CN104497161B - A kind of method of purification of grifolan - Google Patents
A kind of method of purification of grifolan Download PDFInfo
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- CN104497161B CN104497161B CN201510003250.5A CN201510003250A CN104497161B CN 104497161 B CN104497161 B CN 104497161B CN 201510003250 A CN201510003250 A CN 201510003250A CN 104497161 B CN104497161 B CN 104497161B
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Abstract
The present invention relates to the method for purification of a kind of grifolan, Grifola frondosa pulverized, deionized water is as solvent, microwave extraction；Use HD 8 cation exchange resin deproteinization purification, polysaccharide extraction liquid dilution or concentration loading, control certain flow rate, collect effluent, obtain the higher bioactive substance grifolan of purity and purify liquid.This method technological process is short, easy and simple to handle, and efficiency is high, less energy consumption, avoid the consumption of a large amount of organic reagent, isolated and purified process can serialization, and the recyclable recycling of resin, safety and environmental protection, preparation technology process cost is low, advantageously reduces product price, is well suited for large-scale industrial production.
Description
Technical field
The present invention relates to a kind of method purifying bioactive substance from edible fungi, utilize particularly to one micro-
Ripple extracts the method that HD-8 cation exchange resin purification integrated technique purifies grifolan.
Background technology
Grifola frondosa has another name called Semen Castaneae mushroom, Flos Nelumbinis bacterium, thousand Buddhist bacterium etc., is the dietotherapeutic fungus of a kind of preciousness, taste
Deliciousness, rich in various bioactivators, mineral and vitamin etc..Its primary bioactivity material Grifola frondosa
Polysaccharide is the focus of Recent study.
Numerous pharmacology confirm with clinical trial, and grifolan has antitumor, blood pressure lowering, regulates blood glucose, blood
Lipid level, anti HIV-1 virus, strengthen the effect of function of immune system, especially anti-tumor activity, Japan doctor
Learn expert test discovery grifolan tumor control rate up to 86.2%, than internationally recognized PTS Lentinus Edodes
Polysaccharide suppression ratio is high by 32%.Cancerous cell is generated and transfer preventing and treating, hypertension, diabetes, acquired immune deficiency syndrome (AIDS) and immunity
System dysfunction all has certain curative effect, and has no side effect.Therefore, the preparation technology of grifolan is standby
Concerned, there is good DEVELOPMENT PROSPECT.
At present, domestic and international grifolan extract product is the most rarely found, more of uses water extract-alcohol precipitation work in document
Skill method is extracted and the impurity such as deproteinization and pigment, or carries with traditional extracting method such as hot water extraction method, acid
The general deproteinization purification process such as alcohol deposition method of associating such as method, alkaline extraction, Sevage method, trichloroacetic acid method etc.
Purify polysaccharide, there is the shortcoming such as operating time length, organic reagent consumption is big, efficiency is low, weak effect, polysaccharide
Yield and purity are all very restricted, and are unfavorable for that industrialized production prepares polyose.
Therefore, studying grifolan method of purification, develop simple operation, efficiently, the Grifola frondosa of low cost is many
The preparation technology of sugar, for reducing product price, increases added value of product and has great economic benefit, and full
The foot market demand, for society's gain.
Summary of the invention
It is an object of the invention to provide the purification medicine-food two-purpose fungus of the i.e. economical and efficient of a kind of simple operation, low cost
The method of grifolan, to meet industrialized production needs.This invention purifying technique is equally applicable for other foods
Use bacterium.Concrete technical scheme is as follows:
The method of purification of a kind of grifolan, it is characterised in that comprise the steps:
(1) Grifola frondosa being pulverized, the Grifola frondosa selecting particle diameter to be 20 mesh, in the deionized water that pH is 6
Carry out microwave extraction, obtain grifolan extracting solution；
(2) resin deionized water is embathed repeatedly, then soak 18～24h by NaOH solution, then use
Hydrochloric acid solution soaks 18～24h, finally embathes with deionized water, stand-by；
(3) resin that step (2) processed is carried out wet method dress post, after forming resin bed, stands overnight
Standby；Use front deionized water to cross post, resin column is activated；
(4) the grifolan extracting solution that step (1) obtains is taken load the activation tree of step (3) in right amount
Fat post, collects effluent, is deproteinization grifolan after purification and purifies liquid.
In step (1), the output of the device of microwave extraction is 500～650W, and frequency is 2450MHz,
Bubbling stirring in radiative process；Liquid-solid ratio is 20～40mL/1g, and extraction time is 60s～270s.
Microwave extraction a period of time, after extracting solution is cooled to room temperature, need to carry out weighing moisturizing, then sucking filtration.
The resin of pretreatment is separation for amino acids resin dedicated HD-8 cation exchange resin.
The method repeatedly embathed by resin deionized water in step (2) is as follows: every 5～10 points when embathing
Clock changes a water, and frequently stirs, and after changing water 4～5 times, then changed water once every 15～20 minutes, embathes
To embathing water not brown.
Described in step (2), the concentration of NaOH solution is 0.5～1mol/L, and its liquor capacity is resin volume
3～5 times, soak resin the first two hour in constantly stir, with water rinse, until soak water pH value
It is 6～7.
Described in step (2), the concentration of hydrochloric acid solution is 0.5～1mol/L, and its liquor capacity is resin volume
3～5 times, resin is changed into H+Type, after soak with hydrochloric acid, embathes with deionized water, is more than 6 to pH value
Time, can carry out filling post.
Weigh HD-8 cation exchange resin wet method upper prop pretreated for 20g, deionized water is injected chromatography
In post (1.5 × 50cm), drive air away；The HD-8 cation exchange tree that will process through step (2) again
Fat adds deionized water and mixes to suitable denseness, is fed in chromatographic column so that it is free settling；Sedimentation terminates shape
After resin bed, stand overnight standby.
80～100mL deionized waters are flow through resin column with the flow velocity of 0.5～2.5mL/min by step (4),
Repetitive operation 3 times, gets final product loading.
Described in step (4), in grifolan extracting solution, the concentration of protein is 0.15～0.5mg/mL.
The applied sample amount of described grifolan solution is 200～500mL, sample concentration: protein concentration is
0.15～05mg/mL, use Phenol sulfuric acid procedure detection polysaccharide concentration, survey with Coomassie brilliant G-250 staining
Determine protein content.
The method of the present invention, compared with other techniques, has the advantage that
(1) avoiding using organic solvent, liquid-solid ratio 30mL/g, microwave extraction, extraction ratio is up to 22.75%,
Suitable with Enzymatic Extraction rate, but microwave extraction more time-consuming (only 210s), do not use enzyme preparation, reduce
Bringing into of impurity, improves more than 50% than the yield of conventional hot water's extraction, has time-saving and efficiency, reduces power consumption,
Promote the advantages such as productivity.
(2) HD-8 cation exchange resin is that separation for amino acids is resin dedicated, purification effect and routine
Sevage method (deproteinizing rate is 37.95%, and polyoses content is 78.32%) is compared, the reservation of grifolan
Rate and deproteinizing rate all up to more than 90%, purity nearly 60%, and recyclable recycling, safety and environmental protection, and
Being applicable to continuous operation, treating capacity is big.
(3) microwave extraction HD-8 cation exchange resin purification integrated technique has purifying grifolan
Simple operation, efficiently, low cost, the advantage such as good product quality, technique is briefly conducive to industrialized production, and
And this technique can be applicable to purification and the preparation of other edible fungi polysaccharides, there is higher practical value, exploitation meaning
Justice is big.
Detailed description of the invention
Embodiment 1
Extraction purification polysaccharide from Grifola frondosa:
(1) dry Grifola frondosa is pulverized and sieved, accurately weigh the medical material 3g that particle diameter is 20 mesh, be placed in 250mL
In there-necked flask, add the deionized water of 90mL, weigh, microwave extraction 210s, it is cooled to room temperature, then claims
Heavily moisturizing, sucking filtration, i.e. obtain grifolan extracting solution.
(2) weigh 200g HD-8 resin, repeatedly embathe with deionized water 5 times, period frequently stir every
Within 15 minutes, change a water, then changed a water every 30 minutes, embathe 2 times.Then with 400mL, 1mol/L NaOH
Soak 24h, embathe 7 times with deionized water, then with the soak with hydrochloric acid 24h of 400mL, 1mol/L, spend from
Sub-water logging washes 8 times.
(3) weigh 20g and carry out wet method dress post through the resin of pretreatment, about 40mL deionized water is noted
Enter in chromatographic column (1.5 × 50cm), drive air away；Again by the HD-8 cation exchange resin through pretreatment
Add a small amount of deionized water to mix to suitable denseness, be fed in chromatographic column so that it is free settling；Sedimentation terminates
After forming resin bed, left undisturbed overnight is standby.
(4) 80mL deionized water is flow through resin column with the flow velocity of 1mL/min, operate 3 times.
(5) by 400mL, protein content is that the Grifola Frondosa sporophore polysaccharide of 0.33mg/mL microwave extraction is molten
Liquid by HD-8 cation exchange resin column, collects effluent with 1mL/min flow velocity, measures in effluent many
Sugar and protein content.
Grifolan microwave extraction yield is 22.61%, and HD-8 cation exchange resin after purification, protect by polysaccharide
Staying rate is 95.94%, and deproteinizing rate is 92.02%, and purity is 58.7%, and is tested by antioxidant activity,
Gained grifolan liquid antioxidation biology active function is good.
Embodiment 2
Extraction purification polysaccharide from Grifola frondosa:
(1) dry Grifola frondosa is pulverized and sieved, accurately weigh the medical material 3g that particle diameter is 20 mesh, be placed in 250mL
In there-necked flask, add the deionized water of 90mL, weigh, microwave extraction 180s, it is cooled to room temperature, then claims
Heavily moisturizing, sucking filtration, i.e. obtain grifolan extracting solution.
(2) weigh 200g HD-8 resin, repeatedly embathe with deionized water 5 times, period frequently stir every
Within 15 minutes, change a water, then changed a water every 30 minutes, embathe 2 times.Then with 400mL, 1mol/L NaOH
Soak 24h, embathe 7 times with deionized water, then with the soak with hydrochloric acid 24h of 400mL, 1mol/L, spend from
Sub-water logging washes 8 times.
(3) weigh 20g and carry out wet method dress post through the resin of pretreatment, about 40mL deionized water is noted
Enter in chromatographic column (1.5 × 50cm), drive air away；Again by the HD-8 cation exchange resin through pretreatment
Add a small amount of deionized water to mix to suitable denseness, be fed in chromatographic column so that it is free settling；Sedimentation terminates
After forming resin bed, left undisturbed overnight is standby.
(4) 80mL deionized water is flow through resin column with the flow velocity of 1mL/min, operate 3 times.
(5) by 400mL, protein content is the Grifola Frondosa sporophore polysaccharide of 0.309mg/mL microwave extraction
Solution by HD-8 cation exchange resin column, collects effluent with 1mL/min flow velocity, measures in effluent
Polysaccharide and protein content.
Grifolan microwave extraction yield is 21.95%, and HD-8 cation exchange resin after purification, protect by polysaccharide
Staying rate is 93.94%, and deproteinizing rate is 94.08%, and purity is 56.01%, and is tested by antioxidant activity,
Gained grifolan liquid antioxidation biology active function is good.
Embodiment 3
Extraction purification polysaccharide from Grifola frondosa:
(1) dry Grifola frondosa is pulverized and sieved, accurately weigh the medical material 3g that particle diameter is 20 mesh, be placed in 250mL
In there-necked flask, add the deionized water of 90mL, weigh, microwave extraction 210s, it is cooled to room temperature, then claims
Heavily moisturizing, sucking filtration, i.e. obtain grifolan extracting solution.
(2) weigh 200g HD-8 resin, repeatedly embathe with deionized water 5 times, period frequently stir every
Within 15 minutes, change a water, then changed a water every 30 minutes, embathe 2 times.Then with 400mL, 1mol/L NaOH
Soak 24h, embathe 7 times with deionized water, then with the soak with hydrochloric acid 24h of 400mL, 1mol/L, spend from
Sub-water logging washes 8 times.
(3) weigh 20g and carry out wet method dress post through the resin of pretreatment, about 40mL deionized water is noted
Enter in chromatographic column (1.5 × 50cm), drive air away；Again by the HD-8 cation exchange resin through pretreatment
Add a small amount of deionized water to mix to suitable denseness, be fed in chromatographic column so that it is free settling；Sedimentation terminates
After forming resin bed, left undisturbed overnight is standby.
(4) 80mL deionized water is flow through resin column with the flow velocity of 1mL/min, operate 3 times.
(5) by 300mL, protein content is the Grifola Frondosa sporophore polysaccharide of 0.248mg/mL microwave extraction
Solution by HD-8 cation exchange resin column, collects effluent with 1mL/min flow velocity, measures in effluent
Polysaccharide and protein content.
Grifolan microwave extraction yield is 22.57%, and HD-8 cation exchange resin after purification, protect by polysaccharide
Staying rate is 90.08%, and deproteinizing rate is 95.56%, and purity is 54.13%, and is tested by antioxidant activity,
Gained grifolan liquid antioxidation biology active function is good.
Embodiment 4
Extraction purification polysaccharide from Grifola frondosa:
(1) dry Grifola frondosa is pulverized and sieved, accurately weigh the medical material 3g that particle diameter is 20 mesh, be placed in 250mL
In there-necked flask, add the deionized water of 90mL, weigh, microwave extraction 210s, it is cooled to room temperature, then claims
Heavily moisturizing, sucking filtration, i.e. obtain grifolan extracting solution.
(2) weigh 200g HD-8 resin, repeatedly embathe with deionized water 5 times, period frequently stir every
Within 15 minutes, change a water, then changed a water every 30 minutes, embathe 2 times.Then with 400mL, 1mol/L NaOH
Soak 24h, embathe 7 times with deionized water, then with the soak with hydrochloric acid 24h of 400mL, 1mol/L, spend from
Sub-water logging washes 8 times.
(3) weigh 20g and carry out wet method dress post through the resin of pretreatment, about 40mL deionized water is noted
Enter in chromatographic column (1.5 × 50cm), drive air away；Again by the HD-8 cation exchange resin through pretreatment
Add a small amount of deionized water to mix to suitable denseness, be fed in chromatographic column so that it is free settling；Sedimentation terminates
After forming resin bed, left undisturbed overnight is standby.
(4) 80mL deionized water is flow through resin column with the flow velocity of 1mL/min, operate 3 times.
(5) by 500mL, protein content is the Grifola Frondosa sporophore polysaccharide of 0.149mg/mL microwave extraction
Solution by HD-8 cation exchange resin column, collects effluent with 1mL/min flow velocity, measures in effluent
Polysaccharide and protein content.
Grifolan microwave extraction yield is 22.68%, and HD-8 cation exchange resin after purification, protect by polysaccharide
Staying rate is 94.03%, and deproteinizing rate is 94.56%, and purity is 55.02%, and is tested by antioxidant activity,
Gained grifolan liquid antioxidation biology active function is good.
Claims (7)
1. the method for purification of a grifolan, it is characterised in that comprise the steps:
(1) Grifola frondosa is pulverized, the Grifola frondosa selecting particle diameter to be 20 mesh, in the deionized water that pH is 6, carries out microwave extraction,
Obtain grifolan extracting solution；
(2) resin deionized water is embathed repeatedly, then soaks 18～24h by NaOH solution, then soak 18～24h with hydrochloric acid solution,
Finally embathe with deionized water, stand-by；
(3) resin that step (2) processed is carried out wet method dress post, after forming resin bed, stands overnight standby；Use front use
Deionized water crosses post, is activated by resin column；
(4) the grifolan extracting solution that step (1) obtains taken load the activated resin post of step (3) in right amount, collect effluent,
It is deproteinization grifolan after purification and purifies liquid；
Step (2) described resin is separation for amino acids resin dedicated HD-8 cation exchange resin；
Step (3) described HD-8 cation exchange resin wet method dress post refers to inject in chromatographic column by deionized water, drives air away；Again
Through the HD-8 cation exchange resin that step (2) processes, 20g is added deionized water mix to suitable denseness, be fed into layer
In analysis post so that it is free settling is to forming resin bed.
Method of purification the most according to claim 1, it is characterised in that the output of the device of microwave extraction in step (1)
Being 500～650W, frequency is 2450MHz, bubbling stirring in radiative process；Liquid-solid ratio is 20～40mL/g, extraction time
It is 60～270s.
Method of purification the most according to claim 1, it is characterised in that described in step (2) by resin with deionized water repeatedly
The method embathed is as follows: changed a water when embathing every 15 minutes, embathes 5 times, then changed a water every 30 minutes, embathe 2
Secondary.
Method of purification the most according to claim 1, it is characterised in that the concentration of NaOH solution described in step (2) be 0.5～
1mol/L, its liquor capacity is 3～5 times of resin volume, after NaOH soaks, embathes with deionized water, until pH value is
6～7, hydrochloric acid solution immersion treatment can be carried out.
Method of purification the most according to claim 1, it is characterised in that the concentration of hydrochloric acid solution described in step (2) be 0.5～
1mol/L, its liquor capacity is 3～5 times of resin volume, and resin is changed into H+Type, after soak with hydrochloric acid, soaks with deionized water
Wash, to pH value be more than 6 time, can carry out fill post.
Method of purification the most according to claim 1, it is characterised in that deionized water described in step (3) cross post refer to 80～
100mL deionized water flows through resin column, repetitive operation 3 times with the flow velocity of 0.5～2.5mL/min, gets final product loading.
Method of purification the most according to claim 1, it is characterised in that egg in grifolan extracting solution described in step (4)
The concentration of white matter is 0.15～0.5mg/mL.
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| CN104497161B true CN104497161B (en) | 2016-09-14 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1821274A (en) * | 2006-03-17 | 2006-08-23 | 浙江工业大学 | Method for removing protein of Brazil mushroom crude polysaccharide |
| CN101914168A (en) * | 2010-09-06 | 2010-12-15 | 石河子大学 | Medical applications and preparation methods of Pleurotus ferulae Lanzi polysaccharide and composites thereof |
| CN102391387A (en) * | 2011-11-07 | 2012-03-28 | 沈阳科思高科技有限公司 | Microwave chemical extraction method of active polysaccharide in higher plants or edible and medicinal fungi |
| CN102453105A (en) * | 2010-11-02 | 2012-05-16 | 天津济命生生物科技有限公司 | Extraction method for polysaccharide |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59210901A (en) * | 1983-05-17 | 1984-11-29 | Nippon Kinoko Kenkyusho | Glucan having beta-1,6 bond-containing main chain, obtained from maitake and antineoplastic agent comprising same |
-
2015
- 2015-01-04 CN CN201510003250.5A patent/CN104497161B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1821274A (en) * | 2006-03-17 | 2006-08-23 | 浙江工业大学 | Method for removing protein of Brazil mushroom crude polysaccharide |
| CN101914168A (en) * | 2010-09-06 | 2010-12-15 | 石河子大学 | Medical applications and preparation methods of Pleurotus ferulae Lanzi polysaccharide and composites thereof |
| CN102453105A (en) * | 2010-11-02 | 2012-05-16 | 天津济命生生物科技有限公司 | Extraction method for polysaccharide |
| CN102391387A (en) * | 2011-11-07 | 2012-03-28 | 沈阳科思高科技有限公司 | Microwave chemical extraction method of active polysaccharide in higher plants or edible and medicinal fungi |
Non-Patent Citations (1)
| Title |
|---|
| "灰树花多糖提取工艺优化及多糖冲剂的研制";唐小俊等;《食用菌》;20061123(第6期);第58-60页 * |
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