CN106581083A - Extraction method for ganoderma lucidum components, biological feed and preparation method thereof - Google Patents
Extraction method for ganoderma lucidum components, biological feed and preparation method thereof Download PDFInfo
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- CN106581083A CN106581083A CN201611121463.9A CN201611121463A CN106581083A CN 106581083 A CN106581083 A CN 106581083A CN 201611121463 A CN201611121463 A CN 201611121463A CN 106581083 A CN106581083 A CN 106581083A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
- A61K36/074—Ganoderma
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- C—CHEMISTRY; METALLURGY
- 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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- 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
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention relates to an extraction method for ganoderma lucidum components, a biological feed and a preparation method thereof, and belongs to the field of natural product processing. The extraction method comprises the following steps: crushing ganoderma lucidum fruiting bodies, and performing reflux extraction on fat-soluble components to obtain ganoderma lucidum fats and a first residue; performing enzymolysis on the first residue by virtue of cellulase, hemicellulase and papain, adding an ethanol-water solution, performing refluxing treatment to obtain reflux and a second residue, eluting the reflux by virtue of a D101 macroporous adsorption resin chromatographic column, and collecting eluted liquid to obtain ganoderma lucidum triterpene; performing ultrasonic extraction on the second residue to obtain extract and a third residue, and purifying the extract to obtain ganoderma lucidum polysaccharides by virtue of DEAE-cellulose. The preparation method comprises the following steps: mixing the third residue and bran, performing sterilization, inoculating an oyster mushroom liquid strain and an aureobasidium pullulans liquid strain, and performing fermentation. The extraction and preparation methods are simple, mild in condition, easy to operate and high in yield. The biological feed prepared by the preparation method has little side or toxic effects and high nutritional value.
Description
Technical field
The present invention relates to natural products manufacture field, and more particularly to a kind of extracting method, the biological feedstuff of glossy ganoderma composition
And preparation method thereof.
Background technology
Glossy ganoderma (Ganoderma lucidum) is Basidiomycetes, Polyporaceae, Ganoderma (Ganoderma) fungi, is commonly used
Its fructification, is the traditional rare traditional Chinese medicine of China and medicinal fungi.Pharmacological research shows that glossy ganoderma has regulation immunity, resists and swell
Knurl, anti-aging, raising body's hypoxia tolerance isoreactivity, its main composition is ganodenic acid and GL-B.Glossy ganoderma three
Terpene is the main composition of one of which, with obvious protecting liver and expelling toxin, anti-oxidant, antimicrobial antiphlogistic, anti HIV-1 virus, antitumor, town
Bitterly, Angiotensin-converting enzyme inhibition, suppression cholesterol biosynthesis, reduction platelet aggregation, suppression histamine release and regulation immunity
Deng effect, in can be widely applied to medicine and health products.
Because ganodenic acid is present in the cell membrane of glossy ganoderma, ganoderma lucidum fruitbody cell wall constituent is mainly chitin, knot
Structure is complicated and hard, and acid and alkali-resistance, causes fully absorbing by hindering largely for intracellular active principle.At present,
Generally ganodenic acid is extracted using organic solvent method, it usually needs longer extraction time simultaneously consumes substantial amounts of Extraction solvent, together
The use of Shi great Liang organic solvents can give people body and environment brings detrimental effect.Further, since ganoderma lucidum fruitbody close structure,
Active component is attached to intracellular side, and traditional Hot water extraction time length, power consumption height, active component extract yield is low.And
It is relatively low that simple use ultrasound assisted extraction or alcohol reflux such as extract at the triterpene yield that method extracts；Using overcritical
CO2The required equipment investment of extraction is larger, operation difficulty and production cost are also higher.
At present, glossy ganoderma develops and faces two bottlenecks, and the comprehensive utilization of (1) glossy ganoderma raw material is low, there is a large amount of residues after extraction
It is remaining.(2) ganoderma lucidum fruitbody cell wall constituent is mainly chitin, complex structure, hard, and acid and alkali-resistance, causes intracellular to have
Effect material is fully absorbed by hindering largely.Due to glossy ganoderma bioactive ingredients content it is relatively low, residue after extraction
Surplus is larger, and the wasting of resources is more serious.Traditional residue takes stacking, landfill, burning disposal usually as rubbish, and this was both
The waste of resource is caused, and there is potential destruction to natural environment.
The content of the invention
It is an object of the invention to provide a kind of extracting method of glossy ganoderma composition, this extracting method is simple to operate, it is easy to real
It is existing, and the recovery rate of glossy ganoderma composition is higher.
The second object of the present invention is to provide a kind of preparation method of biological feedstuff, and the raw material needed for the preparation method comes
Source is wide, and process is simple, easy to operate, easy to control and time-consuming short.
The third object of the present invention is to provide the biological feeding that a kind of preparation method by above-mentioned biological feedstuff is prepared
Material, the raw material of the biological feedstuff is natural products, and toxic and side effect is little, is of high nutritive value.
The present invention solves its technical problem and employs the following technical solutions to realize：
The present invention proposes a kind of extracting method of glossy ganoderma composition, and it is comprised the following steps：Ganoderma lucidum fruitbody is crushed, with extraction
Agent refluxing extraction liposoluble constituent, obtains glossy ganoderma fat, remainings the first residue.
With cellulase, hemicellulase and the residue of papain mixed enzymolysis first, ethanol-water solution is added, in
Reflow treatment 1-3h under conditions of 75-85 DEG C, obtains phegma and the second residue, is eluted back with D101 macroporous adsorption resin chromatography posts
Flow liquid, collects the liquid after wash-out, removes organic phase, obtains ganodenic acid.
The residue of ultrasonic extraction second, obtains extract and the 3rd residue, removes pigment and protein in extract, Jing
DEAE- cellulose purifications obtain GL-B.
The present invention also proposes a kind of preparation method of biological feedstuff, and it is comprised the following steps：By the 3rd above-mentioned residue with
Wheat bran mixes, sterilizing, by weight 100:10-20 is inoculated with liquid spawn of picking up the ears, and cultivates under the conditions of 25-30 DEG C and is followed by for 5-9 days
Plant and pick up the ears liquid spawn weight ratio for 10-20:The Aureobasidium pullulans liquid spawn of 8-13, fermentation, obtains biological feedstuff.
The present invention also proposes a kind of biological feedstuff, and it is prepared by the preparation method of above-mentioned biological feedstuff.
The beneficial effect of the extracting method of the glossy ganoderma composition of present pre-ferred embodiments is：By carrying out to ganoderma lucidum fruitbody
Crush, increased the contact area between raw material and enzyme and extractant, improve enzymolysis and extraction rate.With cellulase, half fibre
The plain enzyme of dimension and papain, not only beneficial to cell membrane conveying, rupture, make intracellular change collectively as the enzyme in enzymolysis process
Compound separate out, moreover it is possible to the albumen in hydrolyzing plant, promotes cell to discharge more triterpenoids.It is with ethanol-water solution
Extractant, both can guarantee that leaching velocity was fast, extract purity is high, impurity is few, and can also reduces cost.Reflux temperature and return
The stream time is respectively 75-85 DEG C and 1-3h, not only can avoid causing active principle dissolution because return time is too short or temperature is too low
Rate is low, and return time is long or temperature is too high and can cause the excessive dissolution of impurity such as grease, tannin in plant cell, increase
The defects such as the difficulty for isolating and purifying, also can further improve the recovery rate of triterpenoid and keep the activity of triterpenoid optimal.
Desalination and wash-out are carried out with D101 macroporous adsorption resin chromatographies post after filtration to phegma, the type chromatographic column adsorbance is high, fill out
Material particle is uniform, mechanical strength is good, not broken and residue is few, selectively good to organic matter, separation, purifying, the effect of removal of impurities
It is really good, therefore the purity of ganodenic acid can be improved.Second residue is carried out into ultrasonic extraction and obtains GL-B, improve the utilization of raw material
Rate；Using DEAE- cellulose purification polysaccharide, the impurity such as pigment, protein in extract are eliminated, improve gained polysaccharide
Purity.In addition the present invention also mixes the 3rd residue with wheat bran, and is inoculated with pick up the ears liquid spawn and Aureobasidium pullulans liquid spawn,
Biological feedstuff is prepared by the metabolism of microorganism, the utilization rate of glossy ganoderma raw material, and the preparation side is not only further increased
Method is simple, mild condition, easy to operate, low cost, time-consuming short and yield is high.Thus the biological feedstuff poison that preparation method is prepared
Small side effects, are of high nutritive value, environmental protection, great Development volue and application prospect.
Specific embodiment
To make purpose, technical scheme and the advantage of the embodiment of the present invention clearer, below will be in the embodiment of the present invention
Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can pass through the conventional product that commercially available purchase is obtained
Product.
Extracting method, biological feedstuff of glossy ganoderma composition of the embodiment of the present invention and preparation method thereof are carried out specifically below
It is bright.
The extracting method of glossy ganoderma composition provided in an embodiment of the present invention, with ganoderma lucidum fruitbody as raw material, can in extraction process
Pre-treatment, i.e. ganoderma lucidum fruitbody are first carried out to raw material to crush, and to increase the contact area between raw material and enzyme and extractant, are improved
Enzymolysis and extraction rate.Grinding particle size for example can be 30-50 mesh, the active ingredient dissolution rate in the particle size range in raw material compared with
Greatly.Raw material preferably picks up from the fructification of fresh glossy ganoderma, the active constituent content highest in the ganoderma lucidum fruitbody under the conditions of this, favorably
In the content for improving active ingredient in subsequent extracted thing.
As preferred, before pulverising step, the works such as removal of impurities, cleaning and drying can also be carried out to ganoderma lucidum fruitbody
Sequence.Wherein removal of impurities and cleaning can avoid interference of other impurity to extraction effect, drying can partial destruction raw material cell membrane and
Cell membrane, beneficial to extraction.Wherein, dry temperature for example can be selected as 20-45 DEG C, preferably 37 DEG C.In this optimum condition
Under, the active component in raw material can farthest retain not by high temperature.
Due to containing partial fat class material in glossy ganoderma, and the extracting method of lipoid material is relatively simple, therefore, can be first
Fat-extraction is carried out to raw material.For example in ganoderma lucidum fruitbody that can be after being pulverized extractant is added, in order to ensure extraction effect,
Preferably, under conditions of 70-80 DEG C refluxing extraction liposoluble constituent 5-15h, the liposoluble constituent be glossy ganoderma fat, extract
Remaining first residue afterwards.Wherein extractant can for example select the ratio of ether or petroleum ether, extractant and ganoderma lucidum fruitbody can
Think 150-250mL:1g, under the conditions of this ratio, the extraction effect of ether or petroleum ether to lipoid material in ganoderma lucidum fruitbody
Most preferably.
Due to also containing more triterpene substance in the first residue, the embodiment of the present invention is preferably residual to first using enzymatic isolation method
Slag is digested, i.e., using the specificity of organized enzyme come the predetermined substance corresponding to hydrolase.Because ganodenic acid is present in glossy ganoderma
Cell membrane in, ganoderma lucidum fruitbody cell wall constituent is mainly chitin, complex structure and hard, and acid and alkali-resistance, causes cell
Interior active principle is fully absorbed by hindering largely.In recent years, enzyme engineering technology is in Chinese herbal medicine active component
Significantly applied in extraction, from appropriate enzyme effect in chinese medicine material, cell membrane can leniently be decomposed by enzyme reaction
And the composition such as the cellulose in cytoplasm, hemicellulose, pectic substance, destroy the dense construction of cell membrane, cause cell membrane and
Cytoplasm structure produces the changes such as loose local, expansion, collapse, reduces the mass transfer barrier such as cell membrane and cytoplasm to effective
The resistance to mass tranfer that composition spreads from intracellular to Extraction medium, so as to be conducive to the dissolution of active ingredient.Again because of cellulose and half fibre
Dimension element is the main component of cell membrane, with cellulase and hemicellulose ferment treatment, can make that its cell membrane is loose, rupture, favorably
In intracellular compound separate out.Additionally, the albumen in protease energy hydrolyzing plant, promotes cell release more three
Terpene, therefore preferably raw material is digested using complex enzyme in the embodiment of the present invention, with the set time to many kinds of substance simultaneously
Digested, improved enzymolysis efficiency.Wherein complex enzyme can for example include cellulase, hemicellulase and papain,
At least one in bromelain and pectase can also be included.Specifically, for example can by the first residue and cellulase,
Hemicellulase and papain mix, and in pH be 5-6, temperature be to digest 1-3h under conditions of 36-45 DEG C.In order to improve
Enzymolysis efficiency, preferred enzyme activity is respectively 45000-55000u/g, 150000-250000u/g, 750000- in the embodiment of the present invention
The cellulase of 850000u/g, hemicellulase and papain.In order to avoid there is enzyme while enzymolysis efficiency is improved
The phenomenon of waste, it is preferred that the cellulase added in enzymolysis process is 2-4 with the weight ratio of ganoderma lucidum fruitbody:100, half is fine
The plain enzyme of dimension and the weight ratio of ganoderma lucidum fruitbody are 3-5:100, papain is 2-4 with the weight ratio of ganoderma lucidum fruitbody:100.
Additionally, because enzymolysis time is too short or hydrolysis temperature is too low active principle dissolution rate can be caused low, enzymolysis time is long or enzymolysis is warm
Spend high and the excessive dissolutions of impurity such as grease, the tannin in plant cell can be caused, increase the difficulty for isolating and purifying, therefore digest
Time is preferably 2h, and hydrolysis temperature is preferably 37 DEG C.Further, in enzymolysis process, enzymatic hydrolysis system can also be fitted
Work as stirring, to improve the dissolution rate of active principle.Because the optimal pH of enzyme used in the embodiment of the present invention is in the range of 5-6, in order to
The pH of enzymatic hydrolysis system is adjusted, can be adjusted with the addition phosphate buffer in the system.
As preferred, for example enzyme digestion reaction can be stopped using high temperature enzyme activity method of going out in the embodiment of the present invention, you can
8-15min is heated in so that enzymatic hydrolysis system to be placed in 85-100 DEG C of water, preferably, enzymatic hydrolysis system can be placed in 90 DEG C of water
Heating 10min, under the conditions of this, can make the yield highest of enzymolysis product, it is to avoid the insufficient situation of enzymolysis.
After enzymolysis, in order to further improve the recovery rate of triterpenoid, for example, the zymolyte obtained by enzymolysis can also be entered
The process of row alcohol reflux.Specifically, can add in zymolyte mass concentration be 93-97% ethanol-water solution, and in
Reflow treatment 1-3h under the conditions of 75-85 DEG C.Wherein, organic solvent is preferably ethanol-water solution, both can guarantee that extraction rate it is fast,
The purity of extract is high, impurity is few, and low cost.Preferably, the mass concentration of ethanol is 95% in ethanol-water solution, this
The polarity of ethanol solution is moderate under mass fraction so that the most active ingredients in ganoderma lucidum fruitbody are soluble in the solution
In, extract comparatively fast and impurity is less.Additionally, organic solvent can also be methanol-water solution etc..In order that the activity of triterpenoid
Most preferably, the temperature of refluxing extraction is preferably 80 DEG C in the embodiment of the present invention.
After refluxing extraction, for example, phegma can be filtered, obtain filtrate and the second residue, concentration filtrate obtains
Sample thing.Wherein, filtration can be the modes such as suction filtration, or centrifugal filtration.
Further, loading thing is purified.Preferably, the upper quadrat method in the embodiment of the present invention is preferably in wet method
Sample, will loading thing be dissolved in after solvent and carry out again loading, to improve purification effect.Specifically, can be by solid-liquid ratio such as 1g:
1-6mL is dispersed in water loading thing, obtains load solution, then load solution is entered using the column chromatography method in chromatography
Row purifies and separates.Specifically, chromatography is the selectivity distribution using different material in different phase, is relatively fixed with flowing
Mixture in phase is eluted, and different materials can be moved at different rates along fixing phase in mixture, be finally reached point
From effect.Used as preferred, the chromatographic column in the present embodiment selects macroporous adsorption resin chromatography post, and substantially the one of its absorption
Kind of object height dispersion or surface molecular is unequal by active force and the adsorption phenomenon that produces, this absorption property be due to
The result of Van der Waals force or generation hydrogen bond.Simultaneously because the loose structure of macroporous absorbent resin makes it different to molecular size
Material is acted on screening.By above-mentioned this absorption and screening principle, different and molecule of the organic compound according to absorption affinity
The size of amount, the certain solvents of Jing are eluted and reach the difference purpose such as separation, purifying, removal of impurities, concentration on macroporous absorbent resin.
Preferably, the filler in above-mentioned macroporous adsorption resin chromatography post is preferably D101 fillers, it is substantially styrene
The nonpolar EVA of type.The filling adsorption amount of the type is high, particle is uniform, mechanical strength is good, not broken and residue is few, and
It is selectively good to organic matter.Can play a part of desalination to loading thing with it in the present embodiment.
Further, eluant, eluent is added in macroporous adsorption resin chromatography post, and isocratic elution is carried out to load solution.Its
In, eluant, eluent can for example be selected from 94-96wt% alcohol-water solutions, considering cost and separating effect, preferably 95wt%'s
Ethanol-water solution.Preferably, the ratio of eluant, eluent and loading thing can be 400-1000mL:1g, the flow velocity of eluant, eluent can be
0.5-0.8mL/min, the separating effect and recovery rate of active component are optimal under this ratio and flow velocity.Further, it is also possible to using
Whether contain ganodenic acid in vanillin-perchloric acid auxiliary detection eluent.
The liquid after wash-out is collected, the organic equal material in the liquid is removed after being dried, obtain ganodenic acid.Wherein,
Drying can will carry out freeze-drying after the liquid pressure-reducing concentration after wash-out.As preferred, in the embodiment of the present invention for example
Can by the liquid after wash-out in 50-53 DEG C water-bath, rotating speed be 50-55 turn/min and vacuum is 0.07-0.09MPa
Under conditions of rotary evaporation, should under the conditions of the reduced pressure concentration that carries out, be conducive to avoiding the structure of triterpenoid in recycling design
Destroyed.
In order to improve the utilization rate of glossy ganoderma raw material, for example, ultrasonic extraction can also be carried out to the second residue, obtain extract
With the 3rd residue.Wherein, ultrasonic extraction is to accelerate intracellular to have using the cavitation of ultrasonic wave, mechanical effect and fuel factor etc.
Release, diffusion and the dissolving of effect material, can significantly improve extraction efficiency.The power of the ultrasonic extraction for example can be 200-
400W, the time for example can be 30-50min, temperature for example can be 60-90 DEG C, it is preferred that ultrasonic extraction in power 300W,
Temperature be 80 DEG C under conditions of extract 40min.Under the conditions of being somebody's turn to do, extraction effect is optimal.Further, the pigment in extract is removed
And protein, and with the DEAE- cellulose purifications extract, obtain GL-B.Wherein, the adsorbable ion of DEAE- celluloses
Type material, such as protein, acidic polysaccharose etc., and then can smoothly flow out as most neutral polysaccharide, so as to go slightly to take
Essence, reach detached purpose.
Further, the 3rd residue such as can also be made biological feedstuff by the embodiment of the present invention by fermentable.It is micro-
Biological fermentation feed refer to it is artificial it is controllable under the conditions of, with vegetalitas agricultural byproducts as primary raw material, by microorganism
Metabolism, the ANFs in vegetalitas, animality and mineral material is decomposed, is synthesized, and generation more can be adopted by livestock and poultry
Food, the biological feedstuff or feedstuff that absorb nutrient and nonhazardous effect, environment-protecting asepsis.Specifically, for example can be by weight
1:1-5 mixes the 3rd residue with wheat bran, sterilizes to remove miscellaneous bacteria, and by weight 100:10-20 is inoculated with liquid spawn of picking up the ears,
Aureobasidium pullulans liquid spawn, the Aureobasidium pullulans liquid spawn and side are inoculated with after cultivating 5-9 days under the conditions of being preferable over 25-30 DEG C
Ear liquid spawn part by weight such as can be 10-20:8-13, then ferments 5-10 days under conditions of 25-30 DEG C, obtains final product biology
Feed.Wherein, pick up the ears be it is a kind of be grown on broad leaf tree it is dried-up on edible mushroom and medicinal fungus, lignin and cellulose can be decomposed,
Pick up the ears containing 8 kinds of amino acid needed by human, can be used to controlling soreness of waist and ache in legs pain, numb in every limb, grain uncomfortable etc. as Chinese medicine.Go out
It is a kind of yeast-like fungus that the short stalk of bud is mould, can utilize the high Pul of xylose production added value.The Jing preparation methods
The biological feedstuff toxic and side effect being prepared is little, is of high nutritive value, environmental protection, is a kind of great Development volue and application prospect
Multi-functional new bio product.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
It is 30 mesh that 1g fresh glossy ganoderma fructifications are crushed to into granularity, with 1g:The ratio of 150mL adds second in fructification
Ether, the refluxing extraction liposoluble constituent 15h under the conditions of 70 DEG C obtains glossy ganoderma fat, remainings the first residue.By the first residue and enzyme activity point
Not Wei the mixing of the cellulase of 45000u/g, 150000u/g and 750000u/g, hemicellulase and papain, this three kinds
Enzyme is 2 with the weight ratio of ganoderma lucidum fruitbody:3:2:100.In pH be 5, temperature be 36 DEG C under conditions of digest 3h after, body will be digested
System is placed in 85 DEG C of water heats 15min, the ethanol-water solution of addition 93wt% in zymolyte, and in 75 DEG C of conditions next time
Stream extracts 3h, obtains phegma.Suction filtration is carried out to phegma, filtrate and the second residue is obtained, concentration filtrate obtains loading thing.Press
Solid-liquid ratio 1g:1mL is dispersed in water loading thing, obtains load solution.Adopt filler for D101 macroporous adsorption resin chromatography
Post carries out desalination to loading thing, and with 400mL:The ratio of 1g adds the ethanol-water solution conduct of 94wt% in the chromatographic column
Eluant, eluent, isocratic elution is carried out to loading thing, and elution flow rate is 0.5mL/min.The liquid after wash-out is collected, in 50 DEG C of water
Bath, rotating speed are 50 turns/min and vacuum is to carry out freeze-drying again after rotary evaporation under conditions of 0.07MPa, obtain glossy ganoderma
Triterpene.Power be 200W, temperature be 60 DEG C under conditions of ultrasonic extraction 50min is carried out to the second residue, obtain extract and
3rd residue.The pigment and protein in extract is removed, and uses DEAE- cellulose purifications, obtain GL-B.
By weight 1:1 mixes the 3rd residue with wheat bran, by weight 100 after sterilizing:10 are inoculated with liquid spawn of picking up the ears,
The culture under conditions of 25 DEG C inoculates Aureobasidium pullulans liquid spawn after 9 days, the weight ratio of the bacterial classification and liquid spawn of picking up the ears
10:8, then ferment 10 days under conditions of 25 DEG C, obtain biological feedstuff.
Embodiment 2
1g fresh glossy ganodermas fructification is carried out into removal of impurities, cleaning, crushed after being dried is 50 mesh to granularity at 20 DEG C, with 1g:
The ratio of 180mL adds ether in fructification, the refluxing extraction liposoluble constituent 15h under the conditions of 73 DEG C, obtains glossy ganoderma fat, remaining
First residue.First residue and enzyme activity are respectively into the cellulase of 55000u/g, 250000u/g and 850000u/g, half fiber
Plain enzyme and papain mix, and three kinds of enzymes are 4 with the weight ratio of ganoderma lucidum fruitbody:5:4:100.It is that 6, temperature is 45 in pH
Digest after 1h under conditions of DEG C, enzymatic hydrolysis system is placed in 100 DEG C of water and heats 8min, the second of 97wt% is added in zymolyte
Alcohol-water solution, and refluxing extraction 1h under the conditions of 85 DEG C, obtain phegma.Centrifugal filtration is carried out to phegma, obtain filtrate and
Second residue, concentration filtrate obtains loading thing.By solid-liquid ratio 1g:6mL is dispersed in water loading thing, obtains load solution.Adopt
Desalination is carried out to loading thing with the macroporous adsorption resin chromatography post that filler is D101, and with 1000mL:The ratio of 1g is to the chromatography
Add the ethanol-water solution of 96wt% as eluant, eluent in post, isocratic elution is carried out to loading thing, elution flow rate is 0.8mL/
min.The liquid after wash-out is collected, the water-bath, rotating speed in 53 DEG C is 55 turns/min and vacuum is under conditions of 0.09MPa
Carry out freeze-drying after rotary evaporation again, obtain ganodenic acid.Power be 400W, temperature be 90 DEG C under conditions of to the second residue
Ultrasonic extraction 30min is carried out, extract and the 3rd residue is obtained.The pigment and protein in extract is removed, and it is fine with DEAE-
Dimension element purifying, obtains GL-B.
By weight 1:5 mix the 3rd residue with wheat bran, by weight 100 after sterilizing:20 are inoculated with liquid spawn of picking up the ears,
The culture under conditions of 30 DEG C inoculates Aureobasidium pullulans liquid spawn after 5 days, the weight ratio of the bacterial classification and liquid spawn of picking up the ears
20:13, then ferment 5 days under conditions of 30 DEG C, obtain biological feedstuff.
Embodiment 3
1g fresh glossy ganodermas fructification is carried out into removal of impurities, cleaning, crushed after being dried is 40 mesh to granularity at 45 DEG C, with 1g:
The ratio of 250mL adds petroleum ether in fructification, the refluxing extraction liposoluble constituent 10h under the conditions of 80 DEG C, obtains glossy ganoderma fat,
Remaining the first residue.First residue and enzyme activity are respectively into cellulase, half fibre of 50000u/g, 200000u/g and 800000u/g
The plain enzyme of dimension and papain mixing, three kinds of enzymes are 3 with the weight ratio of ganoderma lucidum fruitbody:4:3:100.In pH be 5.5, temperature
To digest after 2h under conditions of 40.5 DEG C, enzymatic hydrolysis system is placed in 92.5 DEG C of water and heats 11.5min, added in zymolyte
The ethanol-water solution of 95wt%, and refluxing extraction 2h under the conditions of 80 DEG C, obtain phegma.Filtered off with suction is carried out to phegma,
Filtrate and the second residue are obtained, concentration filtrate obtains loading thing.By solid-liquid ratio 1g:3.5mL is dispersed in water loading thing, obtains
Load solution.Adopt filler carries out desalination for the macroporous adsorption resin chromatography post of D101 to loading thing, and with 700mL:The ratio of 1g
Example adds the ethanol-water solution of 95wt% as eluant, eluent in the chromatographic column, and to loading thing isocratic elution, elution flow rate are carried out
For 0.65mL/min.The liquid after wash-out is collected, the water-bath, rotating speed in 51.5 DEG C is 53 turns/min and vacuum is
Carry out freeze-drying under conditions of 0.08MPa after rotary evaporation again, obtain ganodenic acid.Power be 300W, temperature be 75 DEG C
Under the conditions of ultrasonic extraction 40min is carried out to the second residue, obtain extract and the 3rd residue.Remove the pigment and egg in extract
White matter, and DEAE- cellulose purifications are used, obtain GL-B.
By weight 1:3 mix the 3rd residue with wheat bran, by weight 100 after sterilizing:15 are inoculated with liquid spawn of picking up the ears,
The culture under conditions of 27 DEG C inoculates Aureobasidium pullulans liquid spawn after 7 days, the weight ratio of the bacterial classification and liquid spawn of picking up the ears
15:11, then ferment 7.5 days under conditions of 27 DEG C, obtain biological feedstuff.
Embodiment 4
1g fresh glossy ganodermas fructification is carried out into removal of impurities, cleaning, crushed after being dried to granularity is 40 mesh at 32.5 DEG C, with
1g:The ratio of 200mL adds ether in fructification, the refluxing extraction liposoluble constituent 10h under the conditions of 75 DEG C, obtains glossy ganoderma fat
Fat, remainings the first residue.First residue is mixed with bromelain, the enzyme is 3 with the weight ratio of ganoderma lucidum fruitbody:100.Add
It is 5.5 that phosphate buffer adjusts the pH of mixed system, and the system is digested after 2h under conditions of 40.5 DEG C, and enzymatic hydrolysis system is put
11.5min is heated in 92.5 DEG C of water, the methanol-water solution of addition 95wt% in zymolyte, and in 80 DEG C of conditions next time
Stream extracts 2h, obtains phegma.Filtered off with suction is carried out to phegma, filtrate and the second residue is obtained, concentration filtrate obtains loading
Thing.By solid-liquid ratio 1g:3.5mL is dispersed in water loading thing, obtains load solution.Adopt filler for D101 macroporous absorption tree
Fat chromatographic column carries out desalination to loading thing, and with 700mL:The ratio of 1g adds the alcohol-water of 95wt% molten in the chromatographic column
Liquid carries out isocratic elution to loading thing as eluant, eluent, and elution flow rate is 0.65mL/min.The liquid after wash-out is collected, in
51.5 DEG C of water-bath, rotating speed is to carry out freezing under conditions of 0.08MPa after rotary evaporation again to do for 53 turns/min and vacuum
It is dry, obtain ganodenic acid.Power be 300W, temperature be 75 DEG C under conditions of ultrasonic extraction 40min is carried out to the second residue, obtain
Extract and the 3rd residue.The pigment and protein in extract is removed, and uses DEAE- cellulose purifications, obtain GL-B.
By weight 1:3 mix the 3rd residue with wheat bran, by weight 100 after sterilizing:15 are inoculated with liquid spawn of picking up the ears,
The culture under conditions of 27 DEG C inoculates Aureobasidium pullulans liquid spawn after 7 days, the weight ratio of the bacterial classification and liquid spawn of picking up the ears
15:11, then ferment 7.5 days under conditions of 27 DEG C, obtain biological feedstuff.
Embodiment 5
1g fresh glossy ganodermas fructification is carried out into removal of impurities, cleaning, crushed after being dried is 40 mesh to granularity at 37 DEG C, with 1g:
The ratio of 200mL adds petroleum ether in fructification, the refluxing extraction liposoluble constituent 10h under the conditions of 75 DEG C, obtains glossy ganoderma fat,
Remaining the first residue.First residue is mixed with pectase, the enzyme is 3 with the weight ratio of ganoderma lucidum fruitbody:100.Phosphoric acid is added to delay
The pH for rushing liquid regulation mixed system is 5.5, and the system is digested after 2h under conditions of 37 DEG C, and enzymatic hydrolysis system is placed in into 90 DEG C
10min is heated in water, the ethanol-water solution of 95wt%, and refluxing extraction 2h under the conditions of 80 DEG C are added in zymolyte, obtained
Phegma.Centrifugal filtration is carried out to phegma, filtrate and the second residue is obtained, concentration filtrate obtains loading thing.By solid-liquid ratio 1g:
4mL is dispersed in water loading thing, obtains load solution.Adopt filler for D101 macroporous adsorption resin chromatography post to loading thing
Desalination is carried out, and with 800mL:The ratio of 1g adds the ethanol-water solution of 95wt% in the chromatographic column as eluant, eluent, to upper
Sample thing carries out isocratic elution, and elution flow rate is 0.65mL/min.The liquid after wash-out is collected, the water-bath, rotating speed in 52 DEG C is 55
Turn/min and vacuum is to carry out freeze-drying again after rotary evaporation under conditions of 0.09MPa, obtains ganodenic acid.In power
Be 300W, temperature be 75 DEG C under conditions of ultrasonic extraction 40min is carried out to the second residue, obtain extract and the 3rd residue.Remove
The pigment gone in extract and protein, and DEAE- cellulose purifications are used, obtain GL-B.
By weight 1:3 mix the 3rd residue with wheat bran, by weight 100 after sterilizing:15 are inoculated with liquid spawn of picking up the ears,
The culture under conditions of 27 DEG C inoculates Aureobasidium pullulans liquid spawn after 7 days, the weight ratio of the bacterial classification and liquid spawn of picking up the ears
15:11, then ferment 7.5 days under conditions of 27 DEG C, obtain biological feedstuff.
Embodiment 6
1g fresh glossy ganodermas fructification is carried out into removal of impurities, cleaning, crushed after being dried is 40 mesh to granularity at 37 DEG C, with 1g:
The ratio of 200mL adds ether in fructification, the refluxing extraction liposoluble constituent 10h under the conditions of 80 DEG C, obtains glossy ganoderma fat, remaining
First residue.First residue and enzyme activity are respectively into the cellulase of 50000u/g, 200000u/g and 800000u/g, half fiber
Plain enzyme, papain, bromelain and pectase mixing, five kinds of enzymes are 3 with the weight ratio of ganoderma lucidum fruitbody:4:3:1:
1:100.Add the pH that phosphate buffer adjusts mixed system to be 5.5, the system is digested enzyme after 2h under conditions of 37 DEG C
Enzymatic hydrolysis system is placed in 90 DEG C of water and heats 10min, and the ethanol-water solution of 95wt% is added in zymolyte, and in 80 DEG C of conditions
Lower refluxing extraction 2h, obtains phegma.Centrifugal filtration is carried out to phegma, filtrate and the second residue is obtained, concentration filtrate obtains
Loading thing.By solid-liquid ratio 1g:4mL is dispersed in water loading thing, obtains load solution.Adopt filler for D101 macroporous absorption
Resin chromatography post carries out desalination to loading thing, and with 800mL:The ratio of 1g adds the alcohol-water of 95wt% in the chromatographic column
Solution carries out isocratic elution to loading thing as eluant, eluent, and elution flow rate is 0.65mL/min.The liquid after wash-out is collected, in
52 DEG C of water-bath, rotating speed is to carry out freezing under conditions of 0.09MPa after rotary evaporation again to do for 55 turns/min and vacuum
It is dry, obtain ganodenic acid.Power be 300W, temperature be 80 DEG C under conditions of ultrasonic extraction 40min is carried out to the second residue, obtain
Extract and the 3rd residue.The pigment and protein in extract is removed, and uses DEAE- cellulose purifications, obtain GL-B.
By weight 1:3 mix the 3rd residue with wheat bran, by weight 100 after sterilizing:15 are inoculated with liquid spawn of picking up the ears,
The culture under conditions of 28 DEG C inoculates Aureobasidium pullulans liquid spawn after 10 days, the weight ratio of the bacterial classification and liquid spawn of picking up the ears
15:11, then ferment 10 days under conditions of 28 DEG C, obtain biological feedstuff.
Repeat to implement above-described embodiment 1-6, extract enough glossy ganoderma fat, ganodenic acid and GL-B, and to press
Glossy ganoderma fat, ganodenic acid and GL-B obtained by the various embodiments described above respectively as test group 1-6, with common extracting method
As a control group, under conditions of material quality is consistent, contrast is each for glossy ganoderma fat, ganodenic acid and GL-B obtained by extraction
The recovery rate of glossy ganoderma fat, ganodenic acid and GL-B in extracting method, its result is as shown in table 1：
The recovery rate of table 1
As can be seen from Table 1, using the ganoderma lucidum fruitbody of identical weight as raw material, the extracting method of the embodiment of the present invention compared with
The recovery rate of the glossy ganoderma fat, ganodenic acid and GL-B of common extracting method will height.Its reason is the embodiment of the present invention
In by with reference to enzymatic isolation method and alcohol reflux the active material in raw material being digested and being extracted, and in enzymolysis process simultaneously
Mixed enzymolysis are carried out using cellulase, hemicellulase and papain, hydrolysis result and recovery rate is improve.Additionally,
By carrying out desalination, decolouring, deproteinization etc. to extract using macroporous absorbent resin and DEAE- celluloses, further to carrying
Take thing to be separated and purified, improve the purity and yield of material.Comparative example 1-6, it can be seen that in embodiment 6
Glossy ganoderma fat, the recovery rate highest of ganodenic acid and GL-B, its reason is, in the embodiment, will in enzymolysis process
Cellulase, hemicellulase, papain, bromelain and pectase collective effect, have fully digested glossy ganoderma real
Triterpenoid in body, additionally, the enzyme activity of complex enzyme is higher in the embodiment, and the addition of each material it is moderate, it is proportioning work as,
Each parameter is also optimal value during enzymolysis, extraction and purifying etc., therefore glossy ganoderma fat, ganodenic acid and glossy ganoderma are more in the embodiment
The recovery rate highest of sugar.
Using the biological feedstuff being prepared by embodiment 1-6 respectively as test group 1-6, do right with commercially available biological feedstuff
According to group, using the pig in the different stages of growth as study subject, the 30% of the birth 1-5 months, the 40% of the birth 6-12 months, it is born
The 20% of the 13-24 months, is born the 10% of the 25-50 months, and study subject is taken food respectively the biological feedstuff of equivalent test group and control group,
The growth rate of subject body weight after taking food 2 months is contrasted, its result is as shown in table 2：
The body weight growth rate of the different stages of growth of table 2
As can be seen from Table 2, the body weight growth rate after the edible test group biological feedstuff of the study subject of the different stages of growth
Relatively eat the body weight growth rate after control group biological feedstuff high, illustrate test group biological feedstuff compared with control group biological feedstuff
Nutrient content is more rich and nutritive value is higher.Wherein, the biological feedstuff of embodiment 6 increases effect the most to subject body weight
Significantly, its reason is the content highest of glossy ganoderma fat, ganodenic acid and GL-B in the embodiment biological feedstuff.
In sum, the extracting method of the glossy ganoderma composition of the embodiment of the present invention is simple to operate, it is easy to accomplish, and glossy ganoderma composition
Recovery rate it is higher；Raw material sources needed for the preparation method of biological feedstuff are wide, and process is simple, easy to operate, easy to control and time-consuming
It is short；The raw material of the biological feedstuff being prepared by the preparation method is natural products, and toxic and side effect is little, is of high nutritive value.
Embodiments described above is a part of embodiment of the invention, rather than the embodiment of whole.The reality of the present invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected enforcement of the present invention
Example.Based on the embodiment in the present invention, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
Claims (10)
1. a kind of extracting method of glossy ganoderma composition, it is characterised in that comprise the following steps：
Ganoderma lucidum fruitbody is crushed, with extractant refluxing extraction liposoluble constituent, glossy ganoderma fat is obtained, the first residue is remaininged；
With the first residue described in cellulase, hemicellulase and papain mixed enzymolysis, ethanol-water solution is added, in
Reflow treatment 1-3h under conditions of 75-85 DEG C, obtains phegma and the second residue, and with D101 macroporous adsorption resin chromatographies post institute is eluted
Phegma is stated, the liquid after wash-out is collected, organic phase is removed, ganodenic acid is obtained；
Second residue described in ultrasonic extraction, obtains extract and the 3rd residue, removes pigment and protein in the extract, Jing
DEAE- cellulose purifications obtain GL-B.
2. the extracting method of glossy ganoderma composition according to claim 1, it is characterised in that the extractant is ether or oil
Ether, the ratio of the ganoderma lucidum fruitbody and the extractant after crushing is 1g:150-250mL.
3. the extracting method of glossy ganoderma composition according to claim 1, it is characterised in that extract the liposoluble constituent be
Temperature is the 5-15h that flows back under conditions of 70-80 DEG C.
4. the extracting method of glossy ganoderma composition according to claim 1, it is characterised in that the enzyme activity of the cellulase is
45000-55000u/g, the cellulase is 2-4 with the weight ratio of the ganoderma lucidum fruitbody:100.
5. the extracting method of glossy ganoderma composition according to claim 1, it is characterised in that the enzyme activity of the hemicellulase is
150000-250000u/g, the hemicellulase is 3-5 with the weight ratio of the ganoderma lucidum fruitbody:100.
6. the extracting method of glossy ganoderma composition according to claim 1, it is characterised in that the enzyme activity of the papain is
750000-850000u/g, the papain is 2-4 with the weight ratio of the ganoderma lucidum fruitbody:100.
7. a kind of preparation method of biological feedstuff, it is characterised in that by the glossy ganoderma composition as described in any one of claim 1-6
The 3rd residue mixes with wheat bran obtained in extracting method, sterilizing, by weight 100:10-20 is inoculated with liquid spawn of picking up the ears,
It is inoculated with after cultivating 5-9 days under the conditions of 25-30 DEG C and picks up the ears liquid spawn weight ratio for 10-20 with described:The short stalk that sprouts of 8-13
Mould liquid spawn, fermentation, obtains biological feedstuff.
8. the preparation method of biological feedstuff according to claim 7, it is characterised in that the 3rd residue and the wheat bran
Weight ratio be 1:1-5.
9. the preparation method of biological feedstuff according to claim 7, it is characterised in that it in temperature is 25-30 DEG C that fermentation is
Under conditions of ferment 5-10 days.
10. a kind of biological feedstuff, it is characterised in that the preparation method of its biological feedstuff by described in any one of claim 7-9
It is prepared.
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Application publication date: 20170426 |