CN106434373A - High-density fermentation medium formula of sparassis crispa and pharmaceutical grade glucan preparation method of high-density fermentation medium formula - Google Patents
High-density fermentation medium formula of sparassis crispa and pharmaceutical grade glucan preparation method of high-density fermentation medium formula Download PDFInfo
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Abstract
The invention discloses a high-density fermentation medium formula of sparassis crispa and a pharmaceutical grade glucan preparation method of the high-density fermentation medium formula. The high-density fermentation medium formula comprises the following raw materials by weight percentage: 1-10% of soluble starch, 0.1-5% of peptone, 0.1-0.5% of KH2PO4, 0.1-0.5% of MgSO4.7H2O, and the balance of glucose solution at a mass concentration percentage of 20-50%, wherein the total weight percentage is 100%. A high biomass sparassis crispa mycelium is obtained by seed liquid preparation and fermentation culture. The pharmaceutical grade glucan preparation method comprises the steps of centrifuging and concentrating a sparassis crispa sporophore with a disc centrifuge, performing homogenization with a high-pressure homogenizer, and performing alkaline extraction, alcohol precipitation, sedimentation and drying to prepare pharmaceutical grade glucan. A glucan content in the mycelium detected by the method is 90%. Compared with the prior art, the formula and the method have the obvious advantages of short production cycle, low cost, simple technology, high product yield, high quality and the like.
Description
Technical field
The present invention relates to the high density fermentation culture medium formula of technical field of microbial fermentation, specifically a kind of Sparassia crispa (Wulf.) Fr. and
Its pharmaceutical grade glucosan preparation method.
Background technology
Sparassia crispa (Wulf.) Fr., is non-pleat pore fungi mesh, silk ball Cordycepps, silk ball Pseudomonas.Medium to the big shape of sporophore, meat is thick by one
Many branches are sent on strong handle, and branch end forms the limb of countless complications, is similar to huge silk ball and gains the name.The maximum of Sparassia crispa (Wulf.) Fr.
Feature is containing a large amount of beta glucans, according to the analysis of japanese food analysis center, contains beta glucan per 100g Sparassia crispa (Wulf.) Fr. and is up to
43.6g, is higher by 3-4 times than Ganoderma and Agaricus blazei Murrill.It can be said that beta glucan contained by Sparassia crispa (Wulf.) Fr. for mushrooms most, be described as " ten thousand
The king of mushroom ".β type glucosan is a kind of bioactive substance, through medical research confirm, with immunomodulating, antitumor, antiinflammatory,
The several functions such as antiviral, antioxidation, radioprotective, blood sugar lowering, blood fat reducing, liver protection.Research also finds, great majority have antitumor
The polysaccharide of activity is all β-(the 1-3)-D- glucosan with β (1-6) glycosidic bond branch.There are some researches prove, from funguses β-
(1-3)-D- glucosan generally has the function of tumor of suppression.More than macrodex % in Sparassia crispa (Wulf.) Fr. is that β-(1-3)-D- Portugal gathers
Sugar, with good antitumor, immunomodulating and improve hemopoietic function the effects such as.
Sparassia crispa (Wulf.) Fr. has gradually been subject to Chinese scholars and consumption in recent years by its good health-care effect and nutritive value
The very big concern of person.However, Sparassia crispa (Wulf.) Fr. is wildly all very limited with yield, cultivation cultural technique is at home and undeveloped,
What literature survey discovery Sparassia crispa (Wulf.) Fr. culture studies were more is artificial solid state cultivation, artificial cultivation condition's harshness, and poor growth is biological
Conversion ratio is low.And liquid fermentation have cycle is short, yield is high, do not limited by environment, low cost, easy to control the quality, without heavy metal
The advantages of pollution, Sparassia crispa (Wulf.) Fr. high density fermentation produces mycelium, need to be significant for market is met.
Content of the invention
The primary and foremost purpose of the present invention is to provide a kind of Sparassia crispa (Wulf.) Fr. high density fermentation culture medium, more can have using the culture medium
Effect fermentation Sparassia crispa (Wulf.) Fr. is to obtain high yield mycelium.
Another object of the present invention is to provide a kind of high density fermentation based on above-mentioned Sparassia crispa (Wulf.) Fr. high density fermentation culture medium
Method, the method utilizes above-mentioned Sparassia crispa (Wulf.) Fr. high density fermentation culture medium, and controls fermentation condition, so as to obtain high yield mycelium.
For achieving the above object, the present invention provides following technical scheme：
A kind of high density fermentation culture medium formula of Sparassia crispa (Wulf.) Fr., by weight percentage, raw material includes：Soluble starch 1-
10%th, peptone 0.1-5%, KH2PO40.1-0.5%, MgSO4·7H2O 0.1-0.5%, balance of mass concentration percentage ratio
The glucose solution of 20-50%, each raw material sum is 100%.
As the further scheme of the present invention：The soluble starch purity is more than 99.9%.
As the further scheme of the present invention：The peptone purity is more than 99.9%.
As the further scheme of the present invention：The glucose solution purity is more than 99.9%.
A kind of preparation method of the high density fermentation culture medium formula of Sparassia crispa (Wulf.) Fr., concretely comprises the following steps：
(1) the silk ball strain inoculation shovel that keeps is inoculated on PDA culture medium flat board, is positioned over 25-28 DEG C of culture
Cultivate in case to growing bacterium colony；Take full bacterium colony block with inoculation shovel in above-mentioned bacterium colony to access in SCF culture medium, 25-28
DEG C, 100-200r/min shake-flask culture 12-24h, obtain required seed liquor；
(2) step (1) is prepared gained seed liquor and Sparassia crispa (Wulf.) Fr. high density fermentation culture medium is accessed with the inoculum concentration of 1-10%
Start fermentation culture in 5L fermentation tank, fermentation culture adopts 28-30 DEG C, 100-280r/min fermentor cultivation；From fermentation culture,
Dissolved oxygen goes back up to more than 50% to start to add glucose solution, is the sterile dextrose of 20-50% using the advance concentration for preparing
Solution, with the feed rate of 3-8ml per hour；Fermentation culture is to weight in wet base for terminating fermentation after 400-600g/L；
(3) by step 2 ferment gained fermentation liquid using obtaining mycelium after disk plate centrifuge centrifugal concentrating, plus single steam
Its homogenizing is become slurry using high pressure homogenisers by aqueous suspension, adjusts pH to 10-11 with sodium hydroxide, and boiling water extracts 30-60min, extraction
Slightly cool down after end, bacterium solution is filtered to obtain, add the alkaline protease of 20-100ppm amount to be digested, add after the completion of enzymolysis
2-4 times of ethanol, standing, it is filtrated to get precipitation；
(4) vacuum that is deposited in of gained in step 3 is done under the conditions of -0.065--0.095Mpa, 70-85 DEG C
Dry.
As the further scheme of the present invention：27 DEG C in concrete steps (1), 150r/min shake-flask culture 18h.
As the further scheme of the present invention：It is specially using disk plate centrifuge centrifugal concentrating in concrete steps (3) and is centrifuged
Speed is to be centrifuged 5-10min under 4000-5000r/min, is digested using alkaline protease, the adding of alkaline protease after centrifugation
Dosage is 20-100ppm, and it is 2-5h that hydrolysis temperature is 40-50 DEG C, enzymolysis time, then carries out precipitate with ethanol.
The present invention is applied to any Sparassia crispa (Wulf.) Fr. strain (Sparassis crispa), is such as preserved in Chinese agriculture microbial bacteria
Resources bank is planted, strain coding 80458 grade of ACCC can all be used.
Silk ball bacteria strain of the present invention is not limited to certain concrete manufacturer, commercially available can all use.
After above-mentioned fermentation ends, the mycelial biomass dry weight of detection Sparassia crispa (Wulf.) Fr. reaches more than 500g/L, and the fermentation side is described
Method can effectively obtain the Sparassia crispa (Wulf.) Fr. mycelium of high-biomass.
After above-mentioned Sparassia crispa (Wulf.) Fr. mycelium high pressure homogenize, alkali carries precipitate with ethanol, detection Sparassia crispa (Wulf.) Fr. mycelium beta glucan content is up to
90%, illustrate that the extracting method can effectively obtain the Sparassia crispa (Wulf.) Fr. beta glucan of high-biomass.
Compared with prior art, the invention has the beneficial effects as follows：
Obtained by after many experiments conceptual design data is analyzed preferably, the collocation of nutrient media components is to Sparassia crispa (Wulf.) Fr.
Growth and breeding and Product formation are highly beneficial.
The Sparassia crispa (Wulf.) Fr. high density fermentation cultural method of the present invention, using batch feeding strategy, the parameter optimization of batch feeding
The formulation optimization of collocation fermentation medium, so as to ensure that the realization of high density, high yield and high-concentration culturing Sparassia crispa (Wulf.) Fr..
The technology of the present invention shortens production cycle, reduces cost：Traditional solid spawn former base is typically needed to sporophore is ripe
10-15 days are taken, and is only needed 3-5 days using liquid spawn high density fermentation.
The technology of the present invention process is simple, easy to operate：The silk ball bacterium culture medium that the present invention is provided, its definite ingredients, simple,
Being easily obtained and low cost, Sparassia crispa (Wulf.) Fr. is produced using the culture medium high density fermentation, process is simple, easy to operate.
The present invention designs product yield high：After the completion of fermentation, concentrated using disk plate centrifuge centrifugal filtration, high pressure homogenisers
Homogenizing becomes slurry, farthest collects Sparassia crispa (Wulf.) Fr. mycelium and is crushed so that the β-Portugal in sporophore gathers in operating process
Sugar dissolution completely.
The technology of the present invention is not limited by area, weather conditions, suitable for mass production.
Specific embodiment
With reference to specific embodiment, the technical scheme of this patent is described in more detail.
By taking Fructus Fragariae Ananssae concentrated juice as an example, embodiment 1-4 carries out the preparation of fermenting fruit juice：
Embodiment 1
The silk ball bacteria strain (Sparassis crispa) of the present embodiment, is preserved in Chinese agriculture microbial resources
Storehouse, strain encodes ACCC 80458.
High density fermentation culture and its preparation of pharmaceutical grade glucosan are carried out to Sparassia crispa (Wulf.) Fr. ACCC 80458, method include as
Lower step：
Step 1：The preparation of seed liquid
The Sparassia crispa (Wulf.) Fr. that keeps is inoculated on PDA culture medium flat board with inoculation shovel, is positioned in 28 DEG C of incubators and cultivates
To growing bacterium colony；Take full bacterium colony block with inoculation shovel in above-mentioned bacterium colony to access in SCF culture medium, 28 DEG C, 150r/min
Shake-flask culture 12h, obtains required seed liquor.
Step 2. fermentation culture
The seed liquor for preparing in step 1 is accessed with 10% inoculum concentration and is opened in Sparassia crispa (Wulf.) Fr. high density fermentation culture medium
Beginning fermentation culture, fermentation culture adopts 28 DEG C, 150r/min fermentor cultivation；More than 50% is gone back up to from fermentation culture dissolved oxygen,
Start to add glucose solution, using the glucose solution that the advance concentration for preparing is 50%, with the feed supplement speed of 5-8mL per hour
Degree；Terminate fermentation after fermentation culture 100h.
Step 3. high pressure homogenize, alkali carries precipitate with ethanol
By step 2 ferment gained fermentation liquid using mycelium is obtained after disk plate centrifuge centrifugal concentrating, using high pressure
Its homogenizing is become slurry by homogenizer, adjusts pH to 10 with sodium hydroxide, and boiling water extracts 30min, and extraction is slightly cooled down after terminating, filtered
Bacterium solution, the alkaline protease of addition 50ppm in bacterium solution, 45 DEG C of water bath heat preservation 5h, in bacterium solution, after terminating, add 2 times of second
Alcohol, standing, it is filtrated to get precipitation.
Step 4. is vacuum dried.
By in step 3 gained be deposited in vacuum in -0.065Mpa, be dried under the conditions of 75 DEG C.
After measured, Sparassia crispa (Wulf.) Fr. mycelial biomass is that 515g/L, in mycelium, beta glucan content is 92%.
Embodiment 2
The silk ball bacteria strain (Sparassis crispa) of the present embodiment, is preserved in Chinese agriculture microbial resources
Storehouse, strain encodes ACCC 51488.
High density fermentation culture and its preparation of pharmaceutical grade glucosan are carried out to Sparassia crispa (Wulf.) Fr. ACCC 51488, method include as
Lower step：
Step 1：The preparation of seed liquid
The Sparassia crispa (Wulf.) Fr. that keeps is inoculated on PDA culture medium flat board with inoculation shovel, is positioned in 28 DEG C of incubators and cultivates
To growing bacterium colony；Take full bacterium colony block with inoculation shovel in above-mentioned bacterium colony to access in SCF culture medium, 25 DEG C, 100r/min
Shake-flask culture 12h, obtains required seed liquor.
Step 2. fermentation culture
The seed liquor for preparing in step 1 is accessed with 10% inoculum concentration and is opened in Sparassia crispa (Wulf.) Fr. high density fermentation culture medium
Beginning fermentation culture, fermentation culture adopts 28 DEG C, 150r/min fermentor cultivation；More than 50% is gone back up to from fermentation culture dissolved oxygen,
Start to add glucose solution, using the glucose solution that the advance concentration for preparing is 50%, with the feed supplement speed of 5ml per hour
Degree；Terminate fermentation after fermentation culture 100h.
Step 3. high pressure homogenize, alkali carries precipitate with ethanol
By step 2 ferment gained fermentation liquid using mycelium is obtained after disk plate centrifuge centrifugal concentrating, using high pressure
Its homogenizing is become slurry by homogenizer, adjusts pH to 10 with sodium hydroxide, and boiling water extracts 30min, and extraction is slightly cooled down after terminating, filtered
Bacterium solution, the alkaline protease of addition 50ppm in bacterium solution, 45 DEG C of water bath heat preservation 5h, in bacterium solution, after terminating, add 2 times of second
Alcohol, standing, it is filtrated to get precipitation.
Step 4. is vacuum dried.
By in step 3 gained be deposited in vacuum in -0.065Mpa, be dried under the conditions of 75 DEG C.
Determine according to People's Republic of China (PRC) agricultural industry criteria NY/T 1676-2008, Sparassia crispa (Wulf.) Fr. mycelial biomass is
520g/L, in mycelium, the content of beta glucan is more than 93%.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of spirit or essential attributes without departing substantially from the present invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit is required rather than described above is limited, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although this specification is been described by according to embodiment, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of description is only that those skilled in the art should for clarity
Using description as an entirety, the technical scheme in each embodiment can also form those skilled in the art through appropriately combined
Understandable other embodiment.
Claims (7)
1. the high density fermentation culture medium formula of a kind of Sparassia crispa (Wulf.) Fr., it is characterised in that by weight percentage, raw material includes：Can
Soluble starch 1-10%, peptone 0.1-5%, KH2PO40.1-0.5%, MgSO4·7H2O 0.1-0.5%, balance of quality
The glucose solution of percentage 20-50%, each raw material sum is 100%.
2. the high density fermentation culture medium formula of a kind of Sparassia crispa (Wulf.) Fr. according to claim 1, it is characterised in that described solvable
Property purity of starch be more than 99.9%.
3. a kind of high density fermentation culture medium formula of Sparassia crispa (Wulf.) Fr. according to claim 1, it is characterised in that the albumen
Peptone purity is more than 99.9%.
4. a kind of high density fermentation culture medium formula of Sparassia crispa (Wulf.) Fr. according to claim 1, it is characterised in that the Fructus Vitis viniferae
Sugar juice purity is more than 99.9%.
5. a kind of preparation method of the high density fermentation culture medium formula of the Sparassia crispa (Wulf.) Fr. as described in claim 1-4 is arbitrary, which is special
Levy and be, concretely comprise the following steps：
(1) the silk ball strain inoculation shovel that keeps is inoculated on PDA culture medium flat board, is positioned in 25-28 DEG C of incubator
Cultivate to growing bacterium colony；Take full bacterium colony block with inoculation shovel in above-mentioned bacterium colony to access in SCF culture medium, 25-28 DEG C,
100-200r/min shake-flask culture 12-24h, obtains required seed liquor；
(2) step (1) is prepared gained seed liquor to send out with the inoculum concentration access Sparassia crispa (Wulf.) Fr. high density fermentation culture medium 5L of 1-10%
Start fermentation culture in fermentation tank, fermentation culture adopts 28-30 DEG C, 100-280r/min fermentor cultivation；From fermentation culture, dissolved oxygen
Go back up to more than 50% to start glucose solution is added, be the sterile dextrose solution of 20-50% using the advance concentration for preparing,
Feed rate with 3-8ml per hour；Fermentation culture is to weight in wet base for terminating fermentation after 400-600g/L；
(3) by step 2 ferment gained fermentation liquid using obtaining mycelium after disk plate centrifuge centrifugal concentrating, plus single water that steams hangs
Floating, using high pressure homogenisers, its homogenizing being become slurry, pH to 10-11 is adjusted with sodium hydroxide, boiling water extracts 30-60min, and extraction terminates
Slightly cool down afterwards, bacterium solution is filtered to obtain, add the alkaline protease of 20-100ppm amount to be digested, after the completion of enzymolysis, add 2-4 times
Ethanol, standing, be filtrated to get precipitation；
(4) vacuum that is deposited in of gained in step 3 is dried under the conditions of -0.065--0.095Mpa, 70-85 DEG C.
6. the preparation method of the high density fermentation culture medium formula of a kind of Sparassia crispa (Wulf.) Fr. according to claim 5, its feature exists
In, 27 DEG C in concrete steps (1), 150r/min shake-flask culture 18h.
7. the preparation method of the high density fermentation culture medium formula of a kind of Sparassia crispa (Wulf.) Fr. according to claim 5, its feature exists
In it is to be centrifuged 5- under 4000-5000r/min to be specially centrifugal speed using disk plate centrifuge centrifugal concentrating in concrete steps (3)
10min, is digested using alkaline protease after centrifugation, and the addition of alkaline protease is 20-100ppm, and hydrolysis temperature is
40-50 DEG C, enzymolysis time is 2-5h, then carries out precipitate with ethanol.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107501429A (en) * | 2017-09-01 | 2017-12-22 | 河南省科学院生物研究所有限责任公司 | A kind of method that bioactivity beta glucan is extracted in the liquid fermentation mycelium from Sparassis crispa |
| CN110184200A (en) * | 2019-06-14 | 2019-08-30 | 福建农林大学 | A kind of high yield Sparassis crispa mycelia fermentation base and preparation method |
| CN110358647A (en) * | 2019-07-01 | 2019-10-22 | 福清市火麒麟食用菌技术开发有限公司 | A kind of preparation method of Sparassis crispa barley wine |
| CN110710669A (en) * | 2019-09-30 | 2020-01-21 | 内蒙古中谷君创生物科技发展有限责任公司 | Preparation method of sparassis crispa mycelium oral liquid |
| CN111685234A (en) * | 2020-07-10 | 2020-09-22 | 融和梦(福建)生物科技有限公司 | Pig feed and method for enhancing immunity by feeding pig feed |
| CN112708648A (en) * | 2020-12-25 | 2021-04-27 | 宁波希诺亚海洋生物科技有限公司 | Preparation method and application of pharmaceutical-grade sparassis crispa polysaccharide for enhancing immunity |
| CN113846021A (en) * | 2021-09-06 | 2021-12-28 | 融和梦(福建)生物科技有限公司 | Liquid culture method of sparassis crispa, freeze-dried powder and application of freeze-dried powder |
| CN114304353A (en) * | 2021-12-30 | 2022-04-12 | 艾苛密（上海）健康科技股份有限公司 | Beta-glucan candy tablet with blood sugar and blood pressure reducing health care functions and preparation method thereof |
| CN114343045A (en) * | 2022-01-20 | 2022-04-15 | 艾苛密（上海）健康科技股份有限公司 | Health-care tabletting candy and preparation method thereof |
| CN114540202A (en) * | 2020-07-10 | 2022-05-27 | 融和梦(福建)生物科技有限公司 | Preparation method of sparassis crispa dry powder, physiological function activator and application thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107501429A (en) * | 2017-09-01 | 2017-12-22 | 河南省科学院生物研究所有限责任公司 | A kind of method that bioactivity beta glucan is extracted in the liquid fermentation mycelium from Sparassis crispa |
| CN110184200B (en) * | 2019-06-14 | 2021-04-27 | 福建农林大学 | High-yield sparassis crispa mycelium fermentation medium and preparation method thereof |
| CN110184200A (en) * | 2019-06-14 | 2019-08-30 | 福建农林大学 | A kind of high yield Sparassis crispa mycelia fermentation base and preparation method |
| CN110358647A (en) * | 2019-07-01 | 2019-10-22 | 福清市火麒麟食用菌技术开发有限公司 | A kind of preparation method of Sparassis crispa barley wine |
| CN110710669A (en) * | 2019-09-30 | 2020-01-21 | 内蒙古中谷君创生物科技发展有限责任公司 | Preparation method of sparassis crispa mycelium oral liquid |
| CN111685234A (en) * | 2020-07-10 | 2020-09-22 | 融和梦(福建)生物科技有限公司 | Pig feed and method for enhancing immunity by feeding pig feed |
| CN114540202A (en) * | 2020-07-10 | 2022-05-27 | 融和梦(福建)生物科技有限公司 | Preparation method of sparassis crispa dry powder, physiological function activator and application thereof |
| CN112708648A (en) * | 2020-12-25 | 2021-04-27 | 宁波希诺亚海洋生物科技有限公司 | Preparation method and application of pharmaceutical-grade sparassis crispa polysaccharide for enhancing immunity |
| CN113846021A (en) * | 2021-09-06 | 2021-12-28 | 融和梦(福建)生物科技有限公司 | Liquid culture method of sparassis crispa, freeze-dried powder and application of freeze-dried powder |
| CN114304353A (en) * | 2021-12-30 | 2022-04-12 | 艾苛密（上海）健康科技股份有限公司 | Beta-glucan candy tablet with blood sugar and blood pressure reducing health care functions and preparation method thereof |
| CN114304353B (en) * | 2021-12-30 | 2023-09-19 | 艾苛密（上海）健康科技股份有限公司 | Beta-glucan candy tablet with blood glucose and blood pressure reducing health care function and preparation method thereof |
| CN114343045A (en) * | 2022-01-20 | 2022-04-15 | 艾苛密（上海）健康科技股份有限公司 | Health-care tabletting candy and preparation method thereof |
| CN114343045B (en) * | 2022-01-20 | 2023-11-17 | 河南省寓生堂食养科技有限公司 | Health care tabletting candy and preparation method thereof |
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Application publication date: 20170222 |