CN115417848B - Method for simultaneously extracting and separating isoflavone, procyanidine and water-soluble polysaccharide from black beans - Google Patents

Method for simultaneously extracting and separating isoflavone, procyanidine and water-soluble polysaccharide from black beans Download PDF

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CN115417848B
CN115417848B CN202211227583.2A CN202211227583A CN115417848B CN 115417848 B CN115417848 B CN 115417848B CN 202211227583 A CN202211227583 A CN 202211227583A CN 115417848 B CN115417848 B CN 115417848B
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isoflavone
procyanidine
ethanol
black
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CN115417848A (en
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唐美玉
李伟
黄华学
吴国亮
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Hunan Huacheng Biotech Inc
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
    • C07D311/36—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only not hydrogenated in the hetero ring, e.g. isoflavones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/40—Separation, e.g. from natural material; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
    • C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P20/10—Process efficiency

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Abstract

The invention relates to a method for simultaneously extracting and separating isoflavone, procyanidine and water-soluble polysaccharide from black beans, which is characterized by comprising the following steps of: (1) pretreatment; (2) extracting; (3) isolation of isoflavones; (4) separation of procyanidins and polysaccharides; and (5) purifying polysaccharide. The invention simultaneously separates and extracts the soybean isoflavone, the procyanidine and the water-soluble polysaccharide from the black bean raw material, fully utilizes the active ingredients of the black bean, improves the comprehensive development level of the black bean, and has good economic and social benefits. The invention also focuses on the taste and flavor of soybean isoflavone and the yield of procyanidine when three products are effectively extracted and separated, and is a process which fully considers the cost, benefit and operation convenience of industrial production and fully utilizes black bean resources of equipment.

Description

Method for simultaneously extracting and separating isoflavone, procyanidine and water-soluble polysaccharide from black beans
Technical Field
The invention relates to the field of natural product extraction, in particular to a method for simultaneously extracting and separating isoflavone, procyanidine and water-soluble polysaccharide from black beans.
Background
Black beans, black seeds of leguminous soybeans are kidney-shaped or round-like, black red or mauve on the surface, glossy, thin and brittle seed coats, and easy to break, and are planted in various places in China. The black beans contain rich nutrients, such as protein, unsaturated fatty acid, cellulose, vitamins, isoflavone, polysaccharide and the like, and the epidermis also contains active ingredients such as procyanidine and the like, so that the black beans have the effects of reducing cholesterol, scavenging free radicals, regulating immune functions and the like.
The isoflavone in black beans is a plant estrogen, can influence hormone secretion, metabolic biological activity, protein synthesis, growth factor activity and the like of human bodies, is a natural cancer chemopreventive agent, can effectively inhibit breast cancer, prostatic cancer and colon cancer, and is also helpful for preventing and treating the osteoporosis of middle-aged and elderly people.
Procyanidins are a general term for a large class of polyphenol compounds widely existing in plants, and Oligomeric Procyanidins (OPC) are internationally recognized natural antioxidants effective in scavenging free radicals in human bodies, and have strong in vivo activity.
The polysaccharide component rich in semen Sojae Atricolor is used as a high molecular carbohydrate, has multiple biological activities, and has good effects on diabetes, cardiovascular and cerebrovascular diseases, cholelithiasis, etc. The water-soluble polysaccharide can also be used as a food additive, can generate stable foam, has the performances of preventing protein precipitation, excellent emulsification, water retention and oil retention, can be used for baking, beverages, functional health-care foods and the like, and has wide market prospect.
In the prior art, soybean isoflavone is mainly prepared by extracting soybean as a raw material. The soybean and the black soybean are beans rich in isoflavone, but compared with the soybean, the soybean isoflavone compound in the black soybean contains the daidzin (D) and the fueliside (G) which are obviously higher than the soybean, and the soybean isoflavone compound in the soybean is mainly malonyl glucoside type (MD and MG are mainly); in addition, the content of free aglycone in black beans is far higher than that in soybeans. This results in lower extraction yield and poor mouthfeel if the isoflavone substances in black beans are extracted according to the general technique of extracting isoflavone from soybeans. In the prior art, when soybean isoflavone is extracted, the extraction rate or yield is mainly concerned, and the taste of soybean isoflavone products is less concerned. In the terminal market, soy isoflavone is mainly used in the fields of nutrition, food and health care products, and the consumers are actually more concerned about the taste and flavor of the products. If one pursues high extraction rate/yield of soybean flavone, but neglects the problem of product taste, the soybean flavone is practically indeterminate.
CN113527246a discloses a method for extracting soybean isoflavone from soybean osmunda, which adopts an aqueous deep eutectic solvent as choline chloride and hydrogen bond donor, and obtains high soybean isoflavone extraction rate. On one hand, the deep eutectic solvent has high cost, and on the other hand, the use of the deep eutectic solvent inevitably introduces solvent residues into the product, so that the deep eutectic solvent has certain potential safety hazard. The patented process lacks industrial applicability.
CN113005157a discloses a preparation method of soybean isoflavone aglycone, which is to add glucosidase solution into soybean isoflavone glycoside solution and to process high-voltage pulse electric field treatment. The method utilizes a high-voltage pulse electric field, is expensive in equipment and complex in operation, and is not suitable for industrialized natural product extraction and food industry.
In the prior art, in order to improve the utilization rate of soybean isoflavone by human body, the soybean isoflavone is hydrolyzed. The method mainly comprises acid hydrolysis, alkali hydrolysis and enzymolysis. The acid and alkaline hydrolysis can damage other active ingredients, and the enzymatic hydrolysis has specificity, so that the enzymatic hydrolysis of the soybean isoflavone can be efficiently completed, the content of isoflavone aglycone is increased, but the enzyme is needed, and the price is high. In the prior art, organic acid such as malic acid/citric acid is adopted for catalytic hydrolysis, so that damage of sulfuric acid and hydrochloric acid inorganic strong acid to other components is avoided, but high temperature above 110 ℃ is needed to achieve the hydrolysis efficiency, and anthocyanin of black soybean seeds is almost damaged at the temperature.
In summary, in the prior art, isoflavone, procyanidine and water-soluble polysaccharide are not extracted and separated at the same time; the extraction and utilization of the black beans are also limited to one product of crude polysaccharide, polypeptide and black bean protein, and anthocyanin products are prepared from black bean peel, so that the waste of black bean raw materials is caused, and the further development of the black bean industry is restricted. The taste and flavor of soybean isoflavone in black beans are not systematically researched, and the blank is filled in the invention.
Disclosure of Invention
The present invention aims to solve at least one of the above technical problems in the prior art. For this purpose, the invention provides a method for simultaneously extracting and separating isoflavone, procyanidine and water-soluble polysaccharide from black beans.
A method for simultaneously extracting and separating isoflavone, procyanidine and water-soluble polysaccharide from black beans comprises the following steps:
(1) Pretreatment: drying black beans, crushing, sieving to obtain black bean powder, and carrying out microwave treatment on the black bean powder;
preferably, in step (1), the mesh number of the sieving screen is 10 to 20 mesh.
Preferably, in step (1), the microwave treatment is performed at a power of 500-700W for a microwave treatment time of 30-60 s. The black beans are subjected to certain microwave treatment, can promote the transformation of isoflavone units, is beneficial to improving the content of aglycone,
(2) Extracting: adding water into the black bean powder obtained in the step (1) for continuous ultrasonic countercurrent extraction to obtain a black bean extract;
Preferably, in the step (2), the continuous countercurrent ultrasonic extraction is carried out for 2-4 hours at the extraction temperature of 45-55 ℃, the ultrasonic power of 200-300W and the frequency of 25-50 kHz.
The inventor finds out through a large number of experiments that the pretreatment of the step (1) has the advantages of high microwave power, high microwave frequency and high microwave treatment time, and the three products have good balance of yield and purity by matching with the conditions of the extraction temperature, the ultrasonic power and the ultrasonic frequency in the ultrasonic extraction of the step (2), and the obtained soybean isoflavone has good taste and flavor, is easy to obtain consumer favor, and is beneficial to the sale and the premium of terminal products.
(3) Isolation of isoflavones: and centrifuging the black bean extract, and then passing through a nanofiltration membrane to obtain trapped fluid and permeate. Allowing the permeate to pass through polar macroporous adsorbent resin, eluting with ethanol, concentrating the eluate under reduced pressure, and drying to obtain semen Sojae Atricolor isoflavone product.
Preferably, in the step (3), the molecular weight cut-off of the nanofiltration membrane is 400-700Da, and the filtration pressure is 0.5 Mpa-1.0 Mpa.
Preferably, in the step (3), the polar macroporous adsorption resin is of NKA-9, ADS-22 and ADS-7, the volume concentration of ethanol is 75-85%, and the dosage is 1.5-2.5 BV.
(4) Separation of procyanidins and polysaccharides: passing the trapped fluid in the step (3) through weak-polarity or nonpolar macroporous adsorption resin, and then passing the effluent through ion exchange resin to remove a small amount of impurities such as pigment, protein and the like, thereby obtaining water-soluble polysaccharide solution; eluting macroporous resin with ethanol, concentrating the eluate under reduced pressure, and drying to obtain procyanidine product;
preferably, in the step (4), the weak polar or nonpolar macroporous adsorption resin is AB-8, LX-12, LSA-10, XDA-6, LX-32, etc., the volume concentration of ethanol is 60-75%, and the dosage is 1.5-2.5 BV.
Preferably, in the step (4), the ion exchange resin is of the type D941, LX-94, LX-T5, LX-T8, LXD-762, etc.
Preferably, in step (4), 0.1-0.2wt% of natural antioxidants such as at least one of astaxanthin, ascorbic acid, vitamin C, beta-carotene is added to the retentate. The anthocyanin is easy to degrade, and part of natural antioxidants are added, so that the anthocyanin can be protected from being oxidized, and the yield is improved; the added antioxidants are all derived from natural products and can be safely eaten.
(6) Polysaccharide purification: concentrating the water-soluble polysaccharide solution in the step (4) under reduced pressure, adding high-grade ethanol into the concentrated solution, precipitating for 3-5 h, performing solid-liquid separation to obtain black soybean polysaccharide precipitate, and vacuum drying to obtain the water-soluble polysaccharide product.
Preferably, in step (5), the concentrate solids concentration is 50-60%.
Preferably, in step (5), the ethanol is 90-100% ethanol, and the polysaccharide solution has an ethanol concentration of 70-80% after the ethanol is added.
In the prior art, microwave and ultrasonic synergistic auxiliary extraction is also adopted, but the purpose of the method is mainly to improve the extraction efficiency and yield of the soybean isoflavone, the problem of taste and flavor of the soybean isoflavone is not concerned, and the effect of extracting soybean isoflavone Huang Tongshi on the yield of anthocyanin is not concerned, because the purpose of the method is to obtain a single product. The invention is characterized in that the parameters of each process step are optimized and selected, so that three products, namely soybean isoflavone, procyanidine and water-soluble polysaccharide, can be continuously and simultaneously obtained, and the taste and flavor of the soybean isoflavone are improved.
The polar macroporous resin, the weak polar macroporous resin or the nonpolar macroporous resin and the ion exchange resin can be regenerated and recycled, and the specific regeneration method of the macroporous resin comprises the following steps: firstly, eluting the resin layer by distilled water until no alcohol smell exists, then eluting the resin layer by sodium hydroxide solution with the mass concentration of 2-4% at the flow rate of 1-2 BV/h, and finally eluting the resin layer by distilled water until the resin layer is neutral at the flow rate of 2-3 BV/h; the specific regeneration method of the ion exchange resin comprises the following steps: the resin layer is leached by 1-2 BV/h of flow rate, 1% of hydrochloric acid solution by mass concentration until the effluent is obviously acidic, 2-3 BV/h of flow rate, distilled water to neutrality, 2-4% of sodium hydroxide solution by mass concentration until the effluent is obviously alkaline, and 2-3 BV/h of flow rate, distilled water to neutrality.
The ethanol used in the invention can be distilled and recovered through decompression concentration operation.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) The invention simultaneously separates and extracts the soybean isoflavone, the procyanidine and the water-soluble polysaccharide from the black bean raw material, fully utilizes the active ingredients of the black bean, improves the comprehensive development level of the black bean, and has good economic and social benefits. The extraction temperature of the black bean raw material is less than 60 ℃, so that the damage of active ingredients such as procyanidine and the like in the black bean raw material is avoided; the extraction temperature is lower, and the energy is saved.
(2) The invention also pays attention to the taste of soybean isoflavone when three products are effectively extracted and separated, which is that the prior art is often used for unilaterally improving the extraction efficiency of soybean isoflavone, and is difficult to consider the taste and the flavor and the yield of procyanidine.
(3) The main reagents used in the preparation method provided by the invention are ethanol and water, and the production process is free of toxic volatile matters and high in safety.
(4) In the invention, the process is simple, the resin, the film and the solvent used in the process can be reused, the process cost is low, and the method is suitable for industrial production.
Detailed Description
The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described with reference to the embodiments, but the present invention is not limited to these embodiments.
The black beans used in the embodiment of the invention are purchased from the black longjiang river for the phytoseization, the isoflavone content in the black beans is 0.62%, the procyanidine content is 0.22%, and the polysaccharide content is 12.15%.
The D941 type, LX-94 type, LX-T8 type anion exchange resin and NKA-9 type, LSA-10 type, ADS-22 type, LX-32 type, ADS-7 type and AB-8 type macroporous adsorption resin used in the embodiment of the invention are all purchased from Sichuan blue and Xiao technology new material Co., ltd; nanofiltration membranes used in the embodiments of the invention are purchased from Nanjing Fulinde environmental protection technology Co., ltd; the materials or chemicals used in the examples of the present invention, unless otherwise specified, were obtained by conventional commercial means.
Example 1
S1, crushing dried black beans, and taking 1000g of 10-mesh undersize; performing microwave treatment on the obtained undersize, wherein the microwave power is 500W, and the microwave treatment time is 60s;
S2, continuously countercurrent ultrasonic extracting 1000g of undersize material obtained in the step S1 with 10L of water for 4 hours at 45 ℃, wherein the ultrasonic power is 200W, the frequency is 25kHz, filtering and centrifuging after extraction to obtain an extracting solution;
S3, passing the extracting solution through a nanofiltration membrane with the aperture of 400Da, passing the permeate through NKA-9 polar macroporous adsorption resin at the flow rate of 2BV/h, eluting with pure water and 70% ethanol water solution at the flow rate of 2BV/h in sequence, collecting ethanol water eluent, concentrating under reduced pressure, and drying to obtain the black bean isoflavone product.
S4, adding 2g of ascorbic acid into the trapped liquid, sequentially passing through LSA-10 macroporous adsorption resin D941 ion exchange resin at a flow rate of 2BV/h after dissolution, washing a column by adopting 2BV pure water at a flow rate of 2BV/h, mixing water washing liquid with effluent of the D941 ion exchange resin column, collecting, concentrating under reduced pressure until the solid concentration is 50%, adding 90% ethanol to enable the ethanol concentration of the solution to be 70%, precipitating for 3h, carrying out solid-liquid separation to obtain black bean polysaccharide precipitate, and carrying out vacuum drying to obtain a water-soluble polysaccharide product.
S5, eluting the LSA-10 macroporous adsorption resin by adopting an ethanol water solution with the volume concentration of 70% at the flow rate of 2BV/h, collecting an ethanol water eluent, concentrating under reduced pressure, and drying to obtain the procyanidine product.
Example 2
Other operations, conditions identical to those of example 1, differ in that: in the step S2, continuous countercurrent ultrasonic extraction is carried out for 4 hours at 50 ℃.
Example 3
Other operations, conditions identical to those of example 1, differ in that: in the step S2, continuous countercurrent ultrasonic extraction is carried out for 4 hours at 55 ℃.
Example 4
Other operations, conditions and example 2 were the same, except: in step S1, the power of the microwave treatment is 700W, and the microwave treatment time is 30S.
Example 5
Other operations, conditions and example 2 were the same, except: in step S1, the power of the microwave treatment is 500W, and the microwave treatment time is 90S.
Example 6
Other operations, conditions and example 2 were the same, except: in step S1, the power of the microwave treatment is 800W, and the microwave treatment time is 30S.
Test example 1
The content and extraction rate of the isoflavone, procyanidin and polysaccharide of the black beans obtained in examples 1 to 3 were examined in this example. The specific test method comprises the following steps: and (2) detecting the standard control solution of isoflavone by an HPLC method, then testing the test solution prepared by the extracting solution obtained in the step (S2), and calculating by the standard control method to obtain a corresponding result. Drawing standard curves of procyanidine and polysaccharide respectively by a UV method, testing the products obtained in the examples, namely the black bean isoflavone, procyanidine and water-soluble polysaccharide products, and calculating by the standard curve method to obtain corresponding results.
Test method
(1) The method for detecting the procyanidine comprises the following steps: spectrophotometry
Drawing a standard curve: accurately sucking 1mL of procyanidine standard series working solutions with the concentrations of 0, 10, 25, 50, 100, 150, 200 and 250 mug/mL, placing the working solutions into ampoule bottles, precisely adding 6mL of hydrochloric acid-n-butanol solution and 0.2mL of ferric ammonium sulfate solution, uniformly mixing, sealing the solutions by using sealing pliers, heating the solutions in boiling water for 40min, taking the solutions out, immediately placing the solutions in ice water for cooling to room temperature, measuring absorbance at a wavelength of 546nm, and stabilizing color development within 1 hour. And drawing a standard curve by taking absorbance as an ordinate and procyanidine concentration as an abscissa.
Determination of sample solution: taking a sample (10-100 mg), precisely weighing, placing into a 50mL volumetric flask, adding 30mL of methanol, performing ultrasonic treatment (with the power of 250W and the frequency of 50 kHz) for 20min, standing to room temperature, adding methanol to a scale, shaking uniformly, centrifuging or placing to clarify, and taking supernatant as a sample solution. Precisely sucking 1mL of the sample solution, placing in an ampoule bottle, and then performing the steps according to a standard curve. And (3) taking the corresponding reagent as a blank, measuring the absorbance of the sample, and calculating the procyanidine content in the sample by using a standard curve. Specifically, the formula (1) is shown.
M: sample amount (mg); v: dilution factor
(2) The isoflavone detection method comprises the following steps: high performance liquid chromatography
Chromatographic conditions: SYMMETRY C18 (3.9X105 mm) reverse-phase chromatography column, mobile phase A: methanol: glacial acetic acid=98:2, mobile phase B: water: methanol: glacial acetic acid=88:10:2, flow rate 0.8mL/min. Gradient elution: 0-13min: mobile phase A5%, mobile phase B95%; 14-30min: mobile phase a 40%, mobile phase B60%; 31-36min: mobile phase a 95%, mobile phase B5%; 37-40min: mobile phase A5% and mobile phase B95%. Detection wavelength: 260nm.
Preparing standard liquid: precisely weighing appropriate amount of daidzin, genistin, daidzein, and genistein reference substance, dissolving in methanol under ultrasonic wave in 50ml volumetric flask, cooling to room temperature, diluting with methanol to scale, and shaking.
Preparation of test solution: precisely weighing a proper amount of soybean extract sample, placing into a 50ml volumetric flask, adding a proper amount of methanol, performing ultrasonic treatment for 30 minutes, cooling to room temperature, fixing volume to scale, mixing, and passing through 0.45um membrane to obtain sample solution.
The isoflavone content is calculated as shown in a formula (2):
A Sample : peak area of the sample; c Label (C) : concentration of standard solution (mg/ml); a Label (C) : peak area of standard; m: sample weight (mg), X is isoflavone content.
(3) The polysaccharide detection method comprises the following steps: spectrophotometry
Drawing a standard curve: glucose (constant weight) 50mg is precisely weighed and placed in a 1000mL volumetric flask, and water is added for dissolution and volume fixing. 0.0,0.1,0.2,0.4,0.6 and 0.8mL of the standard solution are precisely sucked, respectively placed in 10mL test tubes and added with water to be supplemented to 1.0mL. 1.0mL of 5% phenol solution was added, mixed well, 5.0mL of concentrated sulfuric acid was added, and the mixture was left to stand for 0.5h. The absorbance was measured at 485nm with an ultraviolet-visible spectrophotometer. And drawing a standard curve or calculating a regression equation by taking the absorbance value as an ordinate and the concentration (mg) of glucose as an abscissa.
Determination of the samples: accurately weighing about 20mg of the extract sample, placing in a 500mL volumetric flask, adding water for dissolution, fixing the volume, and filtering. Taking 1.0mL of the filtrate, placing the filtrate into a test tube, adding 1.0mL of 5% phenol solution, uniformly mixing, adding 5.0mL of concentrated sulfuric acid, and placing the mixture for 0.5h. The absorbance was measured at 485nm with an ultraviolet-visible spectrophotometer. And (3) taking the corresponding reagent as a blank, measuring the absorbance of the sample, and calculating the polysaccharide content in the sample by using a standard curve.
Polysaccharide content calculation is shown in formula (3):
M: sample amount (mg); v: dilution factor
To ensure accuracy of the experiment, the purity of each sample in this test example was the average of 3 tests.
Test results
The contents and yields of the three effective substances according to the calculation methods of formulas (1) to (3) are shown in table 1.
TABLE 1 results of product content and yield tests
Example 1 Example 2 Example 3 Example 4 Example 5 Example 6
Isoflavone content (%) 65.37 65.51 65.14 65.28 64.45 65.36
Isoflavone yield (%) 87.31 88.64 89.42 89.15 89.52 89.62
Procyanidin content (%) 96.78 95.67 94.89 95.32 95.06 94.35
Procyanidin yield (%) 91.58 92.41 89.12 91.62 90.58 90.84
Polysaccharide content (%) 81.03 82.15 80.81 82.25 81.96 82.18
Polysaccharide yield (%) 82.98 84.25 85.12 84.35 84.42 84.17
In conclusion, the preparation method of the black bean extract provided by the invention can simultaneously separate and extract isoflavone, procyanidine and water-soluble polysaccharide. The yield is that isoflavone is more than or equal to 87%, procyanidine is more than or equal to 91%, and water-soluble polysaccharide is more than or equal to 82%; the purity of the product is that isoflavone is more than or equal to 65.37%, procyanidine is more than or equal to 95.67%, and polysaccharide is more than or equal to 80.81%.
Test example 2
The soybean isoflavone obtained in the example is tested for taste, and the specific method is as follows: soy isoflavones from the different examples were diluted 20-fold and 10 subjects were tasted, scoring the palatable taste, scoring 5 scores, 5 scores being most satisfactory (essentially no bitter and astringent and beany), 1 score indicating the lowest score (bitter and astringent and beany). The results are shown in Table 2 below.
Table 2 taste testing of isoflavone products

Claims (8)

1. A method for simultaneously extracting and separating isoflavone, procyanidine and water-soluble polysaccharide from black beans, which is characterized by comprising the following steps:
(1) Pretreatment: drying black beans, crushing, sieving to obtain black bean powder, and carrying out microwave treatment on the black bean powder; the microwave treatment is carried out at the power of 500-700W for 30s-60s;
(2) Extracting: adding water into the black bean powder obtained in the step (1) for continuous ultrasonic countercurrent extraction to obtain a black bean extract; extracting at 45-55deg.C with ultrasonic power of 200-300W and frequency of 25-50kHz for 2-4 hr;
(3) Isolation of isoflavones: centrifuging the black bean extract, and then passing through a nanofiltration membrane to obtain trapped fluid and permeate; allowing the permeate to pass through polar macroporous adsorbent resin, eluting with ethanol, concentrating the eluate under reduced pressure, and drying to obtain semen Sojae Atricolor isoflavone product; the volume concentration of the ethanol is 75-85%, and the dosage is 1.5-2.5 BV;
(4) Separation of procyanidins and polysaccharides: passing the trapped fluid in the step (3) through weak-polarity or nonpolar macroporous adsorption resin, and passing the effluent through ion exchange resin to remove pigment and protein impurities to obtain water-soluble polysaccharide solution; eluting macroporous resin with ethanol, concentrating the eluate under reduced pressure, and drying to obtain procyanidine product; the volume concentration of the ethanol is 60-75%, and the dosage is 1.5-2.5 BV;
Polysaccharide purification: concentrating the water-soluble polysaccharide solution in the step (4) under reduced pressure, adding high-grade ethanol into the concentrated solution, precipitating 3-5 h, performing solid-liquid separation to obtain black soybean polysaccharide precipitate, and vacuum drying to obtain a water-soluble polysaccharide product; the ethanol concentration of the polysaccharide solution is 70-80% after the ethanol with the concentration of 90-100% is added.
2. The method according to claim 1, wherein in step (3), the nanofiltration membrane has a molecular weight cut-off of 400-700Da and the filtration pressure is 0.5Mpa to 1.0Mpa.
3. The method according to claim 1, wherein in step (3), the polar macroporous adsorption resin is of the type NKA-9, ADS-22, ADS-7.
4. The method according to claim 1, wherein in the step (4), the weakly polar or nonpolar macroporous adsorbent resin is of the type AB-8, LX-12, LSA-10, XDA-6, LX-32.
5. The method according to claim 1, wherein in the step (4), the ion exchange resin is of the type D941, LX-94, LX-T5, LX-T8, LXD-762.
6. The method according to claim 1, wherein in step (4), 0.1 to 0.2wt% of natural antioxidants are added to the retentate of the black bean raw material.
7. The method of claim 1, wherein the natural antioxidant is selected from at least one of astaxanthin, vitamin C, beta-carotene.
8. The method of claim 1, wherein in step (5) the concentrate solids concentration is 50-60%.
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