Preparation method of high-purity cyanidin-3-glucoside-4-vinyl catechol

Abstract

The invention provides a preparation method of high-purity cyanidin-3-glucoside-4-vinyl catechol. The preparation method comprises the following steps: preparing a fermentation system; the activated lactic acid bacteria strain is added into a fermentation system, the inoculation amount is 1 * 10 < 20 > CFU / mL, the black soybean hull crude extract and caffeic acid are added into every 2 L of the fermentation system, the final concentration of caffeic acid is 0.5 mmol / L, the purity of cyanidin-3-glucoside contained in the black soybean hull crude extract reaches 50% or above, and fermentation is carried out at 37 DEG C for at least 24 hours; carrying out suction filtration on the fermentation liquor to obtain supernate; medium-pressure preparation; and carrying out freeze drying on the collected peak to obtain powder, and determining that the powder is cyanidin-3-glucoside-4-vinyl catechol. Compared with cyanidin-3-glucoside, the cyanidin-3-glucoside-4-vinyl catechol prepared by the method disclosed by the invention has higher pH stability, SO2 bleaching stability and thermal stability, and the phenolic pyran anthocyanin has very good in-vitro oxidation resistance and has a relatively great application prospect in the field of food processing.

Description

Technical Field

[0001] The invention belongs to the field of food pigments and functional ingredients, and relates to a method for preparing phenolic pyranoanthocyanin, in particular to a method for preparing high-purity cyanidin-3-glucoside-4-vinyl catechol. Background Art

[0002] Anthocyanins are natural pigments found in many fruits and vegetables (a general term for fruits and vegetables). The presence of anthocyanins allows plants to present bright colors such as red, purple, blue and even orange, but their structure and color stability are poor and are greatly affected by processing conditions such as pH and temperature, which limits their application in food processing and functional ingredients.

[0003] Pyranoanthocyanins are a class of anthocyanin derivatives. Compared with their precursor anthocyanins, they are not only more stable in structure, but also have certain differences in color. As a subtype of pyranoanthocyanins, phenolic pyranoanthocyanins share the same disadvantages as pyranoanthocyanins, such as low yield and long synthesis cycle, which seriously hinders the development and application of pyranoanthocyanins.

[0004] Studies have shown that by incubating malvidin-3-glucoside extracted from grapes with different phenolic acids (caffeic acid, p-coumaric acid, ferulic acid and sinapic acid) at 30°C, phenolic pyranoanthocyanins corresponding to different phenolic acids were finally obtained, including malvidin-3-glucoside-4-vinylcatechol (Sun J, Li X, Luo H, et al. Comparative study on the stability and antioxidant activity of six pyranoanthocyaninsbased Malvidin-3-glucoside[J]. Journal of Agricultural and Food Chemistry, 2020. DOI: 10.1021 / acs.jafc.9b06734.). In addition, when different concentrations of caffeic acid (100 and 200 mg / L) were added to blackberry juice and placed in the dark at 4°C for 60 days, cyanidin-3-glucoside-4-vinylcatechol was detected, and the yield increased with the increase of caffeic acid concentration (Peng Changan, Lu Fengbo, Yuan Ye, et al. Study on the auxiliary color of anthocyanins in blackberry juice by exogenous caffeic acid and ferulic acid [J]. Natural Product Research and Development, 2012, 24(01): 94-97. DOI:, 10.16333 / , j.1001-6880., 2012., 01., 019.). Although these studies can be synthesized through some means, it is costly and difficult to separate them from complex systems. In addition, the anthocyanins in the above-mentioned raw materials (blackberry juice, etc.) are generally rich in variety and low in content, making them difficult to be used as the preferred raw materials for the large-scale preparation of high-purity pyranoanthocyanins. In addition, whether it is natural incubation or yeast fermentation, the cycle is generally one week or even longer. Therefore, it is necessary to research and develop new preparation methods. Summary of the invention

[0005] In order to solve the technical problems of slow preparation and low yield of existing phenolic pyranoanthocyanins, the object of the present invention is to provide a method for preparing phenolic pyranoanthocyanin, namely cyanidin-3-glucoside-4-vinylcatechol (C3G_VC), which can efficiently synthesize phenolic pyranoanthocyanins, and the obtained phenolic pyranoanthocyanins have high stability and in vitro antioxidant capacity.

[0006] The method for preparing high-purity cyanidin-3-glucoside-4-vinyl catechol of the present invention comprises the following steps:

[0007] A. Prepare a fermentation system. Each 2L fermentation system includes: 110.0g glucose, 110.0g fructose, 2.0g potassium dihydrogen phosphate, 0.5g ammonium chloride and 0.2g magnesium sulfate heptahydrate, which are dissolved in water, adjusted to pH 3.5, and sterilized for later use;

[0008] B. Add the activated lactic acid bacteria to the fermentation system described in step A, with an inoculation volume of 1×10 20 CFU / mL, black soybean hull crude extract and caffeic acid were added to each 2L fermentation system, wherein the final concentration of caffeic acid was 0.5mmol / L, the purity of cyanidin-3-glucoside contained in the black soybean hull crude extract reached 50% or more, and fermented at 37°C for at least 24 hours;

[0009] C. Filter the fermentation liquid to obtain the supernatant, and then evaporate and concentrate until the water in the system is basically removed;

[0010] D. Medium pressure preparation: medium pressure preparation liquid chromatography column The column was packed by wet method (C18 packing material), and the column was pressed with methanol and then balanced for use; 50 mL of concentrated fermentation broth was accurately measured and loaded; the medium-pressure preparative liquid chromatography setting conditions were: flow rate of 33 mL / min, 0.1% trifluoroacetic acid aqueous solution (A) and methanol (B) as mobile phases, detection wavelength of 500 nm, collection wavelength of 519 nm, and collection threshold of 1100 mAU;

[0011] E. The peak collected in step D was freeze-dried to obtain a powder, which was detected to be cyanidin-3-glucoside-4-vinylcatechol.

[0012] According to a further feature of the preparation method of the present invention, in step B, the lactic acid bacteria is Lactobacillus plantarum.

[0013] According to a further feature of the preparation method of the present invention, in step B, the molar ratio of the black soybean hull crude extract to caffeic acid is 30:1 to 35:1.

[0014] The method for preparing high-purity cyanidin-3-glucoside-4-vinyl catechol of the present invention has the following beneficial effects: (1) Anthocyanins are extracted from black soybean husks, and a copigment caffeic acid is added to synthesize a novel pyranoanthocyanin, namely cyanidin-3-glucoside-4-vinyl catechol (C3G_VC), and its structure is characterized. (2) Lactic acid bacteria with high efficiency in synthesizing pyranoanthocyanins are screened out, and fermentation with plant lactobacillus can significantly improve the efficiency of pyranoanthocyanin synthesis and effectively shorten the preparation time of novel anthocyanins. (3) The cyanidin-3-glucoside-4-vinyl catechol prepared by the method of the present invention has a purity of 96.33%, has higher pH stability, SO2 bleaching stability and thermal stability than the prototype anthocyanin, and the phenolic pyranoanthocyanin has good in vitro antioxidant capacity, can be used as a material for theoretical research on anthocyanins, and can also be used as a natural food pigment. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the liquid phase diagram of crude extract of black soybean hull.

[0016] Figure 2 Shows the maximum absorption wavelength of cyanidin-3-glucoside, the main component of black soybean hull crude extract.

[0017] Figure 3 This is the liquid phase diagram of cyanidin-3-glucoside standard.

[0018] Figure 4 Shows the maximum absorption wavelength of cyanidin-3-glucoside.

[0019] Figure 5 Shows the production of C3G_VC at different fermentation times by different lactic acid bacteria.

[0020] Figure 6 The production of C3G_VC at different fermentation times under different molar ratios of black soybean hull crude extract:caffeic acid is shown.

[0021] Figure 7 This is the liquid chromatogram of C3G_VC at 280nm.

[0022] Figure 8 This is the liquid chromatogram of C3G_VC at 520nm.

[0023] Fig. 9 Displays the maximum absorption wavelength of C3G_VC.

[0024] Fig.10 It is a schematic diagram of the structure of C3G_VC.

[0025] Fig.11 This is the secondary mass spectrum of C3G_VC.

[0026] Fig.12 This is the C3G_VC H NMR spectrum.

[0027] Fig.13 This is the C3G_VC NMR carbon spectrum.

[0028] Fig.14 Show the color changes of each system solution at different pH.

[0029] Fig.15 It is the full wavelength scanning diagram of each system solution at different pH.

[0030] Fig.16 Shows the color change of the solution at different SO2 concentrations.

[0031] Fig.17 This is a full wavelength scan of the solution at different SO2 concentrations.

[0032] Fig.18 Shows the color change of the solution at different temperatures.

[0033] Fig.19 Shows the changes in anthocyanin retention at different temperatures.

[0034] Fig. 20 Shows the scavenging rate of DPPH free radicals by different system solutions.

[0035] Fig.21 Shows the scavenging rate of ABTS free radicals by different system solutions.

[0036] Fig. 22 This is a graph showing the determination of the iron reducing ability of different system solutions. DETAILED DESCRIPTION

[0037] Example 1: Preparation of cyanidin-3-glucoside-4-vinylcatechol

[0038] Source of bacteria: Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus paracasei, provided by DSM (China) Co., Ltd.; Lactobacillus plantarum, commercial strain Lactobacillus plantarum FEED8, provided by Guangdong Yikewei Biotechnology Co., Ltd.

[0039] In view of past studies, after inoculating two plant lactobacillus subspecies (Lp39 and C8-1) obtained from the China Industrial Microbiological Culture Collection Center into a red wine system for malic acid fermentation for 10-14 days, 11 pyranoanthocyanins were detected in the supernatant (Wang S, Li S, Zhao H, et al. Acetaldehyde released by Lactobacillus plantarum enhances accumulation of pyranoanthocyanins in wine during malolactic fermentation [J]. Food Research International, 2018, 108: 254-263.), it is speculated that plant lactobacillus has the characteristics of synthesizing pyranoanthocyanins, but the synthesis efficiency may be different, so the present embodiment uses plant lactobacillus subspecies FEED8, which can also be used for other plant lactobacilli.

[0040] Sources of materials: MRS culture medium and culture medium were obtained from Qingdao Hope Biotechnology Co., Ltd. Analytical grade reagents of caffeic acid were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. All other chemical reagents and solvents were purchased from China National Pharmaceutical Group Co., Ltd.

[0041] Black soybean husk crude extract: Black soybean husk extract (main component: cyanidin-3-glucoside) was provided by Shaanxi Baichuan Kangze Biotechnology Co., Ltd. Figure 1 and Figure 2 As shown, the main component was cyanidin-3-glucoside after liquid chromatography identification, with a purity of 77.89%. The liquid chromatography diagram of the standard cyanidin-3-glucoside is shown in Figure 3 and Figure 4 As shown. Black soybean hull extracts from other commercial sources may also be used, with a quality standard of cyanidin-3-glucoside purity of 50% or more.

[0042] The following steps were used to prepare cyanidin-3-glucoside-4-vinylcatechol:

[0043] (1) Inoculate the lactic acid bacteria into sterilized MRS broth medium (autoclaved at 121°C for 15 min), culture at 37°C for 24 h, activate for three generations, remove the medium by centrifugation, and set aside;

[0044] (2) Prepare a fermentation system. In this embodiment, a 2 L fermentation system is used, including: 110.0 g of glucose, 110.0 g of fructose, 2.0 g of potassium dihydrogen phosphate, 0.5 g of ammonium chloride and 0.2 g of magnesium sulfate heptahydrate, which are dissolved in water, adjusted to pH 3.5, and sterilized for later use;

[0045] (3) Add the activated bacteria described in step A to the fermentation system described in step A (inoculation amount 1 ╳ 10 20 CFU / mL), black soybean hull crude extract (5.0mmol / L) and caffeic acid (0.5mmol / L) were added to each 2L fermentation system, and fermented at 37℃ for at least 24 hours. The supernatants of all groups were taken separately, and the liquid phase was tested for the generation of C3G_VC;

[0046] Experimental results: Figure 5 As shown in Table 1, no cyanidin-3-glucoside-4-vinyl catechol (C3G_VC) was detected in the supernatant prepared by the Lactobacillus bulgaricus experimental group and the Streptococcus thermophilus experimental group. C3G_VC was detected in the supernatant prepared by the Lactobacillus paracasei experimental group and the Lactobacillus plantarum experimental group, and the Lactobacillus plantarum experimental group had a higher conversion efficiency and reached a peak value after 24 hours of fermentation.

[0047] Table 1: C3G_VC production at different fermentation times by different strains

[0048]

[0049] The plant lactobacillus experimental group (black soybean hull crude extract: caffeic acid = 30:1) was subjected to the following extraction experiment:

[0050] (1) filtering the fermentation liquid to obtain a supernatant, and rotary evaporating the supernatant to concentrate the supernatant until the water in the system is substantially removed;

[0051] (2) Medium pressure preparation: medium pressure preparation liquid chromatography column The column was packed by wet method (C18 packing material), and the column was pressed with methanol and then balanced for use; 50 mL of concentrated fermentation broth was accurately measured and loaded; the medium-pressure preparative liquid chromatography setting conditions were: flow rate of 33 mL / min, 0.1% trifluoroacetic acid aqueous solution (A) and methanol (B) as mobile phases, detection wavelength of 500 nm, collection wavelength of 519 nm, and collection threshold of 1100 mAU;

[0052] (3) The collected peaks are freeze-dried to obtain powder.

[0053] The powder product was dissolved in pure water, and the filtrate obtained by filtering through a 0.22 μm hydrophilic PTFE filter was subjected to liquid phase detection, and it was preliminarily determined that the target product was C3G_VC. Then, secondary mass spectrometry was performed to determine the structural formula of C3G_VC, and finally nuclear magnetic resonance was used to verify again that the obtained powder was C3G_VC.

[0054] Example 2: Optimization of the production amount of cyanidin-3-glucoside-4-vinylcatechol (C3G_VC)

[0055] Caffeic acid is a natural phenolic acid product widely found in plants, mainly in Chinese herbal medicine. Because its structure contains two hydroxyl radicals, it has high antioxidant, anti-cancer and anti-inflammatory biological activities. Past studies have found that the addition of caffeic acid has a significant improvement on the color attenuation during the aging of red wine, and it is a good auxiliary colorant (Alberto AJ, Lizama V, Jose GM, et al. Applying rosemary extract and caffeic acid to modify the composition of Monastrell wines[J]. European Food Research and Technology, 2022, 248: 1787-1802.).

[0056] Experimental method: The plant lactobacillus obtained by screening in Example 1 was used, and the method was the same as that in Example 1.

[0057] A. Add activated Lactobacillus plantarum FEED8 to 2L fermentation system, with an inoculation volume of 1 ╳ 10 20 CFU / mL, caffeic acid (final concentration was 0.5 mmol / L) and black soybean hull crude extract were added.

[0058] B. Divide into 6 groups according to different molar ratios of black soybean hull extract: caffeic acid = 10:1, 15:1, 20:1, 25:1, 30:1 and 35:1, ferment at 37°C for 96 hours, and take samples every 24 hours to detect the amount of C3G_VC generated. No control group was set in this example, because the strain screening control group in Example 1 showed that black soybean hull crude extract and caffeic acid could not spontaneously generate C3G_VC.

[0059] C. Filter the fermentation liquid to obtain the supernatant, and then evaporate and concentrate until the water in the system is basically removed;

[0060] D. Medium pressure preparation: medium pressure preparation liquid chromatography column The column was packed by wet method (C18 packing material), and the column was pressed with methanol and then balanced for standby use; 50 mL of concentrated fermentation broth was accurately measured and loaded; the medium-pressure preparative liquid chromatography was set as follows: the flow rate was 33 mL / min, 0.1% trifluoroacetic acid aqueous solution (A) and methanol (B) were used as mobile phases, and the elution conditions were as follows for phase B: 6%-6%, 0-5 min; 6%-30%, 5-17 min; 30%-40%, 17-77 min; 40%-70%, 77-167 min; 70%-100%, 167-177 min; 100%-100%, 177-197 min; 100%-50%, 197-205 min; 50%-50%, 205-207 min; 50%-6%, 207-211 min; 6%-6%, 211-216 min. The detection wavelength is 500 nm, the collection wavelength is 519 nm, and the collection threshold is 1100 mAU;

[0061] E. The peak collected in step D was freeze-dried to obtain a powder for detection in the following examples.

[0062] Table 2. The production of C3G_VC at different molar ratios (black bean hull crude extract: caffeic acid) at different fermentation times

[0063]

[0064]

[0065] Example 3: Detection and evaluation of the powder product obtained in Example 2

[0066] The powder product obtained from the Lactobacillus plantarum experimental group (black soybean hull crude extract: caffeic acid = 30:1) in Example 2 was subjected to the following tests:

[0067] 1. Purity Identification

[0068] 1) Liquid phase detection: The powder was diluted with ultrapure water and filtered through a 0.22 μm filter membrane for sample preparation. The sample was eluted by LC-MS. The mobile phase was 2% formic acid in water (phase A) and acetonitrile (phase B). The detection conditions were 280 nm and 520 nm. The elution conditions for phase B were: 6%-6%, 0-3 min; 6%-30%, 3-34 min; 30%-95%, 34-35 min; 95%-95%, 35-40 min; 95%-6%, 40-41 min; 6%-6%, 41-45 min.

[0069] Test results: Figure 7 As shown, at 280nm, the test results show that C3G_VC accounts for 96.33%, Figure 8 What is presented is the absorption peak of C3G_VC at 520nm, such as Fig. 9As shown, the absorption wavelength is 505.57nm.

[0070] 2) Mass spectrometry detection: The mass spectrometry conditions were MS-8045 Shimadzu mass spectrometer, electrospray ionization, negative ion mode; nitrogen was used as the collision gas, the nebulizer gas flow rate was 3.0 L / min, the drying gas flow rate was 10.0 L / min; the heater flow rate was 10.0 L / min; the interface temperature was 300°C, the DL temperature was 250°C, the heating block temperature was 400°C, and the collision voltage was 15 V.

[0071] Secondary mass spectrometry structure identification: Fig.10 Structural formula (drawn by software ChemBioDraw 14.0), combined with Fig.11 Secondary mass spectrum (liquid chromatography-mass spectrometry system (LC-MS 8045)), the m / z of C3G_VC is 581, the m / z after removing the glucoside is 162, and the m / z of the parent ion is 419.

[0072] 3) Nuclear magnetic resonance detection: The purified pyranoanthocyanins were 1 H-NMR and 13 C-NMR detection: the detection instrument used was a Bruker 600 MHz superconducting nuclear magnetic resonance instrument, and the solvent was deuterated methanol.

[0073] Test results: Fig.12 As shown, 1 H NMR (300MHz, Methanol-d4) δ7.83 (s, 4H), 7.69 (d, J = 8.3Hz, 1H), 7.60 (s, 1H), 7.1 1(d,J=10.0Hz,2H),6.96(t,J=9.3Hz,3H),3.75-3.64(m,3H),3.48-3.37(m,3H). like Fig.13 As shown, 13 C NMR(600MHz,Methanol-d4)δ167.69,166.55,161.59,152.97,152.59,152.19,151.38,147.28,146.21,145.10,133.43,124.27,1 21.52,121.26,120.58,116.71,115.86,115.02,113.74,107.06,104.45,99.69,99.48,96.81,77.17,76.37,74.12,69.97,61.37.

[0074] 4) pH stability evaluation: Each group of samples and distilled water were used to prepare C3G group and C3G_VC group test solutions at 100 μM, and the pH values ​​were adjusted to 1.0, 2.5, 3.5, 4.5, 5.5, 7.5, 9.0, and 11.0 with 2.0 mol / L disodium hydrogen phosphate and citric acid solutions, respectively. After standing for 15 min under natural light conditions, full-wavelength scanning was performed at 280 nm-800 nm, and the characteristic absorption spectrum was measured and mapped. The color changes of C3G_VC at different pH values ​​were recorded with a camera.

[0075] Test results: Fig.14 As shown in the figure, during the process of C3G_VC changing from acid to base, the color changes from purple-red under acidic conditions to brown under neutral conditions and then to purple under alkaline conditions. Fig.15 As shown, the full wavelength scanning results show that the maximum absorption wavelength of C3G_VC is 564nm in the process of pH from 1.0 to 5.5, but the absorption value shows a gradually decreasing trend. As the pH increases from 7.5 to 11.0, the maximum absorption wavelength decreases (blue shift occurs).

[0076] 5) Evaluation of resistance to sulfur dioxide bleaching: A 125 μM C3G_VC solution was prepared using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH = 2.5. 5 mg / mL NaHSO3 solution was added to make the amount of SO2 0, 5, 10, 25, 50, 150, and 200 ppm, respectively. After standing for 15 minutes under natural light conditions indoors, a full wavelength scan was performed at 280nm-800nm, and the characteristic absorption spectrum was mapped, and the color change of C3G_VC after bleaching with different concentrations of SO2 was recorded with a camera.

[0077] Test results: Fig.16 As shown in the figure, when the sulfur dioxide concentration changes from high to low, the color of C3G_VC does not change significantly, indicating that C3G_VC has strong resistance to sulfur dioxide bleaching. Fig.17 As shown, the full wavelength scanning results show that when the sulfur dioxide concentration changes from high to low, the maximum absorption wavelength of C3G_VC remains constant and the absorption value gradually increases.

[0078] 6) Thermal stability evaluation: The C3G_VC group samples were placed in a water bath at 50, 60, 70, 80, 90, and 100°C according to a 25 μM C3G_VC solution, heated in the dark for 5 h, and the absorbance was measured at a wavelength of 520 nm, and the retention rate of anthocyanins was calculated.

[0079] Test results: Fig.18As shown in the figure, at 100℃, the color shows obvious attenuation as time goes by, gradually changing from orange to light yellow. At 90℃, the color gradually changes from orange to light yellow as time goes by. However, as the temperature gradually decreases, there is no obvious change in color. Fig.19 As shown in the figure, under the conditions of 100℃ and 90℃, the anthocyanin retention rate of C3G_VC decreased significantly with the increase of heating time, but below 80℃, this change trend gradually slowed down.

[0080] 7) Evaluation of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging ability: Take the sample solution of C3G group and C3G_VC group and react with DPPH solution under light-proof conditions for 30 minutes, measure its absorbance at 517nm, and calculate the DPPH free radical scavenging rate. The calculation formula is as follows (1): Where: A1 and A0 are the absorbance of the sample and the control, respectively

[0081] DPPH free radical scavenging rate (%) = (A0-A1) × 100 / A0 (1)

[0082] Test results: Fig. 20 As shown, compared with the prototype anthocyanins, C3G_VC significantly increased the DPPH free radical scavenging rate (P<0.05).

[0083] 8) Evaluation of 2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) free radical scavenging ability: The sample solutions of C3G group and C3G_VC group were reacted with ABTS solution for 10 min under light-proof conditions, and the absorbance was measured at 734 nm to calculate the ABTS free radical scavenging rate. The calculation formula is as follows (2): Where: A1 and A0 are the absorbance of the sample and the control, respectively.

[0084] ABTS free radical scavenging rate (%) = (A0-A1) × 100 / A0 (2)

[0085] Test results: Fig.21 As shown, compared with the prototype anthocyanins, C3G_VC significantly increased the ABTS free radical scavenging rate (P<0.05).

[0086] 9) Evaluation of iron reducing ability (FRAP): 300mM acetate buffer, 10mM 2,4,6-tris(2-pyridine)-1,3,5-triazine (TPTZ), and 20mM FeCl3 were mixed in a ratio of 10:1:1 to form a FRAP solution, and incubated in a water bath at 37°C for 30min. 10μL of the diluted sample was added to 200μL of the FRAP solution, and its absorbance was measured at 593nm. The results were expressed as ascorbic acid equivalents by calibrating the ascorbic acid standard curve (0.01-0.04mM).

[0087] Test results: Fig. 22 As shown, compared with the prototype anthocyanins, C3G_VC significantly increased the iron reducing capacity (P<0.05).

Claims

1. A method for preparing high-purity cyanidin-3-glucoside-4-vinylcatechol, characterized in that: The following steps are involved: A. Prepare a fermentation system. Each 2L fermentation system includes: 110.0g glucose, 110.0g fructose, 2.0g potassium dihydrogen phosphate, 0.5g ammonium chloride and 0.2g magnesium sulfate heptahydrate, which are dissolved in water, adjusted to pH 3.5, and sterilized for later use; B. Add the activated lactic acid bacteria to the fermentation system described in step A, with an inoculation volume of 1×10 20 CFU / mL, black soybean hull crude extract and caffeic acid were added to each 2L fermentation system, wherein the final concentration of caffeic acid was 0.5mmol / L, the purity of cyanidin-3-glucoside contained in the black soybean hull crude extract reached 50% or more, and fermented at 37°C for at least 24 hours; C. Filter the fermentation liquid to obtain the supernatant, and then evaporate and concentrate until the water in the system is basically removed; D. Medium-pressure preparation: A medium-pressure preparative liquid chromatography column (H100 cm×φ6 cm) was wet-packed (C18 filler), and the column was pressed with methanol and then balanced for use; 50 mL of concentrated fermentation broth was accurately measured and loaded; the medium-pressure preparative liquid chromatography setting conditions were: a flow rate of 33 mL / min, 0.1% trifluoroacetic acid aqueous solution and methanol as mobile phases, a detection wavelength of 500 nm, a collection wavelength of 519 nm, and a collection threshold of 1100 mAU; E. The peak collected in step D was freeze-dried to obtain a powder, which was determined to be cyanidin-3-glucoside-4-vinylcatechol.

2. The preparation method according to claim 1, characterized in that: In the step B, the lactic acid bacteria is Lactobacillus plantarum.

3. The preparation method according to claim 1, characterized in that: In the step B, the molar ratio of the black soybean hull crude extract to caffeic acid is 30:1 to 35:1.

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