A method for preparing fermentation filtrate with targeted enrichment of plant active ingredients and multiple skin care benefits

Abstract

This invention relates to a method for preparing a fermentation filtrate with multiple skin-care benefits through targeted enrichment of plant active ingredients. The invention screened a highly active cyclodextrin glucosyltransferase and further improved its enzyme activity through molecular modification. By transferring the cyclodextrin glucosyltransferase into Lactobacillus, the plant is fermented by Lactobacillus. During fermentation, the cyclodextrin glucosyltransferase converts active ingredients such as flavonoids and polyphenols in the plant into more water-soluble glycosides, thereby achieving the enrichment of plant active ingredients.

Description

Technical Field

[0001] This invention relates to the field of skincare technology, and in particular to a method for preparing fermented filtrate with multiple skincare benefits by targeting and enriching plant active ingredients. Background Technology

[0002] In the field of natural skincare, plant-derived active ingredients and extracts are highly favored due to their high safety and diverse efficacy. These plant active ingredients and extracts contain various active components such as polyphenols, flavonoids, and saponins. Polyphenols and flavonoids, in particular, have been widely proven to possess multiple benefits, including antioxidant, anti-inflammatory, whitening, moisturizing, and skin barrier repair effects. However, despite the immense potential of plant-based ingredients, they still face some key challenges in practical applications. First, the active ingredients in plant extracts are often difficult to maintain stably in aqueous solutions. Because plant extracts contain large amounts of polyphenols and flavonoids, which have poor water solubility, they are difficult to maintain stably in water-based skincare products. Second, during the extraction process, organic solvents such as ethanol and methanol are often chosen to ensure the full extraction of plant active ingredients. This is because flavonoids and polyphenols in plants are difficult to extract fully with water; however, using organic solvents such as ethanol and methanol often results in solvent residue. Therefore, how to achieve efficient release of plant active ingredients while maintaining their stable presence in aqueous solutions is a critical issue in plant extraction. Summary of the Invention

[0003] To address the above technical problems, this invention provides a method for preparing a fermentation filtrate that targets and enriches plant active ingredients, resulting in multiple skincare benefits. This fermentation filtrate can target and enrich the active ingredients of plants and stably maintain them in an aqueous solution system, thus providing multiple skincare benefits.

[0004] The first objective of this invention is to provide a cyclodextrin glucosyltransferase mutant, wherein the amino acid sequence of the mutant is the amino acid sequence of SEQ ID NO:1 after being mutated by two or more of F193L, A230I, and G259T.

[0005] Furthermore, the cyclodextrin glucosyltransferase mutant contains the DNA coding sequence shown in SEQ ID No. 2.

[0006] A second object of the present invention is to provide a gene encoding the cyclodextrin glucosyltransferase mutant described above.

[0007] A third objective of this invention is to provide a recombinant bacterium obtained by introducing the aforementioned gene into a host bacterium.

[0008] In some embodiments of the present invention, the host bacteria are selected from Bacillus, lactic acid bacteria, Saccharomyces cerevisiae, Galactomyces-like bacteria, Bifida ferment lysate, Sphingomonas sphingosine monocytogenes, or Pichia pastoris.

[0009] In some embodiments of the present invention, the recombinant bacteria also integrates an amylase gene.

[0010] Furthermore, the amino acid sequence of the amylase gene is shown in SEQ ID No. 3.

[0011] A fourth objective of this invention is to provide a fermentation filtrate for targeted enrichment of plant active ingredients, obtained by fermentation of plants by the recombinant bacteria.

[0012] In some embodiments of the present invention, the plant is selected from one or more of the following: blueberry, cranberry, turmeric, black tea, green tea, soybean, grape, pomegranate, astragalus, scutellaria, angelica, chrysanthemum, licorice, salvia miltiorrhiza, schisandra, polygonatum, milk thistle, gentian, rose, nasturtium, veratrum, safflower, dendrobium, amla, citron, gastrodia, sophora japonica, yew, and larch.

[0013] The fifth objective of this invention is to provide a method for preparing a fermentation filtrate that targets and enriches plant active ingredients, comprising the following steps: Provide a plant and crush it; The recombinant bacteria were fermented and cultured, and an amylase inducer was added for further culture. The obtained fermentation broth was crushed and purified to obtain the fermentation filtrate.

[0014] In some embodiments of the present invention, the fermentation conditions are as follows: the fermentation temperature is 25-37°C, and the fermentation time is 8-24 h.

[0015] Furthermore, the specific procedures for fermentation culture are as follows: S1, Seed culture solution for recombinant bacteria; S2. Inoculate the seed culture of the recombinant bacteria into a fermentation medium containing 0.1-10% plants.

[0016] S3, ferment at 25-37℃ for 8-24 hours.

[0017] In some embodiments of the present invention, the fermentation medium comprises 1-10 g / L glucose, 1-10 g / L soybean extract, and 1-50 g / L starch.

[0018] In some embodiments of the present invention, the inoculum amount of recombinant bacteria is 0.1-10%.

[0019] In some embodiments of the present invention, after the inducing agent is added, the culture time is continued for 8-24 hours.

[0020] The sixth objective of this invention is to provide a multi-functional skincare product, comprising the fermented filtrate that targets and enriches plant active ingredients.

[0021] This invention screened a highly active cyclodextrin glucosyltransferase and further improved its enzyme activity through molecular modification. By transferring the cyclodextrin glucosyltransferase into Lactobacillus, and through Lactobacillus fermentation of plants, the cyclodextrin glucosyltransferase converts active ingredients such as flavonoids and polyphenols in plants into more water-soluble glycosides during fermentation, thereby achieving the enrichment of plant active ingredients.

[0022] By integrating amylase into Lactobacillus, starch can provide a substrate for cyclodextrin glucosyltransferase in the early stage of fermentation, and starch can be degraded by amylase in the later stage of fermentation, while providing nutrients for cell growth.

[0023] Synergistic effects of plant fermentation and modified lactobacillus: 1) Lactobacillus itself can bring significant skin care effects during fermentation; 2) Modified lactobacillus can promote the enrichment of plant active ingredients and promote the stability of these active ingredients in aqueous solution systems; 3) The release of plant active ingredients promotes the growth of lactobacillus, thereby further promoting the production of lactobacillus fermentation active ingredients, thus creating a virtuous cycle.

[0024] The technical solution of the present invention has the following advantages compared with the prior art: A highly active cyclodextrin glucosyltransferase was screened and its activity was further enhanced through molecular modification. The activity of this cyclodextrin glucosyltransferase was significantly enhanced compared with commercially available cyclodextrin glucosyltransferases.

[0025] The co-fermentation of modified lactobacilli and plants has the following advantages: 1) It fully enriches the active ingredients of plants; 2) The active ingredients of plants can be stably present in the aqueous solution system; 3) The release of active ingredients of plants is conducive to the growth of lactobacilli.

[0026] Compared with the traditional method of obtaining flavonoid polyphenols via enzymatic catalysis using cyclodextrin glucosyltransferase, this production method offers the following advantages: 1) This method simultaneously catalyzes and promotes the release of plant active ingredients during fermentation, thereby disrupting the plant structure and ensuring the full release of active ingredients. It also avoids the problem of incomplete extraction due to the solubility of active ingredients during extraction. In contrast, enzymatic catalysis requires prior extraction, concentration, rotary drying, and catalysis, making the process cumbersome and often facing the problem of insufficient extraction due to solvent polarity. 2) Enzymatic catalysis requires cyclodextrin / starch as glycosyl donors, but these sugars can affect cosmetic formulations, necessitating further purification. This method, however, introduces amylase into Lactobacillus, allowing the Lactobacillus to consume starch during fermentation, thus avoiding the generation of byproducts. 3) Synergistic effect of enzyme catalysis-fermentation-plant extraction: In this preparation method, lactic acid bacteria can produce a large number of active ingredients with skin care effects during fermentation. The cyclodextrin glucosyltransferase carried by lactic acid bacteria releases the active ingredients in plants through glycosylation reaction of active substances such as polyphenols and flavonoids during fermentation. This allows lactic acid bacteria to grow well even in simple culture medium (without additional inorganic salts or metal ions), which is very important for skin care products. Attached Figure Description

[0027] 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 is an analysis of the glycosylation activity of different cyclodextrin glucosyltransferase mutant combinations on rutin in Example 2 of the present invention.

[0028] Figure 2 This invention relates to a comparative determination of the total flavonoid content in Scutellaria baicalensis extract and Scutellaria baicalensis fermentation filtrate.

[0029] Figure 3 This invention relates to a comparative determination of the total flavonoid content in the supernatant of Scutellaria baicalensis extract and Scutellaria baicalensis fermentation filtrate after 3 months of storage.

[0030] Figure 4 This is a comparative test of the antioxidant capacity of various samples in this invention.

[0031] Figure 5 This is a comparative test of the antibacterial rate of various samples in this invention.

[0032] Figure 6 This invention involves a comparative determination of the tyrosinase inhibition rate of various samples. Detailed Implementation

[0033] 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.

[0034] Unless otherwise specified, the experimental methods used in this invention are all conventional methods, and the materials and reagents used can be purchased commercially.

[0035] The culture medium used in this invention: The SYP medium consists of 10 g / L soluble starch, 5 g / L yeast extract, 5 g / L peptone, 1 g / L KH2PO4, and 0.2 g / L MgSO4·7H2O.

[0036] The solid screening medium consisted of 10 g / L soluble starch, 5 g / L yeast extract, 5 g / L peptone, 1 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, 0.2 g / L phenolphthalein, 10 g / L Na2CO3, and 15 g / L agar, with a pH of 7.5.

[0037] The fermentation medium consisted of 10 g / L glucose and 10 g / L soybean extract.

[0038] Example 1: Screening of highly active cyclodextrin glucosyltransferases Cyclodextrin glucosyltransferases often react with starch as a substrate, therefore, selecting environments with high starch content makes it easier to screen for highly active cyclodextrin glucosyltransferases. Soil and rice, potato, cypress, lotus root, tea, coffee, nuts, plum, grape, taro, and dendrobium samples were collected from various locations including: rice paddies in Minle Village, Wuchang City, Harbin, Heilongjiang Province; sweet potato fields in Dalukou Town, Sixian County, Suzhou, Anhui Province; the Bajie Giant Cypress Nature Reserve in Linzhi City, Tibet Autonomous Region; the Yangkou Town (Shoubei) Forest Ecological Expo Park in Shouguang City, Weifang, Shandong Province; tea plantations in Huimin Town, Lancang Lahu Autonomous County, Pu'er City, Yunnan Province; coffee and macadamia nut plantations in Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province; plum plantations in Jiashi County, Xinjiang; grape plantations in Turpan City, Xinjiang; taro plantations on Chongming Island, Shanghai; and dendrobium plantations in Yandang Mountain, Zhejiang Province.

[0039] The collected samples were resuspended in SYP medium. The resuspended samples were then plated onto solid screening medium. The solid plates were incubated at 37°C for 24 h, and 100 strains with significant clear zones were selected for further screening.

[0040] The 100 initially screened strains were transferred to SYP medium and cultured at 30℃ for 48 h. After culture, the cyclodextrin glucosyltransferase activity in the culture medium was measured. The method was as follows: 0.1 mL of fermentation supernatant was taken, 0.2 mL of pH 9.0 glycine-NaOH buffer solution was added, followed by 0.2 mL of 0.2% potato starch solution. The mixture was reacted at 40℃ for 10 min, and then 0.5 mL of 0.5 M glacial acetic acid was added immediately to terminate the reaction. 3 mL of 0.005% iodine solution was added for color development. The reaction solution without enzyme solution was used as a blank control. The absorbance (OD) value was measured at 700 nm. One unit of enzyme activity was defined as the amount of enzyme required to reduce the blue value by 10%. The results are as follows. Figure 1 As shown in Table 1, the enzyme activities of the top 20 cyclodextrin glucosyltransferases are listed.

[0041] Table 1. Enzyme activity of cyclodextrin glucosyltransferase in different strains

[0042] Table 1 shows that JJL-CGT68 had the highest cyclodextrin glucosyltransferase activity, at 19.78 U / mL. 16S assay confirmed that this strain is a Bacillus species. Bacillus To further determine the cyclodextrin glucosyltransferase sequence of JJL-CGT68, the genome sequence of JJL-CGT68 was determined by high-throughput sequencing. The amino acid sequence of the cyclodextrin glucosyltransferase in JJL-CGT68 was obtained through protein annotation and sequence alignment, as shown in SEQ ID No. 1.

[0043] Example 2: Modification of highly active cyclodextrin glucosyltransferase After removing the signal peptide sequence from the cyclodextrin glucosyltransferase of Example 1, codon optimization was performed based on the codon preference of *E. coli* to obtain the DNA coding sequence shown in SEQ ID No. 2. Beijing Qingke Biotechnology Co., Ltd. synthesized the gene and cloned it into the NcoI and XhoI sites of the pET-22b plasmid, resulting in a recombinant plasmid named pET-CGT. The recombinant plasmid pET-CGT was transformed into *E. coli* BL21(DE3) competent cells to obtain the recombinant strain *E. coli*CGT.

[0044] Using recombinant plasmid pET-CGT as a template, the plasmid backbone sequence lacking the target gene was amplified using primers 1 and 2 and a high-fidelity enzyme. Error-prone PCR amplification of the gene coding sequence was performed using primers 3 and 4 and Taq DNA polymerase. 0.2 mM Mn was added during the error-prone PCR process. 2+To increase the mutation rate, the purified PCR product was ligated with Gibson and then transformed into E. coli BL21(DE3) competent cells to obtain a mutant library.

[0045] Table 2 Primer Sequences

[0046] Single colonies were randomly picked from the above transformation plates and inoculated into test tubes containing 5 mL LB medium (containing 100 μg / mL ampicillin). The culture was incubated overnight at 37°C and 200 rpm. After incubation, the culture was transferred at a 1% ratio to new LB medium test tubes (containing 100 μg / mL ampicillin). After 2.5 h of incubation, ampicillin was added to a final concentration of 0.2 mM, and the temperature was lowered to 30°C for another 8 h of incubation. Then, 10 g / L soluble starch and 1 g / L rutin were added, and the reaction was catalyzed at 40°C for 24 h. After the reaction, water-insoluble rutin was removed by centrifugation, and the supernatant containing glycosylated rutin was collected. The following components were added sequentially to 96-well plates for initial screening and qualitative comparison of glycosylated rutin content to identify recombinant strains. E. coli CGT served as a control and underwent the same catalytic reaction.

[0047] Sample solution: 50 μL; 30% ethanol: Add to a total volume of approximately 100 μL; 5% NaNO2 solution: 10 μL, mix well, and let stand at room temperature for 6 min; 10% Al(NO3)3 solution: 10 μL, mix well, and let stand at room temperature for 6 min; 1 mol / L NaOH solution: 80 μL, mix well, and let stand at room temperature for 15 min; Finally, the absorbance was measured at 510 nm using an ELISA reader. The higher the absorbance, the higher the content of glycosylated rutin.

[0048] Ten strains with high absorbance values ​​were selected from the initial screening results for secondary screening. These strains were then transferred to 50 mL shake flasks for large-scale culture and induced expression. The induction time was changed to 14 h, while other conditions remained the same as the initial screening. After induction expression, Ni was used... 2+ The protein in the supernatant was purified using a chelation column, and imidazole was removed by dialysis. Protein quantification was performed using the Coomassie Brilliant Blue assay with bovine serum albumin as a control. Equal volumes of purified protein (final concentration 0.05 mg / mL), soluble starch 10 g / L, and rutin 1 g / L were added, and the reaction was carried out at 40 °C for 24 h. After the reaction, water-insoluble rutin was removed by centrifugation, and the supernatant containing glycosylated rutin was collected.

[0049] HPLC was used to quantitatively analyze glycosylated rutin in the catalyst supernatant. The HPLC analysis conditions were as follows: Yuexu AQ-C18 column (4.6x250mm, 5um), UV detector, detection wavelength 254 nm, mobile phase was 18% acetonitrile aqueous solution (containing 0.1% formic acid), column temperature was 30℃, flow rate was 0.8 mL / min, injection volume was 10 μL, and acquisition time was 22 min.

[0050] Sequencing was performed on the positive strains to determine the mutation location. The results are shown in Table 3. The glycosylated rutin yields of mutants 1, 2, and 3 were 246%, 215%, and 134% of those of the wild type, respectively.

[0051] Table 3. Characteristic analysis of high-activity mutants of cyclodextrin glucosyltransferase

[0052] Using plasmid pET-CGT as a template, PCR and plasmid construction were performed using high-fidelity DNA polymerase and primers listed in Table 4. Then, multiple rounds of point mutations were performed using the mutated plasmid as a template to construct double mutants F193L / A230I, F193L / G259T, A230I / G259T, and triple mutant F193L / A230I / G259T, named CGT-1, CGT-2, CGT-3, and CGT-4, respectively. The corresponding plasmids were named pET-CGT-1, pET-CGT-2, pET-CGT-3, and pET-CGT-4. After successful sequencing, the constructed plasmids were transformed into E. coli BL21(DE3) competent cells, and expression induction, protein purification, and activity detection were performed according to the method in Example 2. The detection results are as follows: Figure 1 As shown, the combined mutant CGT-1 exhibited the highest glycosylation activity for rutin, which was 3.48 times that of wild-type CGT.

[0053] Table 4 Primer Sequences

[0054] Example 3: CGT-1 integrated into lactic acid bacteria The lactic acid bacteria signal peptide shown in SEQ ID No. 4 was added to the N-terminus of the CGT-1 mutant obtained in Example 2, and the mutant was transformed into lactic acid bacteria CGMCC No. 24962 to achieve efficient secretion of cyclodextrin glucosyltransferase. For this purpose, the lactic acid bacteria signal peptide DNA was synthesized by Beijing Qingke Biotechnology Co., Ltd., and amplified using primers 11 / 12; the CGT-1 gene was amplified using plasmid pET-CGT-1 as a template, using primers 13 / 14; the pNZ8148-Pnis-MCS1-P32-MCS2 plasmid (purchased from Beijing Solarbio Science & Technology Co., Ltd.) was amplified using primers 15 / 16; the three fragments were ligated using Gibson cloning to obtain the plasmid pNZ8148-CGT-1 for CGT-1 expression. pNZ8148-CGT-1 was transformed into CGMCC No.24962 to obtain a lactic acid bacterium for CGT-1 expression, which was named LpCGT.

[0055] Table 5 Primer Sequences

[0056] Example 4 This embodiment provides the use of LpCGT for the preparation of Scutellaria baicalensis fermentation filtrate, as detailed below: 1) LpCGT was inoculated into MRS medium (purchased from Beijing Solarbio Science & Technology Co., Ltd.) and cultured at 37℃ for 24 h to obtain LpCGT seed culture; 2) Inoculate the LpCGT seed culture into 200 mL of fermentation medium and incubate at 37°C for 8 h; 3) Take 10 g of Scutellaria baicalensis, crush it, add it to the fermentation culture medium in step 2), and add 40 g / L starch to the fermentation culture medium at the same time, and continue fermentation culture for 40 h; 4) After the fermentation culture is completed, the fermentation broth is centrifuged to remove the bacterial cells, filtered using a 0.22 μm membrane, and further filtered using a 10 kD ultrafiltration tube. The clear liquid end is collected to obtain the Scutellaria baicalensis fermentation filtrate.

[0057] Example 5: Preparation of lactic acid bacteria simultaneously expressing CGT-1 and amylase The Scutellaria baicalensis fermentation broth prepared in Example 4 contained a large amount of starch. The presence of this starch affects the skin feel and compatibility of the fermentation filtrate in cosmetics, thus requiring further purification. However, such purification wastes starch and inevitably introduces other solvents, which need to be removed later, adding many steps. Furthermore, purification inevitably results in the loss of active ingredients. The method of this application introduces amylase into Lactobacillus, allowing the Lactobacillus to consume starch during fermentation. This avoids formulation problems caused by starch presence, and the starch is converted into a carbon source usable by the Lactobacillus under the action of amylase, thus promoting Lactobacillus growth and generating more prebiotics. In addition, the growth of Lactobacillus further promotes the release of active ingredients from Scutellaria baicalensis, producing multiple beneficial effects.

[0058] Therefore, the signal peptide shown in SEQ ID No. 4 was added to the N-terminus of the amylase sequence shown in SEQ ID No. 3, and after gene synthesis by Beijing Qingke Biotechnology Co., Ltd., it was inserted into pNZ8148-CGT-1. The specific implementation method is as follows: (1) pNZ8148-CGT-1 usage Nco I and Xba I enzyme digestion yielded plasmid backbone fragments; (2) The synthesized gene fragment was digested with NcoI and XbaI to obtain the gene fragment; (3) The gene fragment and the plasmid backbone fragment were ligated using T4 ligase to obtain the final plasmid pNZ8148-CGT-AMY containing CGT-1 and amylase.

[0059] pNZ8148-CGT-AMY was transformed into CGMCC No. 24962 to obtain a lactic acid bacteria for CGT-1 and amylase expression, which was named LpCA.

[0060] Example 6 This embodiment provides an example of using LpCA to prepare Scutellaria baicalensis fermentation filtrate, as detailed below: 1) LpCA was inoculated into MRS medium (purchased from Beijing Solarbio Science & Technology Co., Ltd.) and cultured at 37℃ for 24 h to obtain LpCA seed culture; 2) Inoculate the LpCA seed culture into 200 mL of fermentation medium. The fermentation medium consists of 10 g / L glucose and 10 g / L soybean extract, and is cultured at 37℃ for 8 h. 3) Take 10 g of Scutellaria baicalensis, crush it, add it to the fermentation culture medium in step 2), and add 40 g / L starch to the fermentation culture medium at the same time, and continue fermentation culture for 16 h; 4) Add nisin to a final concentration of 5 ng / mL and continue fermentation for 24 h; 5) After the fermentation culture is completed, the fermentation broth is centrifuged to remove the bacterial cells, filtered using a 0.22 μm membrane, and further filtered using a 10 kD ultrafiltration tube. The clear liquid end is collected to obtain the Scutellaria baicalensis fermentation filtrate.

[0061] Test Example 1: Determination of Flavonoid Content The flavonoid content of the Scutellaria baicalensis fermentation filtrate prepared in Examples 4 and 6, the Scutellaria baicalensis aqueous extract prepared in Comparative Example 1, and the Scutellaria baicalensis alcohol extract prepared in Comparative Example 2 were determined. The results are as follows: Figure 2 As shown in the figure, the total flavonoid content of the Scutellaria baicalensis fermentation filtrate prepared in Example 6 was 8876 mg Rutin / kg, which was 6.1 times and 2.1 times that of the Scutellaria baicalensis extracts prepared in Comparative Examples 1 and 2, respectively.

[0062] Comparative Example 1 This comparative example provides a method for preparing an aqueous extract of Scutellaria baicalensis, as detailed below: 1) Take 10 g of Scutellaria baicalensis, crush it, add 200 mL of deionized water, and extract at 60℃ for 2 h; 2) Take the extract, centrifuge to collect the supernatant, filter it using a 0.22 μm membrane, and further filter it using a 10 kD ultrafiltration tube. Collect the supernatant end to obtain the Scutellaria baicalensis aqueous extract.

[0063] Comparative Example 2 This comparative example provides a method for preparing a scutellaria baicalensis alcohol extract, as detailed below: 1) Take 10 g of Scutellaria baicalensis, crush it, add 200 mL of 70% ethanol, and extract at 60℃ for 2 h; 2) Take the extract, centrifuge to collect the supernatant, remove the ethanol by rotary evaporation, and obtain a dry powder; Dissolve the dried powder in 200 mL of deionized water, centrifuge to remove insoluble matter, filter the supernatant through a 0.22 μm membrane, and further filter through a 10 kD ultrafiltration tube. Collect the supernatant end to obtain the scutellaria baicalensis alcohol extract.

[0064] Test Example 2: Stability Test Stability is one of the core requirements for raw materials in cosmetics. Stability tests were conducted on the Scutellaria baicalensis fermentation filtrate prepared in Examples 4 and 6, the Scutellaria baicalensis aqueous extract prepared in Comparative Example 1, and the Scutellaria baicalensis ethanol extract prepared in Comparative Example 2. These four samples were placed at 25°C for 3 months. The Scutellaria baicalensis aqueous extract prepared in Comparative Example 1 and the Scutellaria baicalensis ethanol extract prepared in Comparative Example 2 both showed turbidity, while the Scutellaria baicalensis fermentation filtrate prepared in Examples 4 and 6 remained clear and transparent.

[0065] Further centrifugation was used to remove the bottom precipitate, and the total flavonoid content in the supernatant was determined. The results are as follows: Figure 3 As shown in the figure, the total flavonoid content in the Scutellaria baicalensis fermentation filtrate of Examples 6 and 4 did not decrease significantly, while the total flavonoid content in the Scutellaria baicalensis extracts prepared in Comparative Examples 1 and 2 decreased significantly, by 60% and 57%, respectively. This result indicates that the stability of the Scutellaria baicalensis fermentation filtrate is far superior to that of the Scutellaria baicalensis extract.

[0066] Test Example 3: Antioxidant Capacity Test The antioxidant capacity of the Scutellaria baicalensis fermentation filtrate prepared in Examples 4 and 6, the Scutellaria baicalensis aqueous extract prepared in Comparative Example 1, and the Scutellaria baicalensis ethanol extract prepared in Comparative Example 2 were tested using a DPPH free radical scavenging capacity test. The specific test methods are as follows: Samples were added and tested according to Table 6. The sample solutions were the Scutellaria baicalensis fermentation filtrate prepared in Examples 4 and 6, the Scutellaria baicalensis aqueous extract prepared in Comparative Example 1, and the Scutellaria baicalensis ethanol extract prepared in Comparative Example 2. Three parallel experiments were set up for each experimental group.

[0067] Table 6. Sample Addition Information for DPPH Scavenging Ability Determination

[0068] DPPH removal capacity (%) = , C – Absorbance of DPPH tube; C0—DPPH absorbance of blank tube; T—Absorbance of the sample tube; T0—Absorbance value of the blank sample tube; The measurement results are as follows Figure 4 As shown in the figure. Among them, the DPPH free radical scavenging rate of the Scutellaria baicalensis fermentation filtrate prepared in Example 6 was the highest, reaching 95.8%, which was 3.7 times and 2.3 times that of the Scutellaria baicalensis extracts prepared in Comparative Examples 1 and 2, respectively.

[0069] Test Example 4: Antibacterial Ability Test The differences in antibacterial effects of the Scutellaria baicalensis fermentation filtrate prepared in Examples 4 and 6, the Scutellaria baicalensis aqueous extract prepared in Comparative Example 1, and the Scutellaria baicalensis ethanol extract prepared in Comparative Example 2 were evaluated by measuring the antibacterial rate against Propionibacterium acnes. The antibacterial rate of each sample at a 5 wt% concentration against Propionibacterium acnes was measured according to 7.3 of QB / T 2738-2023 "Evaluation Methods for Antibacterial and Antimicrobial Effects of Daily Chemical Products" (Suspension Quantitative Method), with an action time of 30 min. The results are as follows. Figure 5 As shown, the 5% concentration of Scutellaria baicalensis fermentation filtrate prepared in Example 6 showed the highest antibacterial rate against Propionibacterium acnes, reaching 81.2%, while the antibacterial rates of the 5% concentration of Scutellaria baicalensis aqueous extract prepared in Comparative Example 1 and the 5% concentration of Scutellaria baicalensis alcoholic extract prepared in Comparative Example 2 were 35.8% and 41.2%, respectively.

[0070] Test Example 5: Whitening Effect Measurement The whitening effect of each sample was determined by the tyrosinase inhibition rate. The whitening efficacy of each sample was determined using 10 wt% concentrations of the fermented filtrate of *Scutellaria baicalensis* prepared in Examples 4 and 6, the aqueous extract of *Scutellaria baicalensis* prepared in Comparative Example 1, and the ethanolic extract of *Scutellaria baicalensis* prepared in Comparative Example 2. Samples were added and tested according to Table 7. The sample solutions were 10 wt% concentrations of the fermented filtrate of *Scutellaria baicalensis* prepared in Examples 4 and 6, the aqueous extract of *Scutellaria baicalensis* prepared in Comparative Example 1, and the ethanolic extract of *Scutellaria baicalensis* prepared in Comparative Example 2. Three parallel experiments were set up for each experimental group.

[0071] Table 7. Sample addition information for tyrosinase inhibition rate determination.

[0072] Tyrosinase inhibition rate (%) = , C – Control group; C0 – Reference background color group; T—Absorbance of the sample tube; T0—Absorbance value of the blank sample tube; The measurement results are as follows Figure 6 As shown in the figure, the Scutellaria baicalensis fermentation filtrate prepared in Example 6 exhibited the best whitening effect, with a tyrosinase inhibition rate of 81.3% at a concentration of 10 wt%. In contrast, at the same concentration, the tyrosinase inhibition rates of the aqueous extract prepared in Comparative Example 1 and the alcoholic extract prepared in Comparative Example 2 were 11.2% and 24.8%, respectively.

[0073] SEQ ID No.1 amino acid sequence Cyclodextrin glucosyltransferase MRKKTMKRALTLVVGLVILSGLSILDFSITSATQQQATDRSNSVNYSTDVIYQIVTDRFYDGDESNNPSGELYSEDCKNLRKYCGGDWQGIIDKIDDGYLTNMGVTALWISPPVENIFETIDDEFGTTSYHGYWARDYKKTNPFFGSTEDFERLIETAHSHDIKIVIDLAPNHTSPADFDNPDYAENGVLYDDGNYLGSYSDDSDLFLYNGGTDFSNYEDEIYRNLFDLASFNHINPELNNYLEDAVKKWLDLGIDGIRIDAVAHMPPGWQKAYMDTIYDHRAVFTFGEWFTGPSGNEDYTKFANNSGMSVLDFRFAQTTRNVIGNNNGTMYDIEKMLTDTENDYDRPQDQVTFLDNHDMSRFTNGGETRTTDIGLALMLTSRGVPTIYYGTEQYMEGDGDPGSRAMMASFDENKDAYKLIQKLAPLRKSNPAYGYGTTTERWINDDVLIYERHFGENYALIAINRNLNTSYNIQGLQTEMPSNSYDDVLDGLLDGQSIVVDNKGGVSEFQMSPGEVSVWEFEATNVDKPSIGQVGPIIGEAGRTVTISGEGFGSSQGTVHFGSTSAEILSWNDTVITLTVPNNEAGYHDVTVVTEDEQVSNAYEFEVLTADQVTVRFVIDNAETKLGENVFLVGNVHELGNWDPEQSVGRFFNQIVYQYPTWYYDVNVPANTDLEFKFIKIDQDNNVTWQSGANQTYSSPESGTGIIRVDW SEQ ID No.2 DNA sequence Cyclodextrin glucanotransferase Synthetic DNA sequence SEQ ID No.3 Amylase sequence MKKTILALSIGLLSACSQIPENAIVLSVGDDTAVFEPNSNGLLVAEQKLEKGSYTFTIADSNQSCGSSFALAEESRIKFNRPLKMDNCATDAQMPLRIFKANIYQFTLNPTTNELTVRLKPKQNQDITYSCPVATDAPKTINVEQTFNDGTVVRDALTGQETIVTNGSVTMQP GPMSQGLLLLEEVEQQAEKPFSWDNATVYFVMTDRFYNGNPDNDNSYGRSKDGKYEIGTFHGGDLAGLTKKLDYIESLGVNAIWITSPLEQIHGWVGGGDKGDFKHYGYHGYYHQDWTKLDANMGTEDELKTFVDTAHKKGIRVIWDVVMNHTGYATLADMQEFGFGQLYLDDQ EAKELLGEKWTDWQPKSGQSWHSFNDYIKYGDSEAWDKWWGKDWIRTDIGEYDAPGYNDITMSLNYLPDLKTESTQKTGLPNFFRNKDTNAQDELQTPREHLITWLSDWVRDYGIDGFRVDTAKHVEMEAWAELKASTTQALAQWKQNNPDKALDDLPFWMTGEVWAHSVVKS PYFDNGFDSIINFEFQSDVAPKALKCFAQLDSDYRRYAERINSDPEFNVLSYLSSHDTQLFWSARSRSFDDQARAANALMLAPGAVQIYYGDEIARDFGVTGSDPTQGTRSDMPWDKIHGEREDLLQHWQKLGEFRQRHPAVAQGKHITRNQEGYYAFERQYHDDKVLIVYTGE SEQ ID No.4 signal peptide sequence MKKKIISAILMSTVILSAAAPLSGVYA 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 cyclodextrin glucosyltransferase mutant, characterized in that, The amino acid sequence of the mutant is the amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO:1 by two or more of the following mutations: F193L, A230I, and G259T.

2. The gene encoding the cyclodextrin glucosyltransferase mutant of claim 1.

3. A recombinant bacterium, characterized in that, The gene described in claim 2 is obtained by introducing it into a host bacterium.

4. The recombinant bacteria according to claim 3, characterized in that, The host bacteria are selected from Bacillus, lactic acid bacteria, Saccharomyces cerevisiae, galactosomalids, Bifida ferment lysate, Sphingomonas sphingosine monocytogenes, or Pichia pastoris.

5. The recombinant bacteria according to claim 3 or 4, characterized in that, The recombinant bacteria also integrates an amylase gene.

6. A fermentation filtrate for targeted enrichment of plant active ingredients, characterized in that, Obtained by fermentation of plants by the recombinant bacteria described in any one of claims 3 to 5.

7. The fermentation filtrate for targeted enrichment of plant active ingredients according to claim 6, characterized in that, The plants are selected from one or more of the following: blueberry, cranberry, turmeric, black tea, green tea, soybean, grape, pomegranate, astragalus, scutellaria, angelica, chrysanthemum, licorice, salvia miltiorrhiza, schisandra, polygonatum, milk thistle, gentian, rose, nasturtium, veratrum, safflower, dendrobium, amla, citron, gastrodia, sophora japonica, yew, and larch.

8. A method for preparing a fermentation filtrate for targeted enrichment of plant active ingredients, characterized in that, Includes the following steps: Provide a plant and crush it; Fermentation culture was carried out using the recombinant bacteria described in any one of claims 3 to 5, and an amylase inducer was added for further culture. The obtained fermentation broth was crushed and purified to obtain the fermentation filtrate.

9. The preparation method according to claim 8, characterized in that, The fermentation conditions are as follows: the fermentation temperature is 25-37℃, and the fermentation time is 8-24 h.

10. A multi-functional skincare product, characterized in that, The fermentation filtrate includes the targeted enrichment of plant active ingredients as described in claim 6 or 7.

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