CN109439719A - A kind of fungi two-way solid-fermented technique and its application based on Cortex Eucommiae - Google Patents
A kind of fungi two-way solid-fermented technique and its application based on Cortex Eucommiae Download PDFInfo
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- CN109439719A CN109439719A CN201811426185.7A CN201811426185A CN109439719A CN 109439719 A CN109439719 A CN 109439719A CN 201811426185 A CN201811426185 A CN 201811426185A CN 109439719 A CN109439719 A CN 109439719A
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- cortex eucommiae
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Classifications
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Abstract
The invention discloses a kind of fungi two-way solid-fermented technique and its application based on Cortex Eucommiae, culture inclined-plane parent species prepare liquid spawn again after the fermentation technique activates fungal bacterial strain, nutrient solution is added in the Cortex Eucommiae raw material of crushing and stirs homogeneous, it is 50-65% to water content, liquid spawn is uniformly accessed after sterilizing in solid culture primary surface, fermented and cultured, until entire culture medium covers with white hypha, collect tunning, it is dry, Cortex Eucommiae medicinal fungal substance is obtained, product Cortex Eucommiae medicinal fungal substance can be applied to functional food, drug, cosmetics or feed addictive.The present invention plays the role of synergy to Cortex Eucommiae crude drug, expansion is used, and can promote Fungal hyphal growth, significantly improves fermentation efficiency, and mycelium germination point is more, and material feeding ability is strong, and aufwuchsplate is big, and growing way is fast.Fermentation technology process of the invention is simple, and condition is easy to control, can high-volume the factorial production.
Description
[technical field]
The present invention relates to a kind of fungi two-way solid-fermented technique and its application based on Cortex Eucommiae, belong to microbial fermentation skill
Art field.
[background technique]
The solid-fermented technique of medicinal fungi is the key areas of modern biotechnology.Medicinal fungi zymophyte at present
Matter has application in medicine.Locust tree microellobosporia is exactly that Trametes robiniphila Murr Trametes robinioplila Mur. is sought in corn, wheat bran etc.
Resulting dry mycoplasma after cultivation in matrix is supported, national one kind new medicine of Chinese medicine, medicine number WS3-215 (Z- have been approved for
029) the effect of -2001 (Z) has and strengthen the body resistance to consolidate the constitution, eliminating mass of invigorating blood circulation, it can only be known as medicinal mycoplasma.Zhuan Yi (2002) proposes medicine
It is a new starting point with the two-way solid fermentation of fungi, it is different from nutrient matrix in traditional solid fermentation and unidirectionally mentions for fungi
The nutritional ingredients such as high carbon nitrogen source.For two-way solid fermentation using Chinese medicine as matrix, mycothallus therefrom absorbs nutrition, while changing again
Pharmacological property ingredient has amphicheirality.Tunning is referred to as medicinal fungal substance, it is different from the general solid fermentation product of fungi, simultaneous
Have the primitive component and effect of Chinese medicine and medicinal fungi, it is also possible to generate new nature and flavor and effect, play to former Chinese medicine
The effect used, be attenuated is expanded in synergy.Researches show that Trametes robiniphila Murrs to produce medicinal and medicine in nutrient matrix and the top fermentation of pharmacological property matrix respectively
Property two kinds of mycoplasma, compared by drug effect, it was demonstrated that the desired result of " synergy, expand use " occurs in medicinal fungal substance.With different fermentations bacterium
Kind and various fermentation substrate combined crosswises, may be constructed the different fementative composition of a large amount of properties.It is feasible such as to combine science, then through true
All there is new variation in matrix components and drug effect after the biotransformation that bacterium has been fermented.Particularly such as with same fungi
It ferments and different substrates or is fermented same matrix with different strain, theoretically the character function of various mycoplasma is all answered different.
It is directionally fermented to Chinese medicine based on this theoretical foundation using the biological characteristics and drug effect of edible and medicinal fungi, thus
It realizes to the advantageous transformation of the enhancing of Chinese medicine drug effect and purposes expansion etc., enhances the application value of Chinese medicine.
Ganoderma lucidum (Ganoderma lingzhi Sheng H.W et al.) is a kind of rare macro fungi, is had high
Edible and medical value.In more than 2,000 years of the past of China, it is considered as the treasure for prolonging and lengthening one's life by people always.Nowadays ganoderma lucidum conduct
Medicinal material is incorporated into Chinese Pharmacopoeia already, and can be used for health food development by national health department's approval.Ganoderma lucidum is more at present
Sugar, triterpene, sterol are acknowledged as most important active constituent in ganoderma lucidum, have extensive pharmacological activity, such as enhance immune, anti-swollen
Tumor, liver protection, reducing blood lipid etc..Ganoderma lucidum also has good curative effect in clinical application.Research report display ganodenic acid can prolong recently
The development of slow autosomal dominant polycystic kidney (ADPKD)；Ganoderma lucidum spore oil has the function of cardioprotection, can be used as potential heart trouble and protects
Protect agent.The utilization form of ganoderma lucidum is mainly fructification, mycelium and conidia powder at present.In recent years in China, ganoderma lucidum Related product
Market scale be more than 10,000,000,000 yuan, wherein health care product is more than 1000, drug more than 100.But the big multiphase of the raw material of these products
Together, function is close, and homogeneity is very serious, and product newly developed also lacks original innovation, and basic reason is to be limited by original
The unification (mainly using fructification and conidia powder) of material, also without new breakthrough, (fructification uses Ultramicro-powder, water to mention, surpass to technique
Filter, alcohol precipitation etc., conidia powder extract spore oil using supercritical extraction), fail for many years biological characteristics using ganoderma lucidum into
The transformation of row modern biotechnology, this is also urgently to upgrade innovation by the big health industry of representative of ganoderma lucidum.
Hericium erinaceus (Hericium erinaceus (Rull ex F.) Pers.), and a kind of rare edible and medicinal fungi.
Chinese medicine thinks that Hericium erinaceus is sweet natured, have relieving the five internal organs, it is aid digestion, nourish the body and other effects.Pharmacological experiment shows that Hericium erinaceus mentions
The different physiological roles such as high immunity, antitumor, anti-aging, reducing blood lipid.Hericium erinaceus early has application in medicine, by Hericium erinaceus
The culture of mycelium submerged fermentation is through being the raw material of the preparations such as Hericium Evimaccus slice, compound Weining tablets obtained by separation and Extraction.Clinic is answered
With showing that Hericium erinaceus can treat the diseases such as indigestion, gastric ulcer, antral gastritis, stomachache, gasteremphraxis and neurasthenia.But current product
Type is on the low side, and technology is not high.
Grifola frondosus (Grifola frondosa (Dieks.) Gray) is a kind of rare edible and medicinal fungi, both nutrition beauty
Drug effect is significant again for taste food, eats and medical value is high.Grifola frondosus is mild-natured, sweet in flavor, has antitumor, anti-radiation, anticoagulation, drop
The effect of blood glucose and reducing blood lipid.The active polysaccharide main chain extracted in grifola frondosus is combined into side chain with β-(1-3) combination, β-(1-6).
D component (average molecular weight 1,000,000) in grifolan is by activating body immune system that can prevent normal cell lesion, suppression
Malignant cell growth and transfer processed；By the synergistic effect with chemicotherapy, therapeutic effect can be significantly improved, reduces the poison of chemicotherapy
Side effect." grifola frondosa beta-glucan for injection " new drug has been made in the high-purity beta glucan extracted in grifola frondosus, and classification is made a living
Tetramune.It is to replenish qi to invigorate the spleen using Effects of Extracts of Grifola frondosa on Active as grifola frondosus capsule (national drug standard B20020023) major function of raw material.
In phase ii clinical trial application of the oral preparation as anti-cancer drugs of U.S. FDA approved grifolan.
Cortex Eucommiae (Eucommia ulmoides Oliv.) is the distinctive rare traditional Chinese medicine in China, has more than 2000 years medicines
Use history.Shennong's Herbal is classified as top grade, has liver-kidney tonifying, strengthening the muscles and bones, blood pressure lowering, miscarriage prevention, long term usage is made light of one's life by commiting suicide resistance to
It waits for a long time effect." Chinese Pharmacopoeia (2015 editions) " have included folium cortex eucommiae and Bark of Eucommia Ulmoides as Chinese medicine, energy nourishing liver and kidney, strengthening the bones and muscles, use
In kidney deficiency and liver, soreness of waist and knee joint, muscles and bones is powerless, has a dizzy spell.Cortex Eucommiae is rich in flavones, chlorogenic acid, polysaccharide isoreactivity ingredient.It is modern
Pharmacological research show Cortex Eucommiae have anti-aging, strengthening the bones and muscles, inhibiting bacteria and diminishing inflammation, reducing blood lipid blood pressure lowering and other effects.In April, 2018 country
The publication of Wei Jianwei food safety department is about soliciting using 9 kinds of substances such as Radix Codonopsis as being both food according to tradition and Chinese medicine substance
The letter (state, which defends, does food letter (2018) 278) for managing opinion, plans folium cortex eucommiae, ganoderma lucidum is included in food and medicine consangunity item categories, this
Undoubtedly the deep processing for being greatly promoted Cortex Eucommiae, ganoderma lucidum and related industry are developed.
Valuable ingredient of traditional Chinese medicine of the Cortex Eucommiae as Chinese tradition, current industry continues to use always traditional processing technology, and (such as water mentions, alcohol
Mention), primary development utilization only is carried out to Cortex Eucommiae raw material, this makes Cortex Eucommiae industry Related product technology content lower, product form
Single, effect owes significant.Ganoderma lucidum, Hericium erinaceus, grifola frondosus belong to saprophytic form higher fungus, and mycelia decomposes lignin, cellulose
It is very competent, can using many vegetable drugs as fermentation substrate carry out solid fermentation.It, can using this biological characteristics
Secondary innovation exploitation is carried out to medicinal materials such as Cortex Eucommiaes taking human as ground.This process belongs to biotransformation, tunning be rich in
The combination object of fungal mycelium and Eucommia ulmoides, not only belongs to biological products, but also is new traditional Chinese medicinal materials assortment, can significantly improve
The drug activity of crude drug (fermentation substrate) expands purposes.From now on deeply with the research of fungi two-way solid fermentation and application
Development, it is possible that medicinal fungal substance is made by fermented tcm material in more fungies, into food, drug, cosmetics, feed
Additive agent field application.
[summary of the invention]
The purpose of the present invention is overcome the deficiencies in the prior art and disadvantage, provide a kind of two-way solid of the fungi based on Cortex Eucommiae
Fermentation technique efficiently quickly, high yield and high quality can carry out bioconversion.
Another object of the present invention is to provide the applications for the medicinal fungal substance that above-mentioned fermentation technique generates.
The present invention is achieved by the following technical solutions:
A kind of fungi two-way solid-fermented technique based on Cortex Eucommiae, it is characterised in that the following steps are included:
(1) fungal bacterial strain is taken, is switched to after 22-26 DEG C of activation culture on Tube propagation base inclined-plane, inclined-plane mother is prepared
Kind；
(2) from picking fungus block access sterilizing in inclined-plane parent species and cooling fluid nutrient medium, 10-20d, preparation solution are cultivated
Body strain；
(3) the Cortex Eucommiae raw material of crushing are weighed, nutrient solution is added and stirs homogeneous, until water content is 50-65%, after sterilizing
Even access liquid spawn is in solid culture primary surface, fermented and cultured, until entire culture medium covers with white hypha；
(4) tunning is collected, it is dry, obtain Cortex Eucommiae medicinal fungal substance.
Fungi strain in the present invention is one or more of ganoderma lucidum, Hericium erinaceus and grifola frondosus.Liquid spawn be ganoderma lucidum,
The mixed bacteria of one or more of Hericium erinaceus and grifola frondosus.
Actication of culture temperature in step (1) is preferably 24-26 DEG C.
Slant medium in the present invention includes component: peeled potatoes 200g, glucose 20g, peptone 2g, (NH4)2SO42g, MgSO4·7H2O 1g, KH2PO41g, agar 20g, add water to be settled to 1000mL, and pH is adjusted to 6.5.
Fluid nutrient medium is loaded on 250ml triangular flask in step (2), and every bottle of charge weight is 80-150mL.The fluid nutrient medium
Formula are as follows: glucose or sucrose 2-5%, bean cake powder 0.5-2%, peptone 0.3-1%, MgSO4·7H2O 0.1%, KH2PO4
0.1%, vitamin B1 is micro, pH 6.0-6.5.
Preferably, fluid nutrient medium is composed of the following components: glucose sugar or sucrose 3-5%, bean cake powder 0.5-1.5%,
Peptone 0.3-0.5%, MgSO4·7H2O 0.1%, KH2PO40.1%, 20 μ g of vitamin B1, pH6.0-6.5, vitamin
B1 dosage is for total volume 1000mL.
Every bottle of fluid nutrient medium dosage in step (2) is preferably 100mL, and preferred 24-26 DEG C of cultivation temperature, 150rpm shakes
Swing culture.
Cortex Eucommiae raw material in the present invention are folium cortex eucommiae and/or Bark of Eucommia Ulmoides.
Nutrient solution in the present invention contains: glucose 15-30g, CaCO3 2-5g、MgSO4·7H2O 0.4-1g、KH2PO4
0.6-1.5g adds water to 1000mL.The ratio of Cortex Eucommiae raw material and nutrient solution is 1:1.5-1.8 (W:V).
The access amount of step (3) liquid spawn of the present invention be 3-8% (W:V), preferably 5%；The liquid spawn of access is
The mixed bacteria of one of ganoderma lucidum, Hericium erinaceus and grifola frondosus strain or several strains.The preferred bacterium solution of the liquid spawn of access is clear
Clear bright, bacterium ball is tiny uniform, and quantity is more, pure white color, the strong high vigor high density liquid strain of suspending power.
Tunning drying in step (4) is forced air drying or vacuum drying, and drying temperature is 50-65 DEG C, dry
To constant weight.
The Cortex Eucommiae medicinal fungal substance that the present invention is fermented, not only belongs to biological products, but also belongs to Chinese medicine new varieties, nutrition, health care
Or drug effect is significant.
A kind of application of the above-mentioned fungi two-way solid-fermented technique based on Cortex Eucommiae, product Cortex Eucommiae medicinal fungal substance can be applied
In functional food, drug, cosmetics or feed addictive.
Compared with prior art, the present invention has the following advantages:
(1) two kinds of medicinal materials of folium cortex eucommiae and/or Bark of Eucommia Ulmoides of the invention are fermentation substrate, are carried out using edible and medicinal fungi two-way
Solid fermentation, fungal mycelium during the growth process, generate the decomposition such as cellulase, protease, nuclease using in matrix
Ingredient carries out structural modification, realizes bioconversion, plays the role of synergy to Cortex Eucommiae crude drug, expansion is used.
(2) present invention adds the nutrient solution containing glucose and inorganic salts in Cortex Eucommiae, eliminates in culture medium using jade
Rice (or corncob), wheat bran, can promote Fungal hyphal growth, significantly improve fermentation efficiency.
(3) what is accessed on solid medium of the present invention is the liquid spawn of the high vigor of high density, and mycelium germination point is more, material feeding
Ability is strong, and aufwuchsplate is big, and growing way is fast.
(4) Cortex Eucommiae medicinal fungal substance of the invention is the organic combination of fungal organism and Traditional Chinese medicine eucommia bark, not edible medicinal
The physical mixed of fungi and Cortex Eucommiae realizes the promotion of effect.This mycoplasma not only belongs to biological products, but also belongs to Chinese medicine new product
Kind.
(5) fermentation technology process of the invention is simple, and condition is easy to control, can high-volume the factorial production.
(6) present invention gained Cortex Eucommiae medicinal fungal substance, it is safe and non-toxic, it can be widely applied to functional food, drug, makeup
The exploitation of product, feed addictive extremely agrees with the demand of big health industry under the new situation.
The present invention relates to HPLC finger-print it is as Figure 1-10 shows, nucleosides HPLC fingerprint chromatogram technology is as follows:
(1) chromatographic condition: chromatographic column is Aglient TC-C18 column (4.6 × 250mm, 5 μm).Mobile phase is water-methanol:
0 → 20min, methanol 5%；20-35min, methanol 5% → 20%；35-36min, methanol 20% → 5%；36-45min, methanol
5%.Flow velocity 1.0mL/min, 10 μ L of sample volume, UV detector, wavelength 259nm, 30 DEG C of column temperature.
(2) standard curve making: four kinds of nucleosides reference substances of precision weighing 5.00mg or so respectively are placed in 25mL volumetric flask
In, add pure water dissolution and constant volume, the nucleosides hybrid standard product mother liquor of about 200 μ g/mL is made.Prepare the mixing of 6 various concentrations
Standard items working solution is tested by above-mentioned chromatographic condition.
(3) sample preparation and analysis: precision weighs each sample 2g, is placed in 50mL colorimetric cylinder, and appropriate 50% ethyl alcohol is added,
After mixing, ultrasonic (500W, 40kHz) extracts 30min.Taking-up is cooled to room temperature, and is settled to 50mL with 50% ethyl alcohol.It takes and mentions in right amount
Liquid 4000r/m is taken to be centrifuged 10min.Supernatant crosses 0.45 μm of miillpore filter, is transferred in sample injection bottle, carries out by given chromatographic condition
HPLC analysis.
[Detailed description of the invention]
Fig. 1 is folium cortex eucommiae (not accessing strain fermentation) HPLC finger-print
Fig. 2 is Bark of Eucommia Ulmoides (not accessing strain fermentation) HPLC finger-print
Fig. 3 is lucid ganoderma-eucommia bark leaf medicinal fungal substance HPLC finger-print；
Fig. 4 is lucid ganoderma-eucommia bark skin medicinal fungal substance HPLC finger-print；
Fig. 5 is Hericium erinaceus folium cortex eucommiae medicinal fungal substance HPLC finger-print；
Fig. 6 is Hericium erinaceus Bark of Eucommia Ulmoides medicinal fungal substance HPLC finger-print；
Fig. 7 is grifola frondosus folium cortex eucommiae medicinal fungal substance HPLC finger-print；
Fig. 8 is grifola frondosus Bark of Eucommia Ulmoides medicinal fungal substance HPLC finger-print；
Fig. 9 is ganoderma lucidum-grifola frondosus folium cortex eucommiae medicinal fungal substance HPLC finger-print；
Figure 10 is ganoderma lucidum-grifola frondosus Bark of Eucommia Ulmoides medicinal fungal substance HPLC finger-print.
[specific embodiment]
The present invention is more specifically described in detail with attached drawing combined with specific embodiments below, but embodiment party of the invention
Formula is without being limited thereto, for not specifically specified technological parameter, can refer to routine techniques progress.
A kind of fungi two-way solid-fermented technique based on Cortex Eucommiae, it is characterised in that the following steps are included:
(1) ganoderma lucidum, Hericium erinaceus and/or Grifola frondosa strain are taken, Tube propagation is switched to after 22-26 DEG C of activation culture
On base inclined-plane, grow up to the fine and close stalwartness of mycelia, the pure white inclined-plane parent species of color are spare；
(2) choose 250mL triangular flask, per it is bottled enter 80-150mL fluid nutrient medium, sterilizing, from picking number in inclined-plane parent species
Ferfas block is respectively connected to sterilizing and in cooling fluid nutrient medium, and 22-26 DEG C, 150rpm shaken cultivation 10-20d prepare liquid
Strain；
(3) the Cortex Eucommiae raw material for weighing crushing, in Cortex Eucommiae: nutrient solution is added in the ratio of nutrient solution=1:1.5-1.8 (W:V)
Homogeneous is stirred, the culture medium that water content is 50-65% is made, liquid spawn 3-8% (W:V) is uniformly accessed after sterilizing and is trained in solid
Primary surface is supported, 22-26 DEG C is protected from light fermented and cultured, until entire culture medium covers with white hypha；
(4) tunning is collected, it is dry, obtain Cortex Eucommiae medicinal fungal substance；
Wherein inclined-plane culture based formulas are as follows: potato (peeling) 200g, glucose 20g, peptone 2g, (NH4)2SO42g,
MgSO4·7H2O 1g, KH2PO41g, agar 20g, add distilled water to be settled to 1000mL, and pH is adjusted to 6.5；
Wherein fluid nutrient medium are as follows: glucose or sucrose 2-5%, bean cake powder 0.5-2%, peptone 0.3-1%,
MgSO4·7H2O 0.1%, KH2PO40.1%, vitamin B1 is micro, pH6.0-6.5；
Wherein nutrient solution prescription are as follows: glucose 15-30g, CaCO3 2-5g、MgSO4·7H2O0.4-1g、KH2PO4 0.6-
1.5g adds water to 1000mL.
Embodiment 1:
A kind of fungi two-way solid-fermented technique and application thereof based on Cortex Eucommiae, steps are as follows:
(1) ganoderma lucidum is taken to save strain (purchased from Guangdong Province's Culture Collection, strain number GDMCC
5.250) it, is forwarded on the inclined-plane test tube PDA after 26 DEG C of activation cultures, grows up to the fine and close stalwartness of mycelia, the pure white inclined-plane parent species of color are standby
With；
(2) select 250mL triangular flask, per it is bottled enter 100mL fluid nutrient medium, 115 DEG C of sterilizing 20min, from inclined-plane kind
The ganoderma lucidum fungus block access of picking several piece sterilizes and in cooling liquid strain culture medium, 26 DEG C, 150rpm shaken cultivation 12d, preparation
Liquid spawn；
Wherein Liquid Culture based formulas are as follows: glucose 3%, bean cake powder 1%, peptone 0.4%, MgSO4·7H2O
0.1%, KH2PO40.1%, vitamin B1 is micro, pH 6.5；
(3) recipe configuration nutrient solution: glucose 25g, CaCO is pressed3 3.5g、MgSO4·7H2O 0.6g、KH2PO41.2g
Add water to 1000mL；
(4) take folium cortex eucommiae coarse crushing powder in triangular flask, per it is bottled enter 50g, be added 75mL nutrient solution, water content 60%,
It mixes well, is sealed using ventable envelope bottle film, 121 DEG C of sterilizing 30min cool down to room temperature, and it is big to select Peloton density, greatly
Small uniform, not easily settled ganoderma lucidum seed liquor, it is accurate after shaking up to draw 5mL, uniformly it is inoculated in solid culture primary surface, temperature 26
DEG C, culture is until cover with mycelia in the artificial climate incubator of relative humidity 80%；
(5) it after cultivating 52d, collects solid fermentation product and pulverizes and sieves after 55 DEG C of forced air dryings, the fermented product of acquisition
Referred to as lucid ganoderma-eucommia bark leaf medicinal fungal substance.
The lucid ganoderma-eucommia bark leaf drug effect mycoplasma has anti-aging, anti-oxidant, immunological regulation, antitumor, reducing blood lipid, tonifying liver
Kidney, strengthening the bones and muscles and other effects.
Embodiment 2:
(1) ganoderma lucidum is taken to save strain (purchased from Guangdong Province's Culture Collection, strain number GDMCC
5.250) it, is forwarded on the inclined-plane test tube PDA after 26 DEG C of activation cultures, grows up to the fine and close stalwartness of mycelia, the pure white inclined-plane parent species of color are standby
With；
(2) select 250mL triangular flask, per it is bottled enter 100mL fluid nutrient medium, 115 DEG C of sterilizing 20min, from inclined-plane kind
The ganoderma lucidum fungus block access of picking several piece sterilizes and in cooling liquid strain culture medium, 26 DEG C, 150rpm shaken cultivation 10d, preparation
Liquid spawn；
Liquid Culture based formulas are as follows: glucose 3%, bean cake powder 2%, peptone 0.3%, MgSO4·7H2O 0.1%,
KH2PO40.1%, vitamin B1 is micro, pH 6.5；
(3) nutrient solution is configured according to the following formulation: glucose 15g, CaCO3 2g、MgSO4·7H2O 0.4g、KH2PO4
0.8g adds water to 1000mL；
(4) Bark of Eucommia Ulmoides coarse crushing powder 50g is weighed in triangular flask, 85mL nutrient solution is added, and water content 63% is sufficiently mixed
It is even, it is sealed using ventable envelope bottle film, 121 DEG C of sterilizing 30min cool down to room temperature, select that Peloton density is big, and size is equal
One, not easily settled ganoderma lucidum seed liquor is accurate after shaking up to draw 4mL, is uniformly inoculated in solid culture primary surface, and 26 DEG C of temperature,
It is cultivated in the artificial climate incubator of relative humidity 80%, until covering with mycelia；
(5) it cultivates 45 days, collects solid fermentation product, 55 DEG C of forced air dryings pulverize and sieve, and the fermented product of acquisition is known as
Lucid ganoderma-eucommia bark skin medicinal fungal substance.
The lucid ganoderma-eucommia bark skin drug effect mycoplasma has immunological regulation, anti-aging, antitumor, nourishing liver and kidney, strengthening the bones and muscles and other effects.
Embodiment 3:
(1) Hericium erinaceus is taken to save strain (purchased from Guangdong Province's Culture Collection, strain number GDMCC
5.66) it, is forwarded on the inclined-plane test tube PDA after 24 DEG C of activation cultures, grows up to the fine and close stalwartness of mycelia, the pure white inclined-plane parent species of color are standby
With；
(2) select 250mL triangular flask, per it is bottled enter 100mL fluid nutrient medium, 115 DEG C of sterilizing 20min, from inclined-plane kind
The Hericium erinaceus fungus block access of picking several piece sterilizes and in cooling liquid strain culture medium, 24 DEG C, 150rpm shaken cultivation 15d, system
Standby liquid spawn；
Liquid Culture based formulas are as follows: glucose 3%, bean cake powder 1.5%, peptone 0.5%, MgSO4·7H2O 0.1%,
KH2PO40.1%, vitamin B1 is micro, pH 6.5.
(3) nutrient solution is configured according to the following formulation: glucose 25g, CaCO3 5g、MgSO4·7H2O 0.8g、KH2PO4
1.0g, 1000mL is added water to；
(4) take folium cortex eucommiae coarse crushing powder in triangular flask, per it is bottled enter 50g, be added 75mL nutrient solution, water content 60%.
It mixes well, is sealed using ventable envelope bottle film, 121 DEG C of sterilizing 30min cool down to room temperature, and it is big to select Peloton density, greatly
Small uniform, not easily settled Hericium erinaceus seed liquor, it is accurate after shaking up to draw 5mL, uniformly it is inoculated in solid culture primary surface, temperature
It 24 DEG C, cultivates in the artificial climate incubator of relative humidity 80%, until covering with mycelia；
(5) after cultivating 85 days, solid fermentation product is collected, vacuum drying pulverizes and sieves, the fermented product of acquisition is known as monkey
Head bacterium folium cortex eucommiae medicinal fungal substance.
The Hericium erinaceus folium cortex eucommiae medicinal fungal substance has improvement functions of intestines and stomach, alleviates disease of digestive tract, improves immune, antioxygen
Change, delay senescence, enhancing brain function, nourishing liver and kidney, strengthening the bones and muscles and other effects.
Embodiment 4:
(1) grifola frondosus is taken to save strain (purchased from Guangdong Province's Culture Collection, strain number GDMCC
5.63) it, is forwarded on the inclined-plane test tube PDA after 24 DEG C of activation cultures, grows up to the fine and close stalwartness of mycelia, the pure white inclined-plane parent species of color are standby
With；
(2) select 250mL triangular flask, per it is bottled enter 100mL fluid nutrient medium, 115 DEG C of sterilizing 20min, from inclined-plane kind
The grifola frondosus fungus block access of picking several piece sterilizes and in cooling liquid strain culture medium, 24 DEG C, 150rpm shaken cultivation 18d, system
Standby liquid spawn；
Liquid Culture based formulas are as follows: glucose 4%, bean cake powder 1%, peptone 0.5%, MgSO4·7H2O 0.1%,
KH2PO4 0.1%, vitamin B1 are micro, pH 6.5；
(3) nutrient solution is configured according to the following formulation: glucose 25g, CaCO3 4g、MgSO4·7H2O 0.8g、KH2PO4
1.2g adds water to 1000mL；
(4) it takes Bark of Eucommia Ulmoides coarse crushing powder in triangular flask, per bottled 50g, 85mL nutrient solution is added, water content 65% is filled
It point mixes thoroughly, is sealed using ventable envelope bottle film, 121 DEG C of sterilizing 30min cool down to room temperature, and it is big to select Peloton density, size
Uniform, not easily settled grifola frondosus seed liquor, it is accurate after shaking up to draw 4mL, uniformly it is inoculated in solid culture primary surface, temperature 24
DEG C, it is cultivated in the artificial climate incubator of relative humidity 80%, until covering with mycelia；
(5) it cultivates 105 days, collects solid fermentation product, 50-60 DEG C of vacuum drying pulverizes and sieves, the fermented product of acquisition
Referred to as grifola frondosus Bark of Eucommia Ulmoides medicinal fungal substance.
The grifola frondosus Bark of Eucommia Ulmoides medicinal fungal substance has hypoglycemic, immunological regulation, antitumor, strengthening spleen, tonifying kidney, strengthening the bones and muscles etc.
Effect.
Embodiment 5:
(1) ganoderma lucidum, grifola frondosus is taken to save strain (purchased from Guangdong Province's Culture Collection, strain number GDMCC
5.250, GDMCC 5.63) it, is forwarded on the inclined-plane test tube PDA after 24 DEG C of activation cultures, grows up to the fine and close stalwartness of mycelia, color is pure white
Inclined-plane parent species, it is spare；
(2) select 250mL triangular flask, per it is bottled enter 100mL fluid nutrient medium, 115 DEG C of sterilizing 20min, from inclined-plane kind
The ganoderma lucidum of picking several piece, grifola frondosus fungus block respectively access sterilizing and in cooling liquid strain culture mediums, 24 DEG C, 150rpm oscillation training
20d is supported, single liquid strain is prepared；
Liquid Culture based formulas are as follows: glucose 5%, bean cake powder 1%, peptone 0.4%, MgSO4·7H2O 0.1%,
KH2PO4 0.1%, vitamin B1 are micro, pH 6.5.
(3) nutrient solution is configured according to the following formulation: glucose 25g, CaCO3 3.5g、MgSO4·7H2O 1g、KH2PO4
1.2g adds water to 1000mL；
(4) it takes Bark of Eucommia Ulmoides coarse crushing powder in triangular flask, per bottled 50g, 75mL nutrient solution is added, water content 60% is filled
It point mixes thoroughly, is sealed using ventable envelope bottle film, 121 DEG C of sterilizing 30min cool down to room temperature, and it is big to select Peloton density, size
Uniform, not easily settled ganoderma lucidum, grifola frondosus seed liquor respectively access 3mL, are uniformly inoculated in solid culture primary surface, and 25 DEG C of temperature,
It is cultivated in the artificial climate incubator of relative humidity 80%, until covering with mycelia.
(5) after cultivating 65 days, solid fermentation product is collected, vacuum drying pulverizes and sieves, and the fermented product of acquisition is known as spirit
Sesame grifola frondosus Bark of Eucommia Ulmoides medicinal fungal substance.
The ganoderma lucidum grifola frondosus Bark of Eucommia Ulmoides medicinal fungal substance have immunological regulation, hypoglycemic, anti-aging, antitumor, anti-inflammatory,
Strengthening spleen, tonifying kidney, strengthening the muscles and bones and other effects.
Embodiment 6:
(1) ganoderma lucidum, Hericium erinaceus Strains kind are taken, is forwarded to after 22 DEG C of activation cultures on the inclined-plane test tube PDA, mycelia densification is grown up to
Stalwartness, the pure white inclined-plane parent species of color are spare；
(2) select 250mL triangular flask, per it is bottled enter 80mL fluid nutrient medium, 115 DEG C of sterilizing 20min choose from inclined-plane kind
The ganoderma lucidum of block, the Hericium erinaceus fungus block of fetching respectively access sterilizing and in cooling liquid strain culture mediums, 22 DEG C, 150rpm shaken cultivation
21d prepares liquid spawn；
Liquid Culture based formulas are as follows: glucose 2%, bean cake powder 0.5%, peptone 0.3%, MgSO4·7H2O 0.1%,
KH2PO4 0.1%, vitamin B1 are micro, pH 6.0；
(3) nutrient solution is configured according to the following formulation: glucose 30g, CaCO3 4g、MgSO4·7H2O 0.8g、KH2PO4
1.5g adds water to 1000mL；
(4) it takes Bark of Eucommia Ulmoides coarse crushing powder in triangular flask, per bottled 50g, 90mL nutrient solution is added, water content 50% is filled
It point mixes thoroughly, is sealed using ventable envelope bottle film, 121 DEG C of sterilizing 30min cool down to room temperature, and it is big to select Peloton density, size
Uniform, not easily settled ganoderma lucidum, Hericium erinaceus seed liquor are respectively connected to 3mL, are uniformly inoculated in solid culture primary surface, temperature 25
DEG C, it is cultivated in the artificial climate incubator of relative humidity 80%, until covering with mycelia；
(5) after cultivating 65 days, solid fermentation product is collected, vacuum drying pulverizes and sieves, and the fermented product of acquisition is known as spirit
Sesame Hericium erinaceus Bark of Eucommia Ulmoides medicinal fungal substance.
The glossy ganoderma and hericium bacterium Bark of Eucommia Ulmoides medicinal fungal substance have immunological regulation, hypoglycemic, anti-aging, antitumor, anti-inflammatory,
Strengthening spleen, tonifying kidney, strengthening the muscles and bones and other effects.
Embodiment 7:
(1) ganoderma lucidum, Hericium erinaceus, Grifola frondosa strain kind are taken, is forwarded on the inclined-plane test tube PDA, grows up to after 25 DEG C of activation cultures
Mycelia is fine and close healthy and strong, and the pure white inclined-plane parent species of color are spare；
(2) select 250mL triangular flask, per it is bottled enter 150mL fluid nutrient medium, 115 DEG C of sterilizing 20min, from inclined-plane kind
Ganoderma lucidum, Hericium erinaceus and the grifola frondosus fungus block of picking several piece respectively access sterilizing and in cooling liquid strain culture mediums, 26 DEG C,
150rpm shaken cultivation 10d prepares liquid spawn；
Liquid Culture based formulas are as follows: sucrose 2%, bean cake powder 0.5%, peptone 1%, MgSO4·7H2O 0.1%,
KH2PO4 0.1%, vitamin B1 are micro, pH 6.0；
(3) 1 liter of nutrient solution: glucose 20g, CaCO is configured according to the following formulation3 3.5g、MgSO4·7H2O 0.4g、KH2PO4
1.2g adds water to 1000mL；
(4) it takes Bark of Eucommia Ulmoides coarse crushing powder in triangular flask, per bottled 50g, 80mL nutrient solution is added, until water content is 65%,
It mixes well, is sealed using ventable envelope bottle film, 121 DEG C of sterilizing 30min cool down to room temperature, and it is big to select Peloton density, greatly
Small uniform, not easily settled ganoderma lucidum, Hericium erinaceus and grifola frondosus seed liquor, is respectively connected to 3mL, is uniformly inoculated in solid culture base table
Face 25 DEG C of temperature, is cultivated in the artificial climate incubator of relative humidity 80%, until covering with mycelia；
(5) after cultivating 65 days, solid fermentation product is collected, vacuum drying pulverizes and sieves, and the fermented product of acquisition is known as spirit
Sesame Hericium erinaceus Bark of Eucommia Ulmoides medicinal fungal substance.
The made glossy ganoderma and hericium bacterium grifola frondosus Bark of Eucommia Ulmoides medicinal fungal substance of example example 7 have immunological regulation, hypoglycemic, anti-aging,
Antitumor, anti-inflammatory, strengthening spleen, tonifying kidney, strengthening the muscles and bones and other effects.Enhance Evaluation of Immunity test
1 materials and methods
1.1 material
1.1.1 sample treatment
Examples 1 to 7 Cortex Eucommiae Zymocyte and Bark of Eucommia Ulmoides, folium cortex eucommiae crude drug (not fermented) are crushed respectively, cross 100
Mesh, using 50% ethyl alcohol as solvent, solid-liquid ratio 1:20, is extracted twice by 80 DEG C, and combined extract is concentrated under reduced pressure, and 50 DEG C of vacuum are dry
It is dry, obtain embodiment 1-7 extract and Cortex Eucommiae extract, eucommia leaf extract.
1.1.2 experimental animal
SPF grades of BALB/C male mices, 6-8 week old, weight 18-22g are purchased from Guangdong Medical Lab Animal Center, perhaps
It can the number of card: SYXK (Guangdong 2012-0122).Mouse raising is in standard experimental animal room, and 21-23 DEG C of temperature, relative humidity 50-
60%.
1.2 test method
1.2.1 method foundation: " strengthen immunity function evaluation methods " (food medicine supervises guarantorization letter [2012] 470).Specifically
Test operation is carried out according to prescribed requirement.
1.2.2 dosage: dosage is set as 500mg/kg, and is according to dosage dissolved and prepared with distilled water, fills before stomach-filling
Divide and shakes up.Each sample sets a test administration group and 1 negative control group (stomach-filling equivalent solvent).Mouse adapts to after feeding 5d,
Random grouping, every group 10, (extract and Cortex Eucommiae crude drug including embodiment 1-7 extract daily morning stomach-filling tested material
Object).Every 3d weighs a weight, adjusts stomach-filling amount accordingly.Given the test agent continuous gavage 30 days.When off-test, by experiment item
Mesh carries out every immune function measurement.
1.2.3 experimental project
(1) cellular immune function: delayed hair allergy experiment
(2) humoral immune function: antibody-producting cell detection
(3) NK cytoactive detection
1.3 data processing
All data are indicated with mean+SD, are handled using SPSS19.0 statistical software.
2 test results are as shown in table 1.
1 mouse experiment immune function of table, humoral immune function and NK cytoactive detection result
Note: compared with negative control group, P < 0.01 * P < 0.05, * *
As shown in Table 1, compared with negative control group, mentioning for the resulting Cortex Eucommiae Zymocyte of 1-7 of the embodiment of the present invention is given
After taking object 30 days, mouse delayed allergy, antibody tormation, NK cell activity are significantly increased, and difference has statistical significance
(P < 0.01 or P < 0.05)；And the eucommia leaf extract group mouse without the fermentation of ganoderma lucidum, grifola frondosus or Hericium erinaceus is given, delayed
Allergy and antibody tormation increase not significant (P > 0.05), similarly for the NK cell activity liter of Cortex Eucommiae extract group mouse
High not significant (P > 0.05).
Above-mentioned test result shows to ferment Cortex Eucommiae medicinal fungal substance obtained with significant increasing through ganoderma lucidum, grifola frondosus, Hericium erinaceus
Immune function by force, and opposite Cortex Eucommiae crude drug, immune function of mice are obviously improved.Pass through fermentation work of the invention
The immune function of skill, folium cortex eucommiae and Bark of Eucommia Ulmoides further enhances, and potential drug effect is easier to realize.
Cortex Eucommiae medicinal fungal substance under present invention process is applied to functional food food or health care product, cosmetics and feed add
Add in agent, effectiveness is better than simple addition Cortex Eucommiae crude drug, and Cortex Eucommiae medicinal fungal substance produced by the present invention is equally applicable for related disease
Preventative and treatment drug the exploitation of disease.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of fungi two-way solid-fermented technique based on Cortex Eucommiae, it is characterised in that the following steps are included:
(1) fungal bacterial strain is taken, is switched to after 22-26 DEG C of activation culture on Tube propagation base inclined-plane, prepares inclined-plane parent species；
(2) from picking fungus block access sterilizing in inclined-plane parent species and cooling fluid nutrient medium, 10-20d is cultivated, liquid bacteria is prepared
Kind；
(3) the Cortex Eucommiae raw material of crushing are weighed, nutrient solution is added and stirs homogeneous, makes water content 50-65%, is uniformly connect after sterilizing
Enter liquid spawn in solid culture primary surface, fermented and cultured, until entire culture medium covers with white hypha；
(4) tunning is collected, it is dry, obtain Cortex Eucommiae medicinal fungal substance.
2. a kind of fungi two-way solid-fermented technique based on Cortex Eucommiae according to claim 1, it is characterised in that described
Fungi strain is one or more of ganoderma lucidum, Hericium erinaceus and grifola frondosus.
3. a kind of fungi two-way solid-fermented technique based on Cortex Eucommiae according to claim 1, it is characterised in that described
Liquid spawn is one of ganoderma lucidum, Hericium erinaceus and grifola frondosus strain or two or more mixed bacterias.
4. a kind of fungi two-way solid-fermented technique based on Cortex Eucommiae according to claim 1, it is characterised in that the step
Suddenly the fluid nutrient medium in (2) are as follows: glucose or sucrose 2-5%, bean cake powder 0.5-2%, peptone 0.3-1%, MgSO4·
7H2O 0.1%, KH2PO40.1%, vitamin B1 is micro, pH 6.0-6.5.
5. a kind of fungi two-way solid-fermented technique based on Cortex Eucommiae according to claim 1, it is characterised in that described
Cortex Eucommiae raw material are folium cortex eucommiae or Bark of Eucommia Ulmoides.
6. a kind of fungi two-way solid-fermented technique based on Cortex Eucommiae according to claim 1, it is characterised in that described
Nutrient solution contains: glucose 15-30g, CaCO3 2-5g、MgSO4·7H2O 0.4-1g、KH2PO40.6-1.5g is added water to
1000mL。
7. a kind of fungi two-way solid-fermented technique based on Cortex Eucommiae according to claim 1, it is characterised in that Du
The ratio of secondary raw material and nutrient solution is 1:1.5-1.8 (W:V).
8. a kind of fungi two-way solid-fermented technique based on Cortex Eucommiae according to claim 1, it is characterised in that the step
Suddenly the access amount of (3) liquid spawn is 3-8%.
9. a kind of fungi two-way solid-fermented technique based on Cortex Eucommiae according to claim 1, it is characterised in that the step
Suddenly it is forced air drying or vacuum drying that the tunning in (4) is dry, and drying temperature is 50-65 DEG C, until constant weight.
10. a kind of application of fungi two-way solid-fermented technique of any one of the claim 1-9 based on Cortex Eucommiae, it is characterised in that
Obtained Cortex Eucommiae medicinal fungal substance is applied to the functional additive of functional food, drug, cosmetics or feed addictive.
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| CN115886152A (en) * | 2022-11-03 | 2023-04-04 | 中国海洋大学 | Novel eucommia ulmoides fermentation liquor and application thereof in large yellow croaker culture |
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