A method for preparing a stevia extract

Abstract

The application discloses a preparation method of a stevia extract, and belongs to the technical field of plant extract preparation; the preparation method comprises the following steps: S1, crushing stevia raw materials, then soaking the stevia raw materials in water, adding a composite enzyme to perform enzymolysis, and preparing an enzymolysis solution; S2, performing flocculation treatment on the enzymolysis solution; the flocculating agent is a composite flocculating agent, and a crude extract solution is obtained; S3, performing gradient elution on the crude extract solution after adsorption by using a macroporous resin, and collecting eluate of a specific section; and S4, performing elution after adsorption of the eluate by using an aluminum oxide column, concentrating the eluate, and crystallizing; the preparation method only uses water and ethanol as solvents in the whole process, does not use toxic organic reagents, is simple to operate, and is very suitable for industrial mass production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant extracts, and particularly relates to a preparation method of a stevia extract. BACKGROUND

[0002] Stevioside is a kind of glycoside compound synthesized from tetracyclic diterpene in the leaves of plants such as Stevia rebaudiana Bertoni and Rubus suavissimus S. Lee, and has a sweetness of 150 to 300 times that of sucrose due to its close taste to sucrose. The stevia rebaudiana also contains polyphenols, flavonoids, quinic acid, caffeic acid, amino acids and fatty acids and their derivatives, and other impurities such as tannins, tannins, pigments and pectin that need to be removed.

[0003] For the extraction of stevioside, the related technology discloses the following extraction method, including the following steps: first, the stevia rebaudiana leaves are soaked in anhydrous ethanol for 1 h, dried, crushed, and sieved to obtain stevia rebaudiana leaf powder; then the stevia rebaudiana leaf powder is enzymatically extracted and extracted with diethyl ether, and then flocculated with ferric chloride and calcium hydroxide, and the pH is adjusted; then supercritical extraction, macroporous resin adsorption and elution are performed to prepare a crude stevioside product; the crude product is treated with activated alumina adsorption to prepare stevioside; the purity of the stevioside prepared by the method is above 93.6%, but the method needs to use diethyl ether and the purity is relatively low.

[0004] Therefore, the application provides a preparation method of a stevia rebaudiana extract, which prepares stevioside with high purity. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of a stevia rebaudiana extract to solve at least one aspect of the problems and defects in the background art.

[0006] Specifically, the present application discloses a preparation method of a stevia rebaudiana extract, comprising the following steps:

[0007] S1, crushing the stevia rebaudiana raw material and soaking it in water, adding a composite enzyme for enzymatic hydrolysis to prepare an enzymatic hydrolysate;

[0008] S2, flocculating the enzymatic hydrolysate; the flocculant is a composite flocculant, and a crude extract is obtained;

[0009] S3, adsorbing the crude extract with a macroporous resin and performing gradient elution, and collecting the eluate of a specific section;

[0010] S4, adsorbing the eluate with an alumina column and eluting, and crystallizing the eluate after concentration;

[0011] The complex enzyme is composed of amylase, cellulase and pectinase.

[0012] The gradient elution procedure is as follows:

[0013] First elution: water;

[0014] Second elution: 30-40% ethanol aqueous solution;

[0015] Third elution: 70-80% ethanol aqueous solution.

[0016] According to one of the technical solutions of the preparation method of the application, at least the following beneficial effects are achieved:

[0017] The preparation method of the application first uses complex enzyme to enzymatically treat the stevia extract, uses enzyme to change the cell wall of the stevia raw material, thereby changing the permeability of the cell wall of the stevia raw material, and further improves the extraction rate of the effective component (steviol glycoside).

[0018] The application uses a complex flocculant to treat the enzymatic hydrolysate, preliminarily removes the polyphenolic substances in the enzymatic hydrolysate, thereby improving the purity of the finally prepared stevia extract.

[0019] After the crude extract is adsorbed by a macroporous resin, the polar impurities are removed by water, and the polyphenolic impurities are further removed by a low-concentration ethanol aqueous solution (30-40% by volume), thereby realizing the enrichment of steviol glycoside in a high-concentration ethanol aqueous solution (70-80% by volume).

[0020] The preparation method of the application only uses water and ethanol as solvents, does not use toxic organic reagents, is simple to operate, and is very suitable for industrial mass production.

[0021] Finally, the eluate is subjected to adsorption and impurity removal treatment by an aluminum oxide column, thereby realizing further treatment of the polyphenolic impurities, and finally, high-purity stevia extract is prepared through crystallization treatment.

[0022] According to some embodiments of the application, the stevia extract is steviol glycoside.

[0023] According to some embodiments of the application, the stevia raw material is stevia leaf.

[0024] According to some embodiments of the application, the stevia leaf is crushed to prepare stevia leaf powder.

[0025] According to some embodiments of the application, the stevia leaf powder has a mesh number of 60-100.

[0026] The sweet stevia leaf powder is crushed to improve the contact effect of the sweet stevia leaf powder and the composite enzyme, thereby improving the enzymolysis efficiency.

[0027] According to some embodiments of the present application, the enzymolysis time is 120-140 min.

[0028] According to some embodiments of the present application, the enzymolysis temperature is 58-62℃.

[0029] According to some embodiments of the present application, the enzymolysis pH is 4.5-6.0.

[0030] According to some embodiments of the present application, the enzymolysis pH is 5.0-6.0.

[0031] Under certain conditions, temperature improvement is beneficial to improve the enzymolysis efficiency, but too high temperature will affect the enzyme activity, thereby affecting the extraction rate.

[0032] According to some embodiments of the present application, the mass ratio of the composite enzyme to the sweet stevia raw material is 0.6-0.8:100.

[0033] According to some embodiments of the present application, the enzymolysis process adopts ultrasonic assistance; the ultrasonic power in the ultrasonic assistance process is 200-300 W.

[0034] The thermal effect of ultrasonic waves improves the solvent's ability to dissolve effective components, the mechanical effect promotes the solution flow, enhances the mass transfer effect in the extraction process, and the cavitation effect destroys the cell wall of the sweet stevia raw material, accelerating the dissolution of effective components.

[0035] According to some embodiments of the present application, the composite enzyme is composed of the following mass fractions of preparation raw materials:

[0036] 10 parts of cellulase, 3-5 parts of amylase, and 1-3 parts of pectinase.

[0037] According to some embodiments of the present application, the enzyme treatment is followed by enzyme inactivation treatment.

[0038] According to some embodiments of the present application, the enzyme inactivation treatment temperature is 90-100℃.

[0039] According to some embodiments of the present application, the enzyme inactivation treatment time is 6-10 min.

[0040] According to some embodiments of the present application, the enzymolysis liquid in step S1 needs to be reduced pressure concentrated.

[0041] According to some embodiments of the present application, the reduced pressure concentration temperature is 30-50℃.

[0042] According to some embodiments of the present application, the pressure of the reduced pressure concentration is 30mmHg-100mmHg.

[0043] According to some embodiments of the present application, the solid content volume concentration of the reduced pressure concentration is 10%-15%.

[0044] According to some embodiments of the present application, the composite flocculant comprises the following preparation raw materials:

[0045] Activated carbon, chitosan and ferric sulfate.

[0046] According to some embodiments of the present application, the composite flocculant comprises the following preparation raw materials in weight percentage:

[0047] 100 parts of activated carbon, 0.1-0.3 parts of chitosan and 0.5-0.8 parts of ferric sulfate.

[0048] The surface of the composite flocculant is covered with a large amount of modification, the roughness is increased, and the specific surface area is larger, which is beneficial to the adsorption and removal of impurities.

[0049] According to some embodiments of the present application, the mass ratio of the composite flocculant to the stevia raw material is 0.1-0.2:100.

[0050] According to some embodiments of the present application, the activated carbon is subjected to oxidation treatment.

[0051] According to some embodiments of the present application, the activated carbon is subjected to oxidation treatment with potassium permanganate solution.

[0052] According to some embodiments of the present application, the mass-volume ratio of the activated carbon to the potassium permanganate solution is 1g:50mL-100mL.

[0053] According to some embodiments of the present application, the molar concentration of the potassium permanganate solution is 0.1mol / L-1mol / L.

[0054] According to some embodiments of the present application, the oxidation treatment time is 30min-40min.

[0055] According to some embodiments of the present application, the oxidation treatment temperature is 30℃-40℃.

[0056] According to some embodiments of the present application, the activated carbon is subjected to oxidation treatment to obtain oxidized activated carbon.

[0057] According to some embodiments of the present application, the preparation method of the composite flocculant comprises the following steps:

[0058] The oxidized activated carbon is prepared into an oxidized activated carbon dispersion liquid;

[0059] The oxidized activated carbon dispersion liquid, the chitosan solution and the ferric sulfate are mixed and reacted.

[0060] In the present application, the carboxyl group on the surface of the oxidized activated carbon, the chitosan and the ferric ion are used to construct an adsorption network, so as to realize the adsorption and removal of the polyphenol impurities.

[0061] According to some embodiments of the present application, the raw materials for preparing the chitosan solution include chitosan and acetic acid solution.

[0062] According to some embodiments of the present application, the mass-volume ratio of the chitosan and the acetic acid solution is 0.2g-0.5g:500mL.

[0063] According to some embodiments of the present application, the volume fraction of the acetic acid solution is 1.5%-2.5%.

[0064] According to some embodiments of the present application, the volume fraction of the acetic acid solution is 2%.

[0065] According to some embodiments of the present application, the reaction temperature is 65-75℃.

[0066] According to some embodiments of the present application, the reaction time is 2-3h.

[0067] According to some embodiments of the present application, after the reaction, solid-liquid separation is performed, and the solid phase is collected, washed, dried, ground and sieved.

[0068] According to some embodiments of the present application, the drying temperature is 60-70℃.

[0069] According to some embodiments of the present application, the drying time is 12-14h.

[0070] According to some embodiments of the present application, the macroporous resin is D101 resin.

[0071] The present application uses non-polar macroporous resin, which improves the adsorption effect of steviol glycosides, so as to separate steviol glycosides from other impurities, thereby improving the purity of steviol glycosides.

[0072] According to some embodiments of the present application, the amount of water used is 0.5-1.5BV.

[0073] According to some embodiments of the present application, the flow rate of water is 1-2BV / h.

[0074] According to some embodiments of the present application, the amount of the 30%-40% volume fraction of the aqueous ethanol solution used is 2-3BV.

[0075] According to some embodiments of the present application, the flow rate of the 30%-40% ethanol aqueous solution is 1-2 BV / h.

[0076] According to some embodiments of the present application, the amount of the 70%-80% ethanol aqueous solution is 2-3 BV.

[0077] According to some embodiments of the present application, the flow rate of the 70%-80% ethanol aqueous solution is 1-2 BV / h.

[0078] According to some embodiments of the present application, the alumina column is an acidic alumina column.

[0079] According to some embodiments of the present application, the eluent for elution is an acidic ethanol aqueous solution.

[0080] According to some embodiments of the present application, the pH of the acidic ethanol aqueous solution is 6-6.5.

[0081] According to some embodiments of the present application, the volume fraction of ethanol in the acidic ethanol aqueous solution is 90%-95%.

[0082] According to some embodiments of the present application, the flow rate of the acidic ethanol aqueous solution is 1-2 BV / h.

[0083] According to some embodiments of the present application, the amount of the acidic ethanol aqueous solution is 1-2 BV.

[0084] According to some embodiments of the present application, the crystallization temperature is -4-0℃.

[0085] According to some embodiments of the present application, the crystallization time is 8-16 h. DETAILED DESCRIPTION

[0086] The concept and the technical effects of the present application will be described clearly and completely in combination with the embodiments below, so as to fully understand the purposes, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0087] In the description of the present application, the description of the terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the exemplary description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in one or more embodiments or examples.

[0088] Unless otherwise specified, the specific conditions in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are conventional products that can be obtained by commercial purchase, unless otherwise specified.

[0089] The raw material of stevia selected in the embodiments of the present application is stevia leaf, and the mass content of steviol glycoside in the stevia leaf is 11.86% (tested by HPLC).

[0090] Pectinase: Xiasheng FFY-3605.

[0091] Amylase: Xiasheng FDY-2216, with an enzyme activity of 0.3 ten thousand u / g.

[0092] Cellulase: Xiasheng FDY-2243 (CEL-01 type), with an enzyme activity of 1.1 ten thousand u / g.

[0093] Activated carbon: Shandong Lujing Activated Carbon Co., Ltd., model LJHB-A.

[0094] Chitosan: CAS No. 969-33-5.

[0095] The parameters of liquid chromatography in the embodiments of the present application are as follows:

[0096] Chromatographic column: C18 column (250 mm x 4.6 mm, 5 μm);

[0097] Mobile phase: 0.002 mol / L phosphoric acid solution and acetonitrile in a volume ratio of 70:30;

[0098] Flow rate: 1.0 mL / min;

[0099] Column temperature: 30°C;

[0100] Wavelength: 210 nm;

[0101] Injection volume: 10 μL.

[0102] Example 1

[0103] The present embodiment is a preparation method of a stevia extract, which comprises the following steps:

[0104] S1, ultrasonic extraction:

[0105] 500 g of stevia raw material was ground to 80 mesh, 12 times the volume of citric acid solution (pH 5.0, i.e. the mass-volume ratio of stevia raw material to citric acid solution was 1 g: 12 mL) was added; ultrasonic-assisted complex enzyme (the mass ratio of complex enzyme to stevia raw material was 0.6: 100; the complex enzyme was composed of cellulase, amylase and pectinase, and the mass ratio of cellulase, amylase and pectinase was 10:5:2) was used for extraction (the temperature was 60°C), and after extraction, the enzyme was inactivated (the temperature for inactivating the enzyme was 100°C, and the time was 6 min), then solid-liquid separation was performed after inactivation of the enzyme, and the enzyme solution was collected.

[0106] The parameters of ultrasonic assistance were as follows:

[0107] The power of ultrasonic was 200 W, and the time of ultrasonic was 120 min.

[0108] S2, vacuum concentration:

[0109] The extraction solution prepared in step S1 was vacuum concentrated at 40°C and a pressure of 60 mmHg to a solid content concentration of 10%, and then solid-liquid separation was performed, and the concentrated solution was collected.

[0110] S3, flocculation:

[0111] The complex flocculant was added to the enzyme solution prepared in step S1 (the mass ratio of complex flocculant to stevia raw material in step S1 was 0.3: 100), and stirring was performed at 25°C for 60 min, then solid-liquid separation was performed, and the liquid phase was collected to prepare a crude extract.

[0112] The preparation method of the complex flocculant was as follows:

[0113] 100 g of activated carbon was added to an acidic potassium permanganate solution (the molar concentration was 0.5 mol / L, and the mass-volume ratio of activated carbon to acidic potassium permanganate solution was 1 g: 50 mL) for reaction (the reaction temperature was 40°C, and the reaction time was 40 min), then hydrogen peroxide solution (the mass concentration was 30%) was added until no gas bubbles were generated, then solid-liquid separation was performed, and the solid phase was washed to prepare oxidized activated carbon;

[0114] The oxidized activated carbon was uniformly dispersed in 500 mL of water by ultrasonic dispersion to prepare an oxidized activated carbon dispersion;

[0115] 0.2 g of chitosan was dissolved in 500 mL of an ice acetic acid solution with a volume fraction of 2% to prepare a chitosan solution;

[0116] The chitosan solution, the oxidized activated carbon dispersion liquid and ferric sulfate (0.8 g) were stirred uniformly, reacted for 2 h at 70 ℃, filtered, washed with water until neutral, and dried at 65 ℃ under vacuum for 12 h to obtain the composite flocculant.

[0117] S4, elution:

[0118] After the crude extract was adsorbed by the macroporous resin, 1 BV of pure water, 2 BV of 30% ethanol aqueous solution and 2 BV of 80% ethanol aqueous solution were used for elution in sequence, and the eluate of 80% ethanol aqueous solution was collected.

[0119] S5, passing through an acidic alumina column:

[0120] After the eluate obtained in step S4 was adsorbed by the acidic alumina column, 2 BV of 95% acidic ethanol aqueous solution (pH 6.5) was used for elution, and the eluate was collected.

[0121] Then, the eluate was crystallized at 0 ℃ for 12 h to obtain 56.35 g of the stevia extract.

[0122] The mass fraction of steviol glycosides in the stevia extract was 98.5% detected by HPLC.

[0123] Example 2

[0124] The present example is a preparation method of a stevia extract, which comprises the following steps:

[0125] S1, ultrasonic extraction:

[0126] The stevia raw material 500 g was crushed to 80 mesh, 12 times the volume of citric acid solution (pH 5.0, i.e. the mass-volume ratio of the stevia raw material to the citric acid solution was 1 g: 13 mL) was added, and the ultrasonic-assisted composite enzyme (the mass ratio of the composite enzyme to the stevia raw material was 0.6:100; the composite enzyme was composed of cellulase, amylase and pectinase, and the mass ratio of cellulase, amylase and pectinase was 10:5:2) was used for extraction (the temperature was 60 ℃), and after extraction, the enzyme was inactivated (the temperature for inactivation was 100 ℃, and the time was 6 min), and after inactivation, the solid-liquid separation was performed, and the enzyme solution was collected.

[0127] The parameters of the ultrasonic assistance were as follows:

[0128] The power of the ultrasonic was 200 W, and the time of the ultrasonic was 120 min.

[0129] S2, concentration under reduced pressure:

[0130] The extract obtained in step S1 was concentrated under reduced pressure at 40 ℃ and a pressure of 60 mmHg to a solid content concentration of 10%, the solid-liquid separation was performed, and the concentrated solution was collected.

[0131] S3, flocculation:

[0132] The composite flocculant was added to the enzymatic hydrolysate prepared in step S1 (the mass ratio of the composite flocculant to the stevia raw material in step S1 was 0.3:100), and stirred at 25°C for 60 min. The liquid phase was collected after solid-liquid separation to prepare a crude extract.

[0133] The preparation method of the composite flocculant was as follows:

[0134] 100 g of activated carbon was added to an acidic potassium permanganate solution (the molar concentration was 0.5 mol / L, and the mass-volume ratio of the activated carbon to the acidic potassium permanganate solution was 1 g:50 mL) for reaction (the reaction temperature was 40°C, and the reaction time was 40 min). After the reaction, hydrogen peroxide solution (the mass concentration was 30%) was added until no gas bubbles were generated. Then, solid-liquid separation was performed, and the solid phase was washed to prepare oxidized activated carbon.

[0135] The oxidized activated carbon was uniformly dispersed in 500 mL of water by ultrasonic dispersion to prepare an oxidized activated carbon dispersion.

[0136] 0.3 g of chitosan was dissolved in 500 mL of an ice acetic acid solution with a volume fraction of 2% to prepare a chitosan solution.

[0137] The chitosan solution, the oxidized activated carbon dispersion, and iron sulfate (0.5 g) were uniformly stirred, and reacted at 70°C for 2 h. Filtration was performed, and the product was washed with water until neutral. The product was dried at 65°C under vacuum for 12 h to prepare a composite flocculant.

[0138] S4, elution:

[0139] After the crude extract was adsorbed by a macroporous resin, 1 BV of pure water, 2.5 BV of an ethanol aqueous solution with a volume fraction of 30%, and 3 BV of an ethanol aqueous solution with a volume fraction of 80% were sequentially used for elution. The eluate of the ethanol aqueous solution with a volume fraction of 80% was collected.

[0140] S5, passing through an acidic alumina column:

[0141] After the eluate prepared in step S4 was adsorbed by an acidic alumina column, 1 BV of an acidic ethanol aqueous solution (pH 6) with a volume fraction of 95% was used for elution. The eluate was collected.

[0142] The eluate was subjected to crystallization treatment at 0°C for 12 h to obtain 55.78 g of a stevia extract.

[0143] HPLC detection showed that the mass fraction of stevioside in the stevia extract was 96.1%.

[0144] Example 3

[0145] The embodiment is a preparation method of a stevia extract, which comprises the following steps:

[0146] S1, ultrasonic extraction:

[0147] 500 g of stevia raw material is pulverized to 80 mesh, 12 times the volume of a citric acid solution (pH 5.0, i.e. the mass-volume ratio of the stevia raw material to the citric acid solution is 1 g:14 mL) is added, and ultrasonic-assisted composite enzyme (the mass ratio of the composite enzyme to the stevia raw material is 0.6:100; the composite enzyme is composed of cellulase, amylase and pectinase, and the mass ratio of the cellulase, amylase and pectinase is 10:5:2) is used for extraction (the temperature is 60°C), and after extraction, the enzyme is inactivated (the temperature for inactivating the enzyme is 100°C, and the time is 6 min), and after inactivation of the enzyme, solid-liquid separation is performed, and the enzyme hydrolysate is collected.

[0148] The parameters of the ultrasonic assistance are as follows:

[0149] The power of the ultrasonic is 200 W, and the time of the ultrasonic is 120 min.

[0150] S2, vacuum concentration:

[0151] The extraction liquid prepared in step S1 is vacuum concentrated to a solid content concentration of 10% at 40°C and a pressure of 60 mmHg, solid-liquid separation is performed, and the concentrated liquid is collected.

[0152] S3, flocculation:

[0153] The composite flocculant is added to the enzyme hydrolysate prepared in step S1 (the mass ratio of the composite flocculant to the stevia raw material in step S1 is 0.3:100), stirring is performed at 25°C for 60 min, solid-liquid separation is performed, the liquid phase is collected, and a crude extract is prepared.

[0154] The preparation method of the composite flocculant is as follows:

[0155] 100 g of activated carbon is added to an acidic potassium permanganate solution (the molar concentration is 0.5 mol / L, and the mass-volume ratio of the activated carbon to the acidic potassium permanganate solution is 1 g:50 mL) for reaction (the temperature of the reaction is 40°C, and the time of the reaction is 40 min), hydrogen peroxide solution (the mass concentration is 30%) is added after the reaction until no gas bubbles are generated, solid-liquid separation is performed, and the solid phase is washed to prepare oxidized activated carbon;

[0156] The oxidized activated carbon is uniformly dispersed in 500 mL of water by ultrasonic dispersion to prepare an oxidized activated carbon dispersion liquid;

[0157] 0.1 g of chitosan is dissolved in 500 mL of an ice acetic acid solution with a volume fraction of 2% to prepare a chitosan solution;

[0158] The chitosan solution, the oxidized activated carbon dispersion liquid and ferric sulfate (0.7 g) were stirred uniformly, reacted for 2 h at 70°C, filtered, washed with water until neutral, and dried at 65°C under vacuum for 12 h to obtain the composite flocculant.

[0159] S4, elution:

[0160] After the crude extract was adsorbed by macroporous resin, it was eluted with 1.5 BV of pure water, 3 BV of 30% ethanol aqueous solution, and 2.5 BV of 80% ethanol aqueous solution in sequence, and the eluate of 80% ethanol aqueous solution was collected.

[0161] S5, passing through an acidic alumina column:

[0162] After the eluate obtained in step S4 was adsorbed by an acidic alumina column, it was eluted with 2 BV of 95% acidic ethanol aqueous solution (pH 6.5), and the eluate was collected.

[0163] The eluate was crystallized at 0°C for 12 h to obtain 57.17 g of stevia extract.

[0164] HPLC detection showed that the mass fraction of stevioside in the stevia extract was 97.3%.

[0165] Comparative Example 1

[0166] The comparative example is a preparation method of a stevia extract.

[0167] The difference from Example 1 is that the comparative example does not use composite flocculant for impurity removal treatment.

[0168] In the comparative example, 65.53 g of stevia extract was obtained.

[0169] HPLC detection showed that the mass fraction of stevioside in the stevia extract was 71.5%.

[0170] Comparative Example 2

[0171] The comparative example is a preparation method of a stevia extract.

[0172] The difference from Example 1 is that the comparative example replaces the composite flocculant with activated carbon.

[0173] In the comparative example, 63.26 g of stevia extract was obtained.

[0174] HPLC detection showed that the mass fraction of stevioside in the stevia extract was 75.3%.

[0175] Comparative Example 3

[0176] The comparative example is a preparation method of a stevia extract.

[0177] The difference from Example 1 is that the composite flocculant is replaced by oxidized activated carbon in the present comparative example.

[0178] The preparation method of the oxidized activated carbon in the present comparative example comprises the following steps:

[0179] 100 g of activated carbon is added into an acidic potassium permanganate solution (molar concentration of 0.5 mol / L, mass-volume ratio of activated carbon and acidic potassium permanganate solution of 1 g: 50 mL) for reaction (reaction temperature of 40℃, reaction time of 40 min), hydrogen peroxide solution (mass concentration of 30%) is added after reaction until no bubbles are generated, then solid-liquid separation is performed, the solid phase is washed, and the oxidized activated carbon is prepared;

[0180] 62.19 g of stevia extract is obtained in the present comparative example.

[0181] The mass fraction of steviol glycosides in the stevia extract is 77.9% detected by HPLC.

[0182] Comparative Example 4

[0183] The present comparative example is a preparation method of a stevia extract.

[0184] The difference from Example 1 is that the activated carbon is not subjected to oxidation treatment in the preparation process of the composite flocculant in the present comparative example.

[0185] The preparation method of the composite flocculant in the present comparative example comprises the following steps:

[0186] 100 g of activated carbon is added into 500 mL of water for ultrasonic dispersion to prepare an activated carbon dispersion;

[0187] 0.2 g of chitosan is dissolved in 500 mL of 2% ice acetic acid solution to prepare a chitosan solution;

[0188] The chitosan solution, the activated carbon dispersion and iron sulfate (0.8 g) are stirred uniformly, reacted at 70℃ for 2 h, filtered, washed with water until neutral, and dried at 65℃ under vacuum for 12 h to prepare the composite flocculant.

[0189] 61.53 g of stevia extract is obtained in the present comparative example.

[0190] The mass fraction of steviol glycosides in the stevia extract is 85.3% detected by HPLC.

[0191] Comparative Example 5

[0192] The present comparative example is a preparation method of a stevia extract.

[0193] The difference from Example 1 is that no cellulase is added in the present comparative example.

[0194] 43.26 g of stevia extract is obtained in the present comparative example.

[0195] The mass fraction of steviol glycosides in the stevia extract is 94.3% as detected by HPLC.

[0196] Comparative Example 6

[0197] The present comparative example is a preparation method of a stevia extract.

[0198] The difference from Example 1 is that no pectinase is added in the present comparative example.

[0199] 48.71 g of stevia extract is obtained in the present comparative example.

[0200] The mass fraction of steviol glycosides in the stevia extract is 92.6% as detected by HPLC.

[0201] Comparative Example 7

[0202] The present comparative example is a preparation method of a stevia extract.

[0203] The difference from Example 1 is that no amylase is added in the present comparative example.

[0204] 49.36 g of stevia extract is obtained in the present comparative example.

[0205] The mass fraction of steviol glycosides in the stevia extract is 93.1% as detected by HPLC.

[0206] In summary, the preparation method of the present application first uses a composite enzyme to perform enzymatic hydrolysis treatment on the stevia extract, and the enzyme changes the cell wall of the stevia raw material, thereby changing the permeability of the cell wall of the stevia raw material; and the extraction rate of the effective component (steviol glycoside) is further improved. The present application uses a composite flocculant to treat the enzymatic hydrolysis liquid, and preliminarily removes the polyphenolic substances in the enzymatic hydrolysis liquid, so that the purity of the finally prepared stevia extract is improved. After the crude extract is adsorbed by a macroporous resin, the polar impurities are removed by water, and the polyphenolic impurities are further removed by a low-concentration ethanol aqueous solution (30% to 40% by volume), so that the steviol glycosides are enriched in a high-concentration ethanol aqueous solution (70% to 80% by volume). The preparation method of the present application only uses water and ethanol as solvents throughout the process, and does not use toxic organic reagents, and the operation is simple, which is very suitable for industrial mass production.

[0207] The above-described specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application, and it should be understood that the above-described specific embodiments are merely examples of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of preparing a stevia extract, characterized by, The method comprises the following steps: S1, crushing the stevia raw material and then soaking it in water, adding a composite enzyme for enzymolysis to obtain an enzymolysis solution; S2, performing flocculation treatment on the enzymolysis solution; the flocculant is a composite flocculant, and a crude extract solution is obtained; S3, adsorbing the crude extract solution by using a macroporous resin and then performing gradient elution, and collecting the eluate of a specific section; S4, adsorbing the eluate by using an alumina column and then eluting, concentrating the eluate, and crystallizing; The composite enzyme is composed of amylase, cellulase and pectinase; The gradient elution program is as follows: First elution section: water; Second elution section: an ethanol aqueous solution with a volume fraction of 30%-40%; Third elution section: an ethanol aqueous solution with a volume fraction of 70%-80%; The composite flocculant comprises the following preparation raw materials: 100 parts of activated carbon, 0.1-0.3 parts of chitosan and 0.5-0.8 parts of iron sulfate; The activated carbon is subjected to oxidation treatment to obtain oxidized activated carbon; An oxidized activated carbon dispersion liquid is prepared from the oxidized activated carbon; The oxidized activated carbon dispersion liquid, a chitosan solution and iron sulfate are mixed and reacted; The preparation raw materials of the chitosan solution comprise chitosan and an acetic acid solution; The reaction temperature is 65-75 DEG C.

2. The method of preparing a stevia extract according to claim 1, characterized in that, The enzymolysis process is assisted by ultrasonic waves; the power of the ultrasonic waves in the ultrasonic-assisted process is 200-300 W.

3. The method of preparing stevia extract according to claim 1, characterized by, The composite enzyme is composed of the following preparation raw materials in mass fractions: Cellulase 10 parts, amylase 3-5 parts and pectinase 1-3 parts.

4. The method of preparing a stevia extract according to claim 1, characterized in that, The macroporous resin is D101 macroporous resin.

5. The method of preparing a stevia extract according to claim 1, characterized in that, The alumina column is an acidic alumina column.

6. The method of preparing a stevia extract according to claim 5, characterized in that, The eluate for elution is an acidic ethanol aqueous solution.

7. The method of preparing a stevia extract according to claim 6, characterized in that, The pH of the acidic ethanol aqueous solution is 5-6.

8. The method of preparing a stevia extract according to claim 1, characterized in that, The crystallization temperature is -4-0 DEG C.