Antimicrobial composition and application

Abstract

The invention discloses an antimicrobial composition and application thereof. The antimicrobial composition comprises (a) capryloyl hydroxamic acid; and (b) a second antimicrobial preparation, wherein the second antimicrobial preparation is selected from at least one of p-hydroxyacetophenone, glycerol caprylate and ethylhexylglycerin; according to the antimicrobial composition, the synergistic interaction between the capryloyl hydroxamic acid and the second antimicrobial preparation is fully exerted, the antimicrobial effect is remarkably improved, and the antimicrobial composition can be used in daily chemical products, cosmetics, personal care compositions or cleaning compositions and has the advantages of being low in dosage, safe, effective, natural, low in irritation and the like.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial composition technology, and particularly relates to an antimicrobial composition and its application. Background Technology

[0002] Antimicrobial compositions refer to substances added to daily chemical products, cosmetics, personal care compositions, or cleaning compositions for the purpose of inhibiting the growth and reproduction of microorganisms.

[0003] During the production and use of daily chemical products, cosmetics, personal care compositions, or cleaning compositions, it is difficult to avoid the introduction of some microorganisms, which can cause spoilage and deterioration. Antimicrobial compositions can inhibit the growth and reproduction of microorganisms in daily chemical products, cosmetics, personal care compositions, or cleaning compositions, maintaining their stability and preventing them from deteriorating during production and use.

[0004] Traditionally, cosmetics, personal care compositions, and cleaning compositions often employ one or more of the 51 preservatives permitted by cosmetic regulations in combination to address the problem of microbial growth. These 51 preservatives include phenols, esters, halides, and quaternary ammonium salts, with parabens, isothiazolinones, imidazolidinyl ureas, and bromopol being the most commonly used. However, chemical preservatives can easily cause skin problems; for example, isothiazolinones can cause irritation and allergies, and bromopol may lead to the formation of carcinogens. Therefore, many countries have imposed strict limits on their types and dosages. Traditional chemical preservatives can no longer meet people's needs, and research into alternative products is receiving increasing attention.

[0005] Capryloyl hydroxamic acid has the effect of inhibiting the growth of microorganisms such as bacteria and fungi. It is safe and mild, non-irritating to the human body, and can replace traditional preservatives. However, when used alone, its ability to inhibit microbial growth is not strong. Summary of the Invention

[0006] To address the shortcomings of the prior art, the present invention provides an antimicrobial composition that significantly enhances the antimicrobial effect by fully leveraging the synergistic effect between capryloyl hydroxamic acid and a second antimicrobial agent. This antimicrobial composition can be used in daily chemical products, cosmetics, personal care compositions, or cleaning compositions, and has the advantages of low dosage, safety and effectiveness, and natural low irritation.

[0007] The purpose of this invention is to provide an antimicrobial composition containing...

[0008] (a) Capryloyl hydroxamic acid; and

[0009] (b) A second antimicrobial agent, wherein the second antimicrobial agent is selected from at least one of p-hydroxyacetophenone, glyceryl caprylate, and ethylhexylglycerin.

[0010] In some embodiments of the present invention, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:1:0.05-22.

[0011] In some embodiments of the present invention, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.62-10.

[0012] In some embodiments of the present invention, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.05-2.

[0013] In some embodiments of the present invention, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.06-4.

[0014] In some embodiments of the present invention, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.65-11.67.

[0015] In some embodiments of the present invention, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.12-11.67.

[0016] In some embodiments of the present invention, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.34-22.

[0017] In some embodiments of the present invention, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.06-22.

[0018] In some embodiments of the present invention, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:1:0.06-2.

[0019] In some embodiments of the present invention, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:1:0.65-10.50.

[0020] In some embodiments of the present invention, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.12-4.

[0021] In some embodiments of the present invention, the second antimicrobial agent is selected from p-hydroxyacetophenone and glyceryl caprylate, wherein the mass ratio of p-hydroxyacetophenone to glyceryl caprylate is 0.05-10:0.01-2; in some preferred embodiments, the value of p-hydroxyacetophenone in the aforementioned mass ratio may be 0.1-10, 0.5-10, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, or 9-10, and the value of glyceryl caprylate in the aforementioned mass ratio may be 0.02-2, 0.05-2, 0.1-2, 0.2-2, 0.3-2, 0.5-2, 0.6-2, 0.8-2, 1-2, 1.2-2, 1.4-2, 1.6-2, or 1.8-2.

[0022] In some embodiments of the present invention, the second antimicrobial agent is selected from p-hydroxyacetophenone and ethylhexylglycerin in a mass ratio of 0.05-10:0.01-2. In some preferred embodiments, the value of p-hydroxyacetophenone in the aforementioned mass ratio may be 0.1-10, 0.5-10, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, or 9-10, and the value of ethylhexylglycerin in the aforementioned mass ratio may be 0.02-2, 0.05-2, 0.1-2, 0.2-2, 0.3-2, 0.5-2, 0.6-2, 0.8-2, 1-2, 1.2-2, 1.4-2, 1.6-2, or 1.8-2.

[0023] In some embodiments of the present invention, the second antimicrobial agent is selected from glyceryl caprylate and ethylhexylglycerin in a mass ratio of 0.01-2:0.01-2. In some preferred embodiments, the values ​​of glyceryl caprylate and ethylhexylglycerin in the aforementioned mass ratio may be independently 0.02-2, 0.05-2, 0.1-2, 0.2-2, 0.3-2, 0.5-2, 0.6-2, 0.8-2, 1-2, 1.2-2, 1.4-2, 1.6-2, and 1.8-2, respectively.

[0024] In some embodiments of the present invention, the second antimicrobial agent is selected from p-hydroxyacetophenone, glyceryl caprylate, and ethylhexylglycerol in a mass ratio of 0.05-10:0.01-2:0.01-2. In some preferred embodiments, the value of p-hydroxyacetophenone in the aforementioned mass ratio may be 0.1-10, 0.5-10, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, or 9-10, and the values ​​of glyceryl caprylate and ethylhexylglycerol in the aforementioned mass ratio may be independently 0.02-2, 0.05-2, 0.1-2, 0.2-2, 0.3-2, 0.5-2, 0.6-2, 0.8-2, 1-2, 1.2-2, 1.4-2, 1.6-2, or 1.8-2.

[0025] In some embodiments of the present invention, the antimicrobial composition further includes a solvent.

[0026] In some preferred embodiments, the solvent includes at least one selected from 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, isopentanediol, isohexanediol, 1,2-hexanediol, dipropylene glycol, water, glycerol, and methylpropanediol.

[0027] In some preferred embodiments, the lower limit of the mass percentage of the solvent relative to the total mass of the antimicrobial composition is 1%, preferably 5%, 10%, 20%, 30%, or 40%; the upper limit of the mass percentage of the solvent relative to the total mass of the antimicrobial composition is 99%, preferably 95%, 90%, 80%, 70%, or 60%.

[0028] Another object of the present invention is to provide the use of the aforementioned antimicrobial composition as a preservative.

[0029] Another object of the present invention is to provide the application of the aforementioned antimicrobial composition in the preparation of daily chemical products.

[0030] Another object of the present invention is to provide the use of the aforementioned antimicrobial composition in the preparation of cosmetics.

[0031] Another object of the present invention is to provide a personal care composition comprising an effective amount of the aforementioned antimicrobial composition.

[0032] Another object of the present invention is to provide a cleaning composition comprising an effective amount of the aforementioned antimicrobial composition.

[0033] In some embodiments of the present invention, the amount of the antimicrobial composition, by weight percentage, is 0.5-3% of the daily chemical or cosmetic product, including but not limited to 0.5%, 0.8%, 1.0%, 1.5%, 1.7%, 2.0%, 2.3%, 2.6%, and 3%.

[0034] In some embodiments of the present invention, the amount of the antimicrobial composition, by weight percentage, is 0.5-3% of the personal care composition, including but not limited to 0.5%, 0.8%, 1.0%, 1.5%, 1.7%, 2.0%, 2.3%, 2.6%, and 3%.

[0035] In some embodiments of the present invention, the amount of the antimicrobial composition is 0.5-3% of the cleaning composition by weight, including but not limited to 0.5%, 0.8%, 1.0%, 1.5%, 1.7%, 2.0%, 2.3%, 2.6%, and 3%.

[0036] Another object of the present invention is to provide a method for using the aforementioned antimicrobial composition as a preservative.

[0037] Another object of the present invention is to provide a method for preserving daily chemical products, cosmetics, personal care compositions or cleaning compositions by adding the aforementioned antimicrobial composition.

[0038] Another object of the present invention is to provide a method for combating microorganisms by adding the aforementioned antimicrobial composition to daily chemical products, cosmetics, personal care compositions or cleaning compositions.

[0039] Personal care compositions, cleaning compositions, daily chemical products, or cosmetics may contain anti-acne agents, anti-skin aging agents, abrasives, antistatic agents, anti-cellulite agents, anti-dandruff agents, anti-inflammatory agents, antiperspirants, anti-irritants, anti-irritants, antioxidants, astringents, adhesives, bactericides, buffers, carriers, chelating agents, cell stimulants, gelling agents, conditioning agents, depilatory agents, surfactants, deodorants, antiperspirants, emulsifiers, essential oils, color-fixing agents, fibers, emulsifiers, film-forming agents, enzymes, foam forming agents, foam stabilizers, defoamers, stain repellents, foam promoters, gelling agents, hair conditioning agents, hair styling agents, abrasives, silicones, hair straighteners, humectants, moisturizers, humectants, detergents, bleaching agents, strengthening agents, friction reducers, cleansing agents, optical brighteners, and impregnating agents. Additional additives and auxiliaries, including suspending agents, flavoring agents, odorants, skin brightening agents, thickeners, lubricants, polymers, gloss agents, skin healing agents, powders, moisturizing creams, ointments, proteins, fatliquoring agents, skin soothing agents, skin cleansers, skin care agents, skin brighteners, skin protectants, skin softeners, coolants, sunscreens, plasticizers, skin coolants, warming agents, skin warming agents, stabilizers, UV absorbers, covering agents, polishing agents, UV filters, detergents, fabric softeners, vitamins, oils, waxes, fats, phospholipids, saturated fatty acids, dyes, color protectants, monounsaturated or polyunsaturated fatty acids, α-hydroxy acids, polyhydroxy fatty acids, liquefying agents, pigments, polyols, corrosion inhibitors, aromatic substances, surfactants, electrolytes, organic solvents, or silicone derivatives.

[0040] The antimicrobial composition of the present invention has a good inhibitory effect on a variety of bacteria, such as Streptococcus mutans, Porphyromonas gingivalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, Candida albicans, Malassezia, etc.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] (1) This invention significantly improves the antimicrobial effect by giving full play to the synergistic effect between capryloyl hydroxamic acid and the second antimicrobial agent.

[0043] (2) The antimicrobial composition of the present invention can be used in daily chemical products, cosmetics, personal care compositions or cleaning compositions, and has the advantages of low dosage, safety and effectiveness, natural and low irritation. Detailed Implementation

[0044] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0045] 1. Synergistic antibacterial test:

[0046] Streptococcus mutans and Porphyromonas gingivalis were selected as indicator bacteria. The indicator bacteria were washed off with culture medium and resuspended to a concentration of 1×10⁻⁶. 6 CFU / mL bacterial suspension; capryloyl hydroxamic acid was prepared into solutions with concentrations of 800 ppm, 400 ppm, 200 ppm, 100 ppm, and 50 ppm using sterile water, designated as Group 1; p-hydroxyacetophenone was prepared into solutions with concentrations of 4000 ppm, 2000 ppm, 1000 ppm, 500 ppm, and 250 ppm using sterile water, designated as Group 2; glyceryl caprylate was prepared into solutions with concentrations of 200 ppm, 100 ppm, 50 ppm, and 25 ppm using sterile water. A solution of 12.5 ppm was designated as Group 3; ethylhexylglycerin was prepared with sterile water to form solutions with concentrations of 200 ppm, 100 ppm, 50 ppm, 25 ppm, and 12.5 ppm, designated as Group 4; p-hydroxyacetophenone + glyceryl caprylate was prepared with sterile water to form solutions with concentrations of 4000 ppm + 200 ppm, 2000 ppm + 100 ppm, 1000 ppm + 50 ppm, 500 ppm + 25 ppm, and 250 ppm + 12.5 ppm, designated as Group 4; Group 5; Group 6: Solutions of p-hydroxyacetophenone and ethylhexylglycerin with concentrations of 4000 ppm + 200 ppm, 2000 ppm + 100 ppm, 1000 ppm + 50 ppm, 500 ppm + 25 ppm, and 250 ppm + 12.5 ppm were prepared using sterile water. Solutions containing 25ppm and 12.5ppm were designated as Group 7. Solutions containing p-hydroxyacetophenone, glyceryl caprylate, and ethylhexylglycerin were prepared using sterile water to form solutions with concentrations of 4000ppm+200ppm+200ppm, 2000ppm+100ppm+100ppm, 1000ppm+50ppm+50ppm, 500ppm+25ppm+25ppm, and 250ppm+12.5ppm+12.5ppm, respectively, and were designated as Group 8.

[0047] For testing Streptococcus mutans: In columns 1-5 of a 96-well plate, add 100µL of solution from group 1 at different concentrations from high to low, with the same concentration in each column. Add an equal volume of sterile water to column 6. In rows 1-5 of the 96-well plate, add 100µL of solution from any of groups 2-8 at different concentrations from high to low, with the same concentration in each row. Add an equal volume of sterile water to row 6. Finally, add 100µL of Streptococcus mutans suspension to all wells.

[0048] For testing Porphyromonas gingivalis: In columns 1-5 of a 96-well plate, add 50µL of solution from group 1 at different concentrations from high to low, with the same concentration in each column. Add an equal volume of sterile water to column 6. In rows 1-5 of the 96-well plate, add 50µL of solution from any of groups 2-8 at different concentrations from high to low, with the same concentration in each row. Add an equal volume of sterile water to row 6. Finally, add 50µL of Porphyromonas gingivalis suspension to all wells, and then add 100µL of liquid paraffin for sealing.

[0049] After adding the antimicrobial composition and indicator bacteria using the checkerboard method, the absorbance was measured at OD600 nm, and the samples were incubated at 36℃±1℃. The absorbance of *Streptococcus mutans* was measured again at OD600 nm after 1 day of incubation, and the absorbance of *Porphyromonas gingivalis* was measured again after 15 days of incubation. SynergyFinder 3.0 was used to calculate the synergistic antimicrobial coefficient. The results are shown in Table 1.

[0050] The criteria for judgment are as follows: a synergy score greater than 0 indicates a synergistic effect, a synergy score equal to 0 indicates an additive effect, and a synergy score less than 0 indicates an antagonistic effect.

[0051] Table 1. Synergistic antimicrobial effect of antimicrobial compositions.

[0052]

[0053] As shown in Table 1, the octanoyl hydroxamic acid and the second antimicrobial agent of the present invention, wherein the second antimicrobial agent is selected from at least one of p-hydroxyacetophenone, glyceryl caprylate, and ethylhexylglycerin, have a good synergistic antibacterial and synergistic effect against Streptococcus mutans and Porphyromonas gingivalis.

[0054] 2. Antibacterial rate test in toothpaste:

[0055] Refer to WS / T 650-2019, Evaluation Methods for Antibacterial and Bacteriostatic Effects, 5.1.2, Quantitative Antibacterial Experiment with Carrier Immersion.

[0056] 2.1 Test Procedure

[0057] Fresh slant cultures of *Streptococcus mutans* and *Porphyromonas gingivalis* were washed down with PBS and diluted with PBS to approximately 5.0 × 10⁻⁶. 6 CFU / mL ~5.0×10 7 Prepare a bacterial suspension at CFU / mL for later use. Use a micropipette to drop 10 μl of the bacterial suspension onto a sterile carrier, and dry at 36℃±1℃ or air-dry at room temperature. Weigh 5 g / tablet of toothpaste containing the antimicrobial composition into a sterile Petri dish, incubate at 20℃±1℃ for 5 min, and use sterile forceps to completely immerse the carrier in the toothpaste sample containing the antimicrobial composition. Start timing immediately. Allow the carrier to interact with the toothpaste sample containing the antimicrobial composition for 2 h. Add the carrier to a 5.0 mL PBS tube, mix well, shake, wash off the test bacteria, and take 1.0 mL of the sample solution from each tube. Count the viable bacteria using a viable culture method, inoculating two Petri dishes with each sample solution. If the number of colonies growing on the plate is high, perform a 10-fold serial dilution with PBS before viable culture counting. Two tablets of contaminated bacterial carrier were immersed in 10.0 g of a control sample (of the same material as the test sample but without antibacterial components) for parallel testing, serving as a positive control. The recovered bacterial count for the positive control was 1.0 × 10⁻⁶. 4 CFU / tablet ~9.0×10 4 CFU / tablet. PBS and culture medium from the same batch were used as negative controls. All test and control samples were incubated at 36℃±1℃. Results were observed after 2 days of culture for *Streptococcus mutans* and after 15 days for *Porphyromonas gingivalis*. The experiment was repeated three times, and the inhibition rate was calculated. The tested antimicrobial compositions, dosages, and toothpaste formulations are shown in Tables 2 and 3, respectively.

[0058] Table 2. Antimicrobial composition and dosage in toothpaste antibacterial rate test.

[0059]

[0060] Table 3. Toothpaste ingredient list and manufacturing process.

[0061]

[0062] 2.2 Calculation of antibacterial rate:

[0063] X = (A0 - A1) / A0 × 100%

[0064] In the formula:

[0065] X—Antibacterial rate, %

[0066] A0—The average colony count of the control sample, in CFU / plate;

[0067] A1—The average number of colonies in the test sample, in CFU / plate.

[0068] 2.3 Result Evaluation:

[0069] An antibacterial rate of ≥50%~90% indicates antibacterial effect; an antibacterial rate of ≥90% indicates strong antibacterial effect.

[0070] 2.4 Antibacterial rate test results:

[0071] Table 4. Results of toothpaste antibacterial rate test.

[0072]

[0073] The results above show that the antimicrobial composition has an inhibitory effect on Porphyromonas gingivalis and Streptococcus mutans when added to toothpaste.

[0074] 3. Anti-corrosion efficacy test:

[0075] The preservative efficacy test was conducted according to ISO 11930-2019. The bacteria used in this experiment were *Pseudomonas aeruginosa* ATCC 9027, *Escherichia coli* ATCC 8739, and *Staphylococcus aureus* ATCC 6538; the fungi used were *Candida albicans* ATCC 10231 and *Aspergillus niger* ATCC 16404, all of which were obtained from the Guangdong Provincial Microbial Culture Collection Center.

[0076] The final concentration of the bacterial inoculum used in the experiments was 10. 5 -10 6 CFU / g, the final concentration of fungal inoculum was 10. 4 -10 5 The CFU / g test was conducted using a mixed inoculation method involving bacteria and fungi. The bacterial count was measured on days 7, 14, and 28, and the logarithmic decrease in the number of various microorganisms at the corresponding time points was calculated.

[0077] Preservative efficacy tests of antimicrobial compositions in facial mask liquids, moisturizing lotions, and shower creams:

[0078] The tested antimicrobial composition systems and cosmetic dosage forms are shown in Table 5:

[0079] Table 5. Antimicrobial composition systems and cosmetic dosage forms.

[0080]

[0081] Table 6. Ingredients and manufacturing process of facial mask liquid.

[0082]

[0083] Table 7. Ingredients and manufacturing process of moisturizing lotion.

[0084]

[0085] Table 8. Ingredients and manufacturing process of shower gel.

[0086]

[0087] The experimental results were determined as follows: the bacterial count decreased by 3 orders of magnitude on day 7, and decreased by 3 orders of magnitude on days 14 and 28 without increasing. The fungal count decreased by 1 order of magnitude on day 28. The results are shown in Tables 9-11.

[0088] Table 9. Test data on the preservative efficacy of facial mask liquid.

[0089]

[0090] Table 10. Test data on the preservative efficacy of moisturizing lotion.

[0091]

[0092] Table 11. Test data on the preservative efficacy of shower gel.

[0093]

[0094] The results in Tables 9-11 show that the antimicrobial composition of octanoyl hydroxamic acid at a mass ratio of 1:0.05-22 with the second antimicrobial agent can exert excellent preservative effects in aqueous systems, emulsion systems, and washing and conditioning systems.

[0095] 4. Stimulation test:

[0096] 4.1 Experimental Principle: The chicken embryo chorioallantoic membrane (CAM) test is an early and widely used in vitro method for assessing eye irritation. The chorioallantoic membrane (CAM) is a respiratory membrane surrounding the chicken embryo. This experiment utilizes the intact, clear, and transparent vascular system of the mid-stage CAM in hatched chicken embryos. A certain amount of the test substance is directly exposed to the CAM, and after a period of time, changes in CAM toxicity indicators (such as ghost vessels, capillary congestion, and hemorrhage of varying degrees) are observed. Different degrees of vascular damage are scored, with higher scores indicating more severe irritation. This experiment particularly emphasizes the vascular effects of the CAM, such as the resulting ghost vessels, capillary congestion, and hemorrhage of varying degrees.

[0097] The purpose of this test is to assess the ability of the test substance to cause toxic changes in the chorioallantoic membrane of chicken embryos. The standard describes the process for evaluating the potential eye irritation of the substance being evaluated.

[0098] 4.2 Test Methods:

[0099] 4.2.1 CAM Preparation: White Laihang chickens were selected as SPF hatching eggs. The embryos should be transferred or transported without affecting their activity or development, avoiding shaking, unnecessary tilting, knocking, or other mechanical stimulation. Purchased 0-day-old embryos were disinfected and their surfaces cleaned with a prepared benzalkonium chloride solution (1:30) or other disinfectant that does not affect embryo growth and development. They were then placed in an incubator at 37.5℃±0.5℃ and 50%-60% relative humidity. During incubation, the embryos should be placed on an egg rack with the air cell facing upwards. Before the experiment, the embryos to be tested were removed and placed in the dark. An egg candling device was used to examine the embryos, selecting those with abundant and regular blood vessels as experimental materials. At 10 days of age, the air cell locations were marked on the eggshell surface. The marked portion of the eggshell was then peeled off with dental serrated tweezers to expose the white membrane, handling carefully to avoid damaging the membrane's integrity. Add a few milliliters of 0.9wt% sodium chloride (NaCl) solution with a pipette to moisten the egg membrane. You can proceed to the next step immediately; otherwise, place the embryo in an incubator or under a light (to prevent the embryo temperature from dropping), and leave it there for no more than 20 minutes. Pour out the 0.9wt% sodium chloride solution. Carefully remove the inner membrane with tweezers, ensuring the vascular membrane is not damaged. At this point, observe the structure of the vascular system again and assess its integrity and suitability for the experiment.

[0100] 4.2.2 Sample Addition Test: A polytetrafluoroethylene (PTFE) resin ring was placed on the chorioallantoic membrane of a chicken embryo, with the area inside the ring designated as the test area. 40 μL of the test sample (the antimicrobial composition raw materials from Table 5 were prepared as 2 wt% aqueous solutions) was pipetted into the PTFE resin ring. The time of sample addition was recorded, and the air cell was covered with a moistened plastic wrap. The chicken embryo was then transferred to a constant temperature and humidity incubator for 30 min. After 30 min of incubation, the test chicken embryo was removed, photographed directly, and the degree of vascular damage was observed and scored according to the degree of vascular damage. Ten chicken embryos were tested for each sample.

[0101] 4.3 Results Observation

[0102] Table 12. CAMVA scoring criteria.

[0103]

[0104] 4.3.4 Requirements

[0105] Observe and record the degree of ghost vessels, capillary congestion, and hemorrhage of varying degrees. Take photos and preserve experimental images if necessary, and compare the observations with historical laboratory data or relevant reference images.

[0106] 4.4 Results Analysis

[0107] Each egg's result should be evaluated and scored. The highest and lowest scores will be removed from the final ten scores, leaving eight valid scores.

[0108] The stimulus score (NC) is calculated according to formula (1).

[0109] NC=(X1+X2+……+Xn) / n……………………(1).

[0110] In the formula:

[0111] X - Stimulation Score

[0112] N — The number of valid chicken embryos after removing the highest and lowest scores.

[0113] The irritancy of the test substance is classified according to the calculated NC value as shown in Table 13.

[0114] Table 13. Stimulus Evaluation.

[0115]

[0116] 4.5 Test Results

[0117] Table 14. Results of the irritation test.

[0118]

[0119] The data shows that, except for the ratio of capryloyl hydroxamic acid to ethylhexylglycerin = 1:22.5, the other antimicrobial compositions are non-irritating. Based on comprehensive preservative efficacy testing, the antimicrobial compositions of this invention provide excellent preservative performance while also possessing the advantage of being non-irritating.

[0120] 5. Cytotoxicity test:

[0121] This test is based on GB / T 16886.5-2017 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests.

[0122] 5.1 Experimental Principle: The Cell Counting Kit-8 Cytotoxicity Test (CCK-8) is a cell viability marker. Dehydrogenase in the mitochondria of living cells reduces CCK-8 reagent to an orange-yellow formazan dye soluble in the culture medium. The more living cells there are, the more dye is reduced, and the color intensity is linearly related to the number of cells. Cell viability is calculated by measuring the absorbance at a wavelength of 450 nm.

[0123] 5.2 Experimental Cells: Human immortalized epidermal cells (HaCaT) and rabbit corneal epithelial cells (SIRC)

[0124] 5.3 Test Procedure

[0125] 5.3.1 Cell preparation and seeding in 96-well plates

[0126] Cells should be resuscitated and passaged at least 2-3 times before the formal experiment begins, until the cells reach a stable growth state before proceeding to the next step. Take cells from the stable growth state and adjust the cell concentration to 1×10⁻⁶. 5 Cells were seeded at a rate of 100 μl per well into 96-well cell culture plates (excluding the outermost wells). 200 μl of PBS was added to the outermost wells to reduce culture medium evaporation. The plates were then incubated in a CO2 incubator at 37℃±1℃, 90%±5% humidity, and 5%±1% CO2 concentration. After 24 h, the cells were observed. If cell confluence reached 70-80% of the well bottom area and the cells were in good condition, the next step of the experiment could be performed.

[0127] 5.3.2 Sample preparation: The antimicrobial composition raw materials in Table 5 were prepared into test concentrations of 1 g / L using culture medium (such as high-glucose DMEM medium).

[0128] 5.4.3 Sample Loading Test: Remove the 96-well cell culture plate inoculated with cells from the CO2 incubator, disinfect the surface with 75% alcohol, and place it in a biosafety cabinet. Remove the culture medium from the wells using a multi-channel pipette. Perform three replicates for each test sample. Starting from the second column of the 96-well plate, add 100 μl of PBS (blank control), solvent control, prepared sample, and positive control to each well, with three replicates per well. Incubate at 37°C with 5% CO2 for 24 h.

[0129] 5.4.4 Cell Viability Test: Add 100 μl of PBS to each well to wash the test cells twice. When aspirating the liquid from the wells using a pipette, handle carefully, aspirating from the edge of the well to avoid removing viable cells and affecting the experimental results. After washing, remove the PBS. Add 100 μl of DMEM complete culture medium and 10 μl of CCK-8 solution to each well, and incubate in a CO2 incubator for approximately 2 hours. The incubation time should be determined based on the sensitivity of CCK-8, but 2 hours is generally recommended. Measure the absorbance (OD) at a wavelength of 450 nm.

[0130] 5.4.5 Calculation of cell survival rate: The cell survival rate is calculated according to formula (1).

[0131]

[0132] In the formula:

[0133] CSR: Cell survival rate, %

[0134] ODS: Average OD value of the test sample;

[0135] ODC: Solvent reference OD value;

[0136] ODb: Blank control OD value.

[0137] 5.4.6 Results Analysis: When the cell viability is ≥70%, the sample is considered to have no cytotoxicity; when the cell viability is <70%, the sample is considered to have potential cytotoxicity.

[0138] Table 15. Results of toxicity tests on human immortalized epidermal cells (HaCaT).

[0139]

[0140] Table 16. Results of rabbit corneal epithelial cell (SIRC) toxicity test.

[0141]

[0142] Based on the two cytotoxicity results, except for the ratio of capryloyl hydroxamic acid to ethylhexylglycerin = 1:22.5, the other antimicrobial compositions showed no cytotoxicity at a concentration of 1.0 g / L. This indicates that the antimicrobial compositions of the present invention, while improving preservative performance, are not only non-irritating but also retain high cellular safety.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that after reading this application specification, they can still modify or make equivalent substitutions to the specific implementation of the present invention, but these modifications or changes do not depart from the protection scope of the pending claims of the present invention.

Claims

1. An antimicrobial composition, characterized in that, contain (a) Capryloyl hydroxamic acid; and (b) A second antimicrobial agent, wherein the second antimicrobial agent is selected from at least one of p-hydroxyacetophenone, glyceryl caprylate, and ethylhexylglycerin.

2. The antimicrobial composition according to claim 1, characterized in that, The mass ratio of the octanoyl hydroxamic acid to the second antimicrobial agent is 1:0.05-22; Preferably, the mass ratio of capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.62-10; Preferably, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.05-2; Preferably, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.06-4; Preferably, the mass ratio of capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.65-11.67; Preferably, the mass ratio of capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.12-11.67; Preferably, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.34-22.

3. The antimicrobial composition according to claim 2, characterized in that, The mass ratio of the octanoyl hydroxamic acid to the second antimicrobial agent is 1:0.06-22; Preferably, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.06-2; Preferably, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.65-10.50; Preferably, the mass ratio of the capryloyl hydroxamic acid to the second antimicrobial agent is 1:0.12-4.

4. The antimicrobial composition according to any one of claims 1 to 3, wherein the second antimicrobial agent is selected from p-hydroxyacetophenone and glyceryl caprylate in a mass ratio of 0.05-10:0.01-2; Alternatively, the second antimicrobial agent is selected from p-hydroxyacetophenone and ethylhexylglycerin in a mass ratio of 0.05-10:0.01-2; Alternatively, the second antimicrobial agent is selected from p-glycerocaprylate and ethylhexylglycerin in a mass ratio of 0.01-2:0.01-2.

5. The antimicrobial composition according to any one of claims 1 to 3, characterized in that, The antimicrobial composition is selected from p-hydroxyacetophenone, glyceryl caprylate, and ethylhexylglycerin in a mass ratio of 0.05-10:0.01-2:0.01-2.

6. The antimicrobial composition according to any one of claims 1 to 3, characterized in that, The antimicrobial composition further includes a solvent; Preferably, the solvent includes at least one selected from 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, isopentanediol, isohexanediol, 1,2-hexanediol, dipropylene glycol, water, glycerol, and methylpropanediol. Preferably, the lower limit of the mass percentage of the solvent relative to the total mass of the antimicrobial composition is 1%, more preferably 5%, 10%, 20%, 30%, or 40%. Preferably, the upper limit of the mass percentage of the solvent relative to the total mass of the antimicrobial composition is 99%, more preferably 95%, 90%, 80%, 70%, or 60%.

7. The use of the antimicrobial composition according to any one of claims 1 to 6 as a preservative.

8. The use of the antimicrobial composition according to any one of claims 1 to 6 in the preparation of daily chemical products or cosmetics; Preferably, the amount of the antimicrobial composition is 0.5-3% of the daily chemical or cosmetic product by weight percentage.

9. A personal care composition, characterized in that, Containing an effective amount of the antimicrobial composition according to any one of claims 1 to 6; Preferably, the amount of the antimicrobial composition is 0.5-3% of the personal care composition by weight percentage.

10. A cleaning composition, characterized in that, Containing an effective amount of the antimicrobial composition according to any one of claims 1 to 6; Preferably, the amount of the antimicrobial composition is 0.5-3% of the cleaning composition by weight percentage.

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