All-natural biomimetic microcapsules carrying water-soluble active substance oil and preparation method and application thereof

Abstract

The present application relates to a kind of all natural biomimetic microcapsule carrying water-soluble active substance oil and its preparation method and application.The present application constructs all natural biomimetic microcapsule, encapsulates water-soluble active ingredient in its internal hydrophilic cavity, then integrates it into natural vegetable oil, so that the stable coexistence and directional carrying of water and oil components are realized on microscale.The compatibility problem of water-soluble active substance and natural oil is solved, and the natural properties of the formula are retained.

Description

Technical Field

[0001] This invention relates to the field of water-soluble active ingredient efficacy oil technology, and in particular to an all-natural biomimetic microcapsule carrying water-soluble active ingredient efficacy oil, its preparation method and application. Background Technology

[0002] In recent years, the concept of "oil-based skincare" has rapidly evolved from a niche concept into a mainstream trend in the skincare market due to its core functions of repairing the skin barrier and maintaining water-oil balance, spurring a surge in demand for facial serums and skincare oils. The core driver of market growth lies in consumers' pursuit of "natural" and "pure" formulas, making natural plant oils highly sought after for their excellent skin affinity and safety. However, their bioactivity is relatively limited and weak, often falling short in achieving advanced effects such as whitening, anti-wrinkle, and precise repair. To enhance the overall efficacy of products, the industry generally seeks to introduce highly active water-soluble ingredients (such as vitamin C, niacinamide, hyaluronic acid, and peptides) into oil systems. However, the incompatibility of water and oil prevents these water-soluble active ingredients from dissolving in oils. Traditional methods use chemically synthesized emulsifiers to solubilize these ingredients, but this sacrifices the natural properties of the formula and can introduce irritation. Summary of the Invention

[0003] To address the technical challenge of compatibility between water-soluble active ingredients and natural oils, this invention provides an all-natural biomimetic microcapsule-loaded functional oil containing water-soluble active ingredients, along with its preparation method and applications. This invention constructs all-natural biomimetic microcapsules, encapsulating water-soluble active ingredients within their internal hydrophilic cavities, and then integrating them into natural plant oils, thereby achieving stable coexistence and targeted loading of water and oil components at the microscopic scale.

[0004] The first objective of this invention is to provide a Bacillus species that produces high levels of surfactants, using Bacillus subtilis CGMCC No. 36746 as the starting strain, overexpressing one or more of the regulatory factors ComX, ComA, PhrC, and Spo0A; and / or inhibiting the expression of the regulatory factors RapC and RapF.

[0005] In some embodiments of the present invention, the sequence of ComX is as shown in SEQ ID No. 1, the sequence of ComA is as shown in SEQ ID No. 2, the sequence of PhrC is as shown in SEQ ID No. 3, the sequence of Spo0A is as shown in SEQ ID No. 4, the sequence of RapC is as shown in SEQ ID No. 5, and the sequence of RapF is as shown in SEQ ID No. 6.

[0006] In some embodiments of the present invention, one or more of the following genes are overexpressed: dhaBCE gene, dhaF gene, yqhD gene, and PntAB gene from Escherichia coli.

[0007] In some embodiments of the present invention, the sequence of the dhaBCE gene is shown in SEQ ID No. 7; the sequence of the dhaF gene is shown in SEQ ID No. 8; the sequence of the yqhD gene is shown in SEQ ID No. 9; and the sequence of the PntAB gene from Escherichia coli is shown in SEQ ID No. 10.

[0008] The second objective of this invention is to provide an all-natural biomimetic microcapsule carrying a water-soluble active ingredient in an effective oil, the raw materials of which include the high surfactant-producing Bacillus, plant oil, and water-soluble active ingredient.

[0009] In some embodiments of the present invention, the vegetable oils include one or more of the following: soybean oil, coconut oil, sunflower seed oil, jojoba seed oil, rosehip oil, grape seed oil, almond oil, meadowfoam seed oil, macadamia nut oil, olive fruit oil, kukui seed oil, camellia seed oil, sea buckthorn seed oil, peony seed oil, hazelnut oil, rice germ oil, wheat germ oil, borage seed oil, safflower seed oil, plantain leaf artichoke seed oil, blueberry seed oil, baobab seed oil, palm fruit oil, Chilean hazelnut oil, African wild mango oil, white lupin oil, and olive shell oil.

[0010] In some embodiments of the present invention, the water-soluble active ingredient includes 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 officinale extract, Portulaca oleracea extract, polypeptide, tranexamic acid, and camellia extract.

[0011] The third objective of this invention is to provide a method for preparing the all-natural biomimetic microcapsules carrying water-soluble active oils, comprising the following steps: A Bacillus strain that produces high levels of surfactants is provided and fermented, with a first vegetable oil added during the fermentation process; after fermentation, the bacterial cells are removed to obtain the fermentation product. The second vegetable oil is then mixed with the fermentation product to obtain fermented oil; The fermented oil is mixed with a water-soluble active ingredient solution to obtain the all-natural biomimetic microcapsule-loaded water-soluble active ingredient functional oil.

[0012] In some embodiments of the present invention, the fermentation conditions are: temperature 25-40°C, time 12-72 h; The first and second vegetable oils are independently selected from one or more of the following: soybean oil, coconut oil, sunflower seed oil, jojoba seed oil, rosehip oil, grape seed oil, almond oil, meadowfoam seed oil, macadamia nut oil, olive fruit oil, kukui seed oil, camellia seed oil, sea buckthorn seed oil, peony seed oil, hazelnut oil, rice germ oil, wheat germ oil, borage seed oil, safflower seed oil, plantain leaf and blueberry seed oil, bilberry seed oil, baobab seed oil, palm fruit oil, Chilean hazelnut oil, African wild mango oil, white lupin oil, and olive shell oil. The amount of the first vegetable oil added is 1%-10% of the fermentation medium; Removal of bacteria using ceramic membranes and ultrafiltration membranes; The mass ratio of the second vegetable oil to the fermentation product is (4:1)-(20:1).

[0013] The fourth objective of this invention is to provide the application of the high surfactant-producing Bacillus and the all-natural biomimetic microcapsules carrying water-soluble active oils in skin care products.

[0014] The skincare products mentioned have the effects of whitening, anti-wrinkle, and precise repair.

[0015] The core of this invention is to solve the compatibility problem between water-soluble active ingredients and natural oils through all-natural biomimetic microencapsulation technology, while preserving the natural properties of the formula. It mainly includes: By screening strains that produce high levels of surfactants, Bacillus subtilis strain CGMCC No. 36746 was obtained. It has the ability to produce high levels of surfactants and can also produce iturin.

[0016] High-content surfactant production was achieved by overexpressing ComX / ComA / PhrC / Spo0A regulatory factors and inhibiting RapC / RapF expression; further, overexpression of dhaBCE / dhaF / yqhD / PntAB genes enabled the strain to synthesize butanediol simultaneously.

[0017] The fermentation process enables the fermentation products to form all-natural biomimetic microcapsules. The internal hydrophilic cavity encapsulates water-soluble active ingredients, while the external surface is compatible with natural plant oils, achieving stable coexistence and targeted loading of water and oil at the microscopic level.

[0018] The technical solution of the present invention has the following advantages compared with the prior art: 1. Solving the core pain point of water-oil compatibility Without the need for chemically synthesized emulsifiers, this product achieves stable coexistence of water-soluble active ingredients and natural oils through all-natural biomimetic microcapsules, completely solving the traditional water-oil incompatibility problem. The finished product system is clear and transparent, avoiding stability issues such as turbidity and layering caused by emulsifiers, thus improving product appearance and user experience.

[0019] 2. Preserves the natural and pure properties of the formula. The microcapsules are composed of natural products such as surfactants and butylene glycol produced by Bacillus fermentation, without any artificial synthetic additives, meeting consumers' core demands for "natural" and "pure" products. They avoid the irritation of traditional chemical emulsifiers, reducing the risk of product sensitization and making them suitable for a wider range of skin types, including sensitive skin.

[0020] 3. Enhance the overall efficacy of the product The barrier-repairing and water-oil-balancing effects of natural plant oils are combined with the advanced effects of water-soluble active ingredients, such as whitening, anti-wrinkle, and precise repair, to achieve the dual value of "basic care + enhanced efficacy." Biomimetic microcapsules protect the water-soluble active ingredients, reducing their degradation in the oil system and improving the stability and duration of action of the active ingredients. 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 This invention relates to the blood agar plate method for screening high-surfactant-producing strains; Figure 2 The color difference is shown between CPC-BTB when bound (left) and BTB when free (right) according to the present invention. Figure 3 This invention uses the CPC-BTB detection method to determine the concentration of surfactants. Figure 4 This invention describes the effects of 28 regulatory factors on surfactant production. Figure 5 This invention relates to the effect of overexpressing a single regulatory factor on surfactant production; Figure 6 This invention relates to the effect of the combined regulatory factor expression strategy on surfactant production. Figure 7 This invention relates to the effect of the combined regulatory factor expression strategy on ituronidin production. Figure 8 This invention relates to a mixture of fermented oil and vitamin C solution; wherein, A is a mixture of fermented oil and 10% vitamin C solution (System 1), B is a mixture of avocado oil and 10% vitamin C solution (System 2), and C is a mixture of avocado oil, 10% vitamin C, and surfactant solution (System 3). Figure 9 This is the efficacy and oil stability test of Example 7 of the present invention; Figure 10 The efficacy and structure of the oil in the all-natural organic biomimetic microcapsules of this invention are characterized. Figure 11 This invention relates to the difference in DPPH antioxidant capacity between avocado oil and vitamin C-enriched oils. Figure 12 This invention relates to the difference in DPPH antioxidant capacity between rosehip oil and ergothionein-based oils. Figure 13 This invention is a comparative test of the soothing effects of meadowfoam seed oil and centella asiatica oil. Figure 14 This invention is a comparative test of the whitening effects of sea buckthorn seed oil and AA2G functional oils; Figure 15 This invention is a comparative test of the anti-aging effects of ginseng seed oil and PDRN-based functional oils. 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] Example 1: This embodiment provides a screening method for high surfactant-producing strains, as detailed below: Strains are fundamental to achieving high surfactant production; therefore, the first step is to screen for target strains with high production potential, strong environmental adaptability, and stable metabolism. To this end, this invention uses the blood agar plate method for initial selection of surfactant-producing strains, followed by a secondary selection of high-yielding strains using a cetylpyridine chloride-bromothymol blue screening method.

[0024] The blood agar plate method utilizes the hemolytic activity of biosurfactants, which cause red blood cell rupture and produce a clear hemolytic zone on a blood plate for preliminary screening. The presence and size of the clear zone are used as the screening criteria. The blood agar plate method is simple and rapid, making it suitable as a primary screening method. The specific implementation method is as follows: (1) Samples were collected from Qinghai Salt Lake, the Yellow River estuary in Dongying, Shandong, Changbai Mountain, Shangri-La in Yunnan, and Wuyi Mountain. 3 g of each soil sample was weighed and 30 mL of deionized water was added; or 30 mL of water was taken and activated at 30℃ and 200 rpm for 30 min. 5 mL of the sample was transferred to 30 mL of enrichment medium and incubated at 30℃ and 200 rpm for 24 h. The enrichment medium consisted of: 3 g / L glucose, 0.8 g / L peptone, 0.5 g / L yeast extract, 0.2 g / L potassium dihydrogen phosphate, and 0.1 g / L calcium carbonate. The enriched culture solution was then serially diluted 10 times. 5 -10 7Take 100 μL of the solution and spread it onto blood agar plates. Incubate at 30°C for 36 h. Select strains with large hemolytic zones and clear edges, such as... Figure 1 As shown. This process was repeated, and through continuous screening, more than 3,000 strains with large hemolytic zones were selected from approximately 100,000 single colonies on 1,000 screening plates.

[0025] (2) The blood agar plate method is simple and easy to operate as a primary screening method for strains producing surfactants. However, many strains produce hemolysin, resulting in obvious transparent hemolytic zones, which leads to a very high false positive rate and requires rescreening. Here, a complexation colorimetric reaction between bromothymol blue (BTB) and hexadecylpyridine chloride (CPC) is used for screening. The principle of this method is as follows: Figure 2 As shown, BTB itself is blue, but when CPC binds to BTB, a complexation reaction occurs, causing a color change. However, when anionic surfactants are added to the system, they bind to CPC, releasing BTB and causing the system color to change to blue. Based on this phenomenon, the surfactant content can be determined by measuring the change in absorbance.

[0026] The specific method is as follows: Over 3000 bacterial strains obtained from the initial screening were cultured separately using enrichment medium to obtain culture solutions; 0.2 mM CPC solution and 0.2 mM BTB solution were mixed at a volume ratio of 1:1, and 800 μL was added to a 48-well plate. 100 μL of culture supernatant was added and mixed thoroughly. The mixture was allowed to stand for 10 min, and the color change was observed. The absorbance at 600 nm was measured using a microplate reader. Figure 3 As shown, the color of the system changed significantly after each fermentation broth was added. The sample with the deepest blue color was selected for further identification.

[0027] Example 2: This embodiment provides a method for identifying substances that produce high levels of surfactants, as detailed below: The sample with the deepest blue color was selected for strain identification. 16S identification confirmed the selected strain as *Bacillus subtilis*. This strain was deposited with accession number CGMCC No. 36746, deposit date November 24, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Biology, Chinese Academy of Sciences. Classification and nomenclature: *Bacillus subtilis* Bacillus subtilisThe strain was named JJL-SF09. Surfactants commonly produced by Bacillus subtilis include surfactants, iturobrine, and fenbacin. These three substances were identified by HPLC. Surfactants were the predominant component, with a small amount of iturobrine also detected, but no fenbacin was detected. Surfactants have an amphiphilic structure formed by a cyclic heptapeptide and a fatty acid chain, which explains the excellent surface activity of the selected Bacillus subtilis strain.

[0028] Example 3: This embodiment provides a method for identifying genes key to surfactant synthesis using CRISPRi. To further enhance the synthesis of surfactants, this invention utilizes synthetic biology techniques to improve the ability of Bacillus subtilis to synthesize surfactants, with a particular focus on enhancing the synthesis of surfactantin. Surfactant synthesis in Bacillus subtilis is regulated by multiple factors; therefore, this invention first investigates the effects of potential regulatory factors on surfactantin synthesis using the CRISPRi technique. Three sgRNA binding sites were designed for each of 28 regulatory factors to verify their influence on surfactantin synthesis. These 28 regulatory factors include ComX, ComA, RapC, RapF, RapA, RapD, RapG, RapH, RapK, Rap60, RapQ, PhrC, PhrF, PhrG, PhrH, Phr60, PhrQ, PhrA, RsiX, SigX, RghR, PhoR, PhoP, DegU, Spo0A, CodY, SpX, and PerR. To utilize CRISPRi technology, dCas9 was first integrated into the lacA site of JJL-BS03 and expressed via the Pxyl promoter, named JJL-BS03dCas9. Further, specific sgRNA sequences were designed for these genes and integrated into the amyE site of the JJL-BS03dCas9 genome. The specific sgRNA sequences for each gene are shown in Table 1 below.

[0029] Table 1 sgRNA specific sequences

[0030] The construction of the CRISPRi system targeting the ComX-1 site will be used as an example for illustration: (1) pJMP1 (Solepro, Cat: VT002209) was transferred into JJL-BS03 by electroporation, and positive clones were obtained by erythromycin resistance screening to obtain JJL-BS03dCas9; (2) pJMP2 (Solepro, Cat: VT002678) was amplified using primers ComX-1-F / pJMP2-R. sgRNA plasmids targeting the ComX-1 site were obtained using T4 polynucleotide kinase (NEB, Cat: M0201S) and T4 DNA ligase (NEB, Cat: M0202S). The plasmid was then transformed into JJL-BS03dCas9. Positive clones were obtained through chloramphenicol resistance screening. The resulting strain JJL-BS03ComX-1 was used to verify the effect of inhibiting ComX gene expression on surfactant production.

[0031] (3) Production of surfactant by shake-flask fermentation: JJL-BS03ComX-1 was inoculated into LB medium and cultured at 37℃ and 200 rpm for 16 h; the cultured seed liquid was inoculated into shake-flask fermentation medium at 5% and cultured at 37℃ and 200 rpm for 48 h. The shake-flask fermentation medium formula was: sucrose 50 g / L, glycerol 50 g / L, xylose 4 g / L, NH4NO3 8 g / L, KH2PO4 4 g / L, Na2HPO4 7 g / L, MgSO4 0.02 g / L, CaCl2 0.77 mg / L, FeSO4 0.64 mg / L, EDTA 1.5 mg / L.

[0032] Following the above procedure, sgRNA plasmids for other genes were constructed, and the effects of each gene on surfactant production were verified. Primers used for each gene's sgRNA are shown in Table 2 below.

[0033] Table 2 sgRNA plasmid primer sequences

[0034] The effects of various regulatory factors on surfactant synthesis are as follows: Figure 4 As shown. By Figure 4It can be seen that 17 regulatory factors, namely ComX, ComA, PhrC, PhrF, PhrG, PhrH, Phr60, PhrQ, PhrA, RsiX, RghR, Phop, DegU, Spo0A, CodY, SpX, and PerR, play a positive regulatory role in surfactant production. Therefore, when the expression of these genes is downregulated using CRISPRi technology, the surfactant content decreases. Among these positive regulatory factors, ComX, ComA, PhrC, and Spo0A have the most significant impact on surfactant production. Conversely, 7 regulatory factors, namely RapC, RapF, RapD, RapK, Rap60, RapQ, and SigX, play a negative regulatory role in surfactant production. Therefore, when the expression of these genes is downregulated using CRISPRi technology, the surfactant content increases. Among these negative regulatory factors, RapC and RapF have the most significant impact on surfactant production. The regulatory factors that have a relatively small impact on the synthesis of surface activity are RapA, RapG, RapH, and PhoR.

[0035] Example 4: This embodiment provides modifications to key regulatory genes, as detailed below: Example 3 analysis identified six regulatory factors that significantly influence surfactant production. ComX, ComA, PhrC, and Spo0A were identified as key positive regulators, while RapC and RapF were identified as key negative regulators. For the positive regulators, four were overexpressed in Bacillus subtilis strains screened in Example 2 to verify their impact on surfactant synthesis. The construction method was as follows: (1) ComX, ComA, PhrC and Spo0A genes were amplified from Bacillus subtilis JJL-BS03 using ComX-O1F / ComX-O1R, ComA-O1F / ComA-O1R, PhrC-O1F / PhrC-O1R and Spo0A-O1F / Spo0A-O1R (Table 3). The pHT01 plasmid (Solarbio, Cat: VT000077) was digested with BamHI / XbaI and constructed plasmids pHT01-ComXO, pHT01-ComAO, pHT01-PhrCO and pHT01-Spo0AO, respectively, by Gibson ligation (Full Gold, Cat: CU201-02).

[0036] Table 3 Primer sequences used in Example 4

[0037] (2) The plasmid constructed above was transformed into Bacillus subtilis selected in Example 2. The effects of overexpression of the ComX, ComA, PhrC, and Spo0A genes on the surfactant production of Bacillus subtilis were verified by culturing in shake flasks. The results are as follows: Figure 5 As shown. From Figure 5 It can be seen that overexpression of these four positive regulatory factors in Bacillus subtilis significantly increases the content of surfactant, with the surfactant content of Bacillus subtilis overexpressing Spo0A increasing by 160%.

[0038] To further increase the content of surfactant, the above four positive regulatory factors were simultaneously expressed in Bacillus subtilis JJL-BS03, and the expression of two key negative regulatory factors, RapC and RapF, was simultaneously inhibited using CRIPSRi technology. The resulting strain was named JJL-BS6O. The overexpression of the four positive regulatory factors involved the simultaneous integration of the ComX, ComA, PhrC, and Spo0A genes into the pHT01 plasmid. The specific construction method is as follows: (1) Replace chloramphenicol with kanamycin in the pHT01-ComXO plasmid: A kanamycin resistance gene was amplified using primers Kan-F / Kan-R with pJOE8999 plasmid (refer to the literature Altenbuchner J. 2016. Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System. Appl Environ Microbiol 82) as a template; A plasmid backbone fragment was amplified using primers pHT01-F / pHT01-R with pHT01-ComXO as a template; The two fragments were ligated by Gibson (Full Gold, Cat: CU201-02) to construct the pHTKm-ComXO plasmid. Furthermore, the ComA, PhrC, and Spo0A genes were amplified from pHT01-ComAO, pHT01-PhrCO, and pHT01-Spo0AO using primers ComA-O2F / ComA-O2R, PhrC-O2F / PhrC-O2R, and Spo0A-O2F / Spo0A-O1R. The plasmid pHTKm-ComXO was digested with XbaI and constructed via Gibson ligation (Fullmetal Alchemist, Cat: CU201-02) to obtain the plasmid pHTKm-CCPS, which simultaneously overexpresses ComX, ComA, PhrC, and Spo0A.

[0039] (2) Using the sgRNA plasmid targeting the RapC-2 site as a template, the first fragment was obtained by amplification using sgRNA-RapC-2-F / sgRNA-RapC-2-R; using the sgRNA plasmid targeting the RapF-2 site as a template, the second fragment was obtained by amplification using sgRNA-RapF-2-F / sgRNA-RapF-2-R; the first fragment and the second fragment were connected by Gibson (Fullmetal, Cat: CU201-02) to construct the plasmid pJMP2sgRNA2, which simultaneously inhibits the expression of RapC and RapF.

[0040] (3) The plasmid pJMP2sgRNA2 was transformed into JJL-BS03dCas9, and positive clones were obtained through chloramphenicol resistance screening to obtain strain JJL-BS03RCRF. The plasmid pHTKm-CCPS was further transformed into this strain to obtain strain JJL-BS6O, which simultaneously overexpresses ComX, ComA, PhrC, and Spo0A, and inhibits RapC and RapF expression. After culturing JJL-BS6O in shake flasks, the surfactant yield was measured. The shake flask fermentation conditions were the same as in Example 3. The results are as follows: Figure 6 As shown in the figure, the yield of surfactant in JJL-BS6O was further increased from 0.86 g / L in the original strain to 4.35 g / L.

[0041] The ituronine production of the original strain and JJL-BS6O was also determined, and the results are as follows: Figure 7 As shown in the figure, the yield of ituronic acid in JJL-BS6O was significantly increased compared to the original strain.

[0042] Example 5: This embodiment provides a method for preparing fermented oil using JJL-BS6O, as detailed below: (1) JJL-BS6O glycerol tubes were inoculated into LB medium and cultured at 37°C for 16 h. The composition of LB medium was: yeast extract 5 g / L, peptone 10 g / L, and NaCl 10 g / L.

[0043] (2) The cultured JJL-BS6O culture solution was inoculated into the fermentation medium at 5%. The fermentation medium consisted of: sucrose 50 g / L, glycerol 50 g / L, NH4NO3 8 g / L, KH2PO4 4 g / L, Na2HPO4 7 g / L, MgSO4 0.02 g / L, CaCl2 0.77 mg / L, FeSO4 0.64 mg / L, and EDTA 1.5 mg / L.

[0044] (3) After culturing at 37℃ for 8 h, IPTG with a final concentration of 1 mM and avocado oil of 50 g / L were added, and fermentation was continued for 24 h. During fermentation, a dissolved oxygen feedback strategy was used to supplement the culture medium (700 g / L glycerol), and the dissolved oxygen level was controlled at 30%.

[0045] (4) After fermentation, the microbial cells and proteins produced during fermentation are removed by ceramic membrane and ultrafiltration membrane.

[0046] (5) Rotary evaporation concentration was carried out to finally obtain a concentrated solution with a surfactant content of 90 g / L.

[0047] (6) Take 10 times the volume of avocado oil and homogenize it with the concentrate (800 bar, homogenize 3 times). Remove the precipitate by centrifugation to obtain fermented oil.

[0048] Take 100 g of fermented oil and mix it with 2 g of 10% VC solution (System 1). Figure 8 As can be seen from A, the system is in a clear and transparent state. And from... Figure 8 As can be seen from Figure B, when 100 g of avocado oil and 2 g of 10% vitamin C are directly mixed (System II), the two cannot dissolve. Figure 8 As shown in Figure C, directly mixing 100 g of avocado oil, 2 g of 10% vitamin C, and 1 g of surfactant (System 3) also fails to achieve dissolution. This demonstrates that the fermented oil obtained has a significant advantage in dissolving water-soluble active ingredients.

[0049] Example 6: This embodiment provides a method for establishing a butanediol overexpression strain. To enhance the stability of fermented oil, JJL-BS6O was further modified to produce a certain amount of butanediol, thereby increasing the system's stability. For this purpose, JJL-BS6O was modified. The dhaBCE (SEQ ID No. 1) and dhaF (SEQ ID No. 2) genes were integrated into the yvmC site of the JJL-BS6O genome. Simultaneously, to enhance NADPH synthesis, the PntAB (SEQ ID No. 4) gene from *E. coli* was integrated at this site; the yqhD gene (SEQ ID No. 3) was expressed via plasmid using the promoter Pgrac. The resulting strain was named JJL-BS6O06. The specific implementation method is as follows: (1) The pJOE8999 plasmid was amplified using primers yvmC-F / pJOE8999-R, and the plasmid pJyvmC was obtained using T4 polynucleotide kinase (NEB, Cat: M0201S) and T4 DNA ligase (NEB, Cat: M0202S). Using the JJL-BS03 genome as a template, the upstream and downstream fragments of yvmC were amplified using primers yvmC-UF / yvmC-UR and yvmC-DF / yvmC-DR, respectively. pJyvmC was digested with XbaI and the plasmid pJyvmCA was constructed by Gibson ligation (Taiwanese Gold, Cat: CU201-02). The dhaBCE and dhaF genes were synthesized by Shanghai Sangon Biotech and amplified using primers dha-F and dha-R. The Pgrac promoter was amplified using plasmid pHT01 as a template with primers Pgrac-F / Pgrac-R. The two fragments were then fused using overlap PCR to obtain the fragment Pgrac-dhaBCE-dhaF. The PntAB gene was amplified using the E. coli BL21(DE3) genome as a template with primers PntAB-F / PntAB-R. The Pgrac promoter was amplified using plasmid pHT01 as a template with primers Pgrac-F2 / Pgrac-R2. The two fragments were then fused using overlap PCR to obtain the fragment Pgrac-PntAB. The fragments Pgrac-dhaBCE-dhaF and Pgrac-PntAB were further fused using overlap PCR to obtain the fragment Pgrac-dhaBCE-dhaF-Pgrac-PntAB. pJyvmCA was digested with XbaI and ligated with the fragment Pgrac-dhaBCE-dhaF-Pgrac-PntAB via Gibson ligation (TransGold, Cat: CU201-02) to obtain the plasmid pJyvmCdP. The plasmid pJyvmCdP was transformed into JJL-BS03RCRF and incubated on LB agar plates containing 5 μg / mL kanamycin and 0.2% mannose at 30°C. Colonies that grew within 2 days were picked up with a toothpick and inoculated onto antibiotic-free LB agar plates and incubated at 50°C. The next day, colonies were streaked onto LB agar plates and incubated at 42°C to obtain single colonies. Finally, single colonies were inoculated onto LB agar plates containing kanamycin, and plasmid loss was checked again. The correct strain was selected and named JJL-BS03RCRFdP, thus obtaining a strain that overexpressed dhaBCE, dhaF, and PntAB in JJL-BS03RCRF.

[0050] (2) Using plasmid pHT01 as a template, the Pgrac promoter was amplified using primers Pgrac-F3 / Pgrac-R3; using the E. coli BL21(DE3) genome as a template, the yqhD gene was amplified using primers yqhD-F / yqhD-R; plasmid pHTKm-CCPS was digested with XmaI; the three fragments obtained were ligated using Gibson (Full Gold, Cat: CU201-02) to obtain plasmid pHTKm-CCPSY. Plasmid pHTKm-CCPSY was transformed into JJL-BS03RCRFdP and screened for kanamycin resistance to obtain strain JJL-BS6O06.

[0051] Table 4 Primer sequences used in Example 6

[0052] JJL-BS6O06 was cultured in shake flasks under the same fermentation conditions as in Example 3. The butanediol yield was determined to be 5.02 g / L.

[0053] Example 7: This embodiment provides a method for preparing an all-natural, organic, biomimetic microcapsule-loaded, water-soluble active oil, as detailed below: The JJL-BS6O06 strain was used for fermentation culture to prepare all-natural, organic, biomimetic microcapsules containing water-soluble active ingredients for the production of functional oils. The specific culture process is as follows: (1) JJL-BS6O06 glycerol tubes were inoculated into LB medium and cultured at 37°C for 16 h. The composition of LB medium was: yeast extract 5 g / L, peptone 10 g / L, and NaCl 10 g / L.

[0054] (2) The cultured JJL-BS6O06 culture solution was inoculated into the fermentation medium at 3%. The fermentation medium consisted of: sucrose 50 g / L, glycerol 50 g / L, NH4NO3 8 g / L, KH2PO4 4 g / L, Na2HPO4 7 g / L, MgSO4 0.02 g / L, CaCl2 0.77 mg / L, FeSO4 0.64 mg / L, and EDTA 1.5 mg / L.

[0055] (3) After culturing at 37℃ for 8 h, IPTG with a final concentration of 1 mM and avocado oil were added, and fermentation was continued for 48 h. During fermentation, a dissolved oxygen feedback strategy was used to supplement sugar-fed medium (500 g / L glycerol, 200 g / L sucrose) and avocado oil, and dissolved oxygen was controlled at 30%.

[0056] (4) After fermentation, the microbial cells and proteins produced during fermentation are removed by ceramic membrane and ultrafiltration membrane.

[0057] (5) Rotary evaporation concentration was carried out to make the content of surfactant reach 90 g / L.

[0058] (6) Take 10 times the volume of avocado oil and mix it with the concentrate. Remove the precipitate by centrifugation to obtain fermented oil.

[0059] 100 g of fermented oil was mixed with 2 g of 10 wt% vitamin C solution to obtain an effective oil with antioxidant activity. Figure 9 It can be seen that the system is clear and transparent. Stability tests were also conducted on the system under the following conditions: 4°C, 45°C, exposure to light, and room temperature. The results are as follows... Figure 9 As shown in the figure, the fermented oil loaded with VC solution did not show any abnormalities after being placed under various conditions for 90 days, indicating that the fermented oil prepared by this method can be used to stably load water-soluble active substances.

[0060] Test Example 1: Characterization of the efficacy and structure of all-natural organic biomimetic microcapsules carrying water-soluble active ingredients. The structure of the functional oil prepared in Example 7 was characterized using transmission electron microscopy (TEM). The results are as follows: Figure 10 As shown in the figure, in the final functional oil, the water-soluble active ingredients are encapsulated by the surfactants, forming microcapsule structures that are uniformly dispersed in the oil.

[0061] Test Example 2: Antioxidant Capacity Test with Vitamin C 100 g of the fermented oil prepared in Example 7 was mixed with 2 g of 10 wt% vitamin C solution to obtain a vitamin C-loaded functional oil. The difference in DPPH antioxidant capacity between avocado oil and the vitamin C-loaded functional oil was measured separately. The results are as follows: Figure 11 As shown in the figure, the antioxidant capacity of vitamin C-rich oils is 8 times that of avocado oil.

[0062] Test Example 3: Antioxidant Capacity Test with Ergothioneine The avocado oil in Example 7 was replaced with rosehip oil, and the other steps were the same for preparing the fermented oil. 100 g of the obtained fermented oil was mixed with 2 g of 5wt% ergothioneine solution to obtain an ergothioneine-loaded functional oil. The difference in DPPH antioxidant capacity between rosehip oil and this ergothioneine-loaded functional oil was measured, and the results are as follows: Figure 12 As shown in the figure, the DPPH antioxidant capacity of ergothionein-based oil is 4.2 times that of rosehip oil.

[0063] Test Example 4: Soothing Efficacy Test with Centella Asiatica Extract In Example 7, step (3) avocado oil was replaced with camellia seed oil, and step (6) avocado oil was replaced with meadowfoam seed oil, with the other steps remaining the same, to prepare fermented oils. 100 g of the fermented oil was mixed with 2 g of 2wt% Centella asiatica extract solution to obtain Centella asiatica functional oil. The difference in soothing effects between meadowfoam seed oil and this Centella asiatica functional oil was determined.

[0064] The specific testing method is as follows: LPS-induced human keratinocytes are a classic cell model for studying inflammatory factors. By comparing the differences in IL-1α and IL-8 secretion levels in human keratinocytes after treatment with the test substance and those in the control group, the inhibitory effect of the test substance on IL-1α and IL-8 secretion can be evaluated. This assay method was used to determine the soothing effects of meadowfoam seed oil and Centella asiatica oil, and the results are as follows... Figure 13 As shown in the figure, the relative IL-1α content of 0.1% meadowfoam seed oil decreased by 39.87%, while the relative IL-1α content of 0.1% centella asiatica oil decreased by 91.80%. This indicates that the soothing effect of centella asiatica oil is far superior to that of meadowfoam seed oil.

[0065] Test Example 5: Whitening Efficacy Test with AA2G The avocado oil in Example 7 was replaced with sea buckthorn seed oil, and the other steps were the same for preparing the fermented oil. 100 g of the obtained fermented oil was mixed with 2 g of 30wt% AA2G solution to obtain an AA2G-loaded functional oil. The difference in whitening efficacy between the sea buckthorn seed oil and the AA2G-loaded functional oil was determined.

[0066] Based on the B16 mouse melanoma cell model, the whitening efficacy of the samples was evaluated by detecting changes in melanin synthesis after the samples were applied to melanocytes. This testing method was used to determine the whitening efficacy of sea buckthorn seed oil and AA2G functional oil, and the results are as follows: Figure 14 As shown in the figure, the inhibition rate of melanin synthesis in cells of 0.1% sea buckthorn seed oil is 35.2%, while the inhibition rate of melanin synthesis in cells of 0.1% AA2G functional oil is 57.9%. It can be seen that the whitening effect of AA2G functional oil is far superior to that of sea buckthorn seed oil.

[0067] Test Example 6: Anti-aging efficacy test with PDRN The avocado oil in Example 7 was replaced with ginseng seed oil, and the other steps were the same for preparing the fermented oil. 100 g of the obtained fermented oil was mixed with 2 g of 2wt% PDRN solution to obtain PDRN-loaded functional oil. The difference in anti-aging efficacy between ginseng seed oil and PDRN-loaded functional oil was determined separately.

[0068] Using human fibroblasts as the test system, the expression level of type I collagen I in human fibroblasts was detected by immunofluorescence technology to evaluate whether the test samples had anti-wrinkle and firming effects. The anti-aging effects of ginseng seed oil and PDRN-containing oils were determined using this method, and the results are as follows: Figure 15 As shown, treatment with 0.1% ginseng seed oil increased Collagen I content by 88.66 ng / mL, while treatment with 0.1% PDRN-containing active oil increased Collagen I content by 155.16 ng / mL. These results indicate that PDRN-containing active oil has significantly better anti-aging properties than ginseng seed oil.

[0069] SEQ ID No.1 ComX: MQDLINYFLNYPEALKKLKNKEACLIGFDVQETETIIKAYNDYYLADPITRQWGD.

[0070] SEQ ID No.2 ComA: MKKILVIDDHPAVMEGTKTILETDSNLSVDCLSPEPSEQFIKQHDFSSYDLILMDLNLGGEVNGMELSKQILQENPHCKIIVYTGYEVEDYFEEAIRAGLHGAISKT ESKEKITQYIYHVLNGEILVDFAYFKQLMTQQKTKPAPSSQKEQDVLTPRECLILQEVEKGFTNQEIADALHLSKRSIEYSLTSIFNKLNVGSRTEAVLIAKSDGVL.

[0071] SEQ ID No.3 PhrC: MKLKSKLFVICLAAAAIFTAAGVSANAEALDFHVTERGMT.

[0072] SEQ ID No.4 Spo0A: MEKIKVCVADDNRELVSLLSEYIEGQEDMEVIGVAYNGQECLSLFKEKDPDVLVLDIIMPHLDGLAVLERLRESDLKKQPNVIMLTAFGQEDVTKKAVDLGASYFILKPFDMENLVGHIRQVSGNASSVTHRAPSSQSSIIRSSQPEPKKKNLDASITSIIHEIGVPAHIKGYLYLREAISMVYNDIELLGSITKVLYPDIAKKFNTTASRVERAIRHAIEVAWSRGNIDSISSLFGYTVSMTKAKPTNSEFIAMVADKLRLEHKAS。

[0073] SEQ ID No.5 RapC: MKSGVIPSSAVGQKINEWYRYIRTFSVPDAEVLKAEIQQELKHMQHDSNLLLYYSLMEFRHQLMLDYLEPLEKLNIEDQPSLSELSRNIDSNQADLKGLLDYYVNFFRGMYEFDKREFISAITYYKQAEKKLSFVADHIERAEFYFKIAEAYYYMKQTYFSLINIKNAYEIYVEQETYNVRIIQCHFVFGVNLMDERNFEQAARHFKLALNMAQAEQKAQLVGRAYYNLGLCYYNQDLLDPAIDYFEKAVSTFESSRIVNSLPQAYFLITLIYYKQGKHDKASEYHKRGYEYAKETDDADYAVKFEFLQSLYLDQPNEEGIERCFQYLKNKNMYADIEDLALEVAKYYYEQKWFKLSASYFLQVEEARKQIQRSEGLYEIEI。

[0074] SEQ ID No.6 RapF: MTGVISSSSIGEKINEWYMYIRRFSIPDAEYLRREIKQELDQMEEDQDLHLYYSLMEFRHNLMLEYLEPLEKMRIEEQPRLSDLLLEIDKKQARLTGLLEYYFNFFRGMYELDQREYLSAIKFFKKAESKLIFVKDRIEKAEFFFKMSESYYYMKQTYFSMDYARQAYEIYKEHEAYNIRLLQCHSLFATNFLDLKQYEDAISHFQKAYSMAEAEKQPQLMGRTLYNIGLCKNSQSQYEDAIPYFKRAIAVFEESNILPSLPQAYFLITQIHYKLGKIDKAHEYHSKGMAYSQKAGDVIYLSEFEFLKSLYLSGPDEEAIQGFFDFLESKMLYADLEDFAIDVAKYYHERKNFQKASAYFLKVEQVRQLIQGGVSLYEIEV。

[0075] SEQ ID No.7 dhaBCE

[0076] SEQ ID No.8 dhaF ATGGAGAGTAGCGTAGTCGCCCCCGCCATCGTCATTGCCGTCACTGACGAATGCAGCGAACAGTGGCGCGATGTCCTGCTGGGCATTGAAGAGGAAGGCATTCCTTTTGTTCTGCAGCCGCAGACCGGCGGCGATCTTATCCATCACGCCTGGCAGGCGGCGCAGCGTTCGCCGCTGCAGGTAGGCATCGCCTGCGACCGGGAACGGCTCATCGTGCACTACAAAAATTTACCCGCATCAACTCCGTTGTTTTCGCTGATGTATCACCAGAACAGGCTGGCCCGGCGAAACACTGGCAACAATGCGGCTCGTCTCGTCAAAGGGATCCCATTTCGGGATCGCCATGCTTAA。

[0077] SEQ ID No.9 yqhD

[0078] SEQ ID No.10 pntAB

[0079] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made 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.

Claims

1. A type of all-natural biomimetic microcapsule carrying water-soluble active oil, characterized in that, The raw materials include Bacillus subtilis, avocado oil, and water-soluble active ingredients; the Bacillus subtilis strain with high surfactant production is Bacillus subtilis CGMCC No. 36746, which overexpresses regulatory factors ComX, ComA, PhrC, and Spo0A, and inhibits the expression of regulatory factors RapC and RapF; the sequences of ComX, ComA, PhrC, and Spo0A are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively; the sequence of Spo0A is shown in SEQ ID No. 4; the sequence of RapC is shown in SEQ ID No. 5; and the sequence of RapF is shown in SEQ ID No.

6. The water-soluble active ingredient is vitamin C; The all-natural biomimetic microcapsules carrying water-soluble active oils are prepared by the following method: A Bacillus strain that produces high levels of surfactants is provided and fermented, with a first vegetable oil added during the fermentation process; after fermentation, the bacterial cells are removed to obtain the fermentation product. The second vegetable oil is then mixed with the fermentation product to obtain fermented oil; The fermented oil is mixed with a water-soluble active ingredient solution to obtain the all-natural biomimetic microcapsule-loaded water-soluble active ingredient functional oil. The first and second vegetable oils are selected from avocados.

2. The all-natural biomimetic microcapsules carrying water-soluble active oils according to claim 1, characterized in that, The high surfactant-producing Bacillus species also include those overexpressing... dhaBCE Gene, dhaF Gene, yqhD Genes and those derived from E. coli PntAB Genes; the stated dhaBCE The gene sequence is shown in SEQ ID No. 7; dhaF The gene sequence is shown in SEQ ID No. 8; yqhD The gene sequence is shown in SEQ ID No. 9; the gene is derived from *E. coli*. PntAB The gene sequence is shown in SEQ ID No.

10.

3. The all-natural biomimetic microcapsules carrying water-soluble active oils according to claim 1, characterized in that, The fermentation conditions are: temperature 25-40℃, time 12-72 h; The first and second vegetable oils are selected from avocados; The amount of the first vegetable oil added is 1%-10% of the fermentation medium; Removal of bacterial cells using ceramic membranes and ultrafiltration membranes; The ratio of the second vegetable oil to the fermentation product is (4:1) to (20:1).

4. The application of the all-natural biomimetic microcapsules carrying water-soluble active oils as described in any one of claims 1 to 3 in skin care products.

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