CN105505784A - Cordyceps militaris culture medium capable of improving cordycepin content, and preparation method of cordyceps militaris culture medium - Google Patents
Cordyceps militaris culture medium capable of improving cordycepin content, and preparation method of cordyceps militaris culture medium Download PDFInfo
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Abstract
The invention relates to a cordyceps militaris culture medium capable of improving cordycepin content, and a preparation method of the cordyceps militaris culture medium. The method comprises the following steps: (1) preparing stock culture; (2) preparing liquid strain; (3) culturing sporocarp. The cordyceps militaris culture medium is prepared from the following main raw materials: potatoes, millet, barley, soybean meal, cane sugar, cocoon chrysalis powder, milk powder, brown sugar, peptone, purified water and the like. According to the preparation method, adenine is added into the formula of a liquid strain culture medium, and the adenine is coordinated and complementary with the soybean meal, the cane sugar, the cocoon chrysalis powder and other nutrient materials, so that the cordycepin content is effectively improved; the millet and the barley are taken as a sporocarp substrate; the cordycepin content of the cordyceps militaris sporocarp is 8.0-8.5mg/ g. Furthermore, the cordyceps militaris culture is easy in obtaining of components, convenient to prepare, low in cost, simple in practical application and operation and easy to popularize, thus being suitable for large-scale production of cordyceps militaris.
Description
Technical field
The present invention relates to medical, edible bacteria cultivation field, is a kind of Chinese caterpillar fungus culture medium formula and production method of improving cordycepin content specifically.
Background technology
Cordyceps militaris (L.) Link. has another name called Cordyceps militaris (L.) Link., Cordyceps militaris, belong to Mycophyta Ascomycetes Hypocreales Clavicipitaceae Cordyceps, belong to together not of the same race with Cordyceps sinensis, its Chemical Composition and effect closely similar, wherein containing compositions such as cordycepin, Cordyceps polysaccharide, cordycepic acid, adenosines, there is enhancing immunity, endocrine regulation, protect liver guarantor's nutritional health function such as lung, sexual function improving and multiple medicinal efficacy, can as the substitute of Cordyceps sinensis.
Cordycepin (Cordycepin) is the important secondary metabolite of one that Cordyceps militaris (L.) Link. produces, as the bioactive ingredients that Cordyceps militaris (L.) Link. is important, its chemical structure is cordycepin, is found first by Cunningham (1951) from Cordyceps militaris (L.) Link..Because cordycepin has the effects such as antibacterial, antiviral, antitumor, immunomodulatory, the market requirement is increasing, and price is very expensive, and on world market, the price of cordycepin sterling is about up to 2500 dollars/g.
At present, there is many drawbacks in the chemosynthesis of cordycepin, the main source of the nucleosides materials such as cordycepin or Cordyceps militaris (L.) Link. culture, and therefore the optimization of Cordyceps militaris (L.) Link. solid culture method is the focus in Cordyceps militaris (L.) Link. research always.Liquid culture due to short production cycle, condition easily controls, cordycepin is easy to extract and output is more stable, be to obtain the comparatively effective cultural method of cordycepin at present.
Summary of the invention:
The object of the present invention is to provide a kind of Chinese caterpillar fungus culture medium and the production method that improve cordycepin content, be included in liquid nutrient medium and add precursor substance-VITAMIN B4 and carry out the optimization of solid culture method with millet, barley for raw material.
The technical solution used in the present invention is: a kind of Chinese caterpillar fungus culture medium improving cordycepin content, comprises PDA enriched medium, liquid spawn culture medium and sporophore substratum.
Described PDA enriched medium consists of: potato juice 700-800ml, sucrose 18-22g, cocoon pupa juice 200-300ml, milk powder 90-100g, agar 15-20g.
Described liquid spawn culture medium consists of: cocoon pupa juice 200-300ml, brown sugar 10-15g, peptone 5-10g, soya bean juice 200-300ml, pure water 400-600ml.VITAMIN B4 mass concentration 0.8-1.0g/L.
Described sporophore substratum consists of: millet 10-20g, barley 10-15g, liquid spawn culture medium 35-45ml.
Above-mentioned a kind of Chinese caterpillar fungus culture medium improving cordycepin content, described potato juice is added water by potato to boil, and crosses leaching juice, by weight, potato: water=1:5-8.
Above-mentioned a kind of Chinese caterpillar fungus culture medium improving cordycepin content, described cocoon pupa juice is that cocoon pupa powder is added water boil 20-40min, crosses leaching juice, by weight, cocoon pupa powder: water=1:8-10.
Above-mentioned a kind of Chinese caterpillar fungus culture medium improving cordycepin content, described soya bean juice is that analysis for soybean powder is added water boil 20-40min, crosses leaching juice, by weight, analysis for soybean powder: water=1:5-8.
Above-mentioned a kind of Chinese caterpillar fungus culture medium improving cordycepin content, in described liquid spawn culture medium, the Adding Way of VITAMIN B4 is, after first cocoon pupa juice, brown sugar, peptone, soya bean juice and pure water being mixed, autoclaving, to add VITAMIN B4 after 0.20um microfiltration membrane removing miscellaneous bacteria, be adjusted to mass concentration 0.8-1.0g/L.
Above-mentioned a kind of Chinese caterpillar fungus culture medium improving cordycepin content is cultivating the application in Cordyceps militaris (L.) Link..Method is as follows:
1) female kind makes: by Cordyceps militaris spawn, be aseptically inoculated in PDA enriched medium, puts 23 DEG C of constant temperature lucifuges and cultivates, see that light is cultivated after mycelium germination, obtains original mother and plants.
2) liquid spawn makes: get original mother's kind and be inoculated in liquid spawn culture medium, 3-5d cultivated by 23 DEG C of shaking tables, obtain liquid spawn.
3) sporophore is cultivated: sealed by sporophore substratum polypropylene film, in 121 DEG C of-126 DEG C of steam sterilizings, maintains 40min-60min, cultivates by 2-5% inoculum size by the sporophore substratum of liquid-spawn inoculation after sterilizing.
4) cultivation management: vegetative stage requires that lucifuge is cultivated; After mycelia has thorough grasp culture material completely, be 50-100lx, temperature 15-25 DEG C in light intensity, space humidity is under 70%-75%, carries out low light level cultivation; After fruit-body formation, through 8-12 DEG C of thermal stimulation, adding forced ventilation, ventilate every day 2 times-3 times, each 30min, is 200-500lx, temperature 16-20 DEG C in light intensity, under space humidity 85%-95%, cultivates 40d-50d.
The advantage that the present invention has:
1. add precursor substance-VITAMIN B4 in liquid medium within of the present invention, can promptly be transported in cell, effectively absorbed by Cordyceps militaris (L.) Link. and utilize, by cooperative compensating effect, effectively can improve cordycepin content with the nutritive substance such as analysis for soybean powder, sucrose, cocoon pupa powder.
2. the present invention carries out solid culture method optimization with millet, barley for raw material.German millet nutrition enriches, and cost is lower than rice, but particle is little, and after steaming and decocting under high pressure, viscosity is high, hinders mycelial growth.Barley particles is large, and permeability is strong, and used in combination with millet, overcome the shortcoming that millet viscosity is high, and in barley, fatty acid content is higher, external lot of documents reports the growth that vegetables oil can promote mycelia, improves the content of cordycepin.
3. Cordyceps militaris (L.) Link. biological transformation ratio of the present invention increases by 25% than using existing ordinary culture medium and prior art cultural method; In every 1g Cordyccps-militaris-(L.)-link. Sporophore, cordycepin content is 8.0-8.5mg, adenosine content is 17-21mg, apparently higher than use art methods cultivate the Cordyceps militaris (L.) Link. obtained.
4. medium component of the present invention easily obtains, and preparation is convenient, with low cost, and actual operation is simple, is easy to promote, is applicable to the large-scale production of Liaoning Province Cordyceps militaris (L.) Link..
Embodiment
Embodiment 1: a kind of Cordyceps militaris (L.) Link. production method improving cordycepin content
1. cordyceps militaris plantation district requirement
The edible fungus culturing district directly adjoined with conventional farmland must arrange the buffer strip being greater than 30m, to avoid the impact of banned substance.Cultivation place and around prohibit the use chemical synthetic pesticide.Water source should meet the requirement of GB5749.
2. cultivation matrix: the matrix that should adopt organic production or natural materials.
3. female kind makes: by Cordyceps militaris spawn, aseptically be inoculated in PDA enriched medium, put 23 DEG C of constant temperature lucifuges to cultivate, after mycelium germination, see that light is cultivated, selection mycelial growth is neat, and healthy and strong, after seeing light, annesl is fast, examine under a microscope, mycelia bacterial strain that is sturdy, that stand upright is that original mother plants.
PDA enrichment incubation based formulas: potato juice 700ml, sucrose 18g, cocoon pupa juice 300ml, milk powder 90g, agar 15g.
Described potato juice is: boiled by potato to shortcake not rotten, crosses leaching juice, by weight, and potato: water=1:5.
Described cocoon pupa juice is: cocoon pupa powder is added water boil 30min, crosses leaching juice, by weight, and cocoon pupa powder: water=1:8.
4. liquid spawn makes: get 1 piece of 8-10mm
2the original master clock of size is inoculated in the triangular flask (250ml) that 150ml liquid spawn culture medium is housed, and 23 DEG C of shaking tables are cultivated (150r/min) 3-5d and stopped cultivating.Carry out looking into by bottle order from cultivation 2d, find that there is that bacterium liquid is muddy, mycelia slowly long or there is peculiar smell be contaminated bacteria bottle, to sample in cultivation and do slat chain conveyor and microscopy mid-term subsequently, so that Timeliness coverage is with or without the pollution of bacterium and mould, ensure strain quality.
Described liquid spawn culture medium consists of: cocoon pupa juice 200ml, brown sugar 10g, peptone 5g, soya bean juice 300ml, VITAMIN B4 mass concentration 0.8g/L, pure water 500ml.
Described cocoon pupa juice is added water by cocoon pupa powder, boils 30min, crosses leaching juice, by weight, and cocoon pupa powder: water=1:8.
Described soya bean juice is added water at analysis for soybean powder, boils 30min, crosses leaching juice, by weight, and analysis for soybean powder: water=1:5.
The Adding Way of VITAMIN B4 is, after first cocoon pupa juice, brown sugar, peptone, soya bean juice and pure water being mixed, autoclaving, to add VITAMIN B4 after 0.20um microfiltration membrane removing miscellaneous bacteria, is adjusted to mass concentration 0.8g/L.
5. sporophore is cultivated: sporophore substratum polypropylene film is sealed sterilizing.Adopt 121 DEG C of-126 DEG C of high pressure steam sterilizations, hold time 40min-60min, cooling, cultivates in the sporophore substratum of liquid-spawn inoculation after sterilizing by 2% inoculum size.
Described sporophore substratum consists of: millet 10g, barley 10g, step 4) obtained liquid spawn culture medium 35ml.
6. cultivation management: vegetative stage requires that lucifuge is cultivated, and the low light level is cultivated after mycelia has thorough grasp culture material completely, and light intensity is 50-100lx, temperature 15-25 DEG C, and space humidity is 70%-75%; After fruit-body formation, through about 10 DEG C thermal stimulations, forced ventilation need be added, ventilate every day 2 times-3 times, each 30min.Light intensity is 200-500lx, temperature 16-20 DEG C, space humidity 85%-95%, cultivates 40d-50d sporophore maturation and plucks, dry stand-by.
Embodiment 2: a kind of Cordyceps militaris (L.) Link. production method improving cordycepin content
1. cordyceps militaris plantation district requirement
The edible fungus culturing district directly adjoined with conventional farmland must arrange the buffer strip being greater than 30m, to avoid the impact of banned substance.Cultivation place and around prohibit the use chemical synthetic pesticide.Water source should meet the requirement of GB5749.
2. cultivation matrix: the matrix that should adopt organic production or natural materials.
3. female kind makes: by Cordyceps militaris spawn, aseptically be inoculated in PDA enriched medium, put 23 DEG C of constant temperature lucifuges to cultivate, after mycelium germination, see that light is cultivated, selection mycelial growth is neat, and healthy and strong, after seeing light, annesl is fast, examine under a microscope, mycelia bacterial strain that is sturdy, that stand upright is that original mother plants.
PDA enrichment incubation based formulas: potato juice 800ml, sucrose 22g, cocoon pupa juice 200ml, milk powder 100g, agar 20g.
Described potato juice is added water by potato, boils 30min, crosses leaching juice, by weight, and potato: water=1:8.
Described cocoon pupa juice is added water by cocoon pupa powder, boils 30min, crosses leaching juice, by weight, and cocoon pupa powder: water=1:10.
4. liquid spawn makes: get 1 piece of 8-10mm
2the original master clock of size is inoculated in the triangular flask (250ml) that 150ml liquid spawn culture medium is housed, and 23 DEG C of shaking tables are cultivated (150r/min) 3-5d and stopped cultivating.Carry out looking into by bottle order from cultivation 2d, find that there is that bacterium liquid is muddy, mycelia slowly long or there is peculiar smell be contaminated bacteria bottle, to sample in cultivation and do slat chain conveyor and microscopy mid-term subsequently, so that Timeliness coverage is with or without the pollution of bacterium and mould, ensure strain quality.Described liquid spawn culture medium consists of: cocoon pupa juice 300ml, brown sugar 15g, peptone 10g, soya bean juice 250ml, VITAMIN B4 mass concentration 1.0g/L, pure water 450ml.
Described cocoon pupa juice is added water by cocoon pupa powder, boils 30min, crosses leaching juice, by weight, and cocoon pupa powder: water=1:10.
Described soya bean juice is added water at analysis for soybean powder, boils 30min, crosses leaching juice, by weight, and analysis for soybean powder: water=1:8.
The Adding Way of VITAMIN B4 is, after first cocoon pupa juice, brown sugar, peptone, soya bean juice and pure water being mixed, autoclaving, to add VITAMIN B4 after 0.20um microfiltration membrane removing miscellaneous bacteria, is adjusted to mass concentration 0.8g/L.
5. sporophore is cultivated: sporophore substratum polypropylene film is sealed sterilizing.Adopt 121 DEG C of-126 DEG C of high pressure steam sterilizations, hold time 40min-60min, cultivates in the sporophore substratum of liquid-spawn inoculation after sterilizing by 2% inoculum size.
Described sporophore substratum consists of: millet 20g, barley 15g, step 4) obtained liquid spawn culture medium 45ml.
6. cultivation management: vegetative stage requires that lucifuge is cultivated, and the low light level is cultivated after mycelia has thorough grasp culture material completely, and light intensity is 50-100lx, temperature 15-25 DEG C, and space humidity is 70%-75%; After fruit-body formation, through about 10 DEG C thermal stimulations, forced ventilation need be added, ventilate every day 2 times-3 times, each 30min.Light intensity is 200-500lx, temperature 16-20 DEG C, space humidity 85%-95%, cultivates 40d-50d sporophore maturation and plucks, dry stand-by.
Embodiment 3 comparative example
1) female kind makes: PDA substratum: potato juice (200g potato is added the heating of 1000g water and boiled leaching juice), glucose 20g, peptone 3g, MgSO
41.5g, KH
2pO
43g, agar 15g, pure water 1000ml.By Cordyceps militaris spawn, be aseptically inoculated in PDA substratum, put 23 DEG C of constant temperature lucifuges and cultivate, after mycelium germination, see that light is cultivated.Selection mycelial growth is neat, healthy and strong, and after seeing light, annesl is fast, examines under a microscope, and mycelia bacterial strain that is sturdy, that stand upright is that original mother plants.
2) liquid spawn makes: liquid nutrient medium: potato juice (200g potato is added the heating of 1000g water and boiled leaching juice), brown sugar 20g, peptone 3g, MgSO
41.5g, KH
2pO
43g, pure water 1000ml.Get 1 piece of 8-10mm
2the original mother of size plants to be inoculated in is equipped with in 150ml liquid nutrient medium triangular flask (250ml), and 23 DEG C of shaking tables are cultivated (150r/min) 3-5d and stopped cultivating.Carry out looking into by bottle order from cultivation 2d, find that there is that bacterium liquid is muddy, mycelia slowly long or there is peculiar smell be contaminated bacteria bottle, to sample in cultivation and do slat chain conveyor and microscopy mid-term subsequently, so that Timeliness coverage is with or without the pollution of bacterium and mould, ensure strain quality.
3) sporophore is cultivated: every bottle (750ml Cans) add wheat 10g, step 2) obtained liquid nutrient medium 35ml, seals sterilizing with polypropylene film.Adopt high pressure steam sterilization 121 DEG C-126 DEG C, hold time 40min-60min, cultivates in the sporophore substratum of liquid-spawn inoculation after sterilizing by 2% inoculum size.
4) cultivation management: vegetative stage requires that lucifuge is cultivated, and the low light level is cultivated after mycelia has thorough grasp culture material completely, and light intensity is 50-100lx, temperature 15-25 DEG C, and space humidity is 70%-75%; After fruit-body formation, through about 10 DEG C thermal stimulations, forced ventilation need be added, ventilate every day 2 times-3 times, each 30min.Light intensity is 200-500lx, temperature 16-20 DEG C, space humidity 85%-95%, cultivates 40d-50d sporophore maturation and plucks, dry stand-by.
Results and analysis
Table 1
| Cordycepin (mg/g) | Chinese caterpillar fungus adenosine (mg/g) | Biological transformation ratio (%) | |
| Embodiment 1 | 8.2 | 21 | 150 |
| Embodiment 2 | 8.4 | 19 | 148 |
| Comparative example | 2.1 | 7 | 120 |
As can be seen from Table 1, adopt the Cordyceps militaris (L.) Link. that culture medium prescription of the present invention is turned out, its cordycepin, Chinese caterpillar fungus adenosine content are apparently higher than ordinary culture medium, and biological transformation ratio also increases by 25%.
Claims (7)
1. improve a Chinese caterpillar fungus culture medium for cordycepin content, it is characterized in that comprising PDA enriched medium, liquid spawn culture medium and sporophore substratum;
Described PDA enriched medium consists of: potato juice 700-800ml, sucrose 18-22g, cocoon pupa juice 200-300ml, milk powder 90-100g, agar 15-20g;
Described liquid spawn culture medium consists of: cocoon pupa juice 200-300ml, brown sugar 10-15g, peptone 5-10g, soya bean juice 200-300ml, pure water 400-600ml, VITAMIN B4 mass concentration 0.8-1.0g/L;
Described sporophore substratum consists of: millet 10-20g, barley 10-15g, liquid spawn culture medium 35-45ml.
2. a kind of Chinese caterpillar fungus culture medium improving cordycepin content according to claim 1, is characterized in that: described potato juice is added water by potato to boil, and crosses leaching juice, by weight, and potato: water=1:5-8.
3. a kind of Chinese caterpillar fungus culture medium improving cordycepin content according to claim 1, is characterized in that: described cocoon pupa juice is that cocoon pupa powder is added water boil 20-40min, crosses leaching juice, by weight, and cocoon pupa powder: water=1:8-10.
4. a kind of Chinese caterpillar fungus culture medium improving cordycepin content according to claim 1, is characterized in that: described soya bean juice is that analysis for soybean powder is added water boil 20-40min, crosses leaching juice, by weight, and analysis for soybean powder: water=1:5-8.
5. a kind of Chinese caterpillar fungus culture medium improving cordycepin content according to claim 1, it is characterized in that: in described liquid spawn culture medium, the Adding Way of VITAMIN B4 is, after first cocoon pupa juice, brown sugar, peptone, soya bean juice and pure water being mixed, 121 DEG C of-126 DEG C of autoclavings, to add VITAMIN B4 after 0.20um microfiltration membrane removing impurity, be adjusted to mass concentration 0.8-1.0g/L.
6. the arbitrary described a kind of Chinese caterpillar fungus culture medium improving cordycepin content of claim 1-5 is producing the application in Cordyceps militaris (L.) Link..
7. application according to claim 6, is characterized in that method is as follows:
1) female kind makes: by Cordyceps militaris spawn, be aseptically inoculated in PDA enriched medium, puts 23 DEG C of constant temperature lucifuges and cultivates, see that light is cultivated after mycelium germination, obtains original mother and plants;
2) liquid spawn makes: get original mother's kind and be inoculated in liquid spawn culture medium, 3-5d cultivated by 23 DEG C of shaking tables, obtain liquid spawn;
3) sporophore is cultivated: sealed by sporophore substratum polypropylene film, in 121 DEG C of-126 DEG C of steam sterilizings, maintains 40min-60min, cultivates by 2-5% inoculum size by the sporophore substratum of liquid-spawn inoculation after sterilizing;
4) cultivation management: vegetative stage requires that lucifuge is cultivated; After mycelia has thorough grasp culture material completely, be 50-100lx, temperature 15-25 DEG C in light intensity, space humidity is under 70%-75%, carries out low light level cultivation; After fruit-body formation, through 8-12 DEG C of thermal stimulation, adding forced ventilation, ventilate every day 2 times-3 times, each 30min, is 200-500lx, temperature 16-20 DEG C in light intensity, under space humidity 85%-95%, cultivates 40d-50d.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754404A (en) * | 2016-11-21 | 2017-05-31 | 王军 | A kind of Chinese caterpillar fungus culture medium for improving cordycepin content |
| CN108338040A (en) * | 2018-05-17 | 2018-07-31 | 江门市山海堂虫草有限公司 | A kind of Chinese caterpillar fungus culture medium and preparation method thereof |
| CN108617405A (en) * | 2018-05-14 | 2018-10-09 | 安发（福建）生物科技有限公司 | A method of improving cordycepin accumulation in Cordyceps militaris solid state cultivation |
| CN110447463A (en) * | 2019-08-12 | 2019-11-15 | 江门市山海堂虫草有限公司 | A kind of preparation method of cordyceps sporophore |
| CN110637677A (en) * | 2019-08-12 | 2020-01-03 | 江门市山海堂虫草有限公司 | Culture medium for improving cordycepin of cordyceps militaris and preparation method thereof |
| CN113151007A (en) * | 2021-03-30 | 2021-07-23 | 浙江工业大学 | Liquid fermentation medium for increasing cordycepin content in cordyceps militaris |
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| CN102898207A (en) * | 2012-09-12 | 2013-01-30 | 崔学恒 | Liquid medium for cordyceps militaris strain, and preparation and application methods thereof |
| CN104663252A (en) * | 2015-03-19 | 2015-06-03 | 张东海 | Breeding method and production process for cordyceps militaris strain with high cordycepin content |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1104249A (en) * | 1994-08-25 | 1995-06-28 | 俞永信 | Deep fermentation technology for cordyceps sinensis sacc |
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| CN104663252A (en) * | 2015-03-19 | 2015-06-03 | 张东海 | Breeding method and production process for cordyceps militaris strain with high cordycepin content |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754404A (en) * | 2016-11-21 | 2017-05-31 | 王军 | A kind of Chinese caterpillar fungus culture medium for improving cordycepin content |
| CN108617405A (en) * | 2018-05-14 | 2018-10-09 | 安发（福建）生物科技有限公司 | A method of improving cordycepin accumulation in Cordyceps militaris solid state cultivation |
| CN108338040A (en) * | 2018-05-17 | 2018-07-31 | 江门市山海堂虫草有限公司 | A kind of Chinese caterpillar fungus culture medium and preparation method thereof |
| CN110447463A (en) * | 2019-08-12 | 2019-11-15 | 江门市山海堂虫草有限公司 | A kind of preparation method of cordyceps sporophore |
| CN110637677A (en) * | 2019-08-12 | 2020-01-03 | 江门市山海堂虫草有限公司 | Culture medium for improving cordycepin of cordyceps militaris and preparation method thereof |
| CN113151007A (en) * | 2021-03-30 | 2021-07-23 | 浙江工业大学 | Liquid fermentation medium for increasing cordycepin content in cordyceps militaris |
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Application publication date: 20160420 |