CN102220249B - Method for producing Hirsutella sinensis - Google Patents
Method for producing Hirsutella sinensis Download PDFInfo
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Abstract
The invention relates to a method for producing a microbial strain, in particular to a method for producing a new variety of Hirsutella sinensis. In the invention, Hirsutella sinensis screened according to cell dynamic information acquired by rat macrophage culture is subjected to multistage culture and fermentation, then hyphae is filtered by a membrane with a molecular weight between 300 and 4,000 by a solid-liquid separation technique, the bacterial solution is dried, and thus, health-care food and medicines, which can improve an immune function, are manufactured. When the method is used, the instability of industrial Cordyceps sinensis strain is realized, digital standard control is applied in a whole process from strain screening to culture and fermentation to solid-liquid membrane treatment, and the standard production process of industrial Cordyceps sinensis is realized actually; and with an accurate screening process, high production speed, pure production process and low production cost, the method can be widely accepted by people.
Description
Technical field
The present invention relates to the working method of a kind of microbial strains, specifically be meant a kind of working method of China pilose spore new lines.
Technical background
Entomophyte is a kind of rare traditional Chinese medicine of China, and wild resource suffers extremely to gather, and increases day by day with the market requirement; The domestic and international market source of goods is in short supply, and price is higher, therefore; Utilization separates the Cordyceps strain that obtains---China pilose spore from natural cordyceps, cultivate through submerged fermentation, and the tunning filtration drying is processed finished product; Because of its steady quality, reasonable price is more and more favored.With the China pilose spore is strain fermentation article of manufacture (making capsule as hundred), has not only effectively alleviated the entomophyte demand and supply contraction, delays grassland environment and worsens, and make entomophyte become famous but no longer expensive, can let more common people enjoy and afford to use.But also there are many deficiencies; People get angry after using artificial entomophyte fermented product easily, and minority produces gastrointestinal side effect, and natural cordyceps is warm in nature flat; Almost rare, so there is certain distance in artificial entomophyte goods with natural cordyceps on curative effect; In addition, the discharging of artificial entomophyte fermented waste fluid also causes bigger pollution to environment.Address these problems, one side need be set about from the screening of excellent species, improves from zymotechnique and extraction process on the other hand.
Summary of the invention
The present invention is directed to deficiency of the prior art, proposed simple, the easy to operate relatively working method of a kind of production technique.
The present invention is achieved through following technical proposals:
A kind of working method of China pilose spore is characterized in that comprising the steps:
(1) semi-solid strain inclined plane is cultivated:
Substratum is dissolved in the water that boils, and regulates pH to 7.2,121 ℃, sterilization 30 minutes, and the bevel substratum; Put into 28 ℃ of incubators after the cooling and cultivated 3 days, confirms no living contaminants after, insert the China pilose spore bacterial classification, the even coating; 17～19 ℃ of cultivations of controlled temperature, incubation time 30～45 days; Then, putting into 0～4 ℃ of refrigerator preserves;
Wherein the composition of slant medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.1～3.0%, Semen Maydis powder 1.5%, wheat bran 1～5%, glucose 2%, KH
2PO
40.02%, MgSO
40.01%, agar 1.8%, all the other are water;
(2) liquid spawn shake-flask culture:
To the bottle that shakes that liquid nutrient medium is housed, cultivate the bacterial classification inoculation behind the slant culture in the shaking table concussion, 17～19 ℃ of culture temperature, shaking speed 100～120rpm cultivated 8～13 days, inserted in the aseptic inoculation jar, and bacterial classification multiplication culture inoculation is for liquid used;
The composition that shakes bottle bacterium culture medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.1～3.0%, Semen Maydis powder 1.5%, wheat bran 1～5%, glucose 2%, KH
2PO
40.02%, MgSO
40.01%, all the other are water;
(3) liquid spawn multiplication culture:
The liquid spawn multiplication culture is cultivated in culture tank, and culture condition is: tank pressure 0.03～0.05Mpa, and air flow 1: 0.2,17～19 ℃ of jar temperature, stirring velocity 120～140rpm cultivated 7～12 days; Glucose sugar amount descends fast in nutrient solution, and mycelial growth is vigorous, and mycelia is dense, and the mycelium volumetric concentration is not less than 13%, and inspection does not move into fermentor tank after having assorted bacterium;
The substratum of liquid spawn multiplication culture is formed: degreasing dried silkworm chrysalis meal 2.1～3.0%, Semen Maydis powder 1.5%, wheat bran 1～5%, glucose 2%, KH
2PO
40.02%, MgSO
40.01%, all the other are water;
(4) fermentation culture
Fermentation culture is cultivated in culture tank, and culture condition is: tank pressure 0.03～0.05Mpa, air flow 1: 0.2,17～19 ℃ of jar temperature; Stirring velocity 120～140rpm cultivated 9～12 days, and the mycelium volumetric concentration reaches more than 25%; Glucose reduces to 1.0%, is regarded as fermentation termination, puts jar;
The composition of fermention medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.1%, wheat bran 5%, Semen Maydis powder 1.5%, glucose 2%, fish peptone 0.25%, meat peptone 0.75%, yeast extract paste 0.1%, KH
2PO
40.02%, MgSO
40.01%, all the other are water; Regulate pH to 7.2;
(5) solid-liquid separation:
Fermented liquid after fermentation culture separates through solid-liquid separator, isolates mycelium and fermentation clear liquid;
(6) membrane sepn:
The membrane separation apparatus of fermentation clear liquid in the solid-liquid separation process with the 300-4000 molecular weight separated, get the membrane sepn dope; Membrane sepn among the present invention is conventional separation method, selects corresponding mould material, operational condition to get final product, and has a large amount of mould materials to select in the market.
(7) mycelium with separate the dope drying:
The membrane sepn dope is sprayed into moisture eliminator, and together dry with mycelium, drying temperature gets product at 80-100 ℃.
As preferably, the access of the step of aforementioned production method (1) China pilose spore bacterial classification is at 18 ℃, and inoculum size is 10%, and is flame protection aseptic inoculation.
As preferably, the oleaginousness of the degreasing dried silkworm chrysalis meal described in the step of aforementioned production method (1) is lower than 3%.
As preferably, the degreasing dried silkworm chrysalis meal in the aforementioned production method, Semen Maydis powder, wheat bran are with 10-20 times of water extraction, and after filtering, the water extract adds substratum.
As preferably, the China pilose spore bacterial classification in the aforementioned production method is meant that the aqueous extract through above working method products obtained therefrom adds mouse mastocyte RBL-2H3 strain and carried out serum-free culture 60 hours, and its cell index is reduced to the China pilose spore bacterial strain below 1.5.
China pilose spore of the present invention is the formal name of planting of of microbial world fungi, and former name is a Hirsutella hepiali Chen et Shen.
Among the present invention in semisolid medium and liquid the oleaginousness of using＜3% degreasing dried silkworm chrysalis meal, can promote better China pilose spore growth to reach better technique effect.In addition, adding 2.1-3% degreasing dried silkworm chrysalis meal, Semen Maydis powder, bran water extract help the China pilose spore growth in semisolid medium and liquid, make the mycelia amount in the above-mentioned time, reach the requirement of working method regulation.
The liquid spawn multiplication culture is according to the volume of fermentor tank and the requirement of inoculum size among the present invention, can be secondary (inferior), three grades, level Four bacterial classification amplification culture.Indication cell index among the present invention (Cell Index) is the notion that discloses in the prior art, and is specifically disclosed in reference (ASSAY and Drug Development Technologies, Volume 2, Number 4,2004).
Beneficial effect: the method that combines cytobiology through the present invention; The mouse mastocyte strain RBL-2H3 substratum that the aqueous extract of more than 20 separating obtained China pilose spore bacterial classifications is added serum-free culture; Obtain the cell multidate information, compare, in 60 hours of the adding aqueous extract with natural cordyceps; Cell index is reduced to the China pilose spore culture below 1.5, screens to be excellent species.
Through improvement to technology: get the conventional fermented product of China pilose spore, solid-liquid separation, fermentation clear liquid is separated with the membrane separation apparatus of 300～4000 molecular weight, the membrane sepn dope, dope mixes with mycelium or the difference drying, gets the fermentation end product of China pilose spore.
Screening through to excellent species makes the entomophyte goods more approaching with natural cordyceps on effect, experiment showed, to have raise immunity and the strong renal function of tonifying lung through animal pharmacology.
The improvement of China pilose spore fermented product treatment process has improved output greatly, also reduced environmental pollution simultaneously, and controllability strengthens greatly.
Embodiment
Specify in the face of enforcement of the present invention down:
Embodiment 1
A kind of working method of China pilose spore, implement through following step:
(1) semi-solid strain inclined plane is cultivated:
Substratum is dissolved in the water that boils, and regulates pH to 7.2,121 ℃, sterilization 30 minutes, and the bevel substratum; Put into 28 ℃ of incubators after the cooling and cultivated 3 days, confirms no living contaminants after, insert the China pilose spore bacterial classification, the even coating; 17～18 ℃ of cultivations of controlled temperature, incubation time 40 days; Then, putting into 3 ℃ of refrigerators preserves; Wherein the composition of slant medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.1%, Semen Maydis powder 1.5%, wheat bran 1.5%, glucose 2%, KH
2PO
40.02%, MgSO
40.01%, agar 1.8%, all the other are water; Wherein inoculum size is 15%;
(2) liquid spawn shake-flask culture:
To the bottle that shakes that liquid nutrient medium is housed, cultivate the bacterial classification inoculation behind the slant culture in the shaking table concussion, 17 ℃ of culture temperature, shaking speed 100rpm cultivated 9 days, inserted in the aseptic inoculation jar, and bacterial classification multiplication culture inoculation is for liquid used; The composition that shakes bottle bacterium culture medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.1%, Semen Maydis powder 1.5%, wheat bran 2%, glucose 2%, KH
2PO
40.02%, MgSO
40.01%, all the other are water;
(3) liquid spawn multiplication culture:
The liquid spawn multiplication culture is cultivated in culture tank, and culture condition is: tank pressure 0.03Mpa, and air flow 1: 0.2,18 ℃ of jar temperature, stirring velocity 120rpm cultivated 8 days; Glucose sugar amount descends fast in nutrient solution, and mycelial growth is vigorous, and mycelia is dense, and the mycelium volumetric concentration is not less than 13%, and inspection does not move into fermentor tank after having assorted bacterium; The substratum of liquid spawn multiplication culture is formed: degreasing dried silkworm chrysalis meal 2.1%, Semen Maydis powder 1.5%, wheat bran 2%, glucose 2%, KH
2PO
40.02%, MgSO
40.01%, all the other are water;
(4) fermentation culture
Fermentation culture is cultivated in culture tank, and culture condition is: tank pressure 0.04Mpa, and air flow 1: 0.2,18 ℃ of jar temperature, stirring velocity 120rpm cultivated 9 days, and the mycelium volumetric concentration reaches more than 25%, and glucose reduces to 1.0%, is regarded as fermentation termination, puts jar;
The composition of fermention medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.1%, wheat bran 5%, Semen Maydis powder 1.5%, glucose 2%, fish peptone 0.25%, meat peptone 0.75%, yeast extract paste 0.1%, KH
2PO
40.02%, MgSO
40.01%, all the other are water; Regulate pH to 7.2;
(5) solid-liquid separation:
Fermented liquid after fermentation culture separates through solid-liquid separator, isolates mycelium and fermentation clear liquid; This step is the routine operation method in the industry.
(6) membrane sepn:
The membrane separation apparatus of fermentation clear liquid in the solid-liquid separation process with 3000 molecular weight separated, get the membrane sepn dope;
(7) mycelium with separate the dope drying:
The membrane sepn dope is sprayed into moisture eliminator, and together dry with mycelium, drying temperature gets product at 80-100 ℃.
This product has good effect through using checking.
Embodiment 2
The method identical with embodiment implemented through following step
(1) semi-solid strain inclined plane is cultivated:
Substratum is dissolved in the water that boils, and regulates pH to 7.2,121 ℃, sterilization 30 minutes, and the bevel substratum; Put into 28 ℃ of incubators after the cooling and cultivated 3 days, confirms no living contaminants after, insert the China pilose spore bacterial classification, the even coating; 18～19 ℃ of cultivations of controlled temperature, incubation time 45 days; Then, putting into 4 ℃ of refrigerators preserves; Wherein the composition of slant medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.9%, Semen Maydis powder 1.5%, wheat bran 4.5%, glucose 2%, KH
2PO
40.02%, MgSO
40.01%, agar 1.8%, all the other are water; Wherein inoculum size is 15%; The access of China pilose spore bacterial classification is at 18 ℃, and inoculum size is 10%, and is flame protection aseptic inoculation; Wherein the oleaginousness of degreasing dried silkworm chrysalis meal is lower than 3%;
China pilose spore bacterial classification in the present embodiment is meant through the aqueous extract adding mouse mastocyte RBL-2H3 of above working method products obtained therefrom strain and carried out serum-free culture 60 hours that its cell index is reduced to 1.5 o'clock China pilose spore bacterial strain;
(2) liquid spawn shake-flask culture:
To the bottle that shakes that liquid nutrient medium is housed, cultivate the bacterial classification inoculation behind the slant culture in the shaking table concussion, 18 ℃ of culture temperature, shaking speed 120rpm cultivated 13 days, inserted in the aseptic inoculation jar, and bacterial classification multiplication culture inoculation is for liquid used; The composition that shakes bottle bacterium culture medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.8%, Semen Maydis powder 1.5%, wheat bran 4%, glucose 2%, KH
2PO
40.02%, MgSO
40.01%, all the other are water;
(3) liquid spawn multiplication culture:
The liquid spawn multiplication culture is cultivated in culture tank, and culture condition is: tank pressure 0.04Mpa, and air flow 1: 0.2,19 ℃ of jar temperature, stirring velocity 140rpm cultivated 12 days; Glucose sugar amount descends fast in nutrient solution, and mycelial growth is vigorous, and mycelia is dense, and the mycelium volumetric concentration is not less than 13%, and inspection does not move into fermentor tank after having assorted bacterium; The substratum of liquid spawn multiplication culture is formed: degreasing dried silkworm chrysalis meal 2.8%, Semen Maydis powder 1.5%, wheat bran 4%, glucose 2%, KH
2PO
40.02%, MgSO
40.01%, all the other are water;
Degreasing dried silkworm chrysalis meal in the present embodiment, Semen Maydis powder, wheat bran are with 10-20 times of water extraction, and after filtering, the water extract adds substratum;
(4) fermentation culture
Fermentation culture is cultivated in culture tank, and culture condition is: tank pressure 0.05Mpa, and air flow 1: 0.2,18 ℃ of jar temperature, stirring velocity 140rpm cultivated 11 days, and the mycelium volumetric concentration reaches more than 25%, and glucose reduces to 1.0%, is regarded as fermentation termination, puts jar;
The composition of fermention medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.1%, wheat bran 5%, Semen Maydis powder 1.5%, glucose 2%, fish peptone 0.25%, meat peptone 0.75%, yeast extract paste 0.1%, KH
2PO
40.02%, MgSO
40.01%, all the other are water; Regulate pH to 7.2;
(5) solid-liquid separation:
Fermented liquid after fermentation culture separates through solid-liquid separator, isolates mycelium and fermentation clear liquid; This step is the routine operation method in the industry.
(6) membrane sepn:
The membrane separation apparatus of fermentation clear liquid in the solid-liquid separation process with 2000 molecular weight separated, get the membrane sepn dope;
(7) mycelium with separate the dope drying:
This product has good effect through using checking.
Claims (4)
1. the working method of a China pilose spore is characterized in that comprising the steps:
(1) semi-solid strain inclined plane is cultivated:
Substratum is dissolved in the water that boils, and regulates pH to 7.2,121 ℃, sterilization 30 minutes, and the bevel substratum; Put into 28 ℃ of incubators after the cooling and cultivated 3 days, confirms no living contaminants after, insert the China pilose spore bacterial classification, the even coating; 17～19 ℃ of cultivations of controlled temperature, incubation time 30～45 days; Then, putting into 0～4 ℃ of refrigerator preserves;
Wherein the composition of slant medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.1～3.0 %, Semen Maydis powder 1.5 %, wheat bran 1～5 %, glucose 2 %, KH
2PO
40.02%, MgSO
40.01 %, agar 1.8 %, all the other are water;
(2) liquid spawn shake-flask culture:
To the bottle that shakes that liquid nutrient medium is housed, cultivate the bacterial classification inoculation behind the slant culture in the shaking table concussion, 17～19 ℃ of culture temperature, shaking speed 100～120rpm cultivated 8～13 days, inserted in the aseptic inoculation jar, and bacterial classification multiplication culture inoculation is for liquid used;
The composition that shakes bottle bacterium culture medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.1～3.0 %, Semen Maydis powder 1.5 %, wheat bran 1～5 %, glucose 2 %, KH
2PO
40.02%, MgSO
40.01 %, all the other are water;
(3) liquid spawn multiplication culture:
The liquid spawn multiplication culture is cultivated in the multiplication culture jar, and culture condition is: tank pressure 0.03～0.05Mpa, and air flow 1:0.2,17～19 ℃ of jar temperature, stirring velocity 120～140rpm cultivated 7～12 days; Glucose sugar amount descends fast in nutrient solution, and mycelial growth is vigorous, and mycelia is dense, and the mycelium volumetric concentration is not less than 13 %, and inspection does not move into the fermentation culture jar after having assorted bacterium;
The substratum of liquid spawn multiplication culture is formed: degreasing dried silkworm chrysalis meal 2.1～3.0 %, Semen Maydis powder 1.5 %, wheat bran 1～5 %, glucose 2 %, KH
2PO
40.02%, MgSO
40.01 %, all the other are water;
(4) fermentation culture
Fermentation culture is cultivated in the fermentation culture jar, and culture condition is: tank pressure 0.03～0.05Mpa, air flow 1:0.2,17～19 ℃ of jar temperature; Stirring velocity 120～140rpm cultivated 9～12 days, and the mycelium volumetric concentration reaches more than 25%; Glucose reduces to 1.0%, is regarded as fermentation termination, puts jar;
The composition of fermention medium is by weight percentage: degreasing dried silkworm chrysalis meal 2.1%, wheat bran 5%, Semen Maydis powder 1.5%, glucose 2%, fish peptone 0.25%, meat peptone 0.75%, yeast extract paste 0.1%, KH
2PO
40.02%, MgSO
40.01%, all the other are water; Regulate pH to 7.2;
(5) solid-liquid separation:
Fermented liquid after fermentation culture separates through solid-liquid separator, isolates mycelium and fermentation clear liquid;
(6) membrane sepn:
The membrane separation apparatus of fermentation clear liquid in the solid-liquid separation process with the 300-4000 molecular weight separated, get the membrane sepn dope;
(7) mycelium with separate the dope drying:
The membrane sepn dope is sprayed into moisture eliminator, and together dry with mycelium, drying temperature gets product at 80-100 ℃.
2. working method according to claim 1, the access that it is characterized in that step (1) China pilose spore bacterial classification is at 18 ℃, and inoculum size is 10%, and is flame protection aseptic inoculation.
3. working method according to claim 1 is characterized in that the oleaginousness of the degreasing dried silkworm chrysalis meal described in the step (1) is lower than 3 %.
4. working method according to claim 1 is characterized in that described degreasing dried silkworm chrysalis meal, and Semen Maydis powder, wheat bran are with 10-20 times of water extraction, and after filtering, the water extract adds substratum.
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| CN103571757A (en) * | 2012-08-09 | 2014-02-12 | 王云 | Preparation method of pure natural culture medium for deeply fermenting hirsutella sinensis liquid |
| CN103725714B (en) * | 2012-10-16 | 2016-02-10 | 青海珠峰虫草药业有限公司 | A kind of hirsutella sinensis fungal powder production method |
| CN103432174B (en) * | 2013-08-27 | 2015-08-19 | 王辉 | The method of Cordyceps fungus powder produced by a kind of liquid fermentation of hybrid bacterial strain |
| CN103535185B (en) * | 2013-09-30 | 2015-11-25 | 涂峰 | A kind of liquid separation method of China pilose spore bacterial classification |
| CN103881912B (en) * | 2013-12-20 | 2016-05-04 | 云南大学 | A kind of long term storage method of Hirsutella sinensis |
| CN104172149B (en) * | 2014-05-22 | 2016-06-08 | 杭州柯氏生物科技有限公司 | A kind of China pilose spore is applied in medicine, health food or food |
| CN104350949B (en) * | 2014-11-14 | 2017-02-22 | 中山大学 | Cordyceps sinensis liquid-state fermentation process |
| CN104473978B (en) * | 2014-11-26 | 2017-12-15 | 南京中科药业有限公司 | A kind of cordyceps mycelia processing method rich in active component |
| CN107586728B (en) * | 2017-10-26 | 2021-04-27 | 青海珠峰冬虫夏草工程技术研究有限公司 | Cordyceps sinensis culture solution and preparation method thereof |
| CN108118001A (en) * | 2017-12-22 | 2018-06-05 | 青海珠峰冬虫夏草工程技术研究有限公司 | A kind of cordyceps sinensis fluid nutrient medium and preparation method thereof |
| CN108865904B (en) * | 2018-08-30 | 2020-11-10 | 云南大学 | Hirsutella sinensis expanding culture medium, hirsutella sinensis cultured by same and culture method |
| CN111304090A (en) * | 2019-11-26 | 2020-06-19 | 浙江中医药大学 | Application of hirsutella sinensis fermentation product in preparation of medicine for treating heart failure |
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| CN100374542C (en) * | 2003-08-19 | 2008-03-12 | 俞永信 | Cordyceps vegetative stage continuous fermentation technology |
| CN1796539A (en) * | 2004-12-24 | 2006-07-05 | 青海月王青藏药业有限责任公司 | Ferment for producing aweto in large scale and technique for processing power of fungus |
| CN101096641B (en) * | 2007-06-13 | 2011-07-13 | 杭州中美华东制药有限公司 | Culture medium for producing winter worm summer herb mycelium |
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