Antibacterial composition as well as preparation method and application thereof
An antibacterial composition was prepared by combining hop extract, Sargassum polyphenols, and fucoidan, which solved the problem of side effects in existing acne treatment products and achieved highly effective inhibition of acne pathogens and balanced regulation of skin health.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO AGRI UNIV
- Filing Date
- 2026-02-11
- Publication Date
- 2026-05-12
AI Technical Summary
Existing acne treatment products have side effects, such as dry skin, irritation, and drug resistance. They are not effective with long-term use and cannot effectively inhibit acne-related pathogens.
A compound of three natural active ingredients—hope extract, Sargassum polyphenols, and fucoidan—is formulated to create an antibacterial composition that, through synergistic effects, can be used in cosmetics to inhibit the growth of Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus.
Safe and without side effects, it effectively inhibits acne-related pathogens, regulates the skin's flora balance, prevents and improves acne problems, and is suitable for long-term use.
Smart Images
Figure CN122005386A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to an antibacterial composition, its preparation method, and its application. Background Technology
[0002] Acne (pimples) is a common skin condition primarily caused by the overgrowth of microorganisms such as Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus. These bacteria multiply in the hair follicles and sebaceous glands, leading to an inflammatory response and the formation of acne symptoms such as redness, swelling, and pustules. Currently, many acne treatment products on the market contain ingredients such as antibiotics, salicylic acid, and benzoyl peroxide, but these ingredients may have side effects, such as causing dry skin, irritation, and the development of drug resistance.
[0003] Because existing acne treatments, such as antibiotics, can easily lead to antibiotic resistance in pathogens, their antibacterial effect is poor with long-term use. Furthermore, medications such as salicylic acid or benzoyl peroxide can irritate the skin, and long-term use is detrimental to skin health and can easily lead to sensitive skin.
[0004] Therefore, acne treatment products still need further improvement, and safe, healthy, and effective acne treatment products are yet to be developed. Summary of the Invention
[0005] To address the aforementioned problems, this invention provides an antibacterial composition, its preparation method, and its application. The antibacterial composition mainly consists of several key active ingredients, including hop extract, Sargassum polyphenols, and fucoidan. It can effectively inhibit acne-causing bacteria such as Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus. It can be used as an active ingredient in the preparation of antibacterial cosmetics or skincare products to prevent and alleviate acne problems.
[0006] To address the above problems, this application provides the following technical solution: In a first aspect, this application provides an antibacterial composition comprising the following components in parts by weight: 1-5 parts of hop extract, 1-8 parts of Sargassum polyphenols, 1-5 parts of fucoidan oligosaccharides, and 90-110 parts of water.
[0007] In the above antibacterial composition, hop extract, Sargassum polyphenols, and fucoidan are three natural active ingredients that are safe to use and possess antibacterial, anti-inflammatory, and antioxidant effects. Specifically, the flavonoids and polyphenols in hop extract effectively inhibit bacterial growth; Sargassum polyphenols have significant antioxidant and anti-inflammatory effects; and fucoidan, an oligosaccharide extracted from brown algae, regulates the skin's microecology and enhances the skin barrier function. By combining these three active ingredients, their synergistic effect not only enhances the composition's antibacterial effect against acne pathogens but also ensures safe use, non-irritating to the skin, and suitability for long-term use.
[0008] Optionally, the antibacterial composition comprises the following components in parts by weight: 1-4 parts of hop extract, 1-5 parts of Sargassum polyphenols, 1-3 parts of fucoidan oligosaccharides, and 90-110 parts of water.
[0009] Preferably, in the antibacterial composition, the mass ratio of hop extract, Sargassum polyphenols and fucoidan is 1:1:2.
[0010] Preferably, the antibacterial composition comprises the following components in the indicated weight percentages: 1% hop extract, 1% Sargassum polyphenols, 2% fucoidan, and the balance being water.
[0011] During the course of numerous experiments, the applicant unexpectedly discovered that the synergistic effect of the aforementioned hop extract, Sargassum polyphenols, and fucoidan can enhance the inhibitory effect on acne-related pathogens. It can effectively inhibit the growth and proliferation of Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus, thereby inhibiting the deterioration of acne and playing a role in preventing acne.
[0012] Optionally, in the antibacterial composition, the hop extract is prepared by: washing and drying fresh hops, pulverizing them, placing the dried hop powder in an alcohol aqueous solution system, extracting under shaking conditions, and centrifuging to obtain the hop extract.
[0013] Optionally, the preparation method of the Sargassum polyphenol in the antibacterial composition is as follows: fresh Sargassum is washed, dried, and pulverized; the Sargassum powder is placed in a natural eutectic solvent system and extracted under ultrasonic conditions; the crude extract is obtained by centrifugation; and then post-processing is performed to obtain Sargassum polyphenol.
[0014] Optionally, in the antibacterial composition, the fucoidan is prepared by adding fucoidanase to the fucoidan solution, placing it in a water bath for enzymatic hydrolysis, inactivating the enzyme, centrifuging and taking the supernatant; fractionating the supernatant into fucoidan solutions of different molecular weight ranges using an ultrafiltration system, and freeze-drying for later use.
[0015] Secondly, this application also provides a method for preparing the above-mentioned antibacterial composition, which includes the following steps: S1. Preparation of hop extract: Fresh hops are washed, dried and pulverized. The hop powder is placed in an alcohol aqueous solution system and extracted under shaking conditions. After centrifugation, hop extract is obtained.
[0016] S2. Preparation of Sargassum polyphenols: Fresh Sargassum was washed, dried, and pulverized. The Sargassum powder was placed in a natural eutectic solvent system and extracted under ultrasonic conditions. The crude extract was obtained by centrifugation. Then, post-processing was performed to obtain Sargassum polyphenols.
[0017] S3. Preparation of fucoidan oligosaccharides: Dissolve the freeze-dried fucoidan enzyme in distilled water, mix with an equal volume of fucoidan solution at a mass ratio of 0.2%-2%, place in a shaking water bath at 30℃ and 120 rpm for 2 h of enzymatic hydrolysis, then place in a water bath at 100℃ for 10 min to inactivate the enzyme, centrifuge at 4℃ and 8000 r / min for 10 min, and collect the supernatant; fractionate the supernatant into fucoidan oligosaccharide solutions of different molecular weight ranges (molecular weight ranges of less than 5 kDa, 5-10 kDa, and more than 10 kDa) using an ultrafiltration system, freeze-dry for later use.
[0018] S4. The antibacterial composition is prepared by mixing the hop extract, Sargassum polyphenols, and fucoidan. When using, the mixture is dissolved in an appropriate amount of pure water.
[0019] Optionally, in step S1, the aqueous alcohol solution is 45%-60% ethanol or butanediol. The solid-liquid ratio is 1:10-1:40 g / mL. The extraction time is 0.5-1.5 h, the extraction temperature is 40-60 °C, and the rotation speed is 120-200 r / min. After centrifugation at 4 °C and 8000-10000 r / min for 10-15 min, the hop extract is obtained by freeze-drying.
[0020] Preferably, the conditions for S1 are as follows: the alcohol solvent is 45% ethanol; the extraction time is 1.5 h; the extraction temperature is 50℃; the rotation speed is 180 r / min; centrifugation is performed at 4℃ and 10000 r / min for 10 min; and the hop extract is obtained by freeze drying.
[0021] Optionally, in step S2, the natural eutectic solvent system is a mixture of betaine and glycerol in a molar ratio of 1:1, with a water content of 40-60%; the ultrasonic treatment conditions are: ultrasonic time of 15-40 min and ultrasonic temperature of 40-70℃; the centrifugation conditions are: centrifugation at 4℃ and 8000-10000 r / min for 10-15 min.
[0022] Optionally, in step S2, the post-treatment involves removing the eutectic solvent with a macroporous resin and freeze-drying to obtain Sargassum polyphenols.
[0023] Preferably, the natural eutectic solvent system is a mixture of betaine and glycerol in a 1:1 molar ratio, with a water content of 40%, ultrasonication time of 30 min, ultrasonication temperature of 60℃, and centrifugation at 4℃ and 8000 r / min for 15 min. The post-treatment involves removing the eutectic solvent using NKA-9 macroporous resin, followed by freeze-drying to obtain Sargassum polyphenols.
[0024] Optionally, in S3, the fucoidanase is a fucoidanase obtained by fermentation from Flavobacterium RC2-3mut strain with accession number CGMCC No. 14855. The Flavobacterium was deposited on November 3, 2017, at the China General Microbiological Culture Collection Center, No. 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 14855. Information about this strain has been disclosed in patent document CN107988109B.
[0025] Preferably, fucoidan with a molecular weight of 10 kDa or higher is used. Experiments have shown that fucoidan in this molecular weight range has the best antibacterial effect against pathogens.
[0026] Thirdly, this application also provides the use of an antibacterial composition in inhibiting acne pathogens, said pathogens including: Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus.
[0027] Fourthly, this application also provides the application of the above-mentioned antibacterial composition in the preparation of cosmetics or skin care products, wherein the cosmetics are gels, serums, masks, creams, lotions, or cleansing gels. The cosmetics include various conventional skin care products.
[0028] The application method involves adding the antibacterial composition to cosmetics or skincare products. Preferably, the amount of the antibacterial composition added is 1%-6% (by weight).
[0029] The present invention has the following beneficial effects: 1. This invention provides an antibacterial composition that cleverly combines several main active ingredients, including naturally extracted hops extract, Sargassum polyphenols, and fucoidan, to enhance the inhibitory effect on acne-related pathogens (Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus) through the synergistic effect of the three ingredients, thereby inhibiting their proliferation on the skin, regulating the skin flora balance, and preventing and improving acne problems.
[0030] 2. The main components of the antibacterial composition provided by the present invention are all of natural origin, safe to use without side effects, and more easily absorbed by the human body. They can be used as raw materials for antibacterial cosmetics or skin care products to prepare various skin antibacterial products.
[0031] 3. The preparation method of the antibacterial composition of the present invention is simple, has good repeatability, and is easy to scale up production and quality control. Therefore, the antibacterial composition has good market application prospects. Attached Figure Description
[0032] Figure 1 The effects of different extraction methods and three molecular weights of fucoidan on the antibacterial effect of Propionibacterium acnes; Figure 2 The effect of different compound combinations on the antibacterial effect of Propionibacterium acnes; Figure 3 The antibacterial composition of the present invention shows the antibacterial curve against Propionibacterium acnes. Figure 4 The antibacterial composition of the present invention shows the antibacterial curve against Staphylococcus epidermidis; Figure 5 The antibacterial composition of the present invention shows the antibacterial curve against Staphylococcus aureus. Figure 6 The effect of the antibacterial composition of the present invention on the safety of zebrafish embryos is shown in Figure 1; a is the blank control group; b is 0.1 mg / mL salicylic acid; cf are 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, and 0.3 mg / mL compositions, respectively; g and h are 0.4 mg / mL compositions. Figure 7 The effect of the antibacterial composition of the present invention on the surface hydrophobicity of Propionibacterium acnes; different letters indicate significant differences. p <0.05, the same applies below; Figure 8 The effect of the antibacterial composition of the present invention on the adhesiveness of Propionibacterium acnes; Figure 9 The effect of the antibacterial composition of the present invention on the biofilm formation of Propionibacterium acnes; Figure 10 The effect of the antibacterial composition of the present invention on the polysaccharide content of Propionibacterium acnes biofilm; Figure 11 The diameter of the inhibition zone of different substances against Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus. Detailed Implementation
[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.
[0034] Example 1 Preparation of antibacterial composition 1.1 Preparation of hop extract Hops extract was prepared using the organic reagent ethanol. Fresh hops (originating from Gansu) were washed with distilled water, dried, ground into powder, and sieved to prepare a hop solution with a solid-liquid ratio of 1:20 g / mL. The hop solution was extracted with 45% ethanol at 50℃ and 180 r / min on a shaker for 1.5 h. The mixture was then centrifuged at 4℃ and 10000 r / min for 10 min, the supernatant was collected, and the ethanol was removed by rotary evaporation. The resulting product was then freeze-dried for later use.
[0035] Control group: Hops extract was prepared by boiling water extraction (an experiment was also conducted on the extraction solvent to eliminate its influence). Hops and water were mixed at a ratio of 1.0 g: 20 ml, boiled for 30 minutes, and then cooled to a suitable temperature (<45℃). The extract could be centrifuged to precipitate the plant material, and the liquid or filtered and ground hops extract was collected to obtain the hops extract, which was then freeze-dried for later use.
[0036] 1.2 Preparation of Sargassum polyphenols Sargassum polyphenols were prepared using natural eutectic solvents. Fresh Sargassum was washed with distilled water, dried, powdered, and sieved to prepare a Sargassum solution with a solid-liquid ratio of 1:30. The natural eutectic solvent system consisted of betaine and glycerol in a 1:1 molar ratio with a water content of 40%. The extraction conditions were: ultrasonication for 30 min at 70℃, followed by centrifugation at 8000 r / min for 15 min at 4℃. The supernatant was collected, and the eutectic solvent was removed by passing it through NKA-9 macroporous resin. The solution was then freeze-dried for later use.
[0037] 1.3 Preparation of Fucoidooligosaccharides (1) Preparation of biological enzymes Flavobacteriaceae sp. RC2-3mut strain was inoculated into liquid culture medium and cultured in a shaker at 25°C and 150 r / min for 24 h to obtain seed culture. The seed culture was then inoculated into fermentation medium at a 10% inoculation rate and fermented in a 5 L fermenter (4 L of feed) at 25°C and 150 r / min for 48 h to obtain fermentation broth.
[0038] The fermentation broth was centrifuged at 5000 r / min for 10 min at 4℃, the supernatant was discarded and the precipitate was collected. The precipitate was then dissolved in 5 times its volume of seawater solution. The cells were ultrasonically disrupted at 800 W for 10 min with a 5 s interval under ice bath protection. The mixture was then centrifuged at 8000 r / min for 10 min at 4℃. The supernatant was collected and freeze-dried to obtain the bioenzyme, namely fucoidanase.
[0039] (2) Preparation of fucoidan oligosaccharides by degrading fucoidan with bioenzymes: The preparation method of the low-fucoidan oligosaccharide of the present invention is as follows: The freeze-dried fucoidan enzyme is dissolved in distilled water and mixed with an equal volume of fucoidan solution extracted from Sargassum at a mass ratio of 0.2%-2%. The mixture is then placed in a shaking water bath at 30℃ and 120 rpm for 2 h for enzymatic hydrolysis. Afterward, the enzyme is inactivated in a water bath at 100℃ for 10 min, and the mixture is centrifuged at 4℃ and 8000 r / min for 10 min. The supernatant is collected. The supernatant is fractionated into fucoidan oligosaccharide solutions of different molecular weight ranges (less than 5 kDa, 5-10 kDa, and above 10 kDa) using a Vivaflow ultrafiltration system, and then freeze-dried for later use.
[0040] 1.4 Preparation of antibacterial composition An antibacterial composition was prepared by mixing the aforementioned hop extract, Sargassum polyphenols, and fucoidan in a mass ratio of 1:1:2.
[0041] 1.5 Determination of antibacterial properties (1) Experimental method: The hop extract (water extract and alcohol extract), Sargassum polyphenols, fucoidan oligosaccharides of different molecular weight ranges, and antibacterial composition (1:1:2) prepared in this embodiment were subjected to inhibition zone tests against acne-associated pathogens (Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus) to verify their antibacterial activity. The concentration of each component used individually was 10 mg / mL, or 1%; the three components in the composition were formulated to a concentration of 10 mg / mL in a 1:1:2 ratio.
[0042] Inhibition zone test method: Take the well-cultured bacterial solution and dilute it to 1×10⁻⁶. 6 CFU / ml, for later use. Inoculate the test bacterial suspension (1%) into the melted and cooled culture medium (50-55℃), mix well, and pour into Petri dishes, approximately 15-20 mL per dish. Rotate the Petri dish to mix the bacterial suspension with the culture medium, and allow it to solidify. Using a 5 mm punch, evenly punch holes in each plate, add approximately 40-50 μL of the sample to be tested (filtered through a 0.22 μm membrane), cover the Petri dish, and incubate at 37℃ for 18-24 h. P.acnes The bacterial culture should be placed in an anaerobic incubator at 37℃ and incubated for 2-3 days to observe the results. Salicylic acid was used as a positive control. The diameter of the inhibition zone was measured using a colony counter and recorded.
[0043] (2) Experimental results and analysis The results of the antibacterial activity study using the agar perforation method showed that hop extract, Sargassum polyphenols, and fucoidan and their combinations all had varying degrees of inhibitory effects on the three strains mentioned above.
[0044] A. The results are shown in Table 1 and Figure 1As shown, the inhibition zone diameter of alcohol-extracted hops is significantly larger than that of water-extracted hops; and fucoidan with a molecular weight greater than 10 kDa has the largest inhibition zone diameter against Propionibacterium acnes. Therefore, alcohol-extracted hops extract and fucoidan with a molecular weight greater than 10 kDa were selected as components to prepare an antibacterial composition.
[0045] Table 1 Results of the inhibition zone experiment for Propionibacterium acnes
[0046] B. The diameter of the bacterial inhibition zone in the negative control group was 5 mm, the same as the diameter of the agar wells, indicating no inhibition phenomenon; as shown in Table 2 and... Figure 11 The results showed that the diameter of the inhibition zone of bacteria in all experimental groups was greater than 12 mm, indicating that hop extract, Sargassum polyphenols and fucoidan and their combination (1:1:2) had a strong inhibitory effect on the three strains and was higher than that of salicylic acid in the positive control group. Among them, the antibacterial combination had the best antibacterial effect on the three acne pathogens, far exceeding the antibacterial effect of the single raw material.
[0047] Table 2. Results of the inhibition zone experiment on acne-associated pathogens by the three raw materials and their compositions.
[0048] Note: Different letters indicate significant differences between columns. p <0.05, the same applies below; 1.6 Optimization Experiment of Antibacterial Composition (1) Experimental method: Eight antibacterial compositions were prepared according to the proportions in Table 3 below (total concentration of 10 mg / mL), and their antibacterial effects against Propionibacterium acnes, the main pathogen of acne, were determined as described above.
[0049] Table 3. Proportional relationships of components in each combination
[0050] (2) Experimental results and analysis The results are shown in Table 4. Figure 2 As shown, the results indicate that combination 5 (hops extract: Sargassum polyphenols: fucoidan = 1:1:2) exhibits the largest inhibition zone diameter against Propionibacterium acnes, demonstrating a significantly superior antibacterial effect compared to all other combinations. Therefore, the composition used in the experiment was a compound of combination 5 in the optimal ratio. The optimal ratio of hops extract, Sargassum polyphenols, and fucoidan in the antibacterial composition is 1:1:2.
[0051] Table 4. Results of the inhibition zone experiment on Propionibacterium acnes by different combinations.
[0052] Example 2: Determination of minimum inhibitory concentration and minimum bactericidal concentration 1. Determination methods for minimum inhibitory concentration and minimum bactericidal concentration. Take a sterile 96-well plate, inject 100 μL of bacterial suspension and 100 μL of each concentration of sample into each well, and set up 3 replicates for each group; use 200 μL of sterile liquid culture medium as blank control; 100 μL of bacterial suspension and 100 μL of salicylic acid as positive control; 100 μL of bacterial suspension and 100 μL of liquid culture medium as negative control; and 200 μL of sterile water as solvent control. Set up 4 replicates for each group.
[0053] Seal the cultured 96-well plates and place them in a constant temperature incubator at 37℃ for static incubation for 18-24 hours. P.acnes The 96-well plates containing the bacterial culture should be sealed and placed in an anaerobic incubator at 37°C for 2-3 days. After incubation, remove the 96-well plates from the incubator and measure the absorbance at 600 nm using a microplate reader. The concentration in each test solution that completely inhibits bacterial growth is the minimum inhibitory concentration (MIC) for that sample.
[0054] Based on the determined MIC of each drug, 0.1 ml of liquid culture medium at concentrations of 1 / 2 MIC, MIC, 2 MIC, 4 MIC, and 8 MIC were inoculated into the corresponding agar plates, spread, and incubated at 37℃ for 18-24 h. P.acnes After anaerobic culture for 2-3 days to reach the stable growth period of the bacteria, when the number of colonies is ≤5, this concentration is taken as its minimum bactericidal concentration (MBC), and the experiment is conducted in parallel 3 times.
[0055] The three raw materials prepared in Example 1—hope extract (alcohol extract), Sargassum polyphenols, and fucoidan—as well as the antibacterial composition (optimal ratio) were measured according to the above method.
[0056] The extract prepared according to the method disclosed in CN118695867A is considered prior art and is analyzed comparatively: Hops are weighed and added to water at a ratio of 1.0 g hops to 20 ml water. The extract is boiled for 30 minutes. After boiling, the extract is cooled to a suitable temperature (<45°C). The extract can be centrifuged to precipitate the plant material, and the liquid or ground hop extract can be collected, for example, by passing it through a Grade 1 Whitman filter paper to remove the plant material. The liquid extract is then aliquoted into smaller aliquots and frozen at -80°C until use.
[0057] 2. Experimental Results and Analysis The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MIC) of each group were determined by the micro-dilution method. The results are shown in Table 5. The results indicate that hop extract, Sargassum polyphenols, fucoidan and the antibacterial composition have significant inhibitory effects on acne-associated pathogens in vitro. The antibacterial composition has the best antibacterial effect and the lowest MIC, which may be the result of the synergistic effect of the three substances.
[0058] Table 5. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the three raw materials and their compositions against acne-associated pathogens.
[0059] Example 3 Determination of the antibacterial curve of the antibacterial composition The antibacterial curves of the optimally formulated antibacterial composition prepared in Example 1 were determined according to the following method.
[0060] 1. Experimental Methods Pipette 500 μL of bacterial suspension into a deep-well plate, add 500 μL of sample (concentrations of 1 / 2MIC, MIC, and 2MIC, respectively), and use 1 mL of the above bacterial suspension as a control group. Measure the growth curve using a fully automated microbial growth curve analyzer.
[0061] 2. Experimental Results and Analysis The results are as follows Figure 3 , Figure 4 , Figure 5 As shown, antibacterial time curves were plotted for the composition at MIC, 1 / 2MIC, and 1 / 4MIC concentrations. The OD values of the bacterial suspension in the blank control group showed an approximately "S"-shaped upward trend, indicating that the addition of the antibacterial composition effectively inhibited the growth of the three types of bacteria. Compared with the normal bacterial growth in the blank control group, at the MIC concentration, the OD values of each group remained at the initial level over time, indicating that the bacteria were in a lag phase during the observation period, demonstrating that this concentration completely inhibited bacterial growth. At 1 / 2MIC and 1 / 4MIC concentrations, the growth of the experimental group strains was generally delayed. At the same time point, the OD values of the experimental group bacterial suspension were lower than those of the control group, and the duration of the lag phase was longer than that of the blank control group. The process of bacteria entering the logarithmic growth phase was significantly inhibited, and the activity was low during the logarithmic growth phase. Furthermore, the antibacterial effect gradually increased with increasing drug concentration. These results indicate that the antibacterial composition has a good inhibitory effect on acne-related pathogens, and the inhibitory effect is related to the drug concentration.
[0062] Example 4: Effect of the antibacterial composition on the safety of zebrafish embryos The safety of the optimally formulated antibacterial composition prepared in Example 1 was determined according to the following method.
[0063] 1. Experimental Methods Twenty normally developing 6 hpf zebrafish embryos were randomly selected and placed in each well of a 24-well plate. The following experimental groups were set up: a blank control group (containing only culture water), a positive control group (containing culture water with salicylic acid), and a sample group (containing culture water with different concentrations of the compound extract). The 24-well plates were incubated in a 28°C incubator. During incubation, the sample solution was changed every 24 hours. After 72 hours of incubation, the morphology of the embryos was observed under a microscope, and the embryo mortality rate and hatching rate (successful hatching was defined as successful hatching and free swimming under natural conditions) were recorded to evaluate the effect of the compound extract on zebrafish embryos.
[0064] 2. Experimental Results and Analysis The effects of the antibacterial composition on zebrafish embryo growth are shown in Table 6 and... Figure 6 As shown in the figure, the results indicate that no abnormalities were observed in zebrafish embryos at concentrations of 0.3 mg / mL and below, suggesting that low concentrations of the composition had no significant effect on embryonic growth and development. However, when the composition concentration reached 0.4 mg / mL and above, the mortality rate of zebrafish embryos increased significantly, especially at 0.5 mg / mL, where the embryonic mortality rate reached 100%. This may be due to the toxic effects of high concentrations of the composition on the embryos, leading to abnormal embryonic development. Therefore, the maximum experimental concentration of the antibacterial composition for use in zebrafish models was determined to be 0.3 mg / mL.
[0065] Table 6 Effects of the composition on zebrafish embryos
[0066] Example 5: Effect of the antibacterial composition on the hydrophobicity of the Propionibacterium acnes surface. 1. Experimental Methods The hydrophobicity of Propionibacterium acnes cell surface was determined using the microbial adhesion hydrocarbon method. The specific method was as follows: bacterial cultured to the logarithmic growth phase was centrifuged at 8000 r / min for 10 min at 4°C, the cells were washed with sterile PUM buffer, resuspended, and the OD of the bacterial culture was adjusted. 600 Set the concentration to 0.5 and set aside. Mix 3 mL of bacterial culture with 9 mL of the antibacterial composition to achieve final concentrations of 2 MIC, MIC, 1 / 2 MIC, and 1 / 4 MIC. Use the culture without the antibacterial composition as a blank control. After mixing, incubate anaerobically at 37°C for 6 h and measure the OD value. 600 Add 2 mL of n-hexadecane to 3 mL of bacterial suspension, vortex mix for 1 min, and let stand at room temperature for 20 min to separate the two phases. Take the lower aqueous phase and measure the OD. 600 Each group was measured in triplicate. Calculate according to the formula: Hydrophobicity (%) = (1 - OD1 / OD0) × 100% Where OD0 is the absorbance at 600 nm initially, and OD1 is the absorbance of the lower aqueous phase at 600 nm after the addition of hexadecane; 2. Experimental Results and Analysis The results are as follows Figure 7 As shown, the surface hydrophobicity of *Propionibacterium acnes* gradually increased with decreasing concentration of the antibacterial composition (from 2 MIC to 1 / 4 MIC), indicating that the inhibitory effect of the antibacterial composition on the surface hydrophobicity of *Propionibacterium acnes* weakens with decreasing concentration. The 2 MIC group showed the most significant inhibitory effect (8.34%), while the 1 / 4 MIC group showed the least inhibitory effect, almost approaching the level of the control group. These results indicate that the surface hydrophobicity of the antibacterial composition is concentration-dependent; higher concentrations (such as 2 MIC) can more effectively reduce the surface hydrophobicity of bacteria, thereby affecting their adhesion, biofilm formation ability, and other biological characteristics, and reducing the colonization and infection of *Propionibacterium acnes*.
[0067] Example 6: Effect of the antibacterial composition on the adhesiveness of Propionibacterium acnes 1. Experimental Methods Centrifuge the bacterial culture in the logarithmic growth phase at 8000 r / min for 10 min at 4°C, wash the cells with sterile PBS, resuspend, and adjust the bacterial concentration to 10. 6 -10 8 CFU / mL, for later use. Pipette 100 μL of bacterial culture into a 96-well plate, add 100 μL of Propionibacterium acnes liquid medium, and incubate anaerobicly at 37°C for 48 h. Discard the supernatant and wash 2-3 times with sterile PBS. Add 200 μL of antibacterial composition at concentrations of MIC, 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC. Use the culture without antibacterial composition as a blank control and the culture with salicylic acid as a positive control. Incubate anaerobicly at 37°C for 48 h. Discard the upper layer of airborne bacteria, wash 2-3 times with sterile PBS, add 100 μL of MTT (menaquinone) solution, and incubate at 37°C for 4 h. Slowly aspirate the MTT waste liquid, add 200 μL of DMSO solution to each well, and shake on a shaker at 37°C for 10 min. Measure the number of bacteria adhering to the bottom of the wells using a microplate reader at 490 nm.
[0068] Adhesion inhibition rate (%) = [1 - (OD1 / OD0)] × 100% Wherein OD0 is the absorbance value of the control group at 490 nm, and OD1 is the absorbance value of the treatment group at 490 nm.
[0069] 2. Experimental Results and Analysis The results are as follows Figure 8As shown, the adhesion of *Propionibacterium acnes* was inhibited and its metabolic activity decreased after treatment with the antibacterial composition. At the MIC (micron concentration), the inhibition rate reached 72.98%. The inhibition rates at 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC were 39.62%, 30.81%, and 20.88%, respectively. The inhibition rate was directly proportional to the concentration of the antibacterial composition. Inhibiting bacterial growth can alter its surface properties, reduce its adhesion to surfaces, thereby inhibiting biofilm formation and reducing the colonization and infection of *Propionibacterium acnes*.
[0070] Example 10 Effect of antibacterial composition on biofilm formation of Propionibacterium acnes 1. Experimental Method: Centrifuge the bacterial culture in the logarithmic growth phase at 8000 r / min for 10 min at 4°C, wash the cells with sterile PBS, resuspend, and adjust the bacterial concentration to 10. 6 -10 8 CFU / mL, for later use. Using the crystal violet staining method, 100 μL of bacterial culture was pipetted into a 96-well plate, and 100 μL of liquid culture medium containing the antibacterial composition was added to achieve final concentrations of MIC, 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC. A blank control was used without the antibacterial composition, and salicylic acid was used as a positive control. The plates were anaerobic at 37℃ for 48 h. After incubation, the culture medium was slowly aspirated, and the upper layer of floating bacteria was discarded. The plates were washed 2-3 times with sterile PBS, fixed with methanol for 10 min, removed, and air-dried for 5 min. 100 μL of 0.1% crystal violet was added to stain the biofilm for 30 min. After removal, the plates were washed 2-3 times with sterile water, and destained with 95% ethanol by shaking for 15 min. The destaining solution was pipetted into a blank 96-well plate, and the absorbance was measured at 595 nm using a microplate reader. The percentage of biofilm formation in each experimental group was calculated, with the biofilm formation in the control group without the added sample considered as 100%.
[0071] Biofilm inhibition rate (%) = [1 - (OD1 / OD0)] × 100% Wherein OD0 is the absorbance value of the control group at 595 nm, and OD1 is the absorbance value of the treatment group at 595 nm.
[0072] 2. Experimental Results and Analysis The results are as follows Figure 9 As shown, at the MIC, the biofilm formation rate decreased by 85.55%, while at 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC, the biofilm formation rate decreased by only 16.44%, 9.71%, and 1.45%, respectively. This result indicates that the antibacterial composition concentration must be higher than the MIC to effectively inhibit biofilm formation. P.acnes The formation of biofilms reduces the colonization and infection of Propionibacterium acnes.
[0073] Example 11 Effect of antibacterial composition on polysaccharide content in Propionibacterium acnes biofilm 1. Experimental Method: Centrifuge the bacterial culture in the logarithmic growth phase at 8000 r / min for 10 min at 4°C, wash the cells with sterile PBS, resuspend, and adjust the bacterial concentration to 10. 6 -10 8 CFU / mL, for later use. Pipette 0.5 mL of bacterial culture into a 24-well plate, add 0.5 mL of liquid culture medium containing the antibacterial composition, to achieve final concentrations of MIC, 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC. The culture without the compound extract serves as a blank control, while the culture with salicylic acid serves as a positive control. Incubate anaerobically at 37℃ for 48-72 h. After incubation, slowly aspirate the culture medium, wash 2-3 times with sterile PBS, then add 1 mL of sterile PBS to each well and sonicate for 20 min. Centrifuge at 5000 r / min for 10 min at 4℃. Collect the supernatant after centrifugation and determine the polysaccharide content using the phenol-sulfuric acid method. Each group is measured in triplicate.
[0074] 2. Experimental Results and Analysis The results are as follows Figure 10 As shown, the antibacterial composition significantly inhibited the polysaccharide content of Propionibacterium acnes biofilm, and this inhibitory effect weakened with decreasing sample concentration, exhibiting a concentration-dependent effect. At the MIC concentration, the sample showed the strongest inhibitory effect on polysaccharide content, while at 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC concentrations, the inhibitory effect gradually weakened, and the polysaccharide content gradually approached the control group level.
[0075] The above results indicate that the concentration of the antibacterial composition is a key factor affecting its inhibitory effect on the polysaccharide content of Propionibacterium acnes biofilm. High concentration samples more effectively inhibit biofilm formation, thereby reducing polysaccharide content.
[0076] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.
Claims
1. An antibacterial composition, characterized in that, It includes the following components in parts by weight: 1-5 parts hop extract, 1-8 parts Sargassum polyphenols, 1-5 parts fucoidan, and 90-110 parts water.
2. The antibacterial composition according to claim 1, characterized in that, It includes the following components in parts by weight: 1-4 parts hop extract, 1-5 parts Sargassum polyphenols, 1-3 parts fucoidan, and 90-110 parts water.
3. The antibacterial composition according to claim 1, characterized in that, The preparation method of the hop extract is as follows: fresh hops are washed, dried and pulverized. The hop powder is placed in an alcohol aqueous solution system, extracted under shaking conditions, and centrifuged to obtain the hop extract.
4. The antibacterial composition according to claim 1, characterized in that, The preparation method of Sargassum polyphenols is as follows: fresh Sargassum is washed, dried and pulverized. The Sargassum powder is placed in a natural eutectic solvent system and extracted under ultrasonic conditions. The crude extract is obtained by centrifugation. Then, post-processing is performed to obtain Sargassum polyphenols.
5. The antibacterial composition according to claim 1, characterized in that, The preparation method of the fucoidan is as follows: fucoidanase is added to the fucoidan solution, and the solution is placed in a water bath for enzymatic hydrolysis. After enzyme inactivation, the solution is centrifuged and the supernatant is collected. The supernatant is fractionated into fucoidan solutions of different molecular weight ranges using an ultrafiltration system, and then freeze-dried for later use.
6. The method for preparing the antibacterial composition according to claim 1, characterized in that, Includes the following steps: S1. Preparation of hop extract: Fresh hops are washed, dried and pulverized. The hop powder is placed in an alcohol aqueous solution system and extracted under shaking conditions. After centrifugation, hop extract is obtained. S2. Preparation of Sargassum polyphenols: Fresh Sargassum was washed, dried, and pulverized. The Sargassum powder was placed in a natural eutectic solvent system and extracted under ultrasonic conditions. The crude extract was obtained by centrifugation. Then, post-processing was performed to obtain Sargassum polyphenols. S3. Preparation of fucoidan oligosaccharides: The freeze-dried fucoidan enzyme was dissolved in distilled water and mixed with an equal volume of fucoidan solution at a mass ratio of 0.2%-2%. After enzymatic hydrolysis in a water bath for 2 h, the enzyme was inactivated in a 100℃ water bath for 10 min, and then centrifuged at 4℃ and 8000 r / min for 10 min. The supernatant was collected. The supernatant was fractionated into fucoidan oligosaccharide solutions of different molecular weight ranges using an ultrafiltration system, and then freeze-dried for later use. The fucoidan enzyme was obtained by fermentation of Flavobacterium RC2-3mut strain with preservation number CGMCC No.14855. S4. The hop extract, Sargassum polyphenols and fucoidan are mixed to obtain an antibacterial composition.
7. The preparation method according to claim 6, characterized in that, In step S1, the alcohol aqueous solution is 45%-60% ethanol or butanediol, the shaking extraction time is 0.5-1.5h, the extraction temperature is 40-60℃, and the rotation speed is 120-200 r / min; after centrifugation at 4℃ and 8000-10000 r / min for 10-15 min, the hop extract is obtained by freeze drying.
8. The preparation method according to claim 6, characterized in that, In step S2, the natural eutectic solvent system is a mixture of betaine and glycerol in a molar ratio of 1:1, with a water content of 40-60%; the ultrasonic treatment conditions are: ultrasonic time of 15-40 min and ultrasonic temperature of 40-70℃; the centrifugation conditions are: centrifugation at 4℃ and 8000-10000 r / min for 10-15 min.
9. The application of the antibacterial composition as described in claim 1 in inhibiting pathogenic bacteria, characterized in that, The pathogens include Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus.
10. The application of the antibacterial composition according to claim 1 in the preparation of cosmetics, characterized in that, The cosmetic product is a gel, serum, mask, cream, lotion, or cleansing gel.