A composite enzyme and application thereof

Abstract

The application discloses a kind of compound enzyme and its application, belong to the field of biotechnology.The compound enzyme provided by the application includes: pectinase 5-15 parts by weight, glucose glycosidase 5-15 parts, glycerol 2-8 parts and water 15-20 parts, the compound enzyme also includes phytase 2-6 parts, lactic acid dehydrogenase 1-4 parts.The compound enzyme provided by the application can significantly improve the extraction rate of soybean isoflavones, and the content of isoflavones is significantly improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a compound enzyme and application thereof. BACKGROUND

[0002] Soy isoflavones are phytochemicals belonging to the flavonoid family, which are mainly found in legumes, with soybeans having the highest content, 0.1%-0.5%. Soy isoflavones are mainly 3-benzopyrone compounds, and there are 12 natural soy isoflavones in soybeans, which can be divided into three categories: daidzin, genistin, and glycitein. Each category has four forms: free, glucoside, acetylglucoside, and malonylglucoside. The free form accounts for 2%-3% of the total, including genistein, daidzein, and glycitein. The combined form accounts for 97%-98% of the total, mainly in the form of daidzin, genistin, malonylgenistin, and malonyldaidzin, accounting for about 95% of the total. Planting environment, processing method, and genetic factors have certain effects on the content and composition of soy isoflavones, which are manifested as differences in the total amount of isoflavones and the proportion of each component in different soybean varieties. Prevention of cardiovascular disease: epidemiological surveys have found that people who regularly consume soy products can delay the onset of arteriosclerosis, reduce total blood cholesterol concentration, and reduce the incidence of heart disease. The mechanism of action of soy isoflavones in cardiovascular disease is diverse, and the more mature mechanisms include antioxidant effects, receptor regulation, inhibition of vascular smooth muscle cell proliferation, and anti-thrombosis effects. Soy isoflavones can inhibit tyrosine kinase and reduce tyrosine protein phosphorylation in platelets, leading to reduced platelet activity and reduced deposition and aggregation on the vascular wall, preventing thrombosis associated with systemic and atherosclerosis. Soy isoflavones significantly inhibit the increase in plasma triacylglycerol levels caused by high-fat feed in rats, and have a significant antagonistic effect on the increase in body peroxide levels caused by high-fat feed. The main effects are to reduce free radical levels in the liver and myocardium, increase superoxide dismutase activity in the liver, and increase glutathione peroxidase activity in the liver and myocardium, and reduce the content of total antioxidant products in serum, liver, myocardium, and aorta. Prevention of osteoporosis in women: people are looking for a new drug to treat osteoporosis, and soy isoflavones are a highly potential substance with estrogenic effects without the side effects of estrogen. For those with low estrogen levels, soy isoflavones exhibit weak estrogenic effects, bind to estrogen receptors in osteoblasts, enhance osteoblast activity, promote bone matrix production, secretion, and bone mineralization, and can prevent the occurrence of osteoporosis. Research results show that soy protein with high and large doses of soy isoflavones can increase the bone density of the lumbar spine in subjects. Soy isoflavones and diabetes: the level of antioxidant enzymes in pancreatic beta cells not only affects the ability of these cells to resist free radical damage, but also is related to insulin release. Some studies have found that soy isoflavones can inhibit sugar absorption in the small intestine and better regulate glucose metabolism balance when used to intervene in diabetes. The bacterial metabolite equol, which is formed from G, D, and D, can prevent the low-density lipoprotein lipid peroxidation reaction induced by sugar in humans in vitro. Soy isoflavones can inhibit sugar absorption in the rabbit small intestinal mucosa in vitro.In addition, soy isoflavones have weak estrogen-like effects on the metabolic disorders of diabetic patients also have certain regulation function. Soy isoflavones and kidney disease: In recent years, it is found that soy isoflavones have a beneficial effect on the renal function of patients with kidney disease, which is in conflict with the traditional kidney disease of avoiding soy products, which is difficult to explain. Many kidney diseases such as nephrotic syndrome can be complicated with hyperlipidemia, and the most common explanation is that low albuminemia stimulates the compensatory synthesis of liver lipid B to increase, and then produces too much LDL, causing hyperlipidemia. Many years ago, there was a nephrotoxicity theory that high blood lipids would promote the progressive aggravation of glomerular disease, which has been confirmed. Therefore, reducing blood lipids to protect kidney function is an important part of kidney disease treatment, and soy isoflavones can protect kidney function by reducing blood lipids. There are also reports that soy isoflavones exhibit diuretic bioactivity in vitro, can inhibit Na. + K + 2Cl - Transmembrane transport, ensure sufficient blood flow, diuretic effect, can relax the renal blood vessels of the isolated kidney. Therefore, soy isoflavones can also prevent and treat kidney disease.

[0003] The current method for extracting flavonoids is organic solvent extraction, ultrasonic extraction, semi-bionic extraction and the like. These methods are simple to operate, and to some extent, the extraction amount of active substances is improved, but it is far from meeting the demand, and at the same time, it also causes waste of raw materials. For example, the "complex enzyme and its preparation method" provided by Chinese patent CN109913434A uses a complex enzyme composed of cellobiohydrolase, xylanase, cellulase, pectinase, glycerol and sodium chloride to promote the release of flavonoids in Senecio scandens Buchen, and shorten the extraction time; for example, the "production process for extracting eucommia ulmoides flavones from eucommia ulmoides leaves by using a complex enzyme combined with ultrasonic" provided by Chinese patent CN104688801B uses a complex enzyme composed of cellulase and pectinase, and combines ultrasonic to extract flavones in eucommia ulmoides. The method is simple and easy to operate. However, due to the complexity of the components and content of traditional Chinese medicinal materials, the yield of flavones prepared by the existing method is not ideal, and the extraction cost is high. Therefore, it is necessary to explore a complex enzyme capable of efficiently extracting flavones. SUMMARY

[0004] In view of the problems in the prior art related to the flavone extraction process, the present application provides a complex enzyme and its application.

[0005] In one aspect, the present application provides a complex enzyme, which comprises: pectinase 5-15 parts by weight, glucose glycosidase 5-15 parts by weight, glycerol 2-8 parts by weight and water 15-20 parts by weight.

[0006] The complex enzyme further comprises: phytase 2-6 parts by weight.

[0007] The complex enzyme also comprises: lactic acid dehydrogenase 1-4 parts by weight.

[0008] Preferably, the complex enzyme comprises: pectinase 6-12 parts by weight, glucose glycosidase 6-12 parts by weight, glycerol 3-6 parts by weight, phytase 3-5 parts by weight, lactic acid dehydrogenase 2-3 parts by weight and water 16-18 parts by weight.

[0009] Most preferably, the complex enzyme comprises: pectinase 8 parts by weight, glucose glycosidase 10 parts by weight, glycerol 5 parts by weight, phytase 4 parts by weight, lactic acid dehydrogenase 2 parts by weight and water 15 parts by weight.

[0010] The complex enzyme is prepared by mixing raw material powders according to the formula and stirring for 30 minutes.

[0011] In another aspect, the present application also provides a use of the above complex enzyme in the process of extracting flavones from soybeans, comprising the following steps:

[0012] S1: taking 20-mesh soybean powder made of soybeans, adding an extracting agent composed of methanol and 95% ethanol, refluxing and extracting for 30 minutes, recovering the methanol and ethanol to the maximum extent, adding deionized water to obtain a pretreated liquid; preferably, the volume ratio of methanol to 95% ethanol in the extracting agent is 2:8, and the mass-volume ratio of the soybean powder to the extracting agent is 1:10-1:15; most preferably, the mass-volume ratio of the soybean powder to the extracting agent is 1:12, and the weight of the deionized water added is 0.15-0.25 times the weight of the soybean powder.

[0013] S2: adding the complex enzyme to the pretreated liquid obtained in S1 to obtain an enzymatic hydrolysis liquid; preferably, the weight of the complex enzyme added is 4-8‰ of the weight of the soybean powder, the enzymatic hydrolysis temperature is 37-45°C, and the enzymatic hydrolysis time is 48-72 hours; most preferably, the weight of the complex enzyme added is 6‰ of the weight of the soybean powder, the enzymatic hydrolysis temperature is 40°C, and the enzymatic hydrolysis time is 60 hours.

[0014] S3: filtering the enzymatic hydrolysis liquid obtained in S2, centrifuging, collecting the supernatant, and concentrating and drying to obtain an isoflavone extract.

[0015] The present application has the following advantages:

[0016] (1) The present application provides a complex enzyme prepared from pectinase, glucose glycosidase, phytase and lactic acid dehydrogenase, which promotes the release of effective components in soybeans and effectively improves the content of effective components in soybeans.

[0017] (2) The present application first adds phytase and lactic acid dehydrogenase to the complex enzyme, and the addition of these two enzymes significantly promotes the extraction of flavones in soybeans, thereby significantly improving the content of flavones. DETAILED DESCRIPTION

[0018] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the following specific embodiments are further described in the present application, but the following embodiments are only preferred embodiments of the present application, not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0019] The experimental materials, reagents and methods used in the examples 1-5 and comparative examples 1-5 in the specification are as follows:

[0020] 1. Experimental materials, reagents and instruments

[0021] Materials: pectinase, glucose glycosidase, phytase, lactate dehydrogenase, glycerol, water. Among them, pectinase, phytase can be obtained by fermentation of shanghai brewing 336-2 aspergillus, glucose glycosidase can be obtained by fermentation of INVSc1 saccharomyces cerevisiae, lactate dehydrogenase can be obtained by fermentation of lactic acid bacteria.

[0022] Reagents: rutin standard, NaNO2 solution, 30% ethanol

[0023] Instruments: analytical balance, spectrophotometer, water bath, pipette, test tube, etc.

[0024] Experimental method

[0025] 2.1 Preparation method of composite enzyme and its application

[0026] The composite enzyme is prepared by the following steps: mixing and stirring the raw material powder according to the formula amount for 30 minutes to obtain the composite enzyme.

[0027] The above composite enzyme is applied in the process of extracting soybean flavonoids, including the following steps:

[0028] S1: reflux extraction for 30 minutes, recover methanol and ethanol to the end, add deionized water (the weight is 0.2 times the weight of soybean powder), get pretreatment liquid;

[0029] S2: add composite enzyme to the pretreatment liquid obtained in S1 and stand for enzymolysis to obtain enzyme hydrolysis liquid;

[0030] S3: filter the enzyme hydrolysis liquid obtained in S2, centrifuge, collect the supernatant, concentrate and dry to obtain isoflavone extract.

[0031] Detection of soybean isoflavone content

[0032] Chrom ODS-BP column (250 mm x 4.6 mm, 5 μm); mobile phase A: 0.1% acetic acid-water solution, mobile phase B: 0.1% acetic acid-acetonitrile solution, detection wavelength: 260 nm; flow rate: 1.0 mL / min; column temperature: 30°C; injection volume: 10 μL; the gradient elution program is shown in Table 1:

[0033]

[0034] The soy isoflavone standard stock solution was prepared as follows: 0.0050 g of each of six soy isoflavone standards (daidzin, glycitin, genistin, daidzein, glycitein, genistein) was precisely weighed into a 10 mL volumetric flask, dissolved with 70% ethanol and diluted to the calibration mark, to prepare a solution with a concentration of 500 μg / mL. The solution was stored at 0°C-4°C in the dark.

[0035] The soy isoflavone mixed standard solution was prepared as follows: 5 mL of each of the above six kinds of soy isoflavone standard stock solutions was transferred into a 50 mL volumetric flask, dissolved with 70% ethanol and diluted to the calibration mark, to prepare a soy isoflavone standard solution with a concentration of 50 μg / mL. 2 mL of each of the soy isoflavone standard solutions was transferred, mixed, and 1 mL of the mixed standard solution was filtered through a 0.45 μm filter for high performance liquid chromatography detection. The solution was stored at 0°C-4°C in the dark.

[0036] The standard working curve was prepared as follows: 0.0 μL, 50.0 μL, 100.0 μL, 200.0 μL, 300.0 μL, 1000.0 μL, and 2000.0 μL of the mixed standard solution was accurately pipetted into a 10 mL volumetric flask, respectively, and diluted to the calibration mark with 70% ethanol, and mixed uniformly, to prepare a series of soy isoflavone mixed standard working solutions with concentrations of 0.00 μg / mL, 0.25 μg / mL, 0.50 μg / mL, 1.00 μg / mL, 1.50 μg / mL, 5.00 μg / mL, and 10.00 μg / mL of each monomer. 10 μL of each sample was injected in turn, and the standard working curve was plotted with the concentration of each monomer of soy isoflavone as the abscissa and the peak area as the ordinate, to obtain the regression equation of each monomer of the six kinds of soy isoflavones.

[0037] Sample processing and determination: 5 g of isoflavone extract was dried at 50℃, and 70% ethanol was used as the solvent with a material-solvent ratio of 1:15 in an ultrasonic cleaner under the following conditions: temperature 70℃, time 40 min, and power 160 W. The residue was centrifuged at a speed of 7000 r / min for 20 min, and the supernatant was collected. The residue was extracted with ethanol again, and the two supernatants were combined and concentrated to 50 mL. 4-5 mL of the sample solution was centrifuged at a speed of 10000 r / min for 20 min, and filtered through a 0.22 μm filter. The content of isoflavones in the extract obtained by different methods was determined by HPLC, and calculated based on the contents of six isoflavone monomers, i.e., daidzin, glycitein, genistin, daidzein, glycitein, and genistein.

[0038] Examples and comparative examples

[0039]

[0040] Comparative example 1

[0041] In comparison with example 1, the complex enzyme component only lacks phytase and lactate dehydrogenase.

[0042] Comparative example 2

[0043] In comparison with example 1, the complex enzyme component only lacks phytase.

[0044] Comparative example 3

[0045] In comparison with example 1, the complex enzyme component only lacks lactate dehydrogenase.

[0046] Experimental results: see table 3

[0047]

[0048] In summary, the content of isoflavones obtained from 100 g of soybeans is greater than 15 mg when the isoflavones are extracted from soybeans using the complex enzyme provided in the present application. The present application can enrich isoflavones in soybeans, indicating that the complex enzyme of the present application can efficiently enrich isoflavones in soybeans.

[0049] The above description is only a preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification or equivalent change of the above embodiment based on the technical essence of the present application should also be considered as falling within the protection scope of the present application.

Claims

1. A complex enzyme for extracting isoflavones from soybeans, characterized by comprising, The complex enzyme consists of pectinase 5-15 parts by weight, glucose glycosidase 5-15 parts by weight, glycerol 2-8 parts by weight, water 15-20 parts by weight, phytase 2-6 parts by weight and lactate dehydrogenase 1-4 parts by weight.

2. The complex enzyme for extracting isoflavones from soybean according to claim 1, wherein The complex enzyme consists of pectinase 6-12 parts by weight, glucose glycosidase 6-12 parts by weight, glycerol 3-6 parts by weight, phytase 3-5 parts by weight, lactate dehydrogenase 2-3 parts by weight and water 16-18 parts by weight.

3. The complex enzyme for extracting isoflavones from soybean according to claim 2, wherein The complex enzyme consists of pectinase 8 parts by weight, glucose glycosidase 10 parts by weight, glycerol 5 parts by weight, phytase 4 parts by weight, lactate dehydrogenase 2 parts by weight and water 15 parts by weight.

4. The complex enzyme for extracting isoflavones from soybean according to any one of claims 1 to 3, characterized by, The preparation steps of the complex enzyme are as follows: the raw material powder is mixed according to the formula and stirred for 30 minutes to obtain the complex enzyme.

5. Use of the complex enzyme according to any one of claims 1 to 4 in the process of extracting isoflavones from soybeans, characterized in that: The method comprises the following steps: S1: 20 mesh soybean powder made of soybean is taken, an extracting agent composed of methanol and 95% ethanol is added, and reflux extraction is carried out for 30 minutes; methanol and ethanol are recovered to the fullest extent; deionized water is added to obtain a pretreatment liquid; S2: the pretreatment liquid obtained in S1 is added with a complex enzyme and is left to carry out enzymolysis to obtain an enzymolysis liquid; S3: the enzymolysis liquid obtained in S2 is filtered, centrifuged, and the supernatant is collected and concentrated and dried to obtain an isoflavone extract.

6. Use according to claim 5, characterized in that, The volume ratio of methanol and 95% ethanol in the extracting agent in S1 is 2:8, the mass-volume ratio of soybean powder to extracting agent is 1:10-1:15, and the weight of deionized water added is 0.15-0.25 times the weight of soybean powder.

7. Use according to claim 6, characterized in that, The weight of the complex enzyme in S2 is 4-8‰ of the weight of soybean powder.

8. Use according to claim 7, characterized in that, The enzymolysis temperature in S2 is 37-45℃, and the enzymolysis time is 48-72 hours.

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