Application of fermentation of gastrodia elata by using Haliderisiella coagulum

Abstract

The invention discloses application of ahndersia coagulum fermented gastrodia elata and relates to the technical field of biological medicine, and the fermented gastrodia elata is applied to preparation of products for treating medium-wave ultraviolet irradiation acute photoaging skin injury. The gastrodia elata fermented by the Hatherdrikia coagulans is used for treating skin pigmentation caused by UVB irradiation, inhibiting erythema and scale generation and reducing the conditions of skin wrinkle increase and skin thickening, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an application of Gastrodia elata fermented by coagulans bacteria. Background Art

[0002] Human skin is mainly divided into three layers of tissue: epidermis, dermis and subcutaneous fat layer. The solar ultraviolet rays irradiated to human skin contain 90%-99% long-wave ultraviolet rays (UVA, 320-400nm) and 1%-10% medium-wave ultraviolet rays (UVB, 280-320nm). At the same dose, UVB is 1000 times more damaging to the skin than UVA. And UVB mainly acts on the keratinocytes (HaCaT) of the epidermis, causing damage to intracellular proteins, DNA and membrane lipids, causing metabolic disorders and causing light damage to the skin, and in severe cases, causing skin cancer.

[0003] Using traditional sunscreens to resist UV rays may cause side effects such as skin inflammation and allergies. Therefore, the use of natural products produced by plants, animals and microorganisms to resist UV rays has attracted attention. Studies have found that the natural flavonoid silymarin protects cells from UVB damage by upregulating the ER alpha and ERbeta protein pathways; trehalose resists UVB-induced skin aging by inhibiting the expression of MMP; and the exopolysaccharides from Pantoea agglomerans can protect HaCaT cells from UVB damage.

[0004] Fermented Gastrodia elata is a product obtained by fermenting Gastrodia elata with probiotics under appropriate conditions. Simply put, it is a process in which probiotics use Gastrodia elata as a culture medium and ferment and transform Gastrodia elata at an appropriate culture temperature. The entire collection of the final fermentation products is called fermented Gastrodia elata. This fermentation product contains more active substances than Hyndrix coagulans and Gastrodia elata. However, the application of Hyndrix coagulans fermented Gastrodia elata in UVB acute photoaging skin damage has not been reported. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an application of fermenting Gastrodia elata with coagulation of Bacillus heindrix.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: providing an application of Gastrodia elata fermented by coagulating Bacillus hyendriculariae for preparing a product for treating acute photoaging skin damage irradiated by ultraviolet B.

[0007] Furthermore, the above-mentioned Bacillus coagulans is Bacillus coagulans AACE9, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 15, 2024, with the deposit number CGMCCNO.31302.

[0008] Further, the preparation method of Gastrodia elata fermented by coagulating Bacillus hyendriculariae comprises the following steps:

[0009] (1) mixing Gastrodia elata and water in a mass ratio of 1:3-5, crushing and homogenizing, sterilizing, and cooling to below 37° C. to obtain Gastrodia elata slurry;

[0010] (2) inoculating the bacterial liquid of the coagulating bacteria Hyndrixea into the Gastrodia elata slurry obtained in step (1) at a volume ratio of 1:18-22, mixing the mixture evenly, sealing the mixture, and fermenting the mixture at a temperature of 35-40° C. for 5-7 days until the pH value reaches 3.8-4.2, thereby obtaining the Gastrodia elata fermented with the coagulating bacteria Hyndrixea.

[0011] Furthermore, in step (2), the number of viable bacteria in the coagulated Hyndrickella bacterial solution is 1-9×10 8 CFU / mL.

[0012] Furthermore, the above-mentioned product is a medicine, a reagent or a cosmetic.

[0013] Furthermore, the above-mentioned coagulated heindrixella fermented Gastrodia elata has the effect of treating or alleviating skin pigmentation, scaling, erythema, increased wrinkles, skin thickening, reduced elasticity, skin ulceration or edema caused by acute photoaging due to medium-wave ultraviolet radiation.

[0014] Furthermore, the above product is used for external coating.

[0015] The present invention has the following beneficial effects:

[0016] The present invention uses coagulans to ferment Gastrodia elata under suitable conditions, and obtains fermented Gastrodia elata with more active substances than coagulans and Gastrodia elata. Experiments have shown that Gastrodia elata fermented with coagulans can treat skin pigmentation caused by UVB irradiation, inhibit the generation of erythema and scales, and reduce the increase of skin wrinkles and skin thickening, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Macroscopic images of skin lesions of mice in each group;

[0018] Figure 2 HE staining results of skin tissue of mice in each group;

[0019] Figure 3 The results of Masson staining of skin tissue of mice in each group;

[0020] Figure 4 Figure 2 shows the skin scoring results of mice in each group. DETAILED DESCRIPTION

[0021] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0022] Example 1 Isolation, identification and preservation of AACE9 of Bacillus heindrix coagulans

[0023] (1) Isolation of AACE9 from Bacillus heindrix

[0024] Weigh 1 g of the colon sample of Nandan Yao chicken from a farm in Kunming, suspend it in PBS buffer, spread it on LB solid medium after gradient dilution, pick out typical single colonies for expansion culture; take 100 μL of the bacterial solution of each single colony and spread it on a plate containing 5% CaCO 3 The single colony with the calcium-dissolving ring was cultured on MRS solid medium; the single colony with the calcium-dissolving ring was selected and expanded in LB liquid medium. The bacterial solution was purified by zoning and streaking for strain identification, and another 0.5 mL of bacterial solution was centrifuged at 3000 r / min for 10 minutes, the supernatant was discarded, 30% glycerol was added, and the solution was stored in a -80℃ refrigerator.

[0025] (2) Identification of AACE9 of Bacillus heindrix

[0026] Using TSINGKE TSP701-50 Bacteria Genomic DNA Kit (Beijing Qingke Biotechnology Co., Ltd.) was used to extract bacterial DNA, and then universal primers 27F and 1492R were used to amplify the 16S rRNA gene. The PCR product was sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with BLAST in NCBI GenBank to determine the classification and name it Heyndrickxia coagulans AACE9.

[0027] (3) Preservation of strains

[0028] The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection on July 15, 2024, with the deposit registration number CGMCC NO.31302, and the deposit location is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0029] Example 2

[0030] A preparation method of Gastrodia elata fermented with coagulated bacteria Hendrix spp. comprises the following steps:

[0031] (1) Gastrodia elata and water are mixed in a mass ratio of 1:4, crushed and homogenized, sterilized at 70° C. for 30 min, and cooled to below 37° C. to obtain Gastrodia elata slurry;

[0032] (2) The AACE9 bacterial suspension of Hendrixella hyendrica (1×10 8 CFU / mL) was inoculated into the Gastrodia elata slurry obtained in step (1) at a volume ratio of 1:20, mixed evenly, sealed, and fermented at 37°C for 6 days until the pH reached 4.0 to obtain the Gastrodia elata fermented with coagulated Heindrix spp.

[0033] Example 3 Verification of the therapeutic effect of Gastrodia elata fermented with coagulation of Bacillus heindrix

[0034] (1) Establishment of UV-damaged mouse model

[0035] Experimental animals: SPF-grade C57BL / 6J mice, male, 5 weeks old, weighing 18-22 g, were provided by Spefoc (Beijing) Biotechnology Co., Ltd.

[0036] Mice were anesthetized with Avertin (tribromoethanol) (20 μL / g). After the mice were anesthetized, the hair on the back of the mice was removed with a shaver (area 2 cm × 2 cm), and the excess short hair was removed with a depilatory cream. The residual depilatory cream was then scrubbed off with sterile saline, wiped clean, and then disinfected with 75% alcohol. The skin condition of the mice was observed to ensure that the skin of the mice to be modeled had no wounds, damage, or scratches.

[0037] Blindfold the mouse with gauze and fix the mouse on the cardboard with a tie. Use the UV UV aging box ZW-40 (Shaoxing Zhicheng Instrument Co., Ltd.), and preheat the lamp for 10 minutes before modeling. Use two 15W UVB lamps to irradiate the mouse's back skin at a distance of 15 cm for 2 days, 30 minutes each time, and observe the skin damage of the mouse. When the mouse skin shows symptoms such as rashes, edema, and ulcers, the modeling is successful.

[0038] (2) Grouping and drug administration

[0039] Experimental groups: The mice were randomly divided into 6 groups, including a blank group, a model group, a positive control group (carbomer wet gel), a coagulating Bacillus hyendriculariae AACE9 group, a coagulating Bacillus hyendriculariae AACE9 fermented Gastrodia elata group, and a Gastrodia elata group. The drug administration conditions of each group are shown in Table 1.

[0040] Table 1 Drug administration table

[0041]

[0042] Blank group: The mice were depilated on their backs and no other experimental treatment was performed.

[0043] Model group: The mice were depilated on their backs and irradiated with UVB according to the dose to create a model.

[0044] Positive control group: The backs of mice were depilated, and the backs of mice were irradiated with UVB according to the dosage to model the mice. Then, 0.2 g of Carbomer wet gel was evenly applied to the backs of mice at regular intervals every day.

[0045] Bacillus hendrixogenes AACE9 group: The backs of mice were depilated, and the backs of mice were irradiated with UVB according to the dosage for modeling treatment, and then 0.2 mL of Bacillus hendrixogenes AACE9 bacterial liquid was evenly applied to the backs of mice at regular intervals every day.

[0046] Gastrodia elata group: The mice were depilated on their backs, and their backs were irradiated with UVB according to the dosage for modeling. Then, 0.2 mL of Gastrodia elata homogenate was evenly applied to the backs of the mice at regular intervals every day.

[0047] Gastrodia elata fermented with Bacillus hyendriculariae AACE9 group: The backs of mice were depilated, and the backs of mice were irradiated with UVB according to the dosage for modeling treatment, and then 0.2 mL of Gastrodia elata fermented with Bacillus hyendriculariae AACE9 was evenly applied to the backs of mice at regular intervals every day.

[0048] (3) Macroscopic sunburn modeling effect and drug recovery

[0049] During the experiment, the drug administration was strictly carried out, and the changes in the skin on the back of the mice were closely observed. Photos were taken and recorded every day. The macroscopic images of the skin damage of mice in each group are shown in the figure below. Figure 1 shown.

[0050] On the 5th day of modeling and drug administration: except for the blank group, mice in all groups had obvious erythema, accompanied by wrinkles, loss of elasticity and skin thickening, indicating that all groups were successfully modeled.

[0051] On the 10th day after modeling and drug administration: the mice in the model group had large areas of erythema, medium brown precipitation, obvious skin wrinkles, accompanied by edema, scabs and necrosis, and the skin was significantly thickened; the mice in the positive drug group had moderate erythema, scattered light brown pigmentation, obvious skin wrinkles, poor elasticity, and slightly thickened skin; the skin of the mice in the AACE9 bacterial liquid group was accompanied by moderate erythema, medium brown precipitation, accompanied by edema, scabs and necrosis, obvious skin wrinkles, poor elasticity and significantly thickened skin; the skin of the mice in the AACE9 bacterial liquid fermented Gastrodia elata group had no obvious erythema and pigmentation, only mild skin wrinkles and mild skin thickening; the skin of the mice in the Gastrodia elata group was accompanied by scabs and necrosis, medium brown precipitation, moderate skin wrinkles and poor skin elasticity, and the skin was significantly thickened.

[0052] On the 18th day after modeling and drug administration: the mice in the model group still had necrotic, unhealed wounds, thickened skin at the wounds, and light brown pigmentation; the mice in the positive drug group had no obvious wounds in the skin tissue, mild wrinkles on the skin, no obvious pigment erythema, good skin elasticity, and normal thickness; the mice in the AACE9 bacterial solution group still had wounds and necrotic areas in the skin tissue, poor skin elasticity at the wounds, and mild skin thickening; the mice in the AACE9 bacterial solution fermented Gastrodia elata group had intact skin morphology and structure, no pigmentation, no erythema, smooth skin, normal elasticity, thin skin thickness, and good recovery; the mice in the Gastrodia elata group still had necrotic, unhealed wounds, thickened skin at the wounds with mild wrinkles, and light brown pigmentation.

[0053] A comprehensive comparison of the macroscopic mouse skin data shows that the wounds of the AACE9 bacterial solution fermented Gastrodia elata group and the positive drug group had healed on the 18th day, while the model group, Gastrodia elata group and AACE9 bacterial solution still had wounds, and the recovery of the AACE9 bacterial solution fermented Gastrodia elata group was better than that of the positive group.

[0054] (4) Effects of fermented Gastrodia elata on pathological changes in skin cells and collagen fibers in mice with acute UVB sunburn

[0055] In order to study the effect of AACE9 bacterial liquid fermented Gastrodia elata on the skin histopathology of UVB-irradiated mice, the back skin of the mice in the above groups was collected to make tissue sections and performed HE staining and Masson staining.

[0056] HE staining results Figure 2 shown.

[0057] On the 5th day of administration: the skin tissue structure of mice in the model group, positive drug group, AACE9 bacterial liquid group and Gastrodia elata group was disordered and unclear; a large range of necrosis was seen, which was homogeneous and structureless eosinophilic material, and a large number of necrotic cell fragments were seen. A small range of hyperkeratosis was seen around the lesion, and a small amount of incomplete keratinization was seen; a medium range of granulation tissue was seen in the deep skin, and a small number of fibroblasts and new blood vessels were seen in the granulation tissue, accompanied by a large number of lymphocytes and granulocytes infiltration. The skin tissue structure of mice in the fermented Gastrodia elata group was disordered and unclear; a large range of necrosis was seen in the superficial dermis, which was homogeneous and structureless eosinophilic material, and a large number of necrotic cell fragments were seen, and the number of skin appendages at the lesion was small; a medium range of granulation tissue was seen in the deep skin, and a small number of fibroblasts and new blood vessels were seen in the granulation tissue, accompanied by a small number of lymphocytes and granulocytes infiltration. The epidermis of the skin tissue of mice in the blank group was intact; the collagen fibers in the dermis were arranged neatly, the skin appendages hair follicles and sebaceous glands were scattered, and the number of hair follicles was large; the subcutaneous tissue was located below the dermis, composed of loose connective tissue, adipose tissue and muscle layer, and the skin structure was normal.

[0058] On the 18th day of drug administration: the skin tissue structure of mice in the model group was relatively disordered, and the epidermis was thin; hyperplasia of fibrous connective tissue was visible in the superficial layer of the dermis; the subcutaneous tissue was located below the dermis and was composed of loose connective tissue, adipose tissue and muscle layer, and a large number of lymphocyte infiltrations were still visible in the dermis and subcutaneous layer. The epidermis of the skin tissue of mice in the positive drug group was intact and thin; a very small amount of hyperplasia of fibrous connective tissue was visible in the superficial layer of the dermis, and the hair follicles and sebaceous glands of the skin appendages were scattered, and a moderate amount of lymphocyte infiltration was visible in the dermis and subcutaneous layer. The epidermis of the skin tissue of mice in the AACE9 bacterial liquid group was relatively intact and thin; a small amount of bleeding was visible, and the collagen fibers in the dermis were neatly arranged; a moderate amount of lymphocyte infiltration was visible in the dermis and subcutaneous layer. The epidermis of the skin tissue of mice in the fermented Gastrodia elata group was intact and thin; the collagen fibers in the dermis were neatly arranged, the hair follicles of the skin appendages and sebaceous glands were scattered, and the number of hair follicles was large; the subcutaneous tissue was located below the dermis and was composed of loose connective tissue, adipose tissue and muscle layer, and very small amounts of lymphocyte infiltration were observed in the dermis and subcutaneous tissue. The epidermis of the injured part of the skin tissue of mice in the Gastrodia elata group was intact, and parakeratosis in the middle range was observed; the thickness of the epidermis was uneven, and the acanthosis in the middle range was thickened; there was little elongation of the skin process; there was more hyperplasia of new granulation tissue, more hyperplasia of fibrous connective tissue, accompanied by very small amounts of lymphocyte infiltration, a small amount of new blood vessels, and congestion; slight bleeding was observed; the skin appendages in the injured part were reduced, and there were few follicle keratin plugs.

[0059] As can be seen from the above, on the 5th day, the overall modeling was good, and all groups were accompanied by a large amount of cell necrosis and inflammatory cell infiltration. The HE staining results were consistent with the appearance. Figure 1 On the 18th day, the epidermal structures of the positive drug group (carbomer), AACE9 bacterial solution fermented Gastrodia group and AACE9 bacterial solution group were relatively intact, but the HE staining results showed that there was still a moderate amount of inflammatory cell infiltration in the dermis of the skin in the positive drug group (carbomer) and AACE9 bacterial solution group, while there was only a very small amount of lymphocyte infiltration in the AACE9 bacterial solution fermented Gastrodia group, indicating that among all the drug groups, the AACE9 bacterial solution fermented Gastrodia group had the best effect.

[0060] Masson staining results Figure 3 shown.

[0061] On the 5th day of drug administration: the number of collagen fibers in the deep layer of the skin of mice in the model group, positive drug group, Gastrodia group, AACE9 bacterial solution group, and AACE9 bacterial solution fermented Gastrodia group was small, loosely arranged, irregular, and lightly colored. The dermis of the skin tissue of mice in the blank group had abundant collagen fibers, interwoven into a network, stained light blue, and no obvious collagen fiber hyperplasia was observed.

[0062] On the 18th day of drug administration: Masson staining showed a small amount of thin collagen fiber proliferation in the superficial dermis of the skin tissue of the model group, positive drug group, AACE9 bacterial solution group and Gastrodia elata group, which were staggered and stained blue. Masson staining showed that the collagen fiber content in the dermis of the skin tissue of the AACE9 bacterial solution fermented Gastrodia elata group was rich, interwoven and stained light blue, and no obvious collagen fiber proliferation was observed.

[0063] Comprehensive Masson staining results show that, except for the AACE9 bacterial solution fermented Gastrodia group, the model group, positive drug group, AACE9 bacterial solution group and Gastrodia group all had fine collagen fiber hyperplasia, and the external elasticity of the corresponding mouse skin was still poor, or there were lumps, while the AACE9 bacterial solution fermented Gastrodia group had rich collagen fibers in the dermis of the skin tissue, and the interweaving network had returned to normal levels, indicating that it has a very good effect in promoting collagen fiber production.

[0064] (5) Skin appearance scoring of mice with acute UVB photoaging treated with fermented Gastrodia elata

[0065] After the irradiation, in accordance with the "Technical Specifications for Safety of Cosmetics", different experimenters scored the skin damage in the irradiated area on the back of the mice using the double-blind method. The scoring standard table is shown in Table 2.

[0066] Table 2 Mouse skin appearance scoring standard

[0067]

[0068]

[0069] The rating results are as follows Figure 4 As shown in the results, the Gastrodia elata group, model group, positive drug group and AACE9 bacterial liquid group still had a certain degree of skin damage at the end of the experiment; on the 12th day, the skin condition of the mice treated with AACE9 fermented Gastrodia elata recovered well, and its recovery degree was close to that of the blank group. The macroscopic scoring results fully reflect that AACE9 fermented Gastrodia elata has a very good effect in treating UVB skin damage.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An application of Gastrodia elata fermented by coagulating Bacillus hyendriculariae, characterized in that: It is used to prepare products for treating acute photoaging skin damage caused by medium-wave ultraviolet radiation.

2. The use according to claim 1, characterized in that The coagulant Hyendrickella is coagulant Hyendrickella AACE9, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 15, 2024, with the deposit number CGMCC NO.31302.

3. The use according to claim 1, characterized in that The preparation method of Gastrodia elata fermented by coagulating Bacillus hyenderickii comprises the following steps: (1) mixing Gastrodia elata and water in a mass ratio of 1:3-5, crushing and homogenizing, sterilizing, and cooling to below 37° C. to obtain Gastrodia elata slurry; (2) inoculating the bacterial liquid of the coagulating bacteria Hyndrixea into the Gastrodia elata slurry obtained in step (1) at a volume ratio of 1:18-22, mixing the mixture evenly, sealing the mixture, and fermenting the mixture at a temperature of 35-40° C. for 5-7 days until the pH value reaches 3.8-4.2, thereby obtaining the Gastrodia elata fermented with the coagulating bacteria Hyndrixea.

4. The use according to claim 3, characterized in that In step (2), the number of viable bacteria in the coagulated Hyndrickella bacterial solution is 1-9×10 8 CFU / mL.

5. The use according to claim 1, characterized in that The product is a medicine, a reagent or a cosmetic.

6. The use according to claim 1, characterized in that The coagulated heindrixella fermented gastrodia elata has the effect of treating or alleviating skin pigmentation, scaling, erythema, increased wrinkles, skin thickening, reduced elasticity, skin ulceration or edema caused by acute photoaging due to medium-wave ultraviolet irradiation.

7. The use according to claim 1, characterized in that The product is used for external application.

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