Malassezia-resistant theabrownin-selenium nanoparticle-coated malassezia membrane composite system and application thereof

Abstract

The invention discloses a preparation method of a theabrownin-selenium nanoparticle-coated malassezia membrane composite system. The preparation method comprises the following steps: adding a sodium selenite solution and a vitamin C solution into a theabrownin solution, and stirring; carrying out gradient centrifugation on the obtained mixed solution, and preparing the obtained precipitate into a suspension of theabrownin-selenium nanoparticles; preparing a malassezia film by using the malassezia liquid; performing ultrasonic mixing on the turbid liquid of the theabrownin-selenium nanoparticles and the malassezia membrane in an ice bath; and then centrifuging to remove supernatant, and adding deionized water to a constant volume to obtain the theabrownin-selenium nanoparticle / malassezia membrane composite system. The invention also provides an application of the theabrownin-selenium nanoparticle-coated malassezia membrane composite system. The theabrownin-selenium nanoparticle-coated malassezia membrane composite system is used for preparing a malassezia-resistant medicine or reagent.

Description

Technical Field

[0001] The invention belongs to the field of medicine and cosmetics, and in particular relates to a theabrownin-selenium nanoparticle@Malassezia membrane composite system with anti-Malassezia function, and a preparation method and application thereof. Background Art

[0002] Seborrheic dermatitis is a chronic, recurrent and inflammatory skin disease, while dandruff is a mild form of seborrheic dermatitis. The clinical manifestations are excessive fine, gray, dry or slightly greasy bran-like flakes on the scalp, which may be accompanied by itching, and the incidence rate is high in the population. The main pathogenesis of seborrheic dermatitis is that the lipase and phospholipase released by excessive Malassezia and the free unsaturated fatty acids produced by triglyceride hydrolysis cause significant damage to the epidermal barrier, causing abnormal proliferation of keratinocytes, among which restrictive Malassezia is the main dominant species. Malassezia mainly grows on the surface of the scalp and the sebaceous gland area. Therefore, anchoring the microstructure of hair follicles to carry out anti-Malassezia research is the key to the treatment of seborrheic dermatitis. At present, antifungal drugs mainly reduce the number of Malassezia to effectively treat seborrheic dermatitis and dandruff, but long-term use will have certain negative effects on human health. Therefore, it is extremely important to develop safe and effective shampoo and care products that can penetrate into the hair follicles to prevent and treat Malassezia, which can provide patients with effective and convenient treatment methods.

[0003] Compared with conventional therapies, nanomaterials are able to better penetrate into hair follicles and skin layers through their special physical properties and surface characteristics. Studies have shown that particles with medium particle sizes (around 640nm) have the strongest ability to enter hair follicles, and by changing the particle size, different parts of the hair follicle can be selectively targeted. Therefore, nanotechnology is expected to provide a new strategy for the development of anti-Malassezia products that anchor the hair follicle structure. This targeted antibacterial effect can effectively remove the difficult-to-remove Malassezia in the sebaceous glands.

[0004] Theabrownin is a polymer with benzene ring as the main body, complexed with polysaccharides and proteins, and rich in carboxyl, hydroxyl, methyl and other groups. Although they are oxidation products of tea polyphenols, they still maintain relatively high antioxidant activity. Studies have shown that theabrownin has multiple biological activities such as antioxidant, anti-obesity and regulation of intestinal microorganisms. Theabrownin can destroy the cell membrane of Staphylococcus aureus and Escherichia coli and increase the level of intracellular reactive oxygen, affecting the normal metabolic activity of cells through oxidative stress, thereby causing abnormal bacterial death. However, the single theabrownin component is not effective against Malassezia, and a composite system needs to be constructed.

[0005] Studies have shown that selenium nanoparticles have multiple functions such as bactericidal, immune regulation, anti-inflammatory, and antiviral. Some studies further speculate that the possible bactericidal mechanism is that excessive nano-selenium indiscriminately replaces sulfur, causing the thiol required for microbial growth to be converted into selenol, and oxidative stress leads to cell death. However, the exact anti-Malassezia mechanism of nano-selenium still needs further study. Summary of the invention

[0006] The problem to be solved by the present invention is to provide a theabrownin-selenium nanoparticle@Malassezia membrane composite system with anti-Malassezia function and its application.

[0007] In order to solve the above problems, the present invention provides a theabrownin-selenium nanoparticles@Malassezia membrane composite system, comprising the following steps:

[0008] (1) Dissolve 25 mg of theabrownin in 24 ± 2 mL of deionized water and stir evenly;

[0009] (2) Add 90-110 mM (preferably 100 mM) sodium selenite solution 9-11 ml (preferably 10 ml) to the theabrownin solution obtained in step (1), mix well and let stand (standing time is 10-20 min); then add 450-550 mM (preferably 500 mM) vitamin C solution 15-17 ml (preferably 16 ml) and stir for 0.8-1.2 hours (preferably 1 hour);

[0010] (3) Gradient centrifugation:

[0011] The mixed solution obtained in step (2) is first centrifuged at 4000±500 rpm for 15±3 min, and then the supernatant obtained from the first centrifugation is centrifuged for a second time at 6000±500 rpm for 15±3 min; the precipitate obtained from the second centrifugation is washed with deionized water (washed 2 to 4 times, and centrifuged after each washing), and then deionized water is added to make the volume 10 mL to obtain a suspension of theabrownin-selenium nanoparticles (TB-Se);

[0012] Note: The present invention adopts differential centrifugation, and the first centrifugation is to remove larger particles of TB-Se;

[0013] (4) Wash 50±5 ml of Malassezia culture with PBS buffer, then crush it, and then centrifuge (6000±500g for 5±1min) to collect the supernatant; after the supernatant passes through the membrane, take the permeate and centrifuge (12000±1000g for 15±3min) to collect the precipitate, and wash it with sterile water (wash three times) to obtain the Malassezia membrane;

[0014] (5) 5 mL of the suspension of theabrownin-selenium nanoparticles (TB-Se) obtained in step (3) and the Malassezia membrane obtained in step (4) were ultrasonically mixed in an ice bath; then centrifuged (6000±500 rpm for 5±1 min) to remove the supernatant (Malassezia membrane that is not completely coated with TB-Se), and deionized water was added to make the volume 5 mL to obtain a theabrownin-selenium nanoparticle / Malassezia membrane composite system (containing theabrownin selenium nanoparticles coated with Malassezia membrane, TB-Se@M).

[0015] As an improvement of the preparation method of the theabrownin-selenium nanoparticles@Malassezia membrane composite system of the present invention: the bacterial content of the Malassezia liquid is 10 10 Pieces / ml.

[0016] As a further improvement to the preparation method of the theabrownin-selenium nanoparticles@Malassezia membrane composite system of the present invention: the membrane in step (4) is a 0.45 μm water filter membrane.

[0017] As a further improvement of the preparation method of the theabrownin-selenium nanoparticles@Malassezia membrane composite system of the present invention: in the step 3): the precipitate obtained by the secondary centrifugation is washed with deionized water under ultrasonic conditions (40KHz) for 2 to 4 times (preferably 3 times, each washing time is 3±1min, and the precipitate is blown and evenly dispersed during the washing process, and centrifuged after each washing), and after the last washing (and centrifugation), deionized water is added to make the volume 10mL to obtain a suspension of theabrownin-selenium nanoparticles (TB-Se).

[0018] As a further improvement of the preparation method of the theabrownin-selenium nanoparticles@Malassezia membrane composite system of the present invention: in the step 4): glass beads with a diameter of 425 to 600 μm (about 0.5 g) are added for crushing.

[0019] The present invention also provides a theabrownin-selenium nanoparticle@Malassezia membrane composite system for resisting Malassezia prepared by any of the above methods.

[0020] The present invention also provides the use of the theabrownin-selenium nanoparticle@Malassezia membrane composite system prepared by any of the above methods: for preparing drugs or reagents against Malassezia.

[0021] As an improvement of the use of the present invention: it is used for preparing a medicine or reagent for treating seborrheic dermatitis.

[0022] As a further improvement of the use of the present invention: the anti-Malassezia agent can be used in cosmetics.

[0023] That is, the drugs or agents for treating seborrheic dermatitis include pharmaceutical preparations, cosmetics, care products, and beauty products.

[0024] As a further improvement of the use of the present invention: the cosmetic is any one of the following: shampoo, conditioner, hair essence, hair oil, scalp and hair nourishing product, hair gel, hair mask, eyelash / eyebrow care solution.

[0025] The theabrownin-selenium nanoparticle@Malassezia membrane composite system with anti-Malassezia function of the present invention can be prepared into a 100 mg / mL aqueous solution and applied to the scalp 1 to 3 times a day when actually used for scalp washing and care.

[0026] The present invention first considers the preparation of theabrownin-selenium nanoparticles, then separates and extracts Malassezia membranes, constructs a theabrownin-selenium nanoparticle@Malassezia membrane composite system, and utilizes the synergistic effect of theabrownin and nano-selenium in killing Malassezia in the skin and repairing the skin barrier to improve seborrheic dermatitis, providing a new method for solving the anti-Malassezia problem and providing a theoretical basis for related research on the use of nanoparticles to regulate scalp microecology.

[0027] The beneficial effects of the present invention are: successfully preparing a composite nanomaterial based on theabrownin-selenium nanoparticles, expanding the application field of theabrownin-selenium nanoparticles. The nanoscale composite system prepared by the present invention can effectively penetrate into the hair follicles, kill Malassezia, and improve seborrheic dermatitis. It can be developed into a natural cleaning agent for removing dandruff and improving seborrheic dermatitis, replacing artificially synthesized cleaning and anti-Malassezia products; the present invention is scientifically and reasonably designed and has good practical application value.

[0028] In summary, the present invention prepares theabrownin-selenium nanoparticles, combines them with Malassezia membranes, and obtains theabrownin-selenium nanoparticles@Malassezia membrane composite system after centrifugal washing. The composite system has significant effects on killing Malassezia and repairing skin barriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0030] Figure 1 The diagram is a preparation process diagram of theabrownin-selenium nanoparticles and theabrownin-selenium nanoparticles@Malassezia membrane composite system;

[0031] Figure 2 The SEM (upper row) and TEM (lower row) images of theabrownin-selenium nanoparticles and theabrownin-selenium nanoparticles@Malassezia membrane composite system;

[0032] Figure 3 In the figure, a is a graph of nanoparticle size analysis experiment; b is a graph of Zeta potential analysis experiment; c is a graph of UV-visible spectrum experiment; d is a graph of infrared spectrum experiment;

[0033] Figure 4The in vitro bactericidal effects of different nanosystem treatment groups on Malassezia;

[0034] Figure 5 The diagram shows the ear dandruff of mice in different drug-administered groups;

[0035] Figure 6 These are H&E stained sections of ear tissues of mice in different drug-administered groups on day 8;

[0036] Figure 7 is the thickness of the stratum corneum of the ear tissue of mice in different drug administration groups

[0037] Figure 8 The SEM images of TB-Se synthesized using sodium selenite and zinc selenite as raw materials in Comparative Example 1 are shown;

[0038] Fig. 9 The anti-Malassezia effect of TB-Se synthesized from sodium selenite and zinc selenite respectively;

[0039] Fig.10 The anti-Malassezia effect of TB-Se synthesized with different stirring times;

[0040] Fig.11 The SEM images of TB-Se obtained by centrifugation in different methods in Comparative Example 3;

[0041] Fig.12 The anti-Malassezia effect of TB-Se obtained by different centrifugation methods. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0043] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0044] Theabrownin can be purchased from, for example, Zhejiang Zhonglu Songbo Bioengineering Co., Ltd. (the content of the active ingredient theabrownin> 99%).

[0045] The Malassezia was a lyophilized powder of Malassezia restricta No. 185492 of BNCC, and then a conventional activation culture was performed according to the culture conditions informed by the BNCC: 30°C, aerobic, YM medium, 24-48h. YM medium: 3.0g yeast extract, 3.0g malt extract, 10.0g glucose, 5.0g peptone, 20.0g agar (not included in the liquid culture medium), 1.0L distilled water, pH 6.2±0.2. Sterilization at 121°C for 15min.

[0046] The bacterial count was 10 10 / ml of Malassezia culture solution.

[0047] Example 1: A method for preparing a theabrownin-selenium nanoparticle / Malassezia membrane composite system, comprising the following steps:

[0048] (1) Weigh 25 mg of theabrownin into a 50 mL centrifuge tube, add 24 mL of deionized water, and stir evenly;

[0049] (2) Add 10 mL of 100 mM sodium selenite solution to the theabrownin solution obtained in step (1), mix well and let stand for 15 min; then add 16 mL of 500 mM vitamin C solution and stir at 25° C. for 1 h to mix well;

[0050] (3) Gradient centrifugation:

[0051] The mixed solution obtained in step (2) was first centrifuged at 4000 rpm for 15 min, and then the supernatant obtained by the first centrifugation was centrifuged for a second time at 6000 rpm for 15 min; the precipitate obtained by the second centrifugation was washed with deionized water under 40 kHz ultrasonic conditions, and the precipitate was blown at the same time during the washing process to make it evenly dispersed, and the water was removed by centrifugation after each washing; the washing with deionized water was repeated 3 times, and the washing time for each washing was 3 min; after the last washing and centrifugation, deionized water was added to make the volume 10 mL to obtain a suspension of theabrownin-selenium nanoparticles (TB-Se), which was stored at 4°C for subsequent experiments.

[0052] Note: The purpose of the first centrifugation is to remove larger particles (for disposal). The particle size of the theabrownin-selenium nanoparticles obtained in this step is about 60 to 80 nanometers.

[0053] (4) Add 50 ml of Malassezia culture solution (containing 10 10 / ml) in a 2mL round-bottom centrifuge tube, washed three times with PBS buffer (0.01M, pH 7.2-7.4), and then added with 0.5g grinding beads (diameter 425-600μm) for crushing (crushing at a frequency of 60Hz for 3 minutes), centrifuged at 6000g for 5min, collected the supernatant, and discarded the precipitate (this precipitate is the cell wall and organelles of Malassezia). After the supernatant was filtered through a membrane (through a water filter membrane with a diameter of 13mm×0.45μm), the permeate was centrifuged at 12000g for 15min, the precipitate (mainly the cell membrane of Malassezia) was collected, and washed three times with sterile water to obtain the Malassezia membrane (M).

[0054] (5) 5 mL of the suspension of theabrownin-selenium nanoparticles (TB-Se) obtained in step (3) and the Malassezia membrane obtained in step (4) were ultrasonically mixed in an ice bath (so that the Malassezia membrane was loaded onto theabrownin-selenium nanoparticles). Subsequently, the mixture was centrifuged at 6000 rpm for 5 min to remove the supernatant (the Malassezia membrane that was not completely coated with TB-Se), and deionized water was added to make the volume 5 mL to obtain a theabrownin-selenium nanoparticle / Malassezia membrane composite system (i.e., theabrownin-selenium nanoparticles coated with Malassezia membrane, TB-Se@M).

[0055] Description: The present invention also prepares selenium nanoparticles according to the following method: 10 mL of 100 mM sodium selenite solution and 16 mL of 500 mM vitamin C solution are stirred at 25°C for 1 hour to mix. The obtained mixed solution is first centrifuged at 4000 rpm for 15 min, and then the supernatant obtained by the first centrifugation is centrifuged for a second time at 6000 rpm for 15 min. The precipitate obtained by the second centrifugation is washed with deionized water (repeat the washing with deionized water 3 times, each washing time is 3 min, and centrifugation is performed after each washing); after the last washing and centrifugation, water is added to make the volume to 10 mL to obtain a suspension of selenium nanoparticles (Se), which is stored at 4°C for subsequent experiments.

[0056] Experiment 1: Characterization of theabrownin-selenium nanoparticles / Malassezia membrane composite system

[0057] The theabrownin-selenium nanoparticles / Malassezia membrane composite system prepared in Example 1 was characterized by scanning electron microscopy, transmission electron microscopy, ultraviolet absorption spectroscopy, Zeta potential analysis, nanoparticle size analysis, infrared spectroscopy and the like.

[0058] Scanning electron microscopy and transmission electron microscopy were used to characterize the morphology of the nanoparticles. The theabrownin-selenium nanoparticles prepared in Example 1 (the suspension of theabrownin-selenium nanoparticles (TB-Se) obtained in step (3)) and the theabrownin-selenium nanoparticles / Malassezia membrane composite system (TB-Se@M obtained in step (5)) were dropped onto a silicon wafer, and after being naturally air-dried, they were sprayed with gold, and the size and morphology were observed using a scanning electron microscope; the theabrownin-selenium nanoparticles and theabrownin-selenium nanoparticles / Malassezia membrane composite system prepared in Example 1 were dropped onto a copper mesh, and after being naturally air-dried, the size and morphology were observed under a transmission electron microscope. The results showed that the particle size of the theabrownin-selenium nanoparticles was 60 to 80 nanometers, and the particle size of the theabrownin-selenium nanoparticles / Malassezia membrane composite system was 100 to 120 nanometers.

[0059] Ultraviolet absorption spectroscopy is used to characterize the optical properties of nanoparticles. Using a UV-visible spectrophotometer, ultraviolet absorption spectra were measured (200-800 nm) through a quartz microplate. The theabrownin-selenium nanoparticles and the theabrownin-selenium nanoparticles / Malassezia membrane composite system prepared in Example 1 were measured three times.

[0060] Nanoparticle size analysis and Zeta potential analysis were used to characterize the particle size distribution and Zeta potential of nanoparticles. The particle size and Zeta potential of theabrownin-selenium nanoparticles and theabrownin-selenium nanoparticles / Malassezia membrane composite system were measured by a nanoparticle size analyzer. The particles were evenly dispersed by ultrasonic treatment for 3 minutes, and the measurements were performed three times.

[0061] Fourier transform infrared spectroscopy was used to explore the surface chemical groups of different samples. The Malassezia membrane (M), theabrownin (TB), theabrownin-selenium nanoparticle (TB-Se) suspension and theabrownin-selenium nanoparticle / Malassezia membrane composite system (TB-Se@M) solution were freeze-dried and their Fourier transform infrared spectra were measured respectively. The wavelength range was set to 400-4000cm -1 .

[0062] according to Figure 2 and Figure 3 (ad) It can be seen that the addition of Malassezia film changed the ultraviolet spectra, particle size and potential value of theabrownin-selenium nanoparticles, proving the successful preparation of theabrownin-selenium nanoparticles / Malassezia film composite system.

[0063] Experiment 2: Bactericidal effect of theabrownin-selenium nanoparticles / Malassezia membrane composite system on Malassezia

[0064] The plate counting method was used to determine the antibacterial activity of theabrownin group, selenium group, theabrownin-selenium nanoparticle group, and theabrownin-selenium nanoparticle / Malassezia film group (the selenium concentration remained consistent).

[0065] Each group took 1 mL of Malassezia bacterial solution (1×10 5 CFU / mL) was centrifuged at 5500rpm / min for 10min to retain the precipitate, which was washed twice with sterile water. 1.5mL of theabrownin (250mg / L), 1.5mL of the selenium suspension obtained above, 1.5mL of theabrownin-selenium nanoparticle suspension obtained in step (3) of Example 1, and the theabrownin-selenium nanoparticle / Malassezia membrane composite system obtained in step (5) of Example 1 were added as corresponding experimental groups, and a control group (1.5mL of sterile water) was set up at the same time. All treatment groups were placed in a 30°C shaker and sampled after 24 hours. Sterile water was used for gradient dilution according to the ten-fold dilution method, and the number of colonies was recorded after the plate was coated and incubated at 30°C for 24 hours. All experimental groups were counted in three parallel replicates.

[0066] according to Figure 4 It can be seen that compared with the control group (Control), single theabrownin treatment (TB) and selenium treatment (Se) have poor anti-Malassezia effects, while theabrownin-selenium nanoparticles (TB-Se) and theabrownin-selenium nanoparticles / Malassezia film composite system (TB-Se@M) can effectively kill Malassezia, and theabrownin-selenium nanoparticles / Malassezia film composite system (TB-Se@M) has the best anti-Malassezia effect, which confirms the synergistic anti-Malassezia effect of theabrownin and selenium.

[0067] Experiment 3: Effects of theabrownin-selenium nanoparticles / Malassezia membrane composite system on seborrheic dermatitis inflammation

[0068] Eight-week-old male C57BL / 6 mice were used for the experiment. After routine adaptive culture (7 days), the mice were divided into a blank control group, a model group, a theabrownin group (treated with 250 mg / L theabrownin), a selenium group (treated with pure nano-selenium obtained above), a theabrownin-selenium nanoparticle group (treated with the theabrownin-selenium nanoparticles obtained in Example 1), and a theabrownin-selenium nanoparticle / Malassezia membrane group (treated with the theabrownin-selenium nanoparticle / Malassezia membrane composite system obtained in Example 1), with 10 mice in each group. Before the experiment, Malassezia was resuspended in 100 μL of olive oil (10 9 CFU / mL) was used to prepare bacterial suspension, and the ears of mice in each group were taped with medical tape (the degree of treatment was consistent, 5 places per mouse) to destroy the skin barrier. Except for the control group, each mouse in each group was smeared with bacteria once a day, with a dose of 100 μL, and the model was established for 7 consecutive days. Treatment was carried out 2 hours after each modeling, and the ears of mice were fully smeared with cotton wool soaked with theabrownin, selenium, and theabrownin-selenium nanoparticles for 2 minutes; the control group and the model group were smeared with distilled water in the same way, and the treatment was terminated on the 8th day. After the experiment, all mice were euthanized and ear tissues were collected. The tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sliced ​​and stained with hematoxylin and eosin (H&E). The pathological changes of the tissues were observed under a microscope. During the experiment, photos were taken every other day to record the dandruff of the mouse ears. During the experiment, the mice were free to eat and drink.

[0069] according to Figure 5 It can be seen that the model group produced large pieces of dandruff from the fifth to the seventh day. Compared with the model group, the theabrownin group, selenium group, theabrownin-selenium nanoparticle group, and theabrownin-selenium nanoparticle / Malassezia film group only had a small amount of dandruff. This shows that compared with the model group, each material treatment group can improve mouse seborrheic dermatitis and reduce dandruff production, but the theabrownin-selenium nanoparticle / Malassezia film group has the best effect.

[0070] Experiment 4: Effects of theabrownin-selenium nanoparticles / Malassezia membrane composite system on ear tissues of mice with seborrheic dermatitis

[0071] The blank control group (Control), model group (Model), theabrownin group (TB), selenium group (Se), theabrownin-selenium nanoparticle group (TB-Se), and theabrownin-selenium nanoparticle / Malassezia membrane group (TB-Se@M) were set up in the same way as in Experiment 3. After the experiment, all mice were euthanized and ear tissues were collected. The tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sliced, and stained with hematoxylin and eosin (H&E). The pathological changes of the tissues were observed under a microscope.

[0072] according to Figure 6 and Figure 7 It can be seen that compared with the control group, the stratum corneum of the model group was significantly thickened; after treatment with theabrownin, selenium, theabrownin-selenium nanoparticles, and theabrownin-selenium nanoparticles / Malassezia film composite system, the thickness of the stratum corneum became thinner, and theabrownin-selenium nanoparticles / Malassezia film composite system had the best improvement effect, proving the synergistic effect of theabrownin and nano-selenium.

[0073] Comparative Example 1: The sodium selenite in Example 1 was replaced with zinc selenite, and the selenium concentration and dosage remained unchanged, that is, "100 mM sodium selenite solution 10 mL" was replaced with "100 mM zinc selenite solution 10 mL", and the rest was the same as Example 1. The SEM image is compared with that of Example 1. Figure 8 As shown, according to Figure 8 It can be seen that the yield of TB-Se synthesized using sodium selenite as raw material is higher.

[0074] The product obtained in Comparative Example 1 was tested according to the method described in Experiment 2. The results are as follows: Fig. 9 As shown, the sterilization effect of this comparative example 1 is 80% (colony count 2×10 4 CFU), while the sterilization effect of the present invention is 98% (CFU 2×10 3 CFU).

[0075] Comparative Example 2: The stirring time of 1 h in step (2) of Example 1 was changed to 0.5 h and 2 h respectively, and the concentration and amount of each solution remained unchanged, and the rest was the same as Example 1. If the stirring time was too short, the nanoparticles with relatively uniform size described in Example 1 could not be obtained.

[0076] The product obtained in Comparative Example 2 was tested according to the method described in Experiment 2. The results are as follows: Fig.10 As shown in the figure, the sterilization effect corresponding to 0.5h is 86% (colony count 1.4×10 4 CFU), and the sterilization effect corresponding to 2h is 8% (colony count 9.2×10 4CFU), while the sterilization effect of the present invention is 98%.

[0077] Comparative Example 3: The gradient centrifugation in step (3) of Example 1 was changed from "first centrifugation at 4000 rpm to take the supernatant, followed by a second centrifugation at 6000 rpm to take the precipitate" to "centrifugation at 4000 rpm for 15 min to take the precipitate", or changed to "first centrifugation at 6000 rpm for 15 min to take the supernatant, followed by a second centrifugation at 12000 rpm for 15 min to take the precipitate", and the rest was the same as Example 1. The SEM image is shown in FIG. Fig.11 As shown. Fig.11 It can be seen that the TB-Se yield obtained by centrifugation at 4000 rpm is relatively low. The TB-Se obtained by the first centrifugation at 4000 rpm to obtain the supernatant and the second centrifugation at 6000 rpm to obtain the precipitate is uniform in size and has a relatively high yield. However, the TB-Se yield obtained by the second centrifugation at 12000 rpm to obtain the precipitate after the first centrifugation at 6000 rpm to obtain the supernatant is too low and is not recommended.

[0078] The product obtained from comparative example 3 "4000 rpm centrifugation for 15 min to obtain the precipitate" was tested according to the method described in Experiment 2. The results are as follows Fig.12 As shown in the figure, the sterilization effect of "4000 rpm centrifugation to obtain sediment" is 14% (colony count 8.6×10 4 CFU), while the sterilization effect of the present invention is 98%.

[0079] Comparative Example 4: The dosage of the Malassezia liquid in Example 1 was changed to 25 ml and 12.5 ml respectively, and the rest was the same as Example 1. Only Example 1 could obtain TB-Se@M with relatively uniform size, while the two products obtained in Comparative Example 4 could not achieve uniform size, and the subsequent sterilization effect was poor.

[0080] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for preparing a theabrownin-selenium nanoparticle@Malassezia membrane composite system, Features The following steps are involved: (1) Dissolve 25 mg of theabrownin in 24 ± 2 mL of deionized water and stir evenly; (2) Add 90-110 mM sodium selenite solution 9-11 ml to the theabrownin solution obtained in step (1), mix well and let stand; then add 450-550 mM vitamin C solution 15-17 ml and stir for 0.8-1.2 hours; (3) Gradient centrifugation: The mixed solution obtained in step (2) is first centrifuged at 4000±500 rpm for 15±3 min, and then the supernatant obtained from the first centrifugation is centrifuged for a second time at 6000±500 rpm for 15±3 min; the precipitate obtained from the second centrifugation is washed with deionized water, and then deionized water is added to make the volume to 10 mL to obtain a suspension of theabrownin-selenium nanoparticles; (4) 50±5 ml of Malassezia culture was washed with PBS buffer and then crushed, and then the supernatant was collected by centrifugation; after the supernatant passed through the membrane, the permeate was collected by centrifugation and the precipitate was washed with sterile water to obtain the Malassezia membrane; (5) 5 mL of the suspension of theabrownin-selenium nanoparticles obtained in step (3) and the Malassezia membrane obtained in step (4) are ultrasonically mixed in an ice bath; then centrifuged to remove the supernatant, and deionized water is added to make the volume 5 mL to obtain a theabrownin-selenium nanoparticle / Malassezia membrane composite system.

2. The preparation method of the theabrownin-selenium nanoparticles@Malassezia membrane composite system according to claim 1, Features: The bacterial content of Malassezia liquid is 10 10 Pieces / ml.

3. The preparation method of the theabrownin-selenium nanoparticles@Malassezia membrane composite system according to claim 2, Features: The membrane in step (4) is a 0.45 μm water filter membrane.

4. The method for preparing the theabrownin-selenium nanoparticles@Malassezia membrane composite system according to claim 3, Features In the step 3), the precipitate obtained by the secondary centrifugation is washed 2 to 4 times with deionized water under ultrasonic conditions, and after the last washing, deionized water is added to make the volume to 10 mL to obtain a suspension of theabrownin-selenium nanoparticles.

5. The method for preparing the theabrownin-selenium nanoparticles@Malassezia membrane composite system according to any one of claims 1 to 4, Features: In the step 4), glass beads with a diameter of 425 to 600 μm are added for crushing.

6. The theabrownin-selenium nanoparticles@Malassezia membrane composite system prepared by any one of claims 1 to 5.

7. Use of the theabrownin-selenium nanoparticles@Malassezia membrane composite system as claimed in claim 6, Features: Used for preparing drugs or reagents against Malassezia.

8. The use according to claim 7, Features: Used for preparing medicines or reagents for treating seborrheic dermatitis.

9. The use according to claim 7 or 8, Features: The anti-Malassezia agent can be used in cosmetics.

10. The use according to claim 9, Features: Cosmetics are any of the following: shampoo, conditioner, hair serum, hair oil, scalp and hair nourishing products, hair gel, hair mask, eyelash / eyebrow care solution.

Images