CN103897990A - Process for producing high anticancer component-cordyceps militaris L.Fr Link fungus powder in large scale through submerged fermentation - Google Patents
Process for producing high anticancer component-cordyceps militaris L.Fr Link fungus powder in large scale through submerged fermentation Download PDFInfo
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Abstract
The invention belongs to the technical field of fermentation production of traditional Chinese medicines. Cordyceps militaris L.Fr Link and cordyceps sinensis do not have basic difference in terms of pharmacology, properties and clinical effects, so that cordyceps militaris L.Fr Link is applied to adjuvant chemotherapy of cancers as a substitute of cordyceps sinensis. The main anticancer components of cordyceps militaris L.Fr Link are cordycepin and bioactive polysaccharides. Cordyceps militaris L.Fr Link strains preserved by culture media serve as original strains; first-level shake flask seed culture, second-level shake flask seed culture and fermentation tank seed culture are carried out, and finally the seeds are put into a 1.5-ton fermentation tank for production; after the feeds are fermented for 50-84 hours, mycelia in the fermentation liquor can be 1.53% based on dry weight, the cordycepin content and polysaccharide content of dry fungus powder are respectively 1.28% and 5.28% (the polysaccharide content is measured by a sulfuric acid-anthrone method), and the contents of lead and total arsenic in the dry fungus powder are lower than the standard limited by the country. Taking various fermentation indexes into comprehensive consideration, a process has better application prospects.
Description
Technical field
The invention belongs to herb fermenting production field, be specifically related to a kind of technique that can be used for submerged fermentation and produce high anticancer component Cordyceps militaris (L.) Link. fungus powder.
Technical background
Cordyceps militaris (L.) Link. Cordyceps militaris L.Fr Link has another name called Cordyceps militaris (L.) Link., Cordyceps militaris, belongs to Mycophyta Eumycota, Ascomycetes Ascomycotina, meat seat order Clavicipitales, Clavicipitaceae Clavicipitaceae, Cordyceps Cordyceps.Cordyceps militaris (L.) Link. and Cordyceps sinensis is equal belonging to together aspect biology, is the representative species of this genus.Aspect pharmacology, both effects are also close, therefore be recited as " Cordyceps militaris (L.) Link. " in pharmacopeia.Cordyceps sinensis market price in the modern age hundreds thousand of units of per kilogram, and Cordyceps militaris (L.) Link. relative low price.Therefore, the production of Cordyceps militaris (L.) Link. is subject to extensive concern.
Tumour has exceeded cardiovascular and cerebrovascular disease in China city resident becomes the first cause of death.Research shows that Cordyceps militaris (L.) Link. contains various active composition, as cordycepin, adenosine, polysaccharide, cordycepic acid, SOD isoreactivity composition.The research of relevant anti-tumor activity thinks that cordycepin and Cordyceps polysaccharide are antineoplastic essential substance.Cordycepin has better restraining effect to kinds of tumor cells such as Lewis lung cancer cell, melanochrome B16 cell, leukemia HL-60 cells.Jagger once inferred 3 kinds of possible Related Mechanisms: first, it has a free hydroxyl, and this hydroxyl can penetrate in cancer cells DNA and have an effect; Secondly, suppress the derivative of nucleosides or nucleotide phosphodiesteraseization generation diphosphate and triphosphate, thereby inhibition tumor cell nucleic acid is synthetic; Finally, may block inosinic acid amination and become the sweet acid of bird.In tumor growth reproductive process, need a large amount of adenosines, and the structure of cordycepin and adenosine is closely similar, with adenosine competition Phosphatase, nucleotide, stops synthesizing of mRNA.In addition, cordycepin can also strengthen the antitumous effect of endoxan.Research also shows that Polysaccharides in Cultured Cordyceps militaris has stronger restraining effect to Hela cells, and in the time that be 72h action time, inhibiting rate can reach 74.98%.The antitumor action of Cordyceps polysaccharide not comes from direct killing cancer cells, but stimulates its nonspecific defense function.
Along with the function of Cordyceps militaris (L.) Link. is familiar with widely and accepts by people, solid state cultivation Cordyceps militaris (L.) Link. can not meet people's demand far away, and its production cycle is long, occupation of land is many, production efficiency is low, is subject to the threat of disease and pest.Liquid fermentation technology just applies in medicinal fungi fermentation after microbiotic industrial development is got up, its advantage is not only to carry out that industrialization is produced continuously, scale is large, output is high, fermentation period is short, production efficiency is high, more outstanding is that medicinal fungi is in liquid fermenting process, due to the controllability of substratum and culture condition, can make the expression of fungal gene express to the material aspect accumulation property of specific needs, often can and there is food safety compared with several times of the fungi effective active composition height of wild and cultivated.Therefore, to be applied to that Cordyceps militaris (L.) Link. fungus powder produces be the most effectively means of one to liquid fermentation technology.Though Cordyceps militaris fermentation at present has some patents, as patent No. CN101554121B and patent No. CN102630490B, they only concentrate in the research of cordycepin content.So this patent is by a kind of open report production method of high anticancer component Cordyceps militaris (L.) Link. fungus powder, and it not only has higher cordycepin content, also has higher polysaccharide content.
Summary of the invention
The object of the present invention is to provide a kind of technique for the production of high anticancer component Cordyceps militaris (L.) Link. fungus powder, and require the bacterium powder that this technique is produced to there is reliable edible safety and higher production yield.
The present invention is achieved through the following technical solutions:
Cordyceps militaris (L.) Link. seed culture based formulas is: Semen Maydis powder 10-20g/L, glucose 10-20g/L, bean cake powder 5-15g/L, yeast extract paste 5-10g/L, KH
2pO
41-3g/L, MgSO
4.7H
2o0.5-1.5g/L, soya-bean oil 0.5-1.5g/L, bubble enemy 0.5-1g/L, all the other are safe for drinking water, pH value is 5.0-6.5 (with food grade NaOH adjusting pH).
The culture medium prescription of 1.5 tons of fermentor tanks of Cordyceps militaris (L.) Link. is: mulberry silkworm chrysalis powder 5-10g/L, Semen Maydis powder 10-20g/L, glucose 20-40g/L, bean cake powder 10-20g/L, KH
2pO
41-3g/L, MgSO
4.7H
2o0.5-1g/L, soya-bean oil 0.5-1.5g/L, bubble enemy 0.5-1g/L, all the other are safe for drinking water, pH value is 5.0-7.0 (with food grade NaOH adjusting pH).
Concrete fermentation manufacturing technique is:
(1) cultivation of one-level shake-flask seed:
Cordyceps militaris (L.) Link. slant strains is inoculated in the triangular flask that first order seed substratum is housed, puts into 25～28 ℃ of constant-temperature tables, cultivate 56～96h with rotating speed 120～180rpm/min;
(2) cultivation of secondary shake-flask seed:
One-level shake-flask seed is inoculated in the triangular flask that secondary seed medium is housed, and is placed in 25～28 ℃ of constant-temperature tables, cultivate 56～96h with rotating speed 120～180rpm/min;
(3) seeding tank is produced:
After cooling the 200L stirred-tank fermenter sterilizing that substratum is housed, regulate pH value to 5.0～6.5, by in appropriate secondary shake-flask seed access seed fermentation tank, keeping culture temperature is 25～28 ℃, stirring velocity is 50～150rpm/min (2h turns 5-10min), and air flow is 1: 0.8V/Vmin～1: 1.5V/Vmin, tank pressure 0.03～0.06MPa, cultivate through 40～50h, now thalline weight in wet base is 20-30g/100mL fermented liquid;
(4) 1.5 tons of fermentor tanks are produced:
Pack 800L fermention medium into fermentor tank, after sterilizing is cooling, regulate pH value to 6.0～7.0, the seed of seeding tank is moved in fermentation culture tank, keeping culture temperature is 25～28 ℃, stirring velocity is 50～150rpm/min, air flow is 1: 0.5V/Vmin～1: 1.5V/Vmin, tank pressure 0.03～0.06MPa, cultivating to finish to ferment through 60～85h (requires now fermented liquid to become thickness, medium pH drops to 3.8～4.8, more than in fermented liquid, wet mycelia reaches 40g/100mL fermented liquid).
(5) processing of bacterium powder:
Fermented liquid, after Plate Filtration is placed 12-24h, under the oven conditions of 60-85 ℃, is dried to 6-12h by wet thallus, the bacterium powder of oven dry is pulverized with pulverizer, be Cordyceps militaris (L.) Link. fungus powder.
Advantage of the present invention: the one, zymophyte powder has higher cordycepin content, and content reaches 1.28%; The 2nd, zymophyte powder has high intracellular polyse content (sulfuric acid-anthrone method is measured), and polysaccharide content reaches 5.28%; The 2nd, this raw materials technology safety, bacterium powder detects and meets national limit standard completely through plumbous, total arsenic content; The 3rd, to produce bacterium powder yield and reach 1.54%, fermentation period is shorter, and fermention medium cost is lower, and three factors cause holistic cost not high.
Specific embodiments
The mensuration of cordycepin content is with reference to " the mensuration high performance liquid chromatography DB32/T1275-2008 of cordycepin content in Cordyceps militaris (L.) Link. "
The extraction of bacterium powder polysaccharide and determination of polysaccharide are with reference to " Pharmacopoeia of People's Republic of China 2005 editions ".
Lead in bacterium grain weight metal, the mensuration of total arsenic and limit standard are with reference to " edible mushrooms hygienic standard GB7096-2003 ".
Example 1:
Cordyceps militaris (L.) Link. seed culture based formulas is: Semen Maydis powder 10-20g/L, glucose 10-20g/L, bean cake powder 5-15g/L, yeast extract paste 5-10g/L, KH
2pO
41-3g/L, MgSO
4.7H
2o0.5-1.5g/L, soya-bean oil 0.5-1.5g/L, bubble enemy 0.5-1g/L, all the other are safe for drinking water, pH value is 5.0-6.5 (with food grade NaOH adjusting pH).
Screening formulation is: Semen Maydis powder 20g/L, glucose 20g/L, bean cake powder 5g/L, yeast extract paste 5g/L, KH
2pO
43g/L, MgSO
4.7H
2o1.5g/L, soya-bean oil 1g/L, bubble enemy 0.5g/L, all the other are safe for drinking water, pH value is 5.1 (with food grade NaOH adjusting pH).
The culture medium prescription of 1.5 tons of fermentor tanks of Cordyceps militaris (L.) Link. is: mulberry silkworm chrysalis powder 5-10g/L, Semen Maydis powder 10-20g/L, glucose 20-40g/L, bean cake powder 10-20g/L, KH
2pO
41-3g/L, MgSO
4.7H
2o0.5-1g/L, soya-bean oil 0.5-1.5g/L, bubble enemy 0.5-1g/L, all the other are safe for drinking water, pH value is 5.0-7.0 (with food grade NaOH adjusting pH).
Screening formulation is: mulberry silkworm chrysalis powder 5g/L, Semen Maydis powder 10g/L, glucose 30g/L, bean cake powder 15g/L, KH
2pO
41g/L, MgSO
4.7H
2o0.5g/L, soya-bean oil 1g/L, bubble enemy 0.5g/L, all the other are safe for drinking water, pH value is 6.73 (with food grade NaOH adjusting pH).
Concrete fermentation manufacturing technique is:
The whole substratum of the present embodiment adopt more excellent formula, ferment take the Cordyceps militaris spawn of having preservation by oneself as starting strain, specifically comprise the following steps: (1) is inoculated in Cordyceps militaris (L.) Link. slant strains in the 500ml triangular flask that 200ml first order seed substratum is housed, put into 25 ℃ of constant-temperature tables, under rotating speed 150rpm/min, cultivate 84h; (2) one-level shake-flask seed is inoculated in the 1L triangular flask that 400mL secondary seed medium is housed with 5% inoculum size, and is placed in 25 ℃ of constant-temperature tables, cultivate 84h with rotating speed 150rpm/min; (3) 120L seed culture medium will be housed after the sterilizing of 200L stirred-tank fermenter is cooling, regulate pH value to 5.1, by in 800mL secondary shake-flask seed access seed fermentation tank, keeping culture temperature is 25 ℃, stirring velocity is 100rpm/min (2h stirs 5min), and air flow is 1: 1.11V/Vmin, tank pressure 0.4MPa, cultivate through 48h, thalline weight in wet base is 22.9g/100mL fermented liquid; (4) pack 1500L fermention medium into fermentor tank, after sterilizing is cooling, regulate pH value to 6.73, the seed of seeding tank is moved in fermentation culture tank, and keeping culture temperature is 25 ℃, and stirring velocity is 100rpm/min (2h stirs 5min), air flow is 1: 0.72V/Vmin, tank pressure 0.04MPa finishes fermentation after 84h cultivates, and now thalline weight in wet base is 40.63g/100mL fermented liquid (now pH4.13).(5) processing of bacterium powder: after Plate Filtration is placed 24h, wet thallus dries 12h in the baking oven of 85 ℃, and the bacterium powder of oven dry is pulverized with machine, is Cordyceps militaris (L.) Link. fungus powder by fermented liquid.Bacterium powder cordycepin content is 1.28%, and polysaccharide content is 5.28%.Bacterium powder production yield 1.53%.Plumbous, total arsenic content 0.26ppm and 0.32ppm respectively, well below national limit standard (lead≤2ppm, total arsenic≤1ppm).
It should be noted that: the above, be only a preferred embodiments of the present invention, but obviously the invention is not restricted to above example to have many distortion.Those of ordinary skill in the art can be easy to it to make some modifications or improvements.Therefore, any modification or the improvement made without departing from theon the basis of the spirit of the present invention, all should think the scope of protection of present invention.
Claims (3)
1. the technique that high anticancer component Cordyceps militaris (L.) Link. fungus powder is produced in submerged fermentation on a large scale, its feature comprises the steps:
(1) cultivation of one-level shake-flask seed: Cordyceps militaris (L.) Link. slant strains is inoculated in the triangular flask that seed culture medium is housed, puts into 25 ℃～28 ℃ constant-temperature tables, cultivate 56～96h under the rotating speed of 120～180rpm/min;
(2) enlarged culturing of secondary shake-flask seed: one-level shake-flask seed is inoculated in the triangular flask that seed culture medium is housed, is placed in 25 ℃～28 ℃ constant-temperature tables, cultivate 56～96h under the rotating speed of 120～180rpm/min;
(3) enlarged culturing again of seeding tank: after cooling the 200L stirred-tank fermenter sterilizing that seed culture medium is housed, regulate pH value to 5.0～6.5, by in appropriate secondary shake-flask seed access seeding tank, keeping culture temperature is 25～28 ℃, stirring velocity is 50～150rpm/min (2h stirs 5-10min), and air flow is 1: 0.8V/Vmin～1.5V/Vmin, tank pressure 0.03～0.06MPa, cultivate through 40～50h, now thalline weight in wet base is 20-30g/100mL fermented liquid;
(4) 1.5 tons of fermentor tanks are produced: pack 800L fermention medium into fermentor tank, after sterilizing is cooling, regulate pH value to 6.0～7.0, the seed of seeding tank is moved in fermentor tank, keeping culture temperature is 25～28 ℃, stirring velocity is 50～150rpm/min (2h stirs 5-10min), air flow is 1: 0.5V/Vmin～1: 1.5V/Vmin, tank pressure 0.03～0.06MPa, cultivating to finish to ferment through 60～85h (requires now fermented liquid to become thickness, medium pH drops to 3.8～4.8, more than in fermented liquid, wet mycelia reaches 40g/100mL fermented liquid),
(5) preparation of zymophyte powder: fermented liquid is placed after 12-24h through Plate Filtration, wet thallus is placed under the oven conditions of 60 ℃-85 ℃ and dries 6-12h, the bacterium powder of oven dry is pulverized with machine, be Cordyceps militaris (L.) Link. fungus powder.
2. technique according to claim 1, is characterized in that the substratum of described one-level, secondary shake-flask seed and one grade fermemtation tank seed culture based formulas are: Semen Maydis powder 10-20g/L, glucose 10-20g/L, bean cake powder 5-15g/L, yeast extract paste 5-10g/L, KH
2pO
41-3g/L, MgSO
4.7H
2o0.5-1.5g/L, soya-bean oil 0.5-1.5g/L, bubble enemy 0.5-1g/L, all the other are safe for drinking water, pH value is 5.0-7.0 (with food grade NaOH adjusting pH).
3. technique according to claim 1, is characterized in that described fermentor cultivation based formulas is: mulberry silkworm chrysalis powder 5-10g/L, Semen Maydis powder 10-20g/L, glucose 20-40g/L, bean cake powder 10-20g/L, KH
2pO
41-3g/L, MgSO
4.7H
2o0.5-1g/L, soya-bean oil 0.5-1.5g/L, bubble enemy 0.5-1g/L, all the other are safe for drinking water, pH value is 5.0-7.0 (with food grade NaOH adjusting pH).
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Cited By (4)
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| CN104498367A (en) * | 2014-11-28 | 2015-04-08 | 沈阳源康生物技术发展有限公司 | Medium for cordyceps militaris liquid deep fermentation and use 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 |
| CN109007823A (en) * | 2018-08-09 | 2018-12-18 | 张斌 | A kind of preparation method of selenium-enriched cordceps militaris powder |
| CN109010382A (en) * | 2018-09-08 | 2018-12-18 | 河南欧普生物科技有限公司 | A kind of preparation method of fermentation cordyceps oral solution for animals |
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| CN109007823A (en) * | 2018-08-09 | 2018-12-18 | 张斌 | A kind of preparation method of selenium-enriched cordceps militaris powder |
| CN109010382A (en) * | 2018-09-08 | 2018-12-18 | 河南欧普生物科技有限公司 | A kind of preparation method of fermentation cordyceps oral solution for animals |
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