CN106188331B - A kind of method of biomimetic method joint enzymatic isolation method extraction Blackfungus polyhexose - Google Patents
A kind of method of biomimetic method joint enzymatic isolation method extraction Blackfungus polyhexose Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/60—Cultivation rooms; Equipment therefor
- A01G18/64—Cultivation containers; Lids therefor
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention discloses a kind of methods of biomimetic method joint enzymatic isolation method extraction Blackfungus polyhexose.The present invention is digested on the basis of acting synergistically, it is determined that optimum extraction process using bionic extraction.The method includes：1) pre-treatment of black fungus raw material；2) it digests；3) imitation biochemistry extracts；4) it concentrates；5) alcohol precipitation；6) be freeze-dried and etc..Simple using the method for the present invention operating procedure, technique extraction conditions are mild, simulate human consumption's mechanism, the effective polysaccharide being extracted to greatest extent in black fungus, while are also beneficial to improve Blackfungus polyhexose recovery rate, retentive activity polysaccharide complete structure.The it is proposed of the present invention provides a kind of effective technological means for the extraction of Blackfungus polyhexose.
Description
Technical field
It is more particularly to a kind of to be carried using biomimetic method joint enzymatic isolation method the present invention relates to a kind of extracting method of Blackfungus polyhexose
The method for taking Blackfungus polyhexose.The invention belongs to active components of plants extractive technique fields.
Background technology
Black fungus is a kind of integration of drinking and medicinal herbs class edible mushroom, and Blackfungus polyhexose is to isolate and purify to obtain in black fungus fructification
A kind of macromolecule beta glucan, is one of its main composition.Blackfungus polyhexose has QI invigorating salubrity, fills blood, moistening lung, stops
Blood, analgesic, defaecation and other effects.Present pharmacological research shows that Blackfungus polyhexose is a kind of non-specific immunomodulator, can swash
Living person's body immune t-cell, B cell enhance immune cell, have and improve immunity, anti-aging, prevention and cure of cardiovascular disease etc.
Multiple efficacies；Compared with other edible and medical fungis, black fungus fructification, which has, to have a very wide distribution, artificial cultivation technique maturation and yield
Higher advantage；At present, Blackfungus polyhexose has been developed that into plurality kinds of health care food and drug, such as black fungus tea, black fungus crisp chip,
Blackfungus beverage and Auricularia polysaccharide capsule etc..
Bionics extractive technique has imitated transport process of the oral drugs in human gastrointestinal tract, leading under being instructed using activity
To isolation technics, to obtain the biomolecule of high activity；Enzymolysis can effectively destroy fungus sporophore cell wall, will effectively into
Divide and preferably discharge, reduce extraction conditions, improve recovery rate, enzymatic isolation method and semi-bionic extraction method are combined more to fungi
Sugar extracts, it is both comprehensive the advantages of, it is environmentally protective, extraction conditions are mild, recovery rate is high, ensure that polysaccharide structures and activity are complete
Whole property and technical process take short, reduce extraction cost, and the further exploitation for industrialized production provides technical support.
Invention content
The technical problems to be solved by the invention are to provide a kind of using biomimetic method joint enzymatic isolation method extraction Blackfungus polyhexose
Method.
In order to achieve the above object, present invention employs following technological means：
The process flow chart that the biomimetic method joint enzymatic isolation method of the present invention prepares Blackfungus polyhexose is as shown in Figure 1.
Specifically, a kind of method using biomimetic method joint enzymatic isolation method extraction Blackfungus polyhexose of the present invention, according to following
Step carries out：
(1) pre-treatment of black fungus：
Black fungus raw material is cleaned, surface impurity is removed, is put into thermostatic drying chamber that drying to constant weight, is crushed, crosses 60~80
Mesh sieves, spare；
(2) it digests：
The black fungus dry powder of 60~80 mesh will be crushed to, be 1 according to material liquid volume ratio:30~1:80 addition water are soaked
Bubble after impregnating 3-5 hours, is digested according to 10%~20% addition complex cellulase of black fungus dry powder weight, is digested
Temperature is 30~60 DEG C, and enzymolysis time is 90~150min, and 100 DEG C of enzyme-removal temperature obtains black fungus enzymolysis liquid；
(3) imitation biochemistry extracts：
It is 2.0 that above-mentioned enzymolysis liquid first is adjusted pH with hydrochloric acid solution, and temperature is controlled at 35~60 DEG C, extraction 60~
150min, it is 8.0 then to adjust pH with sodium hydroxide solution again, and temperature is controlled at 35~60 DEG C, extracts 60~150min, mistake
Filter, obtains filtrate；
(4) it concentrates：
The filtrate that step (3) obtains is concentrated, collects concentrate, residue is removed, obtains imitation biochemistry concentrate；
(5) alcohol precipitation：
The concentrate and 95% (w/w) ethyl alcohol that step (4) is obtained by volume 1:4 ratio mixing, after mixing
Cord blood left undisturbed overnight centrifuges 15min with 10000r/min, collects precipitation；
(6) it is freeze-dried：
The sediment that step (5) obtains is freeze-dried, as black fungus Thick many candies.
In method of the present invention, it is preferred that the black fungus described in step (1) is to cultivate by the following method
It arrives：
(1) prepared by culture medium：
By the hardwood sawdust of 85~90 parts by weight, the wheat bran of 10~12 parts by weight, the land plaster of 1~2 parts by weight, 1~2
The soya-bean cake of parts by weight and the lime of 1~2 parts by weight water mixing, it is 7~8 to adjust pH value, and water content is maintained at 60~65%, obtains
To the culture medium for domestication wild black fungus；
(2) culture medium pack sterilizing：
The culture medium loading Polypropylene Bag that step (1) is prepared, every bag of 1~1.5kg, 100 DEG C of steam sterilizings 8 are small
When；
(3) it is inoculated with：
By the Bag Material of sterilizing, when material temperature drops to less than 30 DEG C, inoculation black fungus bacterial kind in transfer room or inoculating hood is moved on to；
(4) cultural hypha：
It is 20~25 DEG C that cultivation bag after inoculation, which is placed in temperature, and indoor humidity is 45%-~55%, is carried out under conditions of being protected from light
Culture treats that mycelia is white, thick, stalwartness is advisable, and incubation time is 12~15 days, and it is 15~20 DEG C to adjust room temperature later, appropriate ventilation,
Illumination so that mycelium slowly adapts to external environment, treats that mycelia enters the stage of ripeness, and it is 10-15 DEG C to adjust room temperature, humidity 75%
~85%, ventilation, illumination are identical with external environment, and culture period was controlled at 55~65 days；
(5) scarfing：
Each bag of planting cuts 10-22 V-type scarfing, and deep 0.5cm, length is in 1.5~2.5cm；
(6) it transplants：
It is 30%~40% to select light transmission, humidity be 75%~90%, well-ventilated, ground without ponding and covered with
The broad-leaf forest of trees sawdust moves on to the thalline in maturity period in broad-leaf forest, the growth of pendulum ground；
(7) ear management:
For produce agaric humid control 90%~95%, temperature, ventilation, sunlight and broad-leaf forest are consistent；
(8) ear is adopted：
When auricle is sufficiently spread out, the thinning softening of edge corrugation, color and luster turns light, and the basal part of the ear is shunk, and the outside of belly does not have white powder object
When (conidia powder), harvest in time.
Wherein, it is preferred that the preparation of the culture medium described in step (1) is by the hardwood sawdust of 85 parts by weight, 12 weights
The lime of the wheat bran of amount part, the land plaster of 0.5 parts by weight, the soya-bean cake of 2 parts by weight and 0.5 parts by weight with water mixing, adjusts pH value
It is 7.5, control water content is 65%.
Wherein, it is preferred that the hardwood sawdust in hardwood sawdust described in step (1) forest zone northeast.
Wherein, it is preferred that the size of the Polypropylene Bag described in step (2) is 17cm × 33cm.
Wherein, it is preferred that the month of the transplanting described in step (6) is annual August part.
The present invention is met by simulating the ecological condition needed for wild black fungus growth in black fungus growth and development process
Requirement to temperature, humidity, sunlight, air etc. makes black fungus ear shape, mouthfeel, thickness, color and luster all close to the black wood of the wild state in northeast
Ear, character is stable, resistance, and quality is better than other kinds, and black fungus is more in the black fungus cultivated using this method
The content higher of sugar.
In method of the present invention, it is preferred that the enzymolysis described in step (2) is will to be crushed to the black wood of 60~80 purposes
Ear dry powder is 1 according to material liquid volume ratio:50 addition water are impregnated, after impregnating 4 hours, according to black fungus dry powder weight
15% addition complex cellulase is digested, and hydrolysis temperature is 45 DEG C, enzymolysis time 120min, and 100 DEG C of enzyme-removal temperature obtains
To black fungus enzymolysis liquid.
In method of the present invention, it is preferred that the imitation biochemistry extraction described in step (3) is first to use above-mentioned enzymolysis liquid
Hydrochloric acid solution adjust pH be 2.0, temperature control at 45 DEG C, extract 120min, then again with sodium hydroxide solution section pH be 8.0,
Temperature is controlled at 45 DEG C, extracts 120min, and filtering obtains filtrate.
In method of the present invention, it is preferred that the concentration described in step (4), which refers to filtrate being placed at 50 DEG C, to be concentrated
60min。
Compared to the prior art, the beneficial effects of the present invention are the method for the present invention operating procedure is simple, technique extraction item
Part is mild, simulates human consumption's mechanism, the effective polysaccharide being extracted to greatest extent in black fungus, while be also beneficial to improve black
Auricularia polysaccharide recovery rate, retentive activity polysaccharide complete structure.The it is proposed of the present invention provides one kind for the extraction of Blackfungus polyhexose
Effective technological means.This method is to Blackfungus polyhexose recovery rate up to 30~40%.
Description of the drawings
The imitation biochemistry that Fig. 1 is the present invention combines the process flow chart that enzymolysis prepares Blackfungus polyhexose.
Specific embodiment
With reference to embodiments, the present invention will be described in further detail.The specific embodiment of description described herein is only
Only to explain the present invention, it is not intended to limit the present invention.Technical solution of the present invention is not limited to the specific reality of act set forth below
Mode is applied, further includes the arbitrary combination between each specific embodiment.
Embodiment 1：The imitative wildization cultural method of black fungus
(1) prepared by culture medium：
By the hardwood sawdust of 85 parts by weight, the wheat bran of 12 parts by weight, the land plaster of 0.5 parts by weight, the soya-bean cake of 2 parts by weight
And 0.5 parts by weight lime, with water mixing, it is 7.5 to adjust pH value, and control water content is 65%, is obtained black for domestication wild
The culture medium of agaric；
(2) culture medium pack sterilizing：
The culture medium that step (1) is prepared is packed into the Polypropylene Bag of 17cm × 33cm, every bag of 1kg, steam sterilizing 100
DEG C, it sterilizes 8 hours；
(3) it is inoculated with：
By the Bag Material of sterilizing, when material temperature drops to less than 30 DEG C, move on in transfer room or inoculating hood and start to be inoculated with；
(4) cultural hypha：
Cultivation bag after inoculation is placed in 25 DEG C of temperature, and indoor humidity 50% is cultivated under conditions of being protected from light, treat mycelia it is white,
Slightly, stalwartness is advisable, and incubation time is 14 days, and it is 20 DEG C to adjust room temperature later, appropriate ventilation, illumination, so that mycelium is slowly fitted
External environment is answered, treats that mycelia enters the stage of ripeness, it is 15 DEG C to adjust room temperature, humidity 80%, ventilation, illumination and external environment phase
Together, culture period 60 days；
(5) scarfing：
Each bag of planting cuts 15 V-type scarfings, and deep 0.5cm, length is in 2cm or so；
(6) it transplants：
It is 30%~40% to select light transmission, humidity be 75%~90%, well-ventilated, ground without ponding and covered with
The broad-leaf forest of trees sawdust moves on to the thalline in maturity period in broad-leaf forest, the growth of pendulum ground；The month of transplanting is August part；
(7) ear management:
For produce agaric humid control 90%~95%, temperature, ventilation, sunlight and broad-leaf forest are consistent；
(8) ear is adopted：
When auricle is sufficiently spread out, the thinning softening of edge corrugation, color and luster turns light, and the basal part of the ear is shunk, and the outside of belly does not have white powder object
When (conidia powder), harvest in time.
Imitative wildization cultural method in order to further illustrate the present invention and conventional method (artificial black fungus bag cultivation, people
Work black fungus juggle cultural method) advantage, the present invention using three kinds of methods carried out the experiment in cultivation of black fungus, and right simultaneously
The yield of black fungus has carried out data analysis with character, as a result as shown in table 1 below：
The yield of 1 three kinds of method plantation black fungus of table is compared with character
It is analyzed from upper table, implements the present invention and imitate wild cultivation of auricularia auricula and the cultivation of artificial black fungus bag, artificial black wood
The cultivation of ear juggle is compared, and black fungus yield of the present invention is apparently higher than juggle cultivation, and mouthfeel and shape are close to wild black fungus.
Using the imitative wild biochemical method cultivating black fungus of the present invention, yield is high, quality is good, the cultivation of the suitable wild agaric in northeast.
The extraction of 2 Blackfungus polyhexose of embodiment
(1) pre-treatment of black fungus raw material：
Cleaning embodiment 1 cultivates obtained black fungus raw material, removes surface impurity, is put into 30 DEG C of thermostatic drying chambers and dries
To constant weight, raw material smashes it through 60 mesh sieve, spare；
(2) it digests：
The black fungus dry powder of 60 mesh will be crushed to, be 1 according to material liquid volume ratio:50 addition water are impregnated, and are impregnated 4 hours
Afterwards, 15% according to black fungus dry powder weight adds in complex cellulase (believing (China) Bioisystech Co., Ltd purchased from Novi)
It is digested, hydrolysis temperature is 45 DEG C, enzymolysis time 120min, and 100 DEG C of enzyme-removal temperature obtains black fungus enzymolysis liquid；
(3) imitation biochemistry extracts：
It is 2.0 that above-mentioned enzymolysis liquid first is adjusted pH with hydrochloric acid solution, and temperature is controlled at 45 DEG C, extracts 120min, Ran Houzai
It is 8.0 to adjust pH with sodium hydroxide solution, and temperature is controlled at 45 DEG C, extracts 120min, and filtering obtains filtrate；
(6) it concentrates：
The filtrate that step (3) obtains is placed at 50 DEG C and concentrates 60min, collects concentrate, residue is removed, obtains bionical
Change concentrate；
(7) alcohol precipitation：
By above-mentioned concentrate and 95% (w/w) ethyl alcohol by volume 1:4 ratio is mixed, and low temperature is protected after mixing
Left undisturbed overnight is deposited, 15min is centrifuged with 10000r/min, collects precipitation.
(8) it is freeze-dried：Above-mentioned sediment is freeze-dried, obtains black fungus Thick many candies.
Embodiment 3
The present embodiment is as different from Example 2：According to the 10% compound fibre of addition of black fungus dry powder weight in step (2)
The plain enzyme of dimension.Other steps are same as Example 2.
Embodiment 4
The present embodiment is as different from Example 2：In step (2) according to material liquid volume ratio be 1:60 addition water are soaked
Bubble.Other steps are same as Example 2.
Embodiment 5
The present embodiment is as different from Example 2：Hydrolysis temperature in step (2) is 60 DEG C.Other steps and embodiment 2
It is identical.
Embodiment 6
The present embodiment is as different from Example 2：Enzymolysis time 150min in step (2).Other steps and embodiment 2
It is identical.
Comparative experimental example
The method of biomimetic method joint enzymatic isolation method extraction Blackfungus polyhexose in order to further illustrate the present invention is (according to embodiment
2 the methods carry out) advantage with single method (extraction of single enzymatic isolation method, single imitation biochemistry extract), the present invention uses simultaneously
Three kinds of methods have carried out the extraction of Blackfungus polyhexose, and have carried out data analysis to the yield and content of Blackfungus polyhexose, as a result
It is as shown in table 2 below：
The yield and content of Blackfungus polyhexose that table 2 is extracted using distinct methods
| Serial number | 1 | 2 | 3 |
| Technology | Single enzymatic isolation method extraction | Single imitation biochemistry extraction | The biomimetic method joint enzymatic isolation method of the present invention |
| Recovery rate % | 9~14% | 11~16% | 14~24% |
| Polyoses content % | 10~16% | 25~29% | 34~40% |
As can be seen that extracting Blackfungus polyhexose compared to the method using single enzymolysis and single imitation biochemistry from upper table 2
Content, the method for biomimetic method using the present invention joint enzymatic isolation method, which is extracted to obtain Blackfungus polyhexose content, is nearly higher by one times.
By the two technical tie-up, the recovery rate for implementing the method for the present invention is apparently higher than single two methods, and polysaccharide yield is higher by
10% or so of single imitation biochemistry extraction process.Illustrate the method for the present invention separative efficiency height, and the Blackfungus polyhexose activity extracted
Height, purity are big.
The foregoing is merely presently preferred embodiments of the present invention, is merely illustrative for the purpose of the present invention, and not restrictive
's.All any modification, equivalent and improvement that those skilled in the art is made all within the spirits and principles of the present invention etc.,
It should include within the scope of the present invention.
Claims (9)
1. it is a kind of using biomimetic method joint enzymatic isolation method extraction Blackfungus polyhexose method, it is characterised in that according to following steps into
Row：
(1) pre-treatment of black fungus：
Black fungus raw material is cleaned, surface impurity is removed, is put into thermostatic drying chamber that drying to constant weight, is crushed, crosses 60~80 mesh sieve,
It is spare；
(2) it digests：
The black fungus dry powder of 60~80 mesh will be crushed to, be 1 according to material liquid volume ratio:30~1:80 addition water are impregnated, and are soaked
Bubble is digested, hydrolysis temperature is after 3-5 hours according to 10%~20% addition complex cellulase of black fungus dry powder weight
30~60 DEG C, enzymolysis time is 90~150min, and 100 DEG C of enzyme-removal temperature obtains black fungus enzymolysis liquid；
(3) imitation biochemistry extracts：
It is 2.0 that above-mentioned enzymolysis liquid first is adjusted pH with hydrochloric acid solution, and temperature is controlled at 35~60 DEG C, extracts 60~150min, so
It is 8.0 to adjust pH with sodium hydroxide solution again afterwards, and temperature is controlled at 35~60 DEG C, extracts 60~150min, and filtering is filtered
Liquid；
(4) it concentrates：
The filtrate that step (3) obtains is concentrated, collects concentrate, residue is removed, obtains imitation biochemistry concentrate；
(5) alcohol precipitation：
Concentrate that step (4) is obtained and 95%w/w ethyl alcohol by volume 1:4 ratio mixes, and low temperature is protected after mixing
Left undisturbed overnight is deposited, 15min is centrifuged with 10000r/min, collects precipitation；
(6) it is freeze-dried：
The sediment that step (5) obtains is freeze-dried, as black fungus Thick many candies.
2. the method as described in claim 1, it is characterised in that the black fungus described in step (1) is to cultivate by the following method
It obtains：
(1) prepared by culture medium：
By the hardwood sawdust of 85~90 parts by weight, the wheat bran of 10~12 parts by weight, the land plaster of 1~2 parts by weight, 1~2 weight
The soya-bean cake of part and the lime water mixing of 1~2 parts by weight, it is 7~8 to adjust pH value, and water content is maintained at 60~65%, is used
In the culture medium of domestication wild black fungus；
(2) culture medium pack sterilizing：
The culture medium that step (1) is prepared is packed into Polypropylene Bag, every bag of 1~1.5kg, 100 DEG C of steam sterilizings 8 hours；
(3) it is inoculated with：
By the Bag Material of sterilizing, when material temperature drops to less than 30 DEG C, inoculation black fungus bacterial kind in transfer room or inoculating hood is moved on to；
(4) cultural hypha：
It is 20~25 DEG C that cultivation bag after inoculation, which is placed in temperature, and indoor humidity is 45%-~55%, is trained under conditions of being protected from light
It supports, treats that mycelia is white, thick, stalwartness is advisable, incubation time is 12~15 days, and it is 15~20 DEG C to adjust room temperature later, appropriate ventilation, light
According to, so that mycelium slowly adapts to external environment, treat that mycelia enters the stage of ripeness, it is 10-15 DEG C to adjust room temperature, humidity 75%~
85%th, ventilation, illumination are identical with external environment, and culture period was controlled at 55~65 days；
(5) scarfing：
Each bag of planting cuts 10-22 V-type scarfing, and deep 0.5cm, length is in 1.5~2.5cm；
(6) it transplants：
It is 30%~40% to select light transmission, and humidity is 75%~90%, and well-ventilated, ground is without ponding and covered with trees
The broad-leaf forest of sawdust moves on to the thalline in maturity period in broad-leaf forest, the growth of pendulum ground；
(7) ear management:
For produce agaric humid control 90%~95%, temperature, ventilation, sunlight and broad-leaf forest are consistent；
(8) ear is adopted：
When auricle is sufficiently spread out, edge wrinkles thinning softening, and color and luster turns light, and the basal part of the ear is shunk, when the outside of belly does not have white powder object and
Shi Caishou.
3. method as claimed in claim 2, it is characterised in that the preparation of the culture medium described in step (1) is by 85 parts by weight
Hardwood sawdust, the wheat bran of 12 parts by weight, the land plaster of 0.5 parts by weight, the soya-bean cake of 2 parts by weight and 0.5 parts by weight it is white
Ash, with water mixing, it is 7.5 to adjust pH value, and control water content is 65%.
4. method as claimed in claim 2, it is characterised in that hardwood sawdust described in step (1) forest zone northeast
Hardwood sawdust.
5. method as claimed in claim 2, it is characterised in that the size of the Polypropylene Bag described in step (2) for 17cm ×
33cm。
6. method as claimed in claim 2, it is characterised in that the month of the transplanting described in step (6) is annual August part.
7. the method as described in claim 1, it is characterised in that the enzymolysis described in step (2) is that will to be crushed to 60~80 purposes black
Agaric dry powder is 1 according to material liquid volume ratio:50 addition water are impregnated, after impregnating 4 hours, according to black fungus dry powder weight
15% addition complex cellulase is digested, and hydrolysis temperature is 45 DEG C, enzymolysis time 120min, and 100 DEG C of enzyme-removal temperature obtains
To black fungus enzymolysis liquid.
8. the method as described in claim 1, it is characterised in that the imitation biochemistry extraction described in step (3) is by above-mentioned enzymolysis liquid elder generation
It is 2.0 to adjust pH with hydrochloric acid solution, and temperature is controlled at 45 DEG C, is extracted 120min, is then with sodium hydroxide solution section pH again
8.0, temperature is controlled at 45 DEG C, extracts 120min, and filtering obtains filtrate.
9. the method as described in claim 1, it is characterised in that the concentration described in step (4) refers to filtrate being placed in dense at 50 DEG C
Contracting 60min.
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