CN104278064A - Extracting method of antrodia polysaccharides - Google Patents
Extracting method of antrodia polysaccharides Download PDFInfo
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- CN104278064A CN104278064A CN201410555913.XA CN201410555913A CN104278064A CN 104278064 A CN104278064 A CN 104278064A CN 201410555913 A CN201410555913 A CN 201410555913A CN 104278064 A CN104278064 A CN 104278064A
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- tree sesame
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Abstract
The invention relates to an extracting method of antrodia polysaccharides. The extracting method comprises the following steps: firstly, using an inoculating shovel to take antrodia cultured in a slant culture medium, then putting into a triangular flask together with the culture medium, and cultivating; then, inoculating 10-20% of the cultivated antrodia in a fermentation tank for fermentation cultivation, sieving and filtering culture liquid, crushing and homogenizing filtrate, performing water bath extraction, centrifuging, performing rotary evaporation and concentration, performing alcohol precipitation, centrifuging, taking precipitate and adding distilled water the amount of which is equal to that of the precipitate to dissolve the precipitate; adding chloroform isoamyl alcohol and fiercely oscillating, adding absolute ethyl alcohol into liquid supernatant and performing reflux extraction; centrifugally separating alcohol, freeze-drying precipitate to prepare dry antrodia polysaccharides; filtering rough antrodia polysaccharides by a hollow fiber ultrafiltration membrane to obtain polysaccharides with different molecular weights, and concentrating and drying respectively to obtain a dry product of the antrodia polysaccharides. The extracting method can be used for performing cultivation, water bath extraction, concentration by centrifuging and reflux extraction on a sample in sequence, and has the advantages that the yield of polysaccharides is high, the extraction is sufficient, the polysaccharides are hardly damaged, the energy consumption is low and no pollution is caused.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of extracting method of camphor tree sesame polysaccharide.
Background technology
Camphor tree sesame (Antrodia) has another name called Antrodia camphorata, is the medicinal pore fungus of the distinctive preciousness in Taiwan, it is reported.Research display, camphor tree sesame contains various bioactivators, as camphor tree sesame polysaccharide, triterpenes, SOD, adenosine, immunoglobulin (Ig), VITAMIN, trace element, nucleic acid, hemoglutinin, amino acid, steroid, xylogen and blood pressure stabilization material etc., tool has multiple physiologically active more, as protected the liver, antitumor, anti-oxidant, improve immunity, anti-microbial pathogen and removing toxic substances etc.In Taiwan, camphor tree sesame is widely used in Adjust-blood lipid, removing toxic substances protects the liver, toxin expelling, does not have untoward reaction to occur so far.Research finds, camphor tree sesame polysaccharide is one of health factor main in camphor tree sesame, and it has effects such as improving immune, anticancer, atherosclerosis and the generation of reduction cardiocerebrovasculaevents events.At present, camphor tree sesame polysaccharide has caused the extensive concern of domestic scholars.
Recent study finds, physiologically active and its molecular weight, molecular structure and the extraction process etc. of camphor tree sesame polysaccharide are all closely related, separation-extraction technology can have on the pharmaceutical use of camphor tree sesame polysaccharide the impact really connect, current camphor tree sesame polysaccharide many employings hot water extraction, alcohol such as to analyse at the technique, not only complicated operation, and not easily obtain polysaccharide crude, and it is low to obtain sugared rate, brings difficulty to its industrial applications.For this reason, the invention provides a kind of easy and simple to handle, the extracting method of high, the camphor tree sesame polysaccharide that is suitable for suitability for industrialized production of sugared rate.
Summary of the invention
Object of the present invention be exactly for the defect existed in above-mentioned prior art provide a kind of easy and simple to handle, the extracting method of high, the camphor tree sesame polysaccharide that is suitable for suitability for industrialized production of sugared rate
Technical scheme of the present invention is as follows:
An extracting method for camphor tree sesame polysaccharide, comprises the following steps:
(1) cultivation of camphor tree sesame bacterium seed liquor: get 1-3cm with connecing swollen shovel
2slant medium in cultured band substratum camphor tree sesame bacterium load 500ml triangular flask in cultivate;
(2) the camphor tree sesame bacterium 10%-20% got after cultivating in step (1) is inoculated in fermentor tank and carries out fermentation culture, nutrient solution is crossed 40 mesh sieves, and the mycelium retained filters with 3 times of distilled water washs again, is camphor tree sesame fermented liquid under sieve;
(3) extraction of camphor tree sesame granulose: by camphor tree sesame filtering fermentation liquor filtering thalline obtained in step (2), get filtrate to add 2-3 times amount distilled water and pulverize homogenate in Multi Role Aircraft, water-bath lixiviate, centrifugal, get supernatant liquor carry out rotary evaporation concentrate, get concentrated solution under 4 DEG C of conditions, to carry out alcohol analyse, centrifugal, get precipitation add equivalent distilled water dissolve after, add 2-3 times of chloroform isoamyl alcohol thermal agitation, supernatant liquor adds 4-5 times amount dehydrated alcohol refluxing extraction 3-5 time, each 1.5-2h, centrifugation ethanol, pellet frozen is dry, obtained camphor tree sesame granulose dry product;
(4) camphor tree sesame polysaccharide crude process: adopt hollow fiber ultrafiltration membrane to filter camphor tree sesame granulose crude product obtained in step (3), obtain the polysaccharide body of different molecular weight, obtain the dry products of camphor tree sesame polysaccharide respectively after concentrate drying.
Preferably, the liquid amount in the triangular flask described in step (1) is 100-150ml.
Preferably, the culture temperature described in step (1) is 25-28 DEG C, and incubation time is 3-5d.
Preferably, the fermentation culture conditions described in step (2) is: culture temperature 25-28 DEG C, rotating speed 200-230r/min, incubation time 6-8d, air flow 120-150L/h, liquid amount: 5-7L.
Preferably, during water-bath lixiviate described in step (3), ratio of water to material is (1-3): 1; Water-bath lixiviate time be 1.5-2h, temperature is 120-135 DEG C; The service temperature that rotary evaporation concentrates is 60-65 DEG C.
Preferably, the solvent adopted in process analysed by the alcohol described in step (3) is dehydrated alcohol.
Preferably, the alcohol described in step (3) is analysed in process, and described concentrated solution and the volume ratio of solvent are (1-1.5): (4-5).
Preferably, in the chloroform isoamyl alcohol described in step (3), the volume ratio of chloroform and primary isoamyl alcohol is (4-6): (0.8-1.5).
Preferably, the centrifugation time described in step (3) is 30-60min, and centrifuging temperature is 25-28 DEG C.
Preferably, in the ultra-filtration process described in step (4), pressure is 0.2-0.5Mpa, described hollow fiber ultrafiltration membrane is 1000,100, the three assembly hollow fiber ultrafiltration membrane of 10kDa.
The present invention compared with prior art, has following beneficial effect:
1. the present invention successively adopts water-bath lixiviate, centrifugal concentrating and dehydrated alcohol refluxing extraction, fully can extract polysaccharide in camphor tree sesame; And when water-bath lixiviate, ratio of water to material controls as (1-3) by the present invention: 1, not only ensure that the abundant extraction of polysaccharide, avoid the loss of polysaccharide when the increase of Aqueous extracts total amount causes alcohol precipitation, and significantly reduce the energy consumption in subsequent operations.
2. assay many employings HPLC method of current polysaccharide, in the present invention, before the extraction of polysaccharide, adopt chloroform isoamyl alcohol thermal agitation, effectively can remove albumen precipitation, avoid protein in sample and polysaccharide is gone out to the impact at peak, ensure that HPLC method measures accuracy and the susceptibility of content.
3. the micropore asymmetry structure that has of ultra-filtration membrane, macromole particulate is made to flow through film surface, small-molecule substance flows through ultra-filtration membrane, thus ensure macromole and micromolecular effective separation, and can normal-temperature operation, consume energy low, also have and can use continuously and the advantage such as pollution-free, the demand of industrialized production can be met.
4. polysaccharide is soluble in hot water, is insoluble in organic solvent, in the present invention, first adopts alcohol pre-treatment to sample, can effectively remove the most of pigment of the lipid-soluble substances such as triterpenes, meanwhile, can also effectively soften and destroy cytolemma, be beneficial to the abundant extraction of polysaccharide.
5. first get 1-3cm in the present invention
2slant medium in cultured band substratum camphor tree sesame bacterium load 500ml triangular flask in cultivate; When carrying out fermentation culture, inoculum size is 10%-20%, can ensure the carrying out that the growth and breeding of camphor tree sesame bacterium is more favourable, thus significantly improve its polysaccharide content.
Embodiment
The present invention, by the present invention of following specific embodiment more detailed description, can make those skilled in the art more fully understand the present invention, but not limit the present invention in any way.
Embodiment 1
An extracting method for camphor tree sesame polysaccharide, comprises the following steps:
(1) cultivation of camphor tree sesame bacterium seed liquor: get 1cm with connecing swollen shovel
2slant medium in cultured band substratum camphor tree sesame bacterium load liquid amount be cultivate in the triangular flask of the 500ml of 100ml, culture temperature is 25 DEG C, and incubation time is 3d;
(2) the camphor tree sesame bacterium 10% of getting after cultivating in step (1) is inoculated in fermentor tank and carries out fermentation culture, culture temperature 25 DEG C, rotating speed 200r/min, incubation time 6d, air flow 120-150L/h, liquid amount: 5L; Nutrient solution is crossed 40 mesh sieves, the mycelium retained filters with 3 times of distilled water washs again, is camphor tree sesame fermented liquid under sieve;
(3) extraction of camphor tree sesame granulose: by camphor tree sesame filtering fermentation liquor filtering thalline obtained in step (2), get filtrate and add 2-3 times amount distilled water and pulverize homogenate in Multi Role Aircraft, water-bath lixiviate, during water-bath lixiviate, ratio of water to material is 1:1; Water-bath lixiviate time be 1.5h, temperature is 120 DEG C; Centrifugal, get supernatant liquor carry out rotary evaporation concentrate, get concentrated solution under 4 DEG C of conditions, to carry out alcohol analyse, centrifugal, get precipitation and add after equivalent distilled water dissolves, add 2 times of chloroform isoamyl alcohol thermal agitations, supernatant liquor adds 4 times amount dehydrated alcohol refluxing extraction 3 times, each 1.5h, centrifugation ethanol, pellet frozen is dry, obtained camphor tree sesame granulose dry product;
(4) camphor tree sesame polysaccharide crude process: adopt hollow fiber ultrafiltration membrane to filter camphor tree sesame granulose crude product obtained in step (3), obtain the polysaccharide body of different molecular weight, obtain the dry products of camphor tree sesame polysaccharide respectively after concentrate drying.
The solvent adopted in process analysed by alcohol described in step (3) is dehydrated alcohol.
Alcohol described in step (3) is analysed in process, and described concentrated solution and the volume ratio of solvent are 1:4.
In chloroform isoamyl alcohol described in step (3), the volume ratio of chloroform and primary isoamyl alcohol is 4:0.8.
Centrifugation time described in step (3) is 30min, and centrifuging temperature is 25 DEG C.
In ultra-filtration process described in step (4), pressure is 0.2Mpa, described hollow fiber ultrafiltration membrane is 1000,100, the three assembly hollow fiber ultrafiltration membrane of 10kDa.
Embodiment 2
An extracting method for camphor tree sesame polysaccharide, comprises the following steps:
(1) cultivation of camphor tree sesame bacterium seed liquor: get 3cm with connecing swollen shovel
2slant medium in cultured band substratum camphor tree sesame bacterium load liquid amount be cultivate in the triangular flask of the 500ml of 150ml, culture temperature is 28 DEG C, and incubation time is 5d;
(2) the camphor tree sesame bacterium 20% of getting after cultivating in step (1) is inoculated in fermentor tank and carries out fermentation culture, culture temperature 28 DEG C, rotating speed 230r/min, incubation time 6-8d, air flow 150L/h, liquid amount: 7L; Nutrient solution is crossed 40 mesh sieves, the mycelium retained filters with 3 times of distilled water washs again, is camphor tree sesame fermented liquid under sieve;
(3) extraction of camphor tree sesame granulose: by camphor tree sesame filtering fermentation liquor filtering thalline obtained in step (2), get filtrate and add 3 times amount distilled water and pulverize homogenate in Multi Role Aircraft, water-bath lixiviate, during water-bath lixiviate, ratio of water to material is 3:1; Water-bath lixiviate time be 2h, temperature is 135 DEG C; Centrifugal, get supernatant liquor carry out rotary evaporation concentrate, get concentrated solution under 4 DEG C of conditions, to carry out alcohol analyse, centrifugal, get precipitation and add after equivalent distilled water dissolves, add 3 times of chloroform isoamyl alcohol thermal agitations, supernatant liquor adds 5 times amount dehydrated alcohol refluxing extraction 5 times, each 2h, centrifugation ethanol, pellet frozen is dry, obtained camphor tree sesame granulose dry product;
(4) camphor tree sesame polysaccharide crude process: adopt hollow fiber ultrafiltration membrane to filter camphor tree sesame granulose crude product obtained in step (3), obtain the polysaccharide body of different molecular weight, obtain the dry products of camphor tree sesame polysaccharide respectively after concentrate drying.
The solvent adopted in process analysed by alcohol described in step (3) is dehydrated alcohol.
Alcohol described in step (3) is analysed in process, and described concentrated solution and the volume ratio of solvent are 1.5:5.
In chloroform isoamyl alcohol described in step (3), the volume ratio of chloroform and primary isoamyl alcohol is 6:1.5.
Centrifugation time described in step (3) is 60min, and centrifuging temperature is 28 DEG C.
In ultra-filtration process described in step (4), pressure is 0.5Mpa, described hollow fiber ultrafiltration membrane is 1000,100, the three assembly hollow fiber ultrafiltration membrane of 10kDa.
Embodiment 3
An extracting method for camphor tree sesame polysaccharide, comprises the following steps:
(1) cultivation of camphor tree sesame bacterium seed liquor: get 2cm with connecing swollen shovel
2slant medium in cultured band substratum camphor tree sesame bacterium load liquid amount be cultivate in the triangular flask of the 500ml of 120ml, culture temperature is 26 DEG C, and incubation time is 4d;
(2) the camphor tree sesame bacterium 15% of getting after cultivating in step (1) is inoculated in fermentor tank and carries out fermentation culture, culture temperature 6 DEG C, rotating speed 210r/min, incubation time 7d, air flow 130L/h, liquid amount: 6L; Nutrient solution is crossed 40 mesh sieves, the mycelium retained filters with 3 times of distilled water washs again, is camphor tree sesame fermented liquid under sieve;
(3) extraction of camphor tree sesame granulose: by camphor tree sesame filtering fermentation liquor filtering thalline obtained in step (2), get filtrate and add 3 times amount distilled water and pulverize homogenate in Multi Role Aircraft, water-bath lixiviate, during water-bath lixiviate, ratio of water to material is 2:1; Water-bath lixiviate time be 1.8h, temperature is 125 DEG C; Centrifugal, get supernatant liquor carry out rotary evaporation concentrate, get concentrated solution under 4 DEG C of conditions, to carry out alcohol analyse, centrifugal, get precipitation and add after equivalent distilled water dissolves, add 2 times of chloroform isoamyl alcohol thermal agitations, supernatant liquor adds 5 times amount dehydrated alcohol refluxing extraction 4 times, each 1.8h, centrifugation ethanol, pellet frozen is dry, obtained camphor tree sesame granulose dry product;
(4) camphor tree sesame polysaccharide crude process: adopt hollow fiber ultrafiltration membrane to filter camphor tree sesame granulose crude product obtained in step (3), obtain the polysaccharide body of different molecular weight, obtain the dry products of camphor tree sesame polysaccharide respectively after concentrate drying.
The solvent adopted in process analysed by alcohol described in step (3) is dehydrated alcohol.
Alcohol described in step (3) is analysed in process, and described concentrated solution and the volume ratio of solvent are 1.2:4.5.
In chloroform isoamyl alcohol described in step (3), the volume ratio of chloroform and primary isoamyl alcohol is 5:1.2.
Centrifugation time described in step (3) is 30-60min, and centrifuging temperature is 26 DEG C.
In ultra-filtration process described in step (4), pressure is 0.3Mpa, described hollow fiber ultrafiltration membrane is 1000,100, the three assembly hollow fiber ultrafiltration membrane of 10kDa.
The above embodiment is preferred embodiments of the present invention, is not limit the scope of the present invention, therefore all equivalences done according to structure, feature and the principle described in the present patent application the scope of the claims change or modify, and all should comprise in patent claim of the present invention.
Claims (10)
1. an extracting method for camphor tree sesame polysaccharide, is characterized in that, comprises the following steps:
(1) cultivation of camphor tree sesame bacterium seed liquor: get 1-3cm with connecing swollen shovel
2slant medium in cultured band substratum camphor tree sesame bacterium load 500ml triangular flask in cultivate;
(2) the camphor tree sesame bacterium 10%-20% got after cultivating in step (1) is inoculated in fermentor tank and carries out fermentation culture, nutrient solution is crossed 40 mesh sieves, and the mycelium retained filters with 3 times of distilled water washs again, is camphor tree sesame fermented liquid under sieve;
(3) extraction of camphor tree sesame granulose: by camphor tree sesame filtering fermentation liquor filtering thalline obtained in step (2), get filtrate to add 2-3 times amount distilled water and pulverize homogenate in Multi Role Aircraft, water-bath lixiviate, centrifugal, get supernatant liquor carry out rotary evaporation concentrate, get concentrated solution under 4 DEG C of conditions, to carry out alcohol analyse, centrifugal, get precipitation add equivalent distilled water dissolve after, add 2-3 times of chloroform isoamyl alcohol thermal agitation, supernatant liquor adds 4-5 times amount dehydrated alcohol refluxing extraction 3-5 time, each 1.5-2h, centrifugation ethanol, pellet frozen is dry, obtained camphor tree sesame granulose dry product;
(4) camphor tree sesame polysaccharide crude process: adopt hollow fiber ultrafiltration membrane to filter camphor tree sesame granulose crude product obtained in step (3), obtain the polysaccharide body of different molecular weight, obtain the dry products of camphor tree sesame polysaccharide respectively after concentrate drying.
2. the extracting method of a kind of camphor tree sesame polysaccharide according to claim 1, it is characterized in that, the liquid amount in the triangular flask described in step (1) is 100-150ml.
3. the extracting method of a kind of camphor tree sesame polysaccharide according to claim 1, it is characterized in that, the culture temperature described in step (1) is 25-28 DEG C, and incubation time is 3-5d.
4. the extracting method of a kind of camphor tree sesame polysaccharide according to claim 1, it is characterized in that, the fermentation culture conditions described in step (2) is: culture temperature 25-28 DEG C, rotating speed 200-230r/min, incubation time 6-8d, air flow 120-150L/h, liquid amount: 5-7L.
5. the extracting method of a kind of camphor tree sesame polysaccharide according to claim 1, is characterized in that, during water-bath lixiviate described in step (3), ratio of water to material is (1-3): 1; Water-bath lixiviate time be 1.5-2h, temperature is 120-135 DEG C; The service temperature that rotary evaporation concentrates is 60-65 DEG C.
6. the extracting method of a kind of camphor tree sesame polysaccharide according to claim 1, it is characterized in that, the solvent adopted in process analysed by the alcohol described in step (3) is dehydrated alcohol.
7. the extracting method of a kind of camphor tree sesame polysaccharide according to claim 1, it is characterized in that, the alcohol described in step (3) is analysed in process, and described concentrated solution and the volume ratio of solvent are (1-1.5): (4-5).
8. the extracting method of a kind of camphor tree sesame polysaccharide according to claim 1, is characterized in that, in the chloroform isoamyl alcohol described in step (3), the volume ratio of chloroform and primary isoamyl alcohol is (4-6): (0.8-1.5).
9. the extracting method of a kind of camphor tree sesame polysaccharide according to claim 1, it is characterized in that, the centrifugation time described in step (3) is 30-60min, and centrifuging temperature is 25-28 DEG C.
10. the extracting method of a kind of camphor tree sesame polysaccharide according to claim 1, it is characterized in that, in ultra-filtration process described in step (4), pressure is 0.2-0.5Mpa, described hollow fiber ultrafiltration membrane is 1000,100, the three assembly hollow fiber ultrafiltration membrane of 10kDa.
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Cited By (1)
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| CN111087489A (en) * | 2020-01-15 | 2020-05-01 | 安徽祥云谷现代中药有限公司 | CO (carbon monoxide)2Method for extracting polysaccharide from wall-broken ganoderma lucidum spore powder residue after supercritical extraction of spore oil |
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Cited By (1)
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| CN111087489A (en) * | 2020-01-15 | 2020-05-01 | 安徽祥云谷现代中药有限公司 | CO (carbon monoxide)2Method for extracting polysaccharide from wall-broken ganoderma lucidum spore powder residue after supercritical extraction of spore oil |
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Application publication date: 20150114 |