An antiseptic and antibacterial composition, its preparation method and application

Abstract

This invention belongs to the field of cosmetic raw material technology, and particularly relates to a preservative and antibacterial composition, its preparation method, and its application. The preservative and antibacterial composition comprises the following components in weight percentages: 10-30% antimicrobial lipopeptide, 0.1-1% lysozyme, 0.01-0.05% capryloyl hydroxamic acid, 0.2-0.6% p-hydroxyacetophenone, and the balance being water. This invention screens and extracts natural compounds with antibacterial and preservative activities—antimicrobial lipopeptides and natural antimicrobial substances—lysozyme from natural microbial sources, and combines them with two chemical preservatives, capryloyl hydroxamic acid and p-hydroxyacetophenone, to prepare a novel preservative and antibacterial composition. This compounding method significantly reduces the amount of chemical preservatives used, thereby ensuring the safety and low toxicity of the composition. Furthermore, the components work synergistically to further enhance the preservative and antibacterial properties of the composition.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic raw material technology, and in particular relates to an antiseptic and antibacterial composition, its preparation method and application. Background Technology

[0002] The cosmetics industry has long used chemically synthesized preservatives to extend product shelf life. However, these preservatives have drawbacks, such as causing skin irritation, allergies, and inflammation. To improve the safety and efficacy of cosmetics, researchers have begun searching for safe and effective antibacterial active ingredients from natural products to replace chemically synthesized preservatives.

[0003] Natural microbial sources contain some natural compounds with antibacterial and preservative activities, such as antimicrobial lipopeptides and natural antimicrobial substances like lysozyme. These natural compounds not only have good antibacterial activity but also have no toxic side effects on the human body. By screening and extracting these compounds from natural microorganisms and compounding them with other preservatives to form novel preservative compositions, the amount of chemically synthesized preservatives used can be effectively reduced, thereby reducing their irritation to the human body.

[0004] Therefore, developing a novel preservative composition that combines natural compounds such as antimicrobial lipopeptides and lysozymes derived from natural microorganisms with other preservatives can not only improve the preservative effect but also reduce the amount of chemically synthesized preservatives used, thereby reducing their irritation to human skin and improving the safety and efficacy of cosmetics. This is of great significance to the development of the cosmetics industry. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an antiseptic and antibacterial composition that can effectively improve the antiseptic effect and reduce irritation, as well as its preparation method and application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0007] In a first aspect, the present invention provides an antiseptic and antibacterial composition comprising the following components in weight percentages: 10-30% antimicrobial lipopeptide, 0.1-1% lysozyme, 0.01-0.05% capryloyl hydroxamic acid, 0.2-0.6% p-hydroxyacetophenone, and the balance being water.

[0008] This invention primarily utilizes natural compounds with antibacterial and preservative activities—antimicrobial lipopeptides and natural antimicrobial substances—lysozyme, extracted from natural microbial sources. These are combined with two chemical preservatives, capryloyl hydroxamic acid and p-hydroxyacetophenone, to form a novel antimicrobial and preservative composition. This compounding effectively reduces the amount of chemical preservatives used, achieving a safe and low-toxicity composition. Furthermore, the components synergistically enhance the antimicrobial and preservative properties of the composition. Studies have shown that this antimicrobial and preservative composition exhibits inhibitory activity against fungi such as Candida albicans and Aspergillus niger; it also demonstrates strong inhibitory activity against Gram-positive bacteria such as Staphylococcus aureus and Propionibacterium acnes, as well as Gram-negative plant pathogenic bacteria such as Escherichia coli and Pseudomonas aeruginosa, thus exerting a broad-spectrum and highly effective antimicrobial effect.

[0009] Furthermore, the inventors investigated the dosage of each component and found that when the dosage ratio of the antimicrobial lipopeptide, lysozyme and the other two components is within the above-mentioned preferred range, the components interact with each other and can exert an ideal synergistic effect, thereby effectively improving the antibacterial and preservative effect of the composition. However, when the ratio of the four components exceeds the limited range, the synergistic effect between the components is not obvious or even produces an antagonistic effect, which leads to a decrease in the antibacterial effect of the composition.

[0010] Preferably, the antiseptic and antibacterial composition comprises the following components in weight percentages: 15-25% antimicrobial lipopeptide, 0.3-0.7% lysozyme, 0.01-0.03% capryloyl hydroxamic acid, 0.3-0.5% p-hydroxyacetophenone, and the balance being water.

[0011] Preferably, the antiseptic and antibacterial composition comprises the following components in weight percentages: 20% antimicrobial lipopeptide, 0.4% lysozyme, 0.02% capryloyl hydroxamic acid, 0.3% p-hydroxyacetophenone, and the balance being water.

[0012] Experimental studies have shown that when the components of this invention are combined in the above-defined optimal dosage ratio, the resulting antibacterial and antiseptic composition exhibits the best antibacterial effect and good safety, without causing skin irritation.

[0013] Preferably, the method for preparing the antimicrobial lipopeptide includes the following steps:

[0014] S1. Inoculate Bacillus subtilis into liquid culture medium and culture to prepare seed culture;

[0015] S2. Inoculate the seed culture into a liquid culture medium for fermentation to obtain the fermentation broth;

[0016] S3. Centrifuge the fermentation broth, take the fermentation supernatant, adjust the pH value, centrifuge again, discard the supernatant and take the precipitate, add methanol to the precipitate, vortex, sonicate, extract, centrifuge, take the supernatant and filter to obtain the antimicrobial lipopeptide.

[0017] Preferably, in step S2, the inoculation amount of seed liquid is 2-5%, and the specific fermentation conditions are: temperature of 30-37℃, time of 48-96h, and rotation speed of 160-220rpm.

[0018] Preferably, in step S3, the centrifugation conditions are: centrifugation at 7000-9000 rpm for 10-20 min; the specific method for adjusting the pH value is: adjusting the pH to 1.6-2.4 with 5-7 mol / L hydrochloric acid.

[0019] Preferably, in step S3, the vortex oscillation time is 8-12 min, the ultrasonic time is 10-20 min, and the extraction time is 4-8 h.

[0020] This invention uses Bacillus subtilis to ferment and culture antimicrobial lipopeptides under the above-mentioned preparation process, which can effectively improve the yield, stability and antimicrobial activity of antimicrobial lipopeptides. Therefore, when combined with lysozyme, octanoyl hydroxamic acid and p-hydroxyacetophenone, the antimicrobial and preservative effects of the composition can be further enhanced.

[0021] Secondly, the present invention provides a method for preparing the aforementioned antiseptic and antibacterial composition, comprising the following steps:

[0022] Mix all components and stir until homogeneous to obtain the antiseptic and antibacterial composition.

[0023] Thirdly, the present invention provides the application of the aforementioned antiseptic and antibacterial composition in cosmetics.

[0024] Preferably, the preservative and antibacterial composition has a mass percentage content of 10-25% in the cosmetic.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) This invention screens and extracts natural compounds with antibacterial and preservative activities, such as antimicrobial lipopeptides and natural antimicrobial substances, such as lysozyme, from natural microbial sources. These are combined with two chemical preservatives, octanoyl hydroxamic acid and p-hydroxyacetophenone, to prepare a novel antibacterial and preservative composition. This compounding method can significantly reduce the amount of chemical preservatives used, thereby ensuring the safety and low toxicity of the composition. Moreover, the components cooperate with each other to exert a synergistic effect, thereby enabling the composition to have a broader spectrum and more efficient antibacterial effect.

[0027] (2) The present invention has conducted a microbial preservation challenge test, which verifies that the preservative and antibacterial composition can replace traditional organic synthetic preservatives and the preservative effect is no less than that of traditional preservatives. Furthermore, the chicken embryo chorioallantoic membrane test verifies that the novel preservative and antibacterial composition has low skin irritation, thus providing an effective solution for achieving low-toxicity, safe and efficient preservation and antibacterial properties in the cosmetics field. Attached Figure Description

[0028] Figure 1 This is a finished product appearance diagram of the antiseptic and antibacterial composition described in Example 1. Detailed Implementation

[0029] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0030] Unless otherwise specified, all raw materials used in the following examples and comparative examples are commercially available general-purpose materials.

[0031] The lysozyme described in this invention is derived from egg white and has an activity of 1000 U / mL.

[0032] Examples 1-5

[0033] Examples 1-5 provide an antiseptic and antibacterial composition, the specific formulation of which is shown in Table 1. The preparation method of the composition includes the following steps:

[0034] Lysozyme, antimicrobial lipopeptide, capryloyl hydroxamic acid, and p-hydroxyacetophenone are mixed with water and stirred until homogeneous to obtain the preservative and antibacterial composition.

[0035] The preparation method of the antimicrobial lipopeptide is as follows:

[0036] (1) Preparation of Bacillus subtilis seed culture:

[0037] A single colony of Bacillus subtilis on LB solid medium was inoculated into LB liquid medium using an inoculation loop and cultured at 37°C and 220 rpm for 16-18 h to prepare a seed culture.

[0038] (2) Fermentation by Bacillus subtilis:

[0039] The seed culture was inoculated into liquid LB medium at a ratio of 3% (v / v) of inoculum. The fermentation temperature was 35℃, the fermentation time was 72h, and the shaking speed was 180rpm.

[0040] (3) Preparation of antimicrobial lipopeptides from Bacillus subtilis:

[0041] The fermentation broth was centrifuged at 8000 rpm for 15 min to obtain the fermentation supernatant. The pH of the fermentation supernatant was adjusted to 2.0 with 6 mol / L hydrochloric acid and placed in a refrigerator at 4℃ overnight. The fermentation supernatant was centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and the precipitate was added. After vortexing for 10 min and sonicating for 15 min, the precipitate was extracted for 4-8 h. After centrifugation again, the supernatant was filtered through a 0.22 μm filter membrane to obtain the antimicrobial lipopeptide produced by Bacillus subtilis.

[0042] Table 1. Formulation table (by mass percentage) of the antiseptic and antibacterial compositions described in Examples 1-5.

[0043]

[0044]

[0045] Comparative Examples 1-10

[0046] Comparative Examples 1-10 provide an antiseptic and antibacterial composition, the specific formulation of which is shown in Table 2. The preparation method of the composition is the same as that of the examples, the only difference being the formulation.

[0047] Table 2. Formulation table (by mass percentage) of the antiseptic and antibacterial compositions described in Comparative Examples 1-10

[0048]

[0049] Comparative Example 11

[0050] This comparative example provides an antiseptic and antibacterial composition, which differs from Example 1 only in that an equal amount of phenoxyethanol is used to replace p-hydroxyacetophenone, while the remaining components and contents are the same as in Example 1.

[0051] Comparative Example 12

[0052] This comparative example provides an antiseptic and antibacterial composition, which differs from Example 1 only in that an equal amount of nisin is used to replace the antimicrobial lipopeptide, while the remaining components and contents are the same as in Example 1.

[0053] Example 1: Determination of the inhibition zone

[0054] (1) Test samples: The compositions prepared in Examples 1-5 and Comparative Examples 1-12 were used as test samples, and the inhibition zones were measured to evaluate the antibacterial activity of the samples.

[0055] (2) Preparation of bacterial suspensions: Staphylococcus aureus (CICC 10384), Escherichia coli (CICC 24176), Pseudomonas aeruginosa (CICC 24649), Candida albicans (CICC 1965), and Propionibacterium acnes (ATCC 11827) were scraped from activated bacterial culture tubes and spread onto the corresponding solid culture media. Bacteria were incubated at 30°C for 24 hours, and fungi were incubated at 25°C for 48 hours. The bacterial strains were eluted with sterile physiological saline to prepare bacterial suspensions of different concentrations.

[0056] (3) Preparation of filter paper discs: Select qualitative filter paper with good water absorption, punch out circular filter paper discs with a diameter of 6mm using a punching machine, and sterilize them under high pressure for later use;

[0057] (4) Determination of inhibition zones: The filter paper diffusion method was used. Filter paper was punched into 5mm diameter circular discs, placed in clean beakers, sterilized at 121℃ for 20 minutes, and dried. 5μL of test sample was then pipetted onto each disc to thoroughly wet it. Sterilized nutrient agar or PDA medium was poured into petri dishes and allowed to cool and solidify. 0.1mL of each bacterial or fungal suspension was then evenly spread onto the plate. Sterile forceps were used to pick up the circular filter paper discs soaked in each component and affixed them to the bacterial plate. Four discs were affixed to each plate at equal intervals. One sterile water-soaked filter paper disc served as a blank control. Each petri dish was incubated upside down at 30℃ for 24 hours (bacteria) or 25℃ for 48 hours (fungi). The diameter of the inhibition zone for each strain under different test samples was measured. Each sample was tested in triplicate, and the average value was taken. Specific results are shown in Table 3.

[0058] Table 3. Results of inhibition zone diameter (mm)

[0059]

[0060]

[0061] As shown in Table 3, the antibacterial and antimicrobial compositions prepared in Examples 1-5 of this invention have significantly better antibacterial effects than single components, and have broad-spectrum inhibitory capabilities against Gram-positive bacteria, Gram-negative bacteria, and fungi. They are suitable for various application scenarios, especially the antibacterial and antimicrobial composition described in Example 1, which has the best broad-spectrum antibacterial effect.

[0062] In Comparative Examples 1-4, when only one component of lysozyme, antimicrobial lipopeptide, capryloyl hydroxamic acid, and p-hydroxyacetophenone was used, and in Comparative Examples 5-8, when any one or two of the above four components were omitted, the broad-spectrum antibacterial effect of the prepared antiseptic and antibacterial compositions was significantly reduced compared with the examples, especially the antibacterial effect against Pseudomonas aeruginosa and Propionibacterium acnes was significantly reduced. In Comparative Examples 9-10, when the ratio of the four components exceeded the limits of the present invention, the antibacterial effect of the composition was also weakened. The above results indicate that the four components selected in the composition of the present invention can exert an ideal synergistic effect within a specific dosage range, thereby enhancing the antibacterial effect of the composition and improving the limitation of the poor antibacterial effect of the single components against Gram-negative bacteria such as Pseudomonas aeruginosa and Gram-positive bacteria such as Propionibacterium acnes. This broadens the antibacterial spectrum and application range of the composition, thereby providing a more comprehensive antibacterial effect. In Comparative Examples 11-12, when the components with the same antibacterial effect were replaced with the components selected in this invention, the antibacterial effect of the prepared compositions also deteriorated, indicating that the four components of this invention can exert a unique synergistic effect, which cannot be achieved by conventionally replacing other components.

[0063] Example 2: Determination of Minimum Inhibitory Concentration (MIC)

[0064] This effect example uses the antiseptic and antibacterial composition prepared in Example 1 as the test sample, and its minimum inhibitory concentration is determined. The specific method is as follows:

[0065] (1) Preparation of nutrient broth culture medium;

[0066] (2) MIC determination: The test sample was diluted to five concentration gradients of 5%, 10%, 15%, 20%, and 25%, and bacterial solutions of the corresponding bacterial species were added. After incubation at 36℃ (bacteria) or 28℃ (fungi) for a certain period of time, the growth was observed to determine the MIC value. The specific results are shown in Table 4.

[0067] Table 4

[0068]

[0069] As shown in Table 4, the preservative and antibacterial composition of the present invention exhibits good antibacterial effects against both the tested bacteria and fungi. The minimum inhibitory concentration (MIC) against all tested microorganisms is 10%. This result indicates that the composition has highly efficient antibacterial properties in cosmetics and can effectively inhibit the growth of common cosmetic contaminants.

[0070] Example 3: Microbial Preservative Challenge Test

[0071] This example uses the preservative and antibacterial composition prepared in Example 1 as the test sample, and conducts a 28-day microbial preservative challenge test on it. The test is conducted in accordance with T-GDCDC 010-2019 Cosmetic Preservative Challenge Test Method, and the specific method is as follows:

[0072] (1) Weigh a certain amount of the test sample and add it to the mixed bacterial suspension in proportion, requiring the initial bacterial concentration in the sample to reach 10. 6 cfu / g; initial concentration of yeast and mold reached 10. 5 cfu / g; The standard strains used were: Staphylococcus aureus (CICC 10384), Escherichia coli (CICC 24176), Pseudomonas aeruginosa (CICC 24649), Candida albicans (CICC 1965), and Aspergillus niger (CICC 2487);

[0073] (2) Mix the 10% concentration test sample with the bacterial solution thoroughly, seal the sample bottle with sealing glue, and store it in an incubator at 25°C.

[0074] (3) Samples were taken on days 7, 14, 21 and 28 after inoculation to detect the microbial content and calculate the logarithmic decrease in colony count.

[0075] (4) The anti-corrosion effect is judged based on the logarithmic reduction value. Table 5 shows the background microbial inspection results, Table 6 shows the anti-corrosion challenge test results of the test samples, and Table 7 shows the anti-corrosion effect evaluation criteria.

[0076] Table 5. Background Microbiological Examination Results

[0077] Inspection items unit Test results Limit Total bacterial count CFU / g <10 ≤100 Total number of molds and yeasts CFU / g <10 ≤100

[0078] Table 6 Results of the Corrosion Resistance Challenge Test

[0079]

[0080]

[0081] Table 7 Evaluation Criteria for Corrosion Protection Effect

[0082]

[0083] As shown in Tables 5-7, the preservative and antibacterial composition of the present invention passed the preservative challenge test and met the requirements of Standard I. It can replace traditional organic synthetic preservatives, and its preservative effect is no less than that of traditional preservatives.

[0084] Example 4: Human skin patch test

[0085] This example uses the antiseptic and antibacterial compositions (concentration of 10%) prepared in Examples 1-5 as test samples. Human skin patch tests were conducted to evaluate the safety of the compositions on human skin. The specific method is as follows:

[0086] Human skin patch experiments were conducted on 50 volunteers, using patches with an area <50mm². 2 A qualified spot tester with a depth of 1 mm was used. 0.025 mL of test sample was drawn up with a syringe and placed in the drug chamber of the spot tester. The control well was used as a blank control (without any substance). The spot tester containing the test sample was applied longitudinally from bottom to top to the normal skin on the flexor side of the volunteer's left forearm using hypoallergenic adhesive tape. The palm was gently pressed to ensure even application of the test sample to the skin, and this was continued for 24 hours. Skin reactions were observed 30 minutes (after the indentation disappeared), 24 hours, and 48 hours after removing the spot tester. The results were recorded according to the skin reaction grading standards in the "Cosmetic Safety Technical Specifications" (2015 edition).

[0087] The results of the human skin patch test showed that 0 out of 50 people had a positive reaction. According to the "Cosmetic Safety Technical Specifications" (2015 edition), the test sample will not cause adverse skin reactions and is safe and non-irritating.

[0088] Example 5: Chicken Embryo Villi Allantoic Membrane Test

[0089] This example uses the antiseptic and antibacterial compositions (concentration of 10%) prepared in Examples 1-5 as test samples. A chicken embryo chorioallantoic membrane test was performed to evaluate the composition's irritation to the human body. The specific test method is as follows:

[0090] (1) Chicken embryo strain: Select fertilized chicken embryos of breeds such as White Leghorn chicken, with a weight of 50g-60g. SPF chicken embryos are preferred, and the quality of the chicken embryos must meet the requirements of relevant standards.

[0091] (2) Hatching conditions: room temperature 20-25℃, hatching temperature 37.5℃±0.5℃, relative humidity 55-70%, turntable frequency 3 times / h-6 times / h, 9-month-old chicken embryos do not need to be rotated during hatching;

[0092] (3) CAM preparation:

[0093] 1. Candling of 9-day-old chicken embryos is performed, marking the location of the air cell on the eggshell surface. Using dental serrated forceps, the marked portion of the eggshell is peeled away to expose the white membrane, handling it carefully to avoid damaging its integrity. 1-2 mL of sterile saline solution is added with a pipette to moisten the membrane. The next step can proceed immediately; otherwise, the embryo should be placed in an incubator or under a light (to prevent temperature drop) for no more than 20 minutes. The sterile saline solution is then poured out, and the inner membrane is carefully removed with forceps, ensuring the vascular membrane remains undamaged. The structure of the vascular system is then re-examined, and its integrity and suitability for the experiment are assessed.

[0094] 2. Place a sterile Teflon ring on a clearly visible and intact vascular area on the CAM membrane, add 40 μL of test solution, start timing immediately, observe the reaction, and record the time of occurrence of each toxic effect within 5 minutes (add 0.3 mL of the above transparent liquid directly to the CAM surface, observe the CAM reaction, and record the time of occurrence of each toxic effect within 5 minutes).

[0095] (4) Stimulus rating scale (IS):

[0096] For experiments conducted using the reaction time method, the stimulus score (IS) was calculated using equation (1), and the results were rounded to two decimal places.

[0097]

[0098] In the formula: sec H (hemorrhage time) is the average time at which bleeding begins to occur as observed on the CAM membrane, in seconds (s);

[0099] sec L (vessel lysis time) is the average time at which vascular lysis begins to occur as observed on the CAM membrane, measured in seconds (s).

[0100] sec C (coagulation time) is the average time it takes for coagulation to begin to appear on the CAM membrane, measured in seconds (s).

[0101] The eye irritation of the test substances is classified according to Table 8 based on the calculated IS values.

[0102] Table 8 Evaluation of Stimulus Rating Method Results

[0103] Stimulus rating Irritant Classification IS < 1 Non-irritating 1≤IS<5 Mild irritation 5≤IS<10 moderate irritation IS≥10 Strongly irritating / corrosive

[0104] Testing showed that the antiseptic and antibacterial composition prepared according to this invention exhibited no irritation in the chicken embryo chorioallantoic membrane test, with an irritation score of 0. This result indicates that the composition has good safety in cosmetics and will not cause skin irritation, further verifying its safety in cosmetic applications.

[0105] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An antiseptic and antibacterial composition, characterized in that, The preservative and antibacterial composition comprises the following components in weight percentages: 15-25% antibacterial lipopeptide, 0.3-0.7% lysozyme, 0.01-0.03% capryloyl hydroxamic acid, 0.3-0.5% p-hydroxyacetophenone, and the balance being water; the lysozyme is derived from egg white. The preparation method of the antimicrobial lipopeptide includes the following steps: S1. Take a loopful of a single colony of Bacillus subtilis on LB solid medium and transfer it to LB liquid medium. Incubate at 37°C and 220 rpm for 16-18 h to prepare seed culture. S2. Inoculate the seed culture into liquid LB medium at a ratio of 3v / v%, ferment at 35℃ for 72 hours, and shake at 180 rpm to obtain the fermentation broth. S3. Centrifuge the fermentation broth at 8000 rpm for 15 min to obtain the fermentation supernatant. Adjust the pH of the fermentation supernatant to 2.0 with 6 mol / L hydrochloric acid and place it in a refrigerator at 4℃ overnight. Centrifuge the fermentation supernatant at 8000 rpm for 15 min, discard the supernatant, and keep the precipitate. Add methanol and vortex for 10 min, sonicate for 15 min, extract for 4-8 h, centrifuge again, and filter the supernatant through a 0.22 μm filter membrane to obtain the antimicrobial lipopeptide.

2. The antiseptic and antibacterial composition according to claim 1, characterized in that, The antiseptic and antibacterial composition comprises the following components in weight percentages: 20% antibacterial lipopeptide, 0.4% lysozyme, 0.02% octanoyl hydroxamic acid, 0.3% p-hydroxyacetophenone, and the balance being water.

3. The method for preparing the antiseptic and antibacterial composition according to claim 1 or 2, characterized in that, Includes the following steps: Mix all components and stir until homogeneous to obtain the antiseptic and antibacterial composition.

4. The use of the preservative and antibacterial composition as described in claim 1 or 2 in cosmetics.

5. The application as described in claim 4, characterized in that, The preservative and antibacterial composition has a mass percentage of 10-25% in the cosmetic.

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