CN109748979A - A kind of extracting method of cordyceps flower polysaccharide - Google Patents
A kind of extracting method of cordyceps flower polysaccharide Download PDFInfo
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- CN109748979A CN109748979A CN201711065338.5A CN201711065338A CN109748979A CN 109748979 A CN109748979 A CN 109748979A CN 201711065338 A CN201711065338 A CN 201711065338A CN 109748979 A CN109748979 A CN 109748979A
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- polysaccharide
- cordyceps flower
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Abstract
The present invention provides a kind of extracting method of cordyceps flower polysaccharide, the extracting method includes the following steps: that crushing, water mention, an alcohol precipitation, enzymatic hydrolysis, take off albumen, secondary alcohol precipitation, ultrafiltration, column chromatography；Extracting method product recovery rate of the present invention is high, purity is good, and cordyceps flower polysaccharide yield is greater than 1.8%, and the purity of polysaccharide is greater than 90.0%；Extracting method simple process of the present invention, agents useful for same are easy to get, and production cost is low, it is easy to accomplish industrialization.
Description
(1) technical field
The invention belongs to field of biotechnology, and in particular to a method of polysaccharide is extracted from cordyceps flower.
(2) background technique
" cordyceps flower " is not spent, substantially cordyceps militaris sporocarp, rather than cordyceps sporophore.Culture medium is to copy natural worm
Various nutrients, including cereals, beans, egg-milk etc. contained by son, belong to a kind of Mycophyta.With common mushroom, oyster mushroom etc.
Edible mushroom is much like, only strain, growing environment and growth conditions difference.In order to be distinguished with cordyceps sinensis, businessman is risen
One beautiful name, is called it " cordyceps flower ", and maximum feature is and the only orange without " polypide " to cordyceps flower in appearance
" grass " of color or yellow.
Cordyceps sinensis polysaccharide is nonspecific immunomodulator, has antitumor, anti-inflammatory, anticoagulation, antiviral, anti-puts
It penetrates, is hypoglycemic, the multiple functions such as reducing blood lipid.Polysaccharide is one of most important, the most abundant physiological activator in cordyceps sinensis.Cordyceps sinensis is more
Sugar is considered as that cordyceps sinensis has two-way immunoregulatory effective component, there is antitumor, anti-inflammatory, hypoglycemic, reducing blood lipid and other effects.Worm
Grass polysaccharide even has direct inhibiting effect to AIDS virus, and is expected to become and people living with AIDS is prevented to show symptom
Drug.Furthermore Cordyceps sinensis polysaccharide also has in the industries such as food industry, fermentation industry and petroleum industry in terms of daily cosmetics
It is widely applied.
There are many fungi polysaccharide product currently on the market, but there are no the product of cordyceps flower polysaccharide, more not specifically for
The extracting method of cordyceps flower polysaccharide.
(3) summary of the invention
The present invention provides a kind of extracting method of cordyceps flower polysaccharide, this method combine water extraction and alcohol precipitation method, ultrasonic wave promote and
The strong point of membrane separation technique, the high-quality and low cost for having abandoned single method are difficult to the shortcomings that merging, have both been able to maintain polysaccharide knot
Structure destroys less, purity is high, recovery rate are high, achievees the effect that multi-stage separation, and can be reduced energy consumption reduction processing cost.
The present invention adopts the following technical scheme:
A kind of extracting method of cordyceps flower polysaccharide, the extracting method carry out as follows:
(1) it crushes: dry cordyceps flower being crushed and is sieved, cordyceps flower powder is obtained；
In step (1), the sieve pore of the sieving is 80~120 mesh；
(2) water mentions: cordyceps flower powder obtained by step (1) being mixed with distilled water with feed liquid mass ratio 1:20~80, heating boils 2
~3h, and it is aided with ultrasonic wave assisted extraction, it is centrifuged 10~15min later, takes supernatant, is centrifuged the residue obtained water that repeats and mentions 3~4
It is secondary, merge each gained supernatant, is concentrated into the 1/4~1/10 of original volume (initial volume i.e. before concentration operation), obtains more
Sugared extracting solution；
In step (2), the power of the ultrasonic wave is 500W, frequency 20KHz；The rate of the centrifugation be 8000~
10000rpm；The concentration is concentrated by the way of rotating at 60~80 DEG C；
(3) alcohol precipitations: at 4~25 DEG C of temperature, by polysaccharide extraction liquid obtained by step (2) and edible alcohol with volume ratio 1:1.5
~4 mixing carry out 24~30h of alcohol precipitation, and Precipitation is centrifuged 10~15min, and centrifugation obtained solid substance is taken to repeat alcohol precipitation 2~3
It is secondary, obtain Thick many candies；
In step (3), the rate of the centrifugation is 8000~10000rpm；The centrifugation obtained solid substance repeats the side of alcohol precipitation
Method are as follows: the water of solid matter 30~40 mass times is dissolved, obtains lysate, then by gained lysate and edible alcohol with body
Product is mixed than 1:1.5~4, carries out 24~30h of alcohol precipitation, Precipitation, and 10~15min of centrifugation obtains solid matter, so repeats
Alcohol precipitation 2~3 times, obtain Thick many candies；
(4) it digests: Thick many candies obtained by step (3) being dissolved in the distilled water of 30~40 mass times, adjusted with Na2CO3 aqueous solution
PH value is 7.2~7.8, obtains Thick many candies solution, and trypsase, toluene is added, the constant temperature hydrolysis 18~for 24 hours at 35~37 DEG C,
It is concentrated into the 1/4~1/2 of original volume (initial volume i.e. before concentration operation) later, obtains protein enzymatic hydrolyzate；
In step (4), the concentration of the Na2CO3 aqueous solution is 0.1~0.2mol/L；The activity of the trypsase is 8000
~10000U/g；The additional amount of the trypsase is 0.05~0.1g/1g Thick many candies；The volumetric usage of the toluene is 2.5
~3.5mL/1g Thick many candies；The concentration is concentrated by the way of rotating at 60~80 DEG C；
(5) it takes off albumen: protein enzymatic hydrolyzate obtained by step (4) being mixed with Sevag reagent with volume ratio 1:2.5~3, room temperature (20
~30 DEG C) 1~3h of extracting, it is centrifuged 10~15min later, takes supernatant, which is repeated de- albumen 2~3 times, Zhi Hounong
It is reduced to the 1/4~1/3 of original volume (initial volume i.e. before concentration operation), obtains de- proteoglycan liquid；
In step (5), the Sevag reagent is the mixed liquor that chloroform and n-butanol are formulated with volume ratio 4:1；It is described from
The rate of the heart is 8000~10000rpm；The concentration is concentrated by the way of rotating at 60~80 DEG C；
(6) secondary alcohol precipitation: at 4~25 DEG C of temperature, proteoglycan liquid and edible alcohol will be taken off obtained by step (5) with volume ratio 1:
1.5~4.5 mixing carry out 24~30h of alcohol precipitation, and Precipitation is centrifuged 10~15min, take centrifugation obtained solid substance room temperature (20
~30 DEG C) it dries, obtain polysaccharide crude product；
In step (6), the rate of the centrifugation is 8000~10000rpm；
(7) ultrafiltration, column chromatography: the distilled water of 30~40 mass times of polysaccharide crude product obtained by step (6) is dissolved, then uses ultrafiltration
Film carries out ultrafiltration, collects the component of 10000 dalton of molecular weight or more (generally 10000~1000000 dalton), Yu Changwen
SephadexG-100 column chromatographic purifying is carried out under (20~30 DEG C), eluant, eluent is redistilled water, collects the eluent containing polysaccharide and (adopts
With the polysaccharide in sulfuric acid-phynol method measurement eluent commonly used in the art), freeze-drying obtains cordyceps flower polysaccharide.
In the present invention, the edible alcohol is conventional edible alcohol commercially available on the market, usual second therein
Alcohol content is 95%~98%.
Compared with prior art, the invention has the following beneficial effects:
(1) extracting method product recovery rate of the present invention is high, purity is good, and cordyceps flower polysaccharide yield is greater than 1.8%, and the purity of polysaccharide is big
In 90.0%；
(2) extracting method simple process of the present invention, agents useful for same are easy to get, and production cost is low, it is easy to accomplish industrialization.
(4) specific embodiment
Below by specific embodiment, the invention will be further described, but protection scope of the present invention is not limited to that.
Embodiment 1
(1) dry cordyceps flower is crushed and is sieved (100 mesh), obtain cordyceps flower powder；
(2) insect-taking showy flowers of herbaceous plants powder 10g mixes it with 200mL sterile water, and 2h is boiled in heating, and is aided with ultrasonic wave (power is
500W, frequency 20KHz) assisted extraction, it is centrifuged 10min later, takes supernatant, the residue after centrifugation is repeated by the above process
It extracts 3 times, merges each gained supernatant, be concentrated into 20mL at 60 DEG C using Rotary Evaporators, obtain polysaccharide extraction liquid；
At (3) 4 DEG C, polysaccharide extraction liquid 20mL is mixed with edible alcohol 40mL, carries out alcohol precipitation for 24 hours, Precipitation, centrifugation
10min takes centrifugation obtained solid substance to repeat alcohol precipitation 3 times, obtains Thick many candies 1.32g；
(4) Thick many candies 1g is dissolved in 30mL distilled water, is 7.5 with 0.15mol/LNa2CO3 aqueous solution tune pH value, is added
0.05g trypsase (active 9000U/g), 3mL toluene, 37 DEG C of constant temperature hydrolyze 20h, and 70 DEG C of rotary evaporations are concentrated into 20mL, obtain
To protein enzymatic hydrolyzate.
(5) protein enzymatic hydrolyzate 10mL is mixed with Sevag reagent 30mL, extracts 1h, is centrifuged 10min later, takes supernatant
It repeats de- albumen 2 times, 60 DEG C of rotary evaporations are concentrated into 10mL later, obtain de- proteoglycan liquid.
At (6) 4 DEG C, de- proteoglycan liquid 10mL is mixed with edible alcohol 40mL, carries out alcohol precipitation for 24 hours, Precipitation, from
Heart 10min takes obtained solid substance room temperature to dry, and obtains polysaccharide crude product 0.74g；
(7) polysaccharide crude product 0.5g distilled water 15mL is dissolved, then carries out ultrafiltration with ultrafiltration membrane, collect molecular weight 10000
Then the component of Er Dun or more carries out SephadexG-100 column chromatographic purifying under room temperature, eluant, eluent is redistilled water, and collection contains
The eluent of polysaccharide, freeze-drying, obtains cordyceps flower polysaccharide 0.39g.
Cordyceps flower polysaccharide prepared by the present embodiment be it is filbert, can not be dried using conventional drying methods such as baking ovens, benefit
Powder can be just obtained with freeze-drying, easy to moisture absorption, soluble easily in water, mass content 91.4%.Polyoses content detection: it accurately weighs
Cordyceps flower polysaccharide 10mg sufficiently dissolves in 100mL beaker, draws polysaccharide solution 0.5mL, and 5% phenol 1.0mL and dense sulphur is added
Sour 3.0mL, static 10min, shakes up, and is placed at room temperature for 15min, and using distilled water as blank, light absorption value is surveyed at 490nm.
Embodiment 2
(1) dry cordyceps flower crush and 100 mesh are sieved, obtain cordyceps flower powder；
(2) insect-taking showy flowers of herbaceous plants powder 10g mixes it with 300mL sterile water, and 3h is boiled in heating, and is aided with ultrasonic wave (power is
500W, frequency 20KHz) assisted extraction, it is centrifuged 10min later, takes supernatant, the residue after centrifugation is repeated by the above process
It extracts 3 times, merges each gained supernatant, be concentrated into 20mL at 60 DEG C using Rotary Evaporators, obtain polysaccharide extraction liquid；
At (3) 4 DEG C, polysaccharide extraction liquid 20mL is mixed with edible alcohol 45mL, carries out alcohol precipitation for 24 hours, Precipitation, centrifugation
10min takes centrifugation obtained solid substance to repeat alcohol precipitation 3 times, obtains Thick many candies 1.39g；
(4) Thick many candies 1g is dissolved in 35mL distilled water, is 7.5 with 0.15mol/LNa2CO3 aqueous solution tune pH value, is added
0.05g trypsase (active 9000U/g), 3.5mL toluene, for 24 hours, 70 DEG C of rotary evaporations are concentrated into 20mL for 37 DEG C of constant temperature hydrolysis,
Obtain protein enzymatic hydrolyzate.
(5) protein enzymatic hydrolyzate 10mL is mixed with Sevag reagent 25mL, extracts 1h, is centrifuged 10min later, takes supernatant
It repeats de- albumen 2 times, 60 DEG C of rotary evaporations are concentrated into 10mL later, obtain de- proteoglycan liquid.
At (6) 4 DEG C, de- proteoglycan liquid 10mL is mixed with edible alcohol 45mL, carries out alcohol precipitation for 24 hours, Precipitation, from
Heart 10min takes obtained solid substance room temperature to dry, and obtains polysaccharide crude product 0.81g；
(7) polysaccharide crude product 0.5g distilled water 15mL is dissolved, then carries out ultrafiltration with ultrafiltration membrane, collect molecular weight 10000
Then the component of Er Dun or more carries out SephadexG-100 column chromatographic purifying at 25 DEG C, eluant, eluent is redistilled water, and collection contains
The eluent of polysaccharide, freeze-drying, obtains cordyceps flower polysaccharide 0.41g.
Cordyceps flower polysaccharide prepared by the present embodiment be it is filbert, can not be dried using conventional drying methods such as baking ovens, benefit
Powder can be just obtained with freeze-drying, easy to moisture absorption, soluble easily in water, mass content 89.7%.Polyoses content detection: it accurately weighs
Cordyceps flower polysaccharide 10mg sufficiently dissolves in 100mL beaker, draws polysaccharide solution 0.5mL, and 5% phenol 1.0mL and dense sulphur is added
Sour 3.0mL, static 10min, shakes up, and is placed at room temperature for 15min, and using distilled water as blank, light absorption value is surveyed at 490nm.
Embodiment 3
(1) dry cordyceps flower crush and 120 mesh are sieved, obtain cordyceps flower powder；
(2) insect-taking showy flowers of herbaceous plants powder 10g mixes it with 400mL sterile water, and 3h is boiled in heating, and is aided with ultrasonic wave (power is
500W, frequency 20KHz) assisted extraction, it is centrifuged 10min later, takes supernatant, the residue after centrifugation is repeated by the above process
It extracts 3 times, merges each gained supernatant, be concentrated into 20mL at 60 DEG C using Rotary Evaporators, obtain polysaccharide extraction liquid；
At (3) 4 DEG C, polysaccharide extraction liquid 20mL is mixed with edible alcohol 60mL, carries out alcohol precipitation for 24 hours, Precipitation, centrifugation
10min takes centrifugation obtained solid substance to repeat alcohol precipitation 3 times, obtains Thick many candies 1.43g；
(4) Thick many candies 1g is dissolved in 40mL distilled water, is 7.5 with 0.15mol/LNa2CO3 aqueous solution tune pH value, 0.1g is added
Trypsase (active 9000U/g), 3mL toluene, for 24 hours, 70 DEG C of rotary evaporations are concentrated into 20mL, obtain albumen for 37 DEG C of constant temperature hydrolysis
Enzymolysis liquid.
(5) protein enzymatic hydrolyzate 10mL is mixed with Sevag reagent 30mL, extracts 1h, is centrifuged 10min later, takes supernatant
It repeats de- albumen 2 times, 60 DEG C of rotary evaporations are concentrated into 10mL later, obtain de- proteoglycan liquid.
At (6) 4 DEG C, de- proteoglycan liquid 10mL is mixed with edible alcohol 30mL, carries out alcohol precipitation for 24 hours, Precipitation, from
Heart 10min takes obtained solid substance room temperature to dry, and obtains polysaccharide crude product 0.78g；
(7) polysaccharide crude product 0.5g distilled water 20mL is dissolved, then carries out ultrafiltration with ultrafiltration membrane, collect molecular weight 10000
Then the component of Er Dun or more carries out SephadexG-100 column chromatographic purifying at 25 DEG C, eluant, eluent is redistilled water, and collection contains
The eluent of polysaccharide, freeze-drying, obtains cordyceps flower polysaccharide 0.42g.
Cordyceps flower polysaccharide prepared by the present embodiment be it is filbert, can not be dried using conventional drying methods such as baking ovens, benefit
Powder can be just obtained with freeze-drying, easy to moisture absorption, soluble easily in water, mass content 91.9%.Polyoses content detection: it accurately weighs
Cordyceps flower polysaccharide 10mg sufficiently dissolves in 100mL beaker, draws polysaccharide solution 0.5mL, and 5% phenol 1.0mL and dense sulphur is added
Sour 3.0mL, static 10min, shakes up, and is placed at room temperature for 15min, and using distilled water as blank, light absorption value is surveyed at 490nm.
Claims (7)
1. a kind of extracting method of cordyceps flower polysaccharide, which is characterized in that the extracting method carries out as follows:
(1) it crushes: dry cordyceps flower being crushed and is sieved, cordyceps flower powder is obtained；
(2) water mentions: cordyceps flower powder obtained by step (1) being mixed with distilled water with feed liquid mass ratio 1:20~80, heating boils 2
~3h, and it is aided with ultrasonic wave assisted extraction, it is centrifuged 10~15min later, takes supernatant, is centrifuged the residue obtained water that repeats and mentions 3~4
It is secondary, merge each gained supernatant, is concentrated into the 1/4~1/10 of original volume, obtains polysaccharide extraction liquid；
(3) alcohol precipitations: at 4~25 DEG C of temperature, by polysaccharide extraction liquid obtained by step (2) and edible alcohol with volume ratio 1:1.5
~4 mixing carry out 24~30h of alcohol precipitation, and Precipitation is centrifuged 10~15min, and centrifugation obtained solid substance is taken to repeat alcohol precipitation 2~3
It is secondary, obtain Thick many candies；
(4) it digests: Thick many candies obtained by step (3) being dissolved in the distilled water of 30~40 mass times, adjusted with Na2CO3 aqueous solution
PH value is 7.2~7.8, obtains Thick many candies solution, and trypsase, toluene is added, the constant temperature hydrolysis 18~for 24 hours at 35~37 DEG C,
It is concentrated into the 1/4~1/2 of original volume later, obtains protein enzymatic hydrolyzate；
The activity of the trypsase is 8000~10000U/g；The additional amount of the trypsase is that 0.05~0.1g/1g is thick
Polysaccharide；The volumetric usage of the toluene is 2.5~3.5mL/1g Thick many candies；
(5) it takes off albumen: protein enzymatic hydrolyzate obtained by step (4) being mixed with Sevag reagent with volume ratio 1:2.5~3, room temperature extracting
1~3h is centrifuged 10~15min later, takes supernatant, which is repeated de- albumen 2~3 times, is concentrated into original volume later
1/4~1/3, obtain de- proteoglycan liquid；
The Sevag reagent is the mixed liquor that chloroform and n-butanol are formulated with volume ratio 4:1；
(6) secondary alcohol precipitation: at 4~25 DEG C of temperature, proteoglycan liquid and edible alcohol will be taken off obtained by step (5) with volume ratio 1:
1.5~4.5 mixing carry out 24~30h of alcohol precipitation, and Precipitation is centrifuged 10~15min, and centrifugation obtained solid substance room temperature is taken to dry in the air
It is dry, obtain polysaccharide crude product；
(7) ultrafiltration, column chromatography: the distilled water of 30~40 mass times of polysaccharide crude product obtained by step (6) is dissolved, then uses ultrafiltration
Film carries out ultrafiltration, collects the component of 10000 dalton of molecular weight or more, and it is pure that SephadexG-100 column chromatography is carried out under room temperature
Change, eluant, eluent is redistilled water, collects the eluent containing polysaccharide, and freeze-drying obtains cordyceps flower polysaccharide.
2. the extracting method of cordyceps flower polysaccharide as described in claim 1, which is characterized in that in step (1), the sieve of the sieving
Hole is 80~120 mesh.
3. the extracting method of cordyceps flower polysaccharide as described in claim 1, which is characterized in that in step (2), the ultrasonic wave
Power is 500W, frequency 20KHz.
4. the extracting method of cordyceps flower polysaccharide as described in claim 1, which is characterized in that in step (3), obtained by the centrifugation
The method of solid matter repetition alcohol precipitation are as follows: by the water dissolution of solid matter 30~40 mass times, obtain lysate, then by gained
Lysate is mixed with edible alcohol with volume ratio 1:1.5~4, carries out 24~30h of alcohol precipitation, Precipitation, and 10~15min of centrifugation is obtained
To solid matter, so repeats alcohol precipitation 2~3 times, obtain Thick many candies.
5. the extracting method of cordyceps flower polysaccharide as described in claim 1, which is characterized in that in step (4), the Na2CO3 water
The concentration of solution is 0.1~0.2mol/L.
6. the extracting method of cordyceps flower polysaccharide as described in claim 1, which is characterized in that step (2), (3), (5) or (6)
In, the rate of the centrifugation is 8000~10000rpm.
7. the extracting method of cordyceps flower polysaccharide as described in claim 1, which is characterized in that in step (2), (4) or (5), institute
The concentration stated is concentrated by the way of rotating at 60~80 DEG C.
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| CN201711065338.5A CN109748979A (en) | 2017-11-02 | 2017-11-02 | A kind of extracting method of cordyceps flower polysaccharide |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108503724A (en) * | 2018-04-27 | 2018-09-07 | 华南师范大学 | Chinese caterpillar fungus culture medium polysaccharide and its isolation and purification method and application |
| CN115141290A (en) * | 2022-08-10 | 2022-10-04 | 浙江汇能生物股份有限公司 | Method for separating and purifying cordyceps polysaccharide by adopting membrane |
-
2017
- 2017-11-02 CN CN201711065338.5A patent/CN109748979A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108503724A (en) * | 2018-04-27 | 2018-09-07 | 华南师范大学 | Chinese caterpillar fungus culture medium polysaccharide and its isolation and purification method and application |
| CN108503724B (en) * | 2018-04-27 | 2021-06-01 | 华南师范大学 | Cordyceps militaris culture medium polysaccharide, and separation and purification method and application thereof |
| CN115141290A (en) * | 2022-08-10 | 2022-10-04 | 浙江汇能生物股份有限公司 | Method for separating and purifying cordyceps polysaccharide by adopting membrane |
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Application publication date: 20190514 |