Preparation method of sophorolipid and application thereof in anti-skin aging

Abstract

The application discloses a preparation method of sophorolipids and application of the sophorolipids in anti-skin aging, and belongs to the technical field of fermentation. The sophorolipids are used in cell and animal experiments, and a skin aging cell and a mouse skin photoaging model are established by using a 311 nm UVB ultraviolet lamp. After testing, harmful active oxygen of the cell is reduced after the sophorolipids are used, and the thickening of the surface skin caused by skin photoaging is also relieved, which proves that the sophorolipids can not only relieve the symptoms of skin photoaging caused by long-term ultraviolet irradiation, but also prevent long-term stimulation of the ultraviolet to the mouse skin.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, specifically to a method for preparing sophorolipid and its application in anti-skin aging. Background Technology

[0002] UVA and UVB ultraviolet radiation can penetrate the ozone layer and reach the Earth, triggering oxidative stress in the body. At the same time, it activates collagen hydrolytic enzymes to degrade collagen and elastin fibers in the dermis, damaging skin structure and function, reducing elasticity, causing wrinkles, and leading to skin aging.

[0003] Sophorolipids are microbial secondary metabolites produced by *Candida* yeast through fermentation, using sugars and vegetable oils as carbon sources. Different types of sophorolipids share common structural characteristics: each molecule consists of a hydrophilic and a hydrophobic portion. The hydrophilic portion is sophorose, and the hydrophobic portion is a saturated or unsaturated long-chain hydroxy fatty acid. The differences lie in the length and saturation of the fatty acid chains, as well as the position and degree of acetylation. Based on the presence or absence of 1,4-esterification, they are also classified into lactone and acid types. Lactone-type sophorolipids are better at reducing surface tension and have stronger antibacterial activity; while acid-type sophorolipids have better foam-forming ability and solubility. Sophorolipids are non-toxic, biodegradable, and resistant to high temperatures and salts, making them widely used in various fields. The paper "Optimized Production of Sophorolipids and Their Application in the Petroleum Industry (Shandong University)" points out that in oil extraction, sophorolipids can improve oil recovery by reducing crude oil viscosity and interfacial tension, and can replace chemical surfactants in daily chemical products to form low-foaming, environmentally friendly formulations. The patent "CN109679863A A Microbial Remediation Reagent for Petroleum-Contaminated Soil and Its Preparation Method" indicates that sophorolipids are used in the environmental field to degrade soil organic pollutants and inhibit harmful algae. The paper "Review: Progress and Challenges of Sophorolipids as Anticancer Drugs (Discover Oncology 2.8)" points out that sophorolipids exhibit anti-inflammatory, antitumor, and antibacterial activities in medicine. However, the role of sophorolipids in anti-skin aging remains unknown. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a method for preparing sophorolipid and its application in anti-skin aging.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing sophorolipids by fermentation, comprising the following steps: pretreating a bio-fermentation broth containing lactone-type sophorolipids and acid-type sophorolipids to obtain a sophorolipid layer; mixing the sophorolipid layer with water and separating to remove solid impurities to obtain a sophorolipid solution; adding dihydrogen phosphate and hydrogen phthalate to the sophorolipid solution; and cooling and crystallizing under alkaline conditions to obtain lactone-type sophorolipids; wherein the alkaline conditions are pH 8-9; and the cooling and crystallization is performed by cooling to 4-6°C. The molar ratio of the added hydrogen phthalate to the added dihydrogen phosphate is 1:(1-2); the added amount of hydrogen phthalate is up to a concentration of 0.2-0.4 mol / L; the added amount of dihydrogen phosphate is up to a concentration of 0.2-0.6 mol / L. The hydrogen phthalate is potassium hydrogen phthalate; the dihydrogen phosphate is potassium dihydrogen phosphate. The content of lactone-type sophorolipids in the sophorolipid layer is >60%; the yeast used is Candida tropicalis (…). Candida metapsilosis The accession number is CCTCC WY 2008431.

[0006] Furthermore, the crystallization time is 10 to 20 hours.

[0007] Furthermore, in the process of mixing the sophorolipid layer with water and separating to remove solid impurities to obtain the sophorolipid solution, the amount of water added is 4 to 8 times the volume of the sophorolipid layer.

[0008] Furthermore, the viable cell count in the bacterial strains used in the bio-fermentation broth is 2 × 10⁻⁶. 7 cfu / ml -9×10 9 cfu / ml.

[0009] Furthermore, the sophorose ester layer is mixed with 4 to 8 times its volume of water, stirred thoroughly, and then centrifuged to remove solid impurities.

[0010] In a first aspect, the present invention provides the application of the sophorolipid prepared by the method in the preparation of anti-skin aging products, wherein the anti-skin aging includes: reducing β-galactosidase content and cellular reactive oxygen species levels, maintaining Lamin B1 protein content, alleviating the deepening of wrinkles caused by skin aging, increasing the thickness of the skin epidermis, and reducing collagen fibers.

[0011] Furthermore, the aforementioned anti-skin aging products include physical or chemical sunscreens and topical skin repair products.

[0012] Furthermore, the skin aging mentioned is caused by photoaging, and the light includes natural light and ultraviolet light.

[0013] Furthermore, the sophorolipid is a lactone-type sophorolipid.

[0014] The beneficial effects of this invention are: This invention utilizes the obtained sophorolipids in cell and animal experiments, establishing skin aging cells and mouse skin photoaging models using a 311 nm UVB lamp. Tests showed that the use of sophorolipids reduced harmful reactive oxygen species in cells and alleviated the thickening of the epidermis caused by photoaging, demonstrating that sophorolipids can not only alleviate the symptoms of photoaging caused by long-term ultraviolet radiation but also prevent long-term ultraviolet stimulation of mouse skin. Attached Figure Description

[0015] Figure 1 Figure showing the repair results of senescent HaCaT cells by different concentrations of sophorolipids for β-galactosidase evaluation.

[0016] Figure 2 Figure showing the results of evaluating the prevention of HaCaT cell senescence by different concentrations of sophorolipids for β-galactosidase.

[0017] Figure 3 This is an image showing the results of immunofluorescence assessment of sophorolipid repair of UVB irradiation.

[0018] Figure 4 This is a graph showing the results of immunofluorescence assessment of sophorolipids in preventing UVB irradiation.

[0019] Figure 5 Confocal microscopy results used to evaluate the role of sophorolipids in alleviating elevated ROS levels.

[0020] Figure 6 A diagram illustrating the effects of sophorolipids on skin photoaging using H&E. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0023] Unless otherwise specified, all experimental materials used in the embodiments of this invention are conventional experimental materials in the art and can be purchased through commercial channels. The reagents and instruments used in this invention are as follows: Lamin B1 antibody (catalog number: 12987-1-AP) was purchased from Wuhan Sanying. Alexa Fluor 488 labeled secondary antibody (catalog number: A0423) and β-galactosidase staining kit (catalog number: C0602) were purchased from Beyotime. 0.25% trypsin (catalog number: 40126ES60) and 1% penicillin-streptomycin bispecific antibody (catalog number: 60162ES76) were purchased from Yisheng Biotechnology.

[0024] Ultraviolet phototherapy device (SH1B, Sigma), laser confocal microscope (FV4000, Olympus), fully automated digital slide scanning system (Axio Scan. Z1, Zeiss). The yeast used was *Candida tropicalis*. Candida metapsilosis The accession number is CCTCC WY 2008431.

[0025] Example 1 This embodiment provides a method for preparing lactone-type sophorolipids, as detailed below: (1) Prepare YEPD seed culture medium. Put 50ml of YEPD culture medium into a 300ml shake flask and inoculate a single yeast colony. Incubate for 24h at 160r / min and 30℃.

[0026] (2) Prepare 3L of basic inorganic salt culture medium containing 4% glucose and 6% soybean oil (by mass and volume percentage). Inoculate yeast YEPD seed liquid at 10% of the total fermentation medium. Ferment at an initial temperature of 30℃, stirring at 200r / min, and dissolved oxygen maintained above 30% for 72 hours. Adjust the stirring speed and aeration rate according to the specific situation and maintain pH 5.5.

[0027] (3) After fermentation, the fermentation liquid was heat-treated at 110℃ for 20 minutes, and then the fermentation liquid was poured into a separatory funnel and allowed to stand at 25℃ for 3 hours. The bottom layer of sophorolipid was removed to obtain the sophorolipid layer. The content of lactone-type sophorolipid was 65% by liquid chromatography analysis. (4) Add the sophorolipid layer to 4 times the volume of pure water, stir thoroughly for 10 minutes, and centrifuge at 4500 rpm for 5 minutes to remove solid impurities and obtain a solution of sophorolipid and water; (5) Add potassium hydrogen phthalate to the solution obtained in step (4) to make its concentration 0.2 mol / L, and add potassium dihydrogen phosphate to make its concentration 0.2 mol / L. Adjust the pH to 8.0 with 4M KOH, and place it in a jacketed crystallization bottle for 4℃ low temperature crystallization for 10 hours. (6) Filter to obtain lactone-type sophorolipid crystals, and finally dry and pulverize to obtain lactone-type sophorolipid powder.

[0028] The purity and yield of sophora lipoprotein powder were analyzed by high performance liquid chromatography. The results showed that the purity of the above-mentioned lactone-type sophora lipoprotein powder was 87%, and the yield of lactone-type sophora lipoprotein was 93%.

[0029] Example 2: Construction of a cell experimental model 1. Construction of a cellular senescence model This invention constructs a cellular senescence model by single-stimulation of human keratinocytes (HaCaT) with UVB (311 nm). Specifically, logarithmic-phase HaCaT cells were stimulated at 5.0 × 10⁻⁶ nm. 5 Cells were seeded at a density of 100 cells / well in six-well plates and cultured in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin) for 24 h to allow cell adhesion. The cells were then treated with UVB for 30 s to obtain a cell senescence model.

[0030] Validation of the cell senescence model: The cell senescence model obtained after the above treatment was cultured for 24 h. The DMEM medium in the HaCaT cells was removed, and the HaCaT cells were gently washed three times with PBS. 1 mL of β-gal staining fixative was added, and the cells were fixed at room temperature for 15 min. The β-gal staining fixative was aspirated, and the cells were washed three times with PBS. The PBS buffer was aspirated, and 1 mL of staining working solution was added to each well to adjust the pH to 6.0. To prevent liquid evaporation and pH changes, the six-well plates were sealed with sealing film and wrapped with aluminum foil, and incubated overnight at 37°C in the dark. The cells were then photographed and observed under a microscope.

[0031] β-galactosidase is highly expressed in senescent cells. This invention uses a cell senescence β-galactosidase staining kit to measure intracellular β-galactosidase activity. The kit uses X-Gal as a substrate, which, under the catalysis of senescence-specific β-galactosidase, produces a deep blue product. This allows for easy observation of cells expressing β-galactosidase under a light microscope.

[0032] The results showed that, compared with untreated HaCaT cells, a large number of blue cells appeared in the culture plate treated with UVB, proving that HaCaT cells were successfully induced to senescence and the cell senescence model was successfully constructed.

[0033] 2. Construction of anti-aging models Anti-aging models are divided into prevention models and repair models, as detailed below: Prevention model: HaCaT cells in logarithmic phase were used at a rate of 5.0 × 10⁻⁶.5 Cells were seeded at a density of 100 cells / well in six-well plates and cultured for 24 h in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin) to allow cell adhesion. The medium was then aspirated, and 2 mL of different concentrations (0, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM) of sophorolipid (diluted with DMEM complete medium) were added to each well. Cells were cultured for another 24 h. The medium was then aspirated, and 1 mL of PBS buffer was added to each well. Cells were irradiated with UVB for 30 s, and then the PBS buffer was replaced with DMEM complete medium for another 24 h.

[0034] Repair model: HaCaT cells in logarithmic phase were used at a rate of 5.0 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in six-well plates and cultured in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin) for 24 h to allow cell adhesion. The medium was then aspirated, and 1 mL of PBS buffer was added to each well. Cells were irradiated with UVB for 30 s. The PBS was then aspirated, and 2 mL of sophorolipids (diluted with DMEM complete medium) at different concentrations (0, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM) were added. Cells were cultured for another 24 h.

[0035] Experimental Example 1: Results of Anti-aging Model Cell Experiments In Example 2, cell senescence auxiliary markers were measured in the cell experimental model constructed. The measured items included β-galactosidase activity, intracellular Lamin B1, and cellular reactive oxygen species levels.

[0036] 1. Assay for β-galactosidase activity The results of the β-galactosidase activity assay for the repair effect of sophorolipids on senescent HaCaT cells are shown in the figure. Figure 1 .

[0037] according to Figure 1 As a result, ultraviolet treatment led to the appearance of a large number of blue cells, but after the addition of rhamnolipin, the proportion of blue cells decreased significantly with the increase of sophorolipid concentration, proving that sophorolipid has a significant effect on repairing cell aging.

[0038] The results of β-galactosidase activity assay for the preventive effect of rhamnolipids on senescent HaCaT cells are shown in [the table below]. Figure 2 .

[0039] according to Figure 2 As a result, direct ultraviolet treatment led to the appearance of a large number of blue cells, but when incubated with sophorolipid beforehand, the proportion of blue cells decreased significantly with the increase of sophorolipid concentration, proving that sophorolipid has a concentration-dependent effect in preventing cell senescence.

[0040] 2. Intracellular Lamin B1 Immunofluorescence Staining Assay Place 15 μm diameter climbing plates in a 12-well plate, and inoculate each well with 1 × 10⁻⁶ seed plates. 5 HaCaT cells were cultured for 24 h to allow them to adhere, and then treated with biosurfactants and UVB. After culturing for another 24 h, the cells were washed three times with pre-chilled PBS and fixed with 4% paraformaldehyde for 15 min. After fixation, the cells were washed three times with pre-chilled PBS and then permeabilized with Triton X-100 at room temperature for 20 min. After permeabilization, the cells were blocked with 5% BSA at room temperature for 1 h. The blocking solution was dried, and Lamin B1 primary antibody was added. The cells were incubated at room temperature for 2 h, and then washed with PBST. After washing, Alexa Fluor 488-labeled goat anti-rabbit secondary antibody was added to the cells. After incubation, non-specific binding antibodies were washed away with PBST, and the cells were mounted with anti-fluorescence quenching mounting medium. Finally, the experimental results were photographed using a confocal microscope.

[0041] from Figure 3 The confocal images showed that UVB irradiation caused the loss of the nuclear morphology marker Lamin B1, indicating that the cells were in a senescent state. Treatment of senescent HaCaT cells with 10 μM sophorolipid restored Lamin B1 levels, demonstrating the significant repair effect of sophorolipid on senescent HaCaT cells.

[0042] from Figure 4 The confocal images show that after cells were incubated with sophorolipids before being irradiated with ultraviolet light, the Lamin B1 protein was no longer missing, further demonstrating that sophorolipids have a significant effect in preventing cell senescence.

[0043] 3. Cellular reactive oxygen species level test Regardless of whether skin aging is caused by external or internal factors, the ultimate decline of its cells or tissues is caused by endogenous or exogenous reactive oxygen species (ROS). DCFH-DA (2,7-dichlorofluorescein diacetate) is a commercially available ROS fluorescent probe that can penetrate cells. DCFH-DA can be used for ROS and to determine the overall level of oxidative stress.

[0044] The cell experimental model constructed in Example 2 was incubated with 10 μM DCFH-DA solution for 20 min. The results are shown in [Figure 1]. Figure 5 .

[0045] Confocal images clearly showed an increase in ROS levels in HaCaT cells after UVB stimulation, demonstrating the role of oxidative stress in inducing HaCaT cell senescence. Pre-treatment with the biosurfactant sophorolipid for prevention or repair significantly reduced DCF signal, indicating varying degrees of ROS reduction.

[0046] Example 3: Animal Experiment This invention uses H&E to evaluate the effects of sophorolipids on the increased epidermal thickness caused by photoaging in animal experiments. 1. Establishment of animal models Laboratory animals: BALB / c mice (female; 6-8 weeks old) were all purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and housed at the Experimental Animal Center of Tsinghua University. All animal experiments were approved by the Ethics Committee of Tsinghua University. Six mice were used in each group.

[0047] 1.1 Establishment of a skin photoaging model After removing hair from the backs of mice, they were anesthetized beforehand and placed in a dark box. A UV lamp was then used to irradiate the mice at a height of 15 cm for 15 minutes. Irradiation was performed three times a week, Monday, Wednesday, and Friday, for six consecutive weeks. The appearance of obvious wrinkles on the backs of the mice indicated the successful establishment of the mouse skin photoaging model.

[0048] 1.2 Establishment of Prevention and Remediation Models After hair removal from the backs of mice, they were anesthetized and placed in a dark box. They were then irradiated with a UV lamp at a height of 15 cm for 15 minutes. Five minutes before UV irradiation, a diluted 1 wt% rhamnolipid was applied to the hair-removed area. Irradiation was performed three times a week, Monday, Wednesday, and Friday, for six consecutive weeks to obtain a preventative model.

[0049] After hair removal from the backs of mice, they were anesthetized and placed in a dark box. They were then irradiated with a UV lamp at a height of 15 cm for 15 minutes. Immediately after UV irradiation, diluted 1 wt% rhamnolipin was applied to the irradiated area. Irradiation was performed three times a week, Monday, Wednesday, and Friday, for six consecutive weeks to obtain a repair model.

[0050] 2. Validation of the anti-skin aging properties of rhamnolipids For the skin photoaging model, prevention model, repair model, and untreated mice, skin from the irradiated area on the back of the mice was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E.

[0051] H&E results are as follows Figure 6As shown, the control group without drug treatment exhibited significant epidermal thickening after continuous ultraviolet (UV) irradiation. In the prevention and repair models of sophorolipids, it was observed that sophorolipids could alleviate symptoms of skin aging such as deepened wrinkles, increased epidermal thickness, and reduced collagen fibers. Sophorolipids not only alleviated photoaging symptoms caused by long-term UV irradiation but also prevented long-term UV stimulation of mouse skin.

[0052] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing sophorolipids by fermentation, characterized in that, The process includes the following steps: A bio-fermentation broth containing lactone-type sophorolipids and acid-type sophorolipids is pretreated to obtain a sophorolipid layer; the sophorolipid layer is mixed with water and then separated to remove solid impurities, yielding a sophorolipid solution; dihydrogen phosphate and hydrogen phthalate are added to the sophorolipid solution; and the solution is cooled and crystallized under alkaline conditions to obtain lactone-type sophorolipids; the alkaline conditions are pH 8–9; and the cooling and crystallization is performed at 4–6°C. The molar ratio of the added hydrogen phthalate to the added dihydrogen phosphate is 1:(1-2); the added amount of hydrogen phthalate is up to a concentration of 0.2-0.4 mol / L; the added amount of dihydrogen phosphate is up to a concentration of 0.2-0.6 mol / L. The hydrogen phthalate is potassium hydrogen phthalate; the dihydrogen phosphate is potassium dihydrogen phosphate. The content of lactone-type sophorolipids in the sophorolipid layer is >60%; The yeast used is Candida tropicalis ( Candida metapsilosis The accession number is CCTCC WY2008431.

2. The method for preparing sophorolipids by fermentation according to claim 1, characterized in that, The crystallization time is 10 to 20 hours.

3. The method for preparing sophorolipids by fermentation according to claim 1, characterized in that, In the process of mixing the sophorolipid layer with water and separating to remove solid impurities to obtain the sophorolipid solution, the amount of water added is 4 to 8 times the volume of the sophorolipid layer.

4. The method for preparing sophorolipids by fermentation according to claim 1, characterized in that, The viable cell count in the bacterial strains used in the bio-fermentation broth is 2 × 10⁻⁶. 7 cfu / ml -9×10 9 cfu / ml.

5. The method for preparing sophorolipids by fermentation according to claim 1, characterized in that, The sophorose ester layer was mixed with 4 to 8 times its volume of water, stirred thoroughly, and then centrifuged to remove solid impurities.

6. The use of the sophorolipid prepared by the method according to any one of claims 1-5 in the preparation of anti-skin aging products, characterized in that, The anti-skin aging measures include: reducing β-galactosidase levels and cellular reactive oxygen species levels, maintaining LaminB1 protein levels, alleviating the deepening of wrinkles caused by skin aging, increasing epidermal thickness, and reducing collagen fibers.

7. The application according to claim 6, characterized in that, The anti-skin aging products mentioned include physical or chemical sunscreens and topical skin repair products.

8. The application according to claim 6, characterized in that, The skin aging described is caused by photoaging, and the light mentioned includes natural light and ultraviolet light.

9. The application according to claim 6, characterized in that, The sophorolipid mentioned is a lactone-type sophorolipid.

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