CN107557305A - One kind fermentation synthesis13The method of C flag aweto mycelium - Google Patents
One kind fermentation synthesis13The method of C flag aweto mycelium Download PDFInfo
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Abstract
The present invention relates to one kind fermentation synthesis13The method of C flag aweto mycelium, using Chinese hair spore as starting strain,13C glucose is carbon source, dries, obtains through shake flask fermentation, centrifugation13The mycelium of C flag.Compared with prior art, the inventive method obtains the products such as cordyceps mycelia and Cordyceps sinensis polysaccharide, the cordycepin of isotope marks.This technique is simple, and raw material sources are easy, heavy metal free pollution.
Description
Technical field
The invention belongs to biology, medicine, chemical field, and in particular to the side of fermentation synthesis 13C mark aweto myceliums
Method.
Background technology
Cordyceps sinensis (plantworms or entomophyte) also known as cordyceps sinensis, it is by gang pyrenomycetes Clavicipitaceae cordyceps sinensis
The aweto of category parasitizes the Hepialus larva in alpine meadow soil, larva is ossify, and winter low temperature dry soil keeps worm
Shape is constant (winter worm), under summer warm and humid suitable condition, goes out long bar-shaped ascostome by bombys batryticatus head end pumping and bassets and shape
It is the complex that the fructification of aweto is formed with bombys batryticatus sclerotium (larva corpse) into (summer grass).It is main to originate in Chinese green grass or young crops
Sea, Tibet, Sichuan, Yunnan, Gansu, Guizhou etc. save and 3000 meters of the height above sea level of autonomous region to 5000m severe cold areas snow line near
On careless slope.
Due to its experienced fungal infection bat moth larvae, symbiosis and carry out complex material conversion, mycelia is full of and grows
Stroma, the process for growing up to cordyceps sinensis, many special bioactive substances are formed, cordyceps sinensis is provided with the medicinal of preciousness
Value.
Wherein, Cordyceps sinensis polysaccharide, cordycepin, cordycepic acid etc. can play enhancing immunity of organisms, antibacterial, hypotensive, softening blood
Pipe, eliminating phlegm and relieving asthma and other effects.
Cordyceps sinensis can also be cultivated manually in addition to wild growth, wherein mainly having artificial cultivation and mycelium fermentation
Two kinds.
Artificial cultivation：
Shen Nanying professors have been successfully separated out real Anamorph of Cordyceps Sinensis strain bat in nineteen eighty-three as first
Moth hair spore, and grown and the duplicate stroma of wild cordyceps in triangular flask；Liu's tin Jin, Guo Ying are estranged within 1989
Separate out with Shen Nan scruples from the consistent aweto strain of Hirsutella hepiali Chen et Shen kind, finally named as Hirsutella sinensis
(Hirsutella sinensis), i.e. aweto strain.Mianyang City edible mushroom research institute of Sichuan Province the Spring Festival in 2007 first
Turn out a cordyceps sinensis.
According to the literature, the various chemical composition analysis of ferment cordyceps sinensis D-mannitol and natural cordyceps show, their institutes
Essentially identical containing compound, this instead of cordyceps sinensis using cordyceps mycelia just to provide certain reference.
The content balance (mg/g) of cordyceps sinensis and the mycelium adenosine manually cultivated and cordycepin
Sun Linjie etc., by orthogonal test, is obtained with 4% glucose, 2% beans using aweto Csll to go out bacterium germination
Optimization culture based formulas based on cake powder, 0.2% potassium dihydrogen phosphate and 0.075% magnesium sulfate；Wang Zuhua etc. is to pick up from green grass or young crops
The fresh cordyceps sinensis in sea is mycelium source, is obtained by optimization with 7.5% glucose, 7.5% sucrose, 0.12% egg
White peptone, 0.375% yeast extract, 0.15% potassium dihydrogen phosphate, 0.075% magnesium sulfate and 0.015%VB1 culture medium；Liu Jin
Flower, using 5L fermentation tank cultures, obtains the dry of 17.5g/L using glucose, yeast extract and phosphatic culture medium is optimized
Thalline；Wang Can obtains glucose 2% by optimizing, lactoalbumin hydrolysate 0.5%, the worm summer in winter of dusty yeast 0.15 and milk 20%
Careless culture medium, and for the culture of liquid fermentation.
Chinese patent CN104145719B discloses a kind of aweto mycelium fermentation production method, including worm summer in winter
The preparation of careless strains liquid fermentation special culture media, mycelium introduces a collection preferably and by strain are linked into the step such as fermentation in culture medium
Suddenly；Wherein, mycelium introduces a collection is preferably to be inoculated into the aweto strain mycelium after more than 10 times artificial passages
On artificial solid medium, advantage fructification is selected, preferable Chinese hair spore bacterium, wherein solid are isolated from fructification
The preparation of matrix：Weigh rice 10-40 parts, corn flour 10-30 parts, wheat 15-35 parts, sorghum 10-30 parts, oatmeal 5-15
Part, wheat bran 5-15 parts；The preparation of nutrient solution：Glucose 10-30g/L, sucrose 10-20g/L, peptone 10-20g/L, yeast extract
5-20g/L, composite aminophenol powder 5-20g/L, potassium dihydrogen phosphate 0.1-0.5g/L, dipotassium hydrogen phosphate 0.1-0.5g/L, magnesium sulfate
0.1-0.5g/L, vitamin B1 5mg-50mg/L, vitamin B2 5mg-50mg/L, vitamin B6 10mg-50mg/L, silkworm chrysalis
Or tussah chrysalis tissue fluid 50-150g/L；The preparation of aweto screening and culturing medium：By solid matrix and nutrient solution by weight
Example 1:1.4-1.8 is prepared.
Chinese patent CN104350949B discloses a kind of cordyceps sinensis liquid-state fermentation technology, as follows the step of the technique：
(1) inclined-plane culture：Preservation aweto strain is inoculated in slant medium, 25 DEG C of incubated 5d；(2) seed liquor culture：
The strain of activation is inoculated in seed culture medium, rotating speed 140r/min, 24 DEG C of temperature shaking table in cultivate 3d；(3) ferment
Culture：Fermentation medium is added in 500mL conical flask, liquid amount 100mL, liquid strain is accessed by 3%v/v inoculum concentrations
Son, zymotic fluid initial pH value are 6.51, and in 24 DEG C of temperature, shaking speed cultivates 4d, wherein grape under conditions of being 140r/min
Sugared usage amount is 40.84g/L, peptone usage amount is 21.55g/L.Slant medium volume ratio g/mL by weight meter by
Following components forms：Potato juice 20%, glucose 2%, agar 2%, KH2PO40.3%th, MgSO4·7H2O 0.15%, its
Yu Weishui.Seed culture medium volume ratio g/mL by weight meters are composed of the following components：Potato juice 20%, glucose 2%,
KH2PO40.3%th, MgSO4·7H2O 0.15%, remaining is water.The fermentation medium based on volume ratio g/mL by weight by
Following components forms：Glucose 2%, peptone 1%, KH2PO40.3%th, MgSO4·7H2O 0.15%, vitamin
B10.08%, remaining is water.
As can be seen that the artificial culture of existing cordyceps sinensis, is typically necessary using several kinds of carbon source, as glucose, sucrose,
Potato juice, glutamic acid, glycine, fish meal, yellow meal worm worm oil etc., in order to study the performance of cordyceps sinensis, the present invention
Employ isotope-labelling method, but the synthetic method of common cordyceps sinensis, carbon source is numerous, can not carry out accurate quantitative analysis mark
Know, it is also difficult to obtain the cordyceps sinensis of high abundance.Therefore, the tracer in terms of being not suitable for use in about natural cordyceps is ground
Study carefully.
The content of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of technique is simple, raw material
Source is easy, the fermentation synthesis of heavy metal free pollution13The method of C flag aweto mycelium.
The purpose of the present invention can be achieved through the following technical solutions：One kind fermentation synthesis13C flag Chinese caterpillar fungus bacterial filament
The method of body, it is characterised in that using Chinese hair spore as starting strain,13C glucose is carbon source, is dried through shake flask fermentation, centrifugation
It is dry, obtain13The mycelium of C flag.
Described method specifically includes following steps：
(1) one-level culture：The Chinese ring of hair spore one is accessed in liquid fermentation medium, 105-120 DEG C of sterilizing 15-
At 30min, 15-25 DEG C, 0-200r/min Shaking cultures 288-720h；
(2) two level culture：Zymotic fluid accesses the liquid fermentation medium with 5-30% inoculum concentration obtained by step (1)
In, continue to cultivate 120-300h as secondary medium, 4000r/min centrifugation 10-30min, sediment is in 60 DEG C of vacuum
Dry, obtain13The aweto mycelium of C flag.
Liquid fermentation medium used in one-level culture and two level culture includes：13C flag glucose 1-46g/L, peptone
0.1-7.66g/L, yeast extract 0.1-8.27g/L, dipotassium hydrogen phosphate 0.2-2.13g/L, magnesium sulfate 0.1-1.1g/L, remaining is steaming
Distilled water.
The volume of described shaking flask is 500ml, and liquid amount 20-200ml, cultivation temperature is 15-25 DEG C.
The volume of described shaking flask is 500ml, and the preferred 100ml of liquid amount, preferably 18 DEG C of cultivation temperature, shaking flask rotating speed is preferred
100r/min, cultivate 8 days.
What the above method obtained13The mycelium of C flag may also pass through further extraction and obtain13C flag it is mycelial
Associated extraction thing, such as Cordyceps sinensis polysaccharide, specific method are as follows：
Gained13The mycelium of C flag extracts 2 times after ethanol solution backflow 1-6h extractions, then through water backflow 1-6h, merges
Extract solution, concentrated, crystallization, drying obtain Cordyceps sinensis polysaccharide crude product.
Supernatant of the zymotic fluid after centrifugation adds 1-5 times of ethanol, through left undisturbed overnight, filtering, obtains Cordyceps sinensis polysaccharide and slightly produces
Product.
Crude product after merging is concentrated, dried, obtain again through de- albumen, decolouring, preparation chromatographic isolation13The cordyceps sinensis of C flag is more
Sugar.
The modes such as above-mentioned concentration, crystallization, drying, filtering, de- albumen, decolouring, preparation chromatographic isolation are the routine of this area
Condition and method.
Compared with prior art, the present invention has advantages below：
(1) what the present invention synthesized is cold labeling13The cordyceps sinensis product of C flag, rather than common winter worm
Summer grass product；
(2) due to synthesize13C flag product, therefore required medium component is different, mainly carbon source is different.Need
It can incite somebody to action13C isotopes are incorporated into product13C materials as culture medium raw material, due to13C glucose is to be best suitable for microorganism
Carbon source required for growth, and one of raw material for being conventionally synthesized of applicant, therefore source is easier to.Potato juice fish meal, Huang
Although mealworm oil can also be used as carbon source, their complicated components can not obtain13C flag.
(3) in order to synthesize high abundance13The cordyceps sinensis product of C flag, is preferably selected13C flag carbon source is as sole carbon
Source, otherwise other non-marked carbon sources, which have (such as potato juice), will cause product abundance to dilute, although glutamic acid and glycine have13C flag thing, but product cost can be increased after adding, therefore only use13C glucose is used as same position as sole carbon source
Plain raw material, can just synthesize the product that abundance is more than 98%, and the abundance belongs to high abundance product in product is sold.
(4) using the inventive method synthesis mark cordyceps sinensis with natural cordyceps compared with, without because air with
The pollution in soil causes the problem of natural cordyceps heavy metals exceeding standard, the inventive method heavy metal free raw material, therefore marks production
Product do not have impact of heavy metals naturally.
Embodiment
Embodiments of the invention are elaborated below, the present embodiment is carried out lower premised on technical solution of the present invention
Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
The access of the Chinese ring of hair spore one is fitted into the shaking flask of 100ml liquid fermentation mediums, culture medium includes (1L)：13C
Labelled glucose 1g, peptone 2.8g, yeast extract 5.6g, dipotassium hydrogen phosphate 1.0g, magnesium sulfate 0.5g, remaining is distilled water, 120
DEG C sterilizing 20min.16 DEG C of 100rpm/min Shaking cultures 432h.Above-mentioned liquid fermentation medium is accessed with 10% inoculum concentration
In, continue to cultivate 168h as secondary medium.Zymotic fluid 4000r/min is centrifuged into 20min, washing of precipitate, 60 DEG C true
Sky is dried, and obtains 6.5g13The aweto mycelium of C flag.1g mycelium are flowed back through ethanol solution after 4h, then through water backflow 3h
Extraction 2 times, merges extract solution, and concentrated, crystallization, drying obtain 0.21g Cordyceps sinensis polysaccharide crude products.Zymotic fluid is upper after centrifugation
Clear liquid adds 3 times of ethanol, through left undisturbed overnight, precipitation, filtering, obtains 0.36g Cordyceps sinensis polysaccharide crude products.Crude product after merging passes through again
De- albumen, decolouring, chromatographic isolation is prepared, concentrate, drying, obtain 0.43g13The Cordyceps sinensis polysaccharide of C flag.Purity and abundance are all higher than
90%.
Embodiment 2
The access of the Chinese ring of hair spore one is fitted into the shaking flask of 150ml liquid fermentation mediums, culture medium includes (1L)：13C
Labelled glucose 4g, peptone 7.66g, yeast extract 1.0g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 1.1g, remaining is distilled water,
120 DEG C of sterilizing 20min.22 DEG C of 150rpm/min Shaking cultures 360h.Above-mentioned liquid fermentation is accessed with 10% inoculum concentration to train
Support in base, continue to cultivate 120h as secondary medium.By zymotic fluid 4000r/min centrifuge 20min, washing of precipitate, 60
DEG C vacuum drying, obtain 5.2g13The aweto mycelium of C flag.1g mycelium are flowed back through ethanol solution after 4h, then are returned through water
Flow 3h to extract 2 times, merge extract solution, concentrated, crystallization, drying obtain 0.21g Cordyceps sinensis polysaccharide crude products.Zymotic fluid is after centrifugation
Supernatant add 3 times of ethanol, through left undisturbed overnight, precipitation, filtering, obtain 0.36g Cordyceps sinensis polysaccharide crude products.Crude product after merging
Again through de- albumen, decolouring, preparation chromatographic isolation, concentrate, drying, obtain 0.43g13The Cordyceps sinensis polysaccharide of C flag.Purity and abundance are equal
More than 90%.
Embodiment 3
The access of the Chinese ring of hair spore one is fitted into the shaking flask of 200ml liquid fermentation mediums, culture medium includes (1L)：13C
Labelled glucose 46g, peptone 5.2g, yeast extract 2.7g, dipotassium hydrogen phosphate 0.2g, magnesium sulfate 0.75g, remaining is distilled water,
120 DEG C of sterilizing 20min.18 DEG C of static Shaking culture 720h.Accessed with 10% inoculum concentration in above-mentioned liquid fermentation medium,
Continue to cultivate 240h as secondary medium.Zymotic fluid 4000r/min is centrifuged into 20min, washing of precipitate, 60 DEG C of vacuum are done
It is dry, obtain 7.6g13The aweto mycelium of C flag.1g mycelium are flowed back through ethanol solution after 4h, then through water backflow 3h extractions
2 times, merge extract solution, concentrated, crystallization, drying obtain 0.21g Cordyceps sinensis polysaccharide crude products.Supernatant of the zymotic fluid after centrifugation
3 times of ethanol are added, through left undisturbed overnight, precipitation, filtering, obtain 0.36g Cordyceps sinensis polysaccharide crude products.Crude product after merging is again through de- egg
In vain, decolourize, prepare chromatographic isolation, concentrate, drying, obtain 0.43g13The Cordyceps sinensis polysaccharide of C flag.Purity and abundance are all higher than
90%.
Embodiment 4
The access of the Chinese ring of hair spore one is fitted into the shaking flask of 120ml liquid fermentation mediums, culture medium includes (1L)：13C
Labelled glucose 15g, peptone 0.1g, yeast extract 8.27g, dipotassium hydrogen phosphate 1.8g, magnesium sulfate 0.8g, remaining is distilled water,
120 DEG C of sterilizing 20min.25 DEG C of 200rpm/min Shaking cultures 288h.Above-mentioned liquid fermentation is accessed with 10% inoculum concentration to train
Support in base, continue to cultivate 192h as secondary medium.By zymotic fluid 4000r/min centrifuge 20min, washing of precipitate, 60
DEG C vacuum drying, obtain 3.8g13The aweto mycelium of C flag.1g mycelium are after ethanol solution refluxing extraction, then through water
Refluxing extraction 2 times, merges extract solution, and concentrated, crystallization, drying obtain 0.21g Cordyceps sinensis polysaccharide crude products.Zymotic fluid is after centrifugation
Supernatant add 3 times of ethanol, through it is static, precipitation, filtering, obtain 0.36g Cordyceps sinensis polysaccharide crude products.Crude product again through de- albumen,
Decolourize, prepare chromatographic isolation, concentrate, drying, obtain 0.43g13The Cordyceps sinensis polysaccharide of C flag.Purity and abundance are all higher than 90%.
Embodiment 5
The access of the Chinese ring of hair spore one is fitted into the shaking flask of 50ml liquid fermentation mediums, culture medium includes (1L)：13C
Labelled glucose 23g, peptone 1.5g, yeast extract 0.1g, dipotassium hydrogen phosphate 2.13g, magnesium sulfate 0.1g, remaining is distilled water,
120 DEG C of sterilizing 20min.20 DEG C of 80rpm/min Shaking cultures 600h.Above-mentioned liquid fermentation and culture is accessed with 10% inoculum concentration
In base, continue to cultivate 300h as secondary medium.By zymotic fluid 4000r/min centrifuge 20min, washing of precipitate, 60 DEG C
Vacuum drying, obtains 4.7g13The aweto mycelium of C flag.1g mycelium return after ethanol solution refluxing extraction, then through water
Stream extraction 2 times, merges extract solution, and concentrated, crystallization, drying obtain 0.21g Cordyceps sinensis polysaccharide crude products.Zymotic fluid is after centrifugation
Supernatant adds 3 times of ethanol, through static, precipitation, filtering, obtains 0.36g Cordyceps sinensis polysaccharide crude products.Crude product is again through taking off albumen, taking off
Color, chromatographic isolation is prepared, concentrated, drying, obtain 0.43g13The Cordyceps sinensis polysaccharide of C flag.Purity and abundance are all higher than 90%.
Embodiment 6
The access of the Chinese ring of hair spore one is fitted into the shaking flask of 50ml liquid fermentation mediums, culture medium includes (1L)：13C
Labelled glucose 23g, peptone 1.5g, yeast extract 0.1g, dipotassium hydrogen phosphate 2.13g, magnesium sulfate 0.1g, remaining is distilled water,
105 DEG C of sterilizing 30min.20 DEG C of 80rpm/min Shaking cultures 600h.Above-mentioned liquid fermentation and culture is accessed with 5% inoculum concentration
In base, continue to cultivate 300h as secondary medium.Zymotic fluid 4000r/min is centrifuged into 10min, 60 after sediment washing
DEG C vacuum drying, obtain 4.7g13The aweto mycelium of C flag.1g mycelium are after ethanol solution backflow 6h extractions, then pass through
Water backflow 1h is extracted 2 times, merges extract solution, and concentrated, crystallization, drying obtain 0.21g Cordyceps sinensis polysaccharide crude products.Zymotic fluid pass through from
Supernatant after the heart adds 1 times of ethanol, through static, precipitation, filtering, obtains 0.36g Cordyceps sinensis polysaccharide crude products.Crude product is again through de- egg
In vain, decolourize, prepare chromatographic isolation, concentrate, drying, obtain 0.43g13The Cordyceps sinensis polysaccharide of C flag.Purity and abundance are all higher than
90%.
Embodiment 7
The access of the Chinese ring of hair spore one is fitted into the shaking flask of 50ml liquid fermentation mediums, culture medium includes (1L)：13C
Labelled glucose 23g, peptone 1.5g, yeast extract 0.1g, dipotassium hydrogen phosphate 2.13g, magnesium sulfate 0.1g, remaining is distilled water,
120 DEG C of sterilizing 15min.20 DEG C of 80rpm/min Shaking cultures 600h.Above-mentioned liquid fermentation and culture is accessed with 30% inoculum concentration
In base, continue to cultivate 300h as secondary medium.Zymotic fluid 4000r/min is centrifuged into 30min, 60 after sediment washing
DEG C vacuum drying, obtain 4.7g13The aweto mycelium of C flag.1g mycelium are after ethanol solution backflow 1h extractions, then pass through
Water backflow 6h is extracted 2 times, merges extract solution, and concentrated, crystallization, drying obtain 0.21g Cordyceps sinensis polysaccharide crude products.Zymotic fluid pass through from
Supernatant after the heart adds 5 times of ethanol, through static, precipitation, filtering, obtains 0.36g Cordyceps sinensis polysaccharide crude products.Crude product is again through de- egg
In vain, decolourize, prepare chromatographic isolation, concentrate, drying, obtain 0.43g13The Cordyceps sinensis polysaccharide of C flag.Purity and abundance are all higher than
90%.
Claims (8)
1. one kind fermentation synthesis13The method of C flag aweto mycelium, it is characterised in that using Chinese hair spore as setting out
Bacterial strain,13C glucose is carbon source, dries, obtains through shake flask fermentation, centrifugation13The mycelium of C flag.
2. fermentation synthesis according to claim 113The method of C flag aweto mycelium, it is characterised in that described
Method specifically includes following steps：
(1) one-level culture：The Chinese ring of hair spore one is accessed in liquid fermentation medium, 105-120 DEG C of sterilizing 15-30min,
At 15-25 DEG C, 0-200r/min Shaking cultures 288-720h；
(2) two level culture：Zymotic fluid is accessed in the liquid fermentation medium with 5-30% inoculum concentration obtained by step (1), is made
Continuing to cultivate 120-300h, 4000r/min centrifugation 10-30min for secondary medium, sediment is dried in vacuo at 60 DEG C,
Obtain13The aweto mycelium of C flag.
3. fermentation synthesis according to claim 213The method of C flag aweto mycelium, it is characterised in that one-level is trained
Support includes with the liquid fermentation medium used in two level culture：13C flag glucose 1-46g/L, peptone 0.1-7.66g/L, ferment
Female cream 0.1-8.27g/L, dipotassium hydrogen phosphate 0.2-2.13g/L, magnesium sulfate 0.1-1.1g/L, remaining is distilled water.
4. fermentation synthesis according to claim 1 or 213The method of C flag aweto mycelium, it is characterised in that institute
The volume for the shaking flask stated is 500ml, and liquid amount 20-200ml, cultivation temperature is 15-25 DEG C.
5. fermentation synthesis according to claim 413The method of C flag aweto mycelium, it is characterised in that described
The volume of shaking flask is 500ml, the preferred 100ml of liquid amount, preferably 18 DEG C, the preferred 100r/min of shaking flask rotating speed of cultivation temperature, cultivates 8
My god.
6. fermentation synthesis according to claim 1 or 213The method of C flag aweto mycelium, it is characterised in that institute
13The mycelium of C flag extracts 2 times after ethanol solution backflow 1-6h extractions, then through water backflow 1-6h, merges extract solution, warp
Concentration, crystallization, drying obtain Cordyceps sinensis polysaccharide crude product.
7. fermentation synthesis according to claim 1 or 213The method of C flag aweto mycelium, it is characterised in that hair
Supernatant of the zymotic fluid after centrifugation adds 1-5 times of ethanol, through left undisturbed overnight, filtering, obtains Cordyceps sinensis polysaccharide crude product.
8. fermentation synthesis according to claim 713The method of C flag aweto mycelium, it is characterised in that after merging
Crude product again through de- albumen, decolouring, prepare chromatographic isolation, concentrate, dry, obtain13The Cordyceps sinensis polysaccharide of C flag.
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Cited By (1)
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| CN109321475A (en) * | 2018-11-15 | 2019-02-12 | 重庆农笑农业发展有限公司 | A kind of preparation method of aweto mycelium |
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Application publication date: 20180109 |