CN102219842A - Dioscorea alata glycoprotein and preparation method thereof - Google Patents
Dioscorea alata glycoprotein and preparation method thereof Download PDFInfo
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- CN102219842A CN102219842A CN2011101380324A CN201110138032A CN102219842A CN 102219842 A CN102219842 A CN 102219842A CN 2011101380324 A CN2011101380324 A CN 2011101380324A CN 201110138032 A CN201110138032 A CN 201110138032A CN 102219842 A CN102219842 A CN 102219842A
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Abstract
The invention discloses a dioscorea alata glycoprotein belonging to the field of plant active substances in the phytochemistry. The glycoprotein is prepared from a dioscorea alata raw material. A preparation method comprises the steps of obtaining a crude glycoprotein by using digestion, concentration, heteroproteins removal and alcohol precipitation and obtaining the dioscorea alata glycoprotein through DEAE-52 (Diethylaminoethyl-52) cellulose column chromatography and SephadexG-75 column chromatography purification, concentration and freeze drying steps. A monosaccharide in the dioscorea alata glycoprotein disclosed by the invention comprises L-galactose, D-glucose and D-mannose; and the molecular weight of the dioscorea alata glycoprotein is about 12500-14500 Da. The glycoprotein can be used for preparing functional foods and health care products and can be also used for studying the bioactivity like a function of lowering blood sugar, a function of adjusting immunity and other physiological functions.
Description
Technical field
The present invention relates to seed ginseng potato glycoprotein and preparation method thereof, belonging to vegetable chemistry specifically is about plant function composition and foodstuff additive field.
Technical background
The ginseng potato (
Dioscorea alata L) belong to the Dioscoreaceae plant, have another name called yampi, the red potato etc. of cutting, mainly be distributed in, area such as Guangxi, Jiangxi, Hunan, Hubei, Yunnan, Guangdong, Guizhou, Sichuan.At present in Zhejiang, the existing base plantation of areas such as Jiangsu, Yunnan, Jiangxi, Shandong, resource is abundanter.Ginseng potato stem tuber is sweet flat nontoxic, is traditional dietotherapeutic plant, and tonifying spleen lung, puckery vital essence, detumescence, lenitive effect are arranged.Modern study shows the nutritive ingredient of ginseng potato content abundant starch, amino acid, VITAMIN and mineral substance etc., but also contain glycoprotein (
Glycoprotein) wait composition.Glycoprotein is that a class links form conjugated protein by carbohydrate and polypeptide or protein with covalent linkage.Modern study shows that plant glycoprotein has multiple physiologically active.Nearest Japanese nutritionist finds special physiological functions such as Dioscoreaceae sweet potato glycoprotein tool reducing cholesterol, enhancing organ immunity of organisms, minimizing mortality of hypertension.
At present, the ginseng potato is as the characteristic vegetables, with common methods as boil, stew, cooking methods such as pot eats.But the ginseng potato contains abundant glycoprotein, and the present invention extracts purifying and makes ginseng potato glycoprotein from the ginseng potato.Plant glycoprotein has multiple biological activity; as there are some researches show sweet potato glycoprotein; the nutritionist of Japan finds; sweet potato contains abundant glycoprotein; this is a kind of polysaccharide and protein mixture; human body there is special provide protection; lubricated and the elasticity of blood vessels that can keep digestive tube, respiratory tract, joint cavity, film chamber; owing to can preventing lipid material, this material on ductus arteriosus wall, deposits the arteriosclerosis that causes; can prevent the atrophy of liver and kidney and other organs reticular tissue; can the reducing human organ aging, enhance immunity power ［ 1 ］.Domestic result of study also shows, sweet potato glycoprotein (SPG) has the effect that good reducing blood-fat and strengthening immunity are ridiculed.People such as Kusano-Shuichi have reported the anti-diabetic activity of white skin sweet potato glycoprotein in 2000; And reported the anti-diabetic composition that from white skin sweet potato glycoprotein, extracts in calendar year 2001, its molecular weight is at 2.2x10
5About.The ginseng potato also contains abundant glycoprotein, and the ginseng potato is one of high-quality resource of preparation plant glycoprotein.Do not see bibliographical information about ginseng potato glycoprotein related data so far.
Reference
1, Kan Jianquan, Yan Lei, Chen Zongdao, etc. the Study immune regulation of sweet potato glycoprotein. the west
South agriculture university journal, 2000,22 (3): 257-260.
2, Zhao Li Yana work for bright, Peng Zhiying, etc. the separation of sweet potato glycoprotein, purifying and reducing blood-fat thereof
Function [J1. Food science, 2003,24 (1): 118-121.
Summary of the invention
One of purpose of the present invention provides a seed ginseng potato glycoprotein.
Two of purpose of the present invention provides a kind of preparation method of above-mentioned ginseng potato glycoprotein.
Technical scheme of the present invention
The preparation method of one seed ginseng potato glycoprotein, promptly adopt lixiviate, concentrate, foreigh protein removing, alcohol are analysed, again through DEAE-52 cellulose chromatography and Sephadex G-75 column chromatography purification, concentrate, lyophilize, final ginseng potato glycoprotein of the present invention.。But according to the different mining of raw material with different preparation methods.
Adopt fresh ginseng potato, the preparation method of ginseng potato glycoprotein comprises following preparation process:
(1), the pre-treatment of raw material
Get fresh ginseng potato, wash quiet, peeling, be cut into the long fritter of 10cm;
(2), lixiviate
In the ginseng potato raw material that step (1) is obtained, add earlier 5 times of ginseng potato quality 0.1～2.0%NaCl solution, smash the thick 20-40 of mistake mesh sieve to pieces with stamp mill, after add again 10～15 times of ginseng potato quality 0.1～2.0%NaCl solution, stir well, at 45～50 ℃ of following lixiviate 1～3h, vat liquor is with 3000～5000r/min, centrifugal 10～20 min collect supernatant liquor;
(3), concentrate
The supernatant liquor that step (2) is obtained is evaporated to 1/10～1/15 of original volume, and Concentrating Process Control pressure is-0.08～-0.1Mpa, temperature is 40～45 ℃;
(4), extraction
The chloroform and the propyl carbinol mixed solution that in the concentrated solution that step (3) obtains, add 0.5～0.8 times of volume of concentrated solution, chloroform and propyl carbinol volume ratio are 4:1 in the mixed solution, place 30～40 ℃ water bath chader, 50～100r/m, 10 min that vibrate, then with 4000～5000r/min, centrifugal 10～20min collects supernatant liquid;
The chloroform and the propyl carbinol mixed solution that in supernatant liquid, add its 0.5～0.8 times of volume again, chloroform and propyl carbinol volume ratio are 4:1 in the mixed solution, place 30～40 ℃ water bath chader again, 50-100r/m, vibration 10min, with 4000～5000r/min, centrifugal 10～20min collects final supernatant liquid;
(5), alcohol is analysed
Add 90～95% edible ethanols in the final supernatant liquid that step (4) obtains, making the alcohol final concentration in the solution is 75～85%, stir evenly to leave standstill 24h and separate out precipitation, and with 4000～5000r/min, centrifugal 10～20min, collecting precipitation;
(6), column chromatography
Obtain precipitation to step (5) and add the dissolving of 5mmol/L phosphoric acid buffer,, with 0.05mol/L NaCl wash-out, use 0.1mol/L NaCl wash-out then earlier, collect the raw sugar protein ingredient through last DEAE-52 cellulose chromatography;
Collected raw sugar protein ingredient is gone up Sephadex G-75 column chromatography deionized water wash-out again, collect ginseng potato glycoprotein fraction;
(7) concentrate
It is 20～25% concentrated thing that the ginseng potato glycoprotein fraction that step (6) is obtained is evaporated to water content, Concentrating Process Control pressure is-0.08～-0.1Mpa, temperature is 45～50 ℃;
(8), lyophilize
The thick ginseng potato glycoprotein fraction that obtains in the step (7) is carried out lyophilize, controlled temperature-20～-35 ℃, 30～60Pa vacuumizes freezing 1～2h; Temperature is raised to-5～-10 ℃, keeps 5～20 h, temperature is raised to 30～40 ℃ again and keeps 5～10h, promptly obtains ginseng potato glycoprotein finished product of the present invention.
Adopt the ginseng potato of doing, the preparation method of ginseng potato glycoprotein comprises following preparation process:
(1), the pre-treatment of raw material
Take by weighing ginseng potato dry plate, pulverize, granularity is 40～60 orders;
(2), lixiviate
Add 0.1～2.0%NaCl solution of 15～20 times of ginseng potato quality in the ginseng potato raw material that step (1) is obtained, stir evenly, under 35～50 ℃, lixiviate 1～3h, vat liquor are with 4000～5000r/min, and centrifugal 10～20 min collect supernatant liquor;
(3), concentrate
The supernatant liquor that step (2) is obtained is evaporated to 1/10～1/20 of original volume; Process control pressure is-0.08～-0.1Mpa, temperature is 40～45 ℃;
(4), extraction
The chloroform and the propyl carbinol mixed solution (chloroform and propyl carbinol volume ratio are 4:1 in the mixed solution) that in the concentrated solution that step (3) obtains, add 0.5～0.8 times of volume of concentrated solution, place water bath chader, with 30～40 ℃, 50～100r/m, 10 min that vibrate, then with 4000～5000r/min, centrifugal 10～20min collects supernatant liquid;
The chloroform and the propyl carbinol mixed solution that in supernatant liquid, add its 0.5～0.8 times of volume again, chloroform and propyl carbinol volume ratio are 4:1 in the mixed solution, place 30～40 ℃ of water bath chaders again, 50-100r/m, 10 min vibrate, with 4000～5000r/min, centrifugal 10～20min collects final supernatant liquid;
(5), alcohol is analysed
Add 90～95% edible ethanols in the supernatant liquid that step (4) obtains, making the alcohol final concentration in the solution is 75～85%, stir evenly to leave standstill 24h and separate out precipitation, and with 4000～5000r/min, centrifugal 10～20min, collecting precipitation;
(6), column chromatography
Obtain precipitation to step (5) and add the dissolving of 5mmol/L phosphoric acid buffer,,, use 0.1 mol/L NaCl wash-out then, collect the raw sugar protein ingredient earlier with 0.05 mol/L NaCl wash-out through last DEAE-52 cellulose chromatography;
Collected raw sugar protein ingredient is gone up Sephadex G-75 column chromatography deionized water wash-out again, collect glycoprotein fraction;
(7) concentrate
It is 20～25% concentrated thing that the ginseng potato glycoprotein fraction that step (6) is obtained is evaporated to water content, Concentrating Process Control pressure is-0.08～-0.1Mpa, temperature is 45～50 ℃;
(7), lyophilize
The thick glycoprotein fraction that obtains in the step (6) is carried out lyophilize, controlled temperature-20～-35 ℃, 30～60Pa vacuumizes freezing 1～2h; Temperature is raised to-5～-10 ℃, keeps 5～20h, temperature is raised to 30～40 ℃ again and keeps 5～10h, promptly obtains white cotton-shaped solid ginseng potato of the present invention glycoprotein.
The ginseng potato glycoprotein of gained of the present invention is measured with thin layer chromatography, and the result shows that the composition of monose comprises L-semi-lactosi, D-glucose, D-seminose in the ginseng potato glycoprotein.The ginseng potato glycoprotein of gained is by the molecular weight of SDS-PAGE electrophoretic analysis ginseng potato sugar egg, and its molecular weight is about 12500 Da～14500Da.The ginseng potato glycoprotein fraction of gained is meant that at 280 nm places the protein characteristic absorption value is arranged, and when detecting with the phenolsulfuric acid method simultaneously, the component of polysaccharide feature light absorption value is arranged again at 490 nm.
Beneficial effect of the present invention
Of the present inventionly obtained a kind of glycoprotein from ginseng the potato first, its molecular weight is about 12500 Da～14500Da.Its contained monose is formed and is comprised L-semi-lactosi, D-glucose, D-seminose.
In addition, ginseng potato glycoprotein provided by the invention can be used for manufacturing functional foodstuff and healthcare products.This method has been opened up the new way of ginseng potato processing, and deep processing of farm products is played a positive role.Ginseng potato glycoprotein provided by the invention also can be further in order to study its biological activity such as hypoglycemic, regulate physiological function such as immunizing power.
Description of drawings
The ginseng potato glycoprotein SDS-PAGE electrophoretogram of Fig. 1, embodiment 1 gained.
Embodiment
Below by embodiment the present invention is further set forth, but do not limit the present invention.
The plant and instrument that the present invention is used:
DEAE-52 cellulose chromatography equipment, (Britain Wha tman company);
Sephadex G-75 column chromatography equipment, (Sweden Pharmacia company);
DS-21 high-speed tissue mashing machine (Shanghai Sample Model Factory);
UV-22000 type ultraviolet spectrophotometer (You Nike instrument (Shanghai) Co., Ltd.);
DYY-22C electrophoresis apparatus (Liuyi Instruments Plant, Beijing);
R-201 rotatory evaporator (Shanghai Shen Sheng Bioisystech Co., Ltd);
Automatic Fraction Collector of BSZ one 100 types and HL one 2s type constant flow pump (going up Industrial Co., Ltd. of Nereid section)
LXB type whizzer (Shanghai medical analytical instrument factory);
Reagent used in the present invention and raw material
Lower molecular weight standard protein (Shanghai rises positive Bioisystech Co., Ltd)
Bovine serum albumin (Shanghai sunlight people Bioisystech Co., Ltd)
Other reagent is analytical pure, provides by Shanghai chemical reagents corporation of Chinese Medicine group.
The analysing and detecting method of the ginseng potato glycoprotein that the present invention is used:
The monose composition of ginseng potato glycoprotein is used the thin layer chromatography assay determination, specifically sees document: the research of monose in the beautiful tlc determination paper of the lucky Zou Ning Yao Li of the Li Ji people Wang Yan hydrolyzed solution, Mierocrystalline cellulose science and technology, 2008, (2) 43-47.
The molecular weight of ginseng potato sugar egg is measured by the SDS-PAGE electrophoretic analysis, specifically sees document: Guo Yaojun. protein electrophorese experimental technique, second edition [M]. and Beijing: Science Press, 2005,9 2-95.
Embodiment 1
A kind of preparation method of fresh ginseng potato glycoprotein comprises the steps:
(1), get fresh ginseng potato 100g, wash quiet, peeling, be cut into the long fritter of 10cm;
(2), in the ginseng potato piece that step (1) obtains, add the 0.5%NaCl solution of 500mL, smash the 0.5%NaCl aqueous solution that adds 1000mL again with stamp mill to pieces, pour extraction vessel into after stirring well, get 2h 40 ℃ of following lixiviates, obtain vat liquor.
(3), vat liquor that step (2) is obtained is with 5000r/min, centrifugal 15 min collect supernatant liquor;
(4), the supernatant liquor with step (3) is evaporated to 150mL.Adding chloroform is chloroform and the propyl carbinol mixed solution 125mL of 4:1 with propyl carbinol by volume, places 35 ℃ of water bath chaders, 80r/min 10 min that vibrate, and with 5000r/min, centrifugal 15min, collection supernatant liquid;
Adding chloroform and propyl carbinol again in supernatant liquid is chloroform and the propyl carbinol mixed solution 125mL of 4:1 by volume, places 35 ℃ of water bath chaders, 80r/min 10 min that vibrate, and with 5000r/min, centrifugal 15min collects final supernatant liquid;
(5), in the final supernatant liquor that step (4) obtains, add the 450mL90% edible ethanol, stir evenly and leave standstill 24h, with 5000r/min, centrifugal 15min, collecting precipitation;
(6), obtain precipitation to step (5) and add the dissolving of 100mL 5mmol/L phosphoric acid buffer, earlier with DEAE-52 cellulose chromatography purifying, with 500mL 0.05 mol/L NaCl wash-out, use 500mL 0.1 mol/L NaCl wash-out then, collect the raw sugar protein ingredient;
Collected raw sugar protein ingredient is used Sephadex G-75 column chromatography purification again,, collect ginseng potato glycoprotein fraction with 500 mL deionized water wash-outs;
(7) concentrate
It is 20～25% concentrated thing that the ginseng potato glycoprotein fraction that step (6) is obtained is evaporated to water content, Concentrating Process Control pressure is-0.08～-0.1Mpa, temperature is 45～50 ℃;
(8), lyophilize:
The thick glycoprotein that obtains in the step (7) is carried out lyophilize, controlled temperature-20～-35 ℃, 30 ~ 60Pa vacuumizes freezing 1 ~ 2h; Temperature is raised to-5～-10 ℃, keeps 5 ~ 20h, temperature is raised to 30 ~ 40 ℃ again and keeps 5 ~ 10h, promptly obtains seed ginseng potato sugar egg glycoprotein finished product of the present invention.
The ginseng potato glycoprotein fraction of step (6) gained is meant that at 280 nm places the protein characteristic absorption value is arranged,
When detecting with the phenolsulfuric acid method simultaneously, the component of polysaccharide feature light absorption value is arranged again at 490 nm;
Measure in the ginseng potato glycoprotein of above-mentioned gained monose with thin layer chromatography and form, the result shows that monose is formed in the ginseng potato glycoprotein and comprises L-semi-lactosi, D-glucose, D-seminose.
The molecular weight of the ginseng potato sugar egg by the above-mentioned gained of SDS-PAGE electrophoretic analysis, electrophoretogram is seen shown in Figure 1, as can be seen from Figure 1, the molecular weight of ginseng potato glycoprotein is about 14000Da.
Embodiment 2
A kind of preparation method of trepang potato glycoprotein comprises the steps:
(1), take by weighing 50g ginseng potato dry plate, after the pulverizing, cross 60 mesh sieves;
(2), in ginseng potato powder, add the 0.5%NaCl solution of 250ml, stir evenly, add 1000ml0.5%NaCl solution again and stir evenly, under 45 ℃, lixiviate 1.5h obtains vat liquor;
(3), vat liquor that step (2) is obtained is with 4000r/min, centrifugal 20 min collect supernatant liquor;
(4), the supernatant liquor that step (3) is obtained is evaporated to 125ml, adding chloroform and propyl carbinol is chloroform and the propyl carbinol mixed solution 100ml of 4:1 by volume, place 30 ~ 40 ℃ of vibrations of water bath chader, 80r/min, 10 min, with 5000r/min, centrifugal 10min collects supernatant liquid, adds chloroform and propyl carbinol again and be the chloroform of 4:1 and propyl carbinol mixed solution 100ml by volume in supernatant liquid, place 40 ℃ of water bath chaders, 80r/min, 10 min that vibrate are with 5000r/min, centrifugal 10min collects final supernatant liquid;
(5), in the final supernatant liquor that step (4) obtains, add 660 ml, 95% edible ethanol, leave standstill 24h, with 5000r/min, centrifugal 10min, collecting precipitation;
(6), obtain precipitation to step (5) and add the dissolving of 100mL 5mmol/L phosphoric acid buffer, earlier with DEAE-52 cellulose chromatography purifying, with 400mL 0.05 mol/L NaCl wash-out, use 400mL 0.1 mol/L NaCl wash-out then, collect the raw sugar protein ingredient;
Collected raw sugar protein ingredient is used Sephadex G-75 column chromatography purification again,, collect glycoprotein fraction with 400 mL deionized water wash-outs;
(7), concentrate
It is 20～25% concentrated thing that the ginseng potato glycoprotein fraction that step (6) is obtained is evaporated to water content, Concentrating Process Control pressure is-0.08～-0.1Mpa, temperature is 45～50 ℃;
(8), lyophilize: the thick glycoprotein fraction that obtains in the step (7) is carried out lyophilize, controlled temperature-20～-35 ℃, 30 ~ 60Pa vacuumizes freezing 1 ~ 2h; Temperature is raised to-5～-10 ℃, keeps 5 ~ 20 h, temperature is raised to 30 ~ 40 ℃ again and keeps 5 ~ 10 h, promptly gets ginseng potato glycoprotein finished product of the present invention.
The ginseng potato glycoprotein of above-mentioned gained is analyzed:
Measure the monose of ginseng potato glycoprotein with thin layer chromatography and form, the result shows that the composition of monose in the ginseng potato glycoprotein comprises L-semi-lactosi, D-glucose, D-seminose.
By the molecular weight of SDS-PAGE electrophoretic analysis ginseng potato sugar egg, the result shows that the molecular weight of ginseng potato glycoprotein is about 14500Da.
Above said content only is the basic explanation of the present invention under conceiving, and according to any equivalent transformation that technical scheme of the present invention is done, all should belong to protection scope of the present invention.
Claims (8)
1. a seed ginseng potato glycoprotein is characterized in that this ginseng potato glycoprotein molecule amount is about 12500Da～14500Da, and the composition of monose comprises L-semi-lactosi, D-glucose, D-seminose in this ginseng potato glycoprotein.
2. a seed ginseng potato glycoprotein as claimed in claim 1 is characterized in that this ginseng potato glycoprotein fraction has the protein characteristic absorption value at the 280nm place.
3. the preparation method of a seed ginseng potato glycoprotein as claimed in claim 1 or 2, it is characterized in that adopting lixiviate, concentrate, foreigh protein removing, alcohol analyse, again through DEAE-52 cellulose chromatography and Sephadex G-75 column chromatography purification, concentrate, lyophilize, final ginseng potato glycoprotein of the present invention.
4. the preparation method of a seed ginseng potato glycoprotein as claimed in claim 3 is characterized in that its concrete preparation process comprises the steps:
(1), the pre-treatment of raw material
Get fresh ginseng potato, wash quiet, peeling, be cut into the long fritter of 10cm;
(2), lixiviate
In the ginseng potato raw material that step (1) is obtained, add earlier 5 times of ginseng potato quality 0.1～2.0%NaCl solution, smash to pieces to 20-40 order size with stamp mill, add again 10～15 times of ginseng potato quality 0.1～2.0%NaCl solution, stir well, at 45～50 ℃ of following lixiviate 1～3h, vat liquor is with 3000～5000r/min, centrifugal 10～20 min collect supernatant liquor;
(3), concentrate
The supernatant liquor that step (2) is obtained is evaporated to 1/10～1/15 of original volume, and Concentrating Process Control pressure is-0.08～-0.1Mpa, temperature is 40～45 ℃;
(4), extraction
The chloroform and the propyl carbinol mixed solution that in the concentrated solution that step (3) obtains, add 0.5～0.8 times of volume of concentrated solution, chloroform and propyl carbinol volume ratio are 4:1 in the mixed solution, place 30～40 ℃ water bath chader, 50～100r/m, 10 min that vibrate, then with 4000～5000r/min, centrifugal 10～20min collects supernatant liquid;
The chloroform and the propyl carbinol mixed solution that in supernatant liquid, add its 0.5～0.8 times of volume again, chloroform and propyl carbinol volume ratio are 4:1 in the mixed solution, place 30～40 ℃ water bath chader again, 50-100r/m, vibration 10min, with 4000～5000r/min, centrifugal 10～20min collects final supernatant liquid;
(5), alcohol is analysed
Add 90～95% edible ethanols in the final supernatant liquid that step (4) obtains, making the alcohol final concentration in the solution is 75～85%, stir evenly to leave standstill 24h and separate out precipitation, and with 4000～5000r/min, centrifugal 10～20min, collecting precipitation;
(6), column chromatography
Obtain precipitation to step (5) and add the dissolving of 5mmol/L phosphoric acid buffer,,, use 0.1 mol/L NaCl wash-out then, collect the raw sugar protein ingredient earlier with 0.05 mol/L NaCl wash-out through last DEAE-52 cellulose chromatography;
Collected raw sugar protein ingredient is gone up Sephadex G-75 column chromatography deionized water wash-out again, collect ginseng potato glycoprotein fraction;
(7) concentrate
It is 20～25% concentrated thing that the ginseng potato glycoprotein fraction that step (6) is obtained is evaporated to water content, Concentrating Process Control pressure is-0.08～-0.1Mpa, temperature is 45～50 ℃;
(8), lyophilize
The thick ginseng potato glycoprotein fraction that obtains in the step (7) is carried out lyophilize, controlled temperature-20～-35 ℃, 30 ~ 60Pa vacuumizes freezing 1 ~ 2h; Temperature is raised to-5～-10 ℃, keeps 5 ~ 20 h, temperature is raised to 30～40 ℃ again and keeps 5～10 h, promptly obtains ginseng potato glycoprotein finished product of the present invention.
5. the preparation method of a seed ginseng potato glycoprotein as claimed in claim 4 is characterized in that its concrete preparation process comprises the steps:
(1), get fresh ginseng potato 100g, wash quiet, peeling, be cut into the long fritter of 10cm;
(2), in the ginseng potato piece that step (1) obtains, add the 0.5%NaCl solution of 500mL, smash the 0.5%NaCl aqueous solution that adds 1000mL again with stamp mill to pieces, pour extraction vessel into after stirring well, get 2h 40 ℃ of following lixiviates, obtain vat liquor;
(3), vat liquor that step (2) is obtained is with 5000r/min, centrifugal 15 min collect supernatant liquor;
(4), the supernatant liquor with step (3) is evaporated to 150mL;
Adding chloroform is chloroform and the propyl carbinol mixed solution 125mL of 4:1 with propyl carbinol by volume, places 35 ℃ of water bath chaders, 80r/min 10 min that vibrate, and with 5000r/min, centrifugal 15min, collection supernatant liquid;
Adding chloroform and propyl carbinol again in supernatant liquid is chloroform and the propyl carbinol mixed solution 125mL of 4:1 by volume, places 35 ℃ of water bath chaders, 80r/min 10 min that vibrate, and with 5000r/min, centrifugal 15min collects final supernatant liquid;
(5), in the final supernatant liquor that step (4) obtains, add the 450mL90% edible ethanol, stir evenly and leave standstill 24h, with 5000r/min, centrifugal 15min, collecting precipitation;
(6), obtain precipitation to step (5) and add the dissolving of 100mL 5mmol/L phosphoric acid buffer, earlier with DEAE-52 cellulose chromatography purifying, with 500mL 0.05 mol/L NaCl wash-out, use 500mL 0.1 mol/L NaCl wash-out then, collect the raw sugar protein ingredient;
Collected raw sugar protein ingredient is used Sephadex G-75 column chromatography purification again,, collect ginseng potato glycoprotein fraction with 500 mL deionized water wash-outs;
(7) concentrate
It is 20～25% concentrated thing that the ginseng potato glycoprotein fraction that step (6) is obtained is evaporated to water content, Concentrating Process Control pressure is-0.08～-0.1Mpa, temperature is 45～50 ℃;
(8), lyophilize
The thick glycoprotein that obtains in the step (7) is carried out lyophilize, controlled temperature-20～-35 ℃, 30 ~ 60Pa vacuumizes freezing 1 ~ 2h; Temperature is raised to-5～-10 ℃, keeps 5 ~ 20 h, temperature is raised to 30 ~ 40 ℃ again and keeps 5 ~ 10 h, promptly obtains seed ginseng potato sugar egg glycoprotein of the present invention.
6. the preparation method of a seed ginseng potato glycoprotein as claimed in claim 3 is characterized in that comprising the steps:
(1), the pre-treatment of raw material
Take by weighing ginseng potato dry plate, pulverize, granularity is 40～60 orders;
(2), lixiviate
Add 0.1～2.0%NaCl solution of 15～20 times of ginseng potato quality in the ginseng potato raw material that step (1) is obtained, stir evenly, under 35～50 ℃, lixiviate 1～3h, vat liquor are with 4000～5000r/min, and centrifugal 10～20 min collect supernatant liquor;
(3), concentrate
The supernatant liquor that step (2) is obtained is evaporated to 1/10～1/20 of original volume; Process control pressure is-0.08～-0.1Mpa, temperature is 40～45 ℃;
(4), extraction
The chloroform and the propyl carbinol mixed solution (chloroform and propyl carbinol volume ratio are 4:1 in the mixed solution) that in the concentrated solution that step (3) obtains, add 0.5～0.8 times of volume of concentrated solution, place water bath chader, with 30～40 ℃, 50～100r/m, 10 min that vibrate, then with 4000～5000r/min, centrifugal 10～20min collects supernatant liquid;
The chloroform and the propyl carbinol mixed solution that in supernatant liquid, add its 0.5～0.8 times of volume again, chloroform and propyl carbinol volume ratio are 4:1 in the mixed solution, place 30～40 ℃ of water bath chaders again, 50-100r/m, 10 min vibrate, with 4000～5000r/min, centrifugal 10～20min collects final supernatant liquid;
(5), alcohol is analysed
Add 90～95% edible ethanols in the supernatant liquid that step (4) obtains, making the alcohol final concentration in the solution is 75～85%, stir evenly to leave standstill 24h and separate out precipitation, and with 4000～5000r/min, centrifugal 10～20min, collecting precipitation;
(6), column chromatography
Obtain precipitation to step (5) and add the dissolving of 5mmol/L phosphoric acid buffer,,, use 0.1 mol/L NaCl wash-out then, collect the raw sugar protein ingredient earlier with 0.05 mol/L NaCl wash-out through last DEAE-52 cellulose chromatography;
Collected raw sugar protein ingredient is gone up Sephadex G-75 column chromatography deionized water wash-out again, collect glycoprotein fraction;
(7) concentrate
It is 20～25% concentrated thing that the ginseng potato glycoprotein fraction that step (6) is obtained is evaporated to water content, Concentrating Process Control pressure is-0.08～-0.1Mpa, temperature is 45～50 ℃;
(8), lyophilize
The thick glycoprotein fraction that obtains in the step (6) is carried out lyophilize, controlled temperature-20～-35 ℃, 30～60Pa vacuumizes freezing 1～2h; Temperature is raised to-5～-10 ℃, keeps 5～20 h, temperature is raised to 30～40 ℃ again and keeps 5～10 h, promptly obtains ginseng potato glycoprotein of the present invention.
7. the preparation method of a seed ginseng potato glycoprotein as claimed in claim 6 is characterized in that comprising the steps:
(1), take by weighing 50g ginseng potato dry plate, after the pulverizing, cross 60 mesh sieves;
(2), in ginseng potato powder, add the 0.5%NaCl solution of 250ml, stir evenly, add 1000ml0.5%NaCl solution again and stir evenly, under 45 ℃, lixiviate 1.5h obtains vat liquor;
(3), vat liquor that step (2) is obtained is with 4000r/min, centrifugal 20 min collect supernatant liquor;
(4), the supernatant liquor that step (3) is obtained is evaporated to 125ml, adding chloroform and propyl carbinol is chloroform and the propyl carbinol mixed solution 100ml of 4:1 by volume, place 30 ~ 40 ℃ of vibrations of water bath chader, 80r/min, 10 min, with 5000r/min, centrifugal 10min collects supernatant liquid, adds chloroform and propyl carbinol again and be the chloroform of 4:1 and propyl carbinol mixed solution 100ml by volume in supernatant liquid, place 40 ℃ of water bath chaders, 80r/min, 10 min that vibrate are with 5000r/min, centrifugal 10min collects final supernatant liquid;
(5), in the final supernatant liquor that step (4) obtains, add 660 ml, 95% edible ethanol, leave standstill 24h, with 5000r/min, centrifugal 10min, collecting precipitation;
(6), obtain precipitation to step (5) and add the dissolving of 100mL 5mmol/L phosphoric acid buffer, earlier with DEAE-52 cellulose chromatography purifying, with 400mL 0.05 mol/L NaCl wash-out, use 400mL 0.1 mol/L NaCl wash-out then, collect the raw sugar protein ingredient;
Collected raw sugar protein ingredient is used Sephadex G-75 column chromatography purification again,, collect glycoprotein fraction with 400mL deionized water wash-out;
(7) concentrate
It is 20～25% concentrated thing that the ginseng potato glycoprotein fraction that step (6) is obtained is evaporated to water content, Concentrating Process Control pressure is-0.08～-0.1Mpa, temperature is 40～45 ℃;
(8), lyophilize: the thick glycoprotein fraction that obtains in the step (7) is carried out lyophilize, controlled temperature-20～-35 ℃, 30～60Pa vacuumizes freezing 1～2h; Temperature is raised to-5～-10 ℃, keeps 5～20 h, temperature is raised to 30～40 ℃ again and keeps 5～10 h, promptly gets ginseng potato glycoprotein of the present invention.
8. a seed ginseng potato glycoprotein as claimed in claim 1 or 2 is used to prepare functional food and healthcare products.
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| CN106916869A (en) * | 2017-04-19 | 2017-07-04 | 中国农业科学院农产品加工研究所 | A kind of potato class active peptide and preparation method thereof |
| CN107915772A (en) * | 2016-10-08 | 2018-04-17 | 西南大学 | A kind of new sweet potato glycoprotein and the sweet potato glycoprotein extract comprising the sweet potato glycoprotein |
| WO2020119255A1 (en) * | 2018-12-10 | 2020-06-18 | 江西赣隆药业有限公司 | Dioscorea alata formula granules and preparation method therefor |
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| CN107915772A (en) * | 2016-10-08 | 2018-04-17 | 西南大学 | A kind of new sweet potato glycoprotein and the sweet potato glycoprotein extract comprising the sweet potato glycoprotein |
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