Composition for realizing anti-inflammatory effect by regulating specific skin microorganisms

Abstract

The invention discloses a composition for realizing an anti-inflammatory effect by regulating and controlling specific skin microorganisms. The composition comprises one or more of lactate, saccharides and a nitrogen source, the lactate is selected from one or more of potassium lactate, sodium lactate, zinc lactate, ferrous lactate and calcium lactate; the saccharides are selected from one or more of mannose, lactose, inulin and glycosylglycerol; the nitrogen source is selected from one or more of hydrolyzed rice protein and acetylchitosamine. After the composition of the lactate, the saccharides and the nitrogen source regulates specific skin microorganisms, the composition has an excellent anti-inflammatory effect, especially the composition of the zinc lactate, the saccharides and the nitrogen source. Meanwhile, the anti-inflammatory skin care product has the beneficial effects of skin micro-ecological friendliness, long-acting anti-inflammatory function and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to the field of skin anti-inflammatory technology, specifically to a composition that achieves anti-inflammatory effects by regulating specific skin microorganisms. Background Technology

[0002] Inflammatory factors are various cytokines involved in the inflammatory response, including TNF-α, IL-1β, IL-6, and TGF-β. They play different roles in the inflammatory response, such as inducing acute phase responses and regulating the activation of immune cells. IL-1β is a common cellular inflammatory factor in organisms; almost all nucleated cells can synthesize IL-1β, which mainly originates from activated monocytes / macrophages and lymphocytes. Under normal circumstances, the skin and sweat also contain a certain amount of IL-1β.

[0003] Currently, researchers are adding anti-inflammatory ingredients to skincare products to inhibit IL-1β expression. For example, topical ceramides can inhibit the expression of inflammatory factors such as IL-1β, TSLP, and IL-4, thus suppressing inflammation. Additionally, some natural plant extracts and chemically synthesized compounds also have anti-inflammatory effects, inhibiting the expression of inflammatory factors and alleviating inflammatory responses. However, these methods require sophisticated product formulation processes, and the effective anti-inflammatory ingredients are diluted due to penetration or sweat excretion, preventing them from providing long-lasting and sustained effects.

[0004] Human skin contains a large number and various types of symbiotic microorganisms. These microorganisms play roles such as maintaining the skin's microecological balance, repelling pathogens and protecting the skin from infection, maintaining the skin barrier function, and improving skin physiological and immune functions. Developing new compositions that achieve anti-inflammatory effects by regulating specific skin microorganisms, and further enhancing the anti-inflammatory efficacy and specificity of these compositions, would have significant economic and social value. Summary of the Invention

[0005] Given that existing technologies still have room for expansion and improvement, this invention uses lactate, sugars, and a nitrogen source as a fermentation composition, which can effectively inhibit inflammatory factors in the skin's microbial environment. The specific technical solution adopted by this invention is as follows:

[0006] In a first aspect, the present invention provides a composition that achieves an anti-inflammatory effect by regulating specific skin microorganisms, comprising one or more of lactate, sugars, and nitrogen sources; wherein the lactate is selected from one or more of soluble monovalent, divalent, and trivalent lactate salts.

[0007] In some specific embodiments, the lactate is selected from one or more of potassium lactate, sodium lactate, zinc lactate, ferrous lactate, and calcium lactate.

[0008] In some specific embodiments, the sugar is selected from one or more of mannose, lactose, inulin, and glycerol glucoside.

[0009] In some specific implementations, the nitrogen source is selected from one or more of hydrolyzed rice protein and acetylglucosamine.

[0010] Combinations of zinc lactate, sugars, and nitrogen sources exhibit excellent anti-inflammatory effects after regulating specific skin microbiota.

[0011] Compositions that achieve anti-inflammatory effects by regulating specific skin microorganisms are used as components of fermentation culture media for microbial fermentation, thereby producing substances with inhibitory inflammatory factors through the microorganisms.

[0012] Furthermore, the inflammatory factor that was suppressed was IL-1β.

[0013] In some specific implementations, the skin microorganism is selected from Staphylococcus epidermidis.

[0014] Furthermore, the Staphylococcus epidermidis is one or more of Staphylococcus epidermidis ATCC 12228, Staphylococcus epidermidis ATCC 14990, Staphylococcus epidermidis ATCC 35984, Staphylococcus epidermidis ATCC 700926, and Staphylococcus epidermidis ATCC 51625.

[0015] In some specific embodiments, the composition is used as a component of a fermentation medium to produce a substance that inhibits inflammatory factors by microorganisms; the concentration of the lactate in the fermentation medium is 0.05% to 0.50% by mass fraction; the concentration of the sugar in the fermentation medium is 0.05% to 0.50%; and the concentration of the nitrogen source in the fermentation medium is 0.05% to 0.50%.

[0016] Preferably, the concentration of lactate in the fermentation medium is 0.10% to 0.20% by mass fraction; the concentration of sugar in the fermentation medium is 0.10% to 0.20%; and the concentration of nitrogen source in the fermentation medium is 0.10% to 0.20%.

[0017] A second aspect of the present invention provides a method for evaluating the anti-inflammatory effect of a composition that achieves an anti-inflammatory effect by regulating specific skin microorganisms, comprising the following steps:

[0018] S1. The target strain and the composition are fermented and co-cultured to obtain a fermentation co-culture broth;

[0019] S2. Macrophages are stimulated with LPS to trigger the expression of inflammatory factors in the macrophages. The fermentation co-culture medium is then added to detect the decrease in the expression of inflammatory factors to obtain the inflammatory factor inhibition effect of the fermentation co-culture medium, thereby achieving the purpose of evaluating the skin anti-inflammatory effect of the composition. The evaluation criterion is that the better the inflammatory factor inhibition effect, the better the skin anti-inflammatory effect of the raw material to be screened.

[0020] The target strain is a microorganism derived from the skin or other human tissues. Different compositional components and contents have a regulatory effect on the skin microbiome.

[0021] A third aspect of the invention is the use of the above-described composition in the preparation of anti-inflammatory skin preparations.

[0022] Anti-inflammatory skin preparations can be in any form suitable for adhering to and dispersing on the skin surface, such as lotions, ointments, liniments, gels, creams, etc.

[0023] In a fourth aspect of the invention, a method is provided to achieve an anti-inflammatory effect by regulating specific skin microorganisms, characterized in that a fermentation culture medium is provided to the microorganisms on the skin to achieve the anti-inflammatory effect; said fermentation culture medium comprises a composition as described in any one of the first aspects of the invention.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. Combinations of lactate, sugar, and nitrogen source have excellent anti-inflammatory effects after regulating specific skin microorganisms, especially combinations of zinc lactate, sugar, and nitrogen source.

[0026] 2. Skin-microbiome-friendly: Utilizing selected skin strains, the composition is provided as a fermentation raw material, and the anti-inflammatory effect is exerted through fermentation, with the process exhibiting good biological properties;

[0027] 3. Long-lasting anti-inflammatory effect: Compared with the method of directly adding anti-inflammatory agents, the method of using the raw materials themselves to ferment selected strains to continuously exert anti-inflammatory effects can provide a relatively stable and continuous low-dose stimulation, maintain the skin's biological function for a long time, and effectively avoid the irritation and odor problems caused by excessive concentration due to direct addition.

[0028] The following will further explain the concept and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features and effects of the present invention. Attached Figure Description

[0029] Figure 1 The bar chart shows the IL-1β content results for samples (ES10-1, ES10-9, ES10-11, ES10-14).

[0030] Figure 2 The bar chart shows the IL-1β content results for samples (ES10-18, ES10-26, ES10-28, ES10-32, ES07-21). Detailed Implementation

[0031] To make the technical means, inventive features, objectives, and effects of the invention readily understandable, the invention is further illustrated below with reference to specific figures. However, the invention is not limited to the embodiments described below.

[0032] It should be noted that the accompanying drawings are only used to complement the content disclosed in this specification, so as to enable those skilled in the art to understand and read them, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any adjustments made without creative effort, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0033] All the reagents, antibodies, and other materials used in the examples are commercially available products.

[0034] Macrophages were purchased from Guangdong Boxi Biotechnology Co., Ltd., batch number 210622-1; DMEM culture medium was purchased from Thermo Fisher Scientific; lipopolysaccharide (LPS), dexamethasone, and MTT were purchased from Sigma-Aldrich; PBS was purchased from Solarbio; and IL-6 ELISA kit and IL-1β ELISA kit were purchased from Abcam.

[0035] Example 1

[0036] 1. Prepare the fermentation culture medium

[0037] Prepare 50ml centrifuge tubes, adding the corresponding amount of substrate sample (as shown in Table 1, excluding the bacterial strain) to each tube. Add the corresponding amount of phosphate-buffered saline (PBS) and mix the sample and buffer thoroughly with a mixer. For samples that can withstand high-temperature sterilization, wrap them tightly in aluminum foil and place them in an autoclave at 121°C for 30–60 minutes. After sterilization, temporarily store the samples at 4°C. For samples that cannot withstand high-temperature sterilization, use a 0.22µm filter membrane for filtration. After filtration sterilization, also temporarily store the samples at 4°C. It is important to ensure aseptic technique during sample handling to avoid sample contamination. During high-temperature sterilization, ensure the packaging is tight to prevent sample evaporation or loss due to high temperatures.

[0038]

[0039] Systems containing sodium lactate can maintain the same liquid environment as systems containing zinc lactate.

[0040] 2. Fermentation

[0041] Staphylococcus epidermidis ATCC 12228 underwent necessary activation procedures to activate the strain's growth. Subsequently, enrichment culture was performed to provide the strain with sufficient nutrients within a short period, promoting its reproduction.

[0042] Activated Staphylococcus epidermidis ATCC 12228 was inoculated onto tryptic soy broth (TSB) agar plates and incubated overnight at 37°C to allow the strain to form visible single colonies on the agar plates. These single colonies were then inoculated into TSB medium and incubated at 37°C for approximately 24 hours to allow the strain to proliferate rapidly in the liquid medium.

[0043] After liquid culture, the bacterial culture was centrifuged at 4000 rpm for 10 minutes (at 20°C) to precipitate the bacteria, forming bacterial microspheres. The supernatant was discarded, and the bacterial microspheres were resuspended in PBS buffer.

[0044] Add 1 ml of Staphylococcus epidermidis ATCC 12228 (concentration approximately 10) to each 15 ml tube of fermentation system. 8 (CFU / mL). After thorough mixing, the system was transferred to a 37°C incubator for 24 hours under aerobic conditions, while maintaining mixing with a shaking speed of 220 rpm. A PBS system containing bacteria but without fermentation medium served as a control.

[0045] 3. Sampling

[0046] From a 50ml centrifuge tube after fermentation, accurately aspirate 8-10ml of sample using a disposable syringe. Filter the sample through a 0.22µm filter and transfer the filtered sample to a 15ml centrifuge tube. Immediately freeze the sample to -80°C for subsequent evaluation of its inhibitory effect on UVB-induced expression of inflammatory factors in skin keratinocytes.

[0047] The remaining sample was filtered using a 0.22µm filter, and the filtered sample was transferred to 15ml centrifuge tubes. The pH of the sample was then measured using a pH meter. The filtered and pH-measured sample was then rapidly frozen to -80°C for later use.

[0048] Example 2

[0049] The cytotoxicity of the fermentation broth containing each composition in Table 1 to macrophages was evaluated, and none of them showed significant cytotoxicity.

[0050] Set up blank control (BC), negative control (NC), positive control (PC), and test samples (ES10-1, ES10-9, ES10-11, ES10-14, ES10-18, ES10-26, ES10-28, ES10-32, ES07-21).

[0051] The blank control group contained neither bacteria nor anti-inflammatory substances.

[0052] Negative control: Contains bacteria, but does not contain anti-inflammatory substances;

[0053] Positive control: Dissolve 2 μL of 10% stock solution in 2 mL of culture medium to prepare 0.01% dexamethasone.

[0054] The test groups are set up as shown in Table 2:

[0055]

[0056] The specific experimental procedures for inhibiting the expression of inflammatory factors are as follows:

[0057] 1) Cell seeding: After cell resuscitation, when the cell seeding rate reaches about 60%, seed the cells into 6-well plates, add 2 mL of cell suspension to each well, and place the seeded cell culture plate into a CO2 incubator (37℃, 5% CO2) for 24 hours.

[0058] 2) Discard the old cell culture medium in the wells. Add 1.8 mL of culture medium to each well of the blank control group and negative control group, add 1.8 mL of culture medium containing dexamethasone to each well of the positive control group, and add 1.8 mL of culture medium containing the corresponding concentration of the test substance to each well of the sample group.

[0059] 3) After administration, place the 6-well plate in a CO2 incubator (37℃, 5% CO2) for 2 hours.

[0060] 4) LPS stimulation: 2 hours after administration, the blank control group was supplemented with 200 μL of normal culture medium, and each well of the other groups was supplemented with 200 μL of the prepared working solution containing LPS. The wells were placed in a CO2 incubator (37℃, 5% CO2) and cultured for 22 hours.

[0061] 5) ELISA detection: Collect cell culture supernatant and perform ELISA detection according to the ELISA kit instructions.

[0062] (III) Results Analysis

[0063] Based on the experimental results, the following table is obtained:

[0064]

[0065] Plotting with GraphPad Prism (see) Figure 1 , 2 Results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0066] From Table 3 and Figure 1 , 2 The results show that:

[0067] 1) Compared with the BC group, the IL-1β expression level in the NC group was significantly increased, indicating that the test stimulation conditions were effective and that LPS-stimulated macrophages could normally secrete IL-1β inflammatory factors.

[0068] 2) Compared with the NC group, the expression levels of IL-6 or IL-1β in the PC group were significantly reduced, indicating that the positive control test was effective and that the anti-inflammatory drug dexamethasone can effectively inhibit the occurrence of inflammation.

[0069] 3) Zinc lactate can inhibit IL-1β to some extent in the fermentation system; the fermentation system containing sugars and nitrogen sources (i.e., the group containing sodium lactate) has a negligible inhibitory effect on IL-1β; however, unexpectedly, the fermentation system containing zinc lactate, sugars, and nitrogen sources has an excellent inhibitory effect on IL-1β, significantly stronger than the zinc lactate fermentation system alone. The specific mechanism behind this phenomenon requires further in-depth research.

[0070] Specifically:

[0071] Comparing ES07-21, ES10-1, and ES10-18, it was found that the fermentation system of zinc lactate, mannose, and hydrolyzed rice protein has a synergistic effect in anti-inflammation.

[0072] Comparing ES07-21, ES10-9, and ES10-26, it was found that the fermentation system of zinc lactate, glyceryl glucoside, and hydrolyzed rice protein has a synergistic effect in anti-inflammation.

[0073] Comparing ES07-21, ES10-11, and ES10-28, it was found that the fermentation system of zinc lactate, acetyl glucosamine, and hydrolyzed rice protein has a synergistic effect in anti-inflammation.

[0074] Comparing ES07-21, ES10-14, and ES10-32, it was found that the fermentation system of zinc lactate, inulin, and hydrolyzed rice protein had a synergistic effect in anti-inflammation. Moreover, the ES10-32 group, in particular, achieved the same effect as the other groups when the contents of the three substances were halved, indicating that the fermentation system of zinc lactate, inulin, and hydrolyzed rice protein had the most outstanding anti-inflammatory performance.

[0075] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A composition for regulating specific microorganisms, characterized in that, It contains one or more of lactate, sugar, and nitrogen source; the lactate is selected from one or more of soluble lactate monovalent salt, divalent salt, and trivalent salt.

2. The composition according to claim 1, characterized in that, The lactate is selected from one or more of potassium lactate, sodium lactate, zinc lactate, ferrous lactate, and calcium lactate.

3. The composition according to claim 1, characterized in that, The sugars are selected from one or more of mannose, lactose, inulin, and glycerol glucoside.

4. The composition according to claim 1, characterized in that, The nitrogen source is selected from one or more of hydrolyzed rice protein and acetyl glucosamine.

5. The composition according to any one of claims 1 to 4, characterized in that, It is used as a component of the fermentation medium for microbial fermentation, so that the microorganisms produce substances that inhibit inflammatory factors.

6. The composition according to claim 5, characterized in that, The inflammatory factor that was suppressed was IL-1β.

7. The composition according to claims 1 to 4, characterized in that, The substance used as a component of the fermentation medium is a substance produced by microorganisms to inhibit inflammatory factors; the concentration of lactate in the fermentation medium is 0.05% to 0.50% by mass fraction; the concentration of sugar in the fermentation medium is 0.05% to 0.50%; and the concentration of nitrogen source in the fermentation medium is 0.05% to 0.50%.

8. The composition according to claim 7, characterized in that, The concentration of lactate in the fermentation medium is 0.10% to 0.20% by mass fraction; the concentration of sugar in the fermentation medium is 0.10% to 0.20%; and the concentration of nitrogen source in the fermentation medium is 0.10% to 0.20%.

9. A method for evaluating the anti-inflammatory effect of a composition by modulating specific microorganisms, characterized in that, Includes the following steps: S1. The target strain and the composition are fermented and co-cultured to obtain a fermentation co-culture broth; S2. Macrophages are stimulated with LPS to trigger the expression of inflammatory factors in the macrophages. The fermentation co-culture medium is then added to detect the decrease in inflammatory factor expression to obtain the inflammatory factor inhibition effect of the fermentation co-culture medium, thereby achieving the purpose of evaluating the anti-inflammatory effect of the composition. The evaluation criterion is that the better the inflammatory factor inhibition effect, the better the anti-inflammatory effect of the raw material to be screened.

10. The use of the composition according to any one of claims 1 to 8 in cosmetics and skin care products.

11. A method for achieving anti-inflammatory effects by regulating specific microorganisms, characterized in that, A fermentation medium is provided to specific microorganisms to achieve an anti-inflammatory effect; said fermentation medium comprises the composition as described in any one of claims 1 to 8.

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