CN109182420A - A kind of method that enzymatic modification improves hericium erinaceum polysaccharide bioactivity - Google Patents
A kind of method that enzymatic modification improves hericium erinaceum polysaccharide bioactivity Download PDFInfo
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- CN109182420A CN109182420A CN201811197925.4A CN201811197925A CN109182420A CN 109182420 A CN109182420 A CN 109182420A CN 201811197925 A CN201811197925 A CN 201811197925A CN 109182420 A CN109182420 A CN 109182420A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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Abstract
The present invention provides a kind of method that enzymatic modification improves hericium erinaceum polysaccharide bioactivity, alpha-amylase, inscribe -1 is respectively adopted, 4- 'beta '-mannase, inscribe-Isosorbide-5-Nitrae-beta galactose enzyme, inscribe -1,5- α-arabanase modify hericium erinaceum polysaccharide.The method of such enzymatic hydrolysis polysaccharide can significantly increase the immunocompetence of hericium erinaceum polysaccharide, dramatically increase the phagocytic activity of RAW264.7, the content of immune factor CD86, CD11b significantly increase；After mouse gavaging digests polysaccharide, Thymus and spleen index is dramatically increased, lymphocyte had significant proliferation, and intestinal mucosa sIg A, IL-4 and TNF- γ content significantly improve.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of enzymatic modification improves the side of hericium erinaceum polysaccharide bioactivity
Method.
Background technique
The study found that hericium erinaceum polysaccharide is in blood pressure lowering, antitumor, reducing blood lipid, anti-oxidant, anti-radiation, antiulcer, anti-aging
And have good effect on raising immunity of organisms, but its molecular weight and partial size are bigger, influence utilization of the body to it, it can
Enhance its original bioactivity by method of modifying.Hericium erinaceum polysaccharide, this method are modified using the method for enzyme degradation of polysaccharide herein
It is strong with specificity, it is energy-efficient, the characteristics of safety non-pollution.
Currently, selected modification enzyme is cellulase, alpha-amylase, hemicellulase in the research of enzyme degradation of polysaccharide
With pectase etc., the reason is that starch, cellulose, pectin are the polysaccharide in plant containing individualism in plant cell.(starch
It is a kind of mode of storage energy in plant, cellulose, pectin is the important composition ingredient of plant cell wall).And this paper
In research, before any degrading enzyme is selected in confirmation, first measured with gas chromatography the monosaccharide that contains in hericium erinaceum polysaccharide at
Point, corresponding enzyme is then selected according to the monosaccharide of hericium erinaceum polysaccharide composition, carries out enzyme digestion reaction.In addition, though there is enzyme degradation
The research of polysaccharide, but do not occur also in relation to the research of enzyme modification hericium erinaceum polysaccharide report, so this experiment will do related enzyme drop
The a series of experiments of hericium erinaceum polysaccharide is solved, and proves that the bioactivity of enzymolysis product has and increases significantly.
Summary of the invention
The purpose of the present invention is to provide a kind of methods that enzymatic modification improves hericium erinaceum polysaccharide bioactivity.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of method that enzymatic modification improves hericium erinaceum polysaccharide bioactivity, comprising the following steps:
It is dissolved in distilled water with disodium hydrogen phosphate and sodium dihydrogen phosphate, tune pH is 4.0-7.0, obtains disodium hydrogen phosphate containing 2.8wt.%
With the buffer of 3 wt.% sodium dihydrogen phosphates；36 mg of hericium erinaceum polysaccharide accurately is weighed, is configured to 4 mg/mL solution with buffer；
Taking 45 μ L enzymes with 855 μ L, pH value is that 4.0-7.0 buffer is diluted to enzyme solutions；Polysaccharide solution and enzyme solutions are mixed, respectively
1 h is reacted in 40-75 DEG C of water-bath, then 10 min of boiling water bath；Cooling 1 h, is centrifuged 3000 r/min, and 10 min take
Clear liquid saves after freeze-drying.
Remaining protein is removed with Sevage method, bag filter 24 h of dialysis take out dialyzate, add 3 times of weight
Industrial alcohol carries out alcohol precipitation 1-2 d, then collects precipitating, is centrifuged 6000 rpm, 20 min, saves after freeze-drying.
The enzyme is the alpha-amylase of 5 wt.%, inscribe -1,4- 'beta '-mannase, inscribe -1,4- beta galactose enzyme, interior
Cut -1,5- α-araban enzyme solutions.
The present invention has the advantages that
1. in the research of enzymatic modification polysaccharide, selected modification enzyme is cellulase, alpha-amylase, hemicellulase and fruit
Glue enzyme, seldom with other enzymes.This programme is in addition to alpha-amylase, and there are also inscribe-Isosorbide-5-Nitrae-'beta '-mannases, inscribe-Isosorbide-5-Nitrae-β-
Galactase, inscribe -1,5- α-arabanase are used to modified polysaccharide.
2. having many precipitatings in polysaccharide solution, these impurity may have a great impact after enzymatic hydrolysis modification.This programme is used
Sevage method removes remaining protein, dialysis, alcohol precipitation, centrifugation, and the methods of freeze-drying effectively removes impurity.
3. needing to react 12 h at a certain temperature, the time used is very long when enzymatic hydrolysis.This programme is enzyme in most thermophilic
Under conditions of degree and optimum pH, 1 h is reacted with polysaccharide, can reduce a large amount of time in this way.
Specific embodiment
Embodiment 1
36 mg of hericium erinaceum polysaccharide is taken, with the buffering of 9 mLpH, 6.5 disodium hydrogen phosphate containing 2.8wt.% and 3 wt.% sodium dihydrogen phosphates
Liquid dissolution, take 45 μ L alpha-amylases with 855 μ L, pH value be 6.5 buffers dilute；Room temperature stands 5 min, by polysaccharide solution and
Enzyme solutions mixing, reacts 1 h in 75 DEG C of water-baths respectively, then 10 min of boiling water bath, enzyme deactivation；Cooling 1 h, centrifugation 3000
R/min, 10 min, takes supernatant；Remaining protein is removed with Sevage method, bag filter 24 h of dialysis take out dialyzate, then
The industrial alcohol that 3 times of weight are added carries out 1 d of alcohol precipitation, then collects precipitating, 6000 rpm, 20 min is centrifuged, after freeze-drying
It saves.
Its particle size is detected, hericium erinaceum polysaccharide is 1895.7 nm, and the partial size of enzymolysis product is 1355.7 nm, is dropped
Low, the molecular weight in addition digesting polysaccharide is substantially reduced.After this sample to be configured to the solution of 1 mg/mL, its poison is measured by MTT
Property, find 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3.125 μ g/mL, 1.563 μ g/mL concentration when, can show
The immunocompetence for writing enhancing hericium erinaceum polysaccharide, dramatically increases the phagocytic activity of RAW264.7, immune factor CD86, CD11b's
Content significantly increases；After mouse gavaging digests polysaccharide, Thymus and spleen index is dramatically increased, lymphocyte had significant proliferation, and intestines are viscous
Film sIg A, IL-4 and TNF- γ content significantly improve.
Embodiment 2
36 mg of hericium erinaceum polysaccharide is taken, with the buffering of 9 mLpH, 7.0 disodium hydrogen phosphate containing 2.8wt.% and 3 wt.% sodium dihydrogen phosphates
Liquid dissolution, take 45 μ L inscribes-Isosorbide-5-Nitrae-'beta '-mannase with 855 μ L, pH value be 7.0 buffers dilute；Room temperature stands 5
Polysaccharide solution and enzyme solutions are mixed, react 1 h in 50 DEG C of water-baths respectively, then 10 min of boiling water bath, enzyme deactivation by min；
Cooling 1 h, is centrifuged 3000 r/min, and 10 min take supernatant；Remaining protein, bag filter dialysis are removed with Sevage method
24 h take out dialyzate, and the industrial alcohol for adding 3 times of weight carries out 2 d of alcohol precipitation, then collects precipitating, are centrifuged 6000 rpm
20 min are saved after freeze-drying.
Its particle size is detected, hericium erinaceum polysaccharide is 1895.7 nm, and the partial size of enzymolysis product is 1387.2 nm, is dropped
Low, the molecular weight in addition digesting polysaccharide is substantially reduced.After this sample to be configured to the solution of 1 mg/mL, its poison is measured by MTT
Property, find 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3.125 μ g/mL, 1.563 μ g/mL concentration when, can show
The immunocompetence for writing enhancing hericium erinaceum polysaccharide, dramatically increases the phagocytic activity of RAW264.7, immune factor CD86, CD11b's
Content significantly increases；After mouse gavaging digests polysaccharide, Thymus and spleen index is dramatically increased, lymphocyte had significant proliferation, and intestines are viscous
Film sIg A, IL-4 and TNF- γ content significantly improve.
Embodiment 3
36 mg of hericium erinaceum polysaccharide is taken, with the buffering of 9 mLpH, 4.0 disodium hydrogen phosphate containing 2.8wt.% and 3 wt.% sodium dihydrogen phosphates
Liquid dissolution, take 45 μ L inscribe-Isosorbide-5-Nitrae-beta galactose enzymes with 855 μ L, pH value be 4.0 buffers dilute；Room temperature stands 5 min,
Polysaccharide solution and enzyme solutions are mixed, react 1 h in 50 DEG C of water-baths respectively, then 10 min of boiling water bath, enzyme deactivation；Cooling 1
H, is centrifuged 3000 r/min, and 10 min take supernatant；Remaining protein is removed with Sevage method, bag filter 24 h of dialysis take
Dialyzate out, the industrial alcohol for adding 3 times of weight carry out 1 d of alcohol precipitation, then collect precipitating, are centrifuged 6000 rpm, 20 min,
It is saved after freeze-drying.
Its particle size is detected, hericium erinaceum polysaccharide is 1895.7 nm, and the partial size of enzymolysis product is 789.9 nm, hence it is evident that drop
Low, the molecular weight in addition digesting polysaccharide is substantially reduced.After this sample is configured to the solution of 1mg/mL, its poison is measured by MTT
Property, find 6.25 μ g/mL, 3.125 μ g/mL, 1.563 μ g/mL, 0.781 μ g/mL, 0.391 μ g/mL concentration when,
The immunocompetence that hericium erinaceum polysaccharide can be significantly increased dramatically increases the phagocytic activity of RAW264.7, immune factor CD86,
The content of CD11b significantly increases；After mouse gavaging digests polysaccharide, Thymus and spleen index is dramatically increased, and lymphocyte obviously increases
It grows, intestinal mucosa sIg A, IL-4 and TNF- γ content significantly improve.
Embodiment 4
36 mg of hericium erinaceum polysaccharide is taken, with the buffering of 9 mLpH, 4.0 disodium hydrogen phosphate containing 2.8wt.% and 3 wt.% sodium dihydrogen phosphates
Liquid dissolution, take 45 μ L inscribe -1,5- α-arabanase with 855 μ L, pH value be 4.0 buffers dilute；Room temperature stands 5
Polysaccharide solution and enzyme solutions are mixed, react 1h in 40 DEG C of water-baths respectively, then 10 min of boiling water bath, enzyme deactivation by min；It is cold
But 1 h, is centrifuged 3000 r/min, and 10 min take supernatant；Remaining protein, bag filter dialysis 24 are removed with Sevage method
H takes out dialyzate, and the industrial alcohol for adding 3 times of weight carries out 1 d of alcohol precipitation, then collects precipitating, is centrifuged 6000 rpm 20
Min is saved after freeze-drying.
Its particle size is detected, hericium erinaceum polysaccharide is 1895.7 nm, and the partial size of enzymolysis product is 546.9 nm, hence it is evident that drop
Low, the molecular weight in addition digesting polysaccharide is substantially reduced.After this sample is configured to the solution of 1mg/mL, its poison is measured by MTT
Property, find 6.25 μ g/mL, 3.125 μ g/mL, 1.563 μ g/mL, 0.781 μ g/mL, 0.391 μ g/mL concentration when,
The immunocompetence that hericium erinaceum polysaccharide can be significantly increased dramatically increases the phagocytic activity of RAW264.7, immune factor CD86,
The content of CD11b significantly increases；After mouse gavaging digests polysaccharide, Thymus and spleen index is dramatically increased, and lymphocyte obviously increases
It grows, intestinal mucosa sIg A, IL-4 and TNF- γ content significantly improve.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (2)
1. a kind of method that enzymatic modification improves hericium erinaceum polysaccharide bioactivity, it is characterised in that: the following steps are included: using phosphoric acid
Disodium hydrogen and sodium dihydrogen phosphate are dissolved in distilled water, and tune pH is 4.0-7.0, obtain disodium hydrogen phosphate containing 2.8wt.% and 3 wt.% phosphorus
The buffer of acid dihydride sodium；36 mg of hericium erinaceum polysaccharide accurately is weighed, is configured to 4 mg/mL solution with buffer；Take 45 μ L enzymes
It is that 4.0-7.0 buffer is diluted to enzyme solutions with 855 μ L, pH value；Polysaccharide solution and enzyme solutions are mixed, respectively at 40-75 DEG C
1 h is reacted in water-bath, then 10 min of boiling water bath；Cooling 1 h, is centrifuged 3000 r/min, and 10 min take supernatant, freezes
It is saved after drying.
2. the method that a kind of enzymatic modification according to claim 1 improves hericium erinaceum polysaccharide bioactivity, it is characterised in that:
The enzyme is the alpha-amylase of 5 wt.%, inscribe -1,4- 'beta '-mannase, inscribe -1,4- beta galactose enzyme, inscribe -1,5-
One of α-araban enzyme solutions.
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| CN201811197925.4A CN109182420A (en) | 2018-10-15 | 2018-10-15 | A kind of method that enzymatic modification improves hericium erinaceum polysaccharide bioactivity |
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| CN201811197925.4A CN109182420A (en) | 2018-10-15 | 2018-10-15 | A kind of method that enzymatic modification improves hericium erinaceum polysaccharide bioactivity |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110128563A (en) * | 2019-06-06 | 2019-08-16 | 森田药妆股份有限公司 | A kind of ferment hydrolysis pholiota nameko polysaccharide and its preparation method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101358224A (en) * | 2008-09-04 | 2009-02-04 | 浙江益圣菌物发展有限公司 | Extraction method of hericium erinaceus polysaccharide |
| CN103436571A (en) * | 2013-09-09 | 2013-12-11 | 南京农业大学 | Enzyme method modified preparation method for improving activity of lily polysaccharides |
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2018
- 2018-10-15 CN CN201811197925.4A patent/CN109182420A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101358224A (en) * | 2008-09-04 | 2009-02-04 | 浙江益圣菌物发展有限公司 | Extraction method of hericium erinaceus polysaccharide |
| CN103436571A (en) * | 2013-09-09 | 2013-12-11 | 南京农业大学 | Enzyme method modified preparation method for improving activity of lily polysaccharides |
Non-Patent Citations (3)
| Title |
|---|
| CHOONG YEW KEONG等: "Quantification and identification of polysaccharide contents in Hericium erinaceus", 《NUTRITION & FOOD SCIENCE》 * |
| 张帅 等: "酶法提取猴头菇多糖的研究", 《河南工业大学学报(自然科学版)》 * |
| 李珊珊 等: "不同提取工艺对猴头菇多糖产率、单糖组成及DPPH清除活性的影响", 《江苏农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110128563A (en) * | 2019-06-06 | 2019-08-16 | 森田药妆股份有限公司 | A kind of ferment hydrolysis pholiota nameko polysaccharide and its preparation method and application |
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Application publication date: 20190111 |