CN109402194A - A kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation - Google Patents
A kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation Download PDFInfo
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- CN109402194A CN109402194A CN201811487214.0A CN201811487214A CN109402194A CN 109402194 A CN109402194 A CN 109402194A CN 201811487214 A CN201811487214 A CN 201811487214A CN 109402194 A CN109402194 A CN 109402194A
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Abstract
A kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation includes the following steps: that A, aweto seed culture obtain seed liquor；B, seed liquor obtains Cordyceps mycelium fermented liquid through liquid fermentation；C, Cordyceps mycelium fermented liquid crushes 8-15min and obtains cordyceps sinensis mixed liquor；D, cordyceps sinensis mixed liquor extraction is concentrated to get polysaccharide concentrate；E, the centrifugation of polysaccharide concentrate dries to obtain Chinese caterpillar fungus polysaccharide extract；F, AB-8 type large pore resin absorption column on Chinese caterpillar fungus polysaccharide extract, elution, and collect eluent；G, eluent, upper D-280 large pore resin absorption column, decoloration are concentrated and dried and obtain Chinese caterpillar fungus polysaccharide.Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation of the invention realizes culture, extraction, purifying continuous processing, and extraction efficiency is high, and Chinese caterpillar fungus polysaccharide yield is high, with high purity, and operating process is simpler, and cost is relatively low.
Description
Technical field
The present invention relates to microcultures and technical field of natural product extraction, and in particular to a kind of based on liquid fermentation
Chinese caterpillar fungus polysaccharide extraction and purification process.
Background technique
Cordyceps sinensis polysaccharide or polysaccharide, one of the main active as cordyceps sinensis are content highests in cordyceps sinensis
Pharmacological active substance, content can achieve 10%, be a kind of of great value potential novel dual-purpose of drug and food resource.Cordyceps sinensis polysaccharide
The polysaccharide being made of mannose, cordycepin, adenosine, galactolipin, arabinose, glucose, fucose etc..Its anticancer is living
Property and immunocompetence cause the great interest of people, in addition cordyceps sinensis is also used to control pulmonary tuberculosis, shortness of breath is breathed with cough, impotence, nocturnal emission,
Spontaneous sweating, soreness of waist and knee joint etc..
Cordyceps sinensis polysaccharide is mainly extracted from fruiting bodies of cordyceps militaris in the industrial production at present.But due to cordyceps militaris sporocarp
High production cost, it is high so as to cause the production cost of Cordyceps sinensis polysaccharide.
Mainly there are Hot water extraction, acidleach formulation, alkali method, ultrasonic wave extraction with the extracting method of Cordyceps sinensis polysaccharide at present
Method, microwave loss mechanisms etc..
In addition, being obtained using artificial fermentation's aweto and natural cordyceps chemical composition and pharmacology work
With essentially identical mycelium, become a kind of most viable method for solving worm grass resources shortage instantly.
But existing Chinese caterpillar fungus polysaccharide extraction process, it can not achieve culture extraction purification continuous processing, extraction efficiency
Low, Chinese caterpillar fungus polysaccharide low yield, operating process is complicated, higher cost, and cannot further isolate and purify polysaccharide, can not obtain
To the higher polysaccharide component of purity, the development and utilization of Chinese caterpillar fungus polysaccharide product is made to receive limitation.
Based on the above situation, the invention proposes a kind of, and the Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation can
Effectively solve problem above.
Summary of the invention
The purpose of the present invention is to provide a kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation.The present invention
The Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation, realize culture, extraction, purifying continuous processing, extract effect
Rate is high, and Chinese caterpillar fungus polysaccharide yield is high, with high purity, and operating process is simpler, and cost is relatively low；And polysaccharide can further be separated pure
Change, obtains the higher polysaccharide component of purity, be conducive to the development and utilization of Chinese caterpillar fungus polysaccharide product.
The present invention is achieved through the following technical solutions:
A kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation, includes the following steps:
A, aweto is inoculated on cordyceps sinensis seed culture medium, puts shaking table culture case into, cultivated 3-4d, obtain
Seed liquor；
B, the seed liquor for obtaining step A, is inoculated on the cordyceps sinensis fluid nutrient medium of sterilized processing, and inoculum concentration is
6-9% is put into constant temperature and humidity incubator, under the conditions of 28.5 DEG C of temperature, is fermented 5-8 days, and aweto mycelium hair is obtained
Zymotic fluid；
C, the obtained Cordyceps mycelium fermented liquid of step B is put into wall-breaking machine, the distillation of 2-4 times of quality is added
Water crushes 8-15min, obtains cordyceps sinensis mixed liquor；
D, the cordyceps sinensis mixed liquor for obtaining step C, ultrasonication 40-50min, the refluxing extraction at 70-75 DEG C
4-5h is concentrated after extracting solution filtering, obtains polysaccharide concentrate；
E, the dehydrated alcohol of 3-4.5 times of volume is added in the polysaccharide concentrate that step D is obtained, 4 DEG C are cooled to after stirring,
18-30 hours are stood, carries out centrifugal treating, and precipitating drying is obtained into Chinese caterpillar fungus polysaccharide extract；
F, AB-8 type macroporous absorbent resin and D-280 type macroporous absorbent resin macroporous absorption tree are sufficiently eluted with distilled water
Rouge removes remaining solvent in resin surface and duct with the alcohol solution dipping 2-3h that mass fraction containing ammonia is 8-14g/kg
And reactive resin, it is washed with deionized water to neutrality, no alcohol taste, using wet method dress post, resin is slowly packed into the glass of diameter 10cm
In glass column, the high about 150cm of resin；Distilled water is added to dissolve the Chinese caterpillar fungus polysaccharide extract that step E is obtained, upper AB-8 type macropore
Adsorption resin column is eluted repeatedly, and collects eluent；
G, the eluent for obtaining step F, upper D-280 large pore resin absorption column, decolourizes, after decoloration, vacuum concentration,
Vacuum drying, obtains Chinese caterpillar fungus polysaccharide；
Wherein, in step A cordyceps sinensis seed culture medium described in every kg composition are as follows: glucose sugar 25-35g, yeast extract
1.8-2.2g, mealy potato 185-195g, dried silkworm chrysalis meal 12-15g, acid hydrolyzed casein 1.2-1.4g, potassium dihydrogen phosphate 1.5-1.8g,
Magnesium sulfate 0.9-1.2g, remaining be distilled water；
The composition of cordyceps sinensis fluid nutrient medium described in every kg in step B are as follows: corn flour 20-25g, millet flour 9-12g, soil
Bean powder 7-9g, glucose sugar 38-48g, potassium dihydrogen phosphate 2.1-3.5g, acid hydrolyzed casein 2.8-3.5g, magnesium sulfate 0.9-1.3g,
Remaining is distilled water.
Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation of the invention realizes culture, extraction, purifying company
Continuous processing, extraction efficiency is high, and Chinese caterpillar fungus polysaccharide yield is high, with high purity, and operating process is simpler, and cost is relatively low；And energy will be more
Sugar further isolates and purifies, and obtains the higher polysaccharide component of purity, is conducive to the development and utilization of Chinese caterpillar fungus polysaccharide product.
Preferably, aweto described in step A be Institute of Microorganism, Academia Sinica Paecilomyces hepiali chen or
Hirsutella hepiali Chen et Shen.
It preferably, also include 1-2 piece vitamin B compound in cordyceps sinensis fluid nutrient medium described in every kg in step B.
Preferably, in step A cordyceps sinensis seed culture medium described in every kg composition are as follows: glucose sugar 32.5g, yeast extract
1.95g, mealy potato 190g, dried silkworm chrysalis meal 13.5g, acid hydrolyzed casein 1.3g, potassium dihydrogen phosphate 1.7g, magnesium sulfate 1.1g, remaining
For distilled water.
Preferably, in step B cordyceps sinensis fluid nutrient medium described in every kg composition are as follows: corn flour 22g, millet flour 10g,
Mealy potato 8.5g, glucose sugar 45.5g, potassium dihydrogen phosphate 2.9g, acid hydrolyzed casein 3.3g, magnesium sulfate 1.2g, remaining for distillation
Water.
Preferably, the condition of culture control of shaking table culture case described in step A is the revolving speed of 115-130r/min, 28.5 DEG C
Environment temperature.
Preferably, concentration described in step D is concentrated at 60 DEG C.
Preferably, vacuum concentration described in step G is concentrated in vacuo at 45 DEG C.
Preferably, centrifugation described in step E is 6000r/min centrifugation 10min.
Compared with prior art, the present invention have the following advantages that and the utility model has the advantages that
Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation of the invention realizes culture, extraction, purifying company
Continuous processing, extraction efficiency is high, and Chinese caterpillar fungus polysaccharide yield is high, with high purity, and operating process is simpler, and cost is relatively low；And energy will be more
Sugar further isolates and purifies, and obtains the higher polysaccharide component of purity, is conducive to the development and utilization of Chinese caterpillar fungus polysaccharide product.
Specific embodiment
The present invention is described in further detail below with reference to embodiment, embodiments of the present invention are not limited thereto.
Test method described in following embodiments or test method are unless otherwise specified conventional method；The reagent
And material is obtained from routine business approach, or prepare in conventional manner unless otherwise specified.
Embodiment 1:
A kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation, includes the following steps:
A, aweto is inoculated on cordyceps sinensis seed culture medium, puts shaking table culture case into, cultivated 3-4d, obtain
Seed liquor；
B, the seed liquor for obtaining step A, is inoculated on the cordyceps sinensis fluid nutrient medium of sterilized processing, and inoculum concentration is
6-9% is put into constant temperature and humidity incubator, under the conditions of 28.5 DEG C of temperature, is fermented 5-8 days, and aweto mycelium hair is obtained
Zymotic fluid；
C, the obtained Cordyceps mycelium fermented liquid of step B is put into wall-breaking machine, the distillation of 2-4 times of quality is added
Water crushes 8-15min, obtains cordyceps sinensis mixed liquor；
D, the cordyceps sinensis mixed liquor for obtaining step C, ultrasonication 40-50min, the refluxing extraction at 70-75 DEG C
4-5h is concentrated after extracting solution filtering, obtains polysaccharide concentrate；
E, the dehydrated alcohol of 3-4.5 times of volume is added in the polysaccharide concentrate that step D is obtained, 4 DEG C are cooled to after stirring,
18-30 hours are stood, carries out centrifugal treating, and precipitating drying is obtained into Chinese caterpillar fungus polysaccharide extract；
F, AB-8 type macroporous absorbent resin and D-280 type macroporous absorbent resin macroporous absorption tree are sufficiently eluted with distilled water
Rouge removes remaining solvent in resin surface and duct with the alcohol solution dipping 2-3h that mass fraction containing ammonia is 8-14g/kg
And reactive resin, it is washed with deionized water to neutrality, no alcohol taste, using wet method dress post, resin is slowly packed into the glass of diameter 10cm
In glass column, the high about 150cm of resin；Distilled water is added to dissolve the Chinese caterpillar fungus polysaccharide extract that step E is obtained, upper AB-8 type macropore
Adsorption resin column is eluted repeatedly, and collects eluent；
G, the eluent for obtaining step F, upper D-280 large pore resin absorption column, decolourizes, after decoloration, vacuum concentration,
Vacuum drying, obtains Chinese caterpillar fungus polysaccharide；
Wherein, in step A cordyceps sinensis seed culture medium described in every kg composition are as follows: glucose sugar 25-35g, yeast extract
1.8-2.2g, mealy potato 185-195g, dried silkworm chrysalis meal 12-15g, acid hydrolyzed casein 1.2-1.4g, potassium dihydrogen phosphate 1.5-1.8g,
Magnesium sulfate 0.9-1.2g, remaining be distilled water；
The composition of cordyceps sinensis fluid nutrient medium described in every kg in step B are as follows: corn flour 20-25g, millet flour 9-12g, soil
Bean powder 7-9g, glucose sugar 38-48g, potassium dihydrogen phosphate 2.1-3.5g, acid hydrolyzed casein 2.8-3.5g, magnesium sulfate 0.9-1.3g,
Remaining is distilled water.
Embodiment 2:
A kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation, includes the following steps:
A, aweto is inoculated on cordyceps sinensis seed culture medium, puts shaking table culture case into, cultivated 3-4d, obtain
Seed liquor；
B, the seed liquor for obtaining step A, is inoculated on the cordyceps sinensis fluid nutrient medium of sterilized processing, and inoculum concentration is
6-9% is put into constant temperature and humidity incubator, under the conditions of 28.5 DEG C of temperature, is fermented 5-8 days, and aweto mycelium hair is obtained
Zymotic fluid；
C, the obtained Cordyceps mycelium fermented liquid of step B is put into wall-breaking machine, the distillation of 2-4 times of quality is added
Water crushes 8-15min, obtains cordyceps sinensis mixed liquor；
D, the cordyceps sinensis mixed liquor for obtaining step C, ultrasonication 40-50min, the refluxing extraction at 70-75 DEG C
4-5h is concentrated after extracting solution filtering, obtains polysaccharide concentrate；
E, the dehydrated alcohol of 3-4.5 times of volume is added in the polysaccharide concentrate that step D is obtained, 4 DEG C are cooled to after stirring,
18-30 hours are stood, carries out centrifugal treating, and precipitating drying is obtained into Chinese caterpillar fungus polysaccharide extract；
F, AB-8 type macroporous absorbent resin and D-280 type macroporous absorbent resin macroporous absorption tree are sufficiently eluted with distilled water
Rouge removes remaining solvent in resin surface and duct with the alcohol solution dipping 2-3h that mass fraction containing ammonia is 8-14g/kg
And reactive resin, it is washed with deionized water to neutrality, no alcohol taste, using wet method dress post, resin is slowly packed into the glass of diameter 10cm
In glass column, the high about 150cm of resin；Distilled water is added to dissolve the Chinese caterpillar fungus polysaccharide extract that step E is obtained, upper AB-8 type macropore
Adsorption resin column is eluted repeatedly, and collects eluent；
G, the eluent for obtaining step F, upper D-280 large pore resin absorption column, decolourizes, after decoloration, vacuum concentration,
Vacuum drying, obtains Chinese caterpillar fungus polysaccharide；
Wherein, in step A cordyceps sinensis seed culture medium described in every kg composition are as follows: glucose sugar 25-35g, yeast extract
1.8-2.2g, mealy potato 185-195g, dried silkworm chrysalis meal 12-15g, acid hydrolyzed casein 1.2-1.4g, potassium dihydrogen phosphate 1.5-1.8g,
Magnesium sulfate 0.9-1.2g, remaining be distilled water；
The composition of cordyceps sinensis fluid nutrient medium described in every kg in step B are as follows: corn flour 20-25g, millet flour 9-12g, soil
Bean powder 7-9g, glucose sugar 38-48g, potassium dihydrogen phosphate 2.1-3.5g, acid hydrolyzed casein 2.8-3.5g, magnesium sulfate 0.9-1.3g,
Remaining is distilled water.
Preferably, aweto described in step A be Institute of Microorganism, Academia Sinica Paecilomyces hepiali chen or
Hirsutella hepiali Chen et Shen.
It preferably, also include 1-2 piece vitamin B compound in cordyceps sinensis fluid nutrient medium described in every kg in step B.
Preferably, in step A cordyceps sinensis seed culture medium described in every kg composition are as follows: glucose sugar 32.5g, yeast extract
1.95g, mealy potato 190g, dried silkworm chrysalis meal 13.5g, acid hydrolyzed casein 1.3g, potassium dihydrogen phosphate 1.7g, magnesium sulfate 1.1g, remaining
For distilled water.
Preferably, in step B cordyceps sinensis fluid nutrient medium described in every kg composition are as follows: corn flour 22g, millet flour 10g,
Mealy potato 8.5g, glucose sugar 45.5g, potassium dihydrogen phosphate 2.9g, acid hydrolyzed casein 3.3g, magnesium sulfate 1.2g, remaining for distillation
Water.
Preferably, the condition of culture control of shaking table culture case described in step A is the revolving speed of 115-130r/min, 28.5 DEG C
Environment temperature.
Preferably, concentration described in step D is concentrated at 60 DEG C.
Preferably, vacuum concentration described in step G is concentrated in vacuo at 45 DEG C.
Preferably, centrifugation described in step E is 6000r/min centrifugation 10min.
Embodiment 3:
A kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation, includes the following steps:
A, aweto is inoculated on cordyceps sinensis seed culture medium, puts shaking table culture case into, cultivated 3d, planted
Sub- liquid；
B, the seed liquor for obtaining step A, is inoculated on the cordyceps sinensis fluid nutrient medium of sterilized processing, and inoculum concentration is
9%, it puts into constant temperature and humidity incubator, under the conditions of 28.5 DEG C of temperature, ferments 5 days, obtain Cordyceps mycelium fermented liquid；
C, the obtained Cordyceps mycelium fermented liquid of step B is put into wall-breaking machine, the distilled water of 2 times of quality is added,
8min is crushed, cordyceps sinensis mixed liquor is obtained；
D, the cordyceps sinensis mixed liquor for obtaining step C, ultrasonication 40min, the refluxing extraction 5h at 70 DEG C are extracted
It is concentrated after liquid filtering, obtains polysaccharide concentrate；
E, the dehydrated alcohol of 3 times of volumes is added in the polysaccharide concentrate that step D is obtained, 4 DEG C are cooled to after stirring, stands
18 hours, centrifugal treating is carried out, and precipitating drying is obtained into Chinese caterpillar fungus polysaccharide extract；
F, AB-8 type macroporous absorbent resin and D-280 type macroporous absorbent resin macroporous absorption tree are sufficiently eluted with distilled water
Rouge removes remaining solvent and activity in resin surface and duct with the alcohol solution dipping 3h that mass fraction containing ammonia is 8g/kg
Resin is washed with deionized water to neutrality, no alcohol taste, using wet method dress post, resin is slowly fitted into the glass column of diameter 10cm,
The high about 150cm of resin；Distilled water is added to dissolve the Chinese caterpillar fungus polysaccharide extract that step E is obtained, upper AB-8 type macroporous absorption tree
Rouge column, is eluted repeatedly, and collects eluent；
G, the eluent for obtaining step F, upper D-280 large pore resin absorption column, decolourizes, after decoloration, vacuum concentration,
Vacuum drying, obtains Chinese caterpillar fungus polysaccharide；
Wherein, in step A cordyceps sinensis seed culture medium described in every kg composition are as follows: glucose sugar 25g, yeast extract 1.8g,
Mealy potato 185g, dried silkworm chrysalis meal 12g, acid hydrolyzed casein 1.2g, potassium dihydrogen phosphate 1.5g, magnesium sulfate 0.9g, remaining be distilled water；
The composition of cordyceps sinensis fluid nutrient medium described in every kg in step B are as follows: corn flour 20g, millet flour 9g, mealy potato
7g, glucose sugar 38g, potassium dihydrogen phosphate 2.1g, acid hydrolyzed casein 2.8g, magnesium sulfate 0.9g, remaining be distilled water.
In the present embodiment, aweto described in step A is that the bat moth of Institute of Microorganism, Academia Sinica is quasi-
Mould.
It in the present embodiment, also include 1 tablet of vitamin B compound in cordyceps sinensis fluid nutrient medium described in every kg in step B.
In the present embodiment, the condition of culture control of shaking table culture case described in step A is the revolving speed of 115r/min, 28.5
DEG C environment temperature.
In the present embodiment, concentration described in step D is concentrated at 60 DEG C.
In the present embodiment, vacuum concentration described in step G is concentrated in vacuo at 45 DEG C.
In the present embodiment, centrifugation described in step E is 6000r/min centrifugation 10min.
Embodiment 4:
A kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation, includes the following steps:
A, aweto is inoculated on cordyceps sinensis seed culture medium, puts shaking table culture case into, cultivated 4d, planted
Sub- liquid；
B, the seed liquor for obtaining step A, is inoculated on the cordyceps sinensis fluid nutrient medium of sterilized processing, and inoculum concentration is
6%, it puts into constant temperature and humidity incubator, under the conditions of 28.5 DEG C of temperature, ferments 8 days, obtain Cordyceps mycelium fermented liquid；
C, the obtained Cordyceps mycelium fermented liquid of step B is put into wall-breaking machine, the distilled water of 4 times of quality is added,
15min is crushed, cordyceps sinensis mixed liquor is obtained；
D, the cordyceps sinensis mixed liquor for obtaining step C, ultrasonication 50min, the refluxing extraction 4h at 75 DEG C are extracted
It is concentrated after liquid filtering, obtains polysaccharide concentrate；
E, the dehydrated alcohol of 4.5 times of volumes is added in the polysaccharide concentrate that step D is obtained, is cooled to 4 DEG C after stirring, it is quiet
It sets 30 hours, carries out centrifugal treating, and precipitating drying is obtained into Chinese caterpillar fungus polysaccharide extract；
F, AB-8 type macroporous absorbent resin and D-280 type macroporous absorbent resin macroporous absorption tree are sufficiently eluted with distilled water
Rouge removes remaining solvent and work in resin surface and duct with the alcohol solution dipping 2h that mass fraction containing ammonia is 14g/kg
Property resin, is washed with deionized water to neutrality, no alcohol taste, using wet method dress post, resin is slowly packed into the glass column of diameter 10cm
In, the high about 150cm of resin；Distilled water is added to dissolve the Chinese caterpillar fungus polysaccharide extract that step E is obtained, upper AB-8 type macroporous absorption
Resin column is eluted repeatedly, and collects eluent；
G, the eluent for obtaining step F, upper D-280 large pore resin absorption column, decolourizes, after decoloration, vacuum concentration,
Vacuum drying, obtains Chinese caterpillar fungus polysaccharide；
Wherein, in step A cordyceps sinensis seed culture medium described in every kg composition are as follows: glucose sugar 35g, yeast extract 2.2g,
Mealy potato 195g, dried silkworm chrysalis meal 15g, acid hydrolyzed casein 1.4g, potassium dihydrogen phosphate 1.8g, magnesium sulfate 1.2g, remaining be distilled water；
The composition of cordyceps sinensis fluid nutrient medium described in every kg in step B are as follows: corn flour 25g, millet flour 12g, mealy potato
9g, glucose sugar 48g, potassium dihydrogen phosphate 3.5g, acid hydrolyzed casein 3.5g, magnesium sulfate 1.3g, remaining be distilled water.
In the present embodiment, aweto described in step A is the bat moth quilt of Institute of Microorganism, Academia Sinica
Hair spore.
It in the present embodiment, also include 2 tablets of vitamin B compounds in cordyceps sinensis fluid nutrient medium described in every kg in step B.
In the present embodiment, the condition of culture control of shaking table culture case described in step A is the revolving speed of 130r/min, 28.5
DEG C environment temperature.
In the present embodiment, concentration described in step D is concentrated at 60 DEG C.
In the present embodiment, vacuum concentration described in step G is concentrated in vacuo at 45 DEG C.
In the present embodiment, centrifugation described in step E is 6000r/min centrifugation 10min.
Embodiment 5:
A kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation, includes the following steps:
A, aweto is inoculated on cordyceps sinensis seed culture medium, puts shaking table culture case into, cultivated 3.5d, obtain
Seed liquor；
B, the seed liquor for obtaining step A, is inoculated on the cordyceps sinensis fluid nutrient medium of sterilized processing, and inoculum concentration is
7%, it puts into constant temperature and humidity incubator, under the conditions of 28.5 DEG C of temperature, ferments 6 days, obtain Cordyceps mycelium fermented liquid；
C, the obtained Cordyceps mycelium fermented liquid of step B is put into wall-breaking machine, the distilled water of 3 times of quality is added,
10min is crushed, cordyceps sinensis mixed liquor is obtained；
D, the cordyceps sinensis mixed liquor for obtaining step C, ultrasonication 45min, refluxing extraction 4.5h, mentions at 72 DEG C
It is concentrated after taking liquid to filter, obtains polysaccharide concentrate；
E, the dehydrated alcohol of 3.5 times of volumes is added in the polysaccharide concentrate that step D is obtained, is cooled to 4 DEG C after stirring, it is quiet
It sets 22 hours, carries out centrifugal treating, and precipitating drying is obtained into Chinese caterpillar fungus polysaccharide extract；
F, AB-8 type macroporous absorbent resin and D-280 type macroporous absorbent resin macroporous absorption tree are sufficiently eluted with distilled water
Rouge, with the alcohol solution dipping 2.5h that mass fraction containing ammonia is 11g/kg, remove in resin surface and duct remaining solvent and
Reactive resin is washed with deionized water to neutrality, no alcohol taste, and using wet method dress post, resin is slowly packed into the glass of diameter 10cm
In column, the high about 150cm of resin；Distilled water is added to dissolve the Chinese caterpillar fungus polysaccharide extract that step E is obtained, upper AB-8 type macropore is inhaled
Attached resin column, is eluted repeatedly, and collects eluent；
G, the eluent for obtaining step F, upper D-280 large pore resin absorption column, decolourizes, after decoloration, vacuum concentration,
Vacuum drying, obtains Chinese caterpillar fungus polysaccharide；
Wherein, in step A cordyceps sinensis seed culture medium described in every kg composition are as follows: glucose sugar 32.5g, yeast extract
1.95g, mealy potato 190g, dried silkworm chrysalis meal 13.5g, acid hydrolyzed casein 1.3g, potassium dihydrogen phosphate 1.7g, magnesium sulfate 1.1g, remaining
For distilled water；
The composition of cordyceps sinensis fluid nutrient medium described in every kg in step B are as follows: corn flour 22g, millet flour 10g, mealy potato
8.5g, glucose sugar 45.5g, potassium dihydrogen phosphate 2.9g, acid hydrolyzed casein 3.3g, magnesium sulfate 1.2g, remaining be distilled water.
In the present embodiment, aweto described in step A is that the bat moth of Institute of Microorganism, Academia Sinica is quasi-
Mould.
It in the present embodiment, also include 2 tablets of vitamin B compounds in cordyceps sinensis fluid nutrient medium described in every kg in step B.
In the present embodiment, the condition of culture control of shaking table culture case described in step A is the revolving speed of 120r/min, 28.5
DEG C environment temperature.
In the present embodiment, concentration described in step D is concentrated at 60 DEG C.
In the present embodiment, vacuum concentration described in step G is concentrated in vacuo at 45 DEG C.
In the present embodiment, centrifugation described in step E is 6000r/min centrifugation 10min.
Embodiment 6:
A kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation, includes the following steps:
A, aweto is inoculated on cordyceps sinensis seed culture medium, puts shaking table culture case into, cultivated 3.5d, obtain
Seed liquor；
B, the seed liquor for obtaining step A, is inoculated on the cordyceps sinensis fluid nutrient medium of sterilized processing, and inoculum concentration is
8%, it puts into constant temperature and humidity incubator, under the conditions of 28.5 DEG C of temperature, ferments 7 days, obtain Cordyceps mycelium fermented liquid；
C, the obtained Cordyceps mycelium fermented liquid of step B is put into wall-breaking machine, the distillation of 3.5 times of quality is added
Water crushes 12min, obtains cordyceps sinensis mixed liquor；
D, the cordyceps sinensis mixed liquor for obtaining step C, ultrasonication 48min, refluxing extraction 4.5h, mentions at 73 DEG C
It is concentrated after taking liquid to filter, obtains polysaccharide concentrate；
E, the dehydrated alcohol of 4 times of volumes is added in the polysaccharide concentrate that step D is obtained, 4 DEG C are cooled to after stirring, stands
28 hours, centrifugal treating is carried out, and precipitating drying is obtained into Chinese caterpillar fungus polysaccharide extract；
F, AB-8 type macroporous absorbent resin and D-280 type macroporous absorbent resin macroporous absorption tree are sufficiently eluted with distilled water
Rouge, with the alcohol solution dipping 2.5h that mass fraction containing ammonia is 12g/kg, remove in resin surface and duct remaining solvent and
Reactive resin is washed with deionized water to neutrality, no alcohol taste, and using wet method dress post, resin is slowly packed into the glass of diameter 10cm
In column, the high about 150cm of resin；Distilled water is added to dissolve the Chinese caterpillar fungus polysaccharide extract that step E is obtained, upper AB-8 type macropore is inhaled
Attached resin column, is eluted repeatedly, and collects eluent；
G, the eluent for obtaining step F, upper D-280 large pore resin absorption column, decolourizes, after decoloration, vacuum concentration,
Vacuum drying, obtains Chinese caterpillar fungus polysaccharide；
Wherein, in step A cordyceps sinensis seed culture medium described in every kg composition are as follows: glucose sugar 28g, yeast extract 2.1g,
Mealy potato 188g, dried silkworm chrysalis meal 14g, acid hydrolyzed casein 1.25g, potassium dihydrogen phosphate 1.6g, magnesium sulfate 1.05g, remaining for distillation
Water；
The composition of cordyceps sinensis fluid nutrient medium described in every kg in step B are as follows: corn flour 23g, millet flour 11g, mealy potato
8g, glucose sugar 46g, potassium dihydrogen phosphate 3.1g, acid hydrolyzed casein 3g, magnesium sulfate 1.25g, remaining be distilled water.
In the present embodiment, aweto described in step A is the bat moth quilt of Institute of Microorganism, Academia Sinica
Hair spore.
It in the present embodiment, also include 1 tablet of vitamin B compound in cordyceps sinensis fluid nutrient medium described in every kg in step B.
In the present embodiment, the condition of culture control of shaking table culture case described in step A is the revolving speed of 125r/min, 28.5
DEG C environment temperature.
In the present embodiment, concentration described in step D is concentrated at 60 DEG C.
In the present embodiment, vacuum concentration described in step G is concentrated in vacuo at 45 DEG C.
In the present embodiment, centrifugation described in step E is 6000r/min centrifugation 10min.
Compared with prior art, the present invention have the following advantages that and the utility model has the advantages that
Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation of the invention realizes culture, extraction, purifying company
Continuous processing, extraction efficiency is high, and Chinese caterpillar fungus polysaccharide yield is high, with high purity, and operating process is simpler, and cost is relatively low；And energy will be more
Sugar further isolates and purifies, and obtains the higher polysaccharide component of purity, is conducive to the development and utilization of Chinese caterpillar fungus polysaccharide product.
The above is only presently preferred embodiments of the present invention, not does limitation in any form to the present invention, it is all according to
According to technical spirit any simple modification to the above embodiments of the invention, equivalent variations, protection of the invention is each fallen within
Within the scope of.
Claims (9)
1. a kind of Chinese caterpillar fungus polysaccharide extraction and purification process based on liquid fermentation, characterized by the following steps:
A, aweto is inoculated on cordyceps sinensis seed culture medium, puts shaking table culture case into, cultivated 3-4d, obtain seed
Liquid；
B, the seed liquor for obtaining step A is inoculated on the cordyceps sinensis fluid nutrient medium of sterilized processing, inoculum concentration 6-
9%, it puts into constant temperature and humidity incubator, under the conditions of 28.5 DEG C of temperature, ferments 5-8 days, obtain aweto mycelium fermentation
Liquid；
C, the obtained Cordyceps mycelium fermented liquid of step B is put into wall-breaking machine, the distilled water of 2-4 times of quality, powder is added
Broken 8-15min obtains cordyceps sinensis mixed liquor；
D, the cordyceps sinensis mixed liquor for obtaining step C, ultrasonication 40-50min, the refluxing extraction 4-5h at 70-75 DEG C,
It is concentrated after extracting solution filtering, obtains polysaccharide concentrate；
E, the dehydrated alcohol of 3-4.5 times of volume is added in the polysaccharide concentrate that step D is obtained, 4 DEG C are cooled to after stirring, stands
18-30 hours, centrifugal treating is carried out, and precipitating drying is obtained into Chinese caterpillar fungus polysaccharide extract；
F, AB-8 type macroporous absorbent resin and D-280 type macroporous absorbent resin macroporous absorbent resin are sufficiently eluted with distilled water, are used
Mass fraction containing ammonia is the alcohol solution dipping 2-3h of 8-14g/kg, removes remaining solvent and activity in resin surface and duct
Resin is washed with deionized water to neutrality, no alcohol taste, using wet method dress post, resin is slowly fitted into the glass column of diameter 10cm,
The high about 150cm of resin；Distilled water is added to dissolve the Chinese caterpillar fungus polysaccharide extract that step E is obtained, upper AB-8 type macroporous absorption tree
Rouge column, is eluted repeatedly, and collects eluent；
G, the eluent for obtaining step F, upper D-280 large pore resin absorption column, decolourizes, after decoloration, vacuum concentration, and vacuum
It is dry, obtain Chinese caterpillar fungus polysaccharide；
Wherein, in step A cordyceps sinensis seed culture medium described in every kg composition are as follows: glucose sugar 25-35g, yeast extract 1.8-
2.2g, mealy potato 185-195g, dried silkworm chrysalis meal 12-15g, acid hydrolyzed casein 1.2-1.4g, potassium dihydrogen phosphate 1.5-1.8g, sulfuric acid
Magnesium 0.9-1.2g, remaining be distilled water；
The composition of cordyceps sinensis fluid nutrient medium described in every kg in step B are as follows: corn flour 20-25g, millet flour 9-12g, mealy potato
7-9g, glucose sugar 38-48g, potassium dihydrogen phosphate 2.1-3.5g, acid hydrolyzed casein 2.8-3.5g, magnesium sulfate 0.9-1.3g, remaining
For distilled water.
2. the Chinese caterpillar fungus polysaccharide extraction and purification process according to claim 1 based on liquid fermentation, it is characterised in that: step
Aweto described in rapid A is the Paecilomyces hepiali chen or Hirsutella hepiali Chen et Shen of Institute of Microorganism, Academia Sinica.
3. the Chinese caterpillar fungus polysaccharide extraction and purification process according to claim 1 based on liquid fermentation, it is characterised in that: step
Also comprising 1-2 piece vitamin B compound in cordyceps sinensis fluid nutrient medium described in every kg in rapid B.
4. the Chinese caterpillar fungus polysaccharide extraction and purification process according to claim 1 based on liquid fermentation, it is characterised in that: step
The composition of cordyceps sinensis seed culture medium described in every kg in rapid A are as follows: glucose sugar 32.5g, yeast extract 1.95g, mealy potato 190g, silkworm
Pupa powder 13.5g, acid hydrolyzed casein 1.3g, potassium dihydrogen phosphate 1.7g, magnesium sulfate 1.1g, remaining be distilled water.
5. the Chinese caterpillar fungus polysaccharide extraction and purification process according to claim 1 based on liquid fermentation, it is characterised in that: step
The composition of cordyceps sinensis fluid nutrient medium described in every kg in rapid B are as follows: corn flour 22g, millet flour 10g, mealy potato 8.5g, glucose sugar
45.5g, potassium dihydrogen phosphate 2.9g, acid hydrolyzed casein 3.3g, magnesium sulfate 1.2g, remaining be distilled water.
6. the Chinese caterpillar fungus polysaccharide extraction and purification process according to claim 1 based on liquid fermentation, it is characterised in that: step
The condition of culture control of shaking table culture case described in rapid A is the revolving speed of 115-130r/min, 28.5 DEG C of environment temperature.
7. the Chinese caterpillar fungus polysaccharide extraction and purification process according to claim 1 based on liquid fermentation, it is characterised in that: step
Concentration described in rapid D is concentrated at 60 DEG C.
8. the Chinese caterpillar fungus polysaccharide extraction and purification process according to claim 1 based on liquid fermentation, it is characterised in that: step
Vacuum concentration described in rapid G is concentrated in vacuo at 45 DEG C.
9. the Chinese caterpillar fungus polysaccharide extraction and purification process according to claim 1 based on liquid fermentation, it is characterised in that: step
Centrifugation described in rapid E is 6000r/min centrifugation 10min.
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