CN105294796B - The method that cordycepin and Cordyceps sinensis polysaccharide are extracted from solid culture medium residue of cordyceps militaris - Google Patents
The method that cordycepin and Cordyceps sinensis polysaccharide are extracted from solid culture medium residue of cordyceps militaris Download PDFInfo
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- CN105294796B CN105294796B CN201510767564.2A CN201510767564A CN105294796B CN 105294796 B CN105294796 B CN 105294796B CN 201510767564 A CN201510767564 A CN 201510767564A CN 105294796 B CN105294796 B CN 105294796B
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Abstract
The present invention provide it is a kind of the method for cordycepin and Cordyceps sinensis polysaccharide is extracted from solid culture medium residue of cordyceps militaris, including solid culture medium residue of cordyceps militaris is dried, is crushed, water carries, micro-filtration, concentration, lyophilized and qualitative and quantitative detection.The present invention analyzes the various process conditions using solid culture medium residue of cordyceps militaris extraction Cordyceps sinensis polysaccharide and cordycepin using the methods of Orthogonal Experiment and Design, and determine optimal extracting method using solid culture medium residue of cordyceps militaris as raw material.The method of the present invention slightly carries rate height, easy to operate, can maximally utilise Chinese caterpillar fungus culture medium residue, beneficial to industrialized production, can bring good social benefit, economic benefit and environmental benefit.
Description
Technical field
The present invention relates to cordycepin and Cordyceps sinensis polysaccharide extraction process, specifically, is related to a kind of from Cordyceps militaris solid culture
The method that cordycepin and Cordyceps sinensis polysaccharide are extracted in base residue.
Background technology
Cordyceps militaris (Cordyceps militaris) also known as northern Chinese caterpillar Fungus, Cordceps militaris etc., are rare medicinal true in China
Bacterium, fruiting bodies of cordyceps militaris are the main edible, medicinal effects of Cordyceps militaris.
Solid culture mode has been widely used for the culture of Cordyceps militaris at present, and mycelia is rich in Cordyceps militaris solid medium,
But because no ripe technique carries out extraction separation to active ingredient therein, at present after culture solid medium all by
As hazardous waste, the waste of resource is not only caused, certain influence is also resulted in environment.
With the quickening pace of modern life and improvement of living standard, people increasingly focus on itself health care, Cordyceps sinensis polysaccharide tool
There are antitumor, hypoglycemic, anti-hepatic fibrosis and improve immune function, cordycepin has an antibacterial, in anti-inflammatory, anti-oxidant, adjusting human body
The Cordyceps militaris component extract such as remarkable effect, cordycepin, Cordyceps sinensis polysaccharide such as secretion and enhancing immune function of human body is in medical market
Have good prospects.At the same time as the continuous of Tissue Culture of Cordyceps militaris industry size expands, for how by the pair in incubation
Product is turned waste into wealth, and is improved input-output ratio and is increasingly attracted attention.
The content of the invention
Cordycepin and Cordyceps sinensis polysaccharide are extracted from solid culture medium residue of cordyceps militaris the object of the present invention is to provide a kind of
Method.
It is provided by the invention that cordycepin and worm are extracted from solid culture medium residue of cordyceps militaris in order to realize the object of the invention
The method of grass polysaccharide, including solid culture medium residue of cordyceps militaris is dried, is crushed, water carries, micro-filtration, concentration, lyophilized and fixed
Property quantitatively detects.
Wherein, drying steps are specially：Solid culture medium residue of cordyceps militaris is placed in hot air circulation drying oven in≤80
2h is dried under the conditions of DEG C.
Pulverising step is specially：Dry wild Oryza species residue is crushed with Universalpulverizer, crosses 120 mesh sieves.
Aqueous extraction step is specially：Into the culture medium residue after crushing plus water carries out circumfluence distillation, and material-water ratio presses g：mL
It is calculated as 1：15.Extracted in 65 DEG C 2 times, 2h/ times, extract material-water ratio every time and press g：ML is calculated as 1：15, merge No. 2 extracting solutions.
Water puies forward optimum condition by designing orthogonal experiment, respectively set Extracting temperature (respectively at 65 DEG C, 75 DEG C, 85 DEG C),
Extraction time (extracts 1 time, 2 times, 3 times) respectively, the factor such as single-trial extraction time (1h/ times, 2h/ times, 3h/ times).The worm of acquisition
Grass polysaccharide is with cordycepin optimum extraction condition：Extracted in 65 DEG C 2 times, 2h/ times, extract material-water ratio every time and press g：ML is calculated as 1：
15。
Microfiltration step is specially：The extracting solution after water carries is collected, 6000rpm centrifugation 30min is cooled to room temperature, takes supernatant
With 0.02 μm of ceramic membrane filter, its molecular cut off size is 10000-100000Da, collects filtrate.
Concentration step is specially：The mode (ultrafiltration apparatus) that filtrate is concentrated with ultrafiltration membrane is concentrated into the 1/5 of original volume, receives
Collect concentrate.
The present invention is using the method for vacuum freeze drying in -20 DEG C of lyophilized 6h.
Present invention additionally comprises to the cordycepin in freeze-dried powder and use phend-sulphuric acid to cordyceps sinensis using HPLC methods
Polysaccharide carries out the step of qualitative and quantitative detection.
After testing, about 2.92g cordycepins can be extracted from 100g solid culture medium residue of cordyceps militaris using the method for the present invention
About 4.21g Cordyceps sinensis polysaccharides.
The present invention using solid culture medium residue of cordyceps militaris as raw material, using the methods of Orthogonal Experiment and Design to utilizing Cordyceps militaris
Solid culture medium residue extracts Cordyceps sinensis polysaccharide and the various process conditions of cordycepin are analyzed, and determines optimal extraction side
Method.
The method of the present invention slightly carries rate height, easy to operate, Chinese caterpillar fungus culture medium residue can be maximally utilised, beneficial to work
Industry metaplasia is produced, and can bring good social benefit, economic benefit and environmental benefit.
Embodiment
Following embodiments are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional means that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
It is normal that solid culture medium residue of cordyceps militaris involved in following embodiments is derived from Hunan Yandi Biology Engineering Co., Ltd
Moral subsidiary.0.02 μm of ceramic membrane (molecular cut off size is 10000-100000Da) is purchased from the limited public affairs of Shanghai China film industry
Department.Ultrafiltration apparatus is purchased from Lai Te Riders ultrafiltration apparatus Co., Ltd.
The method that embodiment 1 extracts cordycepin and Cordyceps sinensis polysaccharide from solid culture medium residue of cordyceps militaris
This method includes solid culture medium residue of cordyceps militaris is dried, crushes, water carries, micro-filtration, concentration, lyophilized and fixed
Property quantitatively detects.
1st, it is dry：Solid culture medium residue of cordyceps militaris drying under conditions of≤80 DEG C using hot air circulation drying oven temperature
2h。
2nd, crush：The material of drying is crushed respectively with Universalpulverizer, crosses 120 mesh sieves.
3rd, water carries：Water is added to carry out circumfluence distillation 2 times in 65 DEG C the culture medium residue after crushing, 2h/ times, extraction every time
Material-water ratio presses g：ML is calculated as 1：15.
4th, micro-filtration：30min is centrifuged with 6000rpm after extracting solution is cooled down, then with ceramic membrane filter, collects filtrate.
5th, concentrate：Filtrate is poured into ultrafiltration apparatus and is concentrated into the 1/5 of original volume, collects concentrate.
6th, freeze：Concentrate is dried with the method (- 20 DEG C of lyophilized 6h) of vacuum freeze drying.
7th, cordycepin and Cordyceps sinensis polysaccharide carry out qualitative and quantitative detection.
The detection of cordycepin：
HPLC methods high sensitivity and influenced by sample substrate small, mainly using rp-hplc method, be bonded with octadecyl
Silica gel (C18) is stationary phase, and methanol water, methanol phosphate buffers or acetonitrile water are mobile phase, and common detector has purple
External detector (ultraviolet detector, UVD) and diode array detector (diode array detector,
DAD/photo-diode array, PDA).With citric acid, acetic acid, triethylamine mixed solution-acetonitrile (98：2, volume ratio)
For mobile phase, separated in Capcellpak Cl8 (4.6mmX 150mm, 5um) chromatographic column, UVD is detected at 260nm wavelength.
Cordycepin test limit (1imit of detection, LOD) is 0.2 μ g/mL, and the rate of recovery is 92.03%~97.35%, relatively
Standard deviation (relative standard deviation, RSD) is 0.104%.
After testing, about 2.92g cordycepins can be extracted from 100g solid culture medium residue of cordyceps militaris using this method.
The detection of Cordyceps sinensis polysaccharide：
Using phend-sulphuric acid：Polysaccharide is first hydrolyzed into monose and then rapid dehydration under the action of sulfuric acid and is generated with phenol
Orange-yellow compound, then with spectrophotometry.Phend-sulphuric acid is of less demanding to purity of polysaccharide, more for measuring cordyceps sinensis
Sugared content.Measured using spectrophotometer method at 488nm.Cordyceps sinensis polysaccharide mass concentration is in the range of 7.12~70.08 μ g/mL
Linear good, r values are 0.9995, and the rate of recovery is 95%~105%, RSD 1.09%.
After testing, about 4.21g Cordyceps sinensis polysaccharides can be extracted from 100g solid culture medium residue of cordyceps militaris using this method.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (1)
1. the method for cordycepin and Cordyceps sinensis polysaccharide is extracted from solid culture medium residue of cordyceps militaris, it is characterised in that including to pupa
Cordyceps sinensis solid culture medium residue is dried, crushes, water carries, micro-filtration, concentration, lyophilized and qualitative and quantitative detection the step of；
Drying steps are specially：Solid culture medium residue of cordyceps militaris is placed in hot air circulation drying oven under the conditions of≤80 DEG C and is dried
Dry 2h；
Pulverising step is specially：Dry wild Oryza species residue is crushed with Universalpulverizer, crosses 120 mesh sieves；
Aqueous extraction step is specially：Into the culture medium residue after crushing plus water carries out circumfluence distillation in 65 DEG C, extracts 2 times, 2h/
It is secondary, material-water ratio is extracted every time presses g：ML is calculated as 1：15, merge No. 2 extracting solutions；
Microfiltration step is specially：The extracting solution after water carries is collected, 6000rpm centrifugation 30min is cooled to room temperature, takes supernatant to use
0.02 μm of ceramic membrane filter, its molecular cut off size are 10000-100000Da, collect filtrate；
Concentration step is specially：Filtrate is concentrated into the 1/5 of original volume with the mode that ultrafiltration membrane concentrates, collects concentrate；
Using the method for vacuum freeze drying in -20 DEG C of lyophilized 6h.
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| CN107050067A (en) * | 2016-11-02 | 2017-08-18 | 仪征康海农业发展有限公司 | A kind of industrialized process for preparing of Cordyceps militaris water soluble powder |
| 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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| CN101200481A (en) * | 2006-12-15 | 2008-06-18 | 河南农业大学 | Technique for extracting cordycepin from artificial Chinese caterpillar fungus culture medium residue |
| CN101492483B (en) * | 2008-10-27 | 2012-01-18 | 东莞市生物技术研究所 | Method for extraction and purification of cordycepin, and method for preparation of cordycepin dry powder |
| CN101906127B (en) * | 2010-07-23 | 2016-03-16 | 上海国宝企业发展中心 | A kind of method extracting cordycepin and Cordyceps polysaccharide from north Chinese caterpillar fungus culture medium residue |
| CN102558264B (en) * | 2012-01-18 | 2015-06-10 | 辽宁华夏三宝生物资源科技开发有限责任公司 | Method for extracting cordycepin and cordyceps polysaccharide from cordyceps militaris |
| CN103059086A (en) * | 2012-12-20 | 2013-04-24 | 西藏金稞集团有限责任公司 | Extraction and purification method of cordycepin from cordyceps militaris solid mediums |
| CN103214533A (en) * | 2013-04-19 | 2013-07-24 | 湖南农业大学 | Method for continuously preparing cordycepin and cordyceps polysaccharide by using membrane separation technology |
| CN103467551B (en) * | 2013-09-22 | 2015-11-18 | 上海市农业科学院 | A kind of cordycepin and Cordyceps polysaccharide and preparation method thereof |
| CN104650255A (en) * | 2015-03-20 | 2015-05-27 | 海安县泓寿生物技术有限责任公司 | Method for separating and purifying cordyceps polysaccharide contained in cordyceps militaris solid culture medium |
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