Soybean isoflavone aglycone preparation method based on enzymolysis

Abstract

The invention relates to the technical field of isoflavone biotransformation, in particular to a soybean isoflavone aglycone preparation method based on enzymolysis. Comprising the following steps: inoculating Kluyveromyces marxianus into a basic culture medium for shake cultivation; after culturing, centrifuging, washing and resuspending to obtain a bacterial suspension as a leavening agent, and inoculating the bacterial suspension to a bean dreg composite culture medium for shaking table fermentation for 48-96 hours; centrifuging the fermentation liquor at 0-4 DEG C, and collecting supernate to obtain crude beta-glucosidase; the crude enzyme liquid is sequentially subjected to ammonium sulfate salting-out, ion exchange chromatography and twice sephadex chromatography, and purified beta-glucosidase liquid is obtained; and carrying out enzymatic reaction on soy isoflavone by using the purified beta-glucosidase liquid to obtain the soy isoflavone aglycone. The method has the advantages that the bean dreg culture medium is utilized for fermentation to produce beta-glucosidase, and the combined soy isoflavone is subjected to enzymolysis, so that the utilization of the byproduct bean dregs is improved, the biotransformation of isoflavone is realized, and the obtained product has better activity.

Description

Technical Field

[0001] The invention relates to the technical field of isoflavone biotransformation, and in particular to a method for preparing soybean isoflavone aglycones based on enzymolysis. Background Art

[0002] In recent years, research on the production of a large number of agricultural by-products and their application in the production of energy materials from agricultural waste has been continuously reported. Using cheap by-products to replace expensive raw materials to produce high-value-added biological products has become a research hotspot. my country is one of the major producers of soy products in the world. In the process of producing soy products, a large number of by-products have always been produced, namely dregs. Studies have shown that my country produces about 2 million tons of wet dregs every year. Due to its strong beany smell, rough taste and difficulty in storage, its nutritional value is easily overlooked. It is mainly used to produce feed or discarded as waste, and its utilization rate is extremely low. Studies have shown that dregs are rich in nutrients, such as dietary fiber (about 50%), protein (about 25%), lipids (about 10%), carbohydrates, vitamins and functional nutrients such as soy isoflavones. Therefore, the efficient application of dregs and how to achieve its comprehensive utilization and turn waste into treasure are the problems facing the development of the current soy product industry.

[0003] Soy isoflavones are mainly divided into glycoside isoflavones and aglycone isoflavones, among which free isoflavones can exert higher biological activity. Its degradation process is divided into three steps: first, malonyl glucoside is degraded into acetylglucoside, then acetylglucoside is degraded into glucoside, and finally glucoside is degraded into soy isoflavone aglycones. Its conversion methods include acid degradation, alkaline hydrolysis, Smith degradation and enzymatic hydrolysis. Among them, Smith degradation and acid and alkali methods cause serious corrosion to equipment, produce a large amount of wastewater and pollute the environment, and have high costs. In addition, the hydrolyzed aglycones are very unstable and easily degraded. Therefore, it is necessary to choose a mild and efficient enzyme degradation method to hydrolyze isoflavones. The enzyme most commonly used to hydrolyze soy isoflavone glycosides is β -Glucosidase, its conversion form has the advantages of high efficiency, mildness, safety, non-toxicity and pollution-free.

[0004] β -Glucosidase ( β -D-Glucosidase, EC 3.2.1.21) is a cellulose hydrolase that is an integral part of the sugar metabolism pathway and can hydrolyze the non-reducing end of the substrate. β -D-glucosidic bond, released β -D-glucose and corresponding ligands. β -Glucosidase is widely present in fermentation products of microorganisms, animals and plants. β-Glucosidase has poor operability, and the extraction process is relatively complex and difficult to operate. Microorganisms are one of the important sources of glucosidase. Bacteria such as lactic acid bacteria, Bacillus subtilis and thermophilic Bacillus have strong ability to produce glucosidase; fungi also show great potential in producing glucosidase and cellulase, among which yeast, Penicillium and Trichoderma mainly produce glucosidase. Studies have reported that different sources of β -Glucosidase usually has great differences in enzymatic properties, such as molecular weight, temperature and metal ion stability. β -Glucosidase has different application ranges.

[0005] It has been reported that Kluyveromyces marxianus ( K.Marxianus ) have some characteristics, such as high temperature resistance, high growth rate and the ability to produce rich enzyme systems, such as lipase, protease and β -glucosidase, etc. Therefore, it can be used as an ideal microbial raw material in industrial production and has broad application prospects in industry. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for preparing soybean isoflavone aglycones based on enzymolysis.

[0007] The present invention aims to provide a method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis, comprising the following steps: S1. Inoculate Kluyveromyces marxianus into a basic culture medium and culture it on a shaking table; after culture, centrifuge, wash, and resuspend to obtain a bacterial suspension as a fermentation agent; S2. The fermentation agent was inoculated into the okara composite medium at a mass percentage of 0.1-3% for shaking fermentation for 48-96 hours at a temperature of 35-40°C and a rotation speed of 100-150 r / min; the fermentation broth was centrifuged at 0-4°C, and the supernatant was collected to obtain a crude β-glucosidase enzyme solution; S3. The crude β-glucosidase enzyme solution was sequentially subjected to ammonium sulfate precipitation, ion exchange chromatography and two dextran gel chromatography to obtain a purified β-glucosidase solution; S4. Weigh the prepared soy isoflavone solution, and use purified β-glucosidase solution to perform enzymatic reaction on the soy isoflavones for 60 to 180 minutes to obtain soy isoflavone aglycones.

[0008] Preferably, the okara composite culture medium in step S2 comprises fresh okara and YPD culture medium; the moisture content of the fresh okara is 80-99%; and the amount of glucose added to the okara composite culture medium is 0.25-0.75%.

[0009] Preferably, the amount of glucose added in step S2 is 0.5%; and the shaking fermentation time is 72h.

[0010] Preferably, the components of the basal culture medium are: beef extract powder 8g / L, peptone 10g / L, yeast extract powder 4g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 80 1g / L, deionized water 1000mL, pH 5.7±0.2.

[0011] Preferably, in step S4, the amount of β-glucosidase added is 5-50 U / mg, the reaction temperature is 55-65° C., and the pH value is adjusted to 4.5-5.5.

[0012] Preferably, in step S4, the amount of β-glucosidase added is 5 U / mg, the reaction temperature is 60° C., and the pH value is adjusted to 5.

[0013] Preferably, step S3 includes the following sub-steps: S301. ammonium sulfate graded salting out: adding ammonium sulfate to the crude enzyme solution to obtain a solution with a saturation of 20%; salting out to remove the precipitated enzyme protein; continuing to add ammonium sulfate to obtain a solution with a saturation of 60%; adjusting the pH value of the salting out to 3.0-7.0, and keeping it still for 4-24 hours; collecting the enzyme protein precipitate obtained by salting out by centrifugation; re-dissolving the precipitate with a buffer solution, dialyzing and desalting, and obtaining an enzyme solution purified by ammonium sulfate; S302. Ion exchange chromatography: The enzyme solution after ammonium sulfate purification is concentrated; DEAE-52 anion exchange column material is selected and treated, loaded on the column, loaded, eluted with NaCl solution, and the eluate A is collected; S303. First dextran gel chromatography: concentrate the eluate A; load the eluate onto a dextran G-100 gel column, and collect the eluate B; S304. Second dextran gel chromatography: concentrate the eluate B; load it onto a dextran G-50 gel column, and collect the eluate, which is the purified β-glucosidase solution.

[0014] Preferably, polyethylene glycol 20000 is used during concentration in step S3; the diameter-to-height ratio of the dextran G-100 gel column in step S303 is 1:20, the flow rate is 0.5 mL / min, and the sample loading amount is 1.5%; the diameter-to-height ratio of the dextran G-50 gel column in step S304 is 1:25, the flow rate is 0.25 mL / min, and the sample loading amount is 1.75%.

[0015] Preferably, the deposit number of Kluyveromyces marxianus is CGMCC No.13907.

[0016] Preferably, in step S1, before inoculating the Kluyveromyces marxianus into the basic culture medium, the strain is activated; the shaking culture temperature is 25-35°C, the time is 10-15h; the concentration of the bacterial suspension is 10 7 cfu / mL.

[0017] Compared with the prior art, the present invention can achieve the following beneficial effects: β-glucosidase was produced by fermentation of soybean dregs culture medium, and bound soybean isoflavones were enzymolyzed to improve the utilization of byproduct soybean dregs, realize the biotransformation of isoflavones, and the obtained soybean isoflavone aglycone products had good activity; β The method of preparing aglycones by enzymatic hydrolysis of -glucosidase has the advantages of high hydrolysis rate, high yield, mild conditions, simple operation and green environmental protection, and has good and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the results of the effects of different fermentation medium (okara medium, PDA medium, YPD medium and okara composite medium) on the production of β-glucosidase according to the embodiments of the present invention.

[0019] Figure 2 According to the embodiment of the present invention, different carbon sources are used to treat the okara composite culture medium. K. marxianus The results were affected by the production of β-glucosidase by C21.

[0020] Figure 3 The effect of glucose addition amount and fermentation time on the okara composite culture medium provided by the embodiment of the present invention is as follows: K. marxianus C21 β -glucosidase effect; (A) β -The effect of changes in relative enzyme activity of glucosidase; (B) β -The effect of viable bacterial count of glucosidase; (C) The effect of pH change on the culture system.

[0021] Figure 4 According to the different embodiments of the present invention, β -glucosidase enzyme addition on the enzymatic hydrolysis of soybean isoflavones.

[0022] Figure 5 According to the different embodiments of the present invention, β -glucosidase enzymatic reaction time on the enzymatic hydrolysis of soybean isoflavones.

[0023] Figure 6 The hypoglycemic activity results of soybean isoflavones (ISO) before enzymolysis and soybean isoflavones products (ISO-E) after enzymolysis provided by the embodiments of the present invention; (A)α -Glucosidase inhibition ability; (B) α -Amylase inhibition ability; (C) glucose adsorption ability.

[0024] Figure 7 The lipid-lowering activity results of soybean isoflavones (ISO) before enzymolysis and soybean isoflavones products (ISO-E) after enzymolysis provided by the embodiments of the present invention; (A) cholesterol adsorption capacity; (B) sodium cholate capacity; (C) lipase inhibition capacity.

[0025] Figure 8 The antioxidant activities of soybean isoflavones before enzymolysis (ISO) and soybean isoflavones products after enzymolysis (ISO-E) provided in the embodiments of the present invention are as follows; (A) DPPH scavenging ability; (B) ABTS scavenging ability; (C) -OH free radical scavenging ability; (D) O2 - Scavenging ability; (E)Fe 2+ Chelating capacity; (F) total reducing capacity.

[0026] Fig. 9 The invention discloses the protective effects of soybean isoflavones before enzymolysis (ISO) and soybean isoflavones products after enzymolysis (ISO-E) on HepG2 cells oxidatively damaged by H2O2.

[0027] Fig.10 Provided according to an embodiment of the present invention is the effect of soy isoflavones before enzymatic hydrolysis (ISO) and soy isoflavones product after enzymatic hydrolysis (ISO-E) provided according to an embodiment of the present invention on the ROS level of HepG2 cells induced by H2O2.

[0028] Fig.11 Provided according to an embodiment of the present invention is the effect of soy isoflavones (ISO) before enzymatic hydrolysis and soy isoflavones product (ISO-E) after enzymatic hydrolysis on oxidative stress factors of HepG2 cells induced by H2O2. DETAILED DESCRIPTION

[0029] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the same modules are represented by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, the detailed description thereof will not be repeated.

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0031] Example 1 This embodiment provides a method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis, which specifically comprises the following steps: S1. Inoculate Kluyveromyces marxianus into a basic culture medium and culture it on a shaking table; after culture, centrifuge, wash, and resuspend to obtain a bacterial suspension as a fermentation agent; The preparation method of the starter comprises: activating Kluyveromyces marxianus for 3 generations in a basic culture medium, selecting the activated strain and inoculating it into the basic culture medium, culturing it in a shaking incubator with a rotation speed of 120 r / min, a temperature of 30°C, and a time of 12 hours; centrifuging the bacterial liquid in a high-speed refrigerated centrifuge for 5 minutes, at a temperature of 4°C and a rotation speed of 7500 r / min; washing the centrifuged bacterial bodies twice with sterile water, and then resuspending the bacterial suspension to adjust the concentration of the bacterial suspension to 10 7 cfu / mL, as a fermentation culture; The components of the basic culture medium are: beef extract powder 8g / L, peptone 10g / L, yeast extract powder 4g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 801g / L, deionized water 1000mL, pH 5.7±0.2; Kluyveromyces marxianus ( K. marxianus ) is a C21 strain, deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), with the deposit number CGMCC No.13907, the deposit date is March 21, 2017, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0032] S2. The fermentation agent was inoculated into the okara medium at an inoculum rate of 0.1% for shaking fermentation for 72 hours at a temperature of 35°C and a rotation speed of 120 r / min; the fermentation broth was centrifuged at 4°C and 9000 r / min for 10 minutes, and the supernatant was collected to obtain a crude β-glucosidase enzyme solution; Preparation of okara composite medium: Soybeans and water are added in a volume ratio of 1:5, and the soybeans are soaked at room temperature for 13 hours; the water is drained, and the soaked soybeans and 100°C water are ground in a food processor at medium speed for 3 minutes, filtered, and fresh okara is obtained; the moisture content of the okara is adjusted to about 85%; the pretreated fresh okara and YPD medium are prepared in proportion to form a liquid okara composite medium. Among them, the amount of glucose added is 0.5%.

[0033] S3. The crude β-glucosidase enzyme solution is sequentially subjected to ammonium sulfate fractionation precipitation, ion exchange chromatography and two dextran gel chromatography to obtain a purified β-glucosidase solution; specifically comprising the following steps: S301. ammonium sulfate graded salting out: adding ammonium sulfate to the crude enzyme solution to obtain a solution with a saturation of 20%; salting out to remove the precipitated enzyme protein; continuing to add ammonium sulfate to obtain a solution with a saturation of 60%; adjusting the pH value of the salting out to 3.0-7.0, and keeping it still for 4-24 hours; collecting the enzyme protein precipitate obtained by salting out by centrifugation; re-dissolving the precipitate with a buffer solution, dialyzing and desalting, and obtaining an enzyme solution purified by ammonium sulfate; S302. Ion exchange chromatography: The enzyme solution after ammonium sulfate purification is concentrated; DEAE-52 anion exchange column material is selected and treated, loaded on the column, loaded, eluted with 0.2 mol / L NaCl solution, and the eluate A is collected; S303. First dextran gel chromatography: concentrate the eluate A; load the sample onto a dextran gel column (Sephadex G-100 gel column), and collect the eluate B; the diameter-to-height ratio of the Sephadex G-100 gel column is 1:20, the flow rate is 0.5 mL / min, and the sample loading amount is 1.5%; S304. Second dextran gel chromatography: concentrate the eluate B; load the sample onto a dextran gel column (Sephadex G-50 gel column), collect the eluate, which is the purified β-glucosidase solution; the diameter-to-height ratio of the Sephadex G-50 gel column is 1:25, the flow rate is 0.25 mL / min, and the sample loading amount is 1.75%; In step S3, polyethylene glycol 20000 is used for concentration.

[0034] S4. Weigh soy isoflavones to prepare a solution with a substrate concentration of 5 mg / mL, and perform an enzymatic reaction on soy isoflavones with purified β-glucosidase solution at a reaction temperature of 60°C for 60 min, and adjust the pH value to 5 to obtain soy isoflavone aglycones after the reaction; The amount of β-glucosidase added in this step is 5 U / mg.

[0035] Example 2 The optimization experiment of adding ingredients to the okara composite culture medium is as follows: 1. Preliminary selection of culture medium components YPD and PDA media are commonly used media for yeast. YPD medium is also called yeast extract peptone glucose medium, and its main components are Yeast Extract (yeast extract), Peptone (peptone), and Dextrose (glucose); PDA medium is the abbreviation of Potato Dextrose Agar (Medium), which corresponds to the English words potato, glucose, and agar.

[0036] YPD medium: 2% glucose, 1% yeast powder, 2% peptone, 1000 mL distilled water; PDA medium: 20% potato, 2% glucose, 1000 mL distilled water; Okara (OK for short) culture medium: 3% okara, 1000 mL of distilled water.

[0037] In order to determine the most suitable β -glucosidase okara composite medium, add okara (OK) to the above two culture media respectively, and investigate K. marxianus C21 β -Glucosidase activity, the results are as follows Figure 1 Without adding okara, YPD medium β -Glucosidase activity was 17.7% higher than that in PDA medium, and had no significant difference with that in okara medium ( p >0.05); after adding okara, PDA and YPD β -Glucosidase activity was significantly increased by 211% and 251% respectively ( p <0.05), okara-YPD medium showed stronger enzyme production advantage, and okara as an inducer improved β -glucosidase activity was further demonstrated.

[0038] 2. Effects of different carbon sources on the okara composite culture medium K. marxianus Effect of C21 on β-glucosidase production To investigate the effect of adding different carbon sources on the K. marxianus The production of β-glucosidase by C21 was affected by the effect of glucose, lactose, maltose, sucrose and galactose as carbon sources. The activity of β-glucosidase was compared when the carbon source was added at a rate of 2%. The okara culture medium without carbon source was used as the control group. Figure 2 As shown in the figure, it can be seen that the addition of glucose showed the highest relative enzyme activity, which was 175%, while the addition of lactose, sucrose, maltose and galactose only increased the enzyme activity by about 10%.

[0039] Effect of glucose addition on the growth of soybean dregs composite culture medium K. marxianus C21 β -Glucosidase effect Effect of glucose addition on okara culture medium β -glucosidase plays an important role in the production of glucosidase. This section comprehensively examines the effect of glucose addition on K. marxianus The effects of C21 metabolism, including changes in β-glucosidase activity, viable bacterial count, and system pH ( Figure 3 ).

[0040] 1. Changes in relative enzyme activity The activity of β-glucosidase increased first and then decreased with the increase of fermentation time. The maximum relative enzyme activity was observed when the fermentation time was 72h, which was 72h earlier than the optimal time for β-glucosidase production by Aspergillus flavus produced by Chen et al. The relative enzyme activity was the highest when the addition of glucose was 0.5%, which was 280%. When the addition of glucose was greater than 0.5%, the relative enzyme activity decreased instead, which may be related to the premature aging of the bacteria caused by the imbalance of carbon-nitrogen ratio and the inability to fully metabolize and produce enzymes.

[0041] 2. Changes in viable count The change in viable bacterial count was positively correlated with the change in relative enzyme activity, and it showed a trend of first increasing and then decreasing with the increase in fermentation time. The viable bacterial count was the highest when the fermentation time was 72h, and the highest viable bacterial count was observed in the okara medium with 0.5% glucose, which was 8.17 log cfu / mL. This may be because the carbon-nitrogen ratio of the okara medium with 0.5% glucose was the optimal carbon-nitrogen ratio for K. marxianusC21, which enabled the microorganism to fully grow and metabolize enzymes.

[0042] 3. System pH changes The initial pH of the culture medium was 5, and the pH changes of the culture system were measured at 24, 48, 72, 96, 120, 144 and 168 h of fermentation.

[0043] The pH change of the system can well reflect the growth and metabolism of microorganisms. Figure 3 As can be seen in Figure C, the pH of the fermentation broth shows a trend of first decreasing and then increasing. This is because the bacteria grow rapidly in the early stage of fermentation and use the -NH 3+ , producing organic acids, resulting in a decrease in pH. As fermentation proceeds, metabolites accumulate, bacteria autolyze, and the pH of the fermentation broth increases. After adding glucose, the pH of the culture medium dropped significantly. The higher the amount of glucose added, the faster the pH dropped. This may be because the addition of glucose increases the metabolic capacity of microorganisms, resulting in a decrease in the pH of the system to varying degrees; the suitable pH of the fermentation environment promotes microbial growth, metabolism, and enzyme activity. The pH of the okara culture medium with 0.5% glucose added is maintained at around 5, which is also the optimal pH for β-glucosidase. Therefore, when 0.5% glucose is added, the activity of β-glucosidase is the highest.

[0044] Example 3 β -Optimization experiment of enzymatic hydrolysis of soybean isoflavones by glucosidase, as follows: 1. Preparation of sample and reference solution Accurately weigh soybean isoflavones to prepare a solution; weigh daidzein (abbreviated as De) and genistein to prepare a standard solution. Add 0.1, 0.5, 1, 2, 5, 10, 25, 50 and 100 U / mg respectively.β -glucosidase was used to determine the optimal amount of enzyme for enzymatic hydrolysis of soybean isoflavones; the enzymatic reaction was carried out at 15, 30, 60, 120 and 180 min to determine the optimal enzymatic hydrolysis time. In the subsequent experimental results, soybean isoflavones before enzymatic hydrolysis were expressed as ISO, and the soybean isoflavone products after enzymatic hydrolysis were expressed as ISO-E.

[0045] 2. TLC thin layer test Use GF254 thin layer plate for qualitative and quantitative analysis. Activate the GF254 thin layer, spot 5 μL of sample, put the thin layer plate into the chromatography cylinder, expand it upward for 12 cm, and observe the test results in a UV analyzer at a wavelength of 254 nm.

[0046] In the early experiments, the β -glucosidase and its enzymatic properties were studied. ISO was enzymatically hydrolyzed at the optimal enzymatic hydrolysis temperature and pH conditions. The TLC spectrum results showed that the contents of daidzein and genistein in the ISO-E group were increased compared with those in the ISO group, thus confirming that the enzyme has the ability to hydrolyze the substrate ISO.

[0047] Effect of enzyme addition on enzymatic hydrolysis of soy isoflavones: The aglycone contents of ISO and ISO-E were studied when the soy isoflavone substrate concentration was 5 mg / mL, the hydrolysis temperature was 60°C, the reaction solution pH was 5.0, the shaking reaction time was 30 min, and the enzyme addition amounts were 0.1, 0.5, 1, 2, 5, 10, 25, 50 and 100 U / mg, respectively. The control group was taken without enzyme addition. Figure 4 The TLC spectra of ISO and ISO-E (different enzyme addition amounts) show that the De content increases with the increase of enzyme addition in the reaction system, and the content of conjugated isoflavones decreases with the increase of enzyme addition. When the enzyme addition amount is 5U / mg, the aglycone content increases significantly (the TLC spectra imprint is obviously deepened), so the enzyme addition amount of 5U / mg is selected for subsequent experiments.

[0048] The effect of enzymatic hydrolysis time on the enzymatic hydrolysis of soybean isoflavones: When the enzyme dosage is 5U / mg, the temperature is 60℃, and the pH is 5, the TLC spectrum of soybean isoflavone glycoside conversion obtained at different reaction times is as follows: Figure 5 The relationship between the aglycone content and the reaction time showed a positive correlation. After 60 min of reaction, the change tended to be gentle with the extension of time, and the content of bound glycosides decreased significantly (the thin layer chromatogram imprint became significantly lighter), indicating that the enzymatic reaction had reached a balanced state. Considering efficiency and cost, 60 min was selected as the optimal reaction time for enzymatic hydrolysis.

[0049] Example 4 Activity determination of soybean isoflavones before and after enzymatic hydrolysis, the method and results are as follows: 1. Hypoglycemic activity (1) α -Glucosidase inhibition ability The method of Chen et al. was slightly modified to determine the ISO samples before and after enzymatic hydrolysis. α -Glucosidase inhibition ability. The absorbance was measured at 405 nm and the inhibition percentage was calculated by the following formula:

[0050] Wherein: A1 is the glucose content of the solution after the reaction; A2 is the glucose content of the control group.

[0051] (2) Glucose adsorption capacity The glucose adsorption capacity of ISO samples before and after enzymatic hydrolysis was determined by referring to the method of Wu et al. with slight modifications. The glucose adsorption capacity of the samples was determined at 540 nm and the calculation formula was as follows:

[0052] Where: C1 represents the initial glucose concentration (mmol / L); C2 represents the glucose concentration after adsorption (mmol / L); V represents the volume of glucose solution (L); M represents the sample mass (g).

[0053] (3) α -Amylase inhibition ability The ISO samples before and after enzymatic hydrolysis were determined by slightly modifying the method of Tian et al. α -Amylase inhibition ability. The absorbance was measured at a wavelength of 540nm, and the α-amylase inhibition rate was calculated as follows:

[0054] Wherein: A1 is the glucose content of the solution after the reaction; A2 is the glucose content of the control group.

[0055] result: Figure 6 The results of A~B show that with the increase of ISO and ISO-E sample concentrations, α- The inhibition degree of glucosidase and α-amylase increased continuously. When ISO-E was 10mg / mL, the inhibition rate of ISO-E exceeded 40%. ISO-E had a higher blood sugar lowering ability than ISO. α -Glucosidase and α -Amylase can decompose polysaccharides and starch, inhibit the activity of the two enzymes, slow down the digestion and absorption of sugar in the intestine, and achieve the effect of lowering blood sugar. And as the concentration of glucose increases, the adsorption capacity of glucose is also continuously increased. When the concentration is 200 mmol / mL, the adsorption capacity reaches 1.5 mmol / g ( Figure 6The experimental results show that although ISO and ISO-E have the effect of lowering blood sugar, the extent of lowering blood sugar is not large. Therefore, we believe that the blood sugar lowering effect of soy isoflavones can play a health-care role.

[0056] 2. Cholesterol-lowering activity (1) Cholesterol binding capacity determination The cholesterol adsorption capacity of ISO samples before and after enzymatic hydrolysis was determined by referring to the method of Lindane et al. with slight modifications. The absorbance was measured at a wavelength of 550 nm using the o-phthalaldehyde method. The calculation formula is as follows:

[0057] Where: M represents the mass of the constant weight sample (g); M1 represents the cholesterol content in the supernatant after centrifugation (mg); M2 represents the cholesterol content in the egg yolk emulsion before adsorption (mg).

[0058] (2) Determination of sodium cholate adsorption capacity The sodium cholate adsorption capacity of ISO samples before and after enzymatic hydrolysis was determined by referring to the furfural colorimetric method of Lindane et al. with slight modifications.

[0059] The absorbance was measured at 620 nm and the calculation formula was as follows:

[0060] Where: M represents the mass of the constant weight sample (g); M1 represents the sodium cholate content in the supernatant after adsorption (mg); M2 represents the sodium cholate content before adsorption (mg).

[0061] (3) Lipase inhibition ability The lipase inhibition capacity of ISO samples before and after enzymatic hydrolysis was determined by slightly modifying the method of Truong et al. The calculation formula is as follows:

[0062] Where: A1 is the buffer solution replacing the sample; A2 is the sample group.

[0063] Results: Ge and De are the main active substances in soybeans and soybean foods. In recent years, many in vitro experiments have shown that Ge and De in ISO can enhance energy consumption and achieve the purpose of lowering blood lipids by enhancing the metabolism of fatty acids in liver cells. The adsorption capacity of ISO and ISO-E for cholesterol is shown in Figure 2. Figure 7As shown in the figure, the results show that the adsorption capacity of ISO at pH 2 reaches 4.637 mg / g, the adsorption capacity of ISO-E reaches 8.890 mg / g, the adsorption capacity of ISO at pH 7 reaches 5.191 mg / g, and the adsorption capacity of ISO-E reaches 10.145 mg / g; the adsorption capacity of ISO-E for sodium cholate is 0.671~0.786 mg / g higher than that of ISO. Next, the inhibition of ISO and ISO-E on lipase was compared. The inhibition rate of lipase was more than 60% at the sample concentration of 2 mg / mL, and more than 80% at the concentration of 4 mg / mL. When the concentration of ISO-E was 6 mg / mL, the inhibition rate of lipase reached more than 90%, and the inhibition capacity of ISO-E was significantly higher than that of ISO. The above studies show that soy isoflavones have a certain effect on lowering blood lipids, which can provide a new idea for the research and development of blood lipid-lowering health products.

[0064] 3. Antioxidant Activity (1) Determination of DPPH free radical scavenging ability Referring to Sun's method and modifying it, DPPH standard stock solution was prepared, ascorbic acid was used as a control, and the absorbance was measured at a wavelength of 517nm. The calculation formula is as follows:

[0065] Wherein: A0 represents 100 μL 0.2 mmol / L DPPH solution and 100 μL deionized water; A1 represents 100 μL 0.2 mmol / L DPPH solution and 100 μL sample solution; A2 represents 100 μL anhydrous ethanol and 100 μL sample solution.

[0066] (2) Determination of ABTS free radical scavenging ability The method of Konyalioglu was modified with ascorbic acid as the control, and the absorbance was measured at a wavelength of 734 nm. The calculation formula is as follows:

[0067] Wherein: A0 is 170 μL of ABTS stock solution and 30 μL of deionized water; A1 is 170 μL of ABTS stock solution and 30 μL of sample solution; A2 is 170 μL of deionized water and 30 μL of sample solution.

[0068] (3) O2 - Determination of free radical scavenging ability Refer to Tang's method and modify it, use HCl (10mmol / L) as blank control, ascorbic acid as positive control, and measure the absorbance at 325nm wavelength. The calculation formula is as follows:

[0069] Wherein: A0 is HCl and 180 μL Tris-HCl buffer; A1 is sample solution and 180 μL Tris-HCl buffer.

[0070] (4) Hydroxyl free radical scavenging ability Refer to Chen's method and modify it, use ascorbic acid as the control, and measure the absorbance at a wavelength of 510 nm. The calculation formula is as follows:

[0071] Wherein: A0 represents FeSO4 solution, H2O2 solution, deionized water and salicylic acid solution (all 1 mL); A1 represents sample solution, FeSO4 solution, salicylic acid solution and H2O2 solution; A2 represents sample solution, FeSO4 solution, 50% ethanol solution and H2O2 solution.

[0072] (5) Fe 2+ Determination of chelating capacity Refer to Lin's method and modify it to measure the absorbance at a wavelength of 562nm. The calculation formula is as follows:

[0073] Wherein: A0 is deionized water, 2.5 μL ferrous chloride solution, 10 μL ferrozine solution and 137.5 μL deionized water; A1 is sample solution, 2.5 μL ferrous chloride solution, 10 μL ferrozine solution and 137.5 μL deionized water; A2 is sample solution, 10 μL ferrozine solution and 50 μL deionized water.

[0074] (6) Determination of total reducing power Refer to Mohammed's method and modify it, and measure the absorbance at a wavelength of 700nm. The absorbance value represents the total reducing power of the sample.

[0075] Results: The biotransformation products of soybean isoflavones can protect human cells from oxidative stress damage. Normally, when antioxidants are added to DPPH-ethanol solution, the color of the solution changes from dark purple to light red, and its OD 517 nm decreases accordingly, and the scavenging effect of the sample on DPPH free radicals can be studied based on the absorbance value. C The scavenging ability of ISO and ISO-E on DPPH free radicals increased with the increase of concentration, and ISO-E was significantly higher than ISO ( Figure 8 In A). In addition, DPPH free radicals are determined by H transfer of reducing agents. ABTS free radical scavenging test is widely used in the determination of antioxidant capacity of natural products. After adding antioxidants, the blue-green color of ABTS solvent solution becomes lighter. The lower the absorbance value, the stronger the antioxidant capacity. Figure 8 As shown in B, the positive control V C The results of the scavenging ability of ISO and ISO-E on ABTS free radicals were similar to those of DPPH.

[0076] Hydroxyl free radicals are one of the most toxic, reactive and harmful indicators to organisms. Hydroxyl free radicals can directly or indirectly cause oxidative damage to biological macromolecules such as proteins and nucleic acids, and even cause pathological changes and death of the organism. ISO-E has a strong scavenging ability in hydroxyl free radicals. At a concentration of 0.1 mg / mL, the scavenging ability reaches more than 75% and is greater than V C , the removal capacity of ISO-E increased with the increasing concentration, and ISO-E was significantly higher than ISO ( Figure 8 (C) Excess O2 - It can damage cell DNA, easily lead to lipid peroxidation, and have pathogenic effects on the body. - The scavenging ability is of great significance. When the concentration of ISO-E in O2-free radicals is 0.25mg / mL, its scavenging ability exceeds that of the positive control group. When the concentration reaches 0.5mg / mL, its scavenging ability exceeds 50%. Similar trends were also found in the tests of metal ion chelation ability and total reducing power. When the concentration of ISO-E is 1.0mg / mL, the metal ion chelation ability is more than 40%, which is more than 50% of ISO; the total reducing power reaches 0.63, which is 0.297 higher than ISO. The antioxidant capacity of ISO-E at different concentrations is higher than that of ISO, so β The ability of -glucosidase to hydrolyze ISO was again demonstrated.

[0077] Example 4 Toxicity test of soybean isoflavone aglycones, as follows: In order to determine the optimal sample intervention concentration, the MTT method was used to determine the cytotoxic effects of ISO and ISO-E on HepG2 cells. When the concentration of ISO and ISO-E was in the range of 0-100 µg / mL, the cell survival rate was above 100%. With the increase of ISO and ISO-E concentrations, the cell survival rate decreased. At a concentration of 500 µg / mL, the cell survival rate was above 80%. Based on the above considerations, 100 µg / mL ISO and ISO-E samples were selected as the sample concentration for subsequent experiments.

[0078] 1. Establishment of H2O2-induced oxidative stress injury model in HepG2 cells H2O2 can cause cells to rapidly undergo oxidative stress response and is currently widely used to establish a cell oxidative stress model. In this study, H2O2 was used to establish a model of oxidative stress injury in HepG2 cells.

[0079] After the cells were confluent, the culture medium was removed and replaced with serum-free culture medium containing different concentrations of H2O2 for 4 hours. The culture medium was removed, 100 µL of MTT solution was added, MTT was removed after incubation, 150 µL of DMSO solution was added, and the absorbance was read at 490 nm using a microplate reader after mixing. The results showed that within the concentration range of 0~6mmol / L, the cell survival rate decreased with the increase of H2O2 concentration and approached 40%. The cell survival rates when the H2O2 concentrations were 2mmol / L, 4mmol / L and 6mmol / L were 51.78%, 48.86% and 42.41%, respectively. The half inhibition concentration of 4mmol / L H2O2 was selected to establish the model.

[0080] 2. Determination of the protective effect of ISO and ISO-E on cells damaged by oxidative stress After the cells were confluent, the culture medium was removed and the cells were cultured with serum-free culture medium containing a certain concentration of ISO and ISO-E for 20 h to protect the cells. The culture medium was discarded and serum-free culture medium containing a certain concentration of H2O2 was added. The intervention was continued for 4 h, the culture medium was aspirated, and the cell survival rate was determined using the MTT method.

[0081] Compared with the cell survival rate of the control group, the model group was 48.92%, proving that the HepG2 cell oxidative stress injury model was successfully established using 4mmol / L H2O2 (e.g. Fig. 9 The results show that ISO-E has a stronger protective effect on HepG2 cells than ISO, and reduces the oxidative stress damage of H2O2 to HepG2 cells.

[0082] 3. Determination of intracellular reactive oxygen species (ROS) content The accumulation of ROS is a direct response to oxidative stress damage in the body. Next, the antioxidant activity of ISO and ISO-E was studied by measuring the intracellular ROS level. The determination of intracellular reactive oxygen ROS content was carried out using a reactive oxygen species kit. HepG2 cells in the logarithmic growth phase were inoculated in a 24-well plate. After the cells were fully contaminated, the culture medium was removed. The cells were first protected with a serum-free culture medium containing the sample, and then a serum-free culture medium containing a certain concentration of H2O2 was added, and a DCFH-DA solution diluted in proportion was added. A small amount of trypsin was added to digest the cells, and the fluorescence intensity was measured under the conditions of an emission wavelength of 525nm and an excitation wavelength of 488nm, and then the relative fluorescence intensity was calculated. The formula for calculating the relative fluorescence intensity is as follows:

[0083] Where: FL0 is the fluorescence intensity of the control group; FL1 is the fluorescence intensity of different sample treatment groups.

[0084] like Fig.10 As shown in the figure, the relative fluorescence intensity of the model group increased by 2.48 times compared with the control group, indicating that a large amount of ROS was generated in the cells, causing oxidative stress damage. After intervention with ISO and ISO-E samples, both showed the effect of inhibiting ROS generation. The relative fluorescence intensity of the model group was 1.77 and 2.14 times that of ISO and ISO-E, respectively.

[0085] 4. Determination of intracellular oxidative stress factor levels During the process of oxidative stress in the body, various oxidative stress factors are also changing. SOD (superoxide dismutase) can generate O2 and H2O2 after oxygen free radicals. H2O2 can harm the body. At this time, CAT (catalase) is needed to decompose it into O2 and H2O. CAT is one of the important enzymes in the antioxidant defense system. MDA (malondialdehyde) is the end product of lipid peroxidation and is one of the indicators for evaluating peroxidative damage in the body. Therefore, by measuring the activity of SOD, CAT and the content of MDA, analyzing the changes in the body's oxidative stress factors, and comparing the antioxidant effects of ISO and ISO-E, it can be explained that β- The enzymatic hydrolysis effect of glucosidase.

[0086] After the cells are fully confluent, the culture medium is removed, and the cells are first cultured with a serum-free medium containing ISO and ISO-E for 20 hours to protect the cells, and then a serum-free medium containing a certain concentration of H2O2 is added, and the cells are washed with PBS and resuspended, and the cells are broken and the cell breaking fluid is collected. The determination method of oxidative stress factors such as CAT, SOD, and MDA refers to the instructions of the kit.

[0087] The results are as follows Fig.11 As shown, in the comparison of the three indicators between the model group and the control group, CAT activity decreased by 60.23%, SOD activity decreased by 58.95%, and MDA content increased by 2.72 times, which shows that the HepG2 oxidative stress model was established and the model group produced a large amount of oxidative stress. Next, compared with the model group, after the intervention of ISO and ISO-E samples, SOD activity increased significantly, increasing by 1.67 and 2.11 times, respectively. Fig.11 In B, the CAT activity of ISO and ISO-E increased by 1.90 and 2.28 times, respectively. Fig.11 In C, both ISO and ISO-E greatly reduced the MDA content, which was 2.81 and 4.33 respectively. It can be seen that both ISO and ISO-E have antioxidant activity, and ISO-E is stronger than ISO. This can well explain β -Glucosidase converts isoflavones into free forms, which exert stronger biological activity.

[0088] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps described in the disclosure of the present invention can be performed in parallel, sequentially or in different orders, as long as the desired results of the technical solution disclosed in the present invention can be achieved, and this document does not limit this.

[0089] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis, characterized in that: The steps include: S1. Inoculate Kluyveromyces marxianus into a basic culture medium and culture it on a shaking table; after culture, centrifuge, wash, and resuspend to obtain a bacterial suspension as a fermentation agent; S2. The fermentation agent was inoculated into the okara composite medium at a mass percentage of 0.1-3% for shaking fermentation for 48-96 hours at a temperature of 35-40°C and a rotation speed of 100-150 r / min; the fermentation broth was centrifuged at 0-4°C, and the supernatant was collected to obtain a crude β-glucosidase enzyme solution; S3. The crude β-glucosidase enzyme solution was sequentially subjected to ammonium sulfate precipitation, ion exchange chromatography and two dextran gel chromatography to obtain a purified β-glucosidase solution; S4. Weigh the prepared soy isoflavone solution, and use purified β-glucosidase solution to perform enzymatic reaction on the soy isoflavones for 60 to 180 minutes to obtain soy isoflavone aglycones.

2. The method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis according to claim 1, characterized in that: The okara composite culture medium in step S2 includes fresh okara and YPD culture medium; the moisture content of the fresh okara is 80-99%; and the amount of glucose added to the okara composite culture medium is 0.25-0.75%.

3. The method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis according to claim 2, characterized in that: The amount of glucose added in step S2 is 0.5%; and the shaking fermentation time is 72h.

4. The method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis according to claim 3, characterized in that: The components of the basic culture medium are: 8 g / L beef extract powder, 10 g / L peptone, 4 g / L yeast extract powder, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1 g / L Tween 80, 1000 mL deionized water, pH 5.7±0.

2.

5. The method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis according to claim 4, characterized in that: In step S4, the amount of β-glucosidase added is 5-50 U / mg, the reaction temperature is 55-65° C., and the pH value is adjusted to 4.5-5.

5.

6. The method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis according to claim 5, characterized in that: In step S4, the amount of β-glucosidase added is 5 U / mg, the reaction temperature is 60° C., and the pH value is adjusted to 5.

7. The method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis according to claim 6, characterized in that: The step S3 includes the following sub-steps: S301. ammonium sulfate graded salting out: adding ammonium sulfate to the crude enzyme solution to obtain a solution with a saturation of 20%; salting out to remove the precipitated enzyme protein; continuing to add ammonium sulfate to obtain a solution with a saturation of 60%; adjusting the pH value of the salting out to 3.0-7.0, and keeping it still for 4-24 hours; collecting the enzyme protein precipitate obtained by salting out by centrifugation; re-dissolving the precipitate with a buffer solution, dialyzing and desalting, and obtaining an enzyme solution purified by ammonium sulfate; S302. Ion exchange chromatography: The enzyme solution after ammonium sulfate purification is concentrated; DEAE-52 anion exchange column material is selected and treated, loaded on the column, loaded, eluted with NaCl solution, and the eluate A is collected; S303. First dextran gel chromatography: concentrate the eluate A; load the eluate onto a dextran G-100 gel column, and collect the eluate B; S304. Second dextran gel chromatography: concentrate the eluate B; load it onto a dextran G-50 gel column, and collect the eluate, which is the purified β-glucosidase solution.

8. The method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis according to claim 7, characterized in that: Polyethylene glycol 20000 is used during concentration in step S3; the diameter-to-height ratio of the dextran G-100 gel column in step S303 is 1:20, the flow rate is 0.5 mL / min, and the sample loading amount is 1.5%; the diameter-to-height ratio of the dextran G-50 gel column in step S304 is 1:25, the flow rate is 0.25 mL / min, and the sample loading amount is 1.75%.

9. The method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis according to claim 8, characterized in that: The deposit number of the Kluyveromyces marxianus is CGMCC No.13907.

10. The method for preparing soybean isoflavone aglycones based on enzymatic hydrolysis according to claim 9, characterized in that: In step S1, before inoculating the Kluyveromyces marxianus into the basic culture medium, the strain is activated; the shaking culture temperature is 25-35°C, the time is 10-15h; the concentration of the bacterial suspension is 10 7 cfu / mL.

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