CN103483040B - Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum and fermentation method for producing thereof - Google Patents
Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum and fermentation method for producing thereof Download PDFInfo
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Abstract
The invention discloses a kind of Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum and fermentation method for producing thereof; female kind is made with the Cordyceps sinensis of gathering from Qinghai-Tibet Platean; mother is planted implant in triangular flask and carry out shaking table cultivation; preparation liquid spawn; then access in seeding tank at different levels and carry out fermentation culture, amplify step by step, in fermentor tank, finally realize the large-scale deep liquid fermentation of Cordyceps sinensis; final fermenter volume reaches 5 tons or more, and mycelium dry weight reaches 5-15%.The present invention adopts the Cordyceps strain of high-quality, special culture media and fermenting technique, achieves and promotes Cordyceps fungus bulk-growth, reduction of erection time, decreasing pollution chance, save energy, the object that reduces costs.
Description
Technical field
The invention belongs to edible and medicinal fungi fermentation production technology field, particularly relate to a kind of Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum and fermentation method for producing thereof.
Background technology
Cordyceps sinensis belongs to the fungal species (Classification system is Cordyceps sinensis, also writes Ophiocordyceps sinensis) of Clavicipitaceae, cries again China pilose spore or China by hair spore.Cordyceps sinensis has long-term applicating history at folks of china, and be senior, famous and precious herbal medicine invigorant, 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 aititude such as Qinghai, Tibet, main product in Jinsha jiang River, the Lancang River, Nujiang Three Rivers regions upstream.Excavate and environmental disruption due to excessive, wild Cordyceps Resources falls sharply, and can not meet market demand.
Cordyceps mycelium can be produced in a large number by liquid submerged fermentation, scientist study the chemical composition and pharmacological action that find Cordyceps sinensis fermented hypha and wild cordyceps similar, therefore the Cordyceps mycelium of fermentative production becomes the important substitute of wild cordyceps.More domestic researchers have delivered some Patents and paper at present, disclose substratum and the culture process of some Cordyceps sinensis liquid fermentings, but ubiquity fermentation period is long, easily pollutions, complex steps, energy consumption greatly, problem that mycelium productive rate is not high.
Summary of the invention
The object of this invention is to provide a kind of Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum and fermentation method for producing thereof, fermentation period is short, not easily pollute, step is simple, energy consumption is few, mycelium productive rate is high.
Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum provided by the invention, its each Ingredients Weight ratio is:
1.50-4.00% carbon source, carbon source is glucose or sucrose or maltose, 0.50-2.00% nitrogenous source, nitrogenous source is yeast extract paste or extractum carnis or peptone, 0.05-0.15% magnesium sulfate, 0.10%-0.20 potassium primary phosphate, 0.05-0.10% vitamin complex, 1.00-2.00% agar powder, all the other are moisture;
By above-mentioned heating material to 100 DEG C, make material dissolution, mixing, 0.15MPa sterilized under pressure 30-50 minute at 121 DEG C, cooling is down to after room temperature is solidified and 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 Ingredients Weight ratio is 2.00-6.00% carbon source, carbon source is glucose or sucrose or maltose, 1.00-3.00% nitrogenous source, nitrogenous source is yeast extract paste or extractum carnis or peptone, 0.08-0.20% vitamin complex, all the other are moisture;
By above-mentioned heating material to 100 DEG C, make material dissolution, mixing, 0.15MPa sterilized under pressure 30-50 minute at 121 DEG C, cooling is down to room temperature and can be used.
Above-mentioned vitamin complex is made up 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 utilizing Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum provided by the invention, is characterized in that:
(1) female kind makes: fresh Cordyceps sinensis of gathering from Cordyceps sinensis producing region, from the separate tissue novel bacterial of cut-off footpath 0.5-1cm insect compact structure in solid medium, cultivates and obtains female kind in 25-30 days;
(2) shake-flask culture: be inoculated in the liquid nutrient medium in shaking flask with inoculating needle by mother's kind, shaking speed 100-200rpm, cultivate 8-10 days, 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 obtain seeding tank liquid spawn in 6-8 days, prepare 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 obtain tunning in 8-10 days.
The fermentation method for producing of worm summer grass mass-producing liquid submerged fermentation fermentation substratum provided by the invention, in the bacterial classification access substratum of shake-flask culture, seed tank culture, fermentor cultivation, pH value is 5.0-8.0, and temperature is 10-20 DEG C.
According to the present invention in Cordyceps sinensis fermenting process to nutritive substance and oxygen requirements change research, 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 lower, middle period parameters is higher, thus reaches the object increasing mycelium production, shortening fermentation time, decreasing pollution chance, reduction energy consumption.
Positively effect of the present invention: fermentation period is short, not easily pollute, step is simple, energy consumption is few, 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 kind
Female each Ingredients Weight ratio of solid medium prepared of planting is: 3% carbon source (calculate by molecular weight and obtain carbon element content), carbon source is glucose or sucrose or maltose, 2% nitrogenous source (measure obtain nitrogen element content), nitrogenous source is yeast extract paste or extractum carnis or peptone; 0.1% magnesium sulfate, 0.15% potassium primary phosphate, 0.08% vitamin complex, 1.00% agar powder, all the other are moisture.Vitamin complex is made up 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 DEG C, make material dissolution, mixing, at 121 DEG C, 0.15MPa sterilized under pressure 40 minutes, is cooled to room temperature and solidifies.
Cordyceps sinensis (the Classification system Cordyceps sinensis gathering fresh in Qinghai Tibet plateau cordyceps producing region, also Ophiocordyceps sinensis is write), the early stage grass that, Chinese caterpillar fungus bud sturdy with insect is less is the best, test tube slant carries out conventional organization separation, insect compact structure, the worm tissue of cut-off footpath 0.5-1cm to be received on inclined-plane in solid medium, cultivate 25-30 days, obtain mother's kind that growth conditions is vigorous.
(2) shake-flask culture
The each Ingredients Weight ratio of shake-flask culture 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 DEG C, make material dissolution, mixing, at 121 DEG C, 0.15MPa sterilized under pressure 40 minutes, is cooled to room temperature and solidifies.
Cultivate with the large triangular flask of 1000ml, substratum liquid amount 200-300ml, the mother of access diameter 1cm plants bacterium block, regulate pH to 5.0-8.0 with NaOH and HCl, 10-20 DEG C, under 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 component at different levels, proportioning and collocation method, same to shake-flask culture.
First order seed tankage 50L, substratum liquid amount 30-40L, according to the inoculum size ratio access of 2%, control ph 5.0-8.0, cultivates 10 days at 10-20 DEG C.Wherein 1-3 days stirring velocity 100-150rpm, 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 identical with first class seed pot.
(4) fermentor cultivation
Fermentor cultivation based component, proportioning and collocation method, same to shake-flask culture.
Fermentor tank capacity 5 tons, substratum liquid amount 3-4 ton, according to 5% inoculum size ratio access, control ph 5.0-8.0, cultivates 8 days at 10-20 DEG C.Wherein 1-2 days stirring velocity 100-150rpm, 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) mycelium product obtains
Fermentation-derived product obtains mycelium, and be crushed to 100-500 order fine powder with pulverizer after vacuum-drying, 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 is as secondary and three grades of seeding tanks, and all the other technical process, culture condition are all just the same with embodiment 1, and obtain the dry bacterium powder of pure Cordyceps sinensis, yield is 9.2%.
embodiment 3
Compared with embodiment 1, seed tank culture inoculum size ratio is increased to 5%, and fermentor cultivation inoculum size ratio is increased to 10%, and all the other technical process, culture condition are all just the same with embodiment 2, and obtain the dry bacterium powder of pure Cordyceps sinensis, yield is 11%.
Claims (2)
1. a Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum, it is characterized in that planting for mother the solid culture based formulas prepared as follows: each Ingredients Weight ratio is 1.50-4.00% carbon source, carbon source is glucose or sucrose or maltose, 0.50-2.00% nitrogenous source, nitrogenous source is yeast extract paste or extractum carnis or peptone, 0.05-0.15% magnesium sulfate, 0.10-0.20% potassium primary phosphate, 0.05-0.10% vitamin complex, 1.00-2.00% agar powder, all the other are moisture; By above-mentioned heating material to 100 DEG C, make material dissolution, mixing, 0.15MPa sterilized under pressure 30-50 minute at 121 DEG C, cooling is down to after room temperature is solidified and 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 Ingredients Weight ratio is 2.00-6.00% carbon source, carbon source is glucose or sucrose or maltose, 1.00-3.00% nitrogenous source, nitrogenous source is yeast extract paste or extractum carnis or peptone, 0.08-0.20% vitamin complex, all the other are moisture; By above-mentioned heating material to 100 DEG C, make material dissolution, mixing, 0.15MPa sterilized under pressure 30-50 minute at 121 DEG C, cooling is down to room temperature and can be used.
2. utilize a fermentation method for producing for Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum described in claim 1, it is characterized in that:
(1) female kind makes: fresh Cordyceps sinensis of gathering from Cordyceps sinensis producing region, from the separate tissue novel bacterial of cut-off footpath 0.5-1cm insect compact structure in solid medium, cultivates and obtains female kind in 25-30 days;
(2) shake-flask culture: be inoculated in the liquid nutrient medium in shaking flask with inoculating needle by mother's kind, regulate pH to 5.0-8.0 with NaOH and HCI, temperature is 10-20oC, shaking speed 100-200rpm, and cultivate 8-10 days, 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, the same shake-flask culture of pH value, temperature; 1-3 days stirring velocity 100-150rpm of seed tank culture, 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; Cultivate to obtain seeding tank liquid spawn, prepare 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, the same shake-flask culture of pH value, temperature; 1-2 days stirring velocity 100-150rpm of fermentor cultivation, 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; Cultivate to obtain tunning.
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| CN107409748A (en) * | 2017-05-17 | 2017-12-01 | 左春萌 | It is a kind of can factorial praluction Antrodia camphorata breeding method |
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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 |
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