Application of recombinant fructan sucrase in catalyzing EGCG (epigallocatechin gallate) glycosylation reaction as well as obtained product and application

Abstract

The invention provides application of recombinant fructan sucrase in catalyzing EGCG (epigallocatechin gallate) glycosylation reaction, an obtained product and application. The gene sequence of the recombinant fructan sucrase is shown as SEQ ID NO: 1. The enzyme can efficiently catalyze EGCG with high regioselectivity, through process optimization, the conversion rate reaches 65% or above, the enzyme method is mild in process condition and good in selectivity, the water solubility of the product is remarkably improved compared with that of an EGCG raw material, meanwhile, the antioxidant activity is reserved, the enzyme method shows clear advantages in the aspect of improving inherent defects (poor solubility and instability) of EGCG, the bioavailability of the enzyme is improved, and the enzyme method is suitable for industrial production. A core raw material and a feasible method are provided for developing new products with high stability and high bioavailability in the fields of functional foods, medicines, cosmetics and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a recombinant fructan sucrase in catalyzing the glycosylation reaction of EGCG, the resulting product, and its application. Background Technology

[0002] Epigallocatechin gallate (EGCG), the most abundant polyphenolic active ingredient in tea, possesses excellent antioxidant, anti-inflammatory, and anti-tumor bioactivities. However, EGCG faces technical bottlenecks in practical applications, such as poor water solubility, low chemical stability, and unsatisfactory oral bioavailability, which severely limit its full efficacy.

[0003] Currently, research on enzymatic glycosylation of EGCG mainly focuses on catalytic systems such as glucosyltransferases. Although these methods have achieved some success, they still have limitations in terms of catalytic efficiency, regioselectivity, and product diversity.

[0004] Based on this, the present invention aims to provide a novel method for the efficient and regioselective catalysis of EGCG fructosylation modification using fructan sucrase, in order to overcome the shortcomings of existing EGCG modification methods and provide novel derivatives with better performance for its application in the food, pharmaceutical and cosmetic fields. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides the application of recombinant fructan sucrase in catalyzing the EGCG glycosylation reaction, wherein the gene sequence of the recombinant fructan sucrase is shown in SEQ ID NO: 1.

[0007] As a preferred technical solution for the application described in this invention: the glycosylation reaction is a fructosyl transglycosylation reaction with sucrose as the glycosyl donor.

[0008] As a preferred technical solution for the application described in this invention: the glycosylation reaction is carried out under the conditions of pH = 6.0-7.0 and temperature 20-40℃; the reaction time is 10-30h.

[0009] As a preferred technical solution for the application described in this invention: in the glycosylation reaction, the concentration of EGCG is 5-12.5 g / L and the concentration of sucrose is 100-300 g / L.

[0010] As a preferred technical solution for the application described in this invention: in the glycosylation reaction, the concentration of EGCG is 6.2-6.5 g / L, the concentration of sucrose is 191-200 g / L, and the concentration of recombinant fructan sucrase is 20-25 U / mL.

[0011] As a preferred technical solution for the application described in this invention: the glycosylation reaction is carried out at pH=6.5, 25℃, and 200-300 rpm for 18 h.

[0012] The present invention also provides the reaction product obtained by the EGCG glycosylation reaction catalyzed by the recombinant fructan sucrase.

[0013] The present invention also provides the application of the reaction product obtained by the EGCG glycosylation reaction catalyzed by the recombinant fructan sucrase in the preparation of cosmetics.

[0014] The reaction product has antioxidant activity.

[0015] The present invention also provides the application of the reaction product obtained by the EGCG glycosylation reaction catalyzed by the recombinant fructan sucrase in the preparation of antioxidants.

[0016] The beneficial effects of this invention: This invention provides a novel application of recombinant fructan sucrase (SEQ ID NO:1) in the catalytic fructosylation reaction of EGCG. This enzyme can efficiently and regioselectively catalyze EGCG, achieving a conversion rate of over 65% through process optimization. The enzymatic process is mild, highly selective, and the product exhibits significantly improved water solubility compared to the EGCG raw material, while retaining antioxidant activity. It demonstrates clear advantages in improving the inherent defects of EGCG (poor solubility and instability), enhancing its bioavailability, and providing a core raw material and feasible method for developing new products with high stability and high bioavailability in functional foods, pharmaceuticals, and cosmetics. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein: Figure 1 To detect the enzymatic properties of fructan sucrase.

[0018] Figure 2 Three-dimensional response surface plot optimized for crude enzyme solution catalysis.

[0019] Figure 3 This is the liquid chromatogram of purified EGCG-1F.

[0020] Figure 4 This is the proton NMR spectrum of EGCG-1F.

[0021] Figure 5 This is the carbon NMR spectrum of EGCG-1F.

[0022] Figure 6 The HMBC spectrum of EGCG-1F.

[0023] Figure 7 The COSY spectrum of EGCG-1F.

[0024] Figure 8 The HSQC spectrum of EGCG-1F.

[0025] Figure 9 The structural formula of EGCG-1F Figure 10 Comparison of antioxidant effects between EGCG-1F and EGCG: (a) DPPH inhibition rate (b) ABTS inhibition rate.

[0026] Figure 11 This is a recombinant plasmid map of Example 1 of the present invention. Detailed Implementation

[0027] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0028] Example 1: Cloning of the fructan sucrase encoding gene and construction of its expression vector The Vibrio albus LS gene (ID: WP_109320344.1), encoding fructan sucrase, was optimized according to the codon usage preferences of *E. coli* and synthesized in its entirety by Genewiz Biotechnology Co., Ltd. (Suzhou, China). The Vibrio albus LS gene was expressed using an engineered *E. coli* strain BL21(DE3) carrying the pET28a-LS recombinant plasmid. The sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The recombinant plasmid sequence is shown in SEQ ID NO: 5.

[0029] The steps for expressing recombinant fructan sucrase and detecting its activity in Escherichia coli in Example 2 are as follows: The specific steps for expressing S1, recombinant fructan sucrase are as follows: 1) Inoculate the Escherichia coli strain containing LS into LB liquid medium containing 50 μg / mL kanamycin and incubate at 37℃ and 220 rpm for 16 h; the LB medium consists of 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L NaCl. 2) Inoculate 2% of the culture into TB liquid medium containing 50 μg / mL kanamycin. Incubate at 37℃ and 220 rpm until the bacterial density OD600 ≥ 0.6. Then add isopropyl-β-D-thiopyranoside (IPTG) to a final concentration of 0.5 mM and induce for 24 h at 25℃ and 220 rpm. Collect the bacterial culture. The TB medium consists of: yeast extract 24 g / L, tryptone 12 g / L, glycerol 4 mL / L, KH2PO4 2.3 g / L, and K2HPO4 12.5 g / L. 3) Centrifuge the bacterial culture at 4 ℃ and 10000 rpm for 15 min in a low-temperature refrigerated centrifuge, then wash twice with 50 ml K2HPO4 / KH2PO4 buffer (pH 6.5) and collect the bacterial cells; 4) Resuspend the cells in 50 mM phosphate buffer (pH 6.5) and sonicate to disrupt the cell mass. Use a 400W sonicator for 2 seconds of operation followed by 4 seconds of rest, for a total of 60 minutes. Obtain the supernatant by centrifugation (10,000 rpm, 15 minutes, 4°C), which will be used as the crude recombinant enzyme. S2. The enzyme activity detection system for recombinant fructan sucrase is as follows: 1 mL of 5% sucrose solution and 900 μL of 50 mM phosphate buffer (pH=6.5) are added to 100 μL of crude recombinant fructan sucrase solution. After reacting at 35 ℃ for 10 min, 3 mL of DNS reagent is immediately added and the mixture is boiled in a water bath for 15 min. The absorbance is measured at 540 nm, and the glucose content generated in the reaction solution is calculated. The amount of enzyme required to hydrolyze sucrose to produce 1 μmol of glucose per minute is defined as one enzyme activity unit (U) of fructan sucrase.

[0030] By reacting under different temperature and pH conditions, the optimal reaction temperature for recombinant SPase (recombinant fructan sucrase) was determined to be 40 ℃, and the optimal pH was 6.0. Figure 1 a, Figure 1 b).

[0031] Example 3: The steps for the transglycosylation reaction of EGCG catalyzed by recombinant fructan sucrase are as follows: EGCG (Chengdu Huagao Biological Products Co., Ltd.) and sucrose were added to the crude enzyme solution of recombinant fructan sucrase prepared in Example 1, and reacted at pH 6.5, 25°C, and 220 rpm for 18 h.

[0032] The steps for optimizing the EGCG transglycosylation reaction using response surface methodology in Example 4 are as follows: A three-factor, three-level Box-Behnken design (BBD) was used, with EGCG conversion rate as the response value, to investigate the interaction between EGCG concentration (X1), sucrose concentration (X2), and enzyme (X3). The factor level codes are shown in Table 1.

[0033] Table 1. Factors and levels in the Box-Behnken experimental design

[0034] The experimental results of the crude enzyme solution reaction are shown in Table 2, and the three-dimensional response surface plot of the crude enzyme solution catalyzed reaction is shown in Table 2. Figure 2 The amount of EGCG converted to EGCG-Fs by the crude enzyme solution was represented using a regression equation: Y=62.16-13.82X1+8.28X2+4.96X3+18.28X1X2+16X1X3-10.43X2X3-11.47X1 2 -9.28X2 2 -12.02X3 2 Where Y is the percentage (%) of EGCG converted to EGCG-Fs, X1 is the concentration of EGCG (g / L), X2 is the concentration of sucrose (g / L), and X3 is the concentration of fructan sucrase (U / mL).

[0035] The results of the analysis of variance (ANOVA) for the conversion of EGCG to EGCG-Fs are shown in Table 3. The coefficient of determination (R²) for the crude enzyme solution catalytic experiment data is also shown in Table 3. 2 The coefficient of determination (R²) was 0.9865, indicating that the model used to fit the response variable was significant (p < 0.0001). The F-value of the model was 56.7. The adjusted coefficient of determination (R²) is used to measure the goodness of fit of the regression equation. 2 The value was 0.9691. When the EGCG concentration was 6.2 g / L, the sucrose concentration was 191 g / L, and the fructan sucrase concentration was 20 U / mL, the predicted maximum conversion rate of EGCG to EGCG-Fs was 67.32%. To verify the predicted conversion rate of EGCG to EGCG-Fs, experiments were conducted using the above conditions. The experimental results showed that the conversion rate of EGCG to EGCG-Fs was 65.59 ± 1.71%, which was in good agreement with the predicted value of 67.32%.

[0036] Table 2. Experimental design and results of Box-Behnken (BBD) design for EGCG to EGCG-FS conversion.

[0037] Table 3. Analysis of variance of the quadratic response surface model for EGCG conversion catalyzed by crude enzyme solution.

[0038] The purification steps for EGCG-1F in Example 5 are as follows: S1. Separate the reaction products using a Sephadex LH-20 column (25 mm × 400 mm). After loading the sample, elute with distilled water to remove sugars (sucrose, fructose, and glucose), followed by elution with 70% (v / v) ethanol. Then concentrate the eluent (containing EGCG glycosides) using a rotary evaporator at 40°C. S2. Purification was performed using a TA-C18 (30*250mm 10um, Nanjing HeXi Biotechnology CO.,LTD) column at 30°C, a detection wavelength of 280 nm, a flow rate of 30 mL / min, and an injection volume of 1 mL. The mobile phase consisted of acetonitrile (A) and 0.1% (v / v) trifluoroacetic acid aqueous solution (B), with the following gradient elution program: 0-5 min: 90% B phase, 5-35 min: 90%-87%, 35-50 min: 87% B phase, 50-55 min: 87%-50% B phase, 55-65 min: 50% B phase, with a total run time of 65 min. like Figure 3 As shown, the purified EGCG-1F has a purity of ≥99%.

[0039] The structural identification steps for Example 6 EGCG-1F are as follows: S1. The structure of the product was determined using nuclear magnetic resonance (NMR) technology. Purified EGCG-Fs (10 mg) was dissolved in 500 μL DMSO-d6 and placed in a 5 mm NMR tube. NMR spectra were recorded on an AVANCE III 400 system (Bruker, Germany), which was run at 25°C for 2 minutes at 400 MHz for 1 hour, and then at 100 MHz for 5 hours at 13C. To assign proton and carbon signals, resolve the proton spin system, and determine glycosidic bond connections, HSQC, COSY, and HMBC spectra were further acquired. The specific structure was identified by NMR (1H, 13C, HMBC, COSY, and HSQC). The NMR results and identification process are as follows: 1H NMR (400 MHz, DMSO-d6) δ 6.77 (d, J = 2.0 Hz, 1H), 6.72 (d, J =2.0 Hz, 1H), 5.94 (d, J = 2.3 Hz, 1H), 5.86 (d, J = 2.2 Hz, 1H), 5.39 (s,1H), 5.00 (s, 1H), 4.11 (d, J = 8.4 Hz, 1H), 3.73 (d, J = 7.9 Hz, 1H), 3.65 (dt, J = 7.8, 3.8 Hz, 2H), 3.53 – 3.41 (m, 3H), 3.39 – 3.25 (m, 3H), 2.93(dd, J = 17.4, 4.7 Hz, 1H), 2.68 (d, J = 17.2 Hz, 1H). ( Figure 4 ) 13 C NMR (101 MHz, DMSO-d6) δ 165.11, 156.51, 156.46, 155.53, 145.54,145.36, 141.00, 138.62, 136.97, 127.86, 119.08, 112.68, 109.47, 108.60,107.87, 97.28, 95.54, 94.36, 82.81, 76.32, 75.82, 74.31, 68.01, 62.44, 60.03,25.77. ( Figure 5 ) By comparing the NMR spectra of the raw material EGCG and the product, it can be found that the product has one more fructose ring than the raw material, with signals at δH 4.11 (d, J = 8.4 Hz, 1H), 3.73 (d, J = 7.9 Hz, 1H), 3.65 (dt, J = 7.8, 3.8 Hz, 2H), 3.53 – 3.41 (m, 3H), 3.39 – 3.25 (m, 3H), and δC 107.87, 82.81, 75.82, 74.31, 62.44, and 60.03. Among them, δC 107.87 is the signal of the first carbon of the fructose ring. In order to further determine the connection position of the fructose ring, it is necessary to characterize the two-dimensional NMR signal.

[0040] Two-dimensional NMR signals showed a weak HMBC correlation between the hydrogen δH 4.11 (d, J = 8.4 Hz, 1H) on the fructose ring and the oxygen-bound quaternary carbon δC 141.00 on the flavonoid B ring benzene ring, thus confirming that the fructose ring is attached at position 3' in the structural formula. Analysis of the 1H and 1C NMR spectra of the fructose moiety further clarified its stereoconfiguration. 13 In the 10⁻¹⁰ C NMR spectrum, the anomeric carbon signal appears at δC 107.9, which is within the typical chemical shift range of β-D-fructofuranoside (δC 105–110), suggesting that it has a five-membered furan ring structure. 1 In the 1H NMR spectrum, the anomeric hydrogen signal at δH 4.11 shows a double peak, and its large coupling constant (J = 8.4 Hz) suggests that the furanose ring has a rigid conformation due to the formation of glycosidic bonds, resulting in a dihedral angle of nearly 180° between the anomeric hydrogen and the adjacent hydrogen. This feature is consistent with the stereochemical environment of the β-configuration. 1 H- 1 ¹H COSY and ¹HMBC spectra further confirmed the intact spin system and planar connection of the sugar ring. Based on these characteristics, the sugar unit was determined to be a β-D-fructofuranoside configuration.

[0041] In summary, the structure of the compound is determined as follows, and the key HMBC and COSY related parameters are shown below. Figure 9 .

[0042] Example 7: The procedure for determining the DPPH free radical scavenging activity of EGCG-1F and EGCG is as follows: First, a 50.0 μg / mL DPPH working solution was prepared using anhydrous ethanol. Simultaneously, the test sample was dissolved in deionized water, and six concentration gradients of 7.8125, 15.625, 31.25, 62.5, 125, and 250 μM were set up. The experiment was conducted in a 96-well plate, with three reaction systems: the sample group contained 100 μL of the test sample solution and 100 μL of DPPH ethanol solution; the blank group contained 100 μL of anhydrous ethanol and 100 μL of DPPH ethanol solution as a reference; and the control group contained 100 μL of the test sample solution and 100 μL of anhydrous ethanol to eliminate interference from the sample's own absorbance. After thorough mixing in each well, the mixture was incubated at room temperature in the dark for 30 min. Finally, the absorbance was measured at 517 nm using a multi-functional enzyme-linked immunosorbent assay (ELISA) analyzer (Hangzhou Ausen Instruments Co., Ltd.).

[0043] like Figure 10As shown, both EGCG and EGCG-1F exhibited concentration-dependent DPPH scavenging activity, with EGCG showing stronger free radical scavenging ability (IC50 = 16.29 ± 0.59 μM), while the activity of EGCG-1F was reduced (IC50 = 85.15 ± 9.55 μM).

[0044] Example 8: The steps for determining the ABTS free radical scavenging activity of EGCG-1F and EGCG are as follows: A 7 mmol / L ABTS solution was mixed with a 2.45 mmol / L potassium persulfate solution at a volume ratio of 2:1 and allowed to react in the dark at room temperature for 16 hours to obtain the ABTS stock solution. The ABTS stock solution was diluted with anhydrous ethanol to obtain an absorbance of approximately 0.7 at 734 nm, thus obtaining the ABTS working solution. Samples were dissolved in deionized water at concentrations of 15.625, 31.25, 62.5, 125, 250, and 500 μM. The experiment was conducted in 96-well plates, with sample, blank, and control groups. The sample group consisted of 50 μL of the test sample solution and 150 μL of ABTS ethanol solution; the blank group consisted of 150 μL of anhydrous ethanol instead of the ABTS solution; and the control group consisted of 50 μL of anhydrous ethanol instead of the sample solution. After thorough mixing, the mixture was allowed to react at room temperature in the dark for 15 minutes. Finally, the absorbance was measured at 734 nm using a multi-functional enzyme-linked immunosorbent assay (ELISA) analyzer.

[0045] like Figure 10 As shown, both EGCG and EGCG-1F exhibited concentration-dependent ABTS scavenging activity, with EGCG demonstrating stronger free radical scavenging ability (IC50 = 44.23 ± 17.08 μM), while the activity of EGCG-1F was reduced (IC50 = 206.65 ± 14.28 μM). Notably, despite the decrease in activity, EGCG-1F still maintained a significant free radical scavenging ability, confirming that glycosylation modification did not completely destroy its antioxidant structure.

[0046] Example 9 The high-performance liquid chromatography (HPLC) method was as follows: An XP tC18 (4.6 × 250 mm, 5 μm) column (Micropure Biotechnology (Guangzhou) Co., Ltd.) was used at a column temperature of 30°C, a detection wavelength of 280 nm, a flow rate of 0.5 mL / min, and an injection volume of 10 μL. The mobile phase consisted of acetonitrile (A) and a 0.1% (v / v) trifluoroacetic acid aqueous solution (B). The gradient elution program was as follows: 0–25 min: 95%–70% B phase; 25–35 min: 70%–20% B phase; 35–40 min: 20% B phase; 40–45 min: 20%–95% B phase; 45–55 min: 95% B phase; total run time: 55 min.

[0047] Example 10 Solubility test of EGCG and EGCG-1F: Excess EGCG and EGCG-1F were dissolved in 100 μL of water in Eppendorf tubes at room temperature (25 °C) using ultrasonication for 1 hour. Each dissolved sample was filtered through a 0.45 μm filter membrane, diluted by a certain factor, and the concentration was determined by HPLC analysis. The solubility of EGCG was found to be 5.1 mM, and the solubility of EGCG-1F was found to be 492.6 mM, indicating a highly significant improvement in the solubility of EGCG-1F.

[0048] Comparative Example 1 The C. michiganensis LS gene (ID: WP_011931834.1), encoding fructan sucrase, was optimized according to E. coli codon usage preferences and synthesized in its entirety by Genewiz Biotechnology Co., Ltd. (Suzhou, China). The C. michiganensis LS gene was expressed using an engineered E. coli strain BL21(DE3) carrying the pET28a-LS recombinant plasmid. The sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

[0049] The recombinant fructan sucrase (SEQ ID NO: 1) in Example 3 was replaced with the recombinant fructan sucrose shown in SEQ ID NO: 3. The catalytic reaction was carried out in the same manner as in Examples 3 and 4 above. The experimental results showed that the catalytic reaction failed.

[0050] Comparative Example 1 Experimental Results: The reaction product showed no free radical scavenging activity.

[0051] SEQ ID NO: 1 SEQ ID NO:2:MGSIRMKPVVPVLGVILGVATAGFSQLTSAAQLQPGPEPTLHTQQAYAPESNFTAKWTRADARQIKRMSDPMAGSRENSMPEEYTMPSIPQDFPDMSNGKVWVWDSWPLTDADANQYSVNGQEIIFSLVADRSLGFDDRHVFAKIGYFYRPAGIPADQRPENGGWTYGGLVFDEGVTGQIFEDQSYSHQTQWSGSARIFPGGEVKLFFTDVAFYRDAQGNDIKPYDPRIALSVGKIHANKNGVKFTGFDKVTSLLEADGTYYQTAEQNPYFNFRDPFTFKDPAHPGETFMVFEGNSAMQRGEAKCTEEDLGYQTGDPYAETVRQVNASGATFQIGNVGLARATSDDLTEWEFLPPILSANCVTDQTERPQIYQKDGKYYLFTISHSTTYAAGITGPEGVYGFVGDGIRSDYQPMNQGSGLVLGNPTNLNYYPGTPFDPDYNQPAGHFQSYSHYVMPGGLVQSFIDTVGVKEDFRRGGTLAPTVKIIIDGDSSEVDRSYGFYGNGLGGWADIPASINVNPSGINTKPLK SEQ ID NO:3 SEQ ID NO: 4 MTKRIRRGLSASAAATLVVASALLAGGSAQAAGTTPPRPTTVHTQKAYAPEDDFTAHWTRADAKQIAKLSDPTVAPRTNSMPEALTMPQVPQDFPTMTDQAYVWDTWPLTDSSGQTYSVDGYDVIFALTAPRTLSFDDRHTYAKIGYFTRPTGIPSEQRPENGGWTYQGNVFEDGVTDGIFPDQSFTQQAEWSGSARIMADGTVKLFFTDVAFYRDAKGQDVKPADPVISLSQGRVEKVDGAVALKGFETVTPLLRPDGQRYQTNEQNWSTNFRDPFTFTDPDHPGKTYMVFEANVAGKRGEQECDATDLGYRKGDPAAEDPKEVTARGANYQMASIGLAVADDADLTKWHYLDPLLESACVTDQTERPEVMIENGKHYLFTISHRSTFAAGIDGPEGVYGFVGNGLRSDYKPMNGGSGLVLGNPTNLNYAGGTAYAPDYNQTPGAFQAYSSYILPGGLVESFIDAVGSKESFRRGGTLGPTVKLEFDGDTSELDRGYGEGGLGGYADIPTTRVFDPAHPPPQ SEQ ID NO: 5 It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of a recombinant fructan sucrase in catalyzing the glycosylation reaction of EGCG, characterized in that: The gene sequence of the recombinant fructan sucrase is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that: The glycosylation reaction is a transfructose reaction using sucrose as the glycosyl donor.

3. The application according to claim 2, characterized in that: The glycosylation reaction was carried out at pH 6.0-7.0 and temperature 20-40℃ for 10-30 h.

4. The application according to claim 3, characterized in that: In the glycosylation reaction, the concentration of EGCG is 5-12.5 g / L and the concentration of sucrose is 100-300 g / L.

5. The application according to claim 4, characterized in that: In the glycosylation reaction, the concentration of EGCG is 6.2-6.5 g / L, the concentration of sucrose is 191-200 g / L, and the concentration of recombinant fructan sucrase is 20-25 U / mL.

6. The application according to claim 4, characterized in that: The glycosylation reaction was carried out at pH 6.5, 25°C, and 200-300 rpm for 18 h.

7. The reaction product obtained by the EGCG glycosylation reaction catalyzed by the recombinant fructan sucrase according to any one of claims 1 to 6.

8. The application of the reaction product obtained by the EGCG glycosylation reaction catalyzed by the recombinant fructan sucrase according to claim 7 in the preparation of cosmetics.

9. The application according to claim 8, characterized in that: The reaction product has antioxidant activity.

10. The application of the reaction product obtained by the EGCG glycosylation reaction catalyzed by the recombinant fructan sucrase according to claim 7 in the preparation of antioxidants.

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