CN103483040A - Culture medium for large-scale submerged fermentation for cordyceps sinensis and fermentation production method thereof - Google Patents
Culture medium for large-scale submerged fermentation for cordyceps sinensis and fermentation production method thereof Download PDFInfo
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Abstract
The invention discloses a culture medium for large-scale submerged fermentation for cordyceps sinensis and a fermentation production method thereof. The fermentation production method comprises the following steps of: producing a mother strain by using cordyceps sinensis collected from the Tibet Plateau, planting the mother strain in a triangular flask and performing shake culture, so as to prepare a liquid strain; then inoculating the liquid strain in each stage of a seed tank and performing fermentation culture, and amplifying stage by state; and finally realizing large-scale submerged fermentation for cordyceps sinensis in a fermentation tank, wherein the final volume of the fermentation tank achieves 5 tons or more, and the mycelium dry weight achieves 5-15%. According to the fermentation production method disclosed by the invention, high-quality cordyceps sinensis strains, a special culture medium and a special fermentation process are adopted, thus realizing the purposes of promoting the growth of the cordyceps sinensis strains, reducing work period, reducing pollution opportunity, saving energy, and decreasing cost.
Description
Technical field
The invention belongs to edible and medicinal fungi fermentative production technical field, relate in particular to substratum and fermentation method for producing thereof for a kind of Chinese caterpillar fungus pilot scale liquid submerged fermentation.
Background technology
Cordyceps sinensis belongs to the fungal species (the Latin formal name used at school is Cordyceps sinensis, also writes Ophiocordyceps sinensis) of Clavicipitaceae, cries again China pilose spore or China by the hair spore.Cordyceps sinensis has long-term applicating history at folks of china, is senior, famous and precious herbal medicine invigorant, and its health keeping and medical function has record in " new compilation of materia medica " and the Pharmacopoeia of the People's Republic of China.Cordyceps sinensis is distributed in the cold district of the Qinghai-Tibet high height above sea level such as Qinghai, Tibet, main product in Jinsha jiang River, the upstream in the Lancang River, San Jiang basin, Nujiang.Due to excessive, excavate and environmental disruption, wild Cordyceps Resources falls sharply, and can not meet market demand.
Can produce in a large number Cordyceps mycelium by liquid submerged fermentation, it is similar that scientist studies the chemical composition of finding the Cordyceps sinensis fermented hypha and pharmacological action and wild cordyceps, so the Cordyceps mycelium of fermentative production becomes the important substitute of wild cordyceps.More domestic researchers have been delivered some Patents and paper at present, disclose substratum and the culture process of some Cordyceps sinensis liquid fermentings, but the ubiquity fermentation period is long, easily pollution, complex steps, energy consumption is large, the mycelium productive rate is not high problem.
Summary of the invention
The purpose of this invention is to provide substratum and fermentation method for producing thereof for a kind of Chinese caterpillar fungus pilot scale liquid submerged fermentation, fermentation period is short, be difficult for polluting, step is simple, energy consumption is few, the mycelium productive rate is high.
Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum provided by the invention, its each weight ratio of constituents example is:
The 1.50-4.00% carbon source, carbon source is glucose or sucrose or maltose, the 0.50-2.00% nitrogenous source, nitrogenous source is yeast extract paste or extractum carnis or peptone, 0.05-0.15% sal epsom, 0.10%-0.20 potassium primary phosphate, the 0.05-0.10% vitamin complex, the 1.00-2.00% agar powder, all the other are moisture;
By above-mentioned heating material to 100 ℃, make material dissolution, mix, sterilizing 30-50 minute under 121 ℃ of lower 0.15MPa pressure, cooling being down to after room temperature is solidified can be used.
Liquid culture based component for shake-flask culture, seed tank culture, fermentor cultivation is consistent, culture medium prescription is as follows: each weight ratio of constituents example is the 2.00-6.00% carbon source, carbon source is glucose or sucrose or maltose, the 1.00-3.00% nitrogenous source, nitrogenous source is yeast extract paste or extractum carnis or peptone, the 0.08-0.20% vitamin complex, all the other are moisture;
By above-mentioned heating material to 100 ℃, make material dissolution, mix, sterilizing 30-50 minute under 121 ℃ of lower 0.15MPa pressure, the cooling room temperature of being down to can be used.
Above-mentioned vitamin complex is comprised of VITMAIN B1, Lin Suanna Vitamin B2 Sodium Phosphate, riboflavin, and part by weight is 1:1:1 or 2:2:1.
The fermentation method for producing of Chinese caterpillar fungus pilot scale liquid submerged fermentation with substratum that utilize provided by the invention is characterized in that:
(1) the female kind made: the fresh Cordyceps sinensis of gathering from the Cordyceps sinensis producing region, and the insect compact structure, the separate tissue novel bacterial of cut-off footpath 0.5-1cm, in solid medium, is cultivated and is obtained female kind in 25-30 days;
(2) shake-flask culture: with inoculating needle, mother is planted and is inoculated in the liquid nutrient medium in shaking flask, shaking speed 100-200rpm, cultivate 8-10 days, and mycelium concentration is 30%, seeding tank in preparation;
(3) seed tank culture: according to the inoculum size of 2-5% by the liquid-spawn inoculation of shake-flask culture in the liquid nutrient medium in seeding tank, seeding tank stirring velocity 100-180rpm, tank pressure 0.05-0.10Mpa, air flow quantity 0.60-1.80L/min, cultivate and within 6-8 days, obtain the seeding tank liquid spawn, prepare the top fermentation tank;
(4) fermentor cultivation: according to the inoculum size of 4-10% the liquid-spawn inoculation of shake-flask culture in the liquid nutrient medium in fermentor tank, stirring velocity 100-250rpm, tank pressure 0.05-0.20Mpa, air flow quantity 0.80-2.00L/min, cultivate and within 8-10 days, obtain tunning.
The fermentation method for producing of substratum for worm summer grass mass-producing liquid submerged fermentation provided by the invention fermentation, in the bacterial classification access substratum of shake-flask culture, seed tank culture, fermentor cultivation, the pH value is 5.0-8.0, temperature is 10-20 ℃.
According to the present invention in the Cordyceps sinensis fermenting process, nutritive substance and oxygen being required to the research changed, in seed tank culture and fermentor cultivation process, earlier fermentation, mid-term, later stage adopt different stirring velocitys, tank pressure and air flow quantity, general early stage and rear period parameters are lower, middle period parameters is higher, thereby has reached the purpose that increases mycelium production, shortening fermentation time, minimizing opportunities for contamination, reduces energy consumption.
Positively effect of the present invention: fermentation period is short, be difficult for polluting, step is simple, energy consumption is few, the mycelium productive rate is high.
Embodiment
Below in conjunction with example, method involved in the present invention is described further.
embodiment 1
(1) prepared by female the kind
Female each weight ratio of constituents example of solid medium prepared of planting is: 3% carbon source (press molecular weight and calculate the acquisition carbon element content), carbon source is glucose or sucrose or maltose, 2% nitrogenous source (measure and obtain nitrogen element content), nitrogenous source is yeast extract paste or extractum carnis or peptone; 0.1% sal epsom, 0.15% potassium primary phosphate, 0.08% vitamin complex, 1.00% agar powder, all the other are moisture.Vitamin complex is comprised of VITMAIN B1, Lin Suanna Vitamin B2 Sodium Phosphate, riboflavin, and part by weight is 1:1:1 or 2:2:1;
By above-mentioned mixing of materials, be heated to 100 ℃, make material dissolution, mix, under 121 ℃ of lower 0.15MPa pressure, sterilizing is 40 minutes, is cooled to room temperature and solidifies.
Fresh Cordyceps sinensis (Latin formal name used at school Cordyceps sinensis gathers in the Qinghai Tibet plateau cordyceps producing region, also write Ophiocordyceps sinensis), the early stage grass that the insect of take is sturdy, the Chinese caterpillar fungus bud is less is as best, carry out the conventional organization separation on test tube slant, the insect compact structure, the worm tissue of cut-off footpath 0.5-1cm is received on inclined-plane in solid medium, cultivate 25-30 days, obtain the vigorous mother's kind of growth conditions.
(2) shake-flask culture
Shake-flask culture by each weight ratio of constituents example of substratum is: 5.00% carbon source (carbon source is glucose or sucrose or maltose), 3.00% nitrogenous source (nitrogenous source is yeast extract paste or extractum carnis or peptone), 0.20% vitamin complex (wherein comprises VITMAIN B1, B2 and riboflavin, part by weight is 2:2:1), all the other are moisture.By above-mentioned mixing of materials, be heated to 100 ℃, make material dissolution, mix, under 121 ℃ of lower 0.15MPa pressure, sterilizing is 40 minutes, is cooled to room temperature and solidifies.
With the large triangular flask of 1000ml, cultivate, substratum liquid amount 200-300ml, the mother of access diameter 1cm plants the bacterium piece, regulate pH to 5.0-8.0 with NaOH and HCl, under 10-20 ℃, shaking speed 100-200rpm condition, cultivate 8-10 days, mycelium concentration is 30%, seeding tank in preparation.
(3) seed tank culture
Seed tank culture based components at different levels, proportioning and collocation method, same shake-flask culture.
First order seed tankage 50L, substratum liquid amount 30-40L, the inoculum size ratio access according to 2%, control pH value 5.0-8.0, under 10-20 ℃, cultivates 10 days.1-3 days stirring velocity 100-150rpm wherein, tank pressure 0.05-0.08Mpa, air flow quantity 0.60-1.00L/min; 4-8 days stirring velocity 120-180rpm, tank pressure 0.06-0.10Mpa, air flow quantity 0.80-1.80L/min; 9-10 days stirring velocity 100-150rpm, tank pressure 0.05-0.08Mpa, air flow quantity 0.60-1.00L/min.
Secondary seed tankage 500L, liquid amount 300-400L, inoculum size ratio and fermentation parameter are identical with first class seed pot.
(4) fermentor cultivation
Fermentor cultivation based component, proportioning and collocation method, same shake-flask culture.
5 tons of fermentor tank capacity, substratum liquid amount 3-4 ton, according to 5% inoculum size ratio access, control pH value 5.0-8.0, under 10-20 ℃, cultivates 8 days.1-2 days stirring velocity 100-150rpm wherein, tank pressure 0.05-0.10Mpa, air flow quantity 0.80-1.20L/min; 3-6 days stirring velocity 150-200rpm, tank pressure 0.08-0.20Mpa, air flow quantity 1.00-2.00L/min; 7-8 days stirring velocity 100-150rpm, tank pressure 0.05-0.10Mpa, air flow quantity 0.80-1.20L/min.
(5) the mycelium product obtains
Filter tunning and obtain mycelium, after vacuum-drying, with pulverizer, be crushed to 100-500 order fine powder, obtain pure zymophyte powder of caterpillar fungus, the dry bacterial powder of the present embodiment, yield is 6.8%.
embodiment 2
In embodiment 1, increase the first class seed pot of 5L, 50L and 500L are as secondary and three grades of seeding tanks, and all the other technical process, culture condition are all just the same with embodiment 1, obtain the dry bacterium powder of pure Cordyceps sinensis, and yield is 9.2%.
embodiment 3
With embodiment 1, compare, seed tank culture inoculum size ratio increases to 5%, and fermentor cultivation inoculum size ratio increases to 10%, and all the other technical process, culture condition are all just the same with embodiment 2, obtains the dry bacterium powder of pure Cordyceps sinensis, and yield is 11%.
Claims (5)
1. a Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum, it is characterized in that the solid culture based formulas prepared for mother's kind is as follows: each weight ratio of constituents example is the 1.50-4.00% carbon source, carbon source is glucose or sucrose or maltose, the 0.50-2.00% nitrogenous source, nitrogenous source is yeast extract paste or extractum carnis or peptone, 0.05-0.15% sal epsom, the 0.10-0.20% potassium primary phosphate, the 0.05-0.10% vitamin complex, the 1.00-2.00% agar powder, all the other are moisture;
By above-mentioned heating material to 100 ℃, make material dissolution, mix, sterilizing 30-50 minute under 121 ℃ of lower 0.15MPa pressure, cooling being down to after room temperature is solidified can be used.
Liquid culture based component for shake-flask culture, seed tank culture, fermentor cultivation is consistent, culture medium prescription is as follows: each weight ratio of constituents example is the 2.00-6.00% carbon source, carbon source is glucose or sucrose or maltose, the 1.00-3.00% nitrogenous source, nitrogenous source is yeast extract paste or extractum carnis or peptone, the 0.08-0.20% vitamin complex, all the other are moisture;
By above-mentioned heating material to 100 ℃, make material dissolution, mix, sterilizing 30-50 minute under 121 ℃ of lower 0.15MPa pressure, the cooling room temperature of being down to can be used.
2. Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum according to claim 1, is characterized in that vitamin complex is comprised of VITMAIN B1, Lin Suanna Vitamin B2 Sodium Phosphate, riboflavin, and part by weight is 1:1:1 or 2:2:1.
3. one kind is utilized the fermentation method for producing of the described Chinese caterpillar fungus pilot scale liquid submerged fermentation of claim 1 with substratum, it is characterized in that:
(1) the female kind made: the fresh Cordyceps sinensis of gathering from the Cordyceps sinensis producing region, and the insect compact structure, the separate tissue novel bacterial of cut-off footpath 0.5-1cm, in solid medium, is cultivated and is obtained female kind in 25-30 days;
(2) shake-flask culture: with inoculating needle, mother is planted and is inoculated in the liquid nutrient medium in shaking flask, shaking speed 100-200rpm, cultivate 8-10 days, and mycelium concentration is 30%, seeding tank in preparation;
(3) seed tank culture: according to the inoculum size of 2-5% by the liquid-spawn inoculation of shake-flask culture in the liquid nutrient medium in seeding tank, seeding tank stirring velocity 100-180rpm, tank pressure 0.05-0.10Mpa, air flow quantity 0.60-1.80L/min, cultivate and within 6-8 days, obtain the seeding tank liquid spawn, prepare the top fermentation tank;
(4) fermentor cultivation: according to the inoculum size of 4-10% the liquid-spawn inoculation of shake-flask culture in the liquid nutrient medium in fermentor tank, stirring velocity 100-250rpm, tank pressure 0.05-0.20Mpa, air flow quantity 0.80-2.00L/min, cultivate and within 8-10 days, obtain tunning.
4. worm summer grass mass-producing liquid submerged fermentation according to claim 3 ferments with the fermentation method for producing of substratum; it is characterized in that: in the bacterial classification access substratum of shake-flask culture, seed tank culture, fermentor cultivation; the pH value is 5.0-8.0, and temperature is 10-20 ℃.
5. worm summer grass mass-producing liquid submerged fermentation according to claim 3 ferments with the fermentation method for producing of substratum; it is characterized in that: it is applied in seed tank culture and fermentor cultivation process; the difference required according to different steps Cordyceps sinensis growth, in earlier stage, mid-term, later stage adopt different stirring velocitys, tank pressure and air flow quantity.
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Cited By (6)
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| CN104846018A (en) * | 2015-05-23 | 2015-08-19 | 吉林省方平科技有限公司 | Cordyceps enzyme deep layer liquid fermentation and expanding culture method |
| CN105713848A (en) * | 2016-04-29 | 2016-06-29 | 安发（福建）生物科技有限公司 | Hirsutella sinensis improved culture medium |
| TWI631952B (en) * | 2017-04-24 | 2018-08-11 | 大葉大學 | Nerve cell–like protection of the preparations from ophiocordyceps sinensis submerged culture and production process thereof |
| TWI643627B (en) * | 2017-04-24 | 2018-12-11 | 大葉大學 | Anti-inflammatory application of the extract from ophiocordyceps sinensis mycelium and production process thereof |
| CN112703967A (en) * | 2020-12-07 | 2021-04-27 | 贵州大学 | Method for preparing wood rot fungus liquid strain by using yellow serofluid and bean dregs |
| CN114885748A (en) * | 2022-06-10 | 2022-08-12 | 皖北卫生职业学院 | Rapid culture method of cordyceps sobolifera coreopsis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104846018A (en) * | 2015-05-23 | 2015-08-19 | 吉林省方平科技有限公司 | Cordyceps enzyme deep layer liquid fermentation and expanding culture method |
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| TWI643627B (en) * | 2017-04-24 | 2018-12-11 | 大葉大學 | Anti-inflammatory application of the extract from ophiocordyceps sinensis mycelium and production process thereof |
| CN112703967A (en) * | 2020-12-07 | 2021-04-27 | 贵州大学 | Method for preparing wood rot fungus liquid strain by using yellow serofluid and bean dregs |
| CN114885748A (en) * | 2022-06-10 | 2022-08-12 | 皖北卫生职业学院 | Rapid culture method of cordyceps sobolifera coreopsis |
| CN114885748B (en) * | 2022-06-10 | 2023-06-20 | 皖北卫生职业学院 | Rapid culture method of cordyceps sobolifera stem bundles |
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