Method for preparing rhamnolipid through fermentation and application

Abstract

The invention discloses a method for preparing rhamnolipid through fermentation and application, and belongs to the technical field of fermentation. The dry banana powder and the dry purple cabbage powder are mixed and jointly used as the culture medium additive, the prepared composite culture medium is used for pseudomonas aeruginosa fermentation, and compared with a single culture medium additive or no culture medium additive, the OD value in obtained fermentation liquor is larger, and the composite culture medium is more suitable for fermentation preparation of rhamnolipid. The obtained rhamnolipid is used for cell and animal experiments, and a skin aging cell and mouse skin light aging model is established by using a 311 nm UVB ultraviolet lamp. Tests prove that after the rhamnolipid is used, harmful active oxygen of cells is reduced, surface skin thickening caused by skin photoaging is relieved, and it is proved that the rhamnolipid not only can relieve the skin photoaging symptom caused by long-term ultraviolet irradiation, but also can prevent long-term stimulation of ultraviolet to mouse skin.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, specifically to a method for preparing rhamnolipids by fermentation and its application. Background Technology

[0002] As the outermost organ of the human body, the skin is simultaneously damaged by sunlight and environmental pollutants, leading to aging known as extrinsic aging or photoaging. Studies show that photoaging accounts for over 80% of facial aging. Exposure to ultraviolet (UV) radiation is the primary factor causing photoaging. Sunlight contains UVA (long-wave ultraviolet, 320-400 nm), UVB (medium-wave ultraviolet, 280-320 nm), and UVC (short-wave ultraviolet, 200-280 nm). While UVA has relatively weak mutagenic ability, it has extremely strong penetrating power, reaching the dermis and subcutaneous tissue. UVB has a strong mutagenic ability, directly interacting with DNA to produce thymine dimer photoproducts, causing DNA damage. UVC is the most mutagenic, but it is absorbed by the ozone layer and cannot reach the Earth's surface. Histological and ultrastructural studies indicate that photoaged skin is closely related to increased epidermal thickness and changes at the epidermal-dermal junction. In the epidermis, the vitality and renewal rate of keratinocytes decrease, the barrier function weakens, and this further leads to dry skin and peeling. Meanwhile, in the dermis, the number of fibroblasts gradually decreases, collagen and elastin synthesis slows down and their breakdown accelerates, often resulting in a much more severe form of aging than internal aging.

[0003] Rhamnolipids are typically microbial secondary metabolites produced by *Pseudomonas aeruginosa* through a bio-fermentation process. Rhamnolipids are biodegradable, making them a sustainable alternative. They possess good foaming properties, are less likely to irritate the skin, and can be used in a wide range of applications, from cleansers to personal care products. Rhamnolipids offer advantages such as environmental friendliness and high biosafety. Improving the production efficiency of rhamnolipids and their application in anti-skin aging is a key research direction in the industry. 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 rhamnolipids by fermentation and its application.

[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 rhamnolipin by fermentation, wherein fermentation strains are inoculated into the composite culture medium at an inoculation rate of 1-15% by volume according to the volume of the composite culture medium to obtain rhamnolipin. The fermentation strain is *Pseudomonas aeruginosa* ( Pseudomonas aeruginosa (The accession number is CICC23361;) The composite culture medium comprises the following components in parts by weight: 1-10 parts peptone, 1-10 parts beef extract, 1-10 parts NaCl, 0.1-5 parts culture medium additives, and 900-1100.0 mL distilled water. The culture medium additives include dried banana powder or dried purple cabbage powder.

[0006] Furthermore, in the culture medium additives, the mass ratio of dried banana powder to dried purple cabbage powder is 1:(1-3).

[0007] Further, take fresh, ripe bananas, peel and slice them, dry them at 60-80℃, and grind them until they pass through a 100-300 mesh sieve to obtain banana powder; Take fresh purple cabbage, peel off the outermost leaves, remove the stems, wash and chop it, dry it at 50-70℃, and then grind it through a 100-200 mesh sieve to obtain purple cabbage powder.

[0008] Furthermore, the viable cell count in the fermentation strain was 2 × 10⁻⁶. 7 cfu / ml -9×10 9 cfu / ml.

[0009] Furthermore, during the fermentation process, the fermentation temperature is maintained at 30-40℃, the pH at 6-9, the rotation speed at 200-800 rpm, and sterile air is introduced at a flow rate of 1-2 vvm.

[0010] In a second aspect, the present invention provides the application of the rhamnolipid 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, reducing epidermal thickness, increasing collagen fibers, and reducing skin wrinkles.

[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 rhamnose glycolipid is a disaccharide diester.

[0014] The beneficial effects of this invention are: This invention uses a mixture of dried banana powder and dried purple cabbage powder as a culture medium additive to prepare a composite culture medium for Pseudomonas aeruginosa fermentation. Compared with using a single culture medium additive or no culture medium additive, the fermentation broth has a higher OD value, making it more suitable for the fermentation preparation of rhamnolipids.

[0015] The rhamnolipin obtained in this invention was used in cell and animal experiments. A skin aging cell and mouse skin photoaging model were established using a 311 nm UVB lamp. Tests showed that the use of the rhamnolipin reduced harmful reactive oxygen species in cells and alleviated the thickening of the epidermis caused by photoaging. This demonstrates that rhamnolipin 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

[0016] Figure 1 Figure showing the repair effects of different concentrations of rhamnolipin on senescent HaCaT cells, as evaluated by β-galactosidase.

[0017] Figure 2 Figure showing the results of evaluating the prevention of HaCaT cell senescence by different concentrations of rhamnolipin for β-galactosidase.

[0018] Figure 3 Image showing the results of Western blotting (WB) assessment of rhamnoglycolipid repair of UVB irradiation.

[0019] Figure 4 Figure showing the results of Western blotting (WB) assessment of rhamnolipin's protection against UVB radiation.

[0020] Figure 5 The image shows the results of confocal microscopy assessment of rhamnoglycolipids in alleviating elevated ROS levels.

[0021] Figure 6 A diagram illustrating the effects of rhamnolipin on skin photoaging using H&E. Detailed Implementation

[0022] 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.

[0023] 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.

[0024] Skin aging is a complex biological process caused by both endogenous changes and exogenous damage. After skin cells age, various physiological functions change, including a significant increase in reactive oxygen species (ROS) levels, mitochondrial dysfunction, increased secretion and expression of collagen hydrolase-1 (MMP-1), and decreased synthesis of type I collagen (COL-1), leading to decreased skin elasticity and deeper wrinkles. Excessive ROS disrupts the balance between the body's oxidation and antioxidant systems, causing imbalances in the synthesis of natural antioxidants, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Currently, an increasing number of antioxidants have been proven to delay skin aging, such as vitamin C, fruit acids, vitamin A, and some plant extracts. However, the effects of these preparations are not entirely ideal. Vitamin C is sensitive to oxygen and easily decomposes; retinoids can irritate the skin and are unstable to light; some plant extracts can even cause skin redness and swelling, accompanied by side effects such as allergies and contact dermatitis, affecting their widespread use. In the field of medical aesthetic anti-aging, the focus has previously been solely on increasing collagen production. Although collagen is the most abundant and important protein in the dermis, skin aging actually involves changes in the epidermis, dermis, and subcutaneous tissue. All components of the dermis undergo degeneration, with the most significant being the disruption of the extracellular matrix (ECM). In normal skin, the ECM is in dynamic equilibrium, resulting in elasticity. As skin ages, this dynamic equilibrium is disrupted, leading to ECM disorder, a decrease in collagen and elastin, and consequently, the appearance of wrinkles.

[0025] 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: P21 antibody (catalog number: 10355-1-AP) and GAPDH antibody (catalog number: 814640-5-RR) were purchased from Wuhan Sanying. HRP secondary antibody (catalog number: RS0002) was purchased from Immunoway. QuickBlock™ Western blocking buffer (catalog number: P0226), primary antibody dilution buffer (catalog number: P0103), ECL chemiluminescence kit (catalog number: P0018FM; P0018A), and β-galactosidase staining kit (catalog number: C0602) were purchased from Beyotime. BCA protein concentration assay kit was purchased from Yaxin Biotechnology. Protease inhibitor cocktail was purchased from Taoshu. The 3-color prestained protein marker (catalog number: 20351ES76), SDS-PAGE protein loading buffer (catalog number: 20315ES20), RIPA lysis buffer (catalog number: 20101ES60), 0.25% trypsin (catalog number: 40126ES60) and 1% penicillin-streptomycin antibiotic (catalog number: 60162ES76) were purchased from Yisheng Biotechnology.

[0026] Ultraviolet phototherapy unit (SH1B, Sigma), eBlot rapid wet transfer instrument (eBlot™ L1, GenScript), intelligent image workstation (GelView 6000Plus, Boluteng), laser confocal microscope (FV4000, Olympus), fully automated digital slide scanning system (Axio Scan. Z1, Zeiss). The *Pseudomonas aeruginosa* strain used was... Pseudomonas aeruginosa The strain was purchased from the China Industrial Microbial Culture Collection Center, strain accession number: CICC 23361. The human keratinocytes (HaCaT) used were purchased from Shanghai Jining Industrial Co., Ltd.

[0027] Example 1: Fermentation preparation of rhamnolipids 1. Preparation of composite culture medium 1.1 Preparation of Culture Medium Additives The culture medium additives are dried banana powder and dried purple cabbage powder, and the specific preparation method is as follows: Take fresh, ripe bananas, peel and slice them, dry them at 70℃, and then grind them until they pass through a 200-mesh sieve to obtain banana powder.

[0028] Take fresh purple cabbage, peel off the outermost leaves, remove the stems, wash and chop it, dry it at 60℃, and grind it until it passes through a 150-mesh sieve to obtain purple cabbage powder.

[0029] 1.2 Composite Culture Medium The compound culture medium is based on nutrient broth culture medium, with the addition of culture medium additives.

[0030] The nutrient broth culture medium consists of the following components: 5.0 g peptone, 3.0 g beef extract powder, 5.0 g NaCl, and distilled water to make up to 1000.0 mL, pH=7.0.

[0031] The composition of the compound culture medium is as follows: 5.0 g peptone, 3.0 g beef extract powder, 5.0 g NaCl, 1 g culture medium additive, and distilled water to make up to 1000.0 mL, pH=7.0. In the culture medium additive, the mass ratio of dried banana powder to dried purple cabbage powder is 1:2.

[0032] 2. Fermentation culture The activated *Pseudomonas aeruginosa* was fermented and cultured into the following groups: Basic group (using nutrient broth); Banana group (using nutrient broth with 1g of dried banana powder per 1000ml of medium); Cabbage group (using nutrient broth with 1g of dried cabbage powder per 1000ml of medium); and Complex group (using a compound medium). The bacterial concentration was 3 × 10⁻⁶. 9A 300ml volume of *Pseudomonas aeruginosa* bacterial suspension with cfu / ml was inoculated into a 7.5L fermenter containing 3L of culture medium. During fermentation, the fermentation temperature was maintained at 35℃, pH = 7.0, and the aeration speed was 300rpm. Sterile air was introduced at a flow rate of 1.4 vvm. Fermentation was terminated after 10 hours.

[0033] Experimental Example 1 The fermentation process of Example 1 was monitored. Fermentation broth was taken from the fermenter and the OD 600 value was measured. The OD 600 values ​​(in units of 1) of the fermentation broth after fermentation were completed are shown in Table 1.

[0034] Table 1. OD determination of fermentation broth in Example 1 According to the results in Table 1, compared with using a single culture medium additive or no culture medium additive, the composite culture medium prepared by mixing banana powder and purple cabbage powder together as a culture medium additive, when used for Pseudomonas aeruginosa fermentation, has a larger OD value in the fermentation broth, which is more suitable for the fermentation preparation of rhamnolipids.

[0035] Testing revealed that the main component of the rhamnolipid prepared by this invention is a disaccharide diester, which is an amber-colored liquid with the chemical formula C. 32 H 58 O 13 The molecular weight is 650.79 g / mol, and the CAS number is 869062-42-0.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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 in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin) for 24 h to allow cell adhesion. The medium was then aspirated, and 2 mL of rhamnolipin (diluted with DMEM complete medium) at different concentrations (0, 10 μM, 20 μM) was 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.

[0041] Repair model: HaCaT cells in logarithmic phase were used at a rate of 5.0 × 10⁻⁶. 5Cells 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 then irradiated with UVB for 30 s. The PBS was aspirated, and 2 mL of rhamnolipin at different concentrations (0, 10 μM, 20 μM) (diluted with DMEM complete medium) was added. Cells were cultured for another 24 h.

[0042] Experimental Example 2: Results of Anti-aging Model Cell Experiments The cell experimental model constructed in Example 2 was measured, including β-galactosidase activity and the expression of cell cycle-related factor P21.

[0043] 1. Assay for β-galactosidase activity The results of β-galactosidase activity assay for the repair effect of rhamnolipids on senescent HaCaT cells are shown in the figure. Figure 1 .

[0044] 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 rhamnolipin concentration, proving that rhamnolipin has a significant effect on repairing cell aging.

[0045] 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 .

[0046] 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 rhamnolipin beforehand, the proportion of blue cells decreased significantly with the increase of rhamnolipin concentration, proving that rhamnolipin has a concentration-dependent effect in preventing cell senescence.

[0047] 2. Western blot assay for proteins The expression of the cell cycle-related factor p21 can indicate that cells are in a growth state. This can be detected using Western blotting (WB). The main steps are as follows: Total cell protein extraction: Using the cell experimental model from Example 1, discard the culture medium and wash the cells three times with cold PBS. Scrape off HaCaT cells, carefully aspirate the resulting solution, and centrifuge at 300 g for 5 min at 4 °C to obtain the cells. Lyse the pre-cooled PBS-washed cells in RIPA lysis buffer (containing 1% protease inhibitor) for 30 min, vortexing the cells vigorously multiple times during lysis. After lysis, centrifuge at 11000 g for 15 min at 4 °C, and collect the supernatant as the total cell protein.

[0048] Protein denaturation: Protein concentration was measured using the BCA assay. After quantification, the protein was diluted with protein loading buffer, and the sample was heated at 100°C for 15 min to obtain denatured protein.

[0049] Western blot assay for proteins: 1) Protein electrophoresis: Proteins are separated in a 4-20% Bis-Tris SDS-PAGE gel (GenScript). An appropriate volume of total cell protein extract is loaded into the lanes of the SDS-PAGE gel. Electrophoresis is performed at 80 V for 15 min, then the voltage is adjusted to 120 V and electrophoresis is continued until the bottom of the gel is reached. 2) Transfer: After electrophoresis, the gel is removed, and a suitable NC membrane (0.45 μm) is cut. The transfer apparatus is installed in the following order from negative to positive: sponge pad, gel, NC membrane, sponge pad. After installation, the apparatus is placed in the transfer tank. The liquid level of the transfer solution is checked, the power is turned on, and the transfer is completed after 3 transfer cycles. 3) Blocking: The membrane is placed in QuickBlock™ Western blocking buffer and shaken at room temperature for 20 min. 4) Primary antibody incubation: After blocking, the blocking buffer is discarded, and a primary antibody diluted to an appropriate concentration is added. The membrane is gently shaken overnight at 4 °C. 5) Secondary antibody incubation: Add HRP secondary antibody diluted to an appropriate concentration, and gently shake the NC membrane on a shaker for 1 h at room temperature. After incubation, wash the membrane 4 times with TBST, 5 min each time. 6) Development: Prepare ECL developing solution before development. Lay the NC membrane flat on clean plastic wrap, add developing solution to the membrane and let it stand in the dark for 1 min, then image the NC membrane on the gel system. Results are shown in […]. Figures 3-4 .

[0050] from Figure 3 Western blot (WB) results showed that UVB irradiation increased the expression of the cell cycle-related factor P21, indicating that the cells were in a state of growth arrest. Treatment of senescent HaCaT cells with 20 μM rhamnolipin alleviated the accumulation of P21 caused by senescence, demonstrating the significant repair effect of rhamnolipin on senescent HaCaT cells.

[0051] according to Figure 4 As a result, after cells were incubated with rhamnolipin in advance and then irradiated with ultraviolet light again, P21 protein no longer accumulated, further demonstrating that rhamnolipin has a significant effect in preventing cell senescence.

[0052] 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 to detect ROS and determine the overall level of oxidative stress.

[0053] 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 .

[0054] according to Figure 5 As a result, 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 rhamnolipin, either preventative or restorative, significantly reduced DCF signal, indicating varying degrees of ROS reduction. This demonstrates that rhamnolipin has an anti-aging effect.

[0055] Example 3: Animal Experiment This invention uses H&E to assess the effects of rhamnoglycolipids on the symptoms of increased epidermal thickness caused by photoaging in animal experiments.

[0056] 1. Establishment of animal models The experimental animals were female BALB / c mice (6-8 weeks old), 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.

[0057] 1.1 Establishment of a skin photoaging model After removing hair from the backs of mice, they were anesthetized 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 a total of six weeks. The appearance of noticeable wrinkles on the backs of the mice indicated the successful establishment of the mouse skin photoaging model.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] H&E results are as follows Figure 6 As shown, the control group without drug treatment exhibited significant epidermal thickening after continuous ultraviolet (UV) irradiation. In both the prevention and repair models of rhamnolipin, it was observed that rhamnolipin could alleviate symptoms of skin aging such as deepened wrinkles, increased epidermal thickness, and reduced collagen fibers. Rhamnolipin not only alleviated photoaging symptoms caused by long-term UV irradiation but also prevented long-term UV stimulation of mouse skin.

[0062] 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 rhamnolipids by fermentation, characterized in that, Based on the volume of the compound culture medium, the fermentation strain is inoculated into the compound culture medium at an inoculation rate of 1-15% by volume to obtain rhamnolipin. The fermentation strain is *Pseudomonas aeruginosa* ( Pseudomonas aeruginosa (The accession number is CICC 23361;) The composite culture medium comprises the following components in parts by weight: 1-10 parts peptone, 1-10 parts beef extract, 1-10 parts NaCl, 0.1-5 parts culture medium additives, and 900-1100.0 mL distilled water. The culture medium additives include dried banana powder or dried purple cabbage powder.

2. The method for preparing rhamnolipids by fermentation according to claim 1, characterized in that, In the culture medium additives, the mass ratio of dried banana powder to dried purple cabbage powder is 1:(1-3).

3. The method for preparing rhamnolipids by fermentation according to claim 1, characterized in that, Take fresh, ripe bananas, peel and slice them, dry them at 60-80℃, and then grind them until they pass through a 100-300 mesh sieve to obtain banana powder; Take fresh purple cabbage, peel off the outermost leaves, remove the stems, wash and chop it, dry it at 50-70℃, and then grind it through a 100-200 mesh sieve to obtain purple cabbage powder.

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

5. The method for preparing rhamnolipids by fermentation according to claim 1, characterized in that, During fermentation, maintain the fermentation temperature at 30-40℃, the pH at 6-9, the rotation speed at 200-800 rpm, and introduce sterile air at a flow rate of 1-2 vvm.

6. The use of the rhamnolipin 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 effects include: reducing β-galactosidase levels and cellular reactive oxygen species levels, reducing epidermal thickness, increasing collagen fibers, and reducing the appearance of wrinkles.

7. The application according to claim 6, characterized in that, The anti-skin aging products mentioned include physical or chemical sunscreens and topical skin care and 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 rhamnose glycolipid is a disaccharide diester.

Images