CN105505792B - A kind of Hirsutella sinensis fermentation culture method - Google Patents
A kind of Hirsutella sinensis fermentation culture method Download PDFInfo
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Abstract
The invention discloses a kind of Hirsutella sinensis fermentation culture methods, including：Rhizopus oryzae seed liquor is accessed in the Rhizopus oryzae solid-state fermentation culture medium containing tuniclike psammosilene root and radix polygonati officinalis and is cultivated, is subsequently placed in alternating magnetic field environment and is further cultured for, obtains Rhizopus oryzae product by solid-state fermentation, then through Subcritical Water Extraction, obtain Rhizopus oryzae fermentation subcritical abstraction object I；It will be cultivated in Hirsutella sinensis liquid fermentation medium of the Hirsutella sinensis seed liquor access containing extraction residue II, obtain Hirsutella sinensis tunning III；Composition is Rhizopus oryzae fermentation subcritical abstraction object I and Hirsutella sinensis tunning III, has good liver-protecting function.This method can help to improve the content and activity of active constituent in final products effectively by the nutritional ingredient and effective component progress Biodegradation and biotransformation in each raw material.
Description
Technical field
The present invention relates to biological fermentation fields, and in particular to a kind of Hirsutella sinensis fermentation culture method.
Background technique
Hirsutella sinensis (Hirsutella sinensis) is isolated phorozoon Cordyceps Militaris from natural cs
Strain.Due to the scarcity of wild cordyceps, present people mostly use greatly manual method to separate and cultivate strain to obtain winter worm
The substitute of summer grass.Although the cordyceps mycelia of artificial culture and the cordyceps mycelia of wild are in chemical component and drug effect
Not fully, but its active constituent still has very high drug effect and economic value.If Hirsutella sinensis is with biology
Engineering fermentation, is used as cordyceps sinensis substitute, and a variety of diseases for the treatment of are widely used in the form of folk prescription or compound, is food
The dual-purpose health care product of medicine.
Since natural cordyceps have stringent parasitics and special geographical growing environment, thus limits throughput, add
On in recent years excavation excessively so that Cordyceps Resources fall sharply, be unable to satisfy the demand of health care and medication at all.So using modern
Biofermentation technique Submerged fermentation aweto mycelium is to overcome the optimal path being limited using wild resource biomass
With the basis for realizing desirable metabolites development and utilization.
Currently, the active constituent in Hirsutella sinensis mostly uses traditional extracting and developing method or traditional zymotic culture
Method obtains, if Chinese patent ZL200710050273.7 discloses a kind of production method of hirsutella sinensis fungal powder, using work
The static fermentation technique of body protein medium liquid, living body protein culture medium are made of the raw material of following weight percents：Egg 70-
90%, milk 1-20%, potato fruit 1-5%, carrot juice 2-6%；By the egg in above-mentioned culture medium, potato, carrot
It squeezes, be centrifuged respectively, taking to weigh in proportion with the milk of aseptic filtration after juice, aseptic filtration and be uniformly mixed, being fitted into round.
It then is that inoculum concentration accesses in round by the 5-10% of culture base unit weight by Hirsutella sinensis kind liquid, gas in round,
Liquid volume ratio is 1-3：1, content of soluble protein 13-18%, pH=6-8,15-30 DEG C of fermentation plant temperature, humidity 30-
60%, culture is drying to obtain hirsutella sinensis fungal powder in 7-10 days.
Nowadays, the Hirsutella sinensis product that nutrition characteristic is different in order to obtain and/or purposes is different, people start to China
The traditional fermentation process of coat spore is improved or is innovated, and is desirably to obtain the China of Different Nutrition characteristic and/or different purposes
Coat spore product.Such as：Chinese patent application CN201310455400.7 discloses a kind of with Hirsutella sinensis fermentation fructus lycii
Production method is mainly used in Hirsutella sinensis fungi fermentation fructus lycii；Include the following steps：Step 1：By 30%-60% Chinese holly
Culture medium is made in Qi and 40%-70% cream by weight percentage, is placed in culture vessel, cultivates the 1/ of fiduciary point culture vessel volume
, 115-140 DEG C moist heat sterilization 30 minutes, access accounts for the Hirsutella sinensis strain of culture medium weight 0.5%-1%；
15-22 DEG C culture 3-15 days, media surface covers with mycelia, completes fermentation process；Step 2：Capsule contents are dry,
It crushes, be sieved up to Hirsutella sinensis fermentation fructus lycii product；Its production method for realizing Hirsutella sinensis fermentation fructus lycii for the first time,
Kidney health-care effect is obvious, is easy to large-scale industrial production, is kind of a green safe production method.
As can the new Hirsutella sinensis culture technique and products thereof of exploitation, by can be improved Hirsutella sinensis application range and
Application value.
Summary of the invention
The present invention provides a kind of Hirsutella sinensis fermented and cultured composition, the content and work of active constituent in the composition
Property it is high, there is good liver-protecting function.
The present invention also provides a kind of Hirsutella sinensis fermentation culture methods, utilize a variety of fungi fermentation tuniclike psammosilene roots and jade
Bamboo helps to improve the content and activity of active constituent in final products, obtains the composition with liver-protecting function.
It is a discovery of the invention that according to the fermentation character of Hirsutella sinensis and Rhizopus oryzae, specific fungi fermentation through the invention
And specific technique, can effectively by the nutritional ingredient and effective component progress biodegrade in radix polygonati officinalis and tuniclike psammosilene root and it turn
Change, helps to improve the content and activity of active constituent in final products.
The technical solution adopted by the present invention is that：
A kind of Hirsutella sinensis fermentation culture method, including step：
(1) the Hirsutella sinensis strain after taking activation, which is inoculated into liquid MRS culture medium, to be cultivated -15 days 4 days, is collected by centrifugation
First thallus；First thallus is suspended in the liquid MRS culture medium containing cholate in 18 DEG C of -30 DEG C of incubation 1h-20h, centrifugation is received
Collect the second thallus；Second thallus is resuspended in liquid MRS culture medium after PBS buffer solution cleans up, concussion shakes up
Obtain the second thallus liquid, the second thallus liquid is inoculated into the liquid MRS culture medium containing cholate be further cultured in 18 DEG C -30 DEG C 2 days -
20 days, obtain Hirsutella sinensis seed liquor；
(2) the Rhizopus oryzae strain after taking activation, which is inoculated into liquid MRS culture medium, cultivates 3h-30h, and third bacterium is collected by centrifugation
Third thallus is suspended in the liquid MRS culture medium containing NaCl in 20 DEG C of -30 DEG C of incubation 0.5h-15h by body, is collected by centrifugation
Four thallus；4th thallus is resuspended in liquid MRS culture medium after PBS buffer solution cleans up, concussion shakes up to obtain
4th thallus liquid is inoculated into the liquid MRS culture medium containing NaCl in 20 DEG C of -30 DEG C of culture 3h-30h, obtains by the 4th thallus liquid
To Rhizopus oryzae seed liquor；
(3) by the Rhizopus oryzae seed liquor access Rhizopus oryzae solid-state fermentation culture medium of step (2), 3 are cultivated at 20 DEG C -30 DEG C
It it -30 days, is subsequently placed in alternating magnetic field environment and is further cultured for -20 days 5 days, cultured products obtain after vacuum freeze drying, crushing
Rhizopus oryzae product by solid-state fermentation；Contain tuniclike psammosilene root and radix polygonati officinalis in the Rhizopus oryzae solid-state fermentation culture medium；
(4) the Rhizopus oryzae product by solid-state fermentation of step (3) is obtained into the subcritical extraction of Rhizopus oryzae fermentation through Subcritical Water Extraction
Object I is taken, remaining residue obtains extraction residue II after vacuum freeze drying after extraction；
(5) will step (1) Hirsutella sinensis seed liquor access Hirsutella sinensis liquid fermentation medium in, adjust pH value be
3-7 is cultivated -25 days 5 days at 15 DEG C -25 DEG C, and centrifugation obtains Hirsutella sinensis tunning III；The Hirsutella sinensis liquid
Contain extraction residue II in fermentation medium；
Hirsutella sinensis fermented and cultured composition is Rhizopus oryzae fermentation subcritical abstraction object I and Hirsutella sinensis tunning
Ⅲ。
When the present invention carries out Rhizopus oryzae solid state fermentation, directly adopt through at the two step incubation of liquid MRS culture medium containing NaCl
The Rhizopus oryzae seed liquor of reason, and radix polygonati officinalis and tuniclike psammosilene root are added into culture medium, can effectively by its nutritional ingredient and effectively at
Divide and carry out Biodegradation and biotransformation, helps to improve the content and activity of active constituent in final products.Step (4) and step
(5) in, Rhizopus oryzae product by solid-state fermentation is subjected to Subcritical Water Extraction, enables the activity maximum journey of effective component in extract
Degree retains, content is greatly improved；Again using residue extracted as the main culture medium of Hirsutella sinensis liquid fermentation, and
Using the Hirsutella sinensis seed liquor for being incubated for processing through two step of liquid MRS culture medium containing cholate, recycled by integration,
So that each raw material resources is fully utilized, further improves the content and activity of active constituent in final products.
In order to reach better effect, preferably：
In step (1), the mass percentage of cholate is 0.05%- in the liquid MRS culture medium containing cholate
0.5%.
The volume ratio of first thallus and the liquid MRS culture medium containing cholate for first thallus that suspends is 1:1-3,
The volume ratio of second thallus and the liquid MRS culture medium for second thallus that suspends is 1:The inoculum concentration of 1-3, the second thallus liquid is
5%.
In step (2), the mass percentage of the liquid MRS NaCl in medium containing NaCl is 0.5%-8%.
The volume ratio of the third thallus and the liquid MRS culture medium containing NaCl for the third thallus that suspends is 1:1-3,
The volume ratio of 4th thallus and the liquid MRS culture medium for the 4th thallus that suspends is 1:The inoculum concentration of 1-3, the 4th thallus liquid is
3%.
In step (3), 5.5g-8.5g tuniclike psammosilene root and 0.2g- are contained in the every 1L of Rhizopus oryzae solid-state fermentation culture medium
4.0g radix polygonati officinalis.The Rhizopus oryzae solid-state fermentation culture medium is made of tuniclike psammosilene root, radix polygonati officinalis and fermentation basal medium, the fermentation base
Basal culture medium uses the fermentation basal medium of this field routine, and commercial product can be used, existing preparation method can also be used and match
System.The tuniclike psammosilene root selects tuniclike psammosilene root root.The radix polygonati officinalis selects polygonati rhizoma.
The preparation method of the Rhizopus oryzae solid-state fermentation culture medium, including：By fresh tuniclike psammosilene root, radix polygonati officinalis in tap water undershoot
Wash clean dries, chopping；5.5g-8.5g tuniclike psammosilene root and 0.2g-4.0g radix polygonati officinalis are settled to 1L with fermentation basal medium again,
It is uniformly mixed, pH value is naturally, obtain Rhizopus oryzae solid-state fermentation culture medium.
The access amount of the Rhizopus oryzae seed liquor is the 5%-25% of Rhizopus oryzae solid-state fermentation culture medium volume.
The generally existing fermentation period of solid state fermentation is long at present, mycelia growth sprouting is slow, feature secondary metabolism metabolite contains
Measure the problems such as low.Present invention discover that alternating magnetic field processing technique is applied in solid state fermentation, pass through the suitable of specific cultivation period
When extraneous factors effective stimulus such as processing, the growth metabolism of hypha,hyphae is improved, shortens fermentation time, effectively facilitates secondary metabolism
The generation of product.Magnetic field strength is 0.2mT-6mT, field frequency 3Hz-40Hz in the alternating magnetic field environment.
In step (4), the condition of the Subcritical Water Extraction is：Water material mass ratio is 10-50:1, extracting pressure exists
4MPa-25MPa, extraction temperature are 100 DEG C -180 DEG C, extraction time 10min-90min.
In step (5), residue II is extracted containing 0.2g-1g in the every 1L of Hirsutella sinensis liquid fermentation medium.
The Hirsutella sinensis liquid fermentation medium is made of extraction residue II and fermentation basal medium, the fermentation basis
Culture medium uses the fermentation basal medium of this field routine, and commercial product can be used, and existing preparation method can also be used and prepare.
General fermentation basal medium consists of the following mass percentage components：Glucose 1%-3%,
K2HPO40.1%-0.2%, MgSO4The water of 0.05%-0.1% and surplus.
The preparation method of the Hirsutella sinensis liquid fermentation medium, including：By 0.2g-1g extraction residue II hair
Ferment basal medium is settled to 1L, is uniformly mixed, pH value is naturally, obtain Hirsutella sinensis liquid fermentation medium.
The access amount of the Hirsutella sinensis seed liquor is the 3%-20% of Hirsutella sinensis liquid fermentation medium volume.
Any one existing Hirsutella sinensis strain can be used in the Hirsutella sinensis strain, and commercial product, example can be used
Such as：Hirsutella sinensis (Hirsutella sinensis) strain, is purchased from BeNa Culture Collection Institute of Biotechnology.
Any one existing Rhizopus oryzae strain can be used in the Rhizopus oryzae strain, and commercial product can be used, such as：Rhizopus oryzae
30416 strain of (Rhizopus oryzae) ACCC, is purchased from Chinese agriculture Microbiological Culture Collection administrative center.
The liquid MRS culture medium uses the liquid MRS culture medium of this field routine, and commercial product can be used, can also adopt
It is prepared with existing preparation method.The group of general liquid MRS culture medium becomes：Peptone 10.0g, beef extract 10.0g, yeast leaching
Cream 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, manganese sulfate 0.05g and 1.0 liters of distilled water.
Commercially available production can be used using the PBS buffer solution of this field routine in the PBS buffer solution, that is, phosphate buffered saline solution
Product can also be used existing preparation method and prepare.General 1L PBS buffer solution：Potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 1.42g,
Sodium chloride 8g and potassium chloride 0.2g, adds appropriate amount of deionized water that dissolution is sufficiently stirred, and concentrated hydrochloric acid tune pH to 7.2-7.4 is then added,
Last constant volume is to 1L.
What the Rhizopus oryzae strain after Hirsutella sinensis strain, activation after the activation was cultivated in liquid MRS culture medium
Temperature is natural environment temperature, and preferably 20 DEG C -30 DEG C.1cm is accessed in general 1L liquid MRS culture medium2-4cm2Size
The Rhizopus oryzae strain fungus block after Hirsutella sinensis strain fungus block, activation after activation.The Hirsutella sinensis strain or Rhizopus oryzae
The activation method of strain uses the actication of culture method of this field routine, including：By the Hirsutella sinensis strain of slant preservation or
Rhizopus oryzae strain is inoculated on PDA plate culture medium, progress activation culture, and 20 DEG C -30 DEG C of cultivation temperature, incubation time 3 days -25
It, the Rhizopus oryzae strain after Hirsutella sinensis strain or activation after being activated.The PDA plate culture medium uses ability
The common culture medium of domain seed culture, can be used commercial product.Further preferably, the PDA plate culture medium：Potato
200g, glucose 20g and agar 15g-20g, are settled to 1000mL with water.
The volume for the thallus liquid that the inoculum concentration refers to the accession to and the percentage of the ratio between culture volume.
Culture medium used in the present invention uses after being both needed to sterilizing, and the condition of sterilizing uses the normal condition of this field, such as
Can sterilize 20min-30min at 120 DEG C -125 DEG C.
The Hirsutella sinensis fermented and cultured composition is preferably faced by -55 parts by weight Rhizopus oryzae of 40 parts by weight fermentation Asia
Boundary's extract I and -35 parts by weight Hirsutella sinensis tunning III of 20 parts by weight composition.
Hirsutella sinensis fermented and cultured composition of the present invention has good liver-protecting function, can be used to prepare with protect liver function
The health care product or drug of energy.The health care product or drug can be prepared directly by the composition can also be by the combination
Object is prepared together with the auxiliary material of this field, and product forms can be varied, such as a kind of includes Hirsutella sinensis fermented and cultured
The soft capsule of composition, content is by -55 parts by weight Rhizopus oryzae of 40 parts by weight fermentation subcritical abstraction object I, 20 parts by weight -35
Parts by weight Hirsutella sinensis tunning III and -20 part by weight of vitamin E of 10 parts by weight composition.The preparation of the soft capsule uses
The customary preparation methods of soft capsule.
Tuniclike psammosilene root is the dry of pinkwort tuniclike psammosilene root Psammosilene tunicoides W.C.Wu et C Y.Wu
Dry.Autumn excavation, removes crust and impurity, dries.Alias：RADIX PSAMMOSILENE, to leaf seven, Chuan Shijia.Character：Bitter, pungent, property
Temperature.Effect：Dispelling wind and eliminating dampness, removing blood stasis and acesodyne, removing toxicity for detumescence.It cures mainly：For rheumatic arthralgia, stomachache with cool feeling, traumatic injury, wound goes out
Blood；Boil, snakebite and bugbite are controlled outside.
Radix polygonati officinalis (Polygonatum odoratum (Mill.) Druce) is Liliaceae herbaceos perennial.Rhizome is horizontal
It walks, meat yellow-white, dense majority fibrous root.Blade face green, below grey.Flower axillary, usual 1-3 fasciation.West originating in China
Southern area, but wild distribution is very wide.It is cold-resistant, it is also shade tolerant, wet environment is liked, suitable growth is in the loose soil abundant containing humus
Earth.《Bencao jizhu》" stem is tetanic, like bamboo arrow shaft, there is section for cloud." therefore have the name of radix polygonati officinalis.The rhizome of plant can hyoscine, in
Medicine name is also radix polygonati officinalis, and autumn excavation is cleaned, shines to softness, rub repeatedly, and sunning to no hard-core is dried, or after steaming thoroughly, is rubbed
It is extremely translucent, it dries, cuts sheet or section is used.
Rhizopus oryzae (Rhizopus oryzae Went et Pr.Geerl.) be important mould in middle traditional Chinese medicines and distiller's yeast it
One.Also common on soil, air and other substances.Bacterium colony is loose or dense, becomes grey brown to dark brown after initial white, crawls
Flagellum is creeped, colourless.Rhizoid is flourishing, finger-like or root shape branch.Apophysis wedge shape, mycelia form chlamydospore, and zygosperm has no.
Commercial product can be used in raw material used in the present invention.
Beneficial effects of the present invention：
1, it when the present invention carries out Rhizopus oryzae solid state fermentation, directlys adopt and is incubated for through two step of liquid MRS culture medium containing NaCl
The Rhizopus oryzae seed liquor of processing, and radix polygonati officinalis and tuniclike psammosilene root are added into culture medium, can effectively by its nutritional ingredient and effectively
Ingredient carries out Biodegradation and biotransformation, helps to improve the content and activity of active constituent in final products.Step (4) and step
(5) in, Rhizopus oryzae product by solid-state fermentation is subjected to Subcritical Water Extraction, enables the activity maximum journey of effective component in extract
Degree retains, content is greatly improved；Again using residue extracted as the main culture medium of Hirsutella sinensis liquid fermentation, and
Using the Hirsutella sinensis seed liquor for being incubated for processing through two step of liquid MRS culture medium containing cholate, fermentation period is shortened, is improved
The yield of the fermentation secondary metabolite of Hirsutella sinensis, is recycled by integration, obtains each raw material resources sufficiently
Utilization, further improve the content and activity of active constituent in final products.
2, the method for the present invention raw material sources are natural, quality controllable, no pollution to the environment, are suitable for industrialized production.
3, compared with common Hirsutella sinensis tunning, tuniclike psammosilene root and Rhizoma Polygonati Odorati extract, Hirsutella sinensis hair of the present invention
Ferment culture composition is to CCl4Hepatic tissue alanine aminotransferase (ALT), aspartate transaminase in hepatic injury mice serum
(AST) reduction of content is more significant, to CCl4The improvement of hepatic injury mouse liver histopathology indices is more
Significantly, illustrate Hirsutella sinensis fermented and cultured composition and its soft gel products of the present invention, ALT can be significantly reduced and AST contains
Amount, and hepatic injury mouse liver histopathology indices are significantly improved, there is significant liver-protecting function, can be used to prepare tool
There are the health care product or drug of liver-protecting function.
Specific embodiment
The content of present invention is furtherd elucidate below with reference to some embodiments, but the contents of the present invention and not only
It is limited to the following examples.
Hirsutella sinensis (Hirsutella sinensis) strain, is purchased from BeNa Culture Collection Institute of Biotechnology.
30416 strain of Rhizopus oryzae (Rhizopus oryzae) ACCC, is purchased from Chinese agriculture Microbiological Culture Collection management
Center.
Cholate：No. 3 cholate, i.e. sodium taurocholate, cholic acid-NaTDC salt mixture (remote chemical industry in the perseverance industry of Beijing).
Embodiment 1
One, material prepares
PDA plate culture medium：Potato 200g, glucose 20g and agar 15g are settled to 1000ml, natural pH with water,
Sterilize 20min at 121 DEG C.
The Rhizopus oryzae strain of the Hirsutella sinensis strain of slant preservation, slant preservation is inoculated into PDA plate culture respectively
On base, activation culture is carried out, 22 DEG C of cultivation temperature, incubation time 10 days, respectively obtains the Hirsutella sinensis strain after activating, work
Rhizopus oryzae strain after change.
The group of liquid MRS culture medium becomes：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, sulphur
Sour manganese 0.05g and 1.0 liters of distilled water.
Fermentation basal medium consists of the following mass percentage components：Glucose 1%, K2HPO40.1%,
MgSO40.05% and surplus water.
1L PBS buffer solution：Potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 1.42g, sodium chloride 8g and potassium chloride 0.2g, add
Dissolution is sufficiently stirred in appropriate amount of deionized water, and concentrated hydrochloric acid tune pH to 7.4, last constant volume to 1L is then added.
Two, the preparation of Hirsutella sinensis fermented product
(1) 2cm is taken2Hirsutella sinensis strain after size activation is inoculated into 1L liquid MRS culture medium to be cultivated in 25 DEG C
10 days, culture solution collected the first thallus through 6000rpm centrifugation 10min；First thallus, which is suspended in cholate mass percentage, is
In 25 DEG C of incubation 10h in the 0.2% liquid MRS culture medium containing cholate, the second thallus is collected through 6000rpm centrifugation 10min；It will
Second thallus is resuspended in liquid MRS culture medium after PBS buffer solution cleans up, and concussion shakes up to obtain the second thallus
Second thallus liquid is inoculated into the liquid MRS containing cholate that cholate mass percentage is 0.2% by 5% inoculum concentration and trained by liquid
It supports and is further cultured in 22 DEG C 12 days in base, obtain Hirsutella sinensis seed liquor；
Wherein, the volume ratio of the first thallus and the liquid MRS culture medium containing cholate for first thallus that suspends is 1:1,
The volume ratio of second thallus and the liquid MRS culture medium for second thallus that suspends is 1:1.
(2) 1cm is taken2Rhizopus oryzae strain after size activation is inoculated into 1L liquid MRS culture medium in 25 DEG C of culture 7h, training
Nutrient solution collects third thallus through 6000rpm centrifugation 10min, and it is 5% to contain that third thallus, which is suspended in NaCl mass percentage,
In 25 DEG C of incubation 8h in the liquid MRS culture medium of NaCl, the 4th thallus is collected through 6000rpm centrifugation 10min；4th thallus is passed through
PBS buffer solution is resuspended in liquid MRS culture medium after cleaning up, and concussion shakes up to obtain the 4th thallus liquid, by the 4th bacterium
Body fluid is inoculated into the liquid MRS culture medium containing NaCl that NaCl mass percentage is 5% by 3% inoculum concentration and is trained in 25 DEG C
15h is supported, Rhizopus oryzae seed liquor is obtained；
Wherein, the volume ratio of third thallus and the liquid MRS culture medium containing NaCl for the third thallus that suspends is 1:1,
The volume ratio of 4th thallus and the liquid MRS culture medium for the 4th thallus that suspends is 1:1.
(3) Rhizopus oryzae seed liquor Rhizopus oryzae is accessed by the access amount for accounting for Rhizopus oryzae solid-state fermentation culture medium volume 15% to consolidate
In state fermentation medium, is cultivated 20 days at 25 DEG C, be subsequently placed in the alternating magnetic field that magnetic field strength is 3mT, field frequency is 25Hz
It is further cultured in environment 12 days, cultured products obtain Rhizopus oryzae product by solid-state fermentation after vacuum freeze drying, crushing；
The preparation of Rhizopus oryzae solid-state fermentation culture medium includes：Fresh tuniclike psammosilene root root, polygonati rhizoma are rinsed under tap water
Completely, it dries, cutting is 1cm-2cm long sheet or fritter；Then by 7g tuniclike psammosilene root root and the fermentation basis training of 2g polygonati rhizoma
Feeding base is settled to 1L, is uniformly mixed, pH value is naturally, obtain Rhizopus oryzae solid-state fermentation culture medium.
(4) Rhizopus oryzae product by solid-state fermentation be crushed into 40 meshes, through Subcritical Water Extraction, the condition of Subcritical Water Extraction
For：Water material mass ratio is 30:1, extracting pressure is in 15MPa, and extraction temperature is 150 DEG C, and extraction time 60min obtains a meter root
Mould fermentation subcritical abstraction object I, remaining residue obtains extraction residue II after vacuum freeze drying after extraction.
(5) Hirsutella sinensis seed liquor is accessed by the access amount for accounting for Hirsutella sinensis liquid fermentation medium volume 10%
In Hirsutella sinensis liquid fermentation medium, adjusting pH value is 5, is cultivated 15 days at 20 DEG C, is obtained through 6000rpm centrifugation 10min
State's coat spore tunning III；
The preparation of Hirsutella sinensis liquid fermentation medium includes：By 0.6g extraction residue II fermentation basal medium
It is settled to 1L, is uniformly mixed, pH value is naturally, obtain Rhizopus oryzae liquid fermentation medium.
Hirsutella sinensis fermented and cultured composition is fermented by 40 parts by weight Rhizopus oryzaes in subcritical abstraction object I and 20 parts by weight
State's coat spore tunning III forms.
Three, the preparation of soft gel products：The filler of soft capsule by 40 parts by weight Rhizopus oryzaes fermentation subcritical abstraction object I,
20 parts by weight Hirsutella sinensis tunnings III and 10 part by weight of vitamin E composition.
Embodiment 2
One, material prepares
PDA plate culture medium：Potato 200g, glucose 20g and agar 20g are settled to 1000ml, natural pH with water,
Sterilize 20min at 121 DEG C.
The Rhizopus oryzae strain of the Hirsutella sinensis strain of slant preservation, slant preservation is inoculated into PDA plate culture respectively
On base, activation culture is carried out, 20 DEG C of cultivation temperature, incubation time 25 days, respectively obtains the Hirsutella sinensis strain after activating, work
Rhizopus oryzae strain after change.
Fermentation basal medium consists of the following mass percentage components：Glucose 3%, K2HPO40.2%,
MgSO40.1% and surplus water.
Liquid MRS culture medium and PBS buffer solution are the same as embodiment 1.
Two, the preparation of Hirsutella sinensis fermented product
(1) 1cm is taken2Hirsutella sinensis strain after size activation is inoculated into 1L liquid MRS culture medium to be cultivated in 20 DEG C
15 days, culture solution collected the first thallus through 6000rpm centrifugation 10min；First thallus, which is suspended in cholate mass percentage, is
In 18 DEG C of incubation 20h in the 0.05% liquid MRS culture medium containing cholate, the second thallus is collected through 6000rpm centrifugation 10min；
Second thallus is resuspended in liquid MRS culture medium after PBS buffer solution cleans up, concussion shakes up to obtain the second thallus
Second thallus liquid is inoculated into the liquid MRS containing cholate that cholate mass percentage is 0.05% by 5% inoculum concentration and trained by liquid
It supports and is further cultured in 18 DEG C 20 days in base, obtain Hirsutella sinensis seed liquor；
Wherein, the volume ratio of the first thallus and the liquid MRS culture medium containing cholate for first thallus that suspends is 1:2,
The volume ratio of second thallus and the liquid MRS culture medium for second thallus that suspends is 1:2.
(2) 4cm is taken2Rhizopus oryzae strain after size activation is inoculated into 1L liquid MRS culture medium in 30 DEG C of culture 3h, training
Nutrient solution collects third thallus through 6000rpm centrifugation 10min, and it is 8% to contain that third thallus, which is suspended in NaCl mass percentage,
In 20 DEG C of incubation 15h in the liquid MRS culture medium of NaCl, the 4th thallus is collected through 6000rpm centrifugation 10min；By the 4th thallus
It is resuspended in after PBS buffer solution cleans up in liquid MRS culture medium, concussion shakes up to obtain the 4th thallus liquid, by the 4th
Thallus liquid is inoculated into the liquid MRS culture medium containing NaCl that NaCl mass percentage is 8% in 20 DEG C by 3% inoculum concentration
30h is cultivated, Rhizopus oryzae seed liquor is obtained；
Wherein, the volume ratio of third thallus and the liquid MRS culture medium containing NaCl for the third thallus that suspends is 1:3,
The volume ratio of 4th thallus and the liquid MRS culture medium for the 4th thallus that suspends is 1:3.
(3) Rhizopus oryzae seed liquor Rhizopus oryzae is accessed by the access amount for accounting for Rhizopus oryzae solid-state fermentation culture medium volume 25% to consolidate
In state fermentation medium, is cultivated 30 days at 20 DEG C, be subsequently placed in the alternating magnetic field that magnetic field strength is 0.2mT, field frequency is 3Hz
It is further cultured in environment 20 days, cultured products obtain Rhizopus oryzae product by solid-state fermentation after vacuum freeze drying, crushing；
The preparation of Rhizopus oryzae solid-state fermentation culture medium includes：Fresh tuniclike psammosilene root root, polygonati rhizoma are rinsed under tap water
Completely, it dries, cutting is 1cm-2cm long sheet or fritter；Again by 8.5g tuniclike psammosilene root root and 4.0g polygonati rhizoma fermentation basis
Culture medium is settled to 1L, is uniformly mixed, pH value is naturally, obtain Rhizopus oryzae solid-state fermentation culture medium.
(4) Rhizopus oryzae product by solid-state fermentation be crushed into 40 meshes, through Subcritical Water Extraction, the condition of Subcritical Water Extraction
For：Water material mass ratio is 50:1, extracting pressure is in 25MPa, and extraction temperature is 180 DEG C, and extraction time 10min obtains a meter root
Mould fermentation subcritical abstraction object I, remaining residue obtains extraction residue II after vacuum freeze drying after extraction.
(5) Hirsutella sinensis seed liquor is accessed by the access amount for accounting for Hirsutella sinensis liquid fermentation medium volume 20%
In Rhizopus oryzae liquid fermentation medium, adjusting pH value is 7, is cultivated 5 days at 25 DEG C, obtains Chinese coat through 6000rpm centrifugation 10min
Spore tunning III；
The preparation of Hirsutella sinensis liquid fermentation medium includes：The 1g extraction fermentation basal medium of residue II is determined
Hold to 1L, is uniformly mixed, pH value is naturally, obtain Hirsutella sinensis liquid fermentation medium.
Hirsutella sinensis fermented and cultured composition is fermented by 55 parts by weight Rhizopus oryzaes in subcritical abstraction object I and 35 parts by weight
State's coat spore tunning III forms.
Three, the preparation of soft gel products：The filler of soft capsule by 55 parts by weight Rhizopus oryzaes fermentation subcritical abstraction object I,
35 parts by weight Hirsutella sinensis tunnings III and 20 part by weight of vitamin E composition.
Embodiment 3
One, material prepares
PDA plate culture medium, liquid MRS culture medium and PBS buffer solution are the same as embodiment 1.
The Rhizopus oryzae strain of the Hirsutella sinensis strain of slant preservation, slant preservation is inoculated into PDA plate culture respectively
On base, activation culture is carried out, 30 DEG C of cultivation temperature, incubation time 3 days, respectively obtains the Hirsutella sinensis strain after activating, work
Rhizopus oryzae strain after change.
Fermentation basal medium consists of the following mass percentage components：Glucose 2%, K2HPO40.15%,
MgSO40.08% and surplus water.
Two, the preparation of Hirsutella sinensis fermented product
(1) 4cm is taken2Hirsutella sinensis strain after size activation, which is inoculated into 1L liquid MRS culture medium, cultivates 4 in 30 DEG C
It, culture solution collects the first thallus through 6000rpm centrifugation 10min；First thallus, which is suspended in cholate mass percentage, is
In 30 DEG C of incubation 1h in the 0.5% liquid MRS culture medium containing cholate, the second thallus is collected through 6000rpm centrifugation 10min；It will
Second thallus is resuspended in liquid MRS culture medium after PBS buffer solution cleans up, and concussion shakes up to obtain the second thallus
Second thallus liquid is inoculated into the liquid MRS containing cholate that cholate mass percentage is 0.5% by 5% inoculum concentration and trained by liquid
It supports and is further cultured in 30 DEG C 2 days in base, obtain Hirsutella sinensis seed liquor；
Wherein, the volume ratio of the first thallus and the liquid MRS culture medium containing cholate for first thallus that suspends is 1:3,
The volume ratio of second thallus and the liquid MRS culture medium for second thallus that suspends is 1:3.
(2) 2cm is taken2Rhizopus oryzae strain after size activation is inoculated into 1L liquid MRS culture medium in 20 DEG C of culture 30h,
Culture solution collects third thallus through 6000rpm centrifugation 10min, and it is 0.5% that third thallus, which is suspended in NaCl mass percentage,
The liquid MRS culture medium containing NaCl in 30 DEG C of incubation 0.5h, collect the 4th thallus through 6000rpm centrifugation 10min；By the 4th
Thallus is resuspended in liquid MRS culture medium after PBS buffer solution cleans up, and concussion shakes up to obtain the 4th thallus liquid, will
4th thallus liquid is inoculated into the liquid MRS culture medium containing NaCl that NaCl mass percentage is 0.5% by 3% inoculum concentration
In 30 DEG C of culture 3h, Rhizopus oryzae seed liquor is obtained；
Wherein, the volume ratio of third thallus and the liquid MRS culture medium containing NaCl for the third thallus that suspends is 1:2,
The volume ratio of 4th thallus and the liquid MRS culture medium for the 4th thallus that suspends is 1:2.
(3) Rhizopus oryzae seed liquor Rhizopus oryzae is accessed by the access amount for accounting for Rhizopus oryzae solid-state fermentation culture medium volume 5% to consolidate
In state fermentation medium, is cultivated 3 days at 30 DEG C, be subsequently placed in the alternating magnetic field ring that magnetic field strength is 6mT, field frequency is 40Hz
It is further cultured in border 5 days, cultured products obtain Rhizopus oryzae product by solid-state fermentation after vacuum freeze drying, crushing；
The preparation of Rhizopus oryzae solid-state fermentation culture medium includes：Fresh tuniclike psammosilene root root, polygonati rhizoma are rinsed under tap water
Completely, it dries, cutting is 1cm-2cm long sheet or fritter；Again by 5.5g tuniclike psammosilene root root and 0.2g polygonati rhizoma fermentation basis
Culture medium is settled to 1L, is uniformly mixed, pH value is naturally, obtain Rhizopus oryzae solid-state fermentation culture medium.
(4) Rhizopus oryzae product by solid-state fermentation be crushed into 40 meshes, through Subcritical Water Extraction, the condition of Subcritical Water Extraction
For：Water material mass ratio is 10:1, extracting pressure is in 4MPa, and extraction temperature is 100 DEG C, and extraction time 90min obtains Rhizopus oryzae
Ferment subcritical abstraction object I, and remaining residue obtains extraction residue II after vacuum freeze drying after extraction.
(5) Hirsutella sinensis seed liquor is accessed by the access amount for accounting for Hirsutella sinensis liquid fermentation medium volume 3%
In Rhizopus oryzae liquid fermentation medium, adjusting pH value is 3, is cultivated 25 days at 15 DEG C, obtains Chinese quilt through 6000rpm centrifugation 10min
Hair spore tunning III；
The preparation of Hirsutella sinensis liquid fermentation medium includes：By 0.2g extraction residue II fermentation basal medium
It is settled to 1L, is uniformly mixed, pH value is naturally, obtain Hirsutella sinensis liquid fermentation medium.
Hirsutella sinensis fermented and cultured composition is fermented by 50 parts by weight Rhizopus oryzaes in subcritical abstraction object I and 25 parts by weight
State's coat spore tunning III forms.
Three, the preparation of soft gel products：The filler of soft capsule by 50 parts by weight Rhizopus oryzaes fermentation subcritical abstraction object I,
25 parts by weight Hirsutella sinensis tunnings III and 15 part by weight of vitamin E composition.
Comparative example 1
One, material prepares
Hirsutella sinensis strain, liquid MRS culture medium and fermentation basal medium after PDA plate culture medium, activation is equal
With embodiment 1.
Two, the preparation of Hirsutella sinensis fermented product
(1) 2cm is taken2Hirsutella sinensis strain after size activation, which is inoculated into 1L liquid MRS culture medium, cultivates 5 in 25 DEG C
It, obtains Hirsutella sinensis liquid seeds liquid.
(2) by Hirsutella sinensis liquid seeds liquid by the access amount access fermentation base for accounting for fermentation basal medium volume 15%
It in basal culture medium, is cultivated 10 days at 25 DEG C, cultured products obtain Hirsutella sinensis tunning after vacuum freeze drying, crushing；
Three, the preparation of soft gel products：The filler of soft capsule is by 40 parts by weight Hirsutella sinensis tunnings and 10 weights
Measure part vitamin E composition.
2 tuniclike psammosilene root of comparative example and Rhizoma Polygonati Odorati extract
Fresh tuniclike psammosilene root root and polygonati rhizoma are rinsed well under tap water, dried, cutting be 1cm-2cm long sheet or
Fritter；By feed liquid weight ratio 1:10 add water, extract 10h under fluidized state, filter to obtain first-time filtrate and a filter residue, will be primary
Filter residue presses feed liquid weight ratio 1:5 plus water, 8h is extracted under fluidized state, filters to obtain secondary filtrate, merges first-time filtrate and secondary
Filtrate obtains tuniclike psammosilene root and Rhizoma Polygonati Odorati extract after freeze-dried, crushing.
The preparation of soft gel products：The filler of soft capsule is by 40 parts by weight tuniclike psammosilene roots and Rhizoma Polygonati Odorati extract and 10 parts by weight
Vitamin E composition.
The measurement of active constituent
(1) measurement of the polysaccharide content
Appropriate amount of sample dry powder is weighed, 40 mesh is smashed it through, is placed in a beaker, by solid-liquid ratio 1:Distillation is added in 10 (mass ratioes)
Water, 90 DEG C of extraction 3h are extracted twice, are filtered under diminished pressure and are collected filtrate, be concentrated under reduced pressure at 50 DEG C, it is anhydrous that 4 times of volumes are added in concentrate
Ethyl alcohol, alcohol precipitation overnight, after 3000r/min, 20min centrifugation, precipitating is Thick many candies.Precipitating is settled to 50mL with distilled water,
Polysaccharide in fermentation liquid content is measured with phend-sulphuric acid, is averaged for replication 3 times, i.e. polyoses content.
(2) adenosine and cordycepin content
Dry adenosine and each 4mg of cordycepin standard items (being accurate to 0.0001g) to constant mass is accurately weighed in 50mL
In volumetric flask, uses water dissolution and constant volume is as standard reserving solution, mass concentration is 80 μ g/mL.Be diluted to 2 respectively with ultrapure water,
5, the adenosine standard liquid and cordycepin standard solution of 10,20,40,80 μ g/mL.
Sample is placed in 40 DEG C of drying boxes and is dried, smashes it through 200 meshes through pulverizer, is accurately weighed dry to quality
Constant sample 0.50g is added ultrasonic extraction after 25mL extracting solution, is placed in centrifuge tube after extraction in 50mL centrifuge tube
20 DEG C, be centrifuged 10min, Aspirate supernatant 1mL in the centrifuge of 5000r/min, it is straight after 0.45 μm of water phase filtering with microporous membrane
Injection sample injection bottle is connect for analysis.
Chromatographic column：XBridgeTM C18 (4.6mm × 250mm, 5 μm)；Mobile phase：Ultrapure water-methanol (volume ratio 85:
15)；Flow velocity：1.0mL/min；Sample volume：10μL；Input mode：Automatic sampling；Column temperature：30℃；Detection wavelength：254nm.
(3) measurement of mannitol content：
Mannitol reference substance 500mg is taken, it is accurately weighed, it sets in 100ml measuring bottle, adds 70% methanol of mass percentage concentration water-soluble
Liquid dissolves and is diluted to scale.It shakes up, as reference substance stock solution.
The sample for weighing 2.0g is set in round-bottomed flask, and 70% methanol aqueous solution refluxing extraction of 50ml mass percentage concentration is added
30min.Filtrate is filtered in postposition 50ml volumetric flask, with 70% methanol aqueous solution constant volume of mass percentage concentration to scale.It shakes up, i.e.,
?.
Chromatographic condition：Nh 2 column (5 μm of UG, 250mm × 4.6mm)；Acetonitrile-water (volume ratio 81：19) solution is flowing
Phase；Flow velocity is 1.0ml/min；Drift tube temperature is 90 DEG C；Carrier gas (air) pressure is 2.75Bar；20 μ l of sample volume.
Each embodiment and comparative example main component testing result of table 1
Note：Compared with comparative example 1, comparative example 2, Δ：P<0.05.
The data of table 1 show that Hirsutella sinensis fermented and cultured composition of the present invention significantly improves more in product simultaneously
The content of sugar, adenosine, cordycepin, mannitol isoreactivity ingredient.Compared with 1 conventional culture methods of comparative example, using present invention side
Polyoses content improves 47.9%-52.1% in the Hirsutella sinensis fermented and cultured composition that method obtains, and adenosine content improves
23.1%-38.5%, cordycepin content improve 33.3%-53.3%, and mannitol content improves 30.9%-40%.
Liver-protecting function test
Experimental animal：Male mice in kunming, 18 ± 2g of weight.Sub-cage rearing, free diet, for trying after observation 3 days
It tests.
Experimental design：According to《Health food is examined and assessment technique specification》(2003 editions) evaluation criterion carries out, if low,
Middle and high 3 dose of test groups and 1 negative Basal control group and 1 model control group, basic, normal, high 3 dosage groups distinguish phase
When in 5 times, 10 times, 20 times of people's maximum recommended dosage, i.e., 0.15,0.30,0.60g/kgbw/d, basic, normal, high 3 agent
Measuring compound concentration is respectively 15,30,60mg/mL.Basal control group and model control group give distilled water, all experimental animals
Edible full nutrition mixed feed, drinking-water of freely ingesting.
The tested material of mouse corresponding dosage is orally given once a day, and intragastric administration on mice amount is 10mL/kgbw, continuous gavage
30d, in experiment the 30th day by groups of animals fasting 16h overnight.1%CCl is given in model control group and a tested group of stomach-filling4It plants
Object oil solution (5mL/kgBW), Basal control group give vegetable oil, and tested group is continued to give composition made from embodiment, right
Extract to experiment terminates (with CCl in Hirsutella sinensis tunning or comparative example 2 in ratio 14Stomach-filling interval 4h), it gives
CCl4Mouse wins eyeball and takes blood after for 24 hours, and measurement Serum ALT, AST are horizontal, and liver is taken to carry out histopathological examination.
Experimental result：
1. influence of the composition made from each embodiment to mouse weight
As shown in Table 2, single factor test variance is carried out to the original body mass of each group mouse test/end weight (30d) mean value
Analysis, no significant difference (P>0.05), the results showed that product is to animal in composition made from each embodiment and comparative example
Weight, which has no, to be significantly affected.
2. couple CCl4The influence of hepatic injury mice serum ALT and AST
As shown in Table 3, ALT, AST content of model control group mouse are higher than negative Basal control group, and statistical analysis is poor
Different statistically significant (P < 0.05), composition 0.15 made from embodiment, 0.30, the ALT of 0.60g/kgbw/d dosage group
Value is lower than model control group, and statistical analysis difference is statistically significant (P < 0.05)；The AST value of low middle high dose group is lower than
Model control group, statistical analysis difference are statistically significant (P < 0.05).The result shows that composition made from embodiment 1-3
When being equivalent to 5 times, 10 times of human body recommended intake and 20 multiple dose, having reduces chemical damage mice serum ALT
With the effect of AST, and under same dose composition indices made from embodiment 1-3 compared with than comparative example 1 and comparative example 2
With significant difference (P < 0.05), show the present composition compared with the product that conventional culture methods, extracting method obtain
Protect liver effect is more obvious.
3. composition is to CCl made from embodiment4The influence of hepatic injury mouse liver histopathology
As shown in Table 4, one-way analysis of variance is carried out to the mean value of each group mouse liver histopathological indications quantized value,
Indices with the poor different significance of model control group.The result shows that composition made from embodiment 1-3 is suitable
When 5 times, 10 times of human body recommended intake and 20 multiple dose, zoopery pathological examination is the positive, shows that it has and mitigates
The pathologic lesion of chemical damage mouse liver acts on, and composition items made from embodiment 1-3 refer under same dose
Mark has significant difference (P < 0.05) compared with than comparative example 1 and comparative example 2, shows the present composition and routine culture
The product that method, extracting method obtain is more obvious compared to protect liver effect.
Influence of the capsule to mouse weight made from 2 embodiment and comparative example of table
Influence of the capsule to mice serum ALT and AST made from 3 embodiment and comparative example of table
| Group | AST(U) | ALT(U) |
| Basal control group | 109.32±16.3 | 97.22±3.2 |
| Model control group | 411.23±17.3 | 317.46±4.1 |
| 1 low dose group of embodiment | 363.54±18.4 | 218.23±3.9 |
| 1 middle dose group of embodiment | 306.58±19.3 | 197.31±5.1 |
| 1 high dose group of embodiment | 273.21±15.9 | 179.52±4.9 |
| 2 low dose group of embodiment | 358.53±18.2 | 232.58±4.2 |
| 2 middle dose group of embodiment | 301.26±16.9 | 200.19±4.5 |
| 2 high dose group of embodiment | 257.31±18.2 | 188.52±4.0 |
| 3 low dose group of embodiment | 349.56±16.2 | 249.35±5.2 |
| 3 middle dose group of embodiment | 289.26±18.7 | 222.23±4.0 |
| 3 high dose group of embodiment | 247.54±17.4 | 191.32±5.2 |
| 1 low dose group of comparative example | 391.23±16.5 | 292.34±4.8 |
| 1 middle dose group of comparative example | 380.21±16.8 | 270.56±5.9 |
| 1 high dose group of comparative example | 365..36±18.2 | 259.32±6.2 |
| 2 low dose group of comparative example | 385.12±19.3 | 291.16±3.5 |
| 2 middle dose group of comparative example | 372.46±17.3 | 269.85±4.7 |
| 2 high dose group of comparative example | 359.37±18.5 | 262.31±6.1 |
Capsule made from 4 embodiment of table is to CCl4The influence of hepatic injury mouse liver histopathology
The variation of each parameter has no effect on Chinese coat of the invention in the range of inventive formulation and preparation method limit
Improvement effect and preparation of spore fermented and cultured composition and its soft gel products chemical damage, therefore inventive formulation and system
Hirsutella sinensis fermented and cultured composition and its soft gel products can be obtained in the combination of arbitrary parameter in Preparation Method.Herein no longer
It repeats.
Claims (7)
1. a kind of Hirsutella sinensis fermentation culture method, which is characterized in that including step：
(1) the Hirsutella sinensis strain after taking activation, which is inoculated into liquid MRS culture medium, to be cultivated -15 days 4 days, is collected by centrifugation first
Thallus；First thallus is suspended in the liquid MRS culture medium containing cholate in 18 DEG C of -30 DEG C of incubation 1h-20h, is collected by centrifugation
Two thallus；Second thallus is resuspended in liquid MRS culture medium after PBS buffer solution cleans up, concussion shakes up to obtain
Second thallus liquid is inoculated into the liquid MRS culture medium containing cholate and is further cultured for 2 days -20 in 18 DEG C -30 DEG C by the second thallus liquid
It, obtains Hirsutella sinensis seed liquor；
In step (1), the mass percentage of cholate is 0.05%-0.5% in the liquid MRS culture medium containing cholate；
(2) the Rhizopus oryzae strain after taking activation, which is inoculated into liquid MRS culture medium, cultivates 3h-30h, and third thallus is collected by centrifugation,
Third thallus is suspended in the liquid MRS culture medium containing NaCl in 20 DEG C of -30 DEG C of incubation 0.5h-15h, the 4th bacterium is collected by centrifugation
Body；4th thallus is resuspended in liquid MRS culture medium after PBS buffer solution cleans up, concussion shakes up to obtain the 4th
4th thallus liquid is inoculated into the liquid MRS culture medium containing NaCl in 20 DEG C of -30 DEG C of culture 3h-30h, obtains rice by thallus liquid
Head mold seed liquor；
In step (2), the mass percentage of the liquid MRS NaCl in medium containing NaCl is 0.5%-8%；
(3) will step (2) Rhizopus oryzae seed liquor access Rhizopus oryzae solid-state fermentation culture medium in, 20 DEG C -30 DEG C cultivate 3 days -
It 30 days, is subsequently placed in alternating magnetic field environment and is further cultured for -20 days 5 days, cultured products get meter Gen after vacuum freeze drying, crushing
Mould product by solid-state fermentation；Contain tuniclike psammosilene root and radix polygonati officinalis in the Rhizopus oryzae solid-state fermentation culture medium；
It is beautiful containing 5.5g-8.5g tuniclike psammosilene root and 0.2g-4.0g in the every 1L of Rhizopus oryzae solid-state fermentation culture medium in step (3)
Bamboo；
(4) the Rhizopus oryzae product by solid-state fermentation of step (3) is obtained into Rhizopus oryzae fermentation subcritical abstraction object through Subcritical Water Extraction
I, remaining residue obtains extraction residue II after vacuum freeze drying after extraction；
(5) by the Hirsutella sinensis seed liquor access Hirsutella sinensis liquid fermentation medium of step (1), tune pH value is 3-7,
It is cultivated -25 days 5 days at 15 DEG C -25 DEG C, centrifugation obtains Hirsutella sinensis tunning III；The Hirsutella sinensis liquid fermentation
Contain extraction residue II in culture medium；
In step (5), residue II is extracted containing 0.2g-1g in the every 1L of Hirsutella sinensis liquid fermentation medium；
Hirsutella sinensis fermented and cultured composition is Rhizopus oryzae fermentation subcritical abstraction object I and Hirsutella sinensis tunning III.
2. Hirsutella sinensis fermentation culture method according to claim 1, which is characterized in that in step (1), described first
The volume ratio of thallus and the liquid MRS culture medium containing cholate for first thallus that suspends is 1:1-3, the second thallus with for hanging
The volume ratio of the liquid MRS culture medium of floating second thallus is 1:1-3, the inoculum concentration of the second thallus liquid are 5%；
In step (2), the volume ratio of the third thallus and the liquid MRS culture medium containing NaCl for the third thallus that suspends is
1:The volume ratio of 1-3, the 4th thallus and the liquid MRS culture medium for the 4th thallus that suspends is 1:1-3, the 4th thallus liquid connect
Kind amount is 3%.
3. Hirsutella sinensis fermentation culture method according to claim 1, which is characterized in that in step (3), the alternation
Magnetic field strength is 0.2mT-6mT, field frequency 3Hz-40Hz in magnetic field environment；
In step (4), the condition of the Subcritical Water Extraction is：Water material mass ratio is 10-50:1, extracting pressure is in 4MPa-
25MPa, extraction temperature are 100 DEG C -180 DEG C, extraction time 10min-90min.
4. Hirsutella sinensis fermentation culture method according to claim 1, which is characterized in that in step (3), Rhizopus oryzae kind
The access amount of sub- liquid is the 5%-25% of Rhizopus oryzae solid-state fermentation culture medium volume；
In step (5), the access amount of the Hirsutella sinensis seed liquor is Hirsutella sinensis liquid fermentation medium volume
3%-20%.
5. a kind of Hirsutella sinensis fermented and cultured composition, which is characterized in that use the described in any item China of claim 1-4
Coat spore fermentation culture method culture obtains.
6. Hirsutella sinensis fermented and cultured composition according to claim 5, which is characterized in that the composition is by 40 weights
Measure -55 parts by weight Rhizopus oryzae of part fermentation subcritical abstraction object I and -35 III group of parts by weight Hirsutella sinensis tunning of 20 parts by weight
At.
7. a kind of application of Hirsutella sinensis fermented and cultured composition, which is characterized in that according to claim 5 or 6
Application of state's coat spore fermented and cultured composition in the health care product or drug of preparation protect liver.
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| CN103432174A (en) * | 2013-08-27 | 2013-12-11 | 王辉 | Method for producing cordyceps sinensis powder by virtue of liquid fermentation on mixed bacterial strains |
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