Preparation method of high-purity cyanidin-3-glucoside-4-vinylphenol

Abstract

The invention provides a preparation method of high-purity cyanidin-3-glucoside-4-vinylphenol. 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 p-coumaric acid are added into every 2 L of the fermentation system, the final concentration of p-coumaric 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 conducted for at least 24 hours at 37 DEG C; 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-vinylphenol. Compared with cyanidin-3-glucoside, the cyanidin-3-glucoside 4-vinylphenol 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-vinylphenol. 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 when blueberry juice was fermented with yeast at 30°C for 5 days, liquid phase detection of the fermentation supernatant showed the detection of a variety of phenolic pyranoanthocyanins, including cyanidin-3-glucoside-4-vinylphenol (Zhou J, Tang C, Zou S, et al. Enhancement of pyranoanthocyanin formation in blueberry wine with non-Saccharomyces yeasts[J]. Food Chemistry, 2024(Apr.16):438. DOI:10.1016 / j.foodchem.2023.137956.). In addition, anthocyanins extracted from sugarcane peels were added with organic acids as auxiliary colorants (ferulic acid, vanillic acid, p-coumaric acid, vanillin and syringaldehyde). As the concentration of auxiliary colorants increased, the yield of pyranoanthocyanins increased, among which the corresponding product of p-coumaric acid, cyanidin-3-glucoside-4-vinylphenol, was detected (Yan Huaifeng. Research on the auxiliary color effect of organic acids and phenolic aldehydes on sugarcane anthocyanins [D]. South China University of Technology, 2017.). Although these studies can synthesize cyanidin-3-glucoside-4-vinylphenol through some means, it is costly and difficult to separate it from a complex system. In addition, the above raw materials (blueberry juice, sugarcane peel, etc.) contain rich anthocyanins of low content, which makes it difficult to use them as 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 study 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-vinylphenol (C3G_VP), 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-vinylphenol 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 p-coumaric acid were added to each 2L fermentation system, wherein the final concentration of p-coumaric 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 wet packed (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: a flow rate of 36 mL / min, 0.1% trifluoroacetic acid aqueous solution (A) and methanol (B) as mobile phases, a detection wavelength of 500 nm, a collection wavelength of 519 nm, and a collection threshold of 1700 mAU;

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

[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 p-coumaric acid is 30:1 to 35:1.

[0014] The method for preparing high-purity cyanidin-3-glucoside-4-vinylphenol of the present invention has the following beneficial effects: (1) Anthocyanins are extracted from black soybean hulls, and the copigment p-coumaric acid is added to synthesize a new type of pyranoanthocyanin, namely cyanidin-3-glucoside-4-vinylphenol (C3G_VP), and its structure is characterized. (2) Lactic acid bacteria with high efficiency in synthesizing pyranoanthocyanins are screened out, and fermentation with Lactobacillus plantarum can significantly improve the efficiency of pyranoanthocyanin synthesis and effectively shorten the preparation time of the new type of anthocyanin. (3) The cyanidin-3-glucoside-4-vinylphenol prepared by the method of the present invention has a purity of 96.91%, and has higher pH stability, SO2 bleaching stability and thermal stability than cyanidin-3-glucoside (prototype anthocyanin), and the phenolic pyranoanthocyanin has good in vitro antioxidant ability, and has great application prospects in the field of food processing. 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_VP at different fermentation times by different lactic acid bacteria.

[0020] Figure 6 Shows the production of C3G_VP at different molar ratios (black soybean hull crude extract: p-coumaric acid) and fermentation time.

[0021] Figure 7 This is the liquid chromatogram of C3G_VP at 280nm.

[0022] Figure 8 This is the liquid chromatogram of C3G_VP at 520nm.

[0023] Fig. 9 Displays the maximum absorption wavelength of C3G_VP.

[0024] Fig.10 It is a schematic diagram of the structure of C3G_VP.

[0025] Fig.11 This is the secondary mass spectrum of C3G_VP.

[0026] Fig.12 This is the C3G_VP H NMR spectrum.

[0027] Fig.13 This is the C3G_VP 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-vinylphenol

[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 p-coumaric acid reagents 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] Experimental groups: control group (only black soybean hull extract and p-coumaric acid were added, no inoculation), the experimental group was divided into four groups, black soybean hull extract and p-coumaric acid were added to the simulation system, and then 4 types of lactic acid bacteria were inoculated respectively: Lactobacillus bulgaricus, Streptococcus lactis, Lactobacillus paracasei, and Lactobacillus plantarum.

[0043] Conduct the experiment as follows:

[0044] (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;

[0045] (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;

[0046] (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 p-coumaric 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 formation of C3G_VP;

[0047] Experimental results: Figure 5 As shown in Table 1, no cyanidin-3-glucoside-4-vinylphenol (C3G_VP) was detected in the supernatant prepared by the Lactobacillus bulgaricus experimental group and the Streptococcus thermophilus experimental group. C3G_VP was detected in the supernatant prepared by the Lactobacillus paracasei experimental group and the Lactobacillus plantarum experimental group. The Lactobacillus plantarum experimental group had a higher conversion efficiency and reached a peak value at 24 hours of fermentation.

[0048] Table 1: C3G_VP production at different fermentation times by different strains

[0049]

[0050] The plant lactobacillus experimental group (black soybean hull crude extract: p-coumaric acid = 30:1) was subjected to the following extraction experiment:

[0051] (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;

[0052] (2) Medium pressure preparation: medium pressure preparation liquid chromatography column The column was wet packed (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: a flow rate of 36 mL / min, 0.1% trifluoroacetic acid aqueous solution (A) and methanol (B) as mobile phases, a detection wavelength of 500 nm, a collection wavelength of 519 nm, and a collection threshold of 1700 mAU;

[0053] (3) The collected peaks are freeze-dried to obtain powder.

[0054] 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_VP. Then, secondary mass spectrometry was performed to determine the structural formula of C3G_VP, and finally nuclear magnetic resonance was used to verify again that the obtained powder was C3G_VP.

[0055] Example 2: Optimization of the production amount of cyanidin-3-glucoside-4-vinylphenol (C3G_VP) and its preparation

[0056] p-Coumaric acid is an active ingredient widely found in plants, mainly in sugarcane peel at about 95.781 mg / kg and corn cob at about 10.66 mg / g. p-Coumaric acid is also found in wine, and during the aging process of red wine, p-Coumaric acid has a good auxiliary color effect on its color.

[0057] Experimental method: The plant lactobacillus obtained by screening in Example 1 was used, and the method was the same as that in Example 1.

[0058] A. Add activated Lactobacillus plantarum FEED8 to 2L fermentation system, with an inoculation volume of 1 ╳ 10 20 CFU / mL, p-coumaric acid (final concentration was 0.5 mmol / L) and black soybean hull crude extract were added.

[0059] B. Divide into 6 groups according to different molar ratios of black soybean hull extract: p-coumaric 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_VP 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 p-coumaric acid could not spontaneously generate C3G_VP.

[0060] C. Filter the fermentation liquid to obtain the supernatant, and then evaporate and concentrate until the water in the system is basically removed;

[0061] D. Medium pressure preparation: medium pressure preparation liquid chromatography column The column was wet packed (C18 packing material), and the column was equilibrated after being pressed with methanol for standby 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 36 mL / min, 0.1% trifluoroacetic acid aqueous solution (A) and methanol (B) were used as mobile phases, and the elution conditions for phase B were: 6%-6%, 0-5 min; 6%-30%, 5-17 min; 30%-40%, 17-77 min; 40%-60%, 77-107 min ; 60%-80%, 107-167min; 80%-100%, 167-177min; 100%-100%, 177-202min; 100%-50%, 202-210min; 50%-50%, 210-212min; 50%-6%, 210-218min; 6%-6%, 218-219min; detection wavelength 500nm, collection wavelength 519nm, collection threshold 1700mAU;

[0062] E. Freeze-dry the peak collected in step D to obtain powder.

[0063] Experimental results: Figure 6 As shown in Table 2, when the ratio of crude black soybean hull extract to p-coumaric acid is 30:1, the production of C3G_VP reaches a peak, and the yield is about 18% after 24 hours of fermentation. As the ratio of black soybean hull addition increases to 35:1, the production of C3G_VP remains basically unchanged. Therefore, it can be determined that the ratio of crude black soybean hull extract to p-coumaric acid is 30:1 to 35:1 as the optimal ratio for preparing C3G_VP, and steps C, D and E in the experiment are completed according to this ratio.

[0064] Conclusion: Lactobacillus plantarum has a high efficiency in converting anthocyanins and can achieve rapid synthesis of C3G_VP, which provides a theoretical basis for the efficient preparation of C3G_VP.

[0065] Table 2. The production of C3G_VP at different molar ratios (black soybean hull crude extract: p-coumaric acid) at different fermentation times

[0066]

[0067] Example 3: Detection and evaluation of the powder product obtained in Example 2

[0068] The powder product obtained from the Lactobacillus plantarum experimental group (black soybean hull crude extract: p-coumaric acid = 30:1) in Example 2 was subjected to the following tests:

[0069] 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.

[0070] Test results: Figure 7 As shown, at 280nm, the detection results show that C3G_VP accounts for 96.91%, Figure 8 What is presented is the absorption peak of C3G_VP at 520nm, such as Fig. 9 As shown, the absorption wavelength is 499.32nm.

[0071] 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.

[0072] Secondary mass spectrometry structure identification results: 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_VP is 565, the m / z after removing the glucoside is 162, and the m / z of the parent ion is 403.

[0073] 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.

[0074] Test results: Fig.12 As shown, 1 H NMR (300MHz, Methanol-d4) δ8.15 (d, J = 8.4Hz, 2H), 7.99-7.81 (m, 3H), 7.18-6.91 (m, 5H), 3.74-3.61 (m, 3H), 3.48-3.35 (m, 3H). Such as Fig.13 As shown, 13CNMR(600MHz,Methanol-d4)δ167.63,166.60,163.96,161.69,152.93,152.15,151.46,147.45,145.10,133.49,130.06,124.34, 120.80,120.52,116.71,116.34,115.05,107.05,104.50,99.81,99.53,97.77,96.66,92.55,77.14,76.43,74.12,69.88,61.33.

[0075] 4) pH stability evaluation: Each group of samples and distilled water were used to prepare C3G group and C3G_VP 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_VP at different pH values ​​were recorded with a camera.

[0076] Test results: Fig.14 As shown in the figure, during the process of C3G_VP changing from acid to base, the color changes from red under acidic conditions to orange-red under neutral conditions, and then to dark red under alkaline conditions. Fig.15 As shown, the full wavelength scanning results show that the maximum absorption wavelength of C3G_VP is 558nm when the pH value increases from 1.0 to 5.5, but the absorption value shows a gradually decreasing trend. As the pH value increases from 7.5 to 11.0, the maximum absorption wavelength decreases (blue shift occurs).

[0077] 5) Evaluation of resistance to sulfur dioxide bleaching: A 125 μM C3G_VP 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_VP after bleaching with different concentrations of SO2 was recorded with a camera.

[0078] Test results: Fig.16 As shown in the figure, the color of C3G_VP does not change significantly when the sulfur dioxide concentration changes from high to low, indicating that C3G_VP has strong resistance to sulfur dioxide bleaching. Fig.17As shown, the full wavelength scanning results show that when the sulfur dioxide concentration changes from high to low, the maximum absorption wavelength of C3G_VP remains constant and the absorption value gradually increases.

[0079] 6) Thermal stability evaluation: The C3G_VP group samples were placed in a water bath at 50, 60, 70, 80, 90, and 100°C according to a 25 μM C3G_VP 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.

[0080] Test results: Fig.18 As shown in the figure, under the conditions of 100℃ and 90℃, the color shows obvious attenuation with the extension of time, gradually changing from orange yellow to light yellow. As the temperature gradually decreases, the color change does not change significantly. Fig.19 As shown in the figure, under the conditions of 100℃, 90℃ and 80℃, the anthocyanin retention rate of C3G_VP showed a significant decrease with the increase of heating time, but below 70℃, this change trend gradually slowed down.

[0081] 7) Evaluation of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging ability: Take the sample solution of C3G group and C3G_VP 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

[0082] DPPH free radical scavenging rate (%) = (A0-A1) × 100 / A0(1)

[0083] Test results: Fig. 20 As shown, compared with the prototype anthocyanins, C3G_VP significantly increased the DPPH free radical scavenging rate (P<0.05).

[0084] 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_VP 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.

[0085] ABTS free radical scavenging rate (%) = (A0-A1) × 100 / A0 (2)

[0086] Test results: Fig.21 As shown, compared with the prototype anthocyanins, C3G_VP significantly increased the ABTS free radical scavenging rate (P<0.05).

[0087] 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 Trolox equivalents (TE) by calibrating the Trolox standard curve (0.01-0.04mM).

[0088] Test results: Fig. 22 As shown, compared with the prototype anthocyanins, C3G_VP significantly increased the iron reducing capacity (P<0.05).

Claims

1. A method for preparing high-purity cyanidin-3-glucoside-4-vinylphenol, 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 p-coumaric acid were added to each 2L fermentation system, wherein the final concentration of p-coumaric 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 36 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 1700 mAU; E. The peak collected in step D was freeze-dried to obtain a powder, which was determined to be cyanidin-3-glucoside-4-vinylphenol.

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 ratio of the crude black soybean hull extract to p-coumaric acid is 30:1 to 35:1.

Images