Citrus fermentation liquor with the function of regulating skin inflammation and preparation method thereof

Abstract

The present application relates to a citrus fermentation liquor with the function of regulating skin inflammation. The fermentation raw material of the citrus fermentation liquor comprises: citrus homogenate, white sugar, Saccharomyces cerevisiae culture solution, Lactobacillus plantarum culture solution, and acetic acid bacteria culture solution. The present application also provides a preparation method of the citrus fermentation liquor, and application of the citrus fermentation liquor in skin inflammation regulators. The citrus fermentation liquor with the function of regulating skin inflammation according to the present application can effectively inhibit the production of skin inflammation factors, promote the repair of damaged skin tissue, and is mild and non-irritating, and can be used as a non-hormonal, external preparation for regulating skin inflammation.

Description

Technical Field

[0001] This invention belongs to the fields of fermentation engineering and biomedicine, specifically relating to a citrus fermentation broth with skin inflammation regulating function and its preparation method. Background Technology

[0002] Atopic dermatitis (AD), also known as atopic eczema, is a chronic, relapsing, inflammatory skin disease, belonging to the common dermatitis and eczema category. Atopic dermatitis is characterized by recurrent flare-ups and a prolonged course, often accompanied by intense itching, severely impacting patients' quality of life. At different ages, patients often also have other atopic diseases such as allergic rhinitis and asthma, thus it is considered a systemic disease requiring long-term management as a chronic condition. Atopic dermatitis is currently one of the most common diseases troubling parents and healthcare providers of infants and young children. Besides being considered related to allergies, atopic dermatitis (eczema) is also believed to be closely related to dry skin and poor skin barrier function. Most children with atopic dermatitis develop the condition primarily due to defects in their own skin barrier function, making them more sensitive to external stimuli such as allergens and large temperature differences than the average person.

[0003] The principle of medication for treating atopic eczema is to combine topical and oral medications in a comprehensive approach, based on removing the cause or triggering factors. Commonly used topical medications include corticosteroid ointments, calcineurin inhibitors, and antibiotics. Corticosteroid ointments have anti-inflammatory, anti-allergic, and antirheumatic effects, but long-term use may cause side effects. Calcineurin inhibitors are effective in treating facial eczema with fewer side effects. Antibiotics are used when eczema is complicated by infection to fight bacteria and reduce inflammation. Providing a safe and effective non-steroidal skin inflammation modulator for atopic dermatitis is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] In view of the above problems, the present invention is proposed to provide a citrus fermentation liquid with skin inflammation regulating function and a method for preparing the same, which overcomes or at least partially solves the above problems.

[0005] In a first aspect, embodiments of the present invention provide a citrus fermentation liquid with the function of regulating skin inflammation. The fermentation raw materials of the citrus fermentation liquid include: citrus homogenate, white sugar, brewer's yeast culture medium, plant lactobacillus culture medium, and acetic acid bacteria culture medium.

[0006] Preferably, the fermentation raw materials of the citrus fermentation broth, by weight, include the following components: 990-1010 parts citrus homogenate, 150-250 parts white sugar, 90-110 parts brewer's yeast culture, 90-110 parts plant lactobacillus culture, and 90-110 parts acetic acid bacteria culture.

[0007] Preferably, the raw materials for the brewer's yeast culture broth include brewer's yeast, yeast extract, malt extract, peptone, glucose, and distilled water; the viable count of the brewer's yeast is ≥10. 7 cfu / ml.

[0008] Preferably, the raw materials for the *Lactobacillus plantarum* culture medium include *Lactobacillus plantarum*, casein peptone, beef extract, yeast powder, glucose, sodium acetate, diammonium citrate, Tween 80, K₂HPO₄, MgSO₄·7H₂O, MnSO₄·H₂O, CaCO₃, and distilled water; the viable count of the *Lactobacillus plantarum* is ≥10⁻⁶. 7 cfu / ml.

[0009] Preferably, the raw materials for the acetic acid bacteria culture medium include acetic acid bacteria, glucose, yeast extract, calcium carbonate, distilled water, and anhydrous ethanol; the viable count of the acetic acid bacteria is ≥10. 7 cfu / ml.

[0010] Preferably, the *Lactobacillus plantarum* comprises strain 7024 selected from fermented taro products and strain Y28 selected from fermented cowpea products; the ratio of strain 7024 to strain Y28 in the *Lactobacillus plantarum* is 1 to 2:1.

[0011] Secondly, embodiments of the present invention also provide a method for preparing the citrus fermentation broth with skin inflammation-regulating function as described in the first aspect, comprising the following steps:

[0012] S1. Weigh the fermentation raw materials according to the weight proportions;

[0013] S2, Substrate Preparation

[0014] Place the citrus homogenate and white sugar in a container, mix well, and obtain the substrate;

[0015] S3, First Fermentation

[0016] The activated brewer's yeast culture was added to the substrate obtained in step S2, and fermented at 25-30°C for 12-16 days to obtain the first fermentation product.

[0017] S4, Secondary Fermentation

[0018] The first ferment obtained in step S3 is divided into two portions; one portion of the first ferment is added to an activated plant lactobacillus culture medium and fermented at 25-30℃ for 26-30 days to obtain the second ferment; the other portion of the first ferment is added to an activated acetic acid bacteria culture medium and fermented at 25-30℃ for 26-30 days to obtain the third ferment.

[0019] S5, three-stage fermentation

[0020] The second and third fermentation products prepared in step S4 are mixed in a container and fermented at 25-30°C for 40-44 days. After filtration, citrus fermentation liquid is obtained.

[0021] Preferably, the preparation method further includes the following steps:

[0022] Preparation of citrus homogenate

[0023] Select fully ripe fresh citrus fruits, wash them thoroughly, and remove the peel and seeds; put the pulp into a blender and blend it thoroughly to obtain a citrus homogenate.

[0024] Preparation of brewing yeast culture medium

[0025] Yeast extract, malt extract, peptone, glucose, and distilled water were placed in a container, sterilized, and cooled to room temperature. Then, brewer's yeast was added, stirred evenly, and kept warm to activate the yeast culture.

[0026] Preparation of plant lactobacillus culture medium

[0027] Casein peptone, beef extract, yeast powder, glucose, sodium acetate, diammonium citrate, Tween 80, K2HPO4, MgSO4·7H2O, MnSO4·H2O, CaCO3, and distilled water were placed in a container, stirred evenly, and the pH was adjusted. After sterilization, the mixture was cooled to room temperature. Then, Lactobacillus plantarum was added, stirred evenly, and incubated to activate the culture medium, thus obtaining Lactobacillus plantarum culture medium.

[0028] Preparation of acetic acid bacteria culture medium

[0029] Glucose, yeast extract, calcium carbonate, and distilled water were placed in a container, sterilized, and then anhydrous alcohol was added and cooled to room temperature. Acetic acid bacteria were then added, stirred evenly, and incubated to activate the culture medium, thus obtaining the acetic acid bacteria culture medium.

[0030] Preferably, in step S5, fermentation is stopped when the acidity of the mixed fermentation material in the container is less than 3%, the pH is less than 4, and the ethanol content is less than 0.5%.

[0031] Thirdly, embodiments of the present invention also provide the application of the citrus ferment broth with skin inflammation regulating function described in the first aspect in skin inflammation regulators.

[0032] This invention prepares citrus fermentation broth using microbial fermentation technology, with citrus homogenate as the substrate. This citrus fermentation broth, possessing skin inflammation-regulating functions, features a simple and convenient manufacturing process for skin inflammation regulators, free of chemical reagents and hormones; the preparation process is environmentally friendly and pollution-free. It effectively inhibits the production of skin inflammatory factors, promotes the repair of damaged skin tissue, and is gentle and non-irritating, making it suitable as a non-hormonal topical preparation for regulating skin inflammation. Attached Figure Description

[0033] The accompanying drawings, which form part of the specification, are used to provide a further understanding of the embodiments of this application and illustrate the implementation methods of this application, together with the textual description, to explain the principles of this application. Obviously, the drawings described below are merely some embodiments of this application, and those skilled in the art can obtain other drawings based on these drawings without any creative effort. In the drawings:

[0034] Figure 1 The following figures show the anti-inflammatory test results of the citrus fermentation broth prepared in Example 1 and Comparative Examples 2 to 5 of the present invention, and the citrus homogenate in Comparative Example 1.

[0035] Figure 2 The figures show the oxidative stress test results of the citrus fermentation broth prepared in Example 1 and Comparative Examples 2 to 5 of the present invention, and the citrus homogenate of Comparative Example 1.

[0036] Figure 3 This is a comparative image of mouse skin in an experiment using citrus fermentation broth prepared in Example 1 of the present invention to treat atopic dermatitis.

[0037] Figure 4 The image shows the results of HE staining of mouse skin in a mouse atopic dermatitis test using citrus fermentation broth prepared in Example 1 of this invention. Detailed Implementation

[0038] The following detailed description, in conjunction with specific embodiments, provides a further description of the above-mentioned citrus fermentation broth with skin inflammation regulation function and its preparation method.

[0039] The citrus homogenate described in the embodiments and comparative examples of the present invention is prepared by the following method: select fully ripe fresh citrus fruits, wash them clean, and remove the peel and seeds; put the pulp into a mixer and mix it thoroughly to obtain the citrus homogenate.

[0040] The *Lactobacillus plantarum* used in the embodiments and comparative examples of this invention are strains screened by the applicant from fermented taro and fermented cowpea products. Gene sequencing of these strains and comparison with the DNA database established by the National Center for Biotechnology Information in the United States yielded strains 7024 (GenBank: LC514034.1) and Y28 (GenBank: MT464070.1), respectively. The ratio of strain 7024 to strain Y28 in the *Lactobacillus plantarum* is 2:1. The *Saccharomyces cerevisiae* and *Acetobacterium acetiferum* used in the embodiments and comparative examples of this invention are commercially available, with the *Acetobacterium acetiferum* strain being Shanghai vinegar 1.01.

[0041] The brewer's yeast culture medium described in the embodiments and comparative examples of this invention was prepared as follows: 3.0 g yeast extract, 3.0 g malt extract, 5.0 g peptone, and 10.0 g glucose were dissolved in 1 L of distilled water, sterilized, and cooled to room temperature; brewer's yeast was added and stirred evenly; the mixture was then incubated at 28°C for 48 hours at a controlled rotation speed of 170 rpm. The viable count of the brewer's yeast was ≥10⁻⁶. 7 cfu / ml.

[0042] The *Lactobacillus plantarum* culture medium described in the embodiments and comparative examples of this invention was prepared as follows: 10.0g casein peptone, 10.0g beef extract, 5.0g yeast powder, 5.0g glucose, 5.0g sodium acetate, 2.0g diammonium citrate, 1.0g Tween 80, 2.0g K₂HPO₄, 0.2g MgSO₄·7H₂O, 0.05g MnSO₄·H₂O, and 20.0g CaCO₃ were placed in a container, and distilled water was added to bring the volume to 1.0L. The mixture was stirred evenly, and the pH was adjusted to 6.8. After sterilization, the mixture was cooled to room temperature. *Lactobacillus plantarum* was added, and the mixture was incubated at 35℃ for 48 hours at a controlled rotation speed of 200 rpm. The viable count of *Lactobacillus plantarum* was ≥10⁻⁶. 7 cfu / ml.

[0043] The acetic acid bacteria culture medium described in the embodiments and comparative examples of this invention was prepared as follows: 10.0g glucose, 10.0g yeast extract, and 15.0g calcium carbonate were placed in a container, and distilled water was added to bring the volume to 1.0L; after sterilization, 20.0ml anhydrous ethanol was added; after cooling to room temperature, acetic acid bacteria were added, and the mixture was stirred evenly. The mixture was then incubated at 30℃ for 48 hours at a controlled rotation speed of 2000rpm. The viable count of the acetic acid bacteria was ≥10. 7 cfu / ml.

[0044] Example 1

[0045] A citrus fermentation broth with skin inflammation regulating function, wherein the fermentation raw materials of the citrus fermentation broth include: 1000 parts citrus homogenate, 200 parts white sugar, 100 parts brewer's yeast culture broth, 100 parts plant lactobacillus culture broth, and 100 parts acetic acid bacteria culture broth.

[0046] The citrus fermentation broth was prepared through the following steps:

[0047] S1. Weigh the fermentation raw materials according to the weight proportions;

[0048] S2, Substrate Preparation

[0049] Place the citrus homogenate and white sugar in a container, mix well, and obtain the substrate;

[0050] S3, First Fermentation

[0051] The activated brewer's yeast culture was added to the substrate obtained in step S2 and fermented at 28°C for 14 days to obtain the first fermentation product.

[0052] S4, Secondary Fermentation

[0053] The first ferment obtained in step S3 is divided into two parts; one part of the first ferment is added to activated plant lactobacillus culture medium and fermented at 28°C for 28 days to obtain the second ferment; the other part of the first ferment is added to activated acetic acid bacteria culture medium and fermented at 28°C for 28 days to obtain the third ferment.

[0054] S5, three-stage fermentation

[0055] The second and third fermentation products prepared in step S4 are mixed in a container and fermented at 28°C for 42 days. After filtration, citrus fermentation liquid is obtained.

[0056] In practice, fermentation can be stopped when the acidity of the mixed fermentation material in the container is less than 3%, the pH is less than 4, and the ethanol content is less than 0.5%.

[0057] Example 2

[0058] A citrus fermentation broth with skin inflammation regulating function, wherein the fermentation raw materials of the citrus fermentation broth include: 990 parts citrus homogenate, 150 parts white sugar, 90 parts brewer's yeast culture broth, 90 parts plant lactobacillus culture broth, and 90 parts acetic acid bacteria culture broth.

[0059] The method for preparing citrus fermentation broth in this embodiment is the same as that described in Example 1.

[0060] Example 3

[0061] A citrus fermentation broth with skin inflammation regulating function, wherein the fermentation raw materials of the citrus fermentation broth include: 1010 parts citrus homogenate, 250 parts white sugar, 110 parts brewer's yeast culture broth, 110 parts plant lactobacillus culture broth, and 110 parts acetic acid bacteria culture broth.

[0062] The method for preparing citrus fermentation broth in this embodiment is the same as that described in Example 1.

[0063] Comparative Example 1 is a freshly prepared citrus homogenate.

[0064] Comparative Example 2

[0065] A citrus fermentation broth, by weight, comprises the following fermentation raw materials: 1000 parts citrus homogenate, 200 parts white sugar, 100 parts brewer's yeast culture broth, 110 parts plant lactobacillus culture broth, and 120 parts acetic acid bacteria culture broth.

[0066] The citrus fermentation broth was prepared through the following steps:

[0067] S1. Weigh the fermentation raw materials according to the weight proportions;

[0068] S2, Substrate Preparation

[0069] Place the citrus homogenate and white sugar in a container, mix well, and obtain the substrate;

[0070] S3, First Fermentation

[0071] The activated brewer's yeast culture was added to the substrate obtained in step S2, and fermented at 28°C for 7 days to obtain the first fermentation product;

[0072] S4, Secondary Fermentation

[0073] Add plant lactobacillus culture medium to the first fermentation product prepared in step S3, and ferment at 28°C for 14 days to obtain the second fermentation product;

[0074] S5, three-stage fermentation

[0075] Activated acetic acid bacteria culture was added to the second fermentation product prepared in step S4, and fermentation was stopped after 28 days at 28°C. After filtration, citrus fermentation broth was obtained.

[0076] Comparative Example 3

[0077] A citrus fermentation broth, by weight, comprises the following fermentation raw materials: 1000 parts citrus homogenate, 200 parts white sugar, 100 parts brewer's yeast culture broth, 110 parts plant lactobacillus culture broth, and 120 parts acetic acid bacteria culture broth.

[0078] The citrus fermentation broth was prepared through the following steps:

[0079] S1. Weigh each fermentation raw material according to the weight proportions;

[0080] S2, Substrate Preparation

[0081] Place the citrus homogenate and white sugar in a container, mix well, and obtain the substrate;

[0082] S3, First Fermentation

[0083] The activated brewer's yeast culture and plant lactobacillus culture were added to the substrate obtained in step S2, and fermented at 28°C for 14 days to obtain the first fermentation product.

[0084] S4, Secondary Fermentation

[0085] Activated acetic acid bacteria culture was added to the first fermentation product prepared in step S3, and fermentation was stopped after 28 days at 28°C. After filtration, citrus fermentation broth was obtained.

[0086] Comparative Example 4

[0087] A citrus fermentation broth, by weight, comprises the following fermentation raw materials: 1000 parts citrus homogenate, 200 parts white sugar, 100 parts brewer's yeast culture broth, 110 parts plant lactobacillus culture broth, and 120 parts acetic acid bacteria culture broth.

[0088] The citrus fermentation broth was prepared through the following steps:

[0089] S1. Weigh the fermentation raw materials according to the weight proportions;

[0090] S2, Substrate Preparation

[0091] Place the citrus homogenate and white sugar in a container, mix well, and obtain the substrate;

[0092] S3, First Fermentation

[0093] The activated brewer's yeast culture and plant lactobacillus culture were added to the substrate obtained in step S2, and fermented at 28°C for 28 days to obtain the first fermentation product.

[0094] S4, Secondary Fermentation

[0095] Activated acetic acid bacteria culture was added to the first fermentation product prepared in step S3, and fermentation was stopped after 42 days at 28°C. After filtration, citrus fermentation broth was obtained.

[0096] Comparative Example 5

[0097] A citrus fermentation broth, by weight, comprises the following fermentation raw materials: 1000 parts citrus homogenate, 200 parts white sugar, 100 parts brewer's yeast culture broth, 110 parts plant lactobacillus culture broth, and 120 parts acetic acid bacteria culture broth.

[0098] The citrus fermentation broth was prepared through the following steps:

[0099] S1. Weigh the fermentation raw materials according to the weight proportions;

[0100] S2, Substrate Preparation

[0101] Place the citrus homogenate and white sugar in a container, mix well, and obtain the substrate;

[0102] S3, Fermentation

[0103] Activated brewer's yeast culture, plant lactobacillus culture, and acetic acid bacteria culture were added to the substrate prepared in step S2. Fermentation was stopped after 42 days at 28°C, and the citrus fermentation broth was obtained after filtration.

[0104] Test Experiment

[0105] 1. Anti-inflammatory test

[0106] Example 1 serves as an experimental example. The citrus fermentation broth prepared through Example 1 and Comparative Examples 2 to 5, as well as the citrus homogenate from Comparative Example 1, were subjected to anti-inflammatory tests. The test methods are as follows:

[0107] Construction of a cellular inflammation model: RAW246.7 cells in logarithmic growth phase were cultured at a density of 1 × 10⁶ cells per well. 4 The culture medium was inoculated at a density in 96-well plates and incubated for 24 hours at 37°C with 5% CO2. An equal volume of culture medium was added to each treatment group; the positive control group and treatment group also received an equal volume of 1 μg / mL LPS (lipopolysaccharide); the treatment group also received an equal volume of the drug.

[0108] Negative control group: only RAW246.7 cell-specific culture medium was added;

[0109] Positive control group: only RAW246.7 cell-specific culture medium and LPS were added;

[0110] Treatment group: RAW246.7 cell-specific culture medium, LPS, and drugs were added.

[0111] The drug is the citrus fermentation broth prepared in Example 1, the citrus fermentation broth prepared in Comparative Examples 2 to 5, and the citrus homogenate prepared in Comparative Example 1, all with a concentration of 2 μg / ul.

[0112] Cells in the negative control group were pretreated for 1 hour; cells in the positive control and treatment groups were treated for 24 hours. After the experiment, cell supernatant was collected. OD values ​​were measured and converted to concentrations according to the instructions of the nitric oxide assay kit and the IL-1β, TNF-α, and IL-6 ELISA kits. The effects of different treatments on the inflammatory factors IL-1β, TNF-α, IL-6, and NO in LPS-induced Raw264.7 cells for 24 hours were measured, and the results are as follows: Figure 1 As shown.

[0113] 2. Oxidative stress test

[0114] Example 1 serves as an experimental example. The citrus fermentation broth prepared through Example 1 and Comparative Examples 2 to 5, as well as the citrus homogenate from Comparative Example 1, were subjected to oxidative stress tests. The test methods are as follows:

[0115] Establishment of a cellular oxidative stress model: GES-1 cells in logarithmic growth phase were seeded into 96-well plates. After the cells reached 70% confluence, they were divided into groups for the experiment. Each treatment group received an equal volume of culture medium; the positive control group and treatment group also received an equal volume of 5% ethanol; the treatment group also received an equal volume of the drug.

[0116] Negative control group: only complete culture medium was added;

[0117] Positive control group: only complete culture medium and 5% ethanol were added;

[0118] Treatment group: Add complete culture medium, 5% ethanol, and drugs.

[0119] The drug is the citrus fermentation broth prepared in Example 1, the citrus fermentation broth prepared in Comparative Examples 2 to 5, and the citrus homogenate prepared in Comparative Example 1, all with a concentration of 2 μg / ul.

[0120] Cells in the negative control group were treated for 24 hours. The positive control group was treated with complete culture medium, and the treatment group was treated with complete culture medium and the drug. Cells in both the positive control and treatment groups were treated for 24 hours. Then, 5% ethanol was added to both the positive control and treatment groups, and the cells were treated for another 6 hours. After the experiment, the cell supernatant was collected.

[0121] Cell viability was assessed using CCK-8 reagent, and OD values ​​were measured using a microplate reader, then converted to cell viability. Cellular ROS were detected using a DCFH-DA fluorescent probe, and ROS intensity was expressed as the corresponding fluorescence value. Test results are as follows: Figure 2 As shown.

[0122] 3. Treatment of atopic dermatitis in mice

[0123] An atopic dermatitis model was established using ICR mice: The atopic dermatitis mouse model in this experiment adopted internationally accepted methods. 2,4-Dinitrochlorobenzene (DNCB) is a hapten and a potent sensitizer that readily reacts with tissue proteins in the skin. Initial application of DNCB to the skin can sensitize the body; subsequent challenges with DNCB can induce contact dermatitis and cause delayed-type hypersensitivity reactions. DNCB was completely dissolved in a matrix solution (acetone: corn oil = 1:4) to prepare 1.0% and 0.5% DNCB solutions, respectively.

[0124] Modeling Method: Eight-week-old female SPF-grade ICR mice, weighing 18–20 g, were selected. After one week of acclimatization, they were randomly divided into a model group and a control group. On day 0, a 2 cm × 3 cm hair-removed area was prepared on the back of the mice. From day 1 to day 4, 200 μL of 1% DNCB solution was evenly applied to the hair-removed area on the back of the model group mice daily to elicit an AD-like reaction. After a 3-day interval, starting from week 2, 100 μL of 0.5% DNCB solution was applied twice a week to maintain the AD-like reaction for 3 weeks (modeling was performed on the 3rd and 6th days of each week, starting at 12:00 PM). Mice with successful modeling showed symptoms such as erythema, papules, edema, and scaling on the hair-removed area of ​​their backs.

[0125] Test Method: Mice with successfully modeled skin were divided into two groups (intervention group and model group). The citrus fermentation broth prepared in Example 1 was sprayed onto the damaged skin areas of the intervention group mice 1 to 2 times daily, while the model group received no treatment. During this period, changes in skin damage and recovery of itching were observed and recorded in both groups (see Tables 1 and 2). After approximately two weeks, the skin at the damaged areas of the intervention group mice had largely returned to normal. The skin at the test sites of the blank control group, model group, and intervention group mice was compared, and the results are as follows: Figure 3 As shown in the figure. Skin tissue from the test sites of mice in the blank control group, model group, and intervention group were paraffin-embedded, sectioned, and then subjected to HE staining. The results are shown in the figure. Figure 4 As shown.

[0126]

[0127]

[0128] Table 1

[0129]

[0130] Table 2

[0131] in conclusion

[0132] See Figure 1As shown, comparing the cell concentration data of the positive control group with those of Examples 1 and Comparative Examples 1 to 5, it is evident that the cell concentrations of the treatment groups in Examples 1 and Comparative Examples 1 to 5 are significantly lower than those in the positive control group. Furthermore, comparing the cell concentration data of the treatment groups in Comparative Examples 1 to 5 with those in Example 1, it is evident that the cell concentration in the treatment group in Example 1 is significantly the lowest. Therefore, the citrus fermentation broth of Example 1 can significantly inhibit the production of inflammatory factors such as IL-1β, TNF-α, IL-6, and NO.

[0133] See Figure 2 As shown, comparing the cell viability data and ROS fluorescence values ​​of the positive control group with those of Examples 1 and Comparative Examples 1 to 5, it is evident that the cell viability of the treatment groups in Examples 1 and Comparative Examples 1 to 5 is increased, while the ROS fluorescence value is significantly decreased. This indicates that the drugs in the treatment groups of Examples 1 and Comparative Examples 1 to 5 all possess antioxidant capabilities in terms of oxidative stress. Furthermore, the citrus fermentation broth of Example 1 exhibits even better antioxidant effects.

[0134] See Figure 3 As shown, the skin damage in the model group mice was severe. In contrast, the skin of the intervention group mice, after treatment with the citrus fermentation liquid from Example 1, recovered well, and the itching symptoms largely disappeared (as shown in Tables 1 and 2). Compared with the blank control group, the skin of the intervention group mice was no different from that before the onset of inflammation. Therefore, the citrus fermentation liquid from Example 1 has a significant therapeutic effect on skin lesions and itching in atopic dermatitis model mice.

[0135] See Figure 4 As shown, compared to the model group, the intervention group mice treated with the citrus fermentation broth of Example 1 showed a significant reduction in inflammatory cell infiltration in their skin. Compared with the blank control group, the skin of the intervention group mice essentially returned to the normal skin HE staining state. Therefore, the citrus fermentation broth of Example 1 has a significant effect on regulating skin inflammation and can promote the repair of damaged skin tissue.

[0136] This invention provides a method for preparing citrus fermentation broth using citrus homogenate as a substrate through microbial fermentation technology. The citrus fermentation broth with skin inflammation-regulating function is simple and convenient to prepare, without the addition of chemical reagents or hormones; the preparation process is environmentally friendly and pollution-free. It can effectively inhibit the production of skin inflammatory factors, promote the repair of damaged skin tissue, and is gentle and non-irritating; it can serve as a non-hormonal topical preparation for regulating skin inflammation.

[0137] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing a citrus fermentation broth with skin inflammation-regulating function, wherein the fermentation raw materials of the citrus fermentation broth, by weight, include: The preparation method comprises 990-1010 parts of citrus homogenate, 150-250 parts of white sugar, 90-110 parts of Saccharomyces cerevisiae culture medium, 90-110 parts of Lactobacillus plantarum culture medium, and 90-110 parts of acetic acid bacteria culture medium; characterized in that the preparation method includes the following steps: S1. Weigh the fermentation raw materials according to the above-mentioned weight proportions; S2, Substrate Preparation Place the citrus homogenate and white sugar in a container, mix well, and obtain the substrate; S3, First Fermentation The activated brewer's yeast culture was added to the substrate obtained in step S2, and fermented at 25-30°C for 12-16 days to obtain the first fermentation product. S4, Secondary Fermentation The first ferment obtained in step S3 is divided into two portions; one portion of the first ferment is added to an activated plant lactobacillus culture medium and fermented at 25-30℃ for 26-30 days to obtain the second ferment; the other portion of the first ferment is added to an activated acetic acid bacteria culture medium and fermented at 25-30℃ for 26-30 days to obtain the third ferment. S5, three-stage fermentation The second and third fermentation products prepared in step S4 are mixed in a container and fermented at 25-30°C for 40-44 days. After filtration, citrus fermentation liquid is obtained.

2. The method for preparing citrus fermentation broth with skin inflammation regulating function according to claim 1, characterized in that, The raw materials for the brewer's yeast culture broth include brewer's yeast, yeast extract, malt extract, peptone, glucose, and distilled water; the viable count of the brewer's yeast is ≥10. 7 cfu / ml.

3. The method for preparing the citrus fermentation broth with skin inflammation regulating function according to claim 1, characterized in that, The raw materials for the *Lactobacillus plantarum* culture medium include *Lactobacillus plantarum*, casein peptone, beef extract, yeast powder, glucose, sodium acetate, diammonium citrate, Tween 80, K₂HPO₄, MgSO₄·7H₂O, MnSO₄·H₂O, CaCO₃, and distilled water; the viable count of the *Lactobacillus plantarum* is ≥10⁻⁶. 7 cfu / ml.

4. The method for preparing citrus fermentation broth with skin inflammation regulating function according to claim 1, characterized in that, The raw materials for the acetic acid bacteria culture medium include acetic acid bacteria, glucose, yeast extract, calcium carbonate, distilled water, and anhydrous ethanol; the viable count of the acetic acid bacteria is ≥10. 7 cfu / ml.

5. The method for preparing citrus fermentation broth with skin inflammation regulating function according to claim 1, characterized in that, The preparation method further includes the following steps: Prepare citrus homogenate; Select fully ripe fresh citrus fruits, wash them clean, and remove the peel and seeds; Put the pulp into a blender and blend thoroughly to obtain citrus homogenate; Prepare a brewer's yeast culture medium; place yeast extract, malt extract, peptone, glucose, and distilled water in a container, sterilize, and cool to room temperature; then add brewer's yeast, stir well, and incubate to activate, thus obtaining a brewer's yeast culture medium; Prepare a culture medium for *Lactobacillus plantarum*. Place casein peptone, beef extract, yeast powder, glucose, sodium acetate, diammonium citrate, Tween 80, K₂HPO₄, MgSO₄·7H₂O, MnSO₄·H₂O, CaCO₃, and distilled water in a container, stir well, and adjust the pH. After sterilization, cool to room temperature. Then add *Lactobacillus plantarum*, stir well, and incubate to activate, obtaining the *Lactobacillus plantarum* culture medium. To prepare an acetic acid bacteria culture medium, place glucose, yeast extract, calcium carbonate, and distilled water in a container, sterilize, add anhydrous ethanol, and cool to room temperature; then add acetic acid bacteria, stir well, and incubate to activate, thus obtaining the acetic acid bacteria culture medium.

6. The method for preparing citrus fermentation broth with skin inflammation regulating function according to claim 1, characterized in that, In step S5, fermentation is stopped when the acidity of the mixed fermentation material in the container is less than 3%, the pH is less than 4, and the ethanol content is less than 0.5%.

7. The use of citrus fermentation broth prepared by any one of claims 1 to 6 in the preparation of drugs for skin inflammation.

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