CN105483160A - Antrodia camphorata cultivating composition and preparation method thereof - Google Patents

Antrodia camphorata cultivating composition and preparation method thereof Download PDF

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CN105483160A
CN105483160A CN201511027618.8A CN201511027618A CN105483160A CN 105483160 A CN105483160 A CN 105483160A CN 201511027618 A CN201511027618 A CN 201511027618A CN 105483160 A CN105483160 A CN 105483160A
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liquid
antrodia camphorata
thalline
fermentation
streptomycete
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程俊文
魏海龙
胡传久
贺亮
李海波
付立忠
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Zhejiang Academy of Forestry
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Abstract

The invention discloses an antrodia camphorata cultivating composition and a preparation method thereof. The composition is prepared from an antrodia camphorata fermentation subcritical extract I and a streptomycete fermentation product III and has the good liver protecting function. The preparation method comprises the steps that an antrodia camphorata liquid seed solution is inoculated into an antrodia camphorata solid-state fermentation culture medium containing anoectochilus formosanus and gynura divaricata to be cultured, the culture medium is placed in an alternating magnetic field environment to be cultured again, an antrodia camphorata solid-state fermentation product is obtained, subcritical water extraction is performed, and then the antrodia camphorata fermentation subcritical extract I is obtained; streptomycete seed liquid is inoculated into a streptomycete liquid fermentation culture medium containing extracting residues II to be cultured, and the streptomycete fermentation product III is obtained. According to the method, the nutritional components and effective components in all the raw materials can be effectively biodegraded and converted, and increasing of the content and improving of the activity of the active components in the final product are facilitated.

Description

A kind of Antrodia camphorata cultivates composition and method of making the same
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of Antrodia camphorata and cultivate composition and method of making the same.
Background technology
Antrodia camphorata (Antrodiacamphorata) has another name called Cinnamomum kanahirai hay mushroom, belong to Aphyllophorales, polyporaceae, Antrodia genus, perennial gill fungus mushroom, be parasitize the rare medicinal fungi that China's Taiwan endemic tree Cinnamomum kanahirai hay sets rotten inwall, be called as the ruby of forest.Have detoxify and promote the subsdence of swelling, sedation-analgesia, antibacterial, antiviral, antitumor, promote the effect of immunity of organisms; Unique function is had especially for treatment gastrointestinal pain, diarrhea and vomiting, food poisoning, mushroom poisoning, diabetes, alcoholic liver, fatty liver, liver cirrhosis, liver cancer etc.Antrodia camphorata contains many physiologically active ingredients, as polysaccharide, triterpene compound, sudismase, adenosine, protein (containing immune protein), VITAMIN, trace element, nucleic acid, lectin, amino acid, blood pressure stabilization material etc.The solid state fermentation ubiquity fermentation period of current Antrodia camphorata routine is long, easy microbiological contamination, the problems such as fermenting characteristic product assay is low.
Nowadays, in order to obtain the Antrodia camphorata product that nutrition characteristic is different and/or purposes is different, people start to improve the solid state fermentation of routine or innovate, to obtaining the Antrodia camphorata product of Different Nutrition characteristic and/or different purposes.Such as: Chinese patent application CN201510025535.9 discloses a kind of Antrodia camphorata fermented product and preparation method thereof.The method comprises the steps: 1) Antrodia camphorata is soaked in water extracts, crushing and beating, gets slurries; 2) after adding medium component in slurries, access Leuconostoc mesenteroides carries out the first fermentation, obtained first fermented liquid; 3) to pH value be 4.5-6 fruits and vegetables liquid in add polygalacturonase and carry out enzymolysis processing, obtained enzymolysis fruits and vegetables liquid; 4) after adding medium component in enzymolysis fruits and vegetables liquid, access compound lactobacillus carries out the second fermentation, obtained second fermented liquid; 5) after adding medium component in the second fermented liquid, access mulriple yeasts carries out the 3rd fermentation, obtained 3rd fermented liquid; 6) by centrifugal after the first fermented liquid and the mixing of the 3rd fermented liquid, after centrifuged supernatant homogeneous, sterilizing, obtained Antrodia camphorata fermented product.This fermented product mouthfeel is good, unique flavor, has good immunomodulatory and the function such as anti-oxidant, is suitable for crowd in extensive range.
Chinese patent application CN201410791469.1 discloses a kind of Antrodia camphorata submerged fermentation compound product and preparation method thereof, with preparation submerged fermentation culture mediums such as Poria powder, Rhizoma Dioscoreae powder, powder of Radix Puerariae, bee pollen, soyflour, yeast powder, glucose, dipotassium hydrogen phosphate, magnesium sulfate, the Antrodia camphorata mycelium fermentation broth of Antrodia camphorata polysaccharide is rich in, with employing supercritical CO through Submerged fermentation 2technology extraction, enzymolysis, separation, filtration, the concentrated obtained extracts of Chinese herbal medicine such as Buddha's hand, sealwort, matrimony vine, fructus amomi, phyllanthus emblica, trifoliate orange seed are formulated; It optimizes the content of Antrodia camphorata polysaccharide isoreactivity composition, and output is high, the time is short, cost is low, can suitability for industrialized production, is a kind of biotechnology production technology of high-efficiency environment friendly.
Chinese patent application CN201410850342.2 discloses a kind of method improving Antrodia camphorata polysaccharide anti-oxidative activity, for solving the active low defect of polysaccharide anti-oxidative in current Antrodia camphorata.Mainly utilize the polysaccharide in water seaoning extraction Antrodia camphorata mycelium.
If develop new Antrodia camphorata product and preparation method thereof, range of application and the using value of Antrodia camphorata can be improved.
Summary of the invention
The invention provides a kind of Antrodia camphorata and cultivate composition, in said composition the content of activeconstituents and activity high, there is good liver-protecting function.
Present invention also offers the preparation method that a kind of Antrodia camphorata cultivates composition, utilize multiple fungi fermentation Herba Anoectochili roxburghii and angelica keiskei koidz, contribute to the content and the activity that improve activeconstituents in the finished product, obtain the composition with liver-protecting function.
The present invention finds, according to the fermentation character of Antrodia camphorata, by the specific fungi fermentation of the present invention and specific technique, effectively the nutritive ingredient in angelica keiskei koidz and Herba Anoectochili roxburghii and effective constituent Biodegradation and biotransformation be can be carried out, the content and the activity that improve activeconstituents in the finished product contributed to.
The technical solution used in the present invention is:
Antrodia camphorata cultivates a preparation method for composition, comprises step:
(1) get in the Antrodia camphorata strain inoculation after activation to liquid MRS substratum and cultivate 2 days-8 days, collected by centrifugation first thalline; First thalline is suspended in the liquid MRS substratum containing cholate and hatches 1h-20h in 18 DEG C-30 DEG C, collected by centrifugation second thalline; By the second thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the second thalline liquid, second thalline liquid is inoculated in the liquid MRS substratum containing cholate and cultivates 1 day-10 days again in 18 DEG C-30 DEG C, obtain Antrodia camphorata liquid seeds liquid;
(2) streptomyces species got after activation is inoculated in liquid MRS substratum and cultivates 3h-30h, collected by centrifugation the 3rd thalline, is suspended in by the 3rd thalline in the liquid MRS substratum containing NaCl and hatches 0.5h-15h in 20 DEG C-30 DEG C, collected by centrifugation the 4th thalline; By the 4th thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the 4th thalline liquid, 4th thalline liquid is inoculated in the liquid MRS substratum containing NaCl and cultivates 3h-30h in 20 DEG C-30 DEG C, obtain streptomycete seed liquor;
(3) by the Antrodia camphorata liquid seeds liquid of step (1) access Antrodia camphorata solid-state fermentation culture medium, cultivate 3 days-30 days at 16 DEG C-30 DEG C, then be placed in alternating magnetic field environment and cultivate 5 days-30 days again, cultured products obtains Antrodia camphorata product by solid-state fermentation after vacuum lyophilization, pulverizing; Containing Herba Anoectochili roxburghii and angelica keiskei koidz in described Antrodia camphorata solid-state fermentation culture medium;
(4) by the Antrodia camphorata product by solid-state fermentation of step (3) through Subcritical Water Extraction, obtain Antrodia camphorata fermentation subcritical abstraction thing I, after extraction, remaining residue is extracted residuum II after vacuum lyophilization;
(5) by the streptomycete seed liquor of step (2) access streptomycete liquid fermentation medium, 10h-120h is cultivated at 20 DEG C-30 DEG C, centrifugal, obtain streptomycete fermentation product III, containing extraction residuum II in described streptomycete liquid fermentation medium;
It is Antrodia camphorata fermentation subcritical abstraction thing I and streptomycete fermentation product III that described Antrodia camphorata cultivates composition.
When the present invention carries out Antrodia camphorata solid state fermentation, the direct Antrodia camphorata liquid seeds liquid adopted through hatching process containing liquid MRS substratum two step of cholate, and Herba Anoectochili roxburghii and angelica keiskei koidz is added in substratum, effectively its nutritive ingredient and effective constituent Biodegradation and biotransformation be can be carried out, the content and the activity that improve activeconstituents in the finished product contributed to.In step (4) and step (5), Antrodia camphorata product by solid-state fermentation is carried out Subcritical Water Extraction, make that the activity of effective constituent in extract is at utmost retained, content is greatly improved; Again using extraction after residue as streptomycete liquid fermenting culture medium mainly, and adopt the streptomycete seed liquor through hatching process containing liquid MRS substratum two step of NaCl, by integrated recycle, each raw material resources are fully utilized, further increase content and the activity of activeconstituents in the finished product.
In order to reach better effect, preferably:
In step (1), described is 0.05%-0.5% containing the mass percentage of cholate in the liquid MRS substratum of cholate.
Described first thalline is 1:1-3 with the volume ratio of the liquid MRS substratum containing cholate for first thalline that suspends, and the second thalline is 1:1-3 with the volume ratio for the liquid MRS substratum of second thalline that suspends, and the inoculum size of the second thalline liquid is 5%.
In step (2), the mass percentage of the described liquid MRS NaCl in medium containing NaCl is 0.5%-8%.
Described 3rd thalline is 1:1-3 with the volume ratio of the liquid MRS substratum containing NaCl for the 3rd thalline that suspends, and the 4th thalline is 1:1-3 with the volume ratio for the liquid MRS substratum of the 4th thalline that suspends, and the inoculum size of the 4th thalline liquid is 3%.
In step (3), containing 4.5g-8.0g Herba Anoectochili roxburghii and 0.3g-3.0g angelica keiskei koidz in the every 1L of described Antrodia camphorata solid-state fermentation culture medium.Described Antrodia camphorata solid-state fermentation culture medium is made up of Herba Anoectochili roxburghii, angelica keiskei koidz and fermentation basic medium, and described fermentation basic medium adopts the fermentation basic medium of this area routine, can adopt commercially available prod, and existing compound method also can be adopted to prepare.Described Herba Anoectochili roxburghii adopts Herba Anoectochili roxburghii herb.Described angelica keiskei koidz adopts angelica keiskei koidz herb.
The preparation method of described Antrodia camphorata solid-state fermentation culture medium, comprising: fresh Herba Anoectochili roxburghii herb, angelica keiskei koidz herb are rinsed well under tap water, dry, chopping; Again 4.5g-8.0g Herba Anoectochili roxburghii and 0.3g-3.0g angelica keiskei koidz fermentation basic medium are settled to 1L, mix, pH value nature, obtains Antrodia camphorata solid-state fermentation culture medium.
The access amount of described Antrodia camphorata liquid seeds liquid is the 5%-25% of Antrodia camphorata solid-state fermentation culture medium volume.
Current fungi solid-state fermentation technology ubiquity fermentation period is long, mycelial growth sprouts the problems such as slow, feature secondary metabolism metabolite content is low.The present invention finds alternating magnetic field treatment technology to be applied in solid state fermentation, by extraneous factor effective stimuluss such as the suitable process of specific cultivation period, improve the growth metabolism of hypha,hyphae on Herba Anoectochili roxburghii and angelica keiskei koidz culture base-material, shorten fermentation time, effectively promote the generation of secondary metabolite.In described alternating magnetic field environment, magneticstrength is 0.1mT-5mT, and field frequency is 2Hz-40Hz.
In step (4), the condition of described Subcritical Water Extraction is: water material mass ratio is 10-50:1, and extracting pressure is at 4MPa-25MPa, and extraction temperature is 100 DEG C-180 DEG C, and extraction time is 10min-90min.
In step (5), in the every 1L of described streptomycete liquid fermentation medium, extract residuum II containing 0.2g-1g.Described streptomycete liquid fermentation medium forms with fermentation basic medium by extracting residuum II, and described fermentation basic medium adopts the fermentation basic medium of this area routine, can adopt commercially available prod, and existing compound method also can be adopted to prepare.
General fermentation basic medium is made up of the component of following mass percent: glucose 1%-3%, K 2hPO 40.1%-0.2%, MgSO 4the water of 0.05%-0.1% and surplus.
The preparation method of described streptomycete liquid fermentation medium, comprising: 0.2g-1g is extracted residuum II fermentation basic medium and be settled to 1L, mix, and pH value nature, obtains streptomycete liquid fermentation medium.
The access amount of described streptomycete seed liquor is the 3%-15% of streptomycete liquid fermentation medium volume.
Described Antrodia camphorata bacterial classification can adopt any one Antrodia camphorata bacterial classification existing, can adopt commercially available prod, such as: Antrodia camphorata (Antrodiacamphorata) bacterial classification, is purchased from Beijing North Na Chuanlian Bioteknologisk Institut.
Described streptomyces species can adopt any one streptomyces species existing, can adopt commercially available prod, such as: streptomycete (Streptomyces) ACCC40462 bacterial classification, is purchased from Chinese agriculture Microbiological Culture Collection administrative center.
Described liquid MRS substratum adopts the liquid MRS substratum of this area routine, can adopt commercially available prod, and existing compound method also can be adopted to prepare.Consisting of of general liquid MRS substratum: peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 20.0g, sodium acetate 5.0g, diammonium hydrogen citrate 2.0g, tween-80 1.0ml, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.2g, manganous sulfate 0.05g and distilled water 1.0 liters.
Described PBS damping fluid and phosphate buffered saline(PBS), adopt the PBS damping fluid of this area routine, can adopt commercially available prod, existing compound method also can be adopted to prepare.General 1LPBS damping fluid: potassium primary phosphate 0.27g, Sodium phosphate dibasic 1.42g, sodium-chlor 8g and Repone K 0.2g, adds the abundant stirring and dissolving of appropriate amount of deionized water, and then add concentrated hydrochloric acid and adjust pH to 7.2-7.4, last constant volume is to 1L.
The temperature that streptomyces species after Antrodia camphorata bacterial classification after described activation, activation is cultivated in liquid MRS substratum is physical environment temperature, is preferably 20 DEG C-30 DEG C.1cm is accessed in general 1L liquid MRS substratum 2-4cm 2streptomyces species bacterium block after Antrodia camphorata bacterial classification bacterium block after the activation of size, activation.The activation method of described Antrodia camphorata bacterial classification or streptomyces species adopts the actication of culture method of this area routine, comprise: the Antrodia camphorata bacterial classification of slant preservation or streptomyces species are inoculated on PDA plate culture medium, carry out activation culture, culture temperature 20 DEG C-30 DEG C, incubation time 3 days-25 days, obtains the streptomyces species after the Antrodia camphorata bacterial classification after activating or activation.The substratum that described PDA plate culture medium adopts this area seed culture conventional, can adopt commercially available prod.Further preferably, described PDA plate culture medium: potato 200g, glucose 20g and agar 15g-20g, be settled to 1000mL with water.
The percentage ratio of the volume of the thalline liquid that described inoculum size refers to an access and the ratio of culture volume.
Use after the present invention's substratum used all needs sterilizing, the condition of sterilizing adopts the normal condition of this area, such as can at 120 DEG C-125 DEG C sterilizing 20min-30min.
Described Antrodia camphorata cultivates composition, is preferably made up of 40 weight part-55 weight part Antrodia camphoratas fermentation subcritical abstraction things I and 20 weight part-35 weight part streptomycete fermentation products III.
Antrodia camphorata of the present invention is cultivated composition and is had good liver-protecting function, can be used to prepare the healthcare products or medicine with liver-protecting function.Described healthcare products or medicine directly can be prepared by described composition and also can be prepared together with the auxiliary material of this area by described composition, product forms can be varied, such as comprise the soft capsule that Antrodia camphorata cultivates composition, its content is made up of 40 weight part-55 weight part Antrodia camphoratas fermentation subcritical abstraction thing I, 20 weight part-35 weight part streptomycete fermentation products III and 10 weight part-20 part by weight of vitamin E.The preparation of described soft capsule adopts the customary preparation methods of soft capsule.
Herba Anoectochili roxburghii (Anoectochilusroxburghii (Wall.) Lindl.), has another name called Shorthairy Antenoron, Anoectochilus Roxburghii, gold thread wind.Plant height 8-18 centimetre; Root stock is crawled, and extends, meat, and tool saves, and joint is taken root; Stem is upright, and meat is cylindrical, tool 2-4 piece leaf; Blade oval or avette, above mulberry or black purple, tool golden red with the beautiful net arteries and veins of silky lustre, back side lilac red; Raceme tool 2-6 flower, long 3-5 centimetre; Floral white or incarnadine, be not inverted (lip is positioned at top); The sepal back side is by pubescence, and middle sepal is avette, and depression is in boat shape; Petal quality is thin, nearly sickle shaped, the florescence 8-11 month.Be born in evergreen broad-leaved sylvan life or the dark and damp place of cheuch of height above sea level 50-1600 rice.Originate in China, also there are distribution in Japan, Thailand, Laos, Vietnam, India, Bhutan to Nepal, Bangladesh.Herba Anoectochili roxburghii herb, property is sweet, flat.Clearing heat and cooling blood, detoxify and promote the subsdence of swelling, moistens the lung and relieve the cough.For spitting of blood, cough asthma due to excessive phlegm, difficulty and pain in micturition, quench one's thirst, chyluria, acute infantile convulsion wind, boil on the nape opposite the mouth, heart trouble, venomous snake bite.
Angelica keiskei koidz (Gynuradivaricata (Linn.) DC.), has another name called white back of the body dish, Gynura divaricata, Pterocladia tenuis Okam., root and rhizome of Divaricate Gynura.For composite family Gynura Cass plant, with all herbal medicine, Xia Qiu gathers, and cleans section, using fresh herb or dry.Taste is sweet, light, cold, has clearing heat and detoxicating, the effects such as hemostasis, cool blood, can treat acute conjunctivitis, infantile hyperpyrexia, cardiopulmonary accumulated heat etc.
Streptomycete (Streptomyces) belongs to Gram positive actinomycetes, and substrate mycelium is not ruptured, and aerial hyphae physically well develops usually, forms the fibrillae of spores of long (sometimes short).Spore can not move, and epitheca often has the shape jewelrys such as wart, thorn or hair.
The present invention's raw material used all can adopt commercially available prod.
Beneficial effect of the present invention:
1, when the present invention carries out Antrodia camphorata solid state fermentation, the direct Antrodia camphorata liquid seeds liquid adopted through hatching process containing liquid MRS substratum two step of cholate, and angelica keiskei koidz and Herba Anoectochili roxburghii is added in substratum, effectively the nutritive ingredient in angelica keiskei koidz and Herba Anoectochili roxburghii and effective constituent Biodegradation and biotransformation be can be carried out, the content and the activity that improve activeconstituents in the finished product contributed to.In step (4) and step (5), Antrodia camphorata product by solid-state fermentation is carried out Subcritical Water Extraction, make that the activity of effective constituent in extract is at utmost retained, content is greatly improved; Again using extraction after residue as streptomycete liquid fermenting culture medium mainly, and adopt the streptomycete seed liquor through hatching process containing liquid MRS substratum two step of NaCl, by integrated recycle, each raw material resources are fully utilized, further increase content and the activity of activeconstituents in the finished product.
2, the inventive method raw material sources are natural, quality controllable, and environmentally safe is suitable for suitability for industrialized production.
3, compare with angelica keiskei koidz extract with the Antrodia camphorata tunning of common fermentation, Herba Anoectochili roxburghii, Antrodia camphorata of the present invention cultivates composition to CCl 4hepatic tissue alanine aminotransferase (ALT) in liver injury mice serum, the reduction of aspartate aminotransferase (AST) content are more remarkable, to CCl 4the improvement effect of liver injury mouse liver histopathology indices is more remarkable, illustrate that Antrodia camphorata of the present invention cultivates composition and soft gel products thereof, can significantly reduce ALT and AST content, and significantly improve liver injury mouse liver histopathology indices, there is significant liver-protecting function, can be used to prepare the healthcare products or medicine with liver-protecting function.
Embodiment
Below in conjunction with some embodiments, content of the present invention is illustrated further, but content of the present invention is not limited in the following examples.
Antrodia camphorata (Antrodiacamphorata) bacterial classification, is purchased from Beijing North Na Chuanlian Bioteknologisk Institut.
Streptomycete (Streptomyces) ACCC40462 bacterial classification, is purchased from Chinese agriculture Microbiological Culture Collection administrative center.
Cholate: No. 3 cholate, i.e. Sodium cholic acid, cholic acid-Sodium desoxycholate salt mixture (in the permanent industry in Beijing chemical industry far away).
Embodiment 1
One, material prepares
PDA plate culture medium: potato 200g, glucose 20g and agar 15g, be settled to 1000ml with water, natural pH, sterilizing 20min at 121 DEG C.
The Antrodia camphorata bacterial classification of slant preservation, the streptomyces species of slant preservation are inoculated on PDA plate culture medium respectively, carry out activation culture, culture temperature 22 DEG C, incubation time 10 days, obtain the streptomyces species after the Antrodia camphorata bacterial classification after activating, activation respectively.
Consisting of of liquid MRS substratum: peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 20.0g, sodium acetate 5.0g, diammonium hydrogen citrate 2.0g, tween-80 1.0ml, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.2g, manganous sulfate 0.05g and distilled water 1.0 liters.
Fermentation basic medium is made up of the component of following mass percent: glucose 1%, K 2hPO 40.1%, MgSO 40.05% and the water of surplus.
1LPBS damping fluid: potassium primary phosphate 0.27g, Sodium phosphate dibasic 1.42g, sodium-chlor 8g and Repone K 0.2g, adds the abundant stirring and dissolving of appropriate amount of deionized water, and then add concentrated hydrochloric acid and adjust pH to 7.4, last constant volume is to 1L.
Two, the preparation of Antrodia camphorata fermented product
(1) 2cm is got 2antrodia camphorata strain inoculation after size activation is cultivated 5 days in 25 DEG C in 1L liquid MRS substratum, and nutrient solution collects the first thalline through the centrifugal 10min of 6000rpm; First thalline is suspended in cholate mass percentage be 0.2% containing cholate liquid MRS substratum in hatch 10h in 25 DEG C, collect the second thalline through the centrifugal 10min of 6000rpm; By the second thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the second thalline liquid, by the second thalline liquid by 5% inoculum size be inoculated into cholate mass percentage be 0.2% containing cholate liquid MRS substratum in cultivate 5 days again in 22 DEG C, obtain Antrodia camphorata liquid seeds liquid;
Wherein, the first thalline is 1:1 with the volume ratio of the liquid MRS substratum containing cholate for first thalline that suspends, and the second thalline is 1:1 with the volume ratio for the liquid MRS substratum of second thalline that suspends.
(2) 1cm is got 2streptomyces species after size activation is inoculated in 1L liquid MRS substratum cultivates 15h in 25 DEG C, nutrient solution collects the 3rd thalline through the centrifugal 10min of 6000rpm, 3rd thalline is suspended in NaCl mass percentage be 5% hatch 8h containing in the liquid MRS substratum of NaCl in 25 DEG C, collect the 4th thalline through the centrifugal 10min of 6000rpm; By the 4th thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the 4th thalline liquid, by the 4th thalline liquid by 3% inoculum size be inoculated into NaCl mass percentage be 5% containing NaCl liquid MRS substratum in 25 DEG C cultivate 15h, obtain streptomycete seed liquor;
Wherein, the 3rd thalline is 1:1 with the volume ratio of the liquid MRS substratum containing NaCl for the 3rd thalline that suspends, and the 4th thalline is 1:1 with the volume ratio for the liquid MRS substratum of the 4th thalline that suspends.
(3) Antrodia camphorata liquid seeds liquid is accessed in Antrodia camphorata solid-state fermentation culture medium by the access amount accounting for Antrodia camphorata solid-state fermentation culture medium volume 15%, cultivate 10 days at 25 DEG C, then be placed in the alternating magnetic field environment that magneticstrength is 3mT, field frequency is 15Hz and cultivate 20 days again, cultured products obtains Antrodia camphorata product by solid-state fermentation after vacuum lyophilization, pulverizing;
The preparation of Antrodia camphorata solid-state fermentation culture medium comprises: fresh Herba Anoectochili roxburghii herb, angelica keiskei koidz herb are rinsed well under tap water, dry, chopping; Again 8g Herba Anoectochili roxburghii and 2g angelica keiskei koidz fermentation basic medium are settled to 1L, mix, pH value nature, obtains Antrodia camphorata solid-state fermentation culture medium.
(4) Antrodia camphorata product by solid-state fermentation was pulverized 40 mesh sieves, through Subcritical Water Extraction, the condition of Subcritical Water Extraction is: water material mass ratio is 30:1, extracting pressure is at 15MPa, extraction temperature is 150 DEG C, extraction time is 60min, and obtain Antrodia camphorata fermentation subcritical abstraction thing I, after extraction, remaining residue is extracted residuum II after vacuum lyophilization.
(5) streptomycete seed liquor is accessed in streptomycete liquid fermentation medium by the access amount accounting for streptomycete liquid fermentation medium volume 10%, cultivate 60h at 25 DEG C, obtain streptomycete fermentation product III through the centrifugal 10min of 6000rpm;
The preparation of streptomycete liquid fermentation medium comprises: 0.6g is extracted residuum II fermentation basic medium and be settled to 1L, mix, and pH value nature, obtains streptomycete liquid fermentation medium.
Antrodia camphorata is cultivated composition and is made up of 40 weight part Antrodia camphoratas fermentation subcritical abstraction things I and 20 weight part streptomycete fermentation products III.
Three, the preparation of soft gel products: the weighting material of soft capsule is made up of 40 weight part Antrodia camphoratas fermentation subcritical abstraction thing I, 20 weight part streptomycete fermentation products III and 10 part by weight of vitamin E.
Embodiment 2
One, material prepares
PDA plate culture medium: potato 200g, glucose 20g and agar 20g, be settled to 1000ml with water, natural pH, sterilizing 20min at 121 DEG C.
The Antrodia camphorata bacterial classification of slant preservation, the streptomyces species of slant preservation are inoculated on PDA plate culture medium respectively, carry out activation culture, culture temperature 20 DEG C, incubation time 25 days, obtain the streptomyces species after the Antrodia camphorata bacterial classification after activating, activation respectively.
Fermentation basic medium is made up of the component of following mass percent: glucose 3%, K 2hPO 40.2%, MgSO 40.1% and the water of surplus.
Liquid MRS substratum and PBS damping fluid are with embodiment 1.
Two, the preparation of Antrodia camphorata fermented product
(1) 1cm is got 2antrodia camphorata strain inoculation after size activation is cultivated 8 days in 20 DEG C in 1L liquid MRS substratum, and nutrient solution collects the first thalline through the centrifugal 10min of 6000rpm; First thalline is suspended in cholate mass percentage be 0.05% containing cholate liquid MRS substratum in hatch 20h in 18 DEG C, collect the second thalline through the centrifugal 10min of 6000rpm; By the second thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the second thalline liquid, by the second thalline liquid by 5% inoculum size be inoculated into cholate mass percentage be 0.05% containing cholate liquid MRS substratum in cultivate 10 days again in 18 DEG C, obtain Antrodia camphorata liquid seeds liquid;
Wherein, the first thalline is 1:2 with the volume ratio of the liquid MRS substratum containing cholate for first thalline that suspends, and the second thalline is 1:2 with the volume ratio for the liquid MRS substratum of second thalline that suspends.
(2) 4cm is got 2streptomyces species after size activation is inoculated in 1L liquid MRS substratum cultivates 3h in 30 DEG C, nutrient solution collects the 3rd thalline through the centrifugal 10min of 6000rpm, 3rd thalline is suspended in NaCl mass percentage be 8% hatch 15h containing in the liquid MRS substratum of NaCl in 20 DEG C, collect the 4th thalline through the centrifugal 10min of 6000rpm; By the 4th thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the 4th thalline liquid, by the 4th thalline liquid by 3% inoculum size be inoculated into NaCl mass percentage be 8% containing NaCl liquid MRS substratum in 20 DEG C cultivate 30h, obtain streptomycete seed liquor;
Wherein, the 3rd thalline is 1:3 with the volume ratio of the liquid MRS substratum containing NaCl for the 3rd thalline that suspends, and the 4th thalline is 1:3 with the volume ratio for the liquid MRS substratum of the 4th thalline that suspends.
(3) Antrodia camphorata liquid seeds liquid is accessed in Antrodia camphorata solid-state fermentation culture medium by the access amount accounting for Antrodia camphorata solid-state fermentation culture medium volume 25%, cultivate 30 days at 16 DEG C, then be placed in the alternating magnetic field environment that magneticstrength is 0.1mT, field frequency is 2Hz and cultivate 30 days again, cultured products obtains Antrodia camphorata product by solid-state fermentation after vacuum lyophilization, pulverizing;
The preparation of Antrodia camphorata solid-state fermentation culture medium comprises: fresh Herba Anoectochili roxburghii herb, angelica keiskei koidz herb are rinsed well under tap water, dry, chopping; Again 6.5g Herba Anoectochili roxburghii and 3.0g angelica keiskei koidz fermentation basic medium are settled to 1L, mix, pH value nature, obtains Antrodia camphorata solid-state fermentation culture medium.
(4) Antrodia camphorata product by solid-state fermentation was pulverized 40 mesh sieves, through Subcritical Water Extraction, the condition of Subcritical Water Extraction is: water material mass ratio is 50:1, extracting pressure is at 25MPa, extraction temperature is 180 DEG C, extraction time is 10min, and obtain Antrodia camphorata fermentation subcritical abstraction thing I, after extraction, remaining residue is extracted residuum II after vacuum lyophilization.
(5) streptomycete seed liquor is accessed in streptomycete liquid fermentation medium by the access amount accounting for streptomycete liquid fermentation medium volume 15%, cultivate 120h at 20 DEG C, obtain streptomycete fermentation product III through the centrifugal 10min of 6000rpm;
The preparation of streptomycete liquid fermentation medium comprises: 1g is extracted residuum II fermentation basic medium and be settled to 1L, mix, and pH value nature, obtains streptomycete liquid fermentation medium.
Antrodia camphorata is cultivated composition and is made up of 55 weight part Antrodia camphoratas fermentation subcritical abstraction things I and 35 weight part streptomycete fermentation products III.
Three, the preparation of soft gel products: the weighting material of soft capsule is made up of 55 weight part Antrodia camphoratas fermentation subcritical abstraction thing I, 35 weight part streptomycete fermentation products III and 20 part by weight of vitamin E.
Embodiment 3
One, material prepares
PDA plate culture medium, liquid MRS substratum and PBS damping fluid are with embodiment 1.
The Antrodia camphorata bacterial classification of slant preservation, the streptomyces species of slant preservation are inoculated on PDA plate culture medium respectively, carry out activation culture, culture temperature 30 DEG C, incubation time 3 days, obtain the streptomyces species after the Antrodia camphorata bacterial classification after activating, activation respectively.
Fermentation basic medium is made up of the component of following mass percent: glucose 2%, K 2hPO 40.15%, MgSO 40.08% and the water of surplus.
Two, the preparation of Antrodia camphorata fermented product
(1) 4cm is got 2antrodia camphorata strain inoculation after size activation is cultivated 2 days in 30 DEG C in 1L liquid MRS substratum, and nutrient solution collects the first thalline through the centrifugal 10min of 6000rpm; First thalline is suspended in cholate mass percentage be 0.5% containing cholate liquid MRS substratum in hatch 1h in 30 DEG C, collect the second thalline through the centrifugal 10min of 6000rpm; By the second thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the second thalline liquid, by the second thalline liquid by 5% inoculum size be inoculated into cholate mass percentage be 0.5% containing cholate liquid MRS substratum in cultivate 1 day again in 30 DEG C, obtain Antrodia camphorata liquid seeds liquid;
Wherein, the first thalline is 1:3 with the volume ratio of the liquid MRS substratum containing cholate for first thalline that suspends, and the second thalline is 1:3 with the volume ratio for the liquid MRS substratum of second thalline that suspends.
(2) 2cm is got 2streptomyces species after size activation is inoculated in 1L liquid MRS substratum cultivates 30h in 20 DEG C, nutrient solution collects the 3rd thalline through the centrifugal 10min of 6000rpm, 3rd thalline is suspended in NaCl mass percentage be 0.5% hatch 0.5h containing in the liquid MRS substratum of NaCl in 30 DEG C, collect the 4th thalline through the centrifugal 10min of 6000rpm; By the 4th thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the 4th thalline liquid, by the 4th thalline liquid by 3% inoculum size be inoculated into NaCl mass percentage be 0.5% containing NaCl liquid MRS substratum in 30 DEG C cultivate 3h, obtain streptomycete seed liquor;
Wherein, the 3rd thalline is 1:2 with the volume ratio of the liquid MRS substratum containing NaCl for the 3rd thalline that suspends, and the 4th thalline is 1:2 with the volume ratio for the liquid MRS substratum of the 4th thalline that suspends.
(3) Antrodia camphorata liquid seeds liquid is accessed in Antrodia camphorata solid-state fermentation culture medium by the access amount accounting for Antrodia camphorata solid-state fermentation culture medium volume 5%, cultivate 3 days at 30 DEG C, then be placed in the alternating magnetic field environment that magneticstrength is 5mT, field frequency is 40Hz and cultivate 5 days again, cultured products obtains Antrodia camphorata product by solid-state fermentation after vacuum lyophilization, pulverizing;
The preparation of Antrodia camphorata solid-state fermentation culture medium comprises: fresh Herba Anoectochili roxburghii herb, angelica keiskei koidz herb are rinsed well under tap water, dry, chopping; Again 4.5g Herba Anoectochili roxburghii and 0.3g angelica keiskei koidz fermentation basic medium are settled to 1L, mix, pH value nature, obtains Antrodia camphorata solid-state fermentation culture medium.
(4) Antrodia camphorata product by solid-state fermentation was pulverized 40 mesh sieves, through Subcritical Water Extraction, the condition of Subcritical Water Extraction is: water material mass ratio is 10:1, extracting pressure is at 4MPa, extraction temperature is 100 DEG C, extraction time is 90min, and obtain Antrodia camphorata fermentation subcritical abstraction thing I, after extraction, remaining residue is extracted residuum II after vacuum lyophilization.
(5) streptomycete seed liquor is accessed in streptomycete liquid fermentation medium by the access amount accounting for streptomycete liquid fermentation medium volume 3%, cultivate 10h at 30 DEG C, obtain streptomycete fermentation product III through the centrifugal 10min of 6000rpm;
The preparation of streptomycete liquid fermentation medium comprises: 0.2g is extracted residuum II fermentation basic medium and be settled to 1L, mix, and pH value nature, obtains streptomycete liquid fermentation medium.
Antrodia camphorata is cultivated composition and is made up of 50 weight part Antrodia camphoratas fermentation subcritical abstraction things I and 25 weight part streptomycete fermentation products III.
Three, the preparation of soft gel products: the weighting material of soft capsule is made up of 50 weight part Antrodia camphoratas fermentation subcritical abstraction thing I, 25 weight part streptomycete fermentation products III and 15 part by weight of vitamin E.
Comparative example 1
One, material prepares
Antrodia camphorata bacterial classification after PDA plate culture medium, activation, liquid MRS substratum and fermentation basic medium are all with embodiment 1.
Two, the preparation of Antrodia camphorata fermented product
(1) 2cm is got 2antrodia camphorata strain inoculation after size activation is cultivated 5 days in 25 DEG C in 1L liquid MRS substratum, obtains Antrodia camphorata liquid seeds liquid.
(2) ferment in basic medium by Antrodia camphorata liquid seeds liquid by the access amount access accounting for the basic medium volume 15% that ferments, cultivate 10 days at 25 DEG C, cultured products obtains Antrodia camphorata tunning after vacuum lyophilization, pulverizing;
Three, the preparation of soft gel products: the weighting material of soft capsule is made up of 40 weight part Antrodia camphorata tunnings and 10 part by weight of vitamin E.
Comparative example 2 Herba Anoectochili roxburghii and angelica keiskei koidz extract
Fresh Herba Anoectochili roxburghii herb, angelica keiskei koidz herb are rinsed well under tap water, dries, chopping; Add water by feed liquid weight ratio 1:10,10h is extracted under boiling state, filter to obtain first-time filtrate and a filter residue, a filter residue is added water by feed liquid weight ratio 1:5,8h is extracted under boiling state, filter to obtain secondary filtrate, merge first-time filtrate and secondary filtrate, after lyophilize, pulverizing, obtain Herba Anoectochili roxburghii and angelica keiskei koidz extract.
The preparation of soft gel products: the weighting material of soft capsule is made up of 40 weight part Herba Anoectochili roxburghiis and angelica keiskei koidz extract and 10 part by weight of vitamin E.
The mensuration of activeconstituents
(1) measurement of the polysaccharide content
Take appropriate amount of sample dry powder, cross 40 orders after pulverizing, be placed in beaker, add distilled water by solid-liquid ratio 1:10 (mass ratio), 90 DEG C of lixiviate 3h, extract twice, filtration under diminished pressure collects filtrate, concentrating under reduced pressure at 50 DEG C, concentrated solution adds 4 times of volume dehydrated alcohols, alcohol precipitation overnight, after 3000r/min, 20min are centrifugal, precipitation is Crude polysaccharides.Precipitation distilled water is settled to 50mL, and measure polysaccharide in fermentation liquid content with phend-sulphuric acid, replication is averaged for 3 times, i.e. polysaccharide content.
(2) mensuration of flavones content
Be dried to the rutin standard substance 10.0mg of constant weight under accurately taking 120 DEG C of conditions, dissolve with the aqueous ethanolic solution of mass percentage concentration 70% and be settled to 100mL, being made into 0.1mg/mL standardized solution.Get respectively standardized solution 0.0,1.0,2.0,3.0,4.0,5.0mL in 10mL volumetric flask, after adding mass percentage concentration 70% aqueous ethanolic solution to 5.0mL respectively, add the NaNO of 0.3mL mass percentage concentration 5% 2the aqueous solution, shakes up and places 6min; Add the Al (NO of 0.4mL mass percentage concentration 10% again 3) 3the aqueous solution, shakes up and places 6min; Add the 4.0mL mass percentage concentration 4%NaOH aqueous solution again, mass percentage concentration 60% aqueous ethanolic solution is settled to scale, shake up and place 15min, be cooled to the absorbancy at 510nm place bioassay standard product after room temperature, with standard solution mass concentration C (mg/mL) for X-coordinate, absorbance A is ordinate zou drawing standard curve, obtains regression equation.
Take appropriate amount of sample dry powder, cross 40 orders after pulverizing, first add the aqueous ethanolic solution of mass percentage concentration 70% by solid-liquid ratio 1:30 (mass ratio), at 70 DEG C, extract 2h, be settled to 50ml after extracting solution centrifuging, therefrom get 1ml sample and carry out assay.
(3) mensuration of triterpene content
It is a certain amount of that precision takes Oleanolic Acid reference substance, puts in volumetric flask, with acetic acid ethyl dissolution, and ultrasonic 15min, and be diluted to scale, shake up, make the reference substance solution of 0.1mg/ml.Draw 0.00ml, 0.20ml, 0.40ml, 0.60ml, 0.80ml, 1.00ml and 1.20ml reference substance solution respectively, room temperature is placed after evaporate to dryness in 100 DEG C of water-baths, add Vanillin-glacial acetic acid solution and the 1.00ml perchloric acid of 0.40ml Vanillin mass percentage concentration 5%, heat 15min and move in ice-water bath in 65 DEG C of water-baths and cool 3min, take out and place room temperature, add 5.00ml glacial acetic acid again, shake up and be placed in room temperature.Under 552 ± 2nm wavelength, measure the absorbancy of reference substance solution with spectrophotometer after 15min.Respectively with concentration and absorbancy drawing standard curve.
Sample thief is about 0.50g, accurately weighed, puts in 100ml volumetric flask, uses 85ml acetic acid ethyl dissolution, ultrasonic 30min, and filter, a small amount of ethyl acetate drip washing filter residue, is settled to scale, shakes up.Draw this solution of 1ml, room temperature is placed after evaporate to dryness in 100 DEG C of water-baths, add Vanillin-glacial acetic acid solution and the 1.0ml perchloric acid of 0.40ml Vanillin mass percentage concentration 5%, move into 3min in ice-water bath at 65 DEG C of heating in water bath 15min, take out and place room temperature, add 5.00ml glacial acetic acid again, shake up and be placed in room temperature.By the absorbancy of spectrophotometer working sample solution under 552 ± 2nm wavelength after 15min.
The each embodiment of table 1 and comparative example main component detected result
Note: compare with comparative example 1, comparative example 2, Δ: P<0.05.
The data presentation of table 1, Antrodia camphorata of the present invention cultivates composition, significantly improves the content of polysaccharide in product, flavones, triterpene isoreactivity composition simultaneously.Compared with the conventional culture methods in comparative example 1, the Antrodia camphorata adopting the inventive method to obtain is cultivated polysaccharide content in composition and is improve 60.4%-62.8%, and flavones content improves 38.0%-42.2%, and triterpene content improves 38.7%-45.0%.
Liver-protecting function is tested
Laboratory animal: male mice in kunming, body weight 18 ± 2g.Sub-cage rearing, free diet, observes after 3 days for test.
Experimental design: carry out according to " protective foods inspection and assessment technical specifications " (2003 editions) judgement criteria, if basic, normal, high 3 dose of test groups and 1 negative Basal control group and 1 model control group, basic, normal, high 3 dosage component are not equivalent to people with 5 times, 10 times, 20 times of maximum recommended dosage, 0.15 namely, 0.30,0.60g/kgbw/d, basic, normal, high 3 dosage formulation concentration are respectively 15,30,60mg/mL.Basal control group and model control group all give distilled water, all edible full nutrition mixed feed of all laboratory animal, drinking-water of freely ingesting.
Per os gives the tested material of mouse corresponding dosage once a day, and mouse stomach amount is 10mL/kgbw, continuous gavage 30d, in experiment the 30th day by fasting 16h overnight for each treated animal.Model control group and tested group of gavage give 1%CCl 4vegetable oil solution (5mL/kgBW), Basal control group gives vegetables oil, and tested group is continued to give the obtained composition of embodiment to testing end (with CCl 4gavage interval 4h), give CCl 4after 24h, mouse is won eyeball and gets blood, measures Serum ALT, AST level, and gets liver and carry out histopathological examination.
Experimental result:
1. the composition that obtains of each embodiment is on the impact of Mouse Weight
As shown in Table 2, one-way analysis of variance is carried out to the average of the original body mass/end body weight (30d) of each group of mouse test, no significant difference (P>0.05), result shows that the body weight of composition on animal that each embodiment and comparative example obtain has no obvious impact.
2. couple CCl 4the impact of liver injury mice serum ALT and AST
As shown in Table 3, ALT, AST content of model control group mouse is higher than negative Basal control group, statistical analysis difference has statistical significance (P < 0.05), composition that embodiment obtains 0.15,0.30, the ALT value of 0.60g/kgbw/d dosage group lower than model control group, statistical analysis difference has statistical significance (P < 0.05); The AST value of low middle high dose group is lower than model control group, and statistical analysis difference has statistical significance (P < 0.05).Result shows, the composition that embodiment 1-3 obtains is when being equivalent to 5 times, 10 times of human body recommended intake and 20 multiple dose, there is the effect reducing chemical damage mice serum ALT and AST, and the composition indices that under same dose, embodiment 1-3 is obtained has significant difference (P < 0.05) with comparing with comparative example 2 than comparative example 1, show to protect liver effect compared with the product that the present composition and conventional culture methods, extracting method obtain more obvious.
3. the composition that embodiment is obtained is to CCl 4the histopathologic impact of liver injury mouse liver
As shown in Table 4, one-way analysis of variance is carried out to the average of each group of mouse liver histopathological indications quantized value, indices all with model control group comparing difference significance.Result shows, the composition that embodiment 1-3 obtains is when being equivalent to 5 times, 10 times of human body recommended intake and 20 multiple dose, experimentation on animals pathological examination is positive, show that it has the pathologic lesion effect alleviating chemical damage mouse liver, and the composition indices that under same dose, embodiment 1-3 is obtained has significant difference (P < 0.05) with comparing with comparative example 2 than comparative example 1, show to protect liver effect compared with the product that the present composition and conventional culture methods, extracting method obtain more obvious.
In table 2 embodiment and comparative example, product is on the impact of Mouse Weight
In table 3 embodiment and comparative example, product is on the impact of mice serum ALT and AST
In table 4 embodiment and comparative example, product is to CCl 4the histopathologic impact of liver injury mouse liver
In the scope that formula of the present invention and preparation method limit, the change of each parameter does not affect Antrodia camphorata of the present invention and cultivates composition and soft gel products thereof to improvement effect of chemical damage and preparation, and the Antrodia camphorata that therefore in formula of the present invention and preparation method, the combination of arbitrary parameter all can obtain having good liver-protecting function cultivates composition and soft gel products thereof.Do not repeat them here.

Claims (10)

1. Antrodia camphorata cultivates a preparation method for composition, it is characterized in that, comprises step:
(1) get in the Antrodia camphorata strain inoculation after activation to liquid MRS substratum and cultivate 2 days-8 days, collected by centrifugation first thalline; First thalline is suspended in the liquid MRS substratum containing cholate and hatches 1h-20h in 18 DEG C-30 DEG C, collected by centrifugation second thalline; By the second thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the second thalline liquid, second thalline liquid is inoculated in the liquid MRS substratum containing cholate and cultivates 1 day-10 days again in 18 DEG C-30 DEG C, obtain Antrodia camphorata liquid seeds liquid;
(2) streptomyces species got after activation is inoculated in liquid MRS substratum and cultivates 3h-30h, collected by centrifugation the 3rd thalline, is suspended in by the 3rd thalline in the liquid MRS substratum containing NaCl and hatches 0.5h-15h in 20 DEG C-30 DEG C, collected by centrifugation the 4th thalline; By the 4th thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the 4th thalline liquid, 4th thalline liquid is inoculated in the liquid MRS substratum containing NaCl and cultivates 3h-30h in 20 DEG C-30 DEG C, obtain streptomycete seed liquor;
(3) by the Antrodia camphorata liquid seeds liquid of step (1) access Antrodia camphorata solid-state fermentation culture medium, cultivate 3 days-30 days at 16 DEG C-30 DEG C, then be placed in alternating magnetic field environment and cultivate 5 days-30 days again, cultured products obtains Antrodia camphorata product by solid-state fermentation after vacuum lyophilization, pulverizing; Containing Herba Anoectochili roxburghii and angelica keiskei koidz in described Antrodia camphorata solid-state fermentation culture medium;
(4) by the Antrodia camphorata product by solid-state fermentation of step (3) through Subcritical Water Extraction, obtain Antrodia camphorata fermentation subcritical abstraction thing I, after extraction, remaining residue is extracted residuum II after vacuum lyophilization;
(5) by the streptomycete seed liquor of step (2) access streptomycete liquid fermentation medium, 10h-120h is cultivated at 20 DEG C-30 DEG C, centrifugal, obtain streptomycete fermentation product III, containing extraction residuum II in described streptomycete liquid fermentation medium;
It is Antrodia camphorata fermentation subcritical abstraction thing I and streptomycete fermentation product III that described Antrodia camphorata cultivates composition.
2. preparation method according to claim 1, is characterized in that, in step (3), containing 4.5g-8.0g Herba Anoectochili roxburghii and 0.3g-3.0g angelica keiskei koidz in the every 1L of described Antrodia camphorata solid-state fermentation culture medium.
3. preparation method according to claim 1, is characterized in that, in step (5), extracts residuum II in the every 1L of described streptomycete liquid fermentation medium containing 0.2g-1g.
4. preparation method according to claim 1, is characterized in that, in step (1), described is 0.05%-0.5% containing the mass percentage of cholate in the liquid MRS substratum of cholate;
In step (2), the mass percentage of the described liquid MRS NaCl in medium containing NaCl is 0.5%-8%.
5. preparation method according to claim 1, it is characterized in that, in step (1), described first thalline is 1:1-3 with the volume ratio of the liquid MRS substratum containing cholate for first thalline that suspends, second thalline is 1:1-3 with the volume ratio for the liquid MRS substratum of second thalline that suspends, and the inoculum size of the second thalline liquid is 5%;
In step (2), described 3rd thalline is 1:1-3 with the volume ratio of the liquid MRS substratum containing NaCl for the 3rd thalline that suspends, 4th thalline is 1:1-3 with the volume ratio for the liquid MRS substratum of the 4th thalline that suspends, and the inoculum size of the 4th thalline liquid is 3%.
6. preparation method according to claim 1, is characterized in that, in step (3), in described alternating magnetic field environment, magneticstrength is 0.1mT-5mT, and field frequency is 2Hz-40Hz;
In step (4), the condition of described Subcritical Water Extraction is: water material mass ratio is 10-50:1, and extracting pressure is at 4MPa-25MPa, and extraction temperature is 100 DEG C-180 DEG C, and extraction time is 10min-90min.
7. preparation method according to claim 1, is characterized in that, in step (3), the access amount of Antrodia camphorata liquid seeds liquid is the 5%-25% of Antrodia camphorata solid-state fermentation culture medium volume;
In step (5), the access amount of described streptomycete seed liquor is the 3%-15% of streptomycete liquid fermentation medium volume.
8. Antrodia camphorata cultivates a composition, it is characterized in that, adopts the preparation method described in any one of claim 1-7 to prepare.
9. Antrodia camphorata according to claim 8 cultivates composition, it is characterized in that, described composition is made up of 40 weight part-55 weight part Antrodia camphoratas fermentation subcritical abstraction things I and 20 weight part-35 weight part streptomycete fermentation products III.
10. Antrodia camphorata cultivates an application for composition, it is characterized in that, Antrodia camphorata is according to claim 8 or claim 9 cultivated composition and prepared the application in the healthcare products or medicine protecting liver.
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