Method for preparing dual probiotic fermented exosome with multiple skin care functions and active delivery applications

Abstract

This invention relates to a method for preparing exosomes using dual-strain probiotic fermentation and their application in delivering active ingredients, possessing multiple skincare benefits. The dual-strain probiotics used in this invention include *Lactobacillus plantarum* (CGMCC 24962) and *Bacillus subtilis* (CGMCC 36746). This invention employs both lactic acid bacteria and *Bacillus subtilis* as co-fermentation strains. Both are widely used probiotics with excellent fermentation characteristics and sufficient safety. Furthermore, the exosomes obtained after co-fermentation exhibit synergistic activity, superior to those obtained by fermenting either strain alone. Through the synergistic effect of the dual-strain probiotics, this invention enhances the transdermal absorption efficiency of active ingredients and endows the exosomes with diverse skincare benefits, meeting the high-performance requirements of skincare products.

Description

Technical Field

[0001] This invention relates to the field of exosome preparation technology, and in particular to a method for preparing dual-probiotic fermented exosomes with multiple skin care benefits and its application in delivering active ingredients. Background Technology

[0002] As the skincare industry increasingly focuses on bioactive ingredients, the skin absorption efficiency and synergistic effects of active ingredients (such as antioxidants, peptides, and vitamins) have become core pain points. In traditional skincare products, active ingredients are easily hindered by the skin barrier (stratum corneum), resulting in low transdermal absorption efficiency and poor bioavailability, which makes it difficult to fully realize their skincare effects. At the same time, the skincare effects of a single active ingredient are limited, making it difficult to meet consumers' comprehensive needs for multiple effects such as moisturizing, anti-inflammation, repair, and brightening.

[0003] Probiotic exosomes, as nanoscale vesicle structures secreted by probiotics, are gradually becoming novel active carriers and functional ingredients in the skincare field due to their rich content of bioactive substances such as lipids, proteins, and nucleic acids, as well as their good biocompatibility and skin penetration. Current technologies for preparing probiotic exosomes mostly focus on single strains, resulting in limited skincare efficacy. Furthermore, their function as carriers for active ingredients fails to achieve a synergistic effect between "carrier delivery" and "their own efficacy." Summary of the Invention

[0004] To address the technical challenges in probiotic exosome preparation technology, such as limited efficacy, limited active ingredient delivery efficiency, and low active ingredient loading efficiency, this invention provides a method for preparing binary probiotic fermented exosomes with multiple skincare benefits and its application in active ingredient delivery. This invention, through the synergistic effect of binary strains, not only enhances the transdermal absorption efficiency of active ingredients but also endows the exosomes themselves with diverse skincare benefits, meeting the high-performance requirements of skincare products.

[0005] The first objective of this invention is to provide a binary probiotic, comprising *Lactobacillus plantarum* with accession number CGMCC 24962 and *Bacillus subtilis* with accession number CGMCC 36746, classified as *Bacillus subtilis*, and self-named JJL-SF09. The *Bacillus subtilis* with accession number CGMCC 36746 is deposited at the China General Microbiological Culture Collection Center, with a deposit date of November 24, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] A second objective of this invention is to provide a method for preparing exosomes by fermentation of the aforementioned binary probiotics, comprising the following steps: Seed culture of Lactobacillus plantarum and Bacillus subtilis are provided respectively; Seed cultures of Lactobacillus plantarum and Bacillus subtilis were inoculated into a fermentation medium and fermented to obtain a fermentation broth. The fermentation broth was centrifuged for the first time to obtain the first supernatant. The obtained supernatant was filtered through a filter membrane to obtain the filtrate. The obtained filtrate was centrifuged for the second time to obtain the precipitate and the second supernatant. The precipitate is the exosome, and the second supernatant was collected.

[0007] In some embodiments of the present invention, the inoculation density of the seed solution is 1-5%.

[0008] In some embodiments of the present invention, the fermentation temperature is 20-37°C and the fermentation time is 24-72 h.

[0009] In some embodiments of the present invention, the parameters for the first centrifugation are: 0-10℃, 2000-10000 g centrifugation for 5-20 min; the filter membrane is 0.22-0.45 μm; the parameters for the second centrifugation are: 0-10℃, 50000-200000 g centrifugation for 10-60 min.

[0010] A third objective of this invention is to provide an exosome prepared by the method described above.

[0011] A fourth objective of the present invention is to provide an active substance delivery system, comprising the exosomes and an active substance, prepared by means of the following method: resuspending the exosomes in a second supernatant obtained during the preparation of the exosomes, adding the active substance and mixing to obtain exosomes loaded with the active substance, i.e., the active substance delivery system.

[0012] In some embodiments of the present invention, the active ingredient includes one or more of vitamin C, vitamin C ethyl ether, ascorbate glucoside, nicotinamide, panthenol, hyaluronic acid, collagen, PDRN, Centella asiatica extract, aloe vera extract, Dendrobium nobile extract, Portulaca oleracea extract, polypeptide, tranexamic acid, and camellia extract.

[0013] A third objective of this invention is to provide a skincare product comprising exosomes prepared by the above method, the exosomes, or the active ingredient delivery system.

[0014] In some embodiments of the present invention, the skin care product has the following effects: anti-wrinkle and firming, whitening, soothing, repairing and moisturizing.

[0015] Dual-component probiotic fermentation: This method uses co-fermentation of lactic acid bacteria and Bacillus subtilis. The exosomes prepared by this dual-component probiotic fermentation achieve synergistic effects, which is superior to exosomes obtained by fermentation of single probiotics, and also superior to simple mixtures of exosomes obtained by fermentation of single probiotics.

[0016] When using binary probiotic exosomes for active ingredient delivery, adding the fermentation broth of binary probiotics during the incubation process of exosomes and active ingredients can improve the encapsulation rate of the active ingredients.

[0017] Compared to the method of preparing exosomes by fermentation with a single strain, exosomes prepared by fermentation with binary probiotics are more conducive to the encapsulation of active ingredients.

[0018] The technical solution of the present invention has the following advantages compared with the prior art: 1. Diverse probiotic fermentation enhances skincare benefits. This invention employs lactic acid bacteria and Bacillus subtilis as co-fermentation strains. Both are widely used probiotics with excellent fermentation characteristics and sufficient safety. Furthermore, the exosomes obtained after co-fermentation exhibit synergistic activity, superior to those obtained from fermenting either strain alone. Simultaneously, the strong surfactant (surfactant, iturobrine) secretion capacity of Bacillus subtilis CGMCC36476 is utilized, promoting the loading of active substances into the exosomes. This results in the co-fermentation exosomes carrying more active ingredients from both lactic acid bacteria and Bacillus subtilis, while also enabling the exosomes to carry more water-soluble active ingredients. On the other hand, the prebiotics produced by lactic acid bacteria fermentation promote the growth and active substance secretion of Bacillus subtilis.

[0019] 2. Higher active ingredient loading rate and better penetration effect. The core innovations of this invention are: 1) co-fermentation of lactic acid bacteria and Bacillus subtilis; 2) loading of active substances onto exosomes using a mixture of fermentation supernatant. These two points result in a significantly higher loading efficiency for active substances compared to traditional incubation methods.

[0020] 3. Promotes the absorption of active ingredients Biological probiotic exosomes not only have excellent skin care effects themselves, but can also load active ingredients and promote the penetration and absorption of active ingredients, thereby achieving synergistic enhancement of skin care effects. Attached Figure Description

[0021] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 These are transmission electron microscope images of the exosomes of lactic acid bacteria and Bacillus subtilis of this invention; Figure 2This is a comparison of the effects of various exosomes on promoting Collagen I production according to the present invention; Figure 3 This is a comparison of the effects of various exosomes on inhibiting IL-1α production in this invention; Figure 4 This is a comparison of the encapsulation rates of various encapsulation methods of the present invention; Figure 5 This invention compares the permeation and absorption effects of ExoDi-VCE and VCE aqueous solutions. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0023] The probiotics used in this invention include *Lactobacillus plantarum* (disclosed in patent CN115948442A) with accession number CGMCC 24962 and *Bacillus subtilis* (preserved at the China General Microbiological Culture Collection Center, deposited on November 24, 2025, at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing) with accession number CGMCC 36746.

[0024] All reagents and materials used in this invention are commercially available. The cells used for detection in this invention, such as human fibroblasts and RAW264.7 macrophages, are all conventional cells in the field and are commercially available. Example 1:

[0025] This embodiment provides a method for preparing exosomes of lactic acid bacteria and Bacillus subtilis (preparation of binary probiotic exosomes), as detailed below: Seed culture: Lactic acid bacteria CGMCC 24962 was inoculated into lactic acid bacteria seed culture medium with the following formulation: glucose 10 g / L, rice 20 g / L. After inoculation, it was incubated at 30℃ for 12 h. Bacillus subtilis CGMCC 36746 was inoculated into LB medium with the following formulation: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L. After inoculation, it was incubated at 30℃ for 12 h.

[0026] Lactic acid bacteria were inoculated into the fermentation medium at 2% and Bacillus subtilis at 1%. The fermentation medium consisted of 50 g / L brown sugar, 20 g / L rice flour, 5 g / L yeast extract, and 5 g / L soybean peptone. The mixture was incubated at 30°C for 48 h to obtain the fermentation broth.

[0027] The fermentation broth was centrifuged at 4℃ and 5000 g for 10 min. The supernatant obtained after separating the bacterial cells was filtered through a 0.22 μm filter membrane and then centrifuged at 4℃ and 100000 g for 20 min. The resulting precipitate was the exosome of the binary probiotic. The supernatant was collected as the binary probiotic fermentation broth.

[0028] Exosome morphology analysis: Exosomes obtained after fermentation were observed using transmission electron microscopy (TEM). The results are as follows: Figure 1 As shown, the exosomes are spherical with a double membrane structure and a particle size of 30-130 nm.

[0029] Comparative Example 1: This comparative example provides a method for preparing lactic acid bacteria exosomes, as detailed below: Seed culture: Lactic acid bacteria CGMCC 24962 was inoculated into lactic acid bacteria seed culture medium, the formulation of which was: glucose 10 g / L, rice 20 g / L. After inoculation, the culture was carried out at 30℃ for 12 h.

[0030] Lactic acid bacteria were inoculated into the fermentation medium at a concentration of 2%. The fermentation medium consisted of 50 g / L brown sugar, 20 g / L rice flour, 5 g / L yeast extract, and 5 g / L soybean peptone. The mixture was incubated at 30°C for 48 h to obtain the fermentation broth.

[0031] The fermentation broth was centrifuged at 5000 g for 10 min at 4 °C. The supernatant obtained after separating the bacterial cells was filtered through a 0.22 μm filter membrane and then centrifuged at 100000 g for 20 min at 4 °C. The resulting precipitate was the lactic acid bacteria exosomes, and the supernatant was collected as the lactic acid bacteria fermentation broth.

[0032] Comparative Example 2: This comparative example provides a method for preparing Bacillus subtilis exosomes, as detailed below: Seed culture: Bacillus subtilis CGMCC 36746 was inoculated into LB medium, the formulation of which was: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L. The medium was incubated at 30℃ for 12 h after inoculation.

[0033] Bacillus subtilis was inoculated into the fermentation medium at a concentration of 1%. The fermentation medium consisted of 50 g / L brown sugar, 20 g / L rice flour, 5 g / L yeast extract, and 5 g / L soybean peptone. The mixture was incubated at 30°C for 48 h to obtain the fermentation broth.

[0034] The fermentation broth was centrifuged at 5000 g for 10 min at 4 °C. The supernatant obtained after separating the bacterial cells was filtered through a 0.22 μm filter membrane and then centrifuged at 100000 g for 20 min at 4 °C. The resulting precipitate was the Bacillus subtilis exosome.

[0035] Comparative Example 3: This comparative example provides a method for preparing mixed exosomes from mechanically mixed lactic acid bacteria exosomes and Bacillus subtilis exosomes. The lactic acid bacteria exosomes obtained in Comparative Example 1 and the Bacillus subtilis exosomes obtained in Comparative Example 2 were mechanically mixed at a mass ratio of 1:1.

[0036] Test Example 1: Efficacy in promoting collagen synthesis The exosomes obtained in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were used to determine their collagen-promoting efficacy. Human fibroblasts were used as the test system to measure changes in type I collagen (Collagen I) content before and after treatment. The specific measurement method was as follows: 1) Cell seeding: Seed at an appropriate seeding density (8 × 10⁻⁶). 4 Human fibroblasts were seeded into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0037] 2) Experimental grouping: The experiment included a blank control group, a negative control group, a positive control group, and a sample group. The sample group consisted of 0.01 wt% exosomes from Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0038] 3) Irradiation: Irradiation was carried out when the cell deposition rate in the 24-well plates reached 40%–60%. The negative control group, positive control group, and sample group received a dose of 9 J / cm². 2 The control group was placed under UVA radiation, while the blank control group was placed in the same environment (UVA radiation dose of 0 J / cm²). 2 ).

[0039] 4) Processing: After irradiation, the following processing was performed: 1 mL of cell culture medium was added to each well of the blank control group and negative control group; 1 mL of culture medium containing 100 μg / mL VC and 7 μg / mL VE was added to each well of the positive control group; and 1 mL of culture medium containing the corresponding concentration of exosome test substance was added to each well of the sample group.

[0040] 5) Collect cell supernatant: After 12 h of sample processing, collect the cell culture supernatant in EP tubes and store it frozen at -80℃.

[0041] 6) Collagen I detection: The content of Collagen I was detected and analyzed according to the operating instructions of the ELISA detection kit.

[0042] The measurement results are shown below. Figure 2As can be seen from the figure, the exosomes (binary probiotic exosomes) Collagen I prepared by Example 1 are far superior to the exosomes prepared by fermentation using a single strain in Comparative Examples 1 and 2, and are also far superior to the effect of simply mixing exosomes of a single strain in Comparative Example 3.

[0043] Test Example 2: Determination of Soothing Efficacy The exosomes obtained in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were used to determine their soothing efficacy. Based on the LPS-stimulated RAW264.7 macrophage model, the soothing efficacy of the tested samples was evaluated by detecting changes in the levels of the inflammatory cytokine IL-1α secreted by the cells. The specific method was 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).

[0044] 2) Induction and Drug Administration: When the cell deposition rate in the 24-well plates reached 40%–60%, 100 μL of 10× LPS working solution was added to each well according to the experimental design. The wells were shaken left and right to mix the drug. Simultaneously, the sample was added. The final LPS concentration was 1 μg / mL, with three replicates per group. After drug administration, the wells were placed in an incubator (37℃, 5% CO2) and cultured for another 24 h. The sample included 0.01 wt% exosomes from Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0045] 3) Sample collection: After incubation, collect the cell culture supernatant into EP tubes (Note: Determine the amount of sample to be collected according to the detection indicators). After collection, freeze the sample in a -80℃ freezer.

[0046] 4) IL-1α content detection: The detection was performed according to the instructions of the Mouse IL-1α ELISA kit.

[0047] The measurement results are shown below. Figure 3 As can be seen from the figure, the exosomes prepared in Example 1 can significantly reduce the IL-1α content, and are superior to the exosomes prepared by fermentation using a single strain in Comparative Examples 1 and 2, and are also far superior to the effect of simply mixing exosomes of a single strain in Comparative Example 3.

[0048] Example 2: This embodiment provides an application for the delivery of active substances from binary probiotic exosomes, as detailed below: The exosomes prepared in Example 1 were resuspended and diluted 5 times with the binary probiotic fermentation broth harvested in Example 1. 10 wt% VC ethyl ether was added and mixed well. The mixture was incubated at 37°C for 30 min and then centrifuged at 4°C and 100,000 g for 20 min. The precipitate was harvested to obtain binary probiotic exosomes loaded with VC ethyl ether, which was named ExoDi-VCE.

[0049] Comparative Example 4: This comparative example provides a method for preparing active ingredients without using binary probiotic fermentation broth for delivery, as detailed below: The exosomes prepared in Example 1 were resuspended and diluted 5 times with deionized water, and then mixed with 10 wt% VC ethyl ether. The mixture was incubated at 37°C for 30 min, and then centrifuged at 4°C and 100,000 g for 20 min. The precipitate was harvested to obtain binary probiotic exosomes loaded with VC ethyl ether, which was named ExoDiC-VCE.

[0050] Comparative Example 5: This comparative example provides a method for preparing active substances delivered by lactic acid bacteria exosomes, as detailed below: Take the exosomes prepared in Comparative Example 1, resuspend and dilute them 5 times with deionized water, add 10% VC ethyl ether to the final concentration, mix well, incubate at 37℃ for 30 min, and then centrifuge at 4℃ and 100,000 g for 20 min to collect the precipitate and obtain binary probiotic exosomes loaded with VC ethyl ether, named ExoPL-VCE.

[0051] Test Example 3: Comparison of encapsulation efficiency of exosome active ingredients in Example 2, Comparative Example 4, and Comparative Example 5 The encapsulation efficiency of Example 2 and Comparative Examples 4 and 5 was determined respectively. The content of VC ethyl ether was determined by liquid chromatography. The encapsulation efficiency was calculated using the formula: Encapsulation efficiency = (W... 总量 -W 上清 ) / W 总量 ×100%. The measurement results are as follows: Figure 4 As shown in the figure, the encapsulation efficiency of the active ingredient encapsulation method in Example 2 is far superior to that of the active ingredient encapsulation methods in Comparative Examples 4 and 5.

[0052] Test Example 4: Determination of Active Ingredient Delivery Efficiency in Example 2 Test method: The analysis and determination were performed according to GB / T 27818-2011, "In vitro test method for skin absorption of chemicals". Piglet skin was fixed between the diffusion chamber and the receiving chamber of a Franz cell diffusion cell, with the stratum corneum facing the diffusion chamber and the dermis facing the receiving chamber. The test sample was ExoDi-VCE prepared in Example 2, and the control sample was a PBS solution containing the same concentration of VC ethyl ether (named: VCE aqueous solution). The test results are as follows: Figure 5As shown in the figure, at 4 h, the cumulative permeability of ExoDi-VCE is 6.04 times that of the VCE aqueous solution. This result demonstrates that the binary probiotic exosomes loaded with active ingredients prepared using Example 2 not only have a high encapsulation efficiency but also exhibit excellent permeation performance.

[0053] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention. It is classified and named Bacillus subtilis, and self-designated as JJL-SF09.

Claims

1. A dual-component probiotic, characterized in that, This includes Lactobacillus plantarum with accession number CGMCC 24962 and Bacillus subtilis with accession number CGMCC 36746.

2. A method for preparing exosomes by fermentation of binary probiotics as described in claim 1, characterized in that, Includes the following steps: Seed culture of Lactobacillus plantarum and Bacillus subtilis are provided respectively; Seed cultures of Lactobacillus plantarum and Bacillus subtilis were inoculated into a fermentation medium and fermented to obtain a fermentation broth. The fermentation broth was centrifuged for the first time to obtain the first supernatant. The first supernatant was filtered through a filter membrane to obtain the filtrate. The filtrate was centrifuged for the second time to obtain the precipitate and the second supernatant. The precipitate is the exosome, and the second supernatant was collected.

3. The method according to claim 2, characterized in that, The inoculation density of the seed solution is 1-5%.

4. The method according to claim 2, characterized in that, The fermentation temperature is 20-37℃, and the fermentation time is 24-72 h.

5. The method according to claim 2, characterized in that, The parameters for the first centrifugation separation are: 0-10℃, 2000-10000 g for 5-20 min. The filter membrane has a thickness of 0.22-0.45 μm; The parameters for the second centrifugation were: 0-10℃, 50,000-200,000 g for 10-60 min.

6. An exosome, characterized in that, Exosomes prepared by the method described in any one of claims 2 to 5.

7. An active ingredient delivery system, characterized in that, The system comprises the exosomes and active ingredient as described in claim 6; it is prepared by the following method: resuspending the exosomes in the second supernatant obtained during the exosome preparation process, adding the active ingredient and mixing to obtain exosomes loaded with the active ingredient, i.e., the active ingredient delivery system.

8. The active ingredient delivery system according to claim 7, characterized in that, The active ingredients include one or more of the following: vitamin C, vitamin C ethyl ether, ascorbate glucoside, nicotinamide, panthenol, hyaluronic acid, collagen, PDRN, centella asiatica extract, aloe vera extract, dendrobium extract, purslane extract, polypeptide, tranexamic acid, and camellia extract.

9. A skincare product, characterized in that, It includes exosomes prepared by the method of any one of claims 2 to 5, the exosomes of claim 6, and the active ingredient delivery system of claim 7 or 8.

10. The skincare product according to claim 9, characterized in that, The skincare products are said to have one or more of the following effects: anti-wrinkle and firming, whitening, soothing, repairing, and moisturizing.

Images