CN100447250C - Separation and refining process for rice bran polysaccharide - Google Patents
Separation and refining process for rice bran polysaccharide Download PDFInfo
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- CN100447250C CN100447250C CNB2006101663252A CN200610166325A CN100447250C CN 100447250 C CN100447250 C CN 100447250C CN B2006101663252 A CNB2006101663252 A CN B2006101663252A CN 200610166325 A CN200610166325 A CN 200610166325A CN 100447250 C CN100447250 C CN 100447250C
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Abstract
The invention discloses a separating purifying method of rice bran polysaccharide, which comprises the following steps: leaching the raw material through microwave auxiliary water; enzymolyzing to remove starch and protein; condensing; dialyzing to obtain the rough product of rice bran polysaccharide; purifying the product through Q-Sepharose Big Beads anion exchange column chromatography and Sepharose CL-4B gel filter column chromatography grade by grade; obtaining the single polysaccharide with purity over 90% as raw material or additive of drug, chemical , hygienic and costemics.
Description
Technical field
A kind of separation purification method of rice bran polysaccharide the present invention relates to bioseparation purification technique field, specifically about the technology of rice bran polysaccharide separation and purification.
Background technology
Rice bran polysaccharide is present in the paddy caryopsis cortex, is not to be developed one of main nutrient composition with widespread use at present as yet.Water-soluble rice bran polysaccharide is different with general equal glycan, is a kind of baroque heteroglycan, is made up of wood sugar, seminose, rhamnosyl, semi-lactosi, pectinose and glucose etc.Adopt different rice bran polysaccharide extraction processes can obtain multiple rice bran polysaccharide, they all have significant biological activity and nourishing function, the physiological function that not only has general polysaccharides and had, but also have multiple functions such as antitumor, hypoglycemic, decreasing cholesterol and enhancing immunity.
At present, more and more countries begins to pay attention to the research and development of rice bran polysaccharide in the world, and has obtained many successful experience.It is reported, U.S. paddy Creative Company and Li Pu Man in the extraction of aspects such as rice bran stabilization technology and rice bran nutrients, rice bran nutrient fibre, rice bran protein, rice bran polysaccharide, separate, the existing important breakthrough of purification technique, the level that is in a leading position in the world, multinomial technology can be promoted and be used.
In the last few years, China also attached great importance to the research and development work that rice bran and byproduct thereof is comprised products such as rice bran polysaccharide, rice bran-fiber.On March 15th, 2002, unite the emphasis and the main direction that have spelt out development of food industry in national foodstuffs industry " 15 " development program of promulgation by State Planning Commission, State Economic and Trade Commission and the Ministry of Agriculture, rationally utilize the rice bran resource, improve level of comprehensive utilization, functional food such as active development rice glycoprotein, rice bran nutritional oil, rice bran polysaccharide, food fibre and γ-Hi-Z.
Extract by microwave-assisted, improve the extraction yield of rice bran polysaccharide, and adopt Q-Sepharose Big Beads anion exchange chromatography and Sepharose CL-4B gel filtration chromatography to improve the purity of rice bran polysaccharide, do not see relevant report.
Summary of the invention
The separation purification method that the purpose of this invention is to provide a kind of rice bran polysaccharide, extract by microwave-assisted, improve the extraction yield of rice bran polysaccharide, and adopt Q-Sepharose Big Beads anion exchange chromatography and Sepharose CL-6B gel filtration chromatography to improve the purity of rice bran polysaccharide, make its raw material that can be used for medicine, chemical industry, health care nutriment and makeup or additive.
Technical scheme of the present invention: a kind of separation purification method of rice bran polysaccharide is to be raw material with the rice bran, extracts with the deionized water microwave-assisted of rice bran mass/volume than 10 times of amounts, get centrifugal back supernatant liquor and remove starch and albumen through enzymolysis, get supernatant after centrifugal, concentrate, drying obtains the rice bran polysaccharide crude product; The rice bran polysaccharide crude product is redissolved in phosphate buffer soln, carry out separation and purification through anion-exchange column and gel-filtration column successively, finally obtain the rice bran polysaccharide elaboration, its purity reaches 90%;
1. microwave-assisted water extraction: the deionized water of rice bran with 10 times of mass/volume ratios mixed, and microwave power 100-400W extracted 2-10 minute, and supernatant liquor is got in centrifugation.
2. enzymolysis: get supernatant liquor and use amylase and protease hydrolysis successively, remove starch and albumen.
3. concentrate, alcohol precipitation, freeze-drying: to 1/10 of original volume, the ethanol that adds 95% concentration in concentrated solution stirs, and leaves standstill, and collects the pure hypostasis of ethanol final concentration 60%-90% with supernatant concentration behind the enzymolysis, and lyophilize gets the rice bran polysaccharide crude product.
4. anionresin column purification: the rice bran polysaccharide crude product is dissolved in phosphate buffered saline buffer, with Q-Sepharose Big Beads anion-exchange column the rice bran polysaccharide crude product is carried out purifying then, adopt phosphate buffered saline buffer to carry out balance, use 0 respectively, 0.3,0.5 and 0.7mol/L NaCl solution, each concentration solution is used 1 column volume respectively, flow velocity with 1.5mL/min carries out stepwise elution, 0.5mol/L the NaCl eluant solution gets 2 peaks, all the other concentration NaCl eluant solutions are each got a peak, by measuring each peak sugar content, 0.3mol/L NaCl eluant solution peak P2 component has higher sugared content and lower protein content.
5. gel-filtration column purification: adopt Sepharose CL-4B gel-filtration column to carry out further purifying the P2 component of collecting, carry out wash-out with 0.05mol/L phosphate buffered saline buffer, 3 times of column volumes, flow velocity is 0.5mL/min, obtain a polysaccharide peak P21 component, detecting through high performance liquid phase is simple spike, and purity reaches 90%.
6. lyophilize: the P21 component is carried out lyophilize, is prepared into the cotton-shaped rice bran polysaccharide elaboration of white loose.It is soluble in water, is insoluble to organic solvents such as ethanol, acetone, ether, and no sweet taste, non-volatility, the aqueous solution are faint yellow transparent settled solution.
Beneficial effect of the present invention: the present invention adopts microwave-assisted extraction, enzymolysis destarching and albumen, alcohol precipitation and lyophilize extracting method to obtain the rice bran polysaccharide crude product, adopt Q-Sepharose Big Beads anion exchange chromatography and Sepharose CL-4B gel filtration chromatography to rice bran polysaccharide crude product grading purification progressively again, the rice bran polysaccharide elaboration that it obtains is a single polysaccharide, and purity reaches 90%.Craft science proposed by the invention, rationally, have an advantage quick, easy, that cost is low, yield is high, not only can save energy but also kept many active substances in the rice bran polysaccharide, improve the yield of rice bran polysaccharide to greatest extent, and helped further studying rice bran polysaccharide activity and structure activity relationship.
Description of drawings
The Q-Sepharose Big Beads anion exchange chromatography collection of illustrative plates of Fig. 1 rice bran polysaccharide.
The Sepharose CL-4B gel filtration chromatography collection of illustrative plates of Fig. 2 rice bran polysaccharide.
Embodiment
The preparation of embodiment 1 rice-bran crude polysaccharide
Pressing solid-liquid ratio 1: 10 mixes rice bran with deionized water, adopted the 400W microwave treatment 2 minutes, then with the centrifugal 20min of 3000rpm, get the α-Dian Fenmeishuixie 1h that adds pH 6.0 after the supernatant liquor, the proteolytic enzyme that adds pH 4.5 again after the centrifuging and taking supernatant liquor is removed albumen, continue centrifugal 30min behind the 1h, the gained supernatant liquor adds 95% ethanol sedimentation, and making the ethanol final concentration is 60%-90%.Second day centrifuging and taking precipitation, dialyse with the dialysis tubing of 3500 molecular weight in the dilution back, and then rotary evaporation, obtains rice-bran crude polysaccharide after the vacuum lyophilization.
Adopt Q-Sepharose Big Beads anion exchange chromatography stepwise elution rice-bran crude polysaccharide.
With the phosphate buffered saline buffer of 0.1mol/L, pH 8 as elutriant, last sample 25～30ml Crude polysaccharides sample, use 0 respectively then, 0.3,0.5 and 0.7mol/L NaCl solution, respectively use 1 column volume, with the flow velocity of 1.5mL/min at the enterprising row order section of Q-Sepharose Big Beads anion-exchange column wash-out.0.5mol/L the NaCl eluant solution gets 2 peaks, all the other concentration NaCl eluant solutions are each got a peak.Collect the P2 component that 0.3mol/L NaCl eluant solution obtains, measure each peak sugar content by the phenolsulfuric acid method, 0.3mol/LNaCl eluant solution peak has higher sugared content and lower protein content under comparing.Adopt Sepharose CL-4B gel-filtration column layer to carry out further purifying the P2 component of collecting, use the 0.05mol/L phosphate buffered saline buffer, 3 times of column volumes carry out wash-out, flow velocity is 0.5ml/min, obtain a polysaccharide peak P21 component, detecting through high performance liquid phase is simple spike, and purity reaches 90%.With the lyophilize of P21 component, be prepared into the cotton-shaped rice bran polysaccharide elaboration of white loose.
Claims (1)
1, a kind of separation purification method of rice bran polysaccharide is characterized in that processing step is:
(1) microwave-assisted water extraction: the deionized water of rice bran with 10 times of mass/volume ratios mixed, and microwave-assisted extracts, and microwave power 100-400W extracted 2-10 minute, and supernatant liquor is got in centrifugation;
(2) enzymolysis: get supernatant liquor and use amylase and protease hydrolysis successively, remove starch and albumen;
(3) concentrate, alcohol precipitation, freeze-drying: to 1/10 of original volume, the ethanol that adds 95% concentration in concentrated solution stirs, and leaves standstill, and collects the pure hypostasis of ethanol final concentration 60%-90% with supernatant concentration behind the enzymolysis, and lyophilize gets the rice bran polysaccharide crude product;
(4) anionresin column purification: the rice bran polysaccharide crude product is dissolved in phosphate buffered saline buffer, with Q-SepharoseBig Beads anion-exchange column the rice bran polysaccharide crude product is carried out purifying then, adopt phosphate buffered saline buffer to carry out balance, use 0 respectively, 0.3,0.5 and 0.7mol/L NaCl solution carries out wash-out to the rice bran polysaccharide crude product, the consumption of elutriant is 1 column volume, the flow velocity of elutriant is 1.5mL/min, 0.5mol/L the sample solution that the NaCl eluant solution obtains is after the high performance liquid phase test, its collection of illustrative plates shows two peaks, the sample solution that all the other concentration NaCl eluant solutions obtain is after the high performance liquid phase test, its collection of illustrative plates shows a peak, by measuring the sugared content of each sample solution that wash-out obtains, the sample solution P2 component that obtains with 0.3mol/L NaCl eluant solution is carried out further purifying;
(5) gel-filtration column purification: adopt Sepharose CL-4B gel-filtration column to carry out further purifying the P2 component of collecting, carry out wash-out with 0.05mol/L phosphate buffered saline buffer, 3 times of column volumes, flow velocity is 0.5mL/min, obtains sample solution P21 component;
(6) lyophilize: the P21 component is carried out lyophilize, is prepared into the cotton-shaped rice bran polysaccharide elaboration of white loose.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102212595B (en) * | 2011-04-20 | 2013-04-17 | 江南大学 | Preparation method and application of water-soluble nano grain polysaccharide |
| CN102225161A (en) * | 2011-05-12 | 2011-10-26 | 江南大学 | Method for preparing functional components of antioxidation, blood sugar reduction and cholesterol reduction by using rice bran |
| CN102511552B (en) * | 2011-12-26 | 2013-07-10 | 张树文 | Polysaccharide composite nutritional milk and preparation method thereof |
| CN103290081B (en) * | 2012-02-27 | 2015-04-08 | 潘千水 | Extraction method of rice bran active polysaccharide |
| CN102964466B (en) * | 2012-12-18 | 2014-12-10 | 江苏省中医院 | Abelmoschus manihot polysaccharide with anti-tumor activity and preparation method thereof |
| CN103319617B (en) * | 2013-06-20 | 2016-01-06 | 江苏科技大学 | Utilize method and the application of ultrasonic synergistic Enzymatic Extraction purple potato polysaccharide |
| CN103694368B (en) * | 2013-12-18 | 2015-10-28 | 河北科星药业有限公司 | The extracting method of rice bran polysaccharide |
| CN104306391A (en) * | 2014-09-18 | 2015-01-28 | 河北科星药业有限公司 | Rice bran polysaccharide oral liquid, injection and soluble powder and corresponding preparation method |
| CN108892731A (en) * | 2018-05-02 | 2018-11-27 | 金华市飞凌生物科技有限公司 | A kind of purposes of rice bran active polysaccharide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1560266A (en) * | 2004-03-08 | 2005-01-05 | 江南大学 | Preparation process of rise bran polysaccharose kind active component |
| CN1869075A (en) * | 2006-07-03 | 2006-11-29 | 卢成英 | Resin separation purification method of lentinan |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1560266A (en) * | 2004-03-08 | 2005-01-05 | 江南大学 | Preparation process of rise bran polysaccharose kind active component |
| CN1869075A (en) * | 2006-07-03 | 2006-11-29 | 卢成英 | Resin separation purification method of lentinan |
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| 微波辅助浸提米糠多糖工艺研究. 朱黎萍等.粮食与油脂，第12期. 2005 |
| 微波辅助浸提米糠多糖工艺研究. 朱黎萍等.粮食与油脂，第12期. 2005 * |
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| 米糠多糖及米糠蛋白开发应用研究. 李友林等.粮食与油脂，第2期. 1998 * |
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