CN105766377B - A kind of cultural method improving black fungus flavones content and type - Google Patents
A kind of cultural method improving black fungus flavones content and type Download PDFInfo
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- CN105766377B CN105766377B CN201610243090.6A CN201610243090A CN105766377B CN 105766377 B CN105766377 B CN 105766377B CN 201610243090 A CN201610243090 A CN 201610243090A CN 105766377 B CN105766377 B CN 105766377B
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- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
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Abstract
The invention discloses a kind of cultural methods improving black fungus flavones content and type.Functional additive is added in culture medium and blackfungus culture medium is made by the present invention by a kind of preparation of functional additive for blackfungus culture medium of offer；Using above-mentioned blackfungus culture medium culture black fungus, cultural method includes the following steps：1) it is inoculated with；2) bacterium germination culture：By above-mentioned steps 1) in the black fungus Spawn incubation that is inoculated with, keep mycelia growth, the mycelia continues to cultivate after covering with bacterium bag；3) ear management：It is punched on the bag of the blackfungus culture medium, the mycelia grows up to ear bud, continues to cultivate；4) it harvests：When above-mentioned steps 3) described in ear bud grow up to auricle expansion, ear edge corrugation and soften, be further cultured for, you can picking obtains black fungus.Preparation method of the present invention is simple, is conducive to the recycling to waste mushroom leftover and branch, leaf, bar, and flavones content improves and type increase in the black fungus of culture.
Description
Technical field
The present invention relates to a kind of cultural methods improving black fungus flavones content and type, belong to breed of edible fungus and application
Field.
Background technology
Black fungus is also known as agaric, is the traditional large edible mushroom type in China, is good nutraceutical and dietetic food,
In recent years due to the new discovery that improvement of living standard and black fungus health protection act on, black fungus is as having special health protection function
Rapid development of the black-food in the market sales volume.
Flavone compound has the compound of 2- phenyl chromone (flavone) structure, is to be distributed to pass through by glucose
Shikimic acid pathway and acetate-malonate pathway generate the acetic acid of hydroxyl cinnamic acid and three molecules, then synthesizing chalcone, then spread out
Become all kinds of flavone compounds, have cardiovascular system activity, antibacterial and antiviral activity, antitumor activity, it is anti-oxidant from
By base activity, decompression, reducing blood lipid, anti-aging, raising immunity of organisms etc..
People are concentrated mainly on albumen, polysaccharide, pigment etc., polysaccharide tool for the health protection functional component of black fungus
Have hypoglycemic, reducing fat, inhibit platelet aggregation, antithrombotic etc., but it is less for flavone compound research, research is main
Study on extraction is concentrated on, is not had substantially for the flavones Research on kinds of black fungus, the reason is that the abundant egg of black fungus
In vain, polysaccharide and gum components influence its extraction process, while flavones content is relatively low in 3-5mg/100g, so it studies phase
To lag.It is ground using the wood materials culture black fungus such as wilsonii, sea-buckthorn although also being met in black fungus incubation simultaneously
Study carefully, occurs such as：The ingredients enrichment phenomenon such as eleutheroside, Hippophate flavone black fungus etc., but increase without solving flavone component
With wide variety problem, while a large amount of related wood materials are needed, unreasonable in cost, the general branches and leaves for being rich in Related Component
It is dropped, there are wasting of resources phenomenons, it is most important that due to factors such as resource distributions, be unsuitable for being widely applied.
Invention content
The object of the present invention is to provide a kind of functional additive for blackfungus culture medium, another purpose is to provide
A kind of blackfungus culture medium, further object are to provide a kind of cultural method improving black fungus flavones content and type.This hair
Bright preparation method is simple, is conducive to the recycling to waste mushroom leftover and branch, leaf, bar, and flavones content carries in the black fungus of culture
High and type increases.
Provided by the present invention for the preparation method of the functional additive of blackfungus culture medium, include the following steps：1)
It is extracted using homogenate extraction method after waste mushroom leftover is mixed with extractant, obtains the extracting solution of cellulase and zytase；
2) extracting solution is mixed with solid material in a solvent, digests, obtains enzymatic hydrolysis system；
The solid material is wintercherry, large-fruited Chinese hawthorn, chrysanthemum, dandelion, orange peel, wilsonii, blackcurrant, the sophora bud, Schisandra chinensis
At least one of with bitter buckwheat；
3) in confined conditions, by the enzymatic hydrolysis system microwave treatment, Microwave system is obtained；
4) by the Microwave system ultrasonic extraction to get to the functional additive for blackfungus culture medium.
In above-mentioned preparation method, extraction time of the homogenate extraction method can be 120~240s, concretely 120s,
200s or 120~200s, extraction voltage can be 180~220V, concretely 220V, and Extracting temperature can be 10~30 DEG C, specifically
It can be 25 DEG C, 30 DEG C or 25~30 DEG C；
The quality of the waste mushroom leftover and the volume ratio of the extractant can be 1g：(1~5) mL, concretely 1g:1mL、
1g:4mL or 1g：(1~4) mL；
The waste mushroom leftover and the mixed pH value of extractant can be 4~7, concretely 4.5,4.7,4.5~4.7 or 4
~6；
The waste mushroom leftover is auricuralia auricular bran；The extractant is water；
The enzyme activity of the extracting solution cellulase and zytase respectively can be 6~12IU/g, 5~10IU/g, specifically
The enzyme activity of cellulase can be 8IU/g, 10IU/g or 8~10IU/g, the enzyme activity of zytase can be 8IU/g, 10IU/g or 8~
10IU/g。
In the present invention, the enzyme activity of the extracting solution cellulase refers to 1 enzyme activity unit, refers in specified conditions
Under (25 DEG C, other is optimum condition), it is cellobiose and Portugal that 1 micromolar sodium carboxymethylcellulose can be converted in 1 minute
The required enzyme amount of grape sugar；
The enzyme activity of the zytase refers to 1 enzyme activity unit, refers to that (35 DEG C, other is most suitable in specified conditions
Condition) under, 1 micromolar substrate xylan (sigma reagents) can be converted in 1 minute as the enzyme amount needed for oligosaccharides and monosaccharide.
In above-mentioned preparation method, the solvent is 30~50% ethanol water of mass concentration, concretely 40% second
Alcohol solution；
The quality of the solid material and the volume ratio of the solvent can be 1g：(10~20) mL, concretely 1g:8mL；
The quality of the solid material and the volume ratio of the extracting solution can be 100g：(8~15) mL, concretely
100g：10ml；
The temperature of the enzymolysis is 30~40 DEG C, and concretely 37 DEG C, the time of the enzymolysis is 1~2h, concretely
1.5h, the pH value of the enzymolysis are 4~7, concretely 4.7；
The solid material is grouped as by the group of following mass parts：10~20 parts of the wintercherry；The large-fruited Chinese hawthorn 10~20
Part；2~5 parts of the chrysanthemum；5~10 parts of the dandelion；2~5 parts of the orange peel；10~20 parts of the wilsonii；It is described black
10~20 parts of gallon；5~15 parts of the sophora bud；5~20 parts of the Schisandra chinensis；5~10 parts of the bitter buckwheat；
The wintercherry, the large-fruited Chinese hawthorn, the wilsonii, the blackcurrant, the Schisandra chinensis and the bitter buckwheat are all made of
Its stem, leaf and/or bar.
In the present invention, the solid material can be specifically grouped as by the group of following mass parts：
(1) 10 parts of wintercherry bar；20 parts of sorbic acid wastewater, bar；5 parts of chrysanthemum, 5 parts of dandelion, 5 parts of orange peel, 10 parts of wilsonii bar,
10 parts of blackcurrant bar, 15 parts of the sophora bud, 10 parts of five tastes sub bar, 10 parts of bitter buckwheat bar；
(2) 20 parts of wintercherry bar；20 parts of sorbic acid wastewater；5 parts of chrysanthemum, 5 parts of dandelion, 5 parts of orange peel, 10 parts of wilsonii bar are black
10 parts of bar of gallon, 5 parts of the sophora bud, 10 parts of five tastes sub bar, 10 parts of bitter buckwheat bar.
In the present invention, dandelion is to use complete stool, and the sophora bud is to use seed.
In above-mentioned preparation method, the power of the microwave treatment can be 10~20kW, concretely 15KW, 20KW or 15
The time of~20kW, microwave treatment can be 1~2min, concretely 1min, 1.5min or 1~1.5min；
The time of the ultrasonic extraction can be 20~45min, concretely 30min, 35min or 30~35min；It is described super
The frequency of sound can be 20~30kHz, and the temperature of concretely 20kHz, the ultrasonic extraction can be 25~50 DEG C, concretely 30
DEG C, 40 DEG C or 30~40 DEG C；
It further include the steps that the recycling solvent in step 4).
The present invention also provides the functional additives for blackfungus culture medium that above-mentioned preparation method is prepared.
The present invention also provides a kind of blackfungus culture mediums, it is made of the component of following mass parts：Institute in claim 5
State 10~15 parts of the functional additive for blackfungus culture medium；60~80 parts of sawdust and/or corncob；5~10 parts of rice bran；Stone
1 part of cream powder；0.5 part of quick lime；0.5~1 part of L-phenylalanine；0.5~1 part of L-arginine.
Blackfungus culture medium of the present invention can specifically be made of the component of following mass parts：
1, functional additive 12.5%, linden bits 80%, rice bran 5%, land plaster 1%, quick lime 0.5%；L- phenylpropyl alcohol ammonia
Acid 0.5%, L-arginine 0.5%；
2,12.5 parts of functional additive, 70 parts of corncob, 15.8 parts of rice bran, 1 part of land plaster, 0.5 part of quick lime；L- phenylpropyl alcohols
0.1 part of propylhomoserin, 0.1 part of L-arginine.
The preparation method of the above-mentioned blackfungus culture medium of the present invention, includes the following steps：By the sawdust and/or corncob,
The rice bran, the land plaster and quick lime mixing, are then dissolved in institute by the phenylalanine and the L-arginine
It states the surface for the component for being applied to above-mentioned mixing in the functional additive for blackfungus culture medium, be uniformly mixed to get to black wood
Ear culture medium.
Invention further provides a kind of cultural methods of black fungus, include the following steps：1) it is inoculated with：By claim
The sterilizing of blackfungus culture medium described in 6, pack, are then inoculated with black fungus strain；
2) bacterium germination culture：By above-mentioned steps 1) in be inoculated with the black fungus Spawn incubation, keep mycelia growth, the bacterium
Filament length continues to cultivate after expiring bacterium bag；
3) ear management：It is punched on the bag of the blackfungus culture medium, the mycelia grows up to ear bud, continues to cultivate；
4) it harvests：When above-mentioned steps 3) described in ear bud grow up to auricle expansion, ear edge corrugation and soften, then train
It supports, you can picking obtains black fungus.
In above-mentioned method, in step 1), the pressure of the sterilizing is normal pressure, and the time of the sterilizing can be 15~20h；
The amount that blackfungus culture medium described in per 500g is inoculated with the black fungus strain can be 4~10g, described in specific every 500g
The amount that blackfungus culture medium is inoculated with the black fungus strain can be 5 grams；
In step 2), the temperature of mycelia growth is 20~25 DEG C, concretely 22~25 DEG C, the mycelia growth
Time is 25~40 days；After the mycelia covers with bacterium bag, the temperature can be 20~25 DEG C, concretely 22~25 DEG C, daily
10~30min is exposed, concretely exposure 10min, 20min or 10~20min, the time that the mycelia continues culture are daily
5~7 days, concretely 7 days.
In above-mentioned method, in step 3), the quantity in the hole is 4~8, concretely 6；The strip-shaped hole in the hole
Or " V " type hole, concretely wide 0.1~0.2 centimetre, long 4~7 centimetres of strip-shaped hole or the uniform angle of surrounding in bag are 45
~60 °, cut 0.1~0.2 centimetre 6 wide, length 4~7, deep 0.5 centimetre of V-shape hole；
When growing the ear bud, 0.5% phenylalanine of mass concentration is sprayed 1~2 time to it, it is concretely 1 time, straight daily
Line light application time extended 10~30min by 3~5 hours, concretely extended 20min, by 4 hours time lengthenings by 3 hours
20min extended 15~25min by 3~4 hours；
When the ear bud Cheng little Duo, the temperature for continuing culture can be 15~25 DEG C, and the time that the ear bud continues culture can
It is 6~12 days；
In step 4), when the ear edge wrinkles and softens, the phenylalanine 1 that mass concentration is 0.1% is sprayed to it
Secondary, the time that the auricle is further cultured for is 1~4 day, and the temperature that the auricle is further cultured for is 15~25 DEG C；
When color becomes light brown from dark brown before the auricle expansion, to its ventilation quantity by 120~150m3/ h increases
To 300~500m3/ h, ventilation number are increased to 5~6 times by daily 3~4 times, and concretely ventilation quantity is by 120m3/ h is increased to
400m3/ h or ventilation quantity are by 130m3/ h increases to 350m3/h；Daily straight line light application time extended 30~70min by 4~8 hours,
Concretely extend within 6 hours 30min or extends 50min by 7 hours；
When the picking, the general flavone of the black fungus can be 1.2~1.8%.
In the present invention, using sodium nitrite-aluminum nitrate colorimetric method for determining general flavone；General flavone 1.2~1.8%；Using liquid
Phase chromatography research measures flavones type, and flavones type is specially aurantiamarin, rutin, awns glycosides, Hyperoside, Quercetin, kaempferia galamga
Phenol, Isorhamnetin.
The present invention has the following advantages：
The present invention realizes that bacterium bag resource is again sharp using bacteria residue homogenate extraction method extraction cellulose enzyme, the xylosidase etc. of giving up
With using enzymatic-ultrasonic wave and microwave combined process rapid extraction culture medium function adding ingredient, ensure that active ingredient to the greatest extent may be used
The extraction of energy goes out, and is prepared into reserve liquid and directly uses or obtain medicinal extract storage use, reduces related timber usage amount.Simultaneously logical
With prescription is suitably reformed on medium base, addition is conducive to Flavonoids biosynthesis substance L-phenylalanine, L-arginine, cultivates
Technique only needs to spray Active substance, and basic production technique does not have essential change, is conducive to promote.Extraction is selected in invention
The common branch of material, leaf, bar can be used, and rich reserves, have good development prospect.
Description of the drawings
Fig. 1 is flavones standard solution chromatogram.
Fig. 2 is the agaric chromatogram for the culture medium production for being not added with functional additive.
Fig. 3 is the agaric chromatogram that the embodiment of the present invention 1 adds that the culture medium of functional additive is cultivated.
Fig. 4 is the agaric chromatogram that the embodiment of the present invention 2 adds that the culture medium of functional additive is cultivated.
Fig. 5 is that blank liver HE dyes spectrogram.
Fig. 6 is that model liver HE dyes spectrogram.
Fig. 7 is that low dose of liver HE dyes spectrogram.
Fig. 8 is that large dosage of liver HE dyes spectrogram.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Key instrument used in following embodiments：JHBE-50A flash extracters, Xi'an kylin laboratory apparatus Co., Ltd,
2695e high performance liquid chromatography, waters companies of the U.S., FSH-2 types are adjustable high speed homogenizer, the global science instrument in Jintan City of Jiangsu Province
Device factory；BX41 light microscopic MIcrosope image acquisition systems, Japanese OLYMPUS；JP600 ultrasonic extractors, roc electronics is praised in Wuhan to be had
Limit company；3 types of NJL07-test special microwave oven, Nanjing Jie Quan microwave equipments Co., Ltd；GL20A fully automatic high-speeds freeze
Centrifuge：Thermostat water bath；Vacuum drying chamber；T6 ultraviolet-uisible spectrophotometers, Beijing spectrum analysis；PHS-3C type acidometers, on
Extra large thunder magnetic；Electronic balance, German Sai Duolisi；Agaric strip bundling machine, the vertical automatic packer of edible mushroom, Suizhou Qing Feng；
FY130 pulverizers, Tianjin Stettlen.
Detection method in following embodiments：Using aluminum nitrate-sodium nitrite colorimetric method for determining general flavone；Using liquid chromatogram
Method research measures flavones type；Black fungus acute toxicity is cultivated using general anxious malicious test code research is new, using rat alcohol
Liver injury model studies black fungus to hepatic effects：Its liver index TG, TC, AST, ALT are detected with full automatic biochemical apparatus, MDA
It is measured with TBA methods, SOD is measured with xanthine oxidase, and GSH-PX is measured with DTNB spectrophotometry.
In following embodiments, 1, Determination of Total Flavonoids：
Using one aluminum nitrate method of sodium nitrite.Using rutin as reference substance, general flavone is measured in 500nm.
Extracting solution measures：Extracting solution recycling design is concentrated and dried, and is taken in right amount, is dissolved with a small amount of methanol, takes 70% methanol fixed
Hold into 100mL volumetric flasks, pipettes 1mL mother liquors and measure content according to above method in 25mL colorimetric cylinders.
2, flavones Research on kinds：
Instrument：Liquid chromatogram：waters e2695；Detector：Diode array DAD
Chromatographic condition：30 DEG C of column temperature, chromatographic column are C18 (4.6 × 250um, 5um)；Mobile phase A is water：0.1% phosphoric acid water
Solution, Mobile phase B are acetonitrile；Gradient elution program is 0~15min, and acetonitrile volume fraction is 60%, 15~20min, by 30%
40%, 20~30min are increased to, 50%, 30~45min are increased to by 40%, 60%, 45~55min are increased to by 50%, is increased by 60%
To 80%, 55~65min, constant is 85%；Flow velocity is 1mL/min：Detection wavelength is 360nm.
Reference substance：Aurantiamarin, rutin, awns glycosides, Hyperoside, catechin, Quercetin, Kaempferol, Isorhamnetin, it is above right
Respectively add methanol that the storing solution of 60,200,80,60,80,200,60,60ug/mL is made according to product.0.1% phosphate aqueous solution is added to dilute
To scale, control series product liquid is made.With Mobile phase B volume fraction be 60% be dilution be made into every lmL respectively contain 15,50,
20,15,20,50,15, the catechin of 15ug, rutin, Quercetin, Kaempferol, cyanidenon, aurantiamarin mixed liquor, as right
According to product solution, sample size 10uL, according to the related flavones type of retention time research, as a result such as Fig. 1.
Sample treatment：Addition functional additive cultivates agaric (being not added with functional additive production agaric), naturally cloudy
It is dry, it measures moisture content and differs within 2~5% between the two, crush, cross 30 mesh sieve, accurately weighed 10g agarics powder.20~
30% ethyl alcohol adds certain pectase, cellulase, zytase as solvent, adjusts pH value 4-5, at 35~40 DEG C, enzymatic
Reaction 5h, 1~2min of microwave treatment, adjusting ethyl alcohol to 70~80% 20~30min of supersound process, 50~60 DEG C of ultrasonic temperature,
It filters while hot, it is that 10uL studies related flavones type to take supernatant, sample size, and the results are shown in Figure 2.
In following embodiments, auricuralia auricular bran plants bacterium bag from conventional black fungus upper one year, pedagogical derived from Mudanjiang
Institute edible mushroom research center.
Embodiment 1,
1, prepared by function extractant：
(1) water is extractant, the volume ratio of auricuralia auricular bran quality and water, i.e. solid-to-liquid ratio 1g:4mL, pH 4.5, extraction
Time 120s, extraction voltage 220V, Extracting temperature：30 DEG C, the rapid extraction in flash extracter obtain cellulase and wood
Glycan zyme extract, it is respectively 8IU/g, 8IU/g spare to measure enzyme activity.Wherein, the enzyme activity of cellulase refers to 1 enzyme activity
Unit refers to that 1 micromolar carboxymethyl cellulose can be converted in 1 minute under specified conditions (25 DEG C, other is optimum condition)
Plain sodium is the enzyme amount needed for cellobiose and glucose；The enzyme activity of zytase refers to 1 enzyme activity unit, refers to specific
Under condition (35 DEG C, other is optimum condition), it is widow that 1 micromolar substrate xylan (sigma reagents) can be converted in 1 minute
Enzyme amount needed for sugar and monosaccharide.
(2) 10 parts of wintercherry bar；20 parts of sorbic acid wastewater, bar；5 parts of chrysanthemum, 5 parts of dandelion, 5 parts of orange peel, 10 parts of wilsonii bar,
10 parts of blackcurrant bar, 15 parts of the sophora bud, 10 parts of five tastes sub bar, 10 parts of bitter buckwheat bar.It crushes, crosses 20 mesh sieve, be uniformly mixed spare.
(3) extraction enzyme solution in (1) is added into 10mL according to solid in every 100g (2)；With 30% ethanol water-for solvent, Gu
Liquor ratio 1:10, pH 4.7,35 DEG C of temperature digests 1h.
(4) above-mentioned enzymatic hydrolysis system, sealing condition, microwave power 15KW, microwave treatment 1min；
(5) above-mentioned Microwave system, with ultrasonic time 30min, supersonic frequency 20kHz, the 40 DEG C of progress of ultrasonic wave extraction temperature
Functional additive extracts.Alcohol solvent is recycled, it is spare to be concentrated into original volume 1/10.
2, prepared by culture medium：
(1) the new culture medium of black fungus is：Functional additive 12.5%, linden bits 80%, rice bran 5%, land plaster 1%,
Quick lime 0.5%；L-phenylalanine 0.5%, L-arginine 0.5%.
(2) solid culture medium is strictly prepared in proportion, and spice is uniformly mixed, and phenylalanine, L-arginine is dissolved into work(
In energy additive, even application is uniformly mixed again in solid culture medium surface, normal pressure moist heat sterilization 20 hours, and pack is spare.
3, agaric cultivating process
(1) it is inoculated with：After the material bag cooling of sterilizing, inoculation in the inoculating hood that is put into.Every bag of 500g of inoculum concentration is inoculated with agaric fungus
5 grams of kind；
(2) bacterium germination culture：Keep the growth temperature of mycelia between 22~25 DEG C.After 28 days mycelia cover with bacterium bag,
Exposure 10min daily continues culture 5 days,
(3) ear management：With sterilized blade 0.1~0.2 centimetre 6 wide, length 4~7 is uniformly cut in the surrounding of bag
Centimetre strip-shaped hole, when ear bud occurs, spray concentration 0.5% phenylalanine 1 time；(daily light application time under the premise of sufficient illumination
Extended 20min by 3 hours, other are managed according to normal agaric；
(4) it harvests：When auricle color becomes light brown from dark brown, at this moment certain main more ventilations (ventilation quantity by
120m3/ h increases to 400m3/ h, ventilation number be 5 times), while give more abundant illumination (daily light application time by 6 hours extend
30min), it is sufficiently spread out in auricle, there is apparent corrugation at edge and softens, and is spraying 0.1% phenylalanine of concentration 1 time, culture 2
It, can pick when detecting general flavone ＞ 1.2%.
(5) it examines：According to front flavones detection method, standard curve Y=0.5734x+0.0172, R2=0.9993,
Range of linearity 0.11-0.67mg/mL, general flavone content 1.54%.
Flavones kind liquid-like phase detection spectrogram is as shown in figure 3, as shown in Figure 1 in the black fungus that the present invention cultivates, aurantiamarin, reed
Fourth, awns glycosides, Hyperoside, catechin, Quercetin, Kaempferol, Isorhamnetin successively retention time be 19min, 30min,
33min, 37min, 41min, 50min, 54min, 55min, the black fungus that the foundation retention time present invention cultivates is relative to guarantor
It stays the time corresponding peak occur, illustrates in agaric of the present invention with the presence of above-mentioned substance.Conventional culture methods (Fig. 2) are compareed, are being protected
Time 41min, 50min are stayed, is had compared with small peak, remaining has no apparent appearance.Illustrate that the present invention is had significantly using functional additive
Increase flavones type function, while comparing 41min, 50min, place can obviously increase it and correspond to flavones content.The present invention passes through
Increase functional additive and flavones secondary metabolism precursor phenylalanine and illumination means are appropriately extended, related flavonoid substance is promoted to store
Product and increase.
Embodiment 2,
1, prepared by function extractant：
(1) water is extractant, auricuralia auricular bran solid-to-liquid ratio 1g:1mL, pH 4.7, extraction time 200s, extraction voltage
220v, Extracting temperature：25 DEG C, the rapid extraction in flash extracter obtain cellulase and zytase extracting solution, measure
Enzyme activity (definition is with embodiment 1) is 10IU/g, and 10IU/g is spare；
(2) 20 parts of wintercherry bar；20 parts of sorbic acid wastewater；5 parts of chrysanthemum, 5 parts of dandelion, 5 parts of orange peel, 10 parts of wilsonii bar are black
10 parts of bar of gallon, 5 parts of the sophora bud, 10 parts of five tastes sub bar, 10 parts of bitter buckwheat bar.It crushes, crosses 20 mesh sieve, be uniformly mixed spare.
(3) extraction enzyme solution in (1) is added into 10ml according to solid in every 100g (2)；With 40% ethanol water-for solvent, Gu
Liquor ratio 1g:8mL, pH 4.7,37 DEG C of temperature digest 1.5h.
(4) above-mentioned enzymatic hydrolysis system, sealing condition, microwave power 20KW, microwave treatment 1.5min；
(5) above-mentioned Microwave system, with ultrasonic time 35min, supersonic frequency 20kHz, the 30 DEG C of progress of ultrasonic wave extraction temperature
Functional additive extracts.Alcohol solvent is recycled, it is spare to be concentrated into original volume 1/8.
2, prepared by culture medium：
(1) the new culture medium of black fungus is：Functional additive 12.5%, corncob 70%, rice bran 15.8%, land plaster
1%, quick lime 0.5%；L-phenylalanine 0.1%, L-arginine 0.1%, said components are in terms of mass percentage.
(2) solid culture medium is strictly prepared in proportion, and spice is uniformly mixed, and phenylalanine, L-arginine is dissolved into work(
In energy additive, even application is uniformly mixed again in solid culture medium surface, normal pressure moist heat sterilization 15 hours, and pack is spare.
3, agaric cultivating process
(1) it is inoculated with：After the material bag cooling of sterilizing, inoculation in the inoculating hood that is put into.5 grams or so (every bag of every bag of inoculum concentration
500g blackfungus culture mediums)；
(2) bacterium germination culture：Keep the growth temperature of mycelia between 22~25 DEG C.After 32 days mycelia cover with bacterium bag,
Exposure 20min daily continues culture 7 days between 22~25 DEG C,
(3) ear management：With sterilized blade bag the uniform angle of surrounding be 45~60 °, cut it is 6 wide by 0.1~
0.2 centimetre, length 4~7, deep 0.5 centimetre of V-shape hole, when ear bud occurs, 0.5% phenylalanine of spray concentration 1 time；Sufficient illumination
Under the premise of (daily illumination was by 4 hours time lengthening 20min), other are managed according to normal agaric；
(4) it harvests：When auricle color becomes light brown from dark brown, at this moment certain main more ventilations (ventilation quantity by
130m3/ h increases to 350m3/ h, ventilation number be 6 times), while give more abundant illumination (daily light application time by 7 hours extend
50min), it is sufficiently spread out in auricle, there is apparent corrugation at edge and softens, and is spraying 0.1% phenylalanine of concentration 1 time, is being further cultured for
Growth 1-2 days can pick when detecting general flavone ＞ 1.2%.
(5) it examines：According to front flavones detection method, standard curve Y=0.5734x+0.0172, R2=0.9993,
Range of linearity 0.11-0.67mg/mL, general flavone content 1.42%.
In black fungus prepared by the present invention its flavones kind liquid-like phase detection spectrogram as shown in figure 4, comparison diagram 1,2,4 it is found that
Aurantiamarin, rutin, awns glycosides, Hyperoside, catechin, Quercetin, Kaempferol, Isorhamnetin substance appear in present invention culture wood
In ear, routine culture does not generate related substances.Simultaneously as correlation function extractant matches difference, there are Flavonoid substances and contain
Difference is measured, illustrates that functional additive of the present invention has typical adjusting flavones total amount and type action, changes functional additive composition,
Related flavones amount and type can be flexibly increased or decreased, there is certain theory value and production for exploitation correlation function product
Value.Therefore stalk, stem, leaf different adjustment proportioning are produced in conjunction with locality and realizes that local resources make full use of.Simultaneously according to ratio
It realizes that agaric plants product function, improves agaric health value itself, improve added value of product.And cultivating process is simple, effect
Fruit is more apparent.
Comparative example 1,
Pattern is cultivated to flavones in order to discuss flavones secondary metabolism precursor phenylalanine and arginine and illumination be appropriately extended
The influence of total content, culture medium in case study on implementation 2 is modified, and remove function additive is substituted using corncob.Culture is special
Sign is as follows：
1, the new culture medium of black fungus
(1) the new culture medium of black fungus is：Corncob 82.5%, rice bran 15.8%, land plaster 1%, quick lime 0.5%；
L-phenylalanine 0.1%, L-arginine 0.1%, said components are in terms of mass percentage.
(2) solid culture medium is strictly prepared in proportion, and spice is uniformly mixed, and phenylalanine, L-arginine is dissolved into work(
In energy additive, even application is uniformly mixed again in solid culture medium surface, normal pressure moist heat sterilization 15 hours, and pack is spare.
2, agaric cultivating process
(1) it is inoculated with：After the material bag cooling of sterilizing, inoculation in the inoculating hood that is put into.5 grams or so (every bag of every bag of inoculum concentration
500g blackfungus culture mediums)；
(2) bacterium germination culture：Keep the growth temperature of mycelia between 22~25 DEG C.After 32 days mycelia cover with bacterium bag,
Exposure 20min daily continues culture 7 days between 22~25 DEG C,
(3) ear management：With sterilized blade bag the uniform angle of surrounding be 45~60 °, cut it is 6 wide by 0.1~
0.2 centimetre, length 4~7, deep 0.5 centimetre of V-shape hole, when ear bud occurs, 0.5% phenylalanine of spray concentration 1 time；Sufficient illumination
Under the premise of (daily illumination was by 4 hours time lengthening 20min), other are managed according to normal agaric；
(4) it harvests：When auricle color becomes light brown from dark brown, at this moment certain main more ventilations (ventilation quantity by
130m3/ h increases to 350m3/ h, ventilation number be 6 times), while give more abundant illumination (daily light application time by 7 hours extend
50min), it is sufficiently spread out in auricle, there is apparent corrugation at edge and softens, and is spraying 0.1% phenylalanine of concentration 1 time, is being further cultured for
Growth 1-2 days can pick when detecting general flavone ＞ 1.2%.
(5) it examines：According to front flavones detection method, standard curve Y=0.5734x+0.0172, R2=0.9993,
Range of linearity 0.11-0.67mg/mL, general flavone content 0.28%.It can be seen that its result, which is apparently higher than, does not add flavones precursor
As a result (3-5mg/100g).
The anxious poison experiment of above-described embodiment and alcoholic hepatic injury scale-model investigation：
1, acute toxicity test：Half-and-half by 30 SD rats male and female, it is randomly divided into blank group, control group, experimental group each 9
Only, it tests preceding fasting, can't help water 6h.Experimental group (referring to that above-described embodiment 1 or 2 cultivates obtained agaric) in 1d points 3 times to
Black fungus (crushed 100-200 mesh, sterile purified water, the processing of high-speed homogenization machine obtains suspension) suspension gavage is given, every time
Water 4h is can't help in 0.2g/mL, maximum gastric capacity 4mL/100g, twice gavage interval 6h, fasting after gavage；Control group is given commonly
Black fungus suspension, the same experimental group of method；Blank group gives equivalent distilled water gavage, the same experimental group of method.After gavage, continuous 7d
Observe three groups of Hair of Rats, breathing, feed, two just, mouth and nose secretion, the state of mind and changes of body mass, the upper and lower noon is each daily
1 time；And record the appearance of its symptom, recovery and death time.Measured body weight：From the 1st gavage start recording, 1 after gavage, 3,
5,7d is respectively measured primary.Experiment terminate next day using dislocation method put to death rat, to its appearance and each internal organs of whole body carry out dissection and
It visually observes, the tissue that notes abnormalities carries out pathological examination again.
It is the results show that major organs internal organs have no significant change.
The maximum dosage-feeding of experimental group is 0.2g/mL × 40mL/kg (maximum gastric capacity) × 3 times=24g/kg, is adult
Daily recommend usual amounts (10g/60kg) 140 times.After experimental group is with maximum administration concentration and maximum gastric capacity gavage, feed
Amount and activity significantly reduce, but restore from the 2nd day normal.7d is observed continuously, experimental group, blank group hair, is exhaled at control group
Inhale, feed, two just, mouth and nose secretion, the state of mind and weight be showed no obvious abnormalities, and without dead and other abnormal shapes
State.
Experimental group administration 1,3,5, the equal no difference of science of statistics (P ＞ 0.05) of the weight of 7d in three groups of each periods, knot
Fruit such as table 1.
1 each group rat changes of body mass of table
2, rat alcoholic hepatic injury model：TG, TC, AST, ALT are detected with full automatic biochemical apparatus；MDA is measured with TBA methods；
SOD is measured with xanthine oxidase；GSH-PX is measured with DTNB spectrophotometry.
Experimentation：Normal rats forage feed l weeks.Divide blank group 10, modeling group 40.Blank group is with normal diet
It feeds, distilled water 10mL/ (kgd) gavage freely drinks distilled water；Modeling rat is randomly divided into model group, agaric large dosage
Group, agaric middle dose group and agaric small dose group.Every group 10.Modeling method is 50% ethyl alcohol 10mL/ (kgd) points of 2 fillings
Stomach, periodically 9：00 and 21：00,5% alcohol drink is freely drunk, normal diet is fed.Model group is in modeling simultaneously with distillation
Water 10mL/ (kgd) gavage；Large, medium and small dosage group modeling simultaneously respectively with 20g/ (kgd), 15g/ (kgd) and
10g/ (kgd) suspension gavage；The gavage time is fixed on every afternoon 14：00.Each group is put to death after weighing in 12 weekends, abdominal cavity
Venous blood sampling takes 12h fasting before blood, 5% alcohol drink freely to drink.The rat of execution leaves and takes liver organization and weighs, and observation is outer
Shape, color and luster, quality and section situation take and organize to be made the homogenate of 0.85% sodium chloride in the middle part of liver on a small quantity, centrifuging and taking supernatant, and -20
DEG C refrigerator preserves for use；Take hepatic tissue, neutral formalin liquid, paraffin embedding, slice, hematoxylin eosin staining, light microscopic (microscope
Image capturing system (OLYMPUS, BX41) is observed.As a result such as Fig. 5,6,7,8.
It is typical liver function index below, as shown in table 2.
2 each group rat fat of table, liver function variation
3 each group liver tissues of rats SOD, MDA, GSHpx variation of table
4 each group rat body weight of table changes
The weight of model group rats (P substantially reduced compared with blank group rat body weight<0.01), dosage group rat body weight with
Model group is without significant difference.To the 14th weekend, model group rats liver index dramatically increases (P compared with blank group<0.01).Administration
Group liver index is decreased significantly (P compared with model group<0.05).There is hepatic tissue section and show in model group rats：Fat becomes
Property and inflammatory infiltration；Its Serum ALT, AST, TG, TC activity are dramatically increased compared with blank group.And SOD, GSH-Px activity in hepatic tissue
It significantly reduces.MDA contents significantly increase.No matter which kind of dosage improves administration group in various degree, so making with liver protecting
With.
(4) further aim of the present invention be additive can reduce the miscellaneous bacterias such as mould infection probability have natural environmental-protective it is green
Color feature.
Due to containing chrysanthemum, dandelion, orange peel, bitter buckwheat bar etc., traditional Chinese medicine ingredients have certain bacteriostatic activity, specific real
The reduction of its microbiological contamination probability is obviously found in trampling, specific every 1000 bags lower 1~5%, and microbiological contamination probability again is found in growth course
Lower 5~10%.
Claims (10)
1. a kind of preparation method of functional additive for blackfungus culture medium, includes the following steps：1) by waste mushroom leftover
It is extracted using homogenate extraction method after being mixed with extractant, obtains the extracting solution of cellulase and zytase；
2) extracting solution is mixed with solid material in a solvent, digests, obtains enzymatic hydrolysis system；
The solid material is grouped as by the group of following mass parts：10~20 parts of wintercherry；10~20 parts of large-fruited Chinese hawthorn；Chrysanthemum 2~5
Part；5~10 parts of dandelion；2~5 parts of orange peel；10~20 parts of wilsonii；10~20 parts of blackcurrant；5~15 parts of the sophora bud；Schisandra chinensis 5
~20 parts；5~10 parts of bitter buckwheat；
3) in confined conditions, by the enzymatic hydrolysis system microwave treatment, Microwave system is obtained；
4) by the Microwave system ultrasonic extraction to get to the functional additive for blackfungus culture medium.
2. preparation method according to claim 1, it is characterised in that：The extraction time of the homogenate extraction method be 120~
240s, extraction voltage are 180~220V, and Extracting temperature is 10~30 DEG C；
The quality of the waste mushroom leftover and the volume ratio of the extractant are 1g：(1~5) mL；
The waste mushroom leftover is 4~7 with the mixed pH value of extractant；
The waste mushroom leftover is auricuralia auricular bran；The extractant is water；
The enzyme activity of the extracting solution cellulase and zytase is respectively 6~12IU/g, 5~10IU/g.
3. preparation method according to claim 1 or 2, it is characterised in that：The solvent is 30~50% second of mass concentration
Alcohol solution；
The quality of the solid material and the volume ratio of the solvent are 1g：(10~20) mL；
The quality of the solid material and the volume ratio of the extracting solution are 100g：(8~15) mL；
The temperature of the enzymolysis is 30~40 DEG C, and the time of the enzymolysis is 1~2h, and the pH value of the enzymolysis is 4-7；
The wintercherry, the large-fruited Chinese hawthorn, the wilsonii, the blackcurrant, the Schisandra chinensis and the bitter buckwheat be all made of its stem,
Leaf and/or bar.
4. preparation method according to claim 1 or 2, it is characterised in that：The power of the microwave treatment is 10~20kW,
The time of microwave treatment is 1~2min；
The time of the ultrasonic extraction is 20~45min；The frequency of the ultrasound is 20~30kHz, the temperature of the ultrasonic extraction
Degree is 25~50 DEG C；
It further include the steps that the recycling solvent in step 4).
5. the functional addition for blackfungus culture medium that the preparation method described in any one of claim 1-4 is prepared
Agent.
6. a kind of blackfungus culture medium, it is characterised in that：It is made of the component of following mass parts：It is used described in claim 5
In 10~15 parts of the functional additive of blackfungus culture medium；60~80 parts of sawdust and/or corncob；5~10 parts of rice bran；Land plaster
1 part；0.5 part of quick lime；0.5~1 part of L-phenylalanine；0.5~1 part of L-arginine.
7. a kind of preparation method of blackfungus culture medium, includes the following steps：By sawdust and/or corncob, rice bran, land plaster and
Quick lime mixes, then phenylalanine and L-arginine are dissolved in the work(that blackfungus culture medium is used for described in claim 5
Surface, the mixing of the component of above-mentioned mixing can be applied in additive to get to blackfungus culture medium.
8. a kind of cultural method of black fungus, includes the following steps：1) it is inoculated with：By blackfungus culture medium described in claim 6
Sterilizing, pack, are then inoculated with black fungus strain；
2) bacterium germination culture：By above-mentioned steps 1) in the black fungus Spawn incubation that is inoculated with, keep mycelia growth, the mycelia is long
Continue to cultivate after full bacterium bag；
3) ear management：It is punched on the bag of the blackfungus culture medium, the mycelia grows up to ear bud, continues to cultivate；
4) it harvests：When above-mentioned steps 3) described in ear bud grow up to auricle expansion, ear edge corrugation and soften, be further cultured for,
It can pick to obtain black fungus.
9. according to the method described in claim 8, it is characterized in that：In step 1), the pressure of the sterilizing is normal pressure, described to go out
The time of bacterium is 15~20h；
The amount that blackfungus culture medium described in per 500g is inoculated with the black fungus strain is 4~10g；
In step 2), the temperature of the mycelia growth is 20~25 DEG C, and the time of the mycelia growth is 25~40 days；The bacterium
After filament length expires bacterium bag, the temperature be 20~25 DEG C, daily expose 10~30min, the mycelia continue culture time be 5~
7 days.
10. method according to claim 8 or claim 9, it is characterised in that：In step 3), the quantity in the hole is 4~8；Institute
It is strip-shaped hole or " V " type hole to state hole；
When growing the ear bud, 0.5% phenylalanine of mass concentration is sprayed 1~2 time to it, daily straight line light application time is by 3~5
Hour extends 10~30min；
When the ear bud Cheng little Duo, the temperature for continuing culture is 15~25 DEG C, and the time that the ear bud continues culture is 6~12
It；
In step 4), when the ear edge wrinkles and softens, the phenylalanine 1 time that mass concentration is 0.1%, institute are sprayed to it
It is 1~4 day to state the time that auricle is further cultured for, and the temperature that the auricle is further cultured for is 15~25 DEG C；
When color becomes light brown from dark brown before the auricle expansion, to its ventilation quantity by 120~150m3/ h increases to 300
~500m3/ h, ventilation number are increased to 5~6 times by daily 3~4 times；Daily straight line light application time by extension 30 in 4-8 hour~
70min；
When the picking, the general flavone of the black fungus is 1.2~1.8%.
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| CN110810126A (en) * | 2019-12-05 | 2020-02-21 | 黑龙江黑臻生物科技有限公司 | Yuanmo cultivation method and Yuanmo |
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| CN102630492B (en) * | 2012-04-23 | 2013-05-22 | 国家林业局泡桐研究开发中心 | Production method for cultivating gingko agaric by utilizing medicinal plant residues |
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