Bacillus cereus UAE17 and application thereof

Abstract

The invention discloses a strain of bacillus cereus UAE17 and application thereof, the bacillus cereus UAE17 is preserved in the China Center for Type Culture Collection on February 21, 2025, and the preservation number is CCTCC NO: M 2025274. The bacillus cereus UAE17 has been preserved in the China Center for Type Culture Collection on February 21, 2025. According to the bacillus cereus UAE17, glycoside substances in soybean meal can be converted into soybean isoflavone aglycones, the bacillus cereus can convert the glycoside substances in agricultural and sideline product soybean meal into more effective soybean isoflavone aglycones by producing beta-glucosidase, the yield of isoflavone aglycones can be obviously increased, and the yield of soybean isoflavone aglycones can be increased. The residual value of the agricultural and sideline product soybean meal is improved. Good application prospects are realized in the fields of medical products, breeding industry and health care.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Bacillus cereus UAE17 and applications thereof. Background Art

[0002] Soybean meal, an agricultural byproduct, contains a significant amount of soy isoflavones, which have garnered widespread scientific attention due to their diverse pharmacological activities and potential health benefits. Soy isoflavones can be structurally divided into glycosides and aglycones. Aglycones, primarily genistein, daidzein, and glycitein, are absorbed and utilized by the human body, accounting for only 2%-3% of the total isoflavones. These aglycones exhibit antibacterial, antioxidant, and anti-tumor activities, as well as protection against heart disease and diabetes. They can be used as targeted therapies for anticancer, anti-aging, and osteoporosis prevention, and serve as potential lead compounds for new anticancer drugs. Research indicates that they hold promising prospects in a variety of fields, including food and health care, feed research and development, and pharmaceutical products.

[0003] In recent years, reports on the microbial conversion of glycosides to aglycones have increased year by year. For example, when Lactobacillus plantarum YHG1-155 is fermented in sea buckthorn juice, it can convert polyphenol glycosides into aglycones, thereby improving the antioxidant activity and bioavailability of the juice. During the fermentation process, the glycoside polyphenols in the sea buckthorn juice are converted into aglycones through the biosynthetic pathway of secondary metabolites. Microbial fermentation technology can be used to modify the structure of flavonoids, achieving the mutual conversion of flavonoid glycosides and aglycones, thereby enriching aglycone components. For example, microbial fermentation of flavonoids from plants such as soybeans, dried tangerine peel, and scutellaria baicalensis can produce more novel derivatives and improve their bioavailability. However, there are no reports on the production of soy isoflavone aglycones by converting glycosides from soybean meal through fermentation with Bacillus cereus.

[0004] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0005] The present invention aims to provide a strain of Bacillus cereus UAE17, which can convert glycosides in soybean meal to produce soybean isoflavone aglycones, and the application of Bacillus cereus UAE17 in antibacterial and antioxidant activities.

[0006] To achieve the above objectives, the present invention provides a strain of Bacillus cereus UAE17, which was deposited in the China Center for Type Culture Collection on February 21, 2025, with the deposit number being CCTCC NO: M 2025274.

[0007] The present invention provides the use of the above-mentioned Bacillus cereus UAE17 bacterial agent in fermenting soybean meal.

[0008] Preferably, in the above technical solution, the method for fermenting soybean meal with the inoculum of Bacillus cereus UAE17 comprises:

[0009] (1) placing Bacillus cereus UAE17 in a fermentation medium and culturing it to obtain a bacterial liquid;

[0010] (2) mixing soybean meal, a carbon source, an inorganic salt, and a surfactant to prepare a soybean meal fermentation medium;

[0011] (3) transferring the bacterial culture liquid to a soybean meal fermentation medium, fermenting to obtain fermented soybean meal, extracting the fermented soybean meal product, and obtaining soybean isoflavone aglycone substances.

[0012] Preferably, in the above technical solution, the culture method of the bacterial liquid includes: activating the Bacillus cereus UAE17 and placing it in a fermentation medium at an inoculum amount of 1-5% by volume, carrying out secondary fermentation and expansion culture under the conditions of 34-38°C, 100-300rpm, and pH 7.2-7.4, terminating the fermentation when the culture reaches the logarithmic growth phase, and obtaining the bacterial liquid.

[0013] Preferably, in the above technical solution, the culture method of the bacterial liquid includes: the fermentation medium is BE medium, the main components of which are: 1-5g beef extract powder, 5-15g peptone, 1-10g NaCl, dissolved in 1L deionized water, and adjusted to pH 7.2-7.4; the solid culture medium for bacterial activation is BE medium with 10-30g / L agar added.

[0014] Preferably, in the above technical solution, the soybean meal fermentation medium is mainly composed of the following raw materials: 10-15 g / L cassava flour, 120-180 g / L soybean meal, 0.5-2 g / L NaCl, 0.01-0.1 g / L MgSO4, 0.05-0.2 g / L CaCl2, 0.05-0.2 g / L PEG4000, 0.1-1 g / L Span20, 0.01-0.1% [Emim]Ac, and the pH is adjusted to 5.5-6.5.

[0015] Preferably, in the above technical solution, the fermentation culture conditions in step (3) are as follows: temperature 34-38°C, pH 5.5-6.5, rotation speed 100-300 rpm, liquid volume 40-80 mL / 250 mL, inoculation size 1-5%, and fermentation time 80-120 h.

[0016] Preferably, in the above technical solution, the method for extracting the fermentation product in step (3) comprises: taking a sample from the fermentation broth and mixing it with methanol in a volume ratio of 1:2-10, preferably 1:4; then performing ultrasonic centrifugation and extracting the supernatant, filtering the membrane, and obtaining the purified soybean isoflavone aglycone substance; wherein the extraction temperature is 35-45°C, and the ultrasonic time is 30-120min.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention discloses a strain of Bacillus cereus UAE17, which is capable of converting glycosides in soybean meal into soybean isoflavone aglycones. By producing β-glucosidase, Bacillus cereus can convert glycosides in the agricultural byproduct soybean meal into more effective soybean isoflavone aglycones, thereby greatly maximizing the residual value of soybean meal. The fermented soybean meal products were screened by ultra-high performance liquid chromatography and characterized by mass spectrometry, and were ultimately identified as genistein and daidzein. The invention has good application prospects in pharmaceutical products, aquaculture, and health care.

[0019] (2) The present invention uses ultraviolet mutagenesis, ARTP and EMS continuous mutagenesis, as well as culture medium optimization and fermentation condition optimization, to add Bacillus cereus UAE17 to the soybean meal fermentation medium, thereby converting a large amount of glycoside substances in the soybean meal into more effective genistein and daidzein, which can significantly increase the yield of isoflavone aglycones and improve the surplus value of the agricultural and sideline product soybean meal.

[0020] Preservation Information

[0021] Bacillus cereus UAE17 was deposited in the China Center for Type Culture Collection (CCTCC) on February 21, 2025, with the accession number CCTCC NO: M 2025274. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The morphology and growth curve of Bacillus cereus UAE17;

[0023] Figure 1 In the figure, (A): BE medium; (B): Geniposide colorimetric medium; (C): Gram staining of strains; (D): growth curve of strains.

[0024] Figure 2 Electrophoretogram of Bacillus cereus UAE17;

[0025] Figure 3UPLC-MS / MS analysis of daidzein produced by fermentation of soybean meal by Bacillus cereus UAE17;

[0026] Figure 3 Middle, (AB): extracted ion current chromatograms; (A) is UAE17 extract, (B) is 5 mg / L daidzein standard;

[0027] (CD): Mass-to-charge ratio (M / S) diagram; (C) is the UAE17 extract; (D) is a 5 mg / L daidzein standard.

[0028] Figure 4 UPLC-MS / MS analysis of genistein produced by fermentation of soybean meal by Bacillus cereus UAE17;

[0029] Figure 4 Middle, (AB): extracted ion current chromatograms; (A) is UAE17 extract, (B) is 5 mg / L genistein standard;

[0030] (CD): Mass-to-charge ratio (M / S) diagram; (C) is UAE17 extract, and (D) is 5 mg / L genistein standard.

[0031] Figure 5 The antibacterial effect of the fermentation product of Bacillus cereus UAE17 and optimized soybean meal medium on Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa;

[0032] Figure 5 In the figure, (A): Escherichia coli; (B): Staphylococcus aureus; (C): Pseudomonas aeruginosa.

[0033] The upper left corner of the culture dish is the optimized soybean meal culture medium extract, the upper right corner is methanol, the lower left corner is the fermentation extract of the optimized strain UAE17, and the lower right corner is the fermentation extract of the strain UAE17 before optimization.

[0034] Figure 6 Determination of the antioxidant capacity of extracts fermented with Bacillus cereus UAE17 and optimized soybean meal medium.

[0035] Figure 6 In the figure, (A): DPPH free radical scavenging rate; (B): ABTS free radical scavenging rate; (C): iron reducing power.

[0036] CK represents the optimized soybean meal culture medium extract, GH3 represents the fermentation extract of the starting strain GH3, and UAE17 represents the fermentation extract of the optimized mutagenic strain UAE17. DETAILED DESCRIPTION

[0037] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0038] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0039] Unless otherwise specified, all raw materials and reagents used in the examples are commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0040] In the examples, Bacillus cereus UAE17 was deposited in the China Center for Type Culture Collection (CCTCC) on February 21, 2025, with the deposit number being CCTCC NO: M2025274.

[0041] In the embodiment, BE medium was used: 3 g beef extract powder, 10 g peptone, and 5 g NaCl were dissolved in 1 L deionized water and the pH was adjusted to 7.2-7.4. The solid medium for bacterial activation was BE medium with 20 g / L agar added.

[0042] Example 1

[0043] 1 Screening of strains and identification of soybean isoflavone aglycones produced by transformation

[0044] 1.1 Sampling: Sweet potato leaves were collected in the laboratory, placed in sample bags, and stored in a refrigerator at 4°C.

[0045] 1.2 Screening: Using the plate method, fresh plant leaves were collected and soaked in 75% alcohol for 60 seconds, rinsed with sterile water, and surface-disinfected in 5% sodium hypochlorite solution for 15 seconds. Residues were then rinsed with sterile water. The disinfected samples were cut into small pieces with sterile scissors and placed on beef extract peptone solid medium plates. The plates were incubated in a 37°C incubator for 1-2 days. Several strains with good growth on the leaf cuts were selected and inoculated into geniposide chromogenic medium for 1-2 days for initial screening. Strains showing a blue color around the colonies were selected and inoculated into BE liquid medium at 37°C, 200 rpm, until the late logarithmic growth phase to prepare seed liquid. A 2% portion of the seed liquid was inoculated into a 250 mL Erlenmeyer flask containing 60 mL of soybean meal medium and fermented at 37°C, 200 rpm, for 96 hours. The fermentation broth was mixed with methanol in a 1:4 ratio, sonicated for 50 minutes, and centrifuged twice. The supernatant was filtered through a 0.22 μm filter to prepare the sample solution. Ultra-performance liquid chromatography (UPLC) was used for detection; UPLC parameters were: Kinetex C18 (50 mm × 3 mm, 1.7 μm). Mobile phase A: 0.1% (v / v) formic acid, mobile phase B: acetonitrile. Elution conditions were: 95% A (0-2 min); 5% A (10-11.4 min); and 95% A (11.5-16 min). Flow rate: 0.3 mL / min; column temperature: 35°C; flow rate: 0.3 mL min-1; injection volume: 1 μL; detection wavelength: 260 nm.

[0046] 1.3 Qualitative analysis: Samples with the same elution time as genistein and daidzein standards in UPLC were detected using a Thermo Fisher Q-Exactive HPLC system in the negative ion [MH]- detection mode using an electrospray ionization source (ESI). The results were compared with those of genistein and daidzein standards. The total ion chromatograms of genistein and daidzein in the standards and samples were identical, and the primary and secondary mass spectra showed correct results. A plant endophytic strain with the ability to transform and produce soy isoflavone aglycones was discovered.

[0047] Identification of 2 strains of Bacillus cereus

[0048] 2.1 The obtained strain was subjected to a series of physiological and biochemical identifications, and DNA was extracted for 16S rDNA amplification and sequencing. The results are as follows:

[0049] The strain formed white colonies with irregular edges on beef extract peptone solid medium; Gram staining showed that the strain was a Gram-positive bacterium;

[0050] 2.2 16S rDNA was amplified using primers 27F and 1492R. The primer sequences are as follows:

[0051] 27F:5′-AGAGTTTGATCATGGCTCAG-3′

[0052] 1492R:5′-TAGGGTTACCTTGTTACGACTT-3′

[0053] PCR amplification conditions were 94°C for 3 minutes, 94°C for 30 seconds, 55°C for 1 minute, 72°C for 1.5 minutes, 30 cycles, 72°C for 5 minutes, and storage at 4°C. The PCR product was verified by 1% agarose gel electrophoresis and then sent for sequencing. Sequencing results revealed a 1379-bp 16S rDNA sequence. Based on 16S phylogenetic tree alignment, the strain was most closely related to Bacillus cereus MRS1. Combined with morphological, physiological, and biochemical characteristics, the strain was identified as a strain of the genus Bacillus cereus. Following subsequent mutagenesis, it was named Bacillus cereus UAE17 and deposited with the China Center for Type Culture Collection (CCTCC) under the accession number CCTCCNO: M 2025274.

[0054] 2.3 Inoculate Bacillus cereus UAE17 into 50 mL of basal fermentation medium at a 1% inoculum volume in a 250 mL conical flask and culture at 37°C, 200 rpm in a constant temperature shaker. Take the bacterial solution every 2 hours and use the culture medium without bacterial solution as a control. Measure the absorbance at 600 nm. Draw a growth curve for each group of three replicates. (As shown in the figure, the bacterial solution is taken every 2 hours and the absorbance is measured at 600 nm.) Figure 1-4 )

[0055] Example 2

[0056] A method for producing soybean isoflavone aglycones by fermenting soybean meal with Bacillus cereus UAE17 is as follows:

[0057] 1. Preparation of a bacterial seed solution: After activation of Bacillus cereus UAE17 through continuous mutagenesis, a 2% by volume inoculum is placed in a fermentation medium. Secondary fermentation and expansion are carried out at 37°C, 200 rpm, and a pH of 7.2-7.4. Fermentation is terminated when the culture reaches the logarithmic growth phase to obtain a bacterial seed solution for later use. Preferably, the fermentation medium is BE medium: composed of 3g beef extract powder, 10g peptone, and 5g NaCl dissolved in 1L deionized water, and the pH adjusted to 7.2-7.4. The solid medium for bacterial activation is BE medium supplemented with 20g / L agar.

[0058] 2. Prepare optimized soybean meal culture medium: Grind defatted soybean meal and oven-dried fresh cassava, pass through a 150-mesh sieve, and mix thoroughly with a carbon source, inorganic salts, and surfactant in the appropriate proportions. The optimized culture medium composition is: 12.91 g / L cassava flour, 150.66 g / L soybean meal, 1.0 g / L NaCl, 0.05 g / L MgSO₄, 0.1 g / L CaCl₂, 0.1 g / L PEG4000, 0.37 g / L Span₂O, 0.02% [Emim]Ac. Adjust pH to 6.

[0059] 3. The bacterial culture liquid obtained in step 1 and the optimized soybean meal medium prepared in step 2 were mixed and then fermented. The optimized fermentation conditions were temperature 37°C, pH = 6.0, rotation speed 220 rpm, liquid volume 60 mL / 250 mL, inoculation size 2%, and fermentation time 96 h.

[0060] 4. Product Extraction: The extraction method was a 1:4 ratio of fermentation product to methanol at 40°C, followed by sonication for 50 minutes, followed by centrifugation at 10,000 rpm for 10 minutes. The supernatant was collected and the extraction was repeated once. The supernatant was filtered through a 0.22 μm filter and bottled for ultra-high performance liquid chromatography (UPLC) analysis. UPLC parameters were Kinetex C18 (50 mm × 3 mm, 1.7 μm). Mobile phase A: 0.1% (v / v) formic acid, B: acetonitrile. Elution conditions were: 95% A for 0-2 minutes; 5% A for 10-11.4 minutes; and 95% A for 11.5-16 minutes. Flow rate: 0.3 mL / min; column temperature: 35°C; flow rate: 0.3 mL min-1; injection volume: 1 μL; detection wavelength: 260 nm. The content of soy isoflavone aglycones in the fermentation broth was calculated based on a standard curve.

[0061] 5. Determination of the standard curve: Dissolve 1 mg of genistein and daidzein standards in methanol to prepare a 500 mg / L stock solution. Dilute the stock solution to 1, 5, 10, 25, 50, 100, 200, 300, and 400 mg / L, respectively. Quantify by UPLC. Plot a standard curve using concentration as the abscissa and peak area as the ordinate.

[0062] Example 3

[0063] The specific operation method of the inhibition zone experiment of the fermentation product is as follows:

[0064] 1. First, strain UAE17, grown to the logarithmic growth phase, was fermented using optimized medium composition and fermentation conditions. The strain was inoculated at a 2% (v / v) ratio into optimized soybean meal medium and fermented at 37°C and 220 rpm for 96 hours. Extraction was performed using a 1:4 ratio of fermentation product to methanol at 40°C, ultrasonication for 50 minutes, followed by centrifugation at 10,000 rpm for 10 minutes. The supernatant was then filtered through a 0.22 μm filter. The optimized soybean meal medium and fermentation broth were treated identically, with methanol serving as a blank control.

[0065] 2. Dilute Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa cultured to the logarithmic growth phase to an appropriate concentration and evenly spread them on LB solid medium with a sterile cotton swab. Place a sterilized filter paper on the LB solid medium and draw 10uL of the fermentation product extract on the center of the filter paper. Place it in a 37°C constant temperature incubator and culture for 24 hours. Use methanol solution as a blank control, and use soybean meal medium before optimization and optimized soybean meal medium without bacteria as controls. Observe and calculate the size of the inhibition zone. (If Figure 5 shown)

[0066] Example 4

[0067] The specific operation method of the antioxidant experiment of fermentation products is as follows:

[0068] 1. First, strain UAE17, grown to the logarithmic growth phase, was fermented using optimized medium composition and fermentation conditions. The strain was inoculated at a 2% (v / v) ratio into optimized soybean meal medium and fermented at 37°C and 220 rpm for 96 hours. Extraction was performed using a 1:4 ratio of fermentation product to methanol at 40°C, ultrasonication for 50 minutes, followed by centrifugation at 10,000 rpm for 10 minutes. The supernatant was removed, the mixture was repeated, and the supernatant was filtered through a 0.22 μm filter. The optimized soybean meal medium and fermentation broth were treated identically, with the extract from the optimized soybean meal medium designated as CK.

[0069] 2. Determination of DPPH Radical Scavenging Efficiency: Prepare a 0.2 mmol / L DPPH solution in anhydrous ethanol. Measure 500 μL of each sample solution, add 1500 μL of the DPPH solution, mix thoroughly, and incubate in the dark at room temperature for 30 minutes. Centrifuge at 8000 rpm for 5 minutes, and measure the OD at 517 nm. Perform three replicates for each assay. Use Trolox as the quantitative standard, plot a standard curve, and calculate the DPPH radical scavenging efficiency according to the formula.

[0070] 3. Determination of ABTS scavenging efficiency: Mix equal volumes of 5 ml of ABTS (7.4 mmol / L) and 5 ml of K2S2O8 (2.6 mmol / L) and incubate at room temperature in the dark for 16 hours. Dilute the ABTS mixture with PBS buffer to obtain a final ABTS solution with an OD value of 0.7 ± 0.02 at 734 nm. Finally, add 0.2 ml of sample solution and 0.8 ml of ABTS solution to a centrifuge tube, mix well, and incubate in the dark for 6 minutes. Measure the absorbance of the solution at 734 nm, perform three replicates for each parallel, use Trolox as a quantitative standard, draw a standard curve, and calculate the ABTS clearance rate according to the formula.

[0071] 4. Determination of iron reducing power: Add 0.25 mL of PBS buffer (0.2 Mol / L, pH=6.6), 0.25 mL of 1% potassium ferricyanide solution (K3Fe(CN)6)), and 0.25 mL of sample solution to a 2 mL centrifuge tube. Mix thoroughly and place in a 50°C water bath for 20 minutes. Then quickly cool in an ice-water bath. Quickly add 0.25 mL of 10% (v / v) trichloroacetic acid. Centrifuge at 4000 rpm for 10 minutes. Take the supernatant, then add 0.25 mL of supernatant, 0.25 mL of distilled water, and 0.05 mL of 0.1% FeCl3 solution to a new centrifuge tube in sequence. Mix thoroughly and let stand at room temperature in the dark for 10 minutes. Read the absorbance at 700 nm. Repeat three times for each parallel. Trolox is used as the quantitative standard, and a standard curve is drawn. (e.g. Figure 6 shown)

[0072] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A strain of Bacillus cereus UAE17, characterized in that The Bacillus cereus UAE17 was deposited in the China Center for Type Culture Collection on February 21, 2025, with the deposit number being CCTCC NO: M 2025274.

2. Application of the inoculant of Bacillus cereus UAE17 as claimed in claim 1 in fermented soybean meal.

3. The use according to claim 2, characterized in that The method for fermenting soybean meal with the bacterial agent of Bacillus cereus UAE17 comprises: (1) placing Bacillus cereus UAE17 in a fermentation medium and culturing it to obtain a bacterial liquid; (2) mixing soybean meal, a carbon source, an inorganic salt, and a surfactant to prepare a soybean meal fermentation medium; (3) transferring the bacterial culture liquid to a soybean meal fermentation medium, fermenting to obtain fermented soybean meal, and extracting the fermented soybean meal product.

4. The use according to claim 3, characterized in that The culture method of the bacterial liquid includes: activating the Bacillus cereus UAE17 and placing it in a fermentation medium at an inoculum amount of 1-5% by volume, performing secondary fermentation and expansion culture under the conditions of 34-38° C., 100-300 rpm, and pH 7.2-7.4, terminating the fermentation when the culture reaches the logarithmic growth phase, and obtaining the bacterial liquid.

5. The use according to claim 3, characterized in that The bacterial liquid culture method includes: the fermentation medium is BE medium, which mainly consists of: 1-5g beef extract powder, 5-15g peptone, and 1-10g NaCl, which are dissolved in 1L of deionized water and adjusted to pH 7.2-7.4; the solid culture medium for bacterial activation is BE medium with 10-30g / L agar added.

6. The use according to claim 3, characterized in that The soybean meal fermentation medium mainly consists of the following raw materials: 10-15 g / L of cassava flour, 120-180 g / L of soybean meal, 0.5-2 g / L of NaCl, 0.01-0.1 g / L of MgSO4, 0.05-0.2 g / L of CaCl2, 0.05-0.2 g / L of PEG4000, 0.1-1 g / L of Span20, and 0.01-0.1% of [Emim]Ac, and the pH is adjusted to 5.5-6.

5.

7. The use according to claim 6, characterized in that The fermentation culture conditions in step (3) are as follows: temperature 34-38° C., pH 5.5-6.5, rotation speed 100-300 rpm, liquid volume 40-80 mL / 250 mL, inoculation amount 1-5%, and fermentation time 80-120 h.

8. The use according to claim 3, characterized in that The method for extracting the fermentation product in step (3) comprises: taking a sample from the fermentation broth and mixing it with methanol in a volume ratio of 1:2-10, preferably 1:4; then extracting the supernatant by ultrasonic centrifugation, filtering the membrane, and obtaining the extract; wherein the extraction temperature is 35-45°C and the ultrasonic time is 30-120 min.

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