A fungus-enzyme synergistic method for extracting polysaccharide and aglycone type flavonoids from ginkgo biloba leaf residue

Abstract

The application discloses a kind of from ginkgo leaf residue extraction polysaccharide and aglycone flavonoid bacteria-enzyme synergistic method, belong to ginkgo leaf residue resource comprehensive utilization technical field, including the following steps: (1) high-temperature steam sterilization;(2) aspergillus niger fermentation;(3) ultrasonic wave, hydrothermal extraction;(4) concentration, alcohol precipitation;(5) complex enzyme enzymolysis;(6) homogenization, alcohol hydrothermal extraction.The method of the application, using aspergillus niger and complex enzyme synergism, and combining ultrasonic wave, microjet high pressure homogenization etc., polysaccharide, flavonoid active ingredient in ginkgo leaf residue is extracted, biotransformation, realize that polysaccharide and aglycone flavonoid content in leaf residue is improved, and the "titer" of ginkgo polysaccharide, flavonoid is promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of comprehensive utilization of ginkgo biloba leaf residue resources, and particularly relates to a fungus-enzyme synergistic method for extracting polysaccharides and aglycone flavonoids from ginkgo biloba leaf residue. BACKGROUND

[0002] Ginkgo biloba is widely distributed in China, and the ginkgo resource yield in China accounts for more than 70% of the total amount in the world. Ginkgo biloba leaves contain various active substances such as flavonoids, polysaccharides, lactones and organic acids. Ginkgo biloba extract (GBE) is the main component of "Jin Nuo injection", "Yue Kang Tong injection" and ginkgo biloba tablets, which are widely used in clinical treatment. A large amount of ginkgo biloba leaf residue is generated during the preparation of GBE, accounting for about 60-70% of the ginkgo biloba leaf raw materials. The ginkgo biloba leaf residue contains crude protein, crude fat, crude fiber, chlorophyll and other substances, as well as residual active components such as polysaccharides, flavonoids and lactones. At present, most of the ginkgo biloba leaf residue is discarded, burned, or dried and sold at a low price to feed (or additive) manufacturers after being crushed, which causes resource waste to some extent. The secondary development and utilization of ginkgo biloba leaf residue not only improves the utilization rate of ginkgo biloba leaf resources and broadens the ginkgo industry chain, but also is expected to increase the income of ginkgo planting industry and create income for ginkgo leaf processing enterprises.

[0003] The ginkgo biloba leaf residue of GBE processing enterprises is relatively sticky, and the solvent heating method is directly used to extract active components, which has a large solvent usage amount and high extraction cost. If the enzyme method is used, the active components remaining in the sticky leaf residue are not easy to leach out, and the efficiency of the enzyme method is not ideal. Therefore, these two methods are rarely reported in the secondary utilization of ginkgo biloba leaf residue. For example, Gao Kun et al. prepared copper sodium salt of chlorophyll from ginkgo biloba leaf residue (Journal of Zhejiang Wanli University, 2012; 25(5): 69-73); Wu Qianpan et al. extracted polysaccharides from ginkgo biloba leaf residue by water extraction and alcohol precipitation (China Modern Applied Pharmacy, 2014; 31(1): 9-13); and Jian Yue extracted biflavonoids from ginkgo biloba leaf residue by high-speed homogenization-ultrasonic assisted extraction (Beijing Forestry University 2019 master's thesis).

[0004] In recent years, there are many reports on the fermentation of ginkgo leaves (or leaf residues): Patent Application No. 201110292164.2 reports that the solid fermentation raw materials composed of ginkgo leaves, edible fungus residues, soybean meal, etc. are fermented with β-glucosidase-producing microorganisms as strains. After fermentation, the materials are dried and crushed to obtain the product. The glycoside type flavonoid compound is 0.48 times that before fermentation, while the aglycone type flavonoid is 1.8 times that before fermentation; Patent Application No. 201310067394.8 reports that ginkgo leaves are subjected to bidirectional solid fermentation with Aspergillus niger, and the extraction amount of ginkgo leaf flavonoids is increased by 20-60% compared with the traditional solvent extraction method; Patent Application No. 201410822627.5 reports that ginkgo leaf residues and corn cob powder are mixed with 15-25% cellulase, and after 3-5 days of fermentation and degradation, the base material is obtained. Then, through the steps of bagging, sterilization, inoculation culture, fungus management and debagging harvesting, the golden needle mushroom mycelium is obtained; Gao Shufeng uses ginkgo leaves (or leaf residues) as fermentation substrate, and uses Bacillus licheniformis and Bacillus natto, Candida utilis and Aspergillus niger as strains for solid state fermentation. The fermented product can improve the quality of broiler feed to a certain extent (Nanjing Forestry University Master Thesis, 2014); Zhou H et al. use ginkgo leaf residues for solid state fermentation with Candida tropicalis and Aspergillus oryzae, and the content of ginkgoic acid in the fermentation product is reduced from 14.8 mg / g to 1.5 mg / g (European Food Research and Technology, 2015, 240(2): 379-388); Patent Application No. 201410847739.6 reports that ginkgo leaf processing waste is added to a compound microorganism (Aspergillus niger, Streptococcus faecalis, Bacillus sp., and yeast = 1:1:1:2 by weight) for fermentation culture, followed by Maillard reaction and low-temperature drying to obtain a single-cell protein feed additive; Patent Application No. 201610343614.9 reports that ginkgo leaf residues are subjected to first deep fermentation with Aspergillus niger, Trichoderma viride and Saccharomyces cerevisiae mixed bacteria, and then subjected to second shallow fermentation with Bacillus polymyxa. The agricultural microbial agent prepared by this method can prevent and control pathogenic fungi in soil, reduce soil-borne diseases, promote crop growth, and significantly increase yield; Patent Application No. 201710332351.6 reports that ginkgo leaves are dried, crushed, sterilized and steam exploded, and then fermented with Aspergillus niger. Then, mixed enzyme solution (cellulase, hemicellulase, pectinase = 2:1:5) is added and reacted for 3-6 hours, and protease is added and reacted for 3-4 hours. Then, flavonoid extract is obtained by ethanol extraction, concentration and freeze-drying; Patent Application No. 201710181521.5 reports that lactic acid bacteria, active dry yeast and fresh ginkgo leaves are fermented, and after fermentation, ginkgo leaf polysaccharide and ginkgo leaf flavonoids are obtained by water extraction, alcohol extraction and purification. The content of ginkgo leaf polysaccharide is 48.4%, the extraction rate is 7.4%, the total flavonoid glycoside content is 31.3%, and the extraction rate of ginkgo leaf flavonoids is 87.5% (calculated relative to the flavonoid content in ginkgo leaves).

[0005] In summary, through microbial fermentation, the utilization rate of ginkgo leaf (or leaf residue) protein, cellulose, etc. is improved, which can be used as protein feed (application number 201410822627.5, application number 201410847739.6, Master's thesis of Gao Shu Feng), or mycelium (application number 201410822627.5), or microbial inoculant (application number 201610343614.9), etc. In addition, fermentation treatment can reduce the content of ginkgo acid, a toxic component of ginkgo leaves (Zhou H et al.), and also can dissolve flavonoids and polysaccharides. However, ginkgo flavonoids still exist in the form of glycosides (application number 201710332351.6, application number 201710181521.5), that is, mainly quercetin, kaempferol, and isorhamnetin are connected with glucose, and rutinose, etc. by O-glycoside bond. The bioavailability of glycoside flavonoids is low, and the absorption in the body is far less than that of free aglycone; the water solubility of macromolecular polysaccharides is poor, which is not conducive to absorption into the body to exert biological activity. Therefore, it is necessary to develop a deep processing method for ginkgo leaf residue, which can realize comprehensive extraction and efficient extraction of polysaccharides and aglycone flavonoids, and also improve the "efficacy" of ginkgo polysaccharides and flavonoids. SUMMARY

[0006] The purpose of the present application is to provide a bacteria-enzyme synergistic method for extracting polysaccharides and aglycone flavonoids from ginkgo leaf residue, which aims to realize the comprehensive utilization and efficient utilization of ginkgo leaf residue resources by bacteria-enzyme synergistic effect, on the one hand, polysaccharides and flavonoids in ginkgo leaf residue are effectively dissolved by biological fermentation treatment, on the other hand, low molecular weight polysaccharides and aglycone flavonoids are effectively converted under the assistance of ultrasonic waves and glycosidase, thereby prolonging the ginkgo leaf processing industry chain.

[0007] The present application is realized by the following technical scheme:

[0008] A bacteria-enzyme synergistic method for extracting polysaccharides and aglycone flavonoids from ginkgo leaf residue, comprising the following steps:

[0009] (1) High-temperature steam sterilization:

[0010] The ginkgo leaf residue is loaded into the fermentation tank, then 40-60% water is added and stirred to mix, and then high-temperature steam sterilization is carried out;

[0011] (2) Aspergillus niger fermentation:

[0012] The sterilized material is cooled to 25-30℃, at the same time, Aspergillus niger is activated to form spore solution, then inoculated into the material, and aerated for 3-4d;

[0013] (3) Ultrasonic and hydrothermal extraction:

[0014] The fermented material is taken out, water is added at a solid-liquid ratio of 1:3-5, and ultrasonic pretreatment is carried out for 25-30 in, then water is added at a solid-liquid ratio of 1:10-12, and extraction is carried out at 90-95℃ for 1.5-2h, the extraction liquid is filtered, and the water extraction liquid and residue are obtained and reserved;

[0015] (4) Concentration and alcohol precipitation:

[0016] The water extraction liquid obtained in step (3) is concentrated under reduced pressure to 1 / 5-1 / 6 of the original volume, ethanol is added and mixed, the alcohol concentration is adjusted to 80-90%, alcohol precipitation is carried out, and the precipitate and supernatant are collected by filtration; the precipitate is dried to obtain ginkgo polysaccharide, and the supernatant is reserved for use;

[0017] (5) Complex enzyme hydrolysis:

[0018] The residue obtained in step (3) is added with a buffer solution at pH 4.5-5 and complex enzymes at a solid-liquid ratio of 1:6-8, and reaction is carried out at 50-60℃ for 4-6h, then high-temperature sterilization is carried out, and the obtained enzyme hydrolysis liquid is concentrated under reduced pressure to 1 / 4-1 / 5 of the original volume;

[0019] (6) Homogenization and alcohol-water extraction:

[0020] The supernatant obtained in step (4) and the enzyme hydrolysis concentrated liquid obtained in step (5) are mixed in a micro-jet high-pressure homogenizer, the alcohol concentration is adjusted to 70-80%, and water is added at a solid-liquid ratio of 1:10-12, and extraction is carried out at 70-80℃ for 3-5h, the extraction liquid is filtered, and the filtrate is concentrated under reduced pressure and dried to obtain a crude extract rich in aglycone flavonoids.

[0021] Further, the ginkgo leaf residue in step (1) is the residue after extracting lactones and flavonoids from ginkgo leaves, which is dried and crushed to 4-6mm.

[0022] Further, the high-temperature steam sterilization in step (1) is carried out at a temperature of 115-125℃ for 15-30min.

[0023] Further, the aspergillus niger in step (2) is activated at 4℃, inoculated on a slant medium in a sterile environment, and incubated at 30℃ for 4d to obtain mature spores, which are washed with 0.9% sterile normal saline and adjusted to an OD600 value.

[0024] Further, the spore liquid in step (2) is 5-10% when inoculated.

[0025] Further, the ultrasonic pretreatment in step (3) is carried out at 20-25kHz, 750-800W and 50-60℃.

[0026] Further, the solid-liquid ratio in steps (3), (5) and (6) is calculated based on the dry ginkgo leaf residue.

[0027] Further, the complex enzyme in step (5) is a combination of glycosidase (β-glucosidase or naringinase) and cellulase, pectinase, wherein the complex enzyme is preferably used in an amount of 1 / 500 to 1 / 100 of the weight of the dry ginkgo leaf residue.

[0028] Further, the working pressure in the high-pressure homogenization treatment in step (6) is controlled to be 60 to 100 MPa.

[0029] The present application has the following advantages compared with the prior art:

[0030] 1. The present application uses Aspergillus niger to ferment ginkgo leaf residue, which promotes the leaching of active ingredients such as polysaccharides and flavonoids in ginkgo leaf residue. In order to achieve this effect, the ginkgo leaf residue is mixed with water before fermentation and subjected to high-temperature steam sterilization treatment. This step not only sterilizes the material and the fermentation tank, but also breaks the plant cell wall with the help of steam pressure, promoting the leaching of active ingredients during the fermentation process. Secondly, Aspergillus niger can produce a rich enzyme system, including naringinase, β-glucosidase, cellulase, hemicellulase, etc., which can degrade cellulose, lignin, etc. not only can strengthen the dissolution of active ingredients in plant cells, but also can convert glycoside flavonoids to aglycone to some extent.

[0031] 2. The present application introduces ultrasonic waves and complex enzymes in steps based on fermentation treatment, which not only strengthens the dissolution of active ingredients, but also improves the "potency" of ginkgo polysaccharides and flavonoids. The introduction of ultrasonic waves promotes the dissolution of polysaccharides, shortens the time of hydrothermal extraction of polysaccharides, and more importantly, ultrasonic waves promote the degradation of polysaccharide molecular weight, which can improve the efficacy of polysaccharides to some extent. The introduction of complex enzymes (glycosidase, cellulase, pectinase) promotes the dissolution of active flavonoids in plant cells; more importantly, it can accelerate the conversion of glycoside flavonoids to aglycone flavonoids, thereby improving the "potency" of ginkgo flavonoids by increasing the content of aglycone flavonoids in flavonoid crude extract.

[0032] 3. The present application uses the supernatant after alcohol precipitation of ginkgo polysaccharides as the solvent for subsequent aglycone flavonoid extraction, and uses a micro-jet high-pressure homogenizer for mixing. Through the comprehensive action of strong shearing, high-speed impact, pressure instantaneous release, and vortex in the shock chamber, the solvent is promoted to be reused, the supernatant is uniformly transferred to the flavonoid crude extract, and the degree of cell disruption is further intensified by pressure and other actions, so that the effective components in the cells can quickly reach solubility equilibrium in the solvent, which is beneficial to the extraction process. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The process flowchart of the present application;

[0034] Figure 2 Figure 1 is a liquid chromatogram of the crude ginkgo flavone extract obtained from the ginkgo leaves residue after the synergistic action of Aspergillus niger and complex enzymes according to Example 1;

[0035] Figure 3 Figure 2 is a liquid chromatogram of the crude ginkgo flavone extract obtained by directly using solvent extraction according to Example 2;

[0036] Figure 4 Figure 3 is a liquid chromatogram of the crude ginkgo flavone extract obtained from the fermentation liquor of Aspergillus niger according to Example 3;

[0037] Figure 5 Figure 4 is a liquid chromatogram of the crude ginkgo flavone extract obtained from the ginkgo leaves residue after the synergistic action of Aspergillus niger and complex enzymes according to Example 4. DETAILED DESCRIPTION

[0038] The application will be further described in detail by specific examples, which are only descriptive and not limiting, and cannot limit the protection scope of the application.

[0039] Example 1:

[0040] Extraction of polysaccharide and flavone from ginkgo leaves residue by synergistic action of Aspergillus niger and complex enzymes

[0041] The steps are as follows: about 1 kg of ginkgo leaves residue is loaded into a fermentation tank, mixed with 500 mL of water by stirring, and sterilized by high-temperature steam at 115°C for 30 min; after sterilization, the temperature is lowered to 25-30°C; Aspergillus niger is activated to form a spore solution, which is inoculated at 6% (v / v), and aerated fermentation is carried out for 3 d; after the fermentation is completed, the fermentation product is taken out, about 3.5 L of water is added, and ultrasonic treatment is carried out at 800 W and 55°C for 30 min; water is continuously added to about 10 L, and extraction is carried out at 95°C for 1.5 h; the extract is filtered to obtain a water extract and a residue. The water extract is concentrated under reduced pressure to 2 L, ethanol is added to mix and adjust the alcohol concentration to 85%, and alcohol precipitation is carried out overnight; the precipitate and supernatant are collected, and the precipitate is dried to obtain crude ginkgo polysaccharide 185.8 g, with a yield of 18.6%, and the content of crude polysaccharide (%) is calculated by the phenol-sulfuric acid method.

[0042] To the residue after water extraction, 6 L of phosphate buffer (pH 5) and 3.0 g of complex enzymes (β-glucosidase 0.5 g, cellulase 1.5 g, pectinase 1.0 g) are added, and stirring reaction is carried out at 55°C for 5 h; the enzyme solution is concentrated under reduced pressure to about 1.0 L, the collected supernatant is mixed with it by using the micro-jet high-pressure homogenization method, the ethanol concentration is adjusted to 80%, and it is added at a solid-liquid ratio of 1:10, and extraction is carried out at 80°C for 4 h; after filtration, the filtrate is concentrated under reduced pressure and dried to obtain crude ginkgo flavone extract 60.3 g, with a yield of 6.0%, and the content of total flavone (%) is measured by ultraviolet spectrophotometry, and the content of total aglycone represented by quercetin (%) is calculated by high-performance liquid chromatography (HPLC).

[0043] Test results: The yield of crude polysaccharide prepared by water extraction and alcohol precipitation was 18.6% (based on the weight of Ginkgo leaf residue), and its crude polysaccharide content was 21.8% (based on the weight of crude extract); the yield of crude Ginkgo flavonoids was 6.0% (based on the weight of Ginkgo residue), and its total flavonoid content was 6.3%, and its total aglycone content was 2.5% (based on the weight of crude extract).

[0044] Example 2:

[0045] Ginkgo polysaccharides and flavonoids were obtained from Ginkgo leaf residue using a direct solvent extraction method (control experiment).

[0046] The steps are as follows: Add 10L of water to 1kg of ginkgo leaf residue and extract at 100℃ for 3h. Filter to obtain water extract and residue. Concentrate the water extract to about 2L, add ethanol to adjust the alcohol concentration to about 85%, precipitate overnight, collect the precipitate, dry it to obtain 95.1g of crude ginkgo leaf polysaccharide. Determine the content of crude polysaccharide by phenol-sulfuric acid method. Collect the supernatant for later use.

[0047] The residue was added to the supernatant, and the solid-liquid ratio was adjusted to 1:10 with an 80% alcohol concentration. The mixture was extracted at 80°C for 4 hours, centrifuged and filtered, and the filtrate was concentrated under reduced pressure and dried to obtain 2.2 g of crude ginkgo flavonoid extract. The total flavonoid content and total aglycone content (%) were measured.

[0048] Test results: The yield of crude polysaccharide by water extraction and alcohol precipitation was 9.5% (based on the weight of Ginkgo leaf residue), and the content of crude polysaccharide was 15.3% (based on the weight of crude extract); the yield of crude extract of Ginkgo flavonoids was 2.2% (based on the weight of Ginkgo leaf residue), of which the total flavonoid content was 3.2% and the total aglycone content was 0.6% (based on the weight of crude extract).

[0049] Example 3:

[0050] Extraction of ginkgo polysaccharides and flavonoids from ginkgo leaf residue fermented with Aspergillus niger (control experiment)

[0051] The steps are as follows: Put about 1 kg of ginkgo leaf residue into a fermentation tank, add 500 mL of water and stir to mix, then heat at 115℃.

[0052] High-temperature steam sterilization for 30 minutes; after sterilization, cooling to 25-30℃; activating Aspergillus niger to prepare spore liquid, inoculating with 6% (v / v), and fermenting with aeration for 3 days; after fermentation, the fermentation product was removed, water was added to 3.5L, and ultrasonic treatment was performed at 800W, 60℃ for 30 minutes. Water was then added to 10L at a solid-liquid ratio of 1:10, and extraction was carried out at 95℃ for 2 hours. After centrifugation and filtration, the aqueous extract and residue were obtained. The aqueous extract was concentrated under reduced pressure to 1.5L, ethanol was added and mixed, and the ethanol concentration was adjusted to approximately 85%. The mixture was allowed to stand overnight for alcohol precipitation. The precipitate was collected, dried, and 156.9g of crude Ginkgo biloba polysaccharide was obtained. The supernatant was retained for later use.

[0053] The residue was mixed with the supernatant, adjusted by micro-fluid high pressure homogenization method at a solid-liquid ratio of 1:10 and an ethanol concentration of 80%, and extracted at 80°C for 4h; the extract was filtered, the filtrate was concentrated under reduced pressure and dried to obtain ginkgo flavone crude extract 66.7g.

[0054] Test results: the yield of water extraction and alcohol precipitation crude polysaccharide was 15.7% (based on the weight of ginkgo leaf residue), and the content of crude polysaccharide was 19.8% (based on the weight of crude extract); the yield of ginkgo flavone crude extract was 3.4% (based on the weight of ginkgo leaf residue), and the total flavone content was 3.9% and the total aglycone content was 0.9% (based on the weight of crude extract).

[0055] Example 4:

[0056] Aspergillus niger-complex enzyme synergistic extraction of ginkgo polysaccharide and flavone aglycone from ginkgo leaf residue

[0057] The steps are as follows: 1kg of ginkgo leaf residue is mixed with 0.6L of water; the mixture is sterilized in a fermentation tank at 121°C for 20min; after sterilization, the temperature is lowered to 25°C, and Aspergillus niger is activated to form spore solution, 6% (v / v) inoculation, aeration fermentation for 4d; after fermentation, the fermentation product is taken out, water is added to 4L, treated with ultrasonic wave at 800W and 55°C for 30min, and 10L of water is added for extraction at 95°C for 2h, filtered to obtain water extract and residue, the water extract is concentrated to about 1.5L, ethanol is added and adjusted to an alcohol concentration of 85-90%, and precipitated overnight, the precipitate is collected and dried to obtain ginkgo leaf crude polysaccharide 190.5g, and the supernatant is recovered for use.

[0058] The above residue is added with phosphate buffer (pH 4.5) and 4.0g of complex enzyme (1.0g of naringinase, 2.0g of cellulase, and 1.0g of pectinase) at a ratio of 1:8, and subjected to enzymatic reaction at 50°C for 6h; the enzyme solution is concentrated under reduced pressure to about 1.5L, mixed with the supernatant by micro-fluid high pressure homogenization method, adjusted to an alcohol concentration of 80%, added with 1:12 solid-liquid ratio, and extracted at 80°C for 4h; centrifuged and filtered, the filtrate is concentrated under reduced pressure and dried to obtain ginkgo flavone crude extract 58.1g, and the total flavone and total aglycone contents are measured respectively.

[0059] Test results: the yield of water extraction and alcohol precipitation crude polysaccharide was 19.1% (based on the weight of ginkgo leaf residue), and the content of crude polysaccharide was 20.5% (based on the weight of crude extract); the yield of ginkgo flavone crude extract was 5.8% (based on the weight of ginkgo leaf residue), and the total flavone content was 5.4% and the total aglycone content was 2.3% (based on the weight of crude extract).

[0060] Example 5:

[0061] Aspergillus niger-β-glucosidase synergistic extraction of ginkgo polysaccharide and flavone aglycone from ginkgo leaf residue

[0062] The procedure is as follows: 1 kg of ginkgo leaves residue is mixed with 0.6 L of water; the mixture is sterilized in a fermentation tank at 121°C for 20 min; after sterilization, the temperature is lowered to 25°C, and Aspergillus niger is activated to form spore liquid, which is inoculated at 6% (v / v) and aerated for 4 d; after fermentation, the fermented material is taken out, water is added to 15 L, and extraction is carried out at 95°C for 2 h; filtration is carried out to obtain water extract and residue; the water extract is concentrated to about 1.5 L, ethanol is added, and the alcohol concentration is adjusted to 85-90%; alcohol precipitation is carried out overnight; the precipitate is collected and dried to obtain 170 g of ginkgo leaf crude polysaccharide; and the supernatant is recovered for use.

[0063] The above residue is added with phosphate buffer (pH 4.5) and 2.0 g of β-glucosidase at a ratio of 1:8, and enzymatic hydrolysis is carried out at 50°C for 4 h; the enzymatic hydrolysate is concentrated under reduced pressure to about 1.5 L; the supernatant is added to adjust the alcohol concentration to 80%; the solid-liquid ratio is 1:12; and extraction is carried out at 80°C for 4 h; centrifugal filtration is carried out; the filtrate is concentrated under reduced pressure and dried to obtain 40.3 g of ginkgo flavone crude extract; and the total flavone content and total aglycone content (%) are determined, respectively.

[0064] Test results: the yield of water extraction and alcohol precipitation crude polysaccharide is 17.0% (based on the weight of ginkgo leaf residue); the crude polysaccharide content is 17.3% (based on the weight of crude extract); the yield of ginkgo flavone crude extract is 4.0% (based on the weight of ginkgo leaf residue); the total flavone content is 4.2%; and the total aglycone content is 1.2% (based on the weight of crude extract).

[0065] Ginkgo polysaccharide and flavone aglycone are extracted by the methods of Examples 1-5, respectively, and then the contents and molecular weights are determined. Specifically:

[0066] (1) Determination of crude polysaccharide content by phenol-sulfuric acid method

[0067] Using anhydrous glucose as a standard, a standard curve of glucose mass concentration (mg / ml) versus absorbance (A) is prepared, and the linear regression equation is y=8.379x-0.00124, with a correlation coefficient R 2 =0.9997 (x is the glucose mass concentration, in mg / mL; and y is the absorbance).

[0068] Sample determination: 0.1 g of crude polysaccharide sample is weighed into a 25-ml volumetric flask, and pure water is added to the mark; 2.0 ml of the sample diluent is taken and placed in a colorimetric tube; 1 ml of 5% phenol solution is added, and after shaking, 5 ml of concentrated sulfuric acid is quickly added; water is added to the mark to 25 ml; after standing for 30 min, the absorbance is measured at 490 nm; the polysaccharide concentration is determined by substituting the standard curve; and the total amount of crude polysaccharide in the crude extract (%) is calculated.

[0069] (2) Determination of crude polysaccharide molecular weight

[0070] The above crude polysaccharide is washed with 67% ethanol, deproteinized, decolorized, and dialyzed to obtain a preliminary refined polysaccharide. 2 mg of the polysaccharide sample is dissolved in 1 mL of deionized water, filtered through a 0.22 μm water phase microporous filter, and 20 μL of the filtrate is injected for sampling according to the gel permeation chromatography (GPC) conditions. The molecular weight of the polysaccharide sample is determined according to the standard curve (lgMw=-1.1545t+24.775, R 2 =0.9993, t is the retention time, and lgMw is the logarithm of the molecular weight). The chromatographic detection conditions are as follows: a P230 type GPC gel permeation chromatography system with an RI-201H differential refractometer detector; a PL aquagel-OH MIXED-H chromatographic column; a mobile phase: 0.1 M NaNO3; a flow rate: 1 mL / min; and a column temperature: 40°C.

[0071] (3) Determination of total flavonoid content by ultraviolet spectrophotometry

[0072] Rutin is used as a standard to draw a standard curve of rutin concentration (mg / mL) versus absorbance, Y=0.0146x+0.1238 (R 2 =0.9916) (x is the mass concentration of rutin, in mg / mL; and y is the absorbance).

[0073] Sample determination: 20 mg of the crude flavonoids is dissolved in 10 mL of 95% ethanol, 2 mL of the sample solution is taken, 0.3 mL of 5% NaNO2 aqueous solution is added, and the mixture is shaken and allowed to stand for 6 min. Then, 0.3 mL of 10% Al(NO3)3 aqueous solution is added, the mixture is shaken and allowed to stand for 6 min, 3 mL of 10% NaOH aqueous solution is finally added, the mixture is shaken and allowed to stand for 15 min, the absorbance is measured at 510 nm, the total flavonoid concentration is calculated by substituting the standard curve, and the total amount of total flavonoids in the crude extract is calculated.

[0074] (4) Determination of aglycone flavonoid content by HPLC

[0075] The contents of three main aglycones (quercetin, kaempferol, and isorhamnetin) after enzymatic hydrolysis of the flavonoid glycosides are detected by high performance liquid chromatography (HPLC). The three aglycones all have a basic skeleton of 2-phenyl chromone and have basically the same chemical structure, and thus their ultraviolet absorption is basically the same. Therefore, the standard working curve of quercetin is also applicable to the quantitative calculation of the other two aglycones.

[0076] The total aglycone content is calculated by using the standard working curve of quercetin:

[0077]

[0078]

[0079] In the formula, A1 is the peak area of quercetin; A2 is the peak area of kaempferol; A3 is the peak area of isorhamnetin; K is the slope of the quercetin standard working curve; B is the intercept of the quercetin standard working curve; V is the total volume of the sample solution (mL); N is the injection dilution multiple; and W is the sample weight (mg). The linear fitting equation is:

[0080] y = 96434x - 307.98, the linear range is 0.01-0.16 mg / mL, and the correlation coefficient is 0.9998.

[0081] (HPLC detection method of ginkgo flavonoid glycosides and aglycone components: mobile phase A 0.1% formic acid aqueous solution; mobile phase B methanol; gradient elution conditions are as follows: 0-15 min, A from 65% to 60%, 15-30 min, A from 60% to 55%, 30-45 min, A from 55% to 50%, 45-70 min, A from 50% to 45%; flow rate 0.7 mL / min, detection wavelength 360 nm; column temperature 30°C; injection volume 20 μL.

[0082] The main parameters of the ginkgo polysaccharides and flavones obtained in the above examples are as follows in Table 1:

[0083] Table 1. Ginkgo polysaccharides and flavones obtained by different treatment methods of ginkgo leaf residues

[0084]

[0085] As can be seen from Table 1, the yield and content of the crude polysaccharides and crude flavones and the flavone aglycone ratio of Example 3 are significantly higher than those of Example 2, that is, the fermentation of Aspergillus niger can improve the yield of the product and the content of flavone aglycone. The yield and content of the crude polysaccharides and crude flavones and the flavone aglycone ratio of Examples 1, 4 and 5 are significantly higher than those of Example 2, and the addition amount and technical parameters of Aspergillus niger and complex enzyme have certain effects on the yield and content of the crude polysaccharides and crude flavones and the flavone aglycone ratio, and Example 1 is the best.

Claims

1. A fungus-enzyme synergistic method for extracting polysaccharide and aglycone type flavonoids from ginkgo biloba leaf residue, characterized in that, It comprises the following steps: (1) high-temperature steam sterilization: Put ginkgo leaf residue into a fermentation tank, then add 40-60% water, stir and mix, and then perform high-temperature steam sterilization; (2) Aspergillus niger fermentation: Cool the sterilized material to 25-30℃, activate Aspergillus niger to prepare spore liquid, then inoculate the material, and aerate and ferment for 3-4 days; (3) ultrasonic and hydrothermal extraction: Take out the fermented material, add water according to a solid-liquid ratio of 1:3-5, then perform ultrasonic pretreatment for 25-30 in, then add water according to a solid-liquid ratio of 1:10-12, and extract at 90-95℃ for 1.5-2 hours, filter the extract, and obtain water extract and residue for standby use; (4) concentration and alcohol precipitation: Concentrate the water extract obtained in step (3) under reduced pressure to 1 / 5-1 / 6 of the original volume, add ethanol to mix, adjust the alcohol concentration to 80-90%, stand for alcohol precipitation, filter to collect the precipitate and supernatant; dry the precipitate to obtain ginkgo polysaccharide, and reserve the supernatant for standby use; (5) compound enzyme enzymolysis: Add a buffer solution with pH 4.5-5 and compound enzyme to the residue obtained in step (3) according to a solid-liquid ratio of 1:6-8, and react at 50-60℃ for 4-6 hours; after the reaction is completed, perform high-temperature sterilization, and concentrate the obtained enzymolysis liquid under reduced pressure to 1 / 4-1 / 5 of the original volume; The compound enzyme is a combination of glycosidase, cellulase and pectinase; (6) homogenization and alcohol hydrothermal extraction: Put the supernatant obtained in step (4) and the enzymolysis concentrated liquid obtained in step (5) into a microjet high-pressure homogenizer for high-pressure homogenization mixing, adjust the alcohol concentration to 70-80%, add according to a solid-liquid ratio of 1:10-12, and extract at 70-80℃ for 3-5 hours; filter the extract, concentrate the filtrate under reduced pressure, and dry to obtain a crude extract rich in aglycone flavonoids; The ginkgo leaf residue in step (1) is the residue after extracting lactones and flavonoids from ginkgo leaves, which is dried and crushed to 4-6 mm.

2. The fungus-enzyme synergistic method for extracting polysaccharide and aglycone type flavonoids from ginkgo leaf residue according to claim 1, characterized in that, The high-temperature steam sterilization in step (1) is performed at a temperature of 115-125℃ for 15-30 minutes.

3. The fungus-enzyme synergistic method for extracting polysaccharide and aglycone type flavonoids from ginkgo leaf residue according to claim 1, characterized in that, In step (2), the Aspergillus niger is activated at 4℃, inoculated on slant medium in a sterile environment, and incubated at 30℃ for 4 days to obtain mature spores, which are washed with 0.9% sterile physiological saline and adjusted to OD600 value.

4. The fungus-enzyme synergistic method for extracting polysaccharide and aglycone type flavonoids from ginkgo leaf residue according to claim 1, characterized in that, The spore liquid is 5-10% when inoculated in step (2).

5. The fungus-enzyme synergistic method for extracting polysaccharide and aglycone type flavonoids from ginkgo leaf residue according to claim 1, characterized in that, The ultrasonic pretreatment in step (3) is performed under the following conditions: 20-25 kHz, 750-800 W, and 50-60℃.

6. The fungus-enzyme synergistic method for extracting polysaccharide and aglycone type flavonoids from ginkgo leaf residue according to claim 1, characterized in that, The solid-liquid ratio in steps (3), (5) and (6) is based on the dry weight of ginkgo leaf residue.

7. The fungus-enzyme synergistic method for extracting polysaccharide and aglycone type flavonoids from ginkgo leaf residue according to claim 1, characterized in that, The amount of compound enzyme is 1 / 500-1 / 100 of the dry weight of ginkgo leaf residue.

8. The fungus-enzyme synergistic method for extracting polysaccharide and aglycone type flavonoids from ginkgo leaf residue according to claim 1, characterized in that, In step (6), the high-pressure homogenization is controlled at a working pressure of 60-100 MPa.

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