CN103865913B - Compound enzyme and method for extracting effective ingredients in Chinese herbal medicines by virtue of compound enzyme - Google Patents
Compound enzyme and method for extracting effective ingredients in Chinese herbal medicines by virtue of compound enzyme Download PDFInfo
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Abstract
The invention discloses a compound enzyme and a method for extracting effective ingredients in Chinese herbal medicines by virtue of compound enzyme. The compound enzyme comprises cellulase, acid amylase and pectinase; one gram of compound enzyme contains 500-700U of cellulase, 60-75U of acid amylase and 6000-7000U of pectinase. The compound enzyme can be used for simultaneously extracting two effective ingredients from the Chinese herbal medicines, is obvious in effect, and can be used for extracting effective ingredients in leonurus japonicus houtt, processed epimedium and honeysuckle.
Description
Technical field
The present invention relates to technical field, particularly relate to the method for effective constituent in a kind of prozyme and extraction herbal medicine thereof.
Background technology
From herbal medicine, the method for extracting effective components has multiple, and wherein enzyme extraction method is newer method.Wherein, conventional enzyme has cellulase, polygalacturonase, proteolytic enzyme and amylase.As a rule, the hydrolysis result of the prozyme be made up of multiple enzyme is better than the hydrolysis result being used alone a kind of enzyme.But because the kind of Chinese medicinal materials is different, its biological cell structure and effective constituent have very large difference; And enzyme has unicity, the composition of prozyme, proportioning determine its effect.So that is, the hydrolysis result of prozyme might not be better than the hydrolysis result of single enzyme, different because of the Chinese medicinal materials of wanted enzymolysis.Such as, the prozyme be made up of cellulase and polygalacturonase, extracting the hydrolysis result in Chlorogenic Acid of Flos Lonicerae and single cellulase extracting compared with the hydrolysis result in Chlorogenic Acid of Flos Lonicerae, does not have notable difference.On the other hand, at present the research of Enzymatic Extraction traditional Chinese medicine ingredients is mainly also rested on for some single concrete extraction object.That is, can only study for a kind of Chinese medicinal materials at present, obtain and be only applicable to the prozyme that this Chinese medicinal materials extracts special component.
Summary of the invention
Object of the present invention is exactly defect for above-mentioned existence and provides a kind of prozyme and extract the method for effective constituent in herbal medicine.This prozyme can extract two kinds of effective constituents in herbal medicine simultaneously, Be very effective, can be used for extracting effective constituent in Motherwort Herb, Herba Epimedii Preparata and Japanese Honeysuckle.
A kind of prozyme of the present invention and to extract the method and technology scheme of effective constituent in herbal medicine be that this prozyme is made up of cellulase, acid starch enzyme and polygalacturonase; Cellulase 500-700U, acid starch enzyme 60-75U, polygalacturonase 6000-7000U in 1g prozyme.
Cellulase 667U, acid starch enzyme 66.7U, polygalacturonase 6667U in 1g prozyme.
This prozyme is for extracting reducing sugar in Motherwort Herb and stachydrine hydrochloride; Or the reducing sugar in extraction Herba Epimedii Preparata and icarin; Or the reducing sugar extracted in Japanese Honeysuckle and chlorogenic acid.
The method of effective constituent in this multiplex-enzyme extraction herbal medicine, comprises the following steps:
(1) Chinese herbal medicine powder is broken into powdery;
(2) Chinese herbal medicine powder is added deionized water to mix, PH is adjusted to 4.5, obtains feed liquid;
(3) prozyme aqueous glycerin solution is diluted to obtain prozyme diluent;
(4) by the feed liquid preheating of step (2), add prozyme diluent, filter after enzymolysis, get filtrate.
In step (1), Chinese herbal medicine powder is broken to 40-200 order.
In step (3), aqueous glycerin solution concentration is 20wt%, and prozyme aqueous glycerin solution is diluted to 0.0020g-0.5g/ml, obtains prozyme diluent.
When herbal medicine is Motherwort Herb, prozyme diluent concentration is 0.025-0.30g/ml;
When herbal medicine is Herba Epimedii Preparata, the concentration of prozyme diluent is 0.025-0.28g/ml;
When herbal medicine is Japanese Honeysuckle, the concentration of prozyme diluent is 0.025-0.3g/ml.
Preferably, the preferred concentration of prozyme diluent is 0.25g/ml.
In step (4), by feed liquid preheating 2min in water-bath, add enzyme liquid while stirring, hydrolysis temperature is 40-55 DEG C, time 4-7h.
Preferably, in step (4), hydrolysis temperature is 50 DEG C, time 5h.
Every gram of Motherwort Herb extracts 35.7-181mg reducing sugar and 5.6-10.8mg stachydrine hydrochloride; Every gram of Herba Epimedii Preparata extracts 38.85-97mg reducing sugar and 8.4-10.8g icarin; Every gram of Japanese Honeysuckle extracts 80.13-420.57mg reducing sugar and 13.6-16.8mg chlorogenic acid.
Preferably, concrete steps are as follows: take herbal medicine 5.000g(powdery) be placed in 200ml beaker, the deionized water adding 200mL shakes up; PH value is adjusted to 4.5 by the sulphuric acid soln then adding 0.5mL 1mol/L, obtains feed liquid.Get prozyme (containing cellulase 667u, acid starch enzyme 66.7u and polygalacturonase 6667u in every gram of prozyme) and be diluted to 1ml with 20wt% glycerine dissolved dilution, obtain enzyme liquid.
By feed liquid preheating 2min in 50 ° of C water-baths, add rapidly enzyme liquid while stirring, leave standstill insulation 5h; Then filter, get filtrate.
Wherein, 1g herbal medicine needs prozyme diluent 1ml.
Beneficial effect of the present invention is: prozyme of the present invention destroys the cell walls of herbal medicine, reduces viscosity, improves herbal medicine lixiviate recovery rate, can simultaneously for extracting effective constituent in Motherwort Herb, Herba Epimedii Preparata and Japanese Honeysuckle; Thus the prozyme overcoming a kind of specific composition can only be used for a kind of technical barrier of herbal medicine.In addition, adopt prozyme of the present invention, two kinds of effective constituents in above-mentioned often kind of Chinese medicinal materials can be extracted simultaneously.Specifically, the reducing sugar in Motherwort Herb and stachydrine hydrochloride can be extracted; The reducing sugar in Herba Epimedii Preparata and icarin can be extracted; The reducing sugar in Japanese Honeysuckle and chlorogenic acid can be extracted; And extraction yield is high.
Method of the present invention, simple to operation, can extract two kinds of effective constituents, extraction yield significantly improves simultaneously.
After testing, adopt the inventive method, every gram of Motherwort Herb can extract 35.7-181mg reducing sugar and 5.6-10.8mg stachydrine hydrochloride; Every gram of Herba Epimedii Preparata can extract 35.04-97mg reducing sugar and 8.4-10.8g icarin; Every gram of Japanese Honeysuckle is for can extract 80.13-420.57mg reducing sugar and 13.6-16.8mg chlorogenic acid.As compared to the combinative enzyme hydrolysis not adopting enzymic hydrolysis or adopt cellulase and polygalacturonase to form, the extraction yield of two kinds of effective constituents in often kind of raw material all increases substantially, and extraction yield improves 16.4-495%.
Because the concentration of prozyme can have an impact to the activity of enzyme, thus affect whole leaching process; And its biological cell structure of different material and effective constituent have very large difference; Required optimum prozyme concentration is different.Therefore, so in aforesaid method, when raw material is Motherwort Herb, the preferred concentration of prozyme diluent is 0.025-0.30g/ml; Now, reducing sugar extraction yield is 57.3-181mg/g, and the extraction yield of stachydrine hydrochloride is 8.4-10.8mg/g.Most preferred, the preferred concentration of prozyme diluent is 0.25g/ml; Now, reducing sugar extraction yield is 181mg/g, and the extraction yield of stachydrine hydrochloride is 10.8mg/g.
When raw material is Herba Epimedii Preparata, the preferred concentration of prozyme diluent is 0.025-0.28g/ml; Now, reducing sugar extraction yield is 38.85-97mg/g, and the extraction yield of icarin is 9.6-10.8mg/g.Most preferred, the preferred concentration of prozyme diluent is 0.25g/ml; Now, reducing sugar extraction yield is 97mg/g, and the extraction yield of icarin is 10.8mg/g.
When raw material is Japanese Honeysuckle, the preferred concentration of prozyme diluent is 0.025-0.3g/ml; Now, reducing sugar extraction yield is 168.3-420.57mg/g, and the extraction yield of chlorogenic acid is 14.8-16.8mg/g.Most preferred, the preferred concentration of prozyme diluent is 0.25g/ml; Now, reducing sugar extraction yield is 420.57mg/g, and the extraction yield of chlorogenic acid is 16.8mg/g.
embodiment:
In order to understand the present invention better, describe technical scheme of the present invention in detail with specific examples below.
Polygalacturonase in the present invention, cellulase, acid starch enzyme are:
Polygalacturonase QB1502-92:1g enzyme powder 50 DEG C, under the condition of pH3.5, it is an enzyme activity unit that 1h decompose pectin produces 1mg galacturonic acid.
Cellulase NY/T912-2004: 37 DEG C, under pH is the condition of 5.50, the per minute enzyme amount of degrading from the carboxymethylcellulose sodium solution that concentration is 4mg/mL required for release 1 μm of ol reducing sugar is an enzyme activity unit (U).
Acid starch enzyme: 1g enzyme, in 40 DEG C, pH3.0 condition, 1min liquefaction 1mg Zulkovsky starch, is 1 enzyme activity unit, represents with u/ g.
A kind of prozyme of the present invention and extract the method for effective constituent in herbal medicine, this prozyme is made up of cellulase, acid starch enzyme and polygalacturonase; Cellulase 500-700U, acid starch enzyme 60-75U, polygalacturonase 6000-7000U in 1g prozyme.
This prozyme is for extracting reducing sugar in Motherwort Herb and stachydrine hydrochloride; Or the reducing sugar in extraction Herba Epimedii Preparata and icarin; Or the reducing sugar extracted in Japanese Honeysuckle and chlorogenic acid.
The method of effective constituent in this multiplex-enzyme extraction herbal medicine, comprises the following steps:
(1) Chinese herbal medicine powder is broken into powdery;
(2) Chinese herbal medicine powder is added deionized water to mix, PH is adjusted to 4.5, obtains feed liquid;
(3) prozyme aqueous glycerin solution is diluted to obtain prozyme diluent;
(4) by the feed liquid preheating of step (2), add prozyme diluent, filter after enzymolysis, get filtrate.
In step (1), Chinese herbal medicine powder is broken to 40-200 order.
In step (3), aqueous glycerin solution concentration is 20wt%, and prozyme aqueous glycerin solution is diluted to 0.0020g-0.5g/ml, obtains prozyme diluent.
When herbal medicine is Motherwort Herb, prozyme diluent concentration is 0.025-0.30g/ml;
When herbal medicine is Herba Epimedii Preparata, the concentration of prozyme diluent is 0.025-0.28g/ml;
When herbal medicine is Japanese Honeysuckle, the concentration of prozyme diluent is 0.025-0.3g/ml.
In step (4), by feed liquid preheating 2min in water-bath, add enzyme liquid while stirring, hydrolysis temperature is 40-55 DEG C, time 4-7h.
Mention the mensuration of reducing sugar in embodiment, concrete grammar is:
Glucose solution: take dextrose anhydrous 1.000g, adding distil water dissolves, and is settled to 100ml
DNS reagent: take 3,5-dinitrosalicylic acid 3.15g(chemical pure), add water 500ml, stirs 5s, water-bath to 45 DEG C.Then progressively add 100ml sodium hydroxide solution, constantly stir simultaneously, (note: adding in sodium hydroxide process, solution temperature does not exceed 48 DEG C until solution is as clear as crystal.）。Progressively add Rochelle salt 91.0g, phenol 2.50g and sodium sulphite anhydrous 99.3 2.50g again.Continue 45 DEG C of heating in water bath, add water 300ml simultaneously, constantly stir, until the material added dissolves completely.Stop heating, after being cooled to room temperature, be settled to 1000ml with water.Be stored in brown bottle, keep in Dark Place.Can use after depositing 7 days under room temperature, validity period is 6 months.
Draw distilled water 4.0ml, add DNS reagent 5.0ml, boiling water bath heating 5min.Be cooled to room temperature with tap water, be settled to 25.0ml with water, the blank sample of standard of making.
Get 2ml supernatant liquor and add 2ml distilled water, the rear boiling water bath 5min of 5mlDNS reagent mixing, be cooled to room temperature with tap water, add water after being settled to 25ml and mix.With the blank sample of standard for blank, measure absorbancy at 540nm place.
The drafting of typical curve
Draw glucose solution 1.00,2.00,3.00,4.00,5.00,6.00 and 7.00ml respectively, be settled to 100ml with distilled water respectively, being mixed with concentration is 0.10-0.70mg/ml Glucose standards solution.
The each 2.00ml(of Glucose standards solution drawing above-mentioned concentration series respectively do two parallel), join respectively in scale test tube, then add 2ml water and 5mlDNS reagent respectively.Electromagnetic oscillation 3s, boiling water bath heating 5min.Then use tap water cool to room temperature, then be settled to 25ml with water.With the blank sample of standard for contrast zeroing, measure absorbancy OD value A at 540nm place.
Take glucose concn as Y-axis, absorbancy OD value is X-axis, drawing standard curve.Each new preparation DNS reagent all needs to repaint typical curve.
The calculating of reducing sugar:
X
D = A×K + C
O
X
dconcentration of reduced sugar in-sample diluent, mg/ml;
The absorbancy of A-reaction solution;
The slope of K-typical curve;
C
othe intercept of-typical curve.
Embodiment 1
Take Motherwort Herb 5.000g to be crushed to 100 orders and to be placed in 200ml beaker, the deionized water adding 200mL shakes up; PH value is adjusted to 4.5 by the sulphuric acid soln then adding 0.5mL 1mol/L, obtains Motherwort Herb liquid.Get prozyme (containing cellulase 667u, acid starch enzyme 66.7u and polygalacturonase 6667u in every gram of prozyme) and be diluted to 1ml with 20wt% glycerine dissolved dilution, obtain enzyme liquid.
By Motherwort Herb liquid preheating 2min in 50 ° of C water-baths, add rapidly enzyme liquid while stirring, leave standstill insulation 5h; Then filter, get filtrate.
Aforesaid operations repeats 5 times altogether, and the consumption of each prozyme is all different, is followed successively by 0g, 0.0025g, 0.025g, 0.25g and 0.3g.
Detect the raw sugar content in the filtrate 5 operations obtained and stachydrine hydrochloride content respectively.
Detected result is as table 1.
Table 1
。
Embodiment 2
Take Motherwort Herb 5.000g to be crushed to 180 orders and to be placed in 200ml beaker, the deionized water adding 200mL shakes up; PH value is adjusted to 4.5 by the sulphuric acid soln then adding 0.5mL 1mol/L, obtains Motherwort Herb liquid.Get prozyme 20wt% glycerine dissolved dilution and be diluted to 1ml, obtain enzyme liquid.
By Motherwort Herb liquid preheating 2min in 50 ° of C water-baths, add rapidly enzyme liquid while stirring, leave standstill insulation 5h; Then filter, get filtrate.
Aforesaid operations repeats 3 times altogether, each prozyme consumption 0.25g, and the various enzyme content in prozyme are all different:
1. cellulase 300u, acid starch enzyme 100u and polygalacturonase 200u is contained in every gram of prozyme;
2. cellulase 500u, acid starch enzyme 70u and polygalacturonase 6500u is contained in every gram of prozyme;
3. cellulase 700u, acid starch enzyme 60u and polygalacturonase 6000u is contained in every gram of prozyme.
Detect raw sugar content and stachydrine hydrochloride content respectively.
Detected result is as table 2.
Table 2
。
Embodiment 3
Take Herba Epimedii Preparata 5.000g to be crushed to 200 orders and to be placed in 200ml beaker, the deionized water adding 200mL shakes up, and pH value is adjusted to 4.5 by the sulphuric acid soln then adding 0.25mL 1mol/L, obtains Herba Epimedii Preparata liquid; Get prozyme (containing cellulase 667u, acid starch enzyme 66.7u and polygalacturonase 6667u in every gram of prozyme) and be diluted to 1ml with 20wt% glycerine dissolved dilution, obtain enzyme liquid; By Herba Epimedii Preparata liquid preheating 2min in 50 ° of C water-baths, add rapidly enzyme liquid while stirring, leave standstill insulation 5h; Then filter, filtrate.
Aforesaid operations repeats 5 times altogether, and the consumption of each prozyme is all different, is followed successively by 0g, 0.0025g, 0.025g, 0.25g and 0.3g.
Detect raw sugar content and Icariin content respectively.
Detected result is as table 3.
Table 3
。
Embodiment 4
Take Herba Epimedii Preparata 5.000g to be crushed to 200 orders and to be placed in 200ml beaker, the deionized water adding 200mL shakes up, and pH value is adjusted to 4.5 by the sulphuric acid soln then adding 0.25mL 1mol/L, obtains Herba Epimedii Preparata liquid; Get prozyme 20wt% glycerine dissolved dilution and be diluted to 1ml, obtain enzyme liquid; By Herba Epimedii Preparata liquid preheating 2min in 50 ° of C water-baths, add rapidly enzyme liquid while stirring, leave standstill insulation 5h; Then filter, filtrate.
Aforesaid operations repeats 3 times altogether, each prozyme consumption 0.25g, and the various enzyme content in prozyme are all different:
1. cellulase 300u, acid starch enzyme 100u and polygalacturonase 200u is contained in every gram of prozyme;
2. cellulase 500u, acid starch enzyme 70u and polygalacturonase 6500u is contained in every gram of prozyme;
3. cellulase 700u, acid starch enzyme 60u and polygalacturonase 6000u is contained in every gram of prozyme.
Detect raw sugar content and Icariin content respectively.
Detected result is as shown in table 4:
Table 4
。
Embodiment 5
Take Japanese Honeysuckle 5.000g to be crushed to 40 orders and to be placed in 200ml beaker, the deionized water adding 200mL shakes up, and the pH value of Japanese Honeysuckle liquid is adjusted to 4.5 by the sulphuric acid soln then adding 0.2mL 1mol/L, obtains Japanese Honeysuckle liquid; Get prozyme (containing cellulase 667u, acid starch enzyme 66.7u and polygalacturonase 6667u in every gram of prozyme) and be diluted to 1ml with 20wt% glycerine dissolved dilution, obtain enzyme liquid; By Japanese Honeysuckle liquid preheating 2min in 50 ° of C water-baths, add rapidly enzyme liquid while stirring, leave standstill insulation 5h; Then filter, filtrate.
Aforesaid operations repeats 5 times altogether, and the consumption of each prozyme is all different, is followed successively by 0g, 0.0025g, 0.025g, 0.25g and 0.3g.
Detect the reducing sugar, the chlorogenic acid that operate 5 times in the filtrate obtained respectively, result is as table 5.
Table 5
。
Embodiment 6
Take Japanese Honeysuckle 5.000g to be crushed to 40 orders and to be placed in 200ml beaker, the deionized water adding 200mL shakes up, and the pH value of Japanese Honeysuckle liquid is adjusted to 4.5 by the sulphuric acid soln then adding 0.2mL 1mol/L, obtains Japanese Honeysuckle liquid; Get prozyme (containing cellulase 667u, acid starch enzyme 66.7u and polygalacturonase 6667u in every gram of prozyme) and be diluted to 1ml with 20wt% glycerine dissolved dilution, obtain enzyme liquid; By Japanese Honeysuckle liquid preheating 2min in 50 ° of C water-baths, add rapidly enzyme liquid while stirring, leave standstill insulation 5h; Then filter, filtrate.
Aforesaid operations repeats 3 times altogether, each prozyme consumption 0.25g, and the various enzyme content in prozyme are all different:
1. cellulase 300u, acid starch enzyme 100u and polygalacturonase 200u is contained in every gram of prozyme;
2. cellulase 500u, acid starch enzyme 70u and polygalacturonase 6500u is contained in every gram of prozyme;
3. cellulase 700u, acid starch enzyme 60u and polygalacturonase 6000u is contained in every gram of prozyme.
Detect raw sugar content and chlorogenic acid content respectively.
Detected result is as shown in table 6:
Table 6
。
As can be seen here, adopt the inventive method, every gram of Motherwort Herb can extract 35.7-181mg reducing sugar and 5.6-10.8mg stachydrine hydrochloride; Every gram of Herba Epimedii Preparata can extract 35.04-97mg reducing sugar and 8.4-10.8g icarin; Every gram of Japanese Honeysuckle is for can extract 80.13-420.57mg reducing sugar and 13.6-16.8mg chlorogenic acid.As compared to the combinative enzyme hydrolysis not adopting enzymic hydrolysis or adopt cellulase and polygalacturonase to form, the extraction yield of two kinds of effective constituents in often kind of raw material all increases substantially, and extraction yield improves 16.4-495%.
Because the concentration of prozyme can have an impact to the activity of enzyme, thus affect whole leaching process; And its biological cell structure of different material and effective constituent have very large difference; Required optimum prozyme concentration is different.Therefore, so in aforesaid method, when raw material is Motherwort Herb, the preferred concentration of prozyme diluent is 0.025-0.30g/ml; Now, reducing sugar extraction yield is 57.3-181mg/g, and the extraction yield of stachydrine hydrochloride is 8.4-10.8mg/g.Most preferred, the preferred concentration of prozyme diluent is 0.25g/ml; Now, reducing sugar extraction yield is 181mg/g, and the extraction yield of stachydrine hydrochloride is 10.8mg/g.
When raw material is Herba Epimedii Preparata, the preferred concentration of prozyme diluent is 0.025-0.28g/ml; Now, reducing sugar extraction yield is 38.85-97mg/g, and the extraction yield of icarin is 9.6-10.8mg/g.Most preferred, the preferred concentration of prozyme diluent is 0.25g/ml; Now, reducing sugar extraction yield is 97mg/g, and the extraction yield of icarin is 10.8mg/g.
When raw material is Japanese Honeysuckle, the preferred concentration of prozyme diluent is 0.025-0.3g/ml; Now, reducing sugar extraction yield is 168.3-420.57mg/g, and the extraction yield of chlorogenic acid is 14.8-16.8mg/g.Most preferred, the preferred concentration of prozyme diluent is 0.25g/ml; Now, reducing sugar extraction yield is 420.57mg/g, and the extraction yield of chlorogenic acid is 16.8mg/g.
Claims (8)
1. the method for effective constituent in multiplex-enzyme extraction herbal medicine, it is characterized in that, this prozyme is made up of cellulase, acid starch enzyme and polygalacturonase; Cellulase 500-700U, acid starch enzyme 60-75U, polygalacturonase 6000-7000U in 1g prozyme;
Comprise the following steps:
(1) Chinese herbal medicine powder is broken into powdery;
(2) Chinese herbal medicine powder is added deionized water to mix, PH is adjusted to 4.5, obtains feed liquid;
(3) prozyme aqueous glycerin solution is diluted to obtain prozyme diluent;
(4) by the feed liquid preheating of step (2), add prozyme diluent, filter after enzymolysis, get filtrate.
2. the method for effective constituent in a kind of multiplex-enzyme extraction herbal medicine according to claim 1, is characterized in that, cellulase 667U, acid starch enzyme 66.7U, polygalacturonase 6667U in 1g prozyme.
3. the method for effective constituent in a kind of multiplex-enzyme extraction herbal medicine according to claim 1, it is characterized in that, in step (1), Chinese herbal medicine powder is broken to 40-200 order.
4. the method for effective constituent in a kind of multiplex-enzyme extraction herbal medicine according to claim 1, it is characterized in that, in step (3), aqueous glycerin solution concentration is 20wt%, prozyme aqueous glycerin solution is diluted to 0.0020g-0.5g/ml, obtains prozyme diluent.
5. the method for effective constituent in a kind of multiplex-enzyme extraction herbal medicine according to claim 4, it is characterized in that, when herbal medicine is Motherwort Herb, prozyme diluent concentration is 0.025-0.30g/ml;
When herbal medicine is Herba Epimedii Preparata, the concentration of prozyme diluent is 0.025-0.28g/ml;
When herbal medicine is Japanese Honeysuckle, the concentration of prozyme diluent is 0.025-0.3g/ml.
6. the method for effective constituent in a kind of multiplex-enzyme extraction herbal medicine according to claim 5, it is characterized in that, the concentration of prozyme diluent is 0.25g/ml.
7. the method for effective constituent in a kind of multiplex-enzyme extraction herbal medicine according to claim 1, it is characterized in that, in step (4), by feed liquid preheating 2min in water-bath, add enzyme liquid while stirring, hydrolysis temperature is 40-55 DEG C, time 4-7h.
8. the method for effective constituent in a kind of multiplex-enzyme extraction herbal medicine according to claim 1, it is characterized in that, in step (4), hydrolysis temperature is 50 DEG C, time 5h.
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