CN104045727A - Method for preparing inonotus obliquus (Fr.)Pilat crude polysaccharides by utilizing resin AB-8 with weak polarity - Google Patents
Method for preparing inonotus obliquus (Fr.)Pilat crude polysaccharides by utilizing resin AB-8 with weak polarity Download PDFInfo
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 137
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 137
- 150000004676 glycans Chemical class 0.000 title claims abstract description 135
- 238000000034 method Methods 0.000 title claims abstract description 55
- 239000011347 resin Substances 0.000 title claims abstract description 40
- 229920005989 resin Polymers 0.000 title claims abstract description 40
- 241000414067 Inonotus obliquus Species 0.000 title claims abstract description 13
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 25
- 150000003384 small molecules Chemical class 0.000 claims abstract description 18
- 239000002994 raw material Substances 0.000 claims abstract description 12
- 238000003809 water extraction Methods 0.000 claims abstract description 12
- 238000005238 degreasing Methods 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 35
- 239000000243 solution Substances 0.000 claims description 28
- 241000747105 Fuscoporia Species 0.000 claims description 24
- 239000007864 aqueous solution Substances 0.000 claims description 24
- 238000002203 pretreatment Methods 0.000 claims description 20
- 239000000049 pigment Substances 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 239000003463 adsorbent Substances 0.000 claims description 15
- 238000007670 refining Methods 0.000 claims description 14
- 238000009835 boiling Methods 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 239000012153 distilled water Substances 0.000 claims description 11
- 238000004108 freeze drying Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 8
- 241001558929 Sclerotium <basidiomycota> Species 0.000 claims description 7
- 229920002678 cellulose Polymers 0.000 claims description 7
- 239000001913 cellulose Substances 0.000 claims description 7
- 238000004587 chromatography analysis Methods 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 238000005507 spraying Methods 0.000 claims description 7
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 5
- 235000019441 ethanol Nutrition 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 238000004042 decolorization Methods 0.000 abstract description 2
- 239000012535 impurity Substances 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 abstract description 2
- 238000009777 vacuum freeze-drying Methods 0.000 abstract 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 11
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 230000003247 decreasing effect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
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- 239000007787 solid Substances 0.000 description 2
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- 238000009010 Bradford assay Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
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Abstract
The invention discloses a method for preparing inonotus obliquus (Fr.)Pilat crude polysaccharides by utilizing resin AB-8 with weak polarity. The method comprises the following steps: A. carrying out degreasing pretreatment on raw materials; B. carrying out hot water extraction to prepare crude polysaccharides; C. carrying out enzymolysis on the crude polysaccharides; D. carrying out treatment with the macroporous adsorption resin AB-8; E. carrying out ultrafiltration to remove small molecules; F. carrying out vacuum freeze drying, thus preparing the inonotus obliquus (Fr.)Pilat polysaccharides. The discolored refined inonotus obliquus (Fr.)Pilat polysaccharides from which most protein and small molecule impurities are removed, which are finally obtained by using the process, have higher polysaccharide retention rates and high decolorization rates and protein removal rates. The method is suitable for industrially producing the refined inonotus obliquus (Fr.)Pilat polysaccharides in a large scale.
Description
Technical field
The invention belongs to biological technical field, specifically relate to the method for a kind of AB-8 of utilization low-pole resin for Phaeopoms obliquus Crude polysaccharides.
Background technology
Phaeopoms obliquus [
inonotus obliquus(Fr.) Pilat] be a famous Wild Medicinal fungi among the people.Research shows, Fuscoporia obliqua polysaccharide is one of important activeconstituents, has the immunomodulatory of enhancing, the multiple pharmacologically active such as antitumor, anti-oxidant, hypoglycemic and antiviral, become one of hot fields of domestic and international research.But the research of the separation and purification to Fuscoporia obliqua polysaccharide report is relatively less at present.
Due to the composition complicacy of polysaccharide, the molecular weight distribution of polysaccharide crude extract is wider, polysaccharide kind is also a lot, and previous experiments shows, in Phaeopoms obliquus Crude polysaccharides after extraction, contain a certain amount of albumen and pigment, affect chemical structure and the bioactivity research of polysaccharide, be therefore necessary polysaccharide to carry out separation and purification.But the albumen in removal Crude polysaccharides and pigment, be a great problem in separation of polysaccharides purifying always, traditional Deproteinization and method complex operation, the efficiency of discoloring plain are low.Traditional Deproteinization is taking Sevage method as main, method complex operation, and efficiency is low, and the organic solvent of employing is toxic thereby affect the biological activity of polysaccharide; It is better that activated carbon method is removed pigment effect, but the active carbon powder in later stage is difficult for removal, and it is fine that hydrogen peroxide method is removed pigment effect because of stronger anti-oxidant activity, but larger to the activity influence of polysaccharide, is therefore badly in need of exploring a kind of new process for purification.
Macroporous adsorbent resin is the polymer adsorbing material that a class does not contain cation exchange groups, there is good macroporous netlike structure and larger specific surface area, have good adsorption activity, some macroporous resin has been used in the Deproteinization research in vegetable polysaccharides, has obtained good effect.
Application publication number CN102603906A(application number 201110461635.8) Chinese patent literature a kind of preparation method of the Fuscoporia obliqua polysaccharide aqueous solution is disclosed: a kind of preparation method of the Fuscoporia obliqua polysaccharide aqueous solution, comprise the following steps: trichoroacetic acid(TCA) deproteinated: in Phaeopoms obliquus extract, add trichoroacetic acid(TCA), leave standstill, then solid-liquid separation, gets and separates the solid matter obtaining; Activated carbon decolorizing: to step; Gained solid matter by volume adds the water of 50～100 times, then adds activated carbon decolorizing, and then solid-liquid separation is got gained supernatant liquor; Clarify with natural clarifying agent: gained supernatant liquor adds natural clarifying agent clarification.
Macroporous resin there is not yet bibliographical information in order to remove Phaeopoms obliquus Crude polysaccharides.
Summary of the invention
The object of this invention is to provide a kind of technique of refining Phaeopoms obliquus Crude polysaccharides; core technology of the present invention is: applying biological enzyme and anion exchange absorbing resin substitute traditional Sevage method, activated carbon method and hydrogen peroxide method and remove albumen and pigment, is applicable to mass-producing industrialization and prepares the refining polysaccharide of Phaeopoms obliquus.
Technical scheme of the present invention is as follows:
Utilize the method for AB-8 low-pole resin for Phaeopoms obliquus Crude polysaccharides, it comprises the steps: the degreasing pre-treatment of A. raw material; B. Crude polysaccharides is prepared in hot water lixiviate; C. Crude polysaccharides enzymolysis; D. macroporous adsorbent resin AB-8 processes; E. small molecules is removed in ultrafiltration; F. vacuum lyophilization; Prepare Fuscoporia obliqua polysaccharide.
Foregoing method, preferred scheme is, steps A. the degreasing pre-treatment of raw material refers to, gets dry inonotus obliquus sclerotium, pulverizes, it is preferred that the 60-95% ethanolic soln that is placed in 4-8 times of volume soaks 16-24 h(, the 70-90% ethanolic soln that is placed in 6-7 times of volume soaks 18-20 h, is more preferably, and 80% ethanolic soln that is placed in 6.5 times of volumes soaks 20 h), to remove degrease, single (widow) sugar of part and fat-soluble pigment, the shady and cool place of residue is air-dry.
Foregoing method, preferred scheme is, the lixiviate of step B. hot water is prepared Crude polysaccharides and is referred to, after pretreated residue is air-dry, adds 10-20 times of distilled water (v/w), boiling water extraction 1-3 h, 80-120 order filtered through gauze (preferred, add 13-18 times of distilled water (v/w), boiling water extraction 1.5-2.5h, 85-100 order filtered through gauze, be more preferably, add 15 times of distilled water (v/w), boiling water extraction 2 h, 100 order filtered through gauze), filter residue repeats to extract 1-3 time, and merging filtrate is concentrated, centrifugal, spraying is dried to obtain Crude polysaccharides.
Foregoing method, preferred scheme is that step C. Crude polysaccharides enzymolysis refers to, Crude polysaccharides is dissolved in water, add papoid, 35-45 DEG C of reaction 20-40min(is preferred, 36-42 DEG C of reaction 25-35min, be more preferably 40 DEG C of reaction 30min), obtain the enzymolysis solution of Crude polysaccharides.Be more preferably, after Crude polysaccharides is dissolved in water, be formulated as 0.5-2.0%(w/w) the aqueous solution of the aqueous solution (preferred, be formulated as 0.6-1.8%(w/w), be more preferably, be formulated as 1.5%(w/w) the aqueous solution).Be more preferably, papoid add-on is the 3-5%(w/w of the Crude polysaccharides aqueous solution) (preferred, add-on is the 3.5-4.5%(w/w of the Crude polysaccharides aqueous solution), be more preferably, add-on is the 4.0%(w/w of the Crude polysaccharides aqueous solution)).
Foregoing method, preferred scheme is, step D. macroporous adsorbent resin AB-8 processes and refers to, the enzymolysis solution of Crude polysaccharides adds in the AB-8 resin chromatography column that pre-treatment is good, according to AB-8 Shu Zhi ︰ Crude polysaccharides enzymolysis solution=1.2-2.0 ︰ 1(w/v), flow rate control is preferred at 1.5-2.2 ml/min(, according to AB-8 Shu Zhi ︰ Crude polysaccharides enzymolysis solution=1.5-1.8 ︰ 1(w/v), flow rate control is at 1.5-2 ml/min, be more preferably, according to AB-8 Shu Zhi ︰ Crude polysaccharides enzymolysis solution=1.6 ︰ 1(w/v), flow rate control is at 1.8ml/min), collect effluent liquid.Be more preferably, described pre-treatment refers to, soaked in absolute ethyl alcohol 24 h for macroporous adsorbent resin AB-8, and be eluted to colourlessly with dehydrated alcohol, then wash with water to without alcohol taste.
Foregoing method, preferably scheme is, step e. ultrafiltration is removed small molecules and is referred to, adopts the hollow cellulose film that ultra-filtration membrane molecular weight is 10,000 to carry out ultrafiltration, removes the small-molecule substance below 10,000.
Foregoing method, preferably scheme is, step F. vacuum lyophilization refers to, first Fuscoporia obliqua polysaccharide sample is positioned over to 3 h at-20 DEG C, then proceed at-80 DEG C more than freezing 6 h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
The invention discloses the refining polysaccharide of a kind of Phaeopoms obliquus and preparation technology thereof, preparation technology is as follows: 1, the pre-treatment of raw material; 2, the stripping of Fuscoporia obliqua polysaccharide; 3, protease hydrolysis; 4, resin anion(R.A) processing; 5, ultrafiltration, then dry.Use this technique finally to obtain the refining polysaccharide of Phaeopoms obliquus decolouring and that remove most of albumen and small molecular weight impurity, obtain more much higher sugared retention rate, percent of decolourization and albumen decreasing ratio high, and applicable industrialized scaleization is produced the refining polysaccharide of Phaeopoms obliquus.
Embodiment
Below in conjunction with embodiment, technical solution of the present invention is encyclopaedized, but protection domain is not by this restriction.Raw materials usedly in embodiment all can obtain from market.
embodiment 1utilize the method for AB-8 low-pole resin for Phaeopoms obliquus Crude polysaccharides, comprise the steps:
A. the degreasing pre-treatment of raw material: get dry inonotus obliquus sclerotium, pulverize, 60% ethanolic soln that is placed in 4 times of volumes soaks 16 h, to remove degrease, single (widow) sugar of part and fat-soluble pigment, the shady and cool place of residue is air-dry.
B. Crude polysaccharides is prepared in hot water lixiviate: after pretreated residue is air-dry, add 10 times of distilled water (v/w), and boiling water extraction 1 h, 80 order filtered through gauze, filtrate is concentrated, centrifugal, and spraying is dried to obtain Crude polysaccharides.
C. Crude polysaccharides enzymolysis: Crude polysaccharides is dissolved in water, is formulated as 0.5%(w/w) the aqueous solution, add papoid, papoid add-on is the 3%(w/w of the Crude polysaccharides aqueous solution), 35 DEG C of reaction 20min, obtain the enzymolysis solution of Crude polysaccharides.
D. macroporous adsorbent resin AB-8 processes and refers to, the enzymolysis solution of Crude polysaccharides adds pre-treatment (macroporous adsorbent resin AB-8 soaked in absolute ethyl alcohol 24 h, and be eluted to colourless with dehydrated alcohol, then wash with water to without alcohol taste) in good AB-8 resin chromatography column, according to AB-8 Shu Zhi ︰ Crude polysaccharides enzymolysis solution=1.2 ︰ 1(w/v), flow rate control, at 1.5 ml/min, is collected effluent liquid.
E. small molecules is removed in ultrafiltration: adopt the hollow cellulose film that ultra-filtration membrane molecular weight is 10,000 to carry out ultrafiltration, remove the small-molecule substance below 10,000, obtain Fuscoporia obliqua polysaccharide sample.
F. vacuum lyophilization: first Fuscoporia obliqua polysaccharide sample is positioned over to 3 h at-20 DEG C, then proceeds at-80 DEG C more than freezing 6 h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
embodiment 2utilize the method for AB-8 low-pole resin for Phaeopoms obliquus Crude polysaccharides, comprise the steps:
A. the degreasing pre-treatment of raw material: get dry inonotus obliquus sclerotium, pulverize, 95% ethanolic soln that is placed in 8 times of volumes soaks 24 h, to remove degrease, single (widow) sugar of part and fat-soluble pigment, the shady and cool place of residue is air-dry.
B. Crude polysaccharides is prepared in hot water lixiviate: after pretreated residue is air-dry, add 20 times of distilled water (v/w), and boiling water extraction 3 h, 120 order filtered through gauze, filter residue repeats to extract 2 times, and merging filtrate is concentrated, centrifugal, and spraying is dried to obtain Crude polysaccharides.
C. Crude polysaccharides enzymolysis: Crude polysaccharides is dissolved in water, is formulated as 2.0%(w/w) the aqueous solution, add papoid, papoid add-on is the 5%(w/w of the Crude polysaccharides aqueous solution), 45 DEG C of reaction 40min, obtain the enzymolysis solution of Crude polysaccharides.
D. macroporous adsorbent resin AB-8 processes: soaked in absolute ethyl alcohol 24 h for macroporous adsorbent resin AB-8, and be eluted to colourlessly with dehydrated alcohol, then wash with water to without alcohol taste.
The enzymolysis solution of Crude polysaccharides adds in the AB-8 resin chromatography column that pre-treatment is good, according to AB-8 tree fat ︰ Crude polysaccharides enzymolysis solution=2.0 ︰ 1(w/v), flow rate control, at 2.2 ml/min, is collected effluent liquid.
E. small molecules is removed in ultrafiltration: adopt the hollow cellulose film that ultra-filtration membrane molecular weight is 10,000 to carry out ultrafiltration, remove the small-molecule substance below 10,000.
F. vacuum lyophilization: first Fuscoporia obliqua polysaccharide sample is positioned over to 3 h at-20 DEG C, then proceeds at-80 DEG C more than freezing 6 h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
embodiment 3utilize the method for AB-8 low-pole resin for Phaeopoms obliquus Crude polysaccharides, it comprises the steps:
A. the degreasing pre-treatment of raw material: get dry inonotus obliquus sclerotium, pulverize, 90% ethanolic soln that is placed in 7 times of volumes soaks 20 h, to remove degrease, single (widow) sugar of part and fat-soluble pigment, the shady and cool place of residue is air-dry.
B. Crude polysaccharides is prepared in hot water lixiviate: after pretreated residue is air-dry, add 18 times of distilled water (v/w), and boiling water extraction 2.5h, 100 order filtered through gauze, filter residue repeats to extract 3 times, and merging filtrate is concentrated, centrifugal, and spraying is dried to obtain Crude polysaccharides.
C. Crude polysaccharides enzymolysis: Crude polysaccharides is dissolved in water, is formulated as 1.8%(w/w) the aqueous solution, add papoid, papoid add-on is the 4.5%(w/w of the Crude polysaccharides aqueous solution), 42 DEG C of reaction 35min, obtain the enzymolysis solution of Crude polysaccharides.
D. macroporous adsorbent resin AB-8 processes: the enzymolysis solution of Crude polysaccharides adds in the AB-8 resin chromatography column that pre-treatment (pretreatment mode and embodiment 1-2 are same) is good, according to AB-8 Shu Zhi ︰ Crude polysaccharides enzymolysis solution=1.8 ︰ 1(w/v), flow rate control, at 2 ml/min, is collected effluent liquid.
E. small molecules is removed in ultrafiltration: adopt the hollow cellulose film that ultra-filtration membrane molecular weight is 10,000 to carry out ultrafiltration, remove the small-molecule substance below 10,000.
F. vacuum lyophilization: first Fuscoporia obliqua polysaccharide sample is positioned over to 3 h at-20 DEG C, then proceeds at-80 DEG C more than freezing 6 h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
embodiment 4utilize the method for AB-8 low-pole resin for Phaeopoms obliquus Crude polysaccharides, comprise the steps:
A. the degreasing pre-treatment of raw material: get dry inonotus obliquus sclerotium, pulverize, 70% ethanolic soln that is placed in 6 times of volumes soaks 18 h, to remove degrease, single (widow) sugar of part and fat-soluble pigment, the shady and cool place of residue is air-dry.
B. Crude polysaccharides is prepared in hot water lixiviate: after pretreated residue is air-dry, add 13 times of distilled water (v/w), and boiling water extraction 1.5h, 85 order filtered through gauze, extract 1 time, and filtrate is concentrated, centrifugal, and spraying is dried to obtain Crude polysaccharides.
C. Crude polysaccharides enzymolysis: Crude polysaccharides is dissolved in water, is formulated as 0.5%(w/w) the aqueous solution, add papoid, papoid add-on is the 3.5%(w/w of the Crude polysaccharides aqueous solution), 36 DEG C of reaction 25min, obtain the enzymolysis solution of Crude polysaccharides.
D. macroporous adsorbent resin AB-8 processes: the enzymolysis solution of Crude polysaccharides adds in the AB-8 resin chromatography column that pre-treatment is good, according to AB-8 tree fat ︰ Crude polysaccharides enzymolysis solution=1.5 ︰ 1(w/v), flow rate control, at 1.5 ml/min, is collected effluent liquid.
E. small molecules is removed in ultrafiltration: adopt the hollow cellulose film that ultra-filtration membrane molecular weight is 10,000 to carry out ultrafiltration, remove the small-molecule substance below 10,000.
F. vacuum lyophilization: first Fuscoporia obliqua polysaccharide sample is positioned over to 3 h at-20 DEG C, then proceeds at-80 DEG C more than freezing 6 h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
embodiment 5utilize the method for AB-8 low-pole resin for Phaeopoms obliquus Crude polysaccharides, it comprises the steps:
A. the degreasing pre-treatment of raw material: get dry inonotus obliquus sclerotium, pulverize, 80% ethanolic soln that is placed in 6.5 times of volumes soaks 20 h, to remove degrease, part oligosaccharides and fat-soluble pigment, the shady and cool place of residue is air-dry.
B. Crude polysaccharides is prepared in hot water lixiviate: after pretreated residue is air-dry, add 15 times of distilled water (v/w), and boiling water extraction 2 h, 100 order filtered through gauze, filter residue repeats to extract 2 times, and merging filtrate is concentrated, centrifugal, and spraying is dried to obtain Crude polysaccharides.
C. Crude polysaccharides enzymolysis: Crude polysaccharides is dissolved in water, is formulated as 1.5%(w/w) the aqueous solution, add papoid, papoid add-on is the 4.0%(w/w of the Crude polysaccharides aqueous solution), 40 DEG C of reaction 30min, obtain the enzymolysis solution of Crude polysaccharides.
D. macroporous adsorbent resin AB-8 processes: the enzymolysis solution of Crude polysaccharides adds pre-treatment, and (described pre-treatment refers to, soaked in absolute ethyl alcohol 24 h for macroporous adsorbent resin AB-8, and be eluted to colourless with dehydrated alcohol, then wash with water to without alcohol taste) in good AB-8 resin chromatography column, according to AB-8 Shu Zhi ︰ Crude polysaccharides enzymolysis solution=1.6 ︰ 1(w/v), flow rate control, at 1.8ml/min, is collected effluent liquid.
E. small molecules is removed in ultrafiltration: adopt the hollow cellulose film that ultra-filtration membrane molecular weight is 10,000 to carry out ultrafiltration, remove the small-molecule substance below 10,000.
F. vacuum lyophilization: first Fuscoporia obliqua polysaccharide sample is positioned over to 3 h at-20 DEG C, then proceeds at-80 DEG C more than freezing 6 h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
test examplethe mensuration of embodiment 5 gained Fuscoporia obliqua polysaccharide polysaccharide contents and protein content, and the calculating of albumen decreasing ratio, pigment decreasing ratio and polysaccharide retention rate.The wherein content (%) of content (the %)-reducing sugar of polysaccharide content (%)=total reducing sugar.
Total sugar content is measured the phenolsulfuric acid method that adopts; Reducing sugar content is measured the DNS method that adopts; Determining the protein quantity adopts Bradford method.
In formula: A0, A
11 is respectively the front and absorbancy of rear solution at wavelength 450 nm places of decolouring of decolouring.
In formula: C
0, C
11 is respectively the front and absorbancy of rear solution at wavelength 450 nm places of decolouring of decolouring.
In formula: M0, M11 are respectively the polysaccharide content before and after processing.
Be the result comparison of the refining Phaeopoms obliquus Crude polysaccharides of Sevage method of the present invention, activated carbon method and hydrogen peroxide method, this shows that macroporous resin AB-8 has the ability of removing albumen and pigment concurrently below.See the following form:
The present invention adopts the method for the refining Phaeopoms obliquus Crude polysaccharides of AB-8 resin anion(R.A), not only can remove albumen, also have the ability of removing pigment concurrently, therefore the purity of polysaccharide of preparation is higher, compare with the Crude polysaccharides before processing, pigment decreasing ratio is 90.1%, albumen decreasing ratio is 85.1%, polysaccharide retention rate 72.1%.
Finally it should be noted that, embodiment is the embodiment of optimum of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein equal to replacement.Within the spirit and principles in the present invention all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. utilize the method for AB-8 low-pole resin for Phaeopoms obliquus Crude polysaccharides, it is characterized in that, comprise the steps: the degreasing pre-treatment of A. raw material; B. Crude polysaccharides is prepared in hot water lixiviate; C. Crude polysaccharides enzymolysis; D. macroporous adsorbent resin AB-8 processes; E. small molecules is removed in ultrafiltration; F. vacuum lyophilization; Prepare Fuscoporia obliqua polysaccharide.
2. method according to claim 1, it is characterized in that, steps A. the degreasing pre-treatment of raw material refers to, gets dry inonotus obliquus sclerotium, pulverizes, it is preferred that the 60-95% ethanolic soln that is placed in 4-8 times of volume soaks 16-24 h(, the 70-90% ethanolic soln that is placed in 6-7 times of volume soaks 18-20 h, is more preferably, and 80% ethanolic soln that is placed in 6.5 times of volumes soaks 20 h), to remove degrease, single (widow) sugar of part and fat-soluble pigment, the shady and cool place of residue is air-dry.
3. method according to claim 1, it is characterized in that, the lixiviate of step B. hot water is prepared Crude polysaccharides and is referred to, after pretreated residue is air-dry, adds 10-20 times of distilled water (v/w), boiling water extraction 1-3 h, 80-120 order filtered through gauze (preferred, add 13-18 times of distilled water (v/w), boiling water extraction 1.5-2.5h, 85-100 order filtered through gauze, be more preferably, add 15 times of distilled water (v/w), boiling water extraction 2 h, 100 order filtered through gauze), filter residue repeats to extract 1-3 time, and merging filtrate is concentrated, centrifugal, spraying is dried to obtain Crude polysaccharides.
4. method according to claim 1, it is characterized in that, step C. Crude polysaccharides enzymolysis refers to, Crude polysaccharides is dissolved in water, adds papoid, 35-45 DEG C of reaction 20-40min(is preferred, 36-42 DEG C of reaction 25-35min, be more preferably 40 DEG C of reaction 30min), obtain the enzymolysis solution of Crude polysaccharides.
5. method according to claim 4, is characterized in that, after Crude polysaccharides is dissolved in water, is formulated as 0.5-2.0%(w/w) the aqueous solution of the aqueous solution (preferred, be formulated as 0.6-1.8%(w/w), be more preferably, be formulated as 1.5%(w/w) the aqueous solution).
6. method according to claim 4, it is characterized in that, papoid add-on is the 3-5%(w/w of the Crude polysaccharides aqueous solution) (preferred, add-on is the 3.5-4.5%(w/w of the Crude polysaccharides aqueous solution), be more preferably, add-on is the 4.0%(w/w of the Crude polysaccharides aqueous solution)).
7. method according to claim 1, it is characterized in that, step D. macroporous adsorbent resin AB-8 processes and refers to, the enzymolysis solution of Crude polysaccharides adds in the AB-8 resin chromatography column that pre-treatment is good, according to AB-8 Shu Zhi ︰ Crude polysaccharides enzymolysis solution=1.2-2.0 ︰ 1(w/v), flow rate control is preferred at 1.5-2.2 ml/min(, according to AB-8 Shu Zhi ︰ Crude polysaccharides enzymolysis solution=1.5-1.8 ︰ 1(w/v), flow rate control is at 1.5-2 ml/min, be more preferably, according to AB-8 Shu Zhi ︰ Crude polysaccharides enzymolysis solution=1.6 ︰ 1(w/v), flow rate control is at 1.8ml/min), collect effluent liquid.
8. method according to claim 7, is characterized in that, described pre-treatment refers to, soaked in absolute ethyl alcohol 24 h for macroporous adsorbent resin AB-8, and be eluted to colourlessly with dehydrated alcohol, then wash with water to without alcohol taste.
9. method according to claim 1, is characterized in that, step e. ultrafiltration is removed small molecules and is referred to, adopts the hollow cellulose film that ultra-filtration membrane molecular weight is 10,000 to carry out ultrafiltration, removes the small-molecule substance below 10,000, obtains Fuscoporia obliqua polysaccharide sample.
10. method according to claim 1, is characterized in that, step F. vacuum lyophilization refers to, first Fuscoporia obliqua polysaccharide sample is positioned over to 3 h at-20 DEG C, then proceed at-80 DEG C more than freezing 6 h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
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| CN112521523A (en) * | 2020-12-15 | 2021-03-19 | 山西康欣药业有限公司 | Method for extracting and purifying inonotus obliquus polysaccharide |
| CN114009647A (en) * | 2021-11-18 | 2022-02-08 | 九愿健康产业科技(苏州)有限公司 | Preparation method of inonotus obliquus granule beverage |
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