Co-fermentation method based on sphingomonas and rhodiola rosea extracting solution and application of product

Abstract

The invention discloses a co-fermentation method based on sphingomonas and a rhodiola rosea extracting solution and application of a product, and belongs to the technical field of microorganisms. By utilizing a special rich enzyme system of sphingomonas and combining with an early-stage composite enzymolysis process and synergistic effect, the wall breaking efficiency of rhodiola rosea cell walls and the conversion degree of active components of rhodiola rosea are greatly improved, the bottlenecks of low efficiency and low bioavailability of a traditional rhodiola rosea extraction method are successfully overcome, and the rhodiola rosea extraction method is suitable for industrial production. The functional components such as salidroside and flavone can be fully released and converted. Secondly, different from non-symbiotic bacteria (such as lactic acid bacteria and saccharomycetes) which are commonly used for fermentation in the prior art, a potential symbiotic bacterium of sphingomonas XJL-TP07 from a special environment is innovatively selected and has better metabolic suitability with the rhodiola rosea component, the fermentation process is milder and safer (no irritant is produced), and the rhodiola rosea fermentation method is suitable for large-scale production. And a synergistic interaction effect can be generated through a unique biotransformation path.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of solar energy collectors, and particularly relates to a co-fermentation method based on a sphingomonas and a rhodiola extract and application of a product. BACKGROUND

[0002] The active ingredients of Rhodiola rosea and its anti-aging principle have attracted widespread attention in recent years, especially in the fields of skin care and health care. The anti-aging mechanism mainly realizes the regulation of oxidative stress, inflammatory response and cell metabolism through multiple targets. The core active ingredient of Rhodiola rosea is salidroside, which is the signature component of Rhodiola rosea and accounts for about 0.5%-1.5% of the dry weight, and has strong antioxidant, anti-inflammatory and anti-apoptotic properties. In addition, Rhodiola rosea also has flavonoids, tyrosol, phenolic compounds and polysaccharides, which synergistically enhance the antioxidant capacity, regulate mitochondrial function, inhibit inflammatory factors (such as TNF-α, IL-6), enhance skin barrier function and promote keratinocyte repair. Rhodiola rosea realizes anti-aging through multiple pathways, and is a "all-in-one player" among natural ingredients. However, the natural extraction efficiency of the active ingredients of Rhodiola rosea is low, the bioavailability is low, and at the same time, the extraction is limited by the extraction conditions, and the composition of the extract is limited, which cannot fully exert the beauty effect of Rhodiola rosea.

[0003] Studies have shown that after natural Chinese herbal medicine is fermented by microorganisms, the content of active substances of Chinese herbal medicine is increased, the toxic and side effects are reduced, and the separation, purification and application are easier. The extract of the fermentation product has a regulating function on the skin surface microecology, can regulate the physiological activity of the skin itself, and at the same time, the fermentation method makes the molecules with good moisturizing, whitening, soothing and anti-aging effects in natural raw materials reduce in molecular weight, so that they are easily absorbed by the skin, and at the same time, the toxicity and side effects are greatly reduced through the modification. The above changes benefit from the rich enzyme system and strong biological transformation ability of microorganisms. In addition, the synergistic effect of the metabolic products of microorganisms and drug ingredients may also enhance the efficacy. The mild and inexpensive characteristics of fermentation lay a foundation for its wide development and application.

[0004] Sphingomonas spp. is a group of Gram-negative bacteria widely found in soil, water, and on plant surfaces. Its fermentation products have attracted considerable attention in the anti-aging field in recent years, primarily due to their unique active ingredients and multi-target anti-aging mechanisms. Sphingomonas fermentation products include sphingolipids, sphingosine, ceramides, exopolysaccharides (EPS), and various peptides. These substances can regulate cell proliferation, differentiation, and apoptosis, participate in skin barrier repair, enhance the stratum corneum's water-locking capacity, reduce transepidermal water loss (TEWL), and improve dry skin and fine lines; inhibit inflammatory factors (such as IL-6 and TNF-α), alleviating aging caused by chronic inflammation; directly scavenge free radicals (ROS), reducing oxidative stress damage to collagen and DNA; reduce collagen breakdown, and delay wrinkle formation. Sphingosine monocytogenes cell lysate achieves anti-aging through a quadruple mechanism of antioxidation, barrier repair, collagen regulation, and anti-inflammation, making it especially suitable for dry, sensitive, and photo-aged skin. Its natural origin and low irritation make it a popular candidate for "green anti-aging" ingredients.

[0005] In alpine ecosystems, bacteria such as Sphingomonas often form a "symbiotic network" in the rhizosphere of plants together with other cold-resistant microorganisms. Due to their mutualistic effects on plants, they are commonly found in the rhizosphere of plants at high altitudes. In current studies using Rhodiola rosea as a fermentation substrate, such as CN110772460A, CN112472634A, CN103131730A, CN103127202B, CN104257545B, CN106265846A, CN112754960A, and CN117547566A, all of which use Lactobacillus and yeast, these strains are not natural Rhodiola rosea root symbiotic bacteria. Compared with Lactobacillus or yeast, Sphingomonas has many advantages. (1) Sphingomonas secretes cellulase and lignin peroxidase, which can cleave the cell walls of Chinese herbal medicines (such as the fibrous structure of Astragalus membranaceus and ginseng) and release more fat-soluble active ingredients (such as saponins and flavonoids). Lactic acid bacteria and yeast lack this type of enzyme system and have limited ability to decompose cellulose. (2) Sphingomonas transforms Chinese herbal medicine components into more active forms through reactions such as hydroxylation and glycosylation. In contrast, the biotransformation ability of lactic acid bacteria and yeast is weak, and they mainly stay at the substrate decomposition stage. (3) Sphingomonas is non-pathogenic, has higher safety, does not produce toxins, and does not produce histamine that lactic acid bacteria may secrete or ethanol that yeast may produce (excessive ethanol may irritate the skin). For example, Sphingomonas can increase the conversion rate of total saponins of Panax notoginseng from 30% to 70% in traditional yeast fermentation, and significantly enhance anticoagulant and anti-inflammatory activities. The sphingolipids secreted by Sphingomonas work synergistically with Centella asiatica glycosides to improve the skin barrier repair effect by 2 times compared with lactic acid bacteria fermentation products. However, there are currently no research reports on the use of Rhodiola rosea extract as a fermentation substrate and the supernatant after whole bacteria are broken as active substances in skin care and beauty.

[0006] The problems with existing technology are: (1) Sphingomonas is widely recognized and used as a cosmetic ingredient, but there is no evidence of its application in products co-fermented with Rhodiola rosea. (2) Rhodiola rosea contains a variety of highly active ingredients, but the current technology has low natural extraction efficiency and low bioavailability. At the same time, due to the limitations of extraction conditions, the components contained in the extract are limited, which cannot give full play to the application of Rhodiola rosea in beauty and cosmetic products. (3) The existing technology for fermenting Rhodiola rosea uses yeast or lactobacillus, which are not natural symbiotic strains of Rhodiola rosea; (4) Yeast or lactobacillus have insufficient ability to decompose Rhodiola rosea extract, resulting in poor anti-aging and antioxidant capacity of the product. Summary of the Invention

[0007] To solve the above problems, the technical solution provided by the present invention is as follows: This invention discloses a co-fermentation method based on Sphingomonas paucimobilis and Rhodiola rosea extract. The method involves using Sphingomonas paucimobilis XJL-TP07 (accession number CGMCC:35131) as the fermentation strain, using a culture medium containing Rhodiola rosea extract as the fermentation substrate, and obtaining a fermentation broth after fermentation. The fermentation broth is then filtered through particulate matter, the whole bacteria are broken up, centrifuged, and the supernatant is collected. Finally, the fermentation product is obtained by sterilization filtration.

[0008] Preferably, the fermentation broth is prepared as follows: Step (1) Prepare Rhodiola rosea extract Rhodiola rosea root is ground into powder, mixed with water, and then enzymes are added for enzymatic hydrolysis. The enzymatic hydrolysis is continued until the endpoint is reached, and then the temperature is raised to inactivate the enzymes to end the enzymatic hydrolysis reaction. After sterilization and filtration, Rhodiola rosea extract is obtained. Step (2) Add Rhodiola rosea extract Sphingomonas paucimobilis XJL-TP07 was inoculated into the culture medium for fermentation at an inoculation rate of 1%-10%, and fermented at 15-30℃ for 4-12 hours. Rhodiola rosea extract was added to the culture medium at a fermentation volume of 1-10%. Step (3) Co-fermentation of Sphingomonas and Rhodiola Rosea extract The Sphingomonas fermentation broth inoculated with Rhodiola rosea extract was fermented for 16–42 hours at 15–30°C to obtain the fermentation broth.

[0009] Preferably, specifically After filtering the fermentation broth through gauze to remove insoluble particles, the cells were disrupted by a high-pressure homogenizer at 850 Bar for 3 cycles. The disrupted broth was centrifuged at 10000g for 10 min at 4°C. The supernatant obtained after separation and precipitation was first filtered through a 0.45μm filter membrane and then through a 0.22μm filter membrane. The resulting sterile filtrate was the fermentation product.

[0010] Preferably, in step (1), the Rhodiola rosea is pulverized and then mixed with deionized water in a ratio of (0.5-3):100.

[0011] Preferably, the pulverization operation in step (1) specifically involves using a low-temperature ultra-fine cell-wall breaking pulverizer, pulverizing at 4-10℃ for 10 minutes, and then storing at 4℃.

[0012] Preferably, in step (1), an enzyme is added for enzymatic hydrolysis, wherein the enzyme preparations selected for enzymatic hydrolysis include cellulase, pectinase and papain and mixtures thereof.

[0013] Preferably, the ratio of the enzyme activity of the enzyme preparation to the mass of the Rhodiola rosea root powder is 500,000 U to 5,000,000 U: 1 kg.

[0014] Preferably, the enzymatic hydrolysis reaction is carried out at a temperature of 40-55°C for 2-24 hours, and the pH is controlled at 4.0-5.0.

[0015] Preferably, the fermentation product is used in the preparation of skin care and beauty cosmetics.

[0016] Compared with the prior art, the technical solution provided by this invention has the following advantages: This invention discloses a co-fermentation method and product application based on *Sphingomonas* and *Rhodiola rosea* extract. By co-fermenting *Sphingomonas* and *Rhodiola rosea* extract, significant beneficial effects have been achieved, effectively solving many problems existing in the background technology. Firstly, this method utilizes the rich enzyme system unique to *Sphingomonas*, combined with a prior complex enzymatic hydrolysis process, to synergistically improve the cell wall disruption efficiency of *Rhodiola rosea* and the conversion degree of its active ingredients. This successfully overcomes the bottlenecks of low efficiency and low bioavailability in traditional *Rhodiola rosea* extraction methods, allowing for a more complete release and conversion of rhodioloside, flavonoids, and other active ingredients. Secondly, unlike existing technologies that commonly use non-symbiotic bacteria (such as lactic acid bacteria and yeast) for fermentation, this invention innovatively selects *Sphingomonas* XJL-TP07, a potential symbiotic bacterium originating from a special environment. It has better metabolic compatibility with *Rhodiola rosea* components, resulting in a milder and safer fermentation process (without producing irritants), and can produce a synergistic effect through a unique biotransformation pathway. Experimental results show that the final co-fermentation product far surpasses traditional fermentation products in core efficacy, exhibiting superior soothing (significantly inhibiting TRPV1 expression) and antioxidant repair (significantly enhancing FLG protein) capabilities in cell models. In summary, this invention not only provides a highly efficient and safe new route for preparing Rhodiola rosea active ingredients, but also yields a cosmetic raw material with significantly enhanced efficacy, possessing enormous market application potential.

[0017] Biological deposit notice: Sphingomonas sp. XJL-TP07 was deposited at the Institute of Microbiology, Chinese Academy of Sciences on July 8, 2025. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC: 35131. Attached Figure Description

[0018] Figure 1 The growth curves of Sphingosine mononucleosis from different sources in Example 1 are shown. Figure 2 The growth curves of Sphingosomalidobacterium under different co-fermentation strategies of Rhodiola rosea extracts in Example 1 are shown. Figure 3 The soothing effect of the co-fermentation product of Sphingomonas and Rhodiola rosea extract in Example 4; Figure 4 This study examines the antioxidant effects of the co-fermentation product of Sphingomonas and Rhodiola rosea extract in Example 5. Detailed Implementation

[0019] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0020] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate for the embodiments of this application described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0021] In this application, the terms "upper," "lower," "left," "right," "front," "rear," "top," "bottom," "inner," "outer," "middle," "vertical," "horizontal," "lateral," and "longitudinal" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. These terms are primarily for the purpose of better describing this application and its embodiments, and are not intended to limit the indicated device, element, or component to having a specific orientation, or to be constructed and operated in a specific orientation.

[0022] Furthermore, some of the aforementioned terms, besides indicating orientation or positional relationships, may also have other meanings. For example, the term "above" may, in certain circumstances, indicate a dependency or connection. Those skilled in the art can understand the specific meaning of these terms in this application based on the specific circumstances. In addition, the terms "installation," "set," "equipped with," "connection," "linked," and "socket" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral structure; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium, or an internal connection between two devices, components, or components. Those skilled in the art can understand the specific meaning of the aforementioned terms in this application based on the specific circumstances.

[0023] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] Cellulase, pectinase, and papain were weighed and separately prepared into enzyme solutions with deionized water, each with an activity of 50,000 U / mL. These solutions were then mixed and used in the examples and comparative examples. Both cellulase and pectinase were commercially available.

[0027] The enzyme activity described in this invention is: 1 U / g refers to the enzyme activity of one gram of cellulase. U is defined as the amount of enzyme required to convert 1 micromolar of substrate or 1 micromolar of related groups in the substrate within 1 minute under specific conditions, and is called one International Unit (IU, also known as U).

[0028] Example 1

[0029] Fermentation culture of Sphingomonas XJL-TP07 Preparation of Sphingomonas seed culture Sphingosine monocytogenes was picked from a test tube and inoculated into 50 mL of sterile R2A seed culture medium. After culturing at 25°C and 200 rpm for 13 h, the culture was allowed to reach the logarithmic growth phase.

[0030] 50 ml of R2A medium was placed in a 250 ml Erlenmeyer flask, and 2% (v / v) Sphingomonas XJL-TP07 was inoculated and cultured at 20 °C and 220 rpm. Samples were taken every 4 hours for OD monitoring.

[0031] Comparative Example 1 50 ml of R2A medium was placed in a 250 ml Erlenmeyer flask, and 2% (v / v) Sphingomonas XJL-TP07 was inoculated and cultured at 26 °C and 220 rpm. Samples were taken every 4 hours for OD monitoring.

[0032] Comparative Example 2 50 ml of R2A medium was placed in a 250 ml Erlenmeyer flask, and 2% (v / v) of other natural sphingosine monocytogenes (TP01) was added. The flask was then incubated at 20 °C and 220 rpm, and samples were taken every 4 hours for OD monitoring.

[0033] Comparative Example 3 50 ml of R2A medium was placed in a 250 ml Erlenmeyer flask, and 2% (v / v) of other natural sphingosine monocytogenes (TP01) was added. The flask was then incubated at 26 °C and 220 rpm, and samples were taken every 4 hours for OD monitoring.

[0034] Comparative Example 4 50 ml of R2A medium was placed in a 250 ml Erlenmeyer flask, and 2% (v / v) of other natural sphingosine monocytogenes (TP02) was added. The flask was then incubated at 20 °C and 220 rpm, and samples were taken every 4 hours for OD monitoring.

[0035] Comparative Example 5 50 ml of R2A medium was placed in a 250 ml Erlenmeyer flask, and 2% (v / v) of other natural sphingosine monocytogenes (TP02) was added. The flask was then incubated at 26 °C and 220 rpm, and samples were taken every 4 hours for OD monitoring.

[0036] The growth of the three bacterial strains was tested at two different culture temperatures, and the results are as follows: Figure 1 As shown, the optimal growth temperature of Sphingosomalidobacterium XJL-TP07 is lower than that of other naturally occurring Sphingosomalidobacterium species.

[0037] Example 2 Sphingosomalidone XJL-TP07 co-fermented with Rhodiola rosea extract Sphingosine monocytogenes was picked from a test tube and inoculated into 50 mL of sterile R2A seed culture medium. After culturing at 25°C and 200 rpm for 13 h, the culture was allowed to reach the logarithmic growth phase.

[0038] 50 ml of R2A medium was placed in a 250 ml Erlenmeyer flask, and Sphingomonas XJL-TP07 was inoculated. The flask was then cultured at 20 °C and 220 rpm for 6 h. Once the OD reached 0.15, 2% Rhodiola rosea extract was inoculated. Samples were taken every 4 h to determine the OD600.

[0039] Comparative Example 6 50 ml of R2A medium was placed in a 250 ml Erlenmeyer flask, and Sphingomonas XJL-TP07 was inoculated. The flask was then cultured at 20 °C and 220 rpm for 6 h. Once the OD reached 0.15, 6% Rhodiola rosea extract was inoculated. Samples were taken every 4 h to determine the OD600.

[0040] Comparative Example 7 50 ml of R2A medium was placed in a 250 ml Erlenmeyer flask, and Sphingomonas XJL-TP07 was inoculated. The flask was then cultured at 20 °C and 220 rpm for 6 h. Once the OD reached 0.15, 10% Rhodiola rosea extract was inoculated. Samples were taken every 4 h to determine the OD600.

[0041] Comparative Example 8 50 ml of R2A medium was placed in a 250 ml Erlenmeyer flask. First, 2% Rhodiola rosea extract was added, and then Sphingomonas XJL-TP07 was inoculated. The flask was cultured at 20 °C and 220 rpm. Samples were taken every 4 hours to determine OD600.

[0042] The effects of different Rhodiola rosea extract addition strategies and ratios in Example 2 and Comparative Examples 6, 7, and 8 on the growth of Sphingomonas XJL-TP07 were investigated, and the results are as follows: Figure 2 As shown, with the same 2% Rhodiola rosea extract addition ratio, adding the extract before inoculation of Sphingospora XJL-TP07 significantly inhibits the growth of Sphingospora, making co-fermentation impossible. Adding the Rhodiola rosea extract after Sphingospora XJL-TP07 has grown to OD > 0.3, at a 2% addition ratio, has no effect on the strain's growth; however, a ratio of 10% also significantly inhibits the strain's growth, preventing co-fermentation.

[0043] Example 3

[0044] Cytotoxicity of co-fermentation products of Sphingomonas XJL-TP07 and Rhodiola rosea extract The purpose of cytotoxicity testing is as a basic safety screening method. It rapidly determines whether a raw material has a killing or damaging effect on cells through in vitro experiments, thereby determining its safe usage concentration and replacing animal testing to ensure product safety. The principle is to use a cultured cell model, where the raw material is brought into contact with cells, and the cell viability is quantified by detecting indicators such as metabolic activity (MTT assay) or membrane integrity, thus assessing the potential toxicity of the raw material. The experimental steps are as follows: 1) Cell seeding: Seed cells into 96-well plates at a seeding density of 1×10⁴ cells / well and incubate overnight in an incubator (37℃, 5% CO₂).

[0045] 2) Experimental Groups: The experiment included a zeroing group, a control group, a positive control group, and a sample group. In the sample group, each sample had 8 concentration gradients, and each concentration gradient had 3 replicate wells.

[0046] 3) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table.

[0047] Table 1 Test Concentration Setting Table

[0048]

[0049] 4) Sample loading: Load samples when the cell seeding rate in the 96-well plate reaches 40%–60%. Add 200 μL of culture medium containing 10% PBS to each well of the control group; add 200 μL of culture medium containing 10% DMSO to each well of the positive control group; add 200 μL of culture medium containing the corresponding concentration of the sample to each well of the sample group; add 200 μL of cell culture medium to each well of the zeroing group without cell seeding. After sample loading, place the 96-well plate in an incubator (37℃, 5% CO2) for incubation.

[0050] 5) Detection: After culturing cells for 24 hours, discard the supernatant, add MTT working solution (0.5 mg / mL), and incubate at 37°C in the dark for 4 hours. After incubation, discard the supernatant, add 100 µL LDMSO to each well, and read the OD value at 490 nm.

[0051] 6) Cell viability calculation: Calculated according to the formula. Cell viability = (sample well OD - zeroing well OD) / (solvent control well OD - zeroing well OD) × 100% Results: Table 2. Keratinocyte toxicity of co-fermentation products of Sphingomonas XJL-TP07 and Rhodiola rosea extract.

[0052]

[0053] As shown in Table 2, according to the MTT results, the mussel extract samples did not exhibit keratinocyte toxicity within a concentration range of 10.0000% (V / V). The co-fermentation product of *Sphingomonas* XJL-TP07 and *Rhodiola rosea* extract was not toxic to keratinocytes.

[0054] Example 4

[0055] Evaluation of the soothing efficacy of co-fermentation products of Sphingomonas XJL-TP07 and Rhodiola rosea extract Capsaicin (CAP) is the active ingredient in chili peppers. Contact with the human body produces a burning sensation, primarily because capsaicin activates the TRPV1 sensory receptor, transmitting signals to the central nervous system and causing burning and other discomfort. This test utilizes a CAP-stimulated human immortalized keratinocyte (HaCaT) model. Immunofluorescence technology is used to detect changes in TRPV1 content in keratinocytes to evaluate the soothing efficacy of the tested product. Samples included co-fermentation products of Rhodiola rosea extract and Sphingosine monocytogenes, co-fermentation products of Rhodiola rosea extract and yeast, and co-fermentation products of Rhodiola rosea extract and lactic acid bacteria. The experimental design is as follows: Table 3 Experimental Design for Evaluating Soothing Efficacy

[0056]

[0057] The experimental steps are as follows: 1) Cell seeding: at 1×10 5 Cells were seeded at a density of cells per well into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0058] 2) Solution preparation: Prepare working solutions of different concentrations of the test substance according to the experimental design table.

[0059] 3) Sample loading: According to the experimental design table, when the cell seeding rate in the 24-well plate reaches 40%~60%, samples are loaded in groups, with 1.0 mL loaded per well and 3 replicates per group. The cells are then incubated in an incubator (37℃, 5% CO2) for 24 hours.

[0060] 4) Sample collection: Discard the supernatant and rinse the cells 3 times with PBS.

[0061] 5) Immunofluorescence staining: Perform the routine immunofluorescence staining procedure. The main steps are: fixation, blocking, addition of primary antibody, addition of secondary antibody, and then taking pictures using a fluorescence microscope.

[0062] 6) Results analysis: The fluorescence intensity of TRPV1 was quantitatively analyzed using ImageProPlus software.

[0063] See attached document Figure 3 The results showed that, compared with the blank control group, the fluorescence intensity of TRPV1 protein in keratinocytes of the negative control group was significantly increased after capsaicin stimulation (p<0.01), indicating that the CAP stimulation model was successfully established in this experiment.

[0064] Compared with the negative control group, the fluorescence intensity of TRPV1 protein in keratinocytes of the positive control group was significantly reduced (p<0.01), indicating that the positive control detection was effective.

[0065] Compared with the negative control group, the fluorescence intensity of TRPV1 protein in keratinocytes was significantly reduced at a concentration of 5% (V / V) in the co-fermentation product of Rhodiola rosea extract and Sphingospora. The fluorescence intensity of the co-fermentation product of Rhodiola rosea extract and Sphingospora was also significantly reduced compared with the co-fermentation product of Rhodiola rosea extract and lactic acid bacteria or yeast (p<0.01).

[0066] Conclusion: Based on the CAP-stimulated keratinocyte (HaCaT) model, when the co-fermentation product of Rhodiola rosea extract and Sphingosine monocytogenes was 5% (V / V), the fluorescence intensity of TRPV1 protein in keratinocytes was significantly reduced, and the difference was statistically significant compared with the negative control group and the other two experimental groups (p < 0.01), indicating that the co-fermentation product of Rhodiola rosea extract and Sphingosine monocytogenes has a better soothing effect.

[0067] Example 5 Evaluation of the antioxidant efficacy of co-fermentation products of Sphingomonas XJL-TP07 and Rhodiola rosea extract Skin is frequently exposed to various external stimuli, such as ultraviolet radiation, air pollutants, toxic chemicals, and physical or mechanical damage. These exposures directly or indirectly increase the levels of reactive oxygen species (ROS) in the skin, disrupting the skin's internal environmental balance and leading to oxidative stress. Long-term cumulative effects can result in skin inflammation, aging (photoaging), and even cancer. Therefore, effective antioxidant measures are crucial for maintaining skin health. This test uses a human immortalized keratinocyte (HaCaT) model. An in vitro oxidative damage cell model was established by inducing ROS production through UVB irradiation. The antioxidant capacity of the tested samples was evaluated by detecting their ability to inhibit ROS. Samples included co-fermentation products of Rhodiola rosea extract and Sphingosine monocytogenes, co-fermentation products of Rhodiola rosea extract and yeast, and co-fermentation products of Rhodiola rosea extract and lactic acid bacteria. The experimental design is as follows: Table 4 Experimental Design for Evaluation of Antioxidant Efficacy

[0068]

[0069] The experimental steps are as follows: 1) Cell seeding: Place sterile cell slides at the bottom of a 24-well plate at an appropriate seeding density (1×10⁻⁶). 5 Cells were seeded into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0070] 2) Experimental grouping: The experiment was set up with blank control group, negative control group, positive control group and sample group.

[0071] 3) Solution preparation: Use cell culture medium to prepare working solutions of different concentrations of the test substance according to the concentrations listed in the experimental design.

[0072] 4) Irradiation: Irradiation was carried out when the cell deposition rate in the 24-well plates reached 40%~60%. The total dose received by the negative control group, positive control group, and sample group was 300mJ / cm². 2 The system was exposed to UVB radiation, while a control group was placed in the same environment (UVB radiation dose of 0 J / cm²). 2 ).

[0073] 5) Sample loading: After irradiation, remove the original cell culture medium and load the samples into groups. For the experimental group, add 1.0 mL of sample working solution to each well, with three replicates for each concentration. For the control group (BC group), add 1.0 mL of cell culture medium to each well, with three replicates. Incubate in an incubator (37℃, 5% CO2) for 24 hours.

[0074] 6) Sample collection: Discard the supernatant and rinse the cells 3 times with PBS.

[0075] 7) Immunofluorescence staining: Perform the routine immunofluorescence staining procedure. The main steps are: fixation, blocking, adding primary antibody, adding secondary antibody, DAPI counterstaining, mounting, and then taking pictures using a fluorescence microscope.

[0076] 8) The fluorescence intensity of FLG was quantitatively analyzed using ImageProPlus software.

[0077] See attached document Figure 4 The results showed that, compared with the blank control group, the FLG content in the negative control group was significantly lower (p<0.01), indicating that the UVB stimulation model was successfully established in this experiment.

[0078] Compared with the negative control group, the FLG content in the positive control group was significantly increased at the loading concentration of 100 μg / mL VC + 7 μg / mL VE (p < 0.01), indicating that the positive control in this experiment was effective.

[0079] Compared with the negative control group, the FLG content of the co-fermentation product of Rhodiola rosea extract and Sphingosomalidone was significantly increased at a concentration of 5% (V / V) (p<0.01); the FLG content of the co-fermentation product of Rhodiola rosea extract and Sphingosomalidone was significantly increased compared with the co-fermentation product of Rhodiola rosea extract and yeast (p<0.01).

[0080] Therefore, based on the UVB-stimulated human immortalized keratinocyte (HaCaT) model, the mussel extract sample showed a significant increase in the content of filaggrin (FLG), a cell barrier-related protein, at a concentration of 5% (V / V), which was statistically different from the negative control group or the yeast experimental group (p<0.01), indicating that the co-fermentation product of Rhodiola rosea extract and Sphingosine monocytogenes had better repair effects.

[0081] As mentioned in the background section, although Rhodiola rosea is rich in various highly active ingredients such as rhodioloside and flavonoids, its natural extraction efficiency is low, its bioavailability is not high, and it is limited by traditional extraction processes, resulting in a limited range of product components, thus failing to fully realize its cosmetic and skincare potential. This invention addresses this core problem by creatively employing a synergistic strategy combining "enzymatic pretreatment" and "specific microbial co-fermentation." First, Rhodiola rosea root powder is enzymatically hydrolyzed using carefully selected compound enzyme preparations (cellulase, pectinase, and papain), which efficiently and gently disrupts the plant cell wall structure, maximizing the release of intracellular active substances and providing a rich and easily usable precursor library for subsequent microbial fermentation. Then, the powerful biotransformation capabilities of Sphingomonas XJL-TP07 are utilized to further decompose, modify, and transform the components in the Rhodiola rosea extract. This biotransformation not only degrades macromolecules into smaller, more easily absorbed skin-friendly active peptides and oligosaccharides, but also generates new, more active metabolites through reactions such as hydroxylation and glycosylation. Therefore, this co-fermentation method is not a simple mixing of components, but a process of deep processing and enhancement, which significantly improves the types, content and bioavailability of active ingredients in the final product, fundamentally overcoming the limitations of extraction efficiency and efficacy in the traditional application of Rhodiola rosea.

[0082] Furthermore, in existing technologies, the fermentation of Rhodiola rosea generally uses Lactobacillus or yeast. These strains are not natural symbiotic bacteria of Rhodiola rosea in its natural ecological environment, thus resulting in limited substrate decomposition capacity, a single biotransformation pathway, and the potential production of irritating metabolites such as ethanol or histamine. This invention takes a unique approach, selecting Sphingomonas XJL-TP07, isolated from a specific environment. This strain has a natural ecological connection with the alpine plant Rhodiola rosea, and as its rhizosphere symbiotic bacteria, it exhibits superior metabolic compatibility and transformation efficiency for specific components contained in Rhodiola rosea. Comparative experimental data (e.g.) Figure 2This study fully demonstrates that the strain can grow well in a specific ratio of Rhodiola rosea extract, while traditional strategies of adding substrate before inoculation or adding high-concentration substrate significantly inhibit bacterial growth. This highlights the rationality of the "first incubate bacteria, then co-ferment" process designed in this invention and the specific advantages of the strain. Compared with lactic acid bacteria or yeast fermentation, Sphingomonas does not produce endotoxins or large amounts of lactic acid or ethanol. Its fermentation product itself has higher skin mildness and safety, which was verified in the cytotoxicity test (Example 3). More importantly, efficacy evaluation (Examples 4 and 5) shows that the co-fermentation product is significantly better than the product obtained by lactic acid bacteria or yeast fermentation in terms of soothing (inhibiting TRPV1 expression) and antioxidant repair (increasing FLG protein content). This proves that Sphingomonas and Rhodiola rosea components produce a unique synergistic effect that surpasses that of conventional strains, providing a new solution for developing highly effective and mild "green anti-aging" cosmetic raw materials.

[0083] This invention lies not only in the innovation of strains and formulations, but also in the meticulous design and control of the entire preparation process, thereby transforming the theoretical advantages of co-fermentation into high-quality products suitable for industrial production. Starting with raw material pretreatment, low-temperature ultra-micro cell disruption technology is employed to maximize the retention of heat-sensitive active ingredients. During enzymatic hydrolysis, temperature, pH, and enzyme activity ratio are precisely controlled to ensure efficiency and consistency. In the fermentation stage, the timing of inoculation, temperature range, and the strategy for supplementing Rhodiola rosea extract are precisely controlled to create optimal conditions for cell growth and effective metabolism. In the post-fermentation processing, high-pressure homogenization is used to disrupt cells, efficiently releasing intracellular active substances (such as sphingolipids, polysaccharides, and peptides). The product is then subjected to fractionation centrifugation and sterile filtration through a 0.22μm microporous membrane, ultimately yielding a sterile, clear liquid product. This series of rigorous process steps not only ensures the comprehensive activity of the fermentation product, combining the transformed components of Rhodiola rosea with the microbial metabolites, but also guarantees the product's high purity and excellent physical stability. This allows it to be easily integrated into various cosmetic formulations, directly exerting multiple skincare and beauty benefits such as soothing, anti-oxidation, barrier repair, and anti-aging without complex post-processing. In summary, this invention provides a complete technical solution from strain selection and fermentation to post-processing, successfully preparing an innovative cosmetic raw material with high efficiency, high safety, and promising application prospects, powerfully promoting the in-depth development and application of Rhodiola rosea in the high-end skincare field.

[0084] The above-described embodiments are merely illustrative of certain implementations of the present invention, and are described in a relatively specific and detailed manner. However, 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 are all within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for co-fermentation of Sphingomonas and Rhodiola rosea extract, characterized in that: The method involves using Sphingomonas paucimobilis XJL-TP07 (accession number CGMCC:35131) as the fermentation strain, using a culture medium containing Rhodiola rosea extract as the fermentation substrate, and obtaining the fermentation broth after fermentation culture. The fermentation broth is then filtered through particulate matter, the whole bacteria are broken up, centrifuged, and the supernatant is collected. Finally, the fermentation product is obtained by sterilization filtration.

2. The co-fermentation method based on Sphingomonas and Rhodiola rosea extract according to claim 1, characterized in that: The fermentation broth is prepared as follows: Step (1) Prepare Rhodiola rosea extract Rhodiola rosea root is ground into powder, mixed with water, and then enzymes are added for enzymatic hydrolysis. The enzymatic hydrolysis is continued until the endpoint is reached, and then the temperature is raised to inactivate the enzymes to end the enzymatic hydrolysis reaction. After sterilization and filtration, Rhodiola rosea extract is obtained. Step (2) Add Rhodiola rosea extract Sphingomonas paucimobilis XJL-TP07 was inoculated into the culture medium for fermentation at an inoculation rate of 1%-10%, and fermented at 15-30℃ for 4-12 hours. Rhodiola rosea extract was added to the culture medium at a fermentation volume of 1-10%. Step (3) Co-fermentation of Sphingomonas and Rhodiola Rosea extract The Sphingomonas fermentation broth inoculated with Rhodiola rosea extract was fermented for 16–42 hours at 15–30°C to obtain the fermentation broth.

3. The co-fermentation method based on Sphingomonas and Rhodiola rosea extract according to claim 2, characterized in that: Specifically After filtering the fermentation broth through gauze to remove insoluble particles, the cells were disrupted by a high-pressure homogenizer at 850 Bar for 3 cycles. The disrupted broth was centrifuged at 10000g for 10 min at 4°C. The supernatant obtained after separation and precipitation was first filtered through a 0.45μm filter membrane and then through a 0.22μm filter membrane. The resulting sterile filtrate was the fermentation product.

4. The co-fermentation method based on Sphingomonas and Rhodiola rosea extract according to claim 2, characterized in that: In step (1), the Rhodiola rosea is crushed and then mixed with deionized water in a ratio of (0.5-3):

100.

5. The co-fermentation method based on Sphingomonas and Rhodiola rosea extract according to claim 2, characterized in that: The pulverization operation in step (1) specifically involves using a low-temperature ultra-micro cell-wall breaking pulverizer, pulverizing at 4-10℃ for 10 minutes, and then storing at 4℃.

6. The co-fermentation method based on Sphingomonas and Rhodiola rosea extract according to claim 2, characterized in that: In step (1), enzymes are added for enzymatic hydrolysis. The enzyme preparations used for enzymatic hydrolysis include cellulase, pectinase, papain, and mixtures thereof.

7. The co-fermentation method based on Sphingomonas and Rhodiola rosea extract according to claim 6, characterized in that: The ratio of the enzyme activity of the enzyme preparation to the mass of the Rhodiola rosea root powder is 500,000 U to 5,000,000 U: 1 kg.

8. The co-fermentation method based on Sphingomonas and Rhodiola rosea extract according to claim 6, characterized in that: The enzymatic hydrolysis reaction is carried out at a temperature of 40-55℃ for 2-24 hours, with the pH controlled at 4.0-5.

0.

9. The fermentation product prepared by the co-fermentation method based on *Sphingomonas* and *Rhodiola rosea* extract according to claims 1-8, characterized in that: The application of the fermentation product in the preparation of skin care and beauty cosmetics.

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