations and concentrations

[0114]

[0115] The strains selected in this example include: Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Staphylococcus epidermidis CCSM 0287, and the friendliness of each preservative combination to the above strains is tested. The specific steps are as follows:

[0116] 1) Cultivate the strain using conventional methods and measure the OD of the culture solution 600 , substitute into the standard curve to get the bacterial concentration, dilute with sterile PBS buffer, and dilute with sterile PBS buffer to a bacterial concentration of 1.0×10 8 CFU / mL;

[0117] 2) Continue to dilute the bacterial concentration to 1.0×10 6 CFU / mL (used up within half an hour);

[0118] 3) In a 96-well plate, 100 μL of bacterial solution and 100 μL of combined preservative solution were added to each well of the experimental group, 100 μL of TSB medium and 100 μL of combined preservative solution were added to each well of the blank group, 200 μL of TSB medium were added to each well of the sterile control group, and 100 μL of TSB medium and 100 μL of bacterial solution were added to each well of the growth control group. Four replicate wells were made for each group;

[0119] 4) Incubate at 37°C for 24 hours;

[0120] 5) Calculate the inhibition rate according to the following formula:

[0121]

[0122] The results are as follows Fig.14 As shown, all five preservative combinations can completely inhibit Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, and Pseudomonas aeruginosa ATCC 9027, and only partially inhibit Staphylococcus epidermidis CCSM 0287. In particular, preservative combination 4, while completely inhibiting pathogenic bacteria, has the lowest inhibition rate of 42.46% against Staphylococcus epidermidis CCSM 0287.

Claims

1. A method for screening cosmetic preservatives, characterized in that: The SVS value and / or CVS value are used as screening indicators to screen cosmetic preservatives that can inhibit pathogenic bacteria and are friendly to skin bacteria; among them: SVS value = MIC value of Staphylococcus epidermidis / MIC value of Staphylococcus aureus, CVS value = MIC value of Propionibacterium acnes / MIC value of Staphylococcus aureus.

2. The screening method according to claim 1, characterized in that The CVS value is a standard for measuring the friendliness of a preservative to Propionibacterium acnes. A value ≥1 indicates that the MIC value for inhibiting Propionibacterium acnes is greater than or equal to the MIC value of Staphylococcus aureus, and is friendly to Propionibacterium acnes, and the larger the value, the friendlier it is; a value <1 indicates that the friendliness to Propionibacterium acnes is poor; the SVS value is a standard for measuring the friendliness of a preservative to Staphylococcus epidermidis. A value ≥1 indicates that the MIC value for inhibiting Staphylococcus epidermidis is greater than or equal to the MIC value of Staphylococcus aureus, and is friendly to Staphylococcus epidermidis, and the larger the value, the friendlier it is; a value <1 indicates that the friendliness to Staphylococcus epidermidis is poor.

3. The screening method according to claim 1, characterized in that The Staphylococcus epidermidis is Staphylococcus epidermidis CCSM 0287, Staphylococcus epidermidis ATCC 12228 or Staphylococcus epidermidis ATCC 35984; more preferably Staphylococcus epidermidis CCSM 0287.

4. The screening method according to claim 1, wherein The Propionibacterium acnes is Propionibacterium acnes CCSM0331 or Propionibacterium acnes ATCC 6919; more preferably Propionibacterium acnes CCSM 0331.

5. The screening method according to claim 1, characterized in that The Staphylococcus aureus is Staphylococcus aureus ATCC 6538.

6. Use of the screening method according to claim 1 in screening cosmetic preservatives.

7. A cosmetic preservative screened by the screening method according to claim 1, characterized in that: Includes at least one of 1,2-hexanediol, phenoxyethanol and ethylhexylglycerin.

8. The cosmetic preservative according to claim 7, characterized in that The cosmetic preservative is a combination of 1,2-hexanediol and phenoxyethanol.

9. The cosmetic preservative according to claim 8, characterized in that The concentration of 1,2-hexanediol is 0.5%-1%, and the concentration of phenoxyethanol is 0.25%-0.5%, both of which are mass percentages.

10. The cosmetic preservative according to claim 7, characterized in that The cosmetic preservative is a combination of 1,2-hexanediol and ethylhexylglycerin; More preferably, the concentration of 1,2-hexanediol is 0.5%-1%, and the concentration of ethylhexylglycerol is 0.125%-0.25%, both of which are mass percentages.

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