CN103467618B - A kind of method of Cordyceps mycelium separation of polysaccharides purifying - Google Patents
A kind of method of Cordyceps mycelium separation of polysaccharides purifying Download PDFInfo
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- CN103467618B CN103467618B CN201310434343.4A CN201310434343A CN103467618B CN 103467618 B CN103467618 B CN 103467618B CN 201310434343 A CN201310434343 A CN 201310434343A CN 103467618 B CN103467618 B CN 103467618B
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Abstract
The method of a kind of Cordyceps mycelium separation of polysaccharides of the present invention purifying, belongs to technical field of bioengineering.After mycelium powder of sinensis petroleum ether degreasing, 95 DEG C of hot water returns extract, and centrifugal concentrating obtains Cordyceps mycelium Aqueous extracts; Be 95% ethanol precipitation with different multiples massfraction again, centrifugal collecting precipitation, dialysis, lyophilize, obtains the Cordyceps mycelium Crude polysaccharides of different components; By ionic liquid 1-butyl-3-methyl imidazolium a tetrafluoro borate ([Bmim] BF
4) and inorganic phosphate NaH
2pO
4the ionic liquid double-aqueous phase system of composition carries out extracting and separating, and the inorganic salt face dialysis of lower floor, concentrated, lyophilize, obtains the Cordyceps mycelium polysaccharide of separation and purification.The Cordyceps mycelium polysaccharide utilizing the present invention to prepare can separation and purification Cordyceps mycelium polysaccharide to greatest extent, neither affect polysaccharide natural structure and can keep again its original biological activity, and the inventive method is easy, cost is low, practicality flexibly, is beneficial to popularization, development and application.
Description
Technical field
The present invention relates to a kind of method of separation and purification Cordyceps mycelium polysaccharide from Cordyceps mycelium, belong to technical field of bioengineering.
Background technology
Cordyceps sinensis (
cordycepssinensis), have another name called Cordyceps or Cordyceps sinensis, be the distinctive a kind of famous and precious medicinal fungi of China and traditional excellent tonic product, include abundant biologically active substance, there is strengthening immunity, antitumor, the function such as delay senility.Current research is thought, polysaccharide is one of main composition of Cordyceps sinensis, and Cordyceps polysaccharide, except antitumor and immunoregulatory activity, has again hypoglycemic blood fat and suppresses diabetes effect, anti-oxidant activity, so most develops multiple product innovation, and the potentiality of novelty teabag.Due to natural cordyceps resource-constrained, price is high, cannot meet the widespread demand of people to rare worm grass resources, Cordyceps artificial fermentation is cultivated to obtain Cordyceps mycelium be that the industrialization development of Chinese caterpillar fungus provides possibility.Artificial culture Chinese caterpillar fungus thalline gathers in the crops Cordyceps mycelium for target, and at present, business-like worm grass product is Cordyceps mycelium body capsule or the healthy products that mix with other traditional Chinese medicine ingredients mainly.
Research for Cordyceps mycelium separation of polysaccharides purifying process is a lot, as ethanol precipitation, ion-exchange chromatography, gel filtration chromatography and membrane filtration etc.Wherein, column chromatography applies a kind of the most general method in separation and purification process, but purge process has length consuming time, costly, product recovery rate is low, solvent load is large and the shortcoming such as complex operation, limits suitability for industrialized production and the exploitation of Cordyceps mycelium polysaccharide.In addition, ethanol precipitation is a kind of simple and method of effective separation and purification, but protein also precipitates thereupon while extraction polysaccharide, and protein content increases with alcohol concn and increases, and this brings certain difficulty for follow-up polysaccharide purification.Therefore, a kind of method that is efficient, fast separating and purifying Cordyceps mycelium polysaccharide seems particularly important.
Ionic liquid double-aqueous phase system combines the advantage of ionic liquid and double-aqueous phase system, opens new extracting and separating system.Compared with traditional double-aqueous phase system, the ionic liquid double-aqueous phase system phase-splitting time is short, viscosity is low, extraction process not easily can recycle by emulsification and ionic liquid, these advantages just overcome the shortcoming of traditional double aqueous phase system, are widely used in the abstraction and purification of protein, microbiotic, alkaloid, medicine and small molecular organic compounds.But, about ionic liquid double-aqueous phase system in conjunction with ordinary method as the Research Literature of the fast separating and purifying such as ethanol precipitation, dialysis polysaccharide or patent have not been reported.
Summary of the invention
The problem to be solved in the present invention is to provide a kind of method of Cordyceps mycelium separation of polysaccharides purifying.
The invention provides the method for Cordyceps mycelium separation of polysaccharides purifying, do not affect structure and the biological activity of natural polysaccharide, and can as medicinal, edible (healthcare products) raw material.
The concrete steps of this technique obtain different components polysaccharide mixture by hot water extraction, ethanol precipitation from liquid fermenting gained Cordyceps mycelium, by ionic liquid double-aqueous phase system extracting and separating, dialysis, lyophilize, obtains Cordyceps mycelium polysaccharide.
A method for Cordyceps mycelium separation of polysaccharides purifying, carry out according to following step:
(1) degreasing: mycelium powder of sinensis petroleum ether degreasing process 2 times, dries naturally;
(2) hot water extraction: refluxing extraction 8h in mycelium powder of sinensis 95 DEG C of waters bath with thermostatic control after the degreasing of step (1) gained, repeats 2 times, with the centrifugal 15min of 5000rpm, collects supernatant liquor, concentrated, obtains Cordyceps mycelium Aqueous extracts, 4 DEG C of refrigerator storage, for subsequent use;
(3) ethanol precipitation: it is the alcohol settling of 95% that the Aqueous extracts of step (2) gained adds 1/5 times of massfraction, 4 DEG C of refrigerator hold over night, with the centrifugal 30min of 8000rpm, collecting precipitation, distill water dialysis 48h, lyophilize; Continuing in centrifugal gained supernatant liquor to add 1/5,2/5,3/5,1 and 2 times of massfraction is successively the ethanol of 95%, repeats above operation, obtains the Cordyceps mycelium Crude polysaccharides of different multiples alcohol settling;
(4) ionic liquid double-aqueous phase system extracting and separating: the Cordyceps mycelium Crude polysaccharides of step (3) gained is made into the aqueous solution that mass concentration is 10mg/mL, gets 1.0mL and is added to by 1.0mL1-butyl-3-methyl imidazolium tetrafluoroborate ([Bmim] BF
4), 1.25gNaH
2pO
4with in the ionic liquid double-aqueous phase system of 3.0mL water composition, fully vibration, dissolving, 25 DEG C of waters bath with thermostatic control leave standstill 30min, are separated upper and lower two-phase;
(5) dialysis and lyophilize: the inorganic salt face dialysis of lower floor, concentrated, lyophilize, obtains white flock Cordyceps mycelium polysaccharide;
(6) ionic liquid reclaims: upper strata ionic liquid phase dichloromethane extraction, reduction vaporization, reclaims ionic liquid [Bmim] BF
4.
The present invention sets up the method for Cordyceps mycelium separation of polysaccharides purifying, obtains the polysaccharide of homogeneous molecular weight, and composition is glucose, seminose and semi-lactosi, has immunomodulatory and anti-oxidant activity.Significant to studying its chemical structure, comformation in solution further and illustrating its pharmacologically active mechanism.
Accompanying drawing explanation
Fig. 1 is 1 times of volume mass mark is the GPC-MALLS color atlas that 95% alcohol settling component purifying obtains Cordyceps mycelium polysaccharide.
Embodiment
Embodiment 1:
1. degreasing: mycelium powder of sinensis loads in degreasing cloth bag, puts into 500mL apparatus,Soxhlet's, adds sherwood oil, 45 DEG C of water bath with thermostatic control degreasing 6h, repeats degreasing 1 time, pours out mycelium powder of sinensis, naturally dry.
2. hot water extraction: after the degreasing of step 1 gained, mycelium powder of sinensis is by 1:10(g/L) solid-liquid ratio add distilled water, fully after mixing, refluxing extraction 8h in 95 DEG C of waters bath with thermostatic control, repeats extraction 2 times.After extraction terminates, mixture 500mL plastic centrifuge cup, centrifugal with 5000rpm, 15min, supernatant liquor 45 DEG C of rotatory evaporator concentrating under reduced pressure, are concentrated into 1/3 of original volume, obtain Cordyceps mycelium Aqueous extracts, and 4 DEG C of refrigerator storage are for subsequent use.
3. ethanol precipitation: the massfraction that the Cordyceps mycelium Aqueous extracts of step 2 gained adds 1/5 times of precooling is 95% alcohol settling, abundant vibration, 4 DEG C of refrigerator hold over night, centrifugal with 8000rpm, 30min, collecting precipitation, water-soluble, distill water dialysis 48h, removes the small molecular weight impurities such as oligosaccharides, inorganic salt, monose, pigment, lyophilize further; It is the ethanol of 95% that centrifugal gained supernatant liquor continues to add 1/5,2/5,3/5,1 and 2 times of massfraction successively, repeats above operation, obtains the Cordyceps mycelium Crude polysaccharides of different multiples 95% alcohol settling.
4. ionic liquid double-aqueous phase system extracting and separating: the Cordyceps mycelium Crude polysaccharides of step 3 gained is water-soluble is made into the aqueous solution that mass concentration is 10mg/mL, get 1.0mL polysaccharide soln to be added in 10mL tool scale test tube, then add 1.0mL1-butyl-3-methyl imidazolium tetrafluoroborate ([Bmim] BF
4), 1.25g inorganic phosphate NaH
2pO
4with 3.0mL water, fully vibration, dissolving, 25 DEG C of waters bath with thermostatic control leave standstill 30min, are separated upper and lower two-phase.
5. dialysis and lyophilize: it is in the dialysis tubing of 8000 ~ 12000Da that step 4 gained lower floor inorganic salt are added to molecular weight cut-off, distill water dialysis 48h, removes inorganic phosphate NaH
2pO
4dialyzate 45 DEG C of concentrating under reduced pressure, lyophilize, obtains the Cordyceps mycelium polysaccharide sterling of white flock, be detected as simple spike (see figure 1) through gel permeation chromatography-multi-angle laser light scattering (GPC-MALLS), phend-sulphuric acid detects neutral sugar content more than 96%.
6. ionic liquid reclaims: the ionic liquid phase that the impurity such as protein are contained on upper strata adds certain volume methylene dichloride, is placed in separating funnel, fully vibrates, and collect methylene dichloride phase, 45 DEG C of rotatory evaporator reduction vaporizations, obtain ionic liquid [Bmim] BF
4.
Claims (1)
1. a method for Cordyceps mycelium separation of polysaccharides purifying, is characterized in that carrying out according to following step:
(1) degreasing: mycelium powder of sinensis petroleum ether degreasing process 2 times, dries naturally;
(2) hot water extraction;
(3) ethanol precipitation;
(4) ionic liquid double-aqueous phase system extracting and separating:
(5) dialysis and lyophilize: the inorganic salt face dialysis of lower floor, concentrated, lyophilize, obtains white flock Cordyceps mycelium polysaccharide;
(6) ionic liquid reclaims: upper strata ionic liquid phase dichloromethane extraction, reduction vaporization, reclaims ionic liquid [Bmim] BF
4;
Wherein step (2) hot water extraction step is as follows: refluxing extraction 8h in mycelium powder of sinensis 95 DEG C of waters bath with thermostatic control after the degreasing of step (1) gained, repeats 2 times, with the centrifugal 15min of 5000rpm, collect supernatant liquor, concentrated, obtain Cordyceps mycelium Aqueous extracts, 4 DEG C of refrigerator storage, for subsequent use;
Wherein step (3) ethanol precipitation step is as follows: it is the alcohol settling of 95% that the Aqueous extracts of step (2) gained adds 1/5 times of massfraction, 4 DEG C of refrigerator hold over night, with the centrifugal 30min of 8000rpm, and collecting precipitation, distill water dialysis 48h, lyophilize; Continuing in centrifugal gained supernatant liquor to add 1/5,2/5,3/5,1 and 2 times of massfraction is successively the ethanol of 95%, repeats above operation, obtains the Cordyceps mycelium Crude polysaccharides of different multiples alcohol settling;
Wherein step (4) ionic liquid double-aqueous phase system extracting and separating step is as follows: the Cordyceps mycelium Crude polysaccharides of step (3) gained is made into the aqueous solution that mass concentration is 10mg/mL, gets 1.0mL polysaccharide soln and joins by 1.0mL1-butyl-3-methyl imidazolium tetrafluoroborate ([Bmim] BF
4), 1.25gNaH
2pO
4with in the ionic liquid double-aqueous phase system of 3.0mL water composition, fully vibration, dissolving, 25 DEG C of waters bath with thermostatic control leave standstill 30min, are separated upper and lower two-phase.
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| CN104007201A (en) * | 2014-05-29 | 2014-08-27 | 华南理工大学 | Analysis method for measuring carbohydrate composition of polysaccharide |
| CN104861082B (en) * | 2015-06-03 | 2017-02-01 | 江苏大学 | Method for separating polysaccharide and protein by using choline ionic liquid two-phase aqueous system |
| CN106511401A (en) * | 2015-09-09 | 2017-03-22 | 遵义医学院附属医院 | Preparation method and application of cordyceps taii mycelia polysaccharides |
| CN110156902A (en) * | 2017-12-26 | 2019-08-23 | 浦江县美泽生物科技有限公司 | Tuber of multiflower knotweed polysaccharide and preparation method thereof |
| CN108102003A (en) * | 2017-12-26 | 2018-06-01 | 浦江县美泽生物科技有限公司 | The preparation method of matrimony vine refined polysaccharide with antioxidation activity |
| CN110713550B (en) * | 2018-07-12 | 2022-03-15 | 浙江省亚热带作物研究所 | Method for preparing refined polysaccharide with antibacterial activity by using cordyceps culture |
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| Siumultaneous extraction and purification of aloe polysaccharides and proteins using ionic liquid based aqueous two-phase system coupled with dialysis membrane;Tan et al.;《Desalination》;20111222;第389页第2栏第3段、第390页第1栏第3段、第391页表2、393页结论部分 * |
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