CN106810618A - A kind of extraction from Chinese caterpillar fungus culture medium and the method for continuous polysaccharide enrichment - Google Patents
A kind of extraction from Chinese caterpillar fungus culture medium and the method for continuous polysaccharide enrichment Download PDFInfo
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- CN106810618A CN106810618A CN201710009796.0A CN201710009796A CN106810618A CN 106810618 A CN106810618 A CN 106810618A CN 201710009796 A CN201710009796 A CN 201710009796A CN 106810618 A CN106810618 A CN 106810618A
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention discloses a kind of extraction from Chinese caterpillar fungus culture medium and the method for continuous polysaccharide enrichment, the drying of step including Cordyceps militaris rice residual media, ultramicro grinding, biological multi-enzyme system enzymatic hydrolysis, centrifugal filtration, backflow control, multifunctional membrane enrichment method, spray drying, qualitative, quantitative, establish Cordyceps sinensis polysaccharide it is quick, efficient, be economically separated method.The present invention prepares Cordyceps sinensis polysaccharide using green extraction joint dysfunction type membrane separation techniques such as enzyme process, that is, ensure that the purity and quality of Cordyceps sinensis polysaccharide, fast enriching concentration is realized again, and ensure that the bioactivity of polysaccharide.The present invention effectively make use of discarded Chinese caterpillar fungus culture medium, save resource and cost.The suitable large-scale industrial production of the present invention.By Cordyceps sinensis polysaccharide content in the isolated polysaccharide concentrate of the method for the present invention up to more than 80%, product recovery rate is up to more than 85%.
Description
Technical field
The invention belongs to medicines and health protection, food processing technology field, particularly belong to one kind and extracted from Chinese caterpillar fungus culture medium
And the method for continuous polysaccharide enrichment.
Background technology
Cordyceps militaris [Cordyceps militaris (Fr) Link.], also known as northern Chinese caterpillar Fungus, Cordceps militaris, belong to sac fungus
Subphylum, gang pyrenomycetes, Sphaerials, Clavicipitaceae, Cordyceps sinensis fungus.It is the type sepecies of Cordyceps, widely distributed, is countries in the world
Scholar recognizes and receives and complete stroma has been bred as under artificial condition and Cordyceps militaris has the Cordyceps sinensis polysaccharide of higher amount
With the active ingredient such as cordycepin, its unique pharmacological action increasingly caused the great attention of pharmacy circle.Due to Cordyceps militaris have with
Upper advantage, the outstanding person as medicinal Cordyceps Militaris in Cordyceps.The success of the artificial culture technique of Cordyceps militaris, is that China opens name
Another sky of precious medicinal material industrialization production.At present, the artificial culture of domestic Cordyceps militaris has enter into the industrialization stage.
Cordyceps sinensis polysaccharide as Cordyceps militaris one of main active, be content highest pharmacological actives in Cordyceps militaris
Matter, content can reach 10%, be a kind of of great value potential new medicine-food two-purpose resource.Cordyceps sinensis polysaccharide be by mannose,
The polysaccharide of the compositions such as cordycepin, adenosine, galactolipin, arabinose, glucose, fucose.Its active anticancer and immune work
Property causes the great interest of people, in addition Cordyceps sinensis be additionally operable to control pulmonary tuberculosis, shortness of breath breath with cough, impotence, nocturnal emission, spontaneous sweating,
Soreness of waist and knee joint etc..
Cordyceps sinensis polysaccharide is main in the industrial production at present extracts from fruiting bodies of cordyceps militaris.But due to cordyceps militaris sporocarp
Production cost is high, so as to cause the production cost of Cordyceps sinensis polysaccharide high.Nowadays the artificial cultivation technique of Cordyceps militaris is mutually treated as
It is ripe, it is widely used in production, certain economic benefit is achieved, substantial amounts of culture medium is generated the problem that therewith（Leftover bits and pieces）It is useless
Abandon, fouling of turning sour causes environmental pollution.Also there is researcher as animal feed, but it is not comprehensively utilized.Root
Found according to the measure of our early stages, mycelia is full of in Cordyceps militaris solid medium, wherein rich in substantial amounts of Cordyceps sinensis polysaccharide, but because not having
The technique for having maturation carries out extraction separation to effective ingredient therein, and solid medium is all by as useless generally after culture terminates
Thing is discarded.
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., due to there is low Cordyceps sinensis polysaccharide dissolution rate, many product impurity, complex process, production in above-mentioned these methods
The shortcomings of cycle is long, makes its industrialized production and larger scale clinical application receive limitation, and utilizes amylase, protein and fiber
The green extractions such as the compound ferment treatment such as plain enzyme carry out pre-treatment to cordyceps mycelium or culture medium, by Enzymatic Extraction
Condition is controlled, and can at utmost reduce the influence that wherein starch, cellulose etc. are extracted to Cordyceps sinensis polysaccharide, also can be at utmost
The yield and purity of Cordyceps sinensis polysaccharide are improved, while excluding influence tool of the non-functional polysaccharide such as starch, cellulose to product quality
Have the advantages that extraction conditions are gentle, recovery rate is high.And recent studies have shown that, the pharmacological activity of the fungi polysaccharide such as Chinese caterpillar fungus is tied with it
Structure, relative molecular weight etc. are closely related, and general molecular weight just has stronger biology living in the macromolecular components of 30-250 KDa
Property.However, the extracting method of traditional Cordyceps sinensis polysaccharide, such as water extraction and alcohol precipitation method, Thick many candies complex, the molecular weight of composition for obtaining
After scope is wide, and polysaccharide is acted on through ethanol, higher structure is easy to be destroyed, it is difficult to ensure that quality per batch products and
Curative effect, therefore, the polysaccharide with bioactivity is efficiently extracted and is enriched with, the quality tool to controlling Cordyceps sinensis polysaccharide product
It is of great significance.
The content of the invention
For the deficiency that background technology is proposed, present invention research one kind is extracted from Chinese caterpillar fungus culture medium and continuous enrichment is more
The method of sugar, its object is to：This method prepares Chinese caterpillar fungus using green extraction joint dysfunction type membrane separation techniques such as enzyme process
Polysaccharide, that is, ensure that the purity and quality of Cordyceps sinensis polysaccharide, fast enriching concentration be realized again, and ensure that the biology of polysaccharide is living
Property.
Technical solution of the invention：
A kind of extraction from Chinese caterpillar fungus culture medium and the method for continuous polysaccharide enrichment, comprise the following steps：
Cordyceps militaris rice residual media is dried, ultramicro grinding, biological multi-enzyme system enzymatic hydrolysis, centrifugal filtration, is returned
The steps such as flow control, multifunctional membrane enrichment method, spray drying, qualitative and quantitative detection.
Drying steps are specially：The harvesting of same kind is cut into sheet with the Cordyceps militaris rice medium after Cordyceps militaris,
It is respectively dried 2-4 times at 70-100 DEG C in vacuum drying chamber, each 1.5-3h, is≤5% to culture medium section water content.
Ultramicro grinding step is specially：The Cordyceps militaris rice medium section hammer leaf powder of water content≤5% that will be ready for
Broken machine is crushed, and crosses 100 mesh sieves, Cordyceps militaris rice medium meal is obtained, further by it through supersonic airstream rotary collision formula
After pulverizer is crushed, particle delivers to cyclone separator by air stream, after varigrained Cordyceps militaris rice medium powder is separated
Primary collection device and two grades of collectors are respectively enterd, air-flow crushing one-level separator and the second-order separation thing is obtained（Acquisition amount compared with
It is few）, air-flow crushing one-level separator.The air-flow crushing one-level separator that will have been prepared is put into planetary ball mill tank, using thing
Material and mill ball 1:1-1:3 ratio carries out planetary ball mill and crushes 1-3h, crushes 2-3 times altogether（Grinding time is total to be no more than
6h）, even-grained Cordyceps militaris rice medium Ultramicro-powder is obtained, size distribution is 20-60 μm, content of powder≤3%.
Biological multi-enzyme system enzyme process aqueous extraction step is specially：Additional proportion is 10-30 times in above-mentioned culture medium Ultramicro-powder
Water after, first extract 1-4h at 60-100 DEG C, be subsequently adding cellulase, amylase and protease（These three enzymes need to be most suitable
Temperature and optimal pH are close, and optimal pH scope is 6.0-7.5）, the addition of three kinds of enzymes is the 0.03%- for extracting system weight
0.20%（m/v）, three kinds of ratios of enzyme are 1:1:1.Leaching liquor after above-mentioned pre- extraction is supreme with HCl or NaOH regulations pH
The most suitable scope of enzyme is stated, and by temperature adjustment to 35-45 DEG C, after stirring enzymolysis 2-6h, pH is adjusted to neutrality, and in 90-95
Go out enzyme 3-6min at DEG C, is cooled to room temperature.
Centrifugal filtration and backflow rate-determining steps are specially：Extract solution containing precipitation obtained above is filtered, and
Filtrate is centrifuged 10-30min under the speed of 3000-5000r/min using vertical automatic scraper bottom discharge centrifugal machine, is obtained
To the supernatant extract solution containing Polysaccharides in Cultured Cordyceps militaris;The precipitation additional proportion that will be obtained is 5-15 times of water, according to biological multienzyme
It is that enzyme dosage in enzyme process aqueous extraction step, usage are extracted 1-2 times again, gained filtrate can merge with said extracted liquid and use.
Multifunctional membrane enrichment method step is specially：Polysaccharides in Cultured Cordyceps militaris extract solution obtained above is utilized into ceramic membrane equipment
Carry out activity enrichment.0.1-0.45 μm of membrane microfiltration was first used before ultrafiltration enrichment is carried out, it is a small amount of after continuous filter press to remove
Suspension solid content and finely ground particle substance, feed temperature are 30-60 DEG C, between pH 6-8, operating pressure 0.1-0.4MPa, during micro-filtration
Between 0.5-3.5h, collect micro-filtrate it is stand-by；Micro-filtrate obtained above is placed in Multi-function ultrafiltration equipment liquid storing barrel, according to work
Property polysaccharide molecular weight distribution, choose molecular cut off（MwCO）It is the organic film ultrafiltration of 30-300 KDa, ultrafiltration pressure choosing
0.1-0.4MPa is selected as, between pH 6-8, micro-filtration time 0.5-3.0h；The trapped fluid for obtaining is again with 0.1-0.45 μm of microfiltration membranes
Enrichment method 0.5-3.5h is carried out, now gained polysaccharide concentrate cycles of concentration is 5-15 times, and polysaccharide molecular weight distribution is in
The field of activity of 30-250 KDa.
Spray drying step is specially：Dry-type Cordyceps sinensis polysaccharide product such as to be produced, dries using closed cycle spray
Machine is dried treatment, and it is 150-190 DEG C to set spray drying device inlet temperature, and outlet temperature is 60-100 DEG C, is adjusted
Polysaccharides in Cultured Cordyceps militaris Ultramicro-powder can be collected after spray drying.
Qualitative and quantitative detection step is specially：
（1）High effective liquid chromatography for measuring Cordyceps sinensis polysaccharide content, purity and molecular weight
By the successive sample introductions of standard Dextran of different molecular weight, retention time is recorded（TR）, with the retention time of each standard items
（TR）It is abscissa, its molecular weight（M）For ordinate draws standard curve, molecular weight and retention time are drawn（TR）Recurrence side
Journey.
High-efficient liquid phase chromatogram condition is：Chromatograph：The high performance liquid chromatographs of Agilent 1200；Chromatographic column：TSK-GEL
G4000 PWXL chromatographic columns（300 × 7.8 mm）；Detector：The type EISDs of Alltech 3300（ELSD）,
Sensitivity is 7,55 DEG C of atomization temperature, the bar of atomizing pressure 3.06；Column temperature：30℃；Mobile phase：ddH2O；Flow velocity：0.8 mL/
min；Sample size：20 μL
Each Cordyceps sinensis polysaccharide sample presses above-mentioned steps sample introduction, is identified in the number and shape of chromatogram superiors according to each Cordyceps sinensis polysaccharide
The purity of polysaccharide；In addition, according to the retention time of gained（TR）, by the relative of each polysaccharide of the regression equation calculation of standard curve
Molecular weight；
（2）Sulfuric acid-phynol method determines Cordyceps sinensis polysaccharide content
The making of standard curve：Prepare the dextrose standard sample solution of 0.1 mg/mL, by above-mentioned standard product solution 0.0,50.0,
100.0th, 150.0,200.0,250.0,300.0 μ L are respectively placed in eight teat glasses of equivalent specifications, add water benefit extremely
500.0 μL.The distilled water of 500 μ L, the 6% μ L of the phenol 500.0 and mL of the concentrated sulfuric acid 2.5 are then respectively adding, vibration shakes up,
Room temperature place 20 min after, using ultraviolet-uisible spectrophotometer at 490 nm mensuration absorbance value.With monose standard items
Concentration makees abscissa, and absorbance makees ordinate and draws standard curve, and draws regression equation.
Sample determination：0.5 mL Cordyceps sinensis polysaccharide solution is taken in test tube, alternate standard product solution, subsequent operation is the same, put down
Row three times, determines respective absorbance, seeks the average value of three pipe A490（If measured value can be to sample beyond standard curve range
Carry out appropriate dilution）.
After testing, by Cordyceps sinensis polysaccharide content in the isolated polysaccharide concentrate of the method for the present invention up to more than 80%, produce
The product rate of recovery is up to more than 85%.
Beneficial effects of the present invention：
The yield of Cordyceps sinensis polysaccharide is farthest improved, the rice medium discarded after being harvested with Cordyceps militaris（Leftover bits and pieces）It is original
Material, extracts, using green extractions such as cellulase, amylase and protease Combined Processings to Cordyceps sinensis polysaccharide therein
Pre-treatment is carried out to Chinese caterpillar fungus culture medium, is controlled by Enzymatic Extraction condition, at utmost reduce wherein cellulose, shallow lake
The influence that powder and protein are extracted to Cordyceps sinensis polysaccharide, improves the yield of Cordyceps sinensis polysaccharide;Using equipment such as micro-filtration, milipore filters to carrying
The Cordyceps sinensis polysaccharide taken in liquid is enriched with, and by optimizing the key parameters such as membrane flux, reaches optimal concentration effect, is substituted traditional
The high pollutions such as organic solvent deposit, spin concentration, the method for highly energy-consuming, at utmost reduce the dirt of energy consumption and processing to environment
Dye, has saved the energy, without phase transformation in whole membrane separating process, the limited activity for protecting Cordyceps sinensis polysaccharide.It is of the invention effective
Discarded Chinese caterpillar fungus culture medium is make use of, resource and cost has been saved.The suitable large-scale industrial production of the present invention.
Specific embodiment
Embodiment 1
The harvesting of same kind is cut into sheet with the Cordyceps militaris rice medium after Cordyceps militaris, in the vacuum drying chamber at 70 DEG C point
Not Hong Gan 4 times, each 2h, to culture medium section water content be≤5%；The Cordyceps militaris rice training of water content≤5% that will be ready for
Support base section hammer crusher to crush, cross 100 mesh sieves, obtain Cordyceps militaris rice medium meal, whirlwind is delivered to by air stream
Separator, obtains air-flow crushing one-level separator and the second-order separation thing, and material and mill ball 1 are used after adopting:3 ratio enters every trade
Star ball mill grinding 1h, crushes 3 times altogether, obtains even-grained Cordyceps militaris rice medium Ultramicro-powder, and size distribution is 60 μm, powder
Last content≤3%；After additional proportion is 10 times of water in the above-mentioned culture medium Ultramicro-powder, 4h first is extracted at 60 DEG C, be subsequently adding
Cellulase, amylase and protease（Optimal pH scope is 6.0-7.5）, the addition of three kinds of enzymes is to extract system weight
0.03%（m/v）, and adding proportion is 1:1:1.Leaching liquor after above-mentioned pre- extraction is adjusted into pH to above-mentioned with HCl or NaOH
The most suitable scope of enzyme, and by temperature adjustment to 35 DEG C, after stirring enzymolysis 6h, pH is adjusted to neutrality, and in the enzyme that gone out at 90 DEG C
6min, is cooled to room temperature；By it is obtained above containing precipitation extract solution filtered, and by filtrate 3000 r/min speed
The lower centrifugation 30min of degree, obtains the supernatant extract solution containing Polysaccharides in Cultured Cordyceps militaris, and the precipitation additional proportion that will be obtained is 5 times of water, is pressed
Extracted again 1 time according to enzyme dosage, usage in biological multi-enzyme system enzyme process aqueous extraction step, gained filtrate can merge with said extracted liquid and make
With；0.1 μm of membrane microfiltration was first used before ultrafiltration enrichment is carried out, to remove suspension solid content a small amount of after continuous filter press and small
Grain material, feed temperature is 30 DEG C, pH 6, operating pressure 0.1MPa, and micro-filtration time 3.5h collects micro-filtrate stand-by；Will be above-mentioned
The micro-filtrate for obtaining is placed in Multi-function ultrafiltration equipment liquid storing barrel, according to active polysaccharide range of molecular weight distributions, chooses retention point
Son amount（MwCO）It is the organic film ultrafiltration of 30 KDa, ultrafiltration pressure selection is 0.1MPa, pH 6, micro-filtration time 3.0h；Obtain
Trapped fluid carries out enrichment method 3.5h with 0.1 μm of microfiltration membranes again, and now gained polysaccharide concentrate cycles of concentration is 7 times, and many
Glycan molecule amount field of activity of the distribution in 30-250 KDa；Dry-type Cordyceps sinensis polysaccharide product such as to be produced, follows using enclosed
Ring spray dryer is dried treatment, and it is 190 DEG C to set spray drying device inlet temperature, and outlet temperature is 100 DEG C, is carried out
Polysaccharides in Cultured Cordyceps militaris Ultramicro-powder can be collected after adjusting spray drying.
Embodiment 2
The harvesting of same kind is cut into sheet with the Cordyceps militaris rice medium after Cordyceps militaris, in the vacuum drying chamber at 80 DEG C point
Not Hong Gan 3 times, each 2h, to culture medium section water content be≤5%；The Cordyceps militaris rice training of water content≤5% that will be ready for
Support base section hammer crusher to crush, cross 100 mesh sieves, obtain Cordyceps militaris rice medium meal, whirlwind is delivered to by air stream
Separator, obtains air-flow crushing one-level separator and the second-order separation thing, and material and mill ball 1 are used after adopting:2 ratio enters every trade
Star ball mill grinding 1h, crushes 3 times altogether, obtains even-grained Cordyceps militaris rice medium Ultramicro-powder, and size distribution is 50 μm, powder
Last content≤3%；After additional proportion is 15 times of water in the above-mentioned culture medium Ultramicro-powder, 3h first is extracted at 60 DEG C, be subsequently adding
Cellulase, amylase and protease（Optimal pH scope is 6.0-7.5）, the addition of three kinds of enzymes is to extract system weight
0.06%（m/v）, and adding proportion is 1:1:1.Leaching liquor after above-mentioned pre- extraction is adjusted into pH to above-mentioned with HCl or NaOH
The most suitable scope of enzyme, and by temperature adjustment to 40 DEG C, after stirring enzymolysis 4h, pH is adjusted to neutrality, and in the enzyme that gone out at 95 DEG C
3min, is cooled to room temperature；By it is obtained above containing precipitation extract solution filtered, and by filtrate 3000 r/min speed
The lower centrifugation 30min of degree, obtains the supernatant extract solution containing Polysaccharides in Cultured Cordyceps militaris, and the precipitation additional proportion that will be obtained is 5 times of water, is pressed
Extracted again 1 time according to enzyme dosage, usage in biological multi-enzyme system enzyme process aqueous extraction step, gained filtrate can merge with said extracted liquid and make
With；0.2 μm of membrane microfiltration was first used before ultrafiltration enrichment is carried out, to remove suspension solid content a small amount of after continuous filter press and small
Grain material, feed temperature is 40 DEG C, pH 7, operating pressure 0.2MPa, and micro-filtration time 3.0h collects micro-filtrate stand-by；Will be above-mentioned
The micro-filtrate for obtaining is placed in Multi-function ultrafiltration equipment liquid storing barrel, according to active polysaccharide range of molecular weight distributions, chooses retention point
Son amount（MwCO）It is the organic film ultrafiltration of 50 KDa, ultrafiltration pressure selection is 0.2MPa, pH 6, micro-filtration time 2.5h；Obtain
Trapped fluid carries out enrichment method 3.0h with 0.2 μm of microfiltration membranes again, and now gained polysaccharide concentrate cycles of concentration is 9 times, and many
Glycan molecule amount field of activity of the distribution in 30-250 KDa；Dry-type Cordyceps sinensis polysaccharide product such as to be produced, follows using enclosed
Ring spray dryer is dried treatment, and it is 190 DEG C to set spray drying device inlet temperature, and outlet temperature is 100 DEG C, is carried out
Polysaccharides in Cultured Cordyceps militaris Ultramicro-powder can be collected after adjusting spray drying.
Embodiment 3
The harvesting of same kind is cut into sheet with the Cordyceps militaris rice medium after Cordyceps militaris, in the vacuum drying chamber at 90 DEG C point
Not Hong Gan 3 times, each 1.5h, to culture medium section water content be≤5%；The Cordyceps militaris rice of water content≤5% that will be ready for
Culture medium section is crushed with hammer crusher, crosses 100 mesh sieves, obtains Cordyceps militaris rice medium meal, and rotation is delivered to by air stream
Wind separator, obtains air-flow crushing one-level separator and the second-order separation thing, and material and mill ball 1 are used after adopting:2 ratio is carried out
Planetary ball mill crushes 1h, crushes 3 times altogether, obtains even-grained Cordyceps militaris rice medium Ultramicro-powder, and size distribution is 50 μm,
Content of powder≤3%；After additional proportion is 20 times of water in the above-mentioned culture medium Ultramicro-powder, 2h, Ran Houjia are first extracted at 80 DEG C
Enter cellulase, amylase and protease（Optimal pH scope is 6.0-7.5）, the addition of three kinds of enzymes is to extract system weight
0.06%（m/v）, and adding proportion is 1:1:1.Leaching liquor after above-mentioned pre- extraction is adjusted into pH to above-mentioned with HCl or NaOH
The most suitable scope of enzyme, and by temperature adjustment to 40 DEG C, after stirring enzymolysis 4h, pH is adjusted to neutrality, and in the enzyme that gone out at 95 DEG C
3min, is cooled to room temperature；By it is obtained above containing precipitation extract solution filtered, and by filtrate 4000 r/min speed
The lower centrifugation 20min of degree, obtains the supernatant extract solution containing Polysaccharides in Cultured Cordyceps militaris, and the precipitation additional proportion that will be obtained is 10 times of water,
Extracted again 1 time according to enzyme dosage, usage in biological multi-enzyme system enzyme process aqueous extraction step, gained filtrate can merge with said extracted liquid
Use；0.2 μm of membrane microfiltration was first used before ultrafiltration enrichment is carried out, to remove suspension solid content a small amount of after continuous filter press and small
Particulate matter, feed temperature is 50 DEG C, pH 7, operating pressure 0.2MPa, and micro-filtration time 2.5h collects micro-filtrate stand-by；Will be upper
State the micro-filtrate for obtaining to be placed in Multi-function ultrafiltration equipment liquid storing barrel, according to active polysaccharide range of molecular weight distributions, choose retention
Molecular weight（MwCO）It is the organic film ultrafiltration of 100 KDa, ultrafiltration pressure selection is 0.2MPa, pH 6, micro-filtration time 2.5h；Obtain
Trapped fluid carry out enrichment method 2.5h with 0.3 μm of microfiltration membranes again, now gained polysaccharide concentrate cycles of concentration is 10 times, and
Polysaccharide molecular weight field of activity of the distribution in 30-250 KDa；Dry-type Cordyceps sinensis polysaccharide product such as to be produced, using enclosed
Circulation spray dryer is dried treatment, and it is 180 DEG C to set spray drying device inlet temperature, and outlet temperature is 90 DEG C, is entered
Row can collect Polysaccharides in Cultured Cordyceps militaris Ultramicro-powder after adjusting spray drying.
Embodiment 4
The harvesting of same kind is cut into sheet with the Cordyceps militaris rice medium after Cordyceps militaris, in the vacuum drying chamber at 100 DEG C
It is respectively dried 2 times, each 1.5h, is≤5% to culture medium section water content；The Cordyceps militaris of water content≤5% that will be ready for is big
Rice culture medium section is crushed with hammer crusher, crosses 100 mesh sieves, obtains Cordyceps militaris rice medium meal, is delivered to by air stream
Cyclone separator, obtains air-flow crushing one-level separator and the second-order separation thing, and material and mill ball 1 are used after adopting:3 ratio is entered
Every trade star ball mill grinding 1h, crushes 2 times altogether, obtains even-grained Cordyceps militaris rice medium Ultramicro-powder, and size distribution is 30 μ
M, content of powder≤3%；After additional proportion is 20 times of water in the above-mentioned culture medium Ultramicro-powder, 1.5h is first extracted at 90 DEG C, so
Cellulase, amylase and protease are added afterwards（Optimal pH scope is 6.0-7.5）, three kinds of additions of enzyme are extraction system weight
The 1.0% of amount（m/v）, and adding proportion is 1:1:1.Leaching liquor after above-mentioned pre- extraction is supreme with HCl or NaOH regulations pH
The most suitable scope of enzyme is stated, and by temperature adjustment to 42 DEG C, after stirring enzymolysis 3h, pH is adjusted to neutrality, and in the enzyme that gone out at 95 DEG C
3min, is cooled to room temperature；By it is obtained above containing precipitation extract solution filtered, and by filtrate 5000 r/min speed
The lower centrifugation 10min of degree, obtains the supernatant extract solution containing Polysaccharides in Cultured Cordyceps militaris, and the precipitation additional proportion that will be obtained is 12 times of water,
Extracted again 1 time according to enzyme dosage, usage in biological multi-enzyme system enzyme process aqueous extraction step, gained filtrate can merge with said extracted liquid
Use；0.2 μm of membrane microfiltration was first used before ultrafiltration enrichment is carried out, to remove suspension solid content a small amount of after continuous filter press and small
Particulate matter, feed temperature is 55 DEG C, pH 7, operating pressure 0.3MPa, and micro-filtration time 1.0h collects micro-filtrate stand-by；Will be upper
State the micro-filtrate for obtaining to be placed in Multi-function ultrafiltration equipment liquid storing barrel, according to active polysaccharide range of molecular weight distributions, choose retention
Molecular weight（MwCO）It is the organic film ultrafiltration of 100 KDa, ultrafiltration pressure selection is 0.3MPa, pH 7, micro-filtration time 1.0h；Obtain
Trapped fluid carry out enrichment method 2.5h with 0.3 μm of microfiltration membranes again, now gained polysaccharide concentrate cycles of concentration is 12 times, and
Polysaccharide molecular weight field of activity of the distribution in 30-250 KDa；Dry-type Cordyceps sinensis polysaccharide product such as to be produced, using enclosed
Circulation spray dryer is dried treatment, and it is 180 DEG C to set spray drying device inlet temperature, and outlet temperature is 90 DEG C, is entered
Row can collect Polysaccharides in Cultured Cordyceps militaris Ultramicro-powder after adjusting spray drying.
Embodiment 5
The harvesting of same kind is cut into sheet with the Cordyceps militaris rice medium after Cordyceps militaris, in the vacuum drying chamber at 100 DEG C
It is respectively dried 2 times, each 1.5h, is≤5% to culture medium section water content；The Cordyceps militaris of water content≤5% that will be ready for is big
Rice culture medium section is crushed with hammer crusher, crosses 100 mesh sieves, obtains Cordyceps militaris rice medium meal, is delivered to by air stream
Cyclone separator, obtains air-flow crushing one-level separator and the second-order separation thing, and material and mill ball 1 are used after adopting:3 ratio is entered
Every trade star ball mill grinding 1h, crushes 2 times altogether, obtains even-grained Cordyceps militaris rice medium Ultramicro-powder, and size distribution is 30 μ
M, content of powder≤3%；After additional proportion is 30 times of water in the above-mentioned culture medium Ultramicro-powder, 1.0h first is extracted at 1000 DEG C,
It is subsequently adding cellulase, amylase and protease（Optimal pH scope is 6.0-7.5）, three kinds of additions of enzyme are extraction system
The 2.0% of weight（m/v）, and adding proportion is 1:1:1.By the leaching liquor after above-mentioned pre- extraction with HCl or NaOH adjust pH to
The most suitable scope of above-mentioned enzyme, and by temperature adjustment to 45 DEG C, after stirring enzymolysis 2h, pH is adjusted to neutrality, and gone out at 95 DEG C
Enzyme 3min, is cooled to room temperature；Extract solution containing precipitation obtained above is filtered, and by filtrate 5000 r/min's
10min is centrifuged under speed, the supernatant extract solution containing Polysaccharides in Cultured Cordyceps militaris is obtained, the precipitation additional proportion that will be obtained is 12 times
Water, extracts 1 time again according to enzyme dosage, usage in biological multi-enzyme system enzyme process aqueous extraction step, and gained filtrate can be with said extracted liquid
Merging is used；0.1 μm of membrane microfiltration was first used before ultrafiltration enrichment is carried out, to remove a small amount of suspension solid content after continuous filter press
And finely ground particle substance, feed temperature is 60 DEG C, and pH 7, operating pressure 0.4MPa, micro-filtration time 0.5h collect micro-filtrate stand-by；
Micro-filtrate obtained above is placed in Multi-function ultrafiltration equipment liquid storing barrel, according to active polysaccharide range of molecular weight distributions, is chosen
Molecular cut off（MwCO）It is the organic film ultrafiltration of 300 KDa, ultrafiltration pressure selection is 0.4MPa, pH 7, micro-filtration time 0.5h；
The trapped fluid for obtaining carries out enrichment method 1.0h with 0.45 μm of microfiltration membranes again, and now gained polysaccharide concentrate cycles of concentration is 14
Times, and polysaccharide molecular weight field of activity of the distribution in 30-250 KDa；Dry-type Cordyceps sinensis polysaccharide product such as to be produced, it is available
Closed cycle spray drying machine is dried treatment, and it is 190 DEG C to set spray drying device inlet temperature, and outlet temperature is 70
DEG C, collect Polysaccharides in Cultured Cordyceps militaris Ultramicro-powder by carrying out after tune spray drying.
Have been described in detail above embodiments of the present invention, but this is only to facilitate the example for understanding and lifting, should not be by
It is considered as and limits the scope of the present invention.Equally, any person of ordinary skill in the field can technology according to the present invention
Scheme and its description of preferred embodiment, make various possible equivalent changes or replacement, but all these changes or replacement are all
Scope of the claims of the invention should be belonged to.
Claims (6)
1. a kind of extraction from Chinese caterpillar fungus culture medium and the continuously method of polysaccharide enrichment, it is characterised in that：Comprise the following steps：
Step one：Cordyceps militaris fermentation residual media is dried：Cordyceps militaris rice medium is cut into sheet, in vacuum drying chamber
It is respectively dried 2-4 times at 70-100 DEG C, each 1.5-3h, produces Cordyceps militaris fermentation residual media, water content is≤5%；
Step 2：Ultramicro grinding：The Cordyceps militaris rice medium section that step one is produced is crushed, and crosses 100 mesh sieves, obtains pupa worm
Careless rice medium meal, respectively enters primary collection device and two grades after varigrained Cordyceps militaris rice medium powder is separated
Collector, obtains air-flow crushing one-level, the second-order separation thing and air-flow crushing one-level separator；
The air-flow crushing one-level separator that will have been prepared is put into planetary ball mill tank, using material and mill ball 1:1-1:3
Ratio carries out planetary ball mill and crushes 1-3h, crushes 2-3 times altogether（Grinding time is total to be no more than 6h）, obtain even-grained pupa worm
Careless rice medium Ultramicro-powder, size distribution is 20-60 μm, content of powder≤3%；
Step 3：Biological multi-enzyme system enzymatic hydrolysis:Additional proportion is 10-30 times in the culture medium Ultramicro-powder that step 2 is obtained
Water after, first extract 1-4h at 60-100 DEG C, be subsequently adding cellulase, amylase and protease；After above-mentioned pre- extraction
Leaching liquor adjust pH to 6.0-7.5 with HCl or NaOH, and by temperature adjustment to 35-45 DEG C, after stirring enzymolysis 2-6h, will
PH is adjusted to neutrality, and in the enzyme 3-6min that gone out at 90-95 DEG C, is cooled to room temperature；
Step 4：Centrifugal filtration, backflow control：Produce Polysaccharides in Cultured Cordyceps militaris；
Step 5：Multifunctional membrane enrichment method：
A, the Polysaccharides in Cultured Cordyceps militaris extract solution for obtaining step 4 carry out activity enrichment using ceramic membrane equipment；
B, using 0.1-0.45 μm of membrane microfiltration, to remove a small amount of suspension solid content and finely ground particle substance after step A continuous filter press,
Feed temperature is 30-60 DEG C, and between pH 6-8, operating pressure 0.1-0.4MPa, micro-filtration time 0.5-3.5h collect micro-filtrate and treat
With；
C, micro-filtrate is placed in Multi-function ultrafiltration equipment liquid storing barrel, organic film ultrafiltration；
D, the trapped fluid for obtaining carry out enrichment method 0.5-3.5h with 0.1-0.45 μm of microfiltration membranes again, and polysaccharide molecular weight is distributed
Field of activity in 30-250 KDa；
Step 6：Spray drying：Produce collection Polysaccharides in Cultured Cordyceps militaris Ultramicro-powder.
2. extracted in a kind of fermentation residual media from Cordyceps militaris according to claim 1, be enriched with, refine Cordyceps sinensis polysaccharide
Method, it is characterised in that：The step 3 cellulase, amylase and protease, three kinds of ratios of enzyme are 1:1:1, and add
Dosage is the 0.03%-0.20% for extracting system weight（m/v）.
3. extracted in a kind of fermentation residual media from Cordyceps militaris according to claim 1, be enriched with, refine Cordyceps sinensis polysaccharide
Method, it is characterised in that：The step 5 step is：Extract solution containing precipitation obtained above is filtered, and will filter
Liquid is centrifuged 10-30min under the speed of 3000-5000r/min using vertical automatic scraper bottom discharge centrifugal machine, is contained
There is the supernatant extract solution of Polysaccharides in Cultured Cordyceps militaris;The precipitation additional proportion that will be obtained is 5-15 times of water, according to biological multi-enzyme system enzyme
Enzyme dosage, usage are extracted 1-2 times again in method aqueous extraction step.
4. extracted in a kind of fermentation residual media from Cordyceps militaris according to claim 1, be enriched with, refine Cordyceps sinensis polysaccharide
Method, it is characterised in that：The organic film ultrafiltration molecular cut off is（MwCO）It is 30-300 KDa, the ultrafiltration pressure selection
It is 0.1-0.4MPa, between pH 6-8, micro-filtration time 0.5-3.0h.
5. extracted in a kind of fermentation residual media from Cordyceps militaris according to claim 1, be enriched with, refine Cordyceps sinensis polysaccharide
Method, it is characterised in that：The micro-filtrate that will be collected in step 5 B combines the mode of micro-filtration membrane concentration, cycles of concentration with milipore filter
Original 5-15 times is reached, Polysaccharides in Cultured Cordyceps militaris concentrate is obtained.
6. extracted in a kind of fermentation residual media from Cordyceps militaris according to claim 1, be enriched with, refine Cordyceps sinensis polysaccharide
Method, it is characterised in that：Step 6 step is：Spray drying device inlet temperature is 150-190 DEG C, and outlet temperature is 60-100
℃。
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| CN110250496A (en) * | 2019-06-25 | 2019-09-20 | 泸州品创科技有限公司 | Cordyceps sinensis powder and preparation method thereof |
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