CN1181186C - Deel-layer fermentation process for culturing Brazilian cordyceps mycelia - Google Patents
Deel-layer fermentation process for culturing Brazilian cordyceps mycelia Download PDFInfo
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Abstract
The present invention discloses a submerged fermentation and culture method for Brazilian caterpillar fungus mycelium, which takes Brazilian caterpillar fungus as starting fungus and adopts a new-researched formulation of a culture medium for producing caterpillar fungus mycelium under the culture conditions of proper temperature, proper stirring speed, proper pH value, etc., pure caterpillar fungus mycelial powder is produced after the mycelium is dried. The main components and the pharmacological action of caterpillar fungus mycelial powder produced by the method are basically consistent to those of natural caterpillar fungus. The technology can be used for large-scale production and has the advantages of low cost and high developing value.
Description
Technical field
The invention belongs to the biological fermentation field, more specifically say so about a kind of Deel-layer fermentation process for culturing Brazilian cordyceps mycelia.
Background technology
Cordyceps sinensis (Cordyceps Sinesis (Berk) Sacc) is a kind of famous and precious strengthening by means of tonics medicine, and it has pharmaceutical use widely, as strengthening cellular immunization and the body fluid function of exempting from service, the effect of male hormone sample etc.But the natural cs source is limited, costs an arm and a leg.
Summary of the invention
The invention provides the Brazilian cordyceps species of a kind of usefulness and produce the method for mycelium powder of sinensis through submerged fermentation.
The objective of the invention is to solve by the following technical solutions:
Utilize Brazilian cordyceps species, adopt the culture medium prescription of development newly, aerated culture in fermentor tank, culture condition such as the temperature that control suits, stirring velocity, potential of hydrogen, fermentative production Cordyceps mycelium, mycelium make pure mycelium powder of sinensis after drying.Method mainly comprises shake-flask culture, seed tank culture, fermentor cultivation, and concrete technology is as follows:
(1) shake-flask culture: insert Brazilian cordyceps species,, under shaking speed 120-300 rev/min the condition, cultivate after 3-6 days, insert seed tank culture at 22-32 ℃;
(2) seed tank culture: the shake-flask seed that will shake inserts in the seeding tank, inoculum size 1-15%, at 22-32 ℃, stirring velocity 150-500 rev/min, tank pressure 0.03-0.12 MPa, air quantity 1: 0.3-1: 1.2 volume/volume/minute condition under, cultivated 1-4 days;
(3) fermentor cultivation: cultured seed inserts fermentor tank, inoculum size is 5-20%, at 22-32 ℃, stirring velocity 150-350 rev/min, tank pressure 0.02-0.12 MPa, air quantity 1: 0.3-1: 1.2 volume/volume/minute, under the condition of pH6.5-8.0, cultivated 3-6 days, and got Cordyceps mycelium, the dry mycelium powder of sinensis that gets;
Wherein used nutrient media components and weight percentage are: starch 0.5-4%, sugared 0.75-2%, pupa casein 0.5-4%, inorganic salt 0.01-0.2%, pH6.5-8.0, solid medium add 1-2% agar.
The preferred version of the component of substratum and weight percentage is in the method for the present invention: starch 0.8-3.5%, glucose 0.8-1.8%, pupa casein 0.8-3%, MgSO
4And K
2HPO
40.03-0.1%, pH6.5-8.0, solid medium add 1-2% agar.
Used Brazilian cordyceps species is to cultivate and obtain through separate tissue, the domestication of routine from natural cordyceps among the present invention.
The mycelium powder of sinensis major ingredient and pharmacological action and the natural cordyceps basically identical that utilize this processing method to obtain: the content of micro-Cu, Zn, Sr etc. is unanimous on the whole; In 19 kinds of essential amino acids that are rich in, except that Aspartic Acid, Histidine, arginine, all the other basically identicals; The content mycelium powder of sinensis that mainly contains effective constituent D-N.F,USP MANNITOL, ergosterol, Cordyceps polysaccharide is a little more than natural cordyceps; Both are close in the pharmacological action that strengthens cellular immunization, humoral immunization and male hormone sample prescription face; The mycelium powder of sinensis of this explained hereafter is to CCl
4The liver injury of bringing out has the certain protection effect.
This technology can be carried out scale operation, and production cost is low, has higher development and is worth.The mycelium powder of sinensis of this explained hereafter and the main component of natural cs and pharmacological action basically identical are laid a good foundation for it replaces natural cs.
Embodiment
Below in conjunction with embodiment method of the present invention is further described.
Embodiment 1
(1) shake-flask culture
50 liters seeding tank, in 30 liters of loading amounts, inoculum size 3%, the amount of then joining the shake-flask culture base are 30 liters * 3%=0.9 liter.Press starch 1%, glucose 1%, pupa casein 1%, MgSO
40.05%, K
2HPO
40.05%, the prescription of pH6.8 is joined 0.9 liter of nutrient solution, is divided in 500 milliliters of triangular flasks, and every bottled amount is 100 milliliters, installs the back sterilization, is cooled to 30 ℃, inserts the test tube kind.Every bottle graft is gone into 2 about 0.5 square centimeter of kind of piece.Shaking speed is 180 rev/mins, and controlled temperature is 25 ℃, after cultivating in 4 days, is seeded to 50 liters of seed tank culture.
(2) seed tank culture
50 liters seeding tank declared content is 30 liters, deducts 0.9 liter of bottle kind amount of shaking, and then is the 30-0.9=29.1 liter.Press the described prescription of present embodiment (1), join 29.1 liters of substratum (reality only adds water to 26 liters, and 3 liters steam condensate is arranged when estimating sterilization), behind the medium sterilization, treat to insert when temperature is reduced to 30 ℃ the shake-flask seed that shakes, inoculum size is 10%, and the temperature of control seeding tank is 25 ℃, and mixing speed is 300 rev/mins, ventilation be 1: 0.5 volume/volume/minute, tank pressure is 0.05 MPa, cultivates after 2 days, and the seeding tank seed is seeded to 500 liters of fermentation cylinder for fermentation and cultivates.
(3) fermentor cultivation
500 liters of fermentor tanks are 300 liters in actual loading amount, deduct 30 liters of the amounts of seeding tank, and the amount that the real attenuation jar need be joined nutrient solution is: the 300-30=270 liter.The cultivation of joining 270 liters by the described prescription of present embodiment (1) is based on (reality only adds water to 260 liters in 500 liters of fermentor tanks, estimate about 10 liters steam condensate), sterilization, when treating that temperature drops to 30 ℃ in the seeding tank cultured seed insert in the fermentor tank, inoculum size 10%, the control fermentation jar temperature is 25 ℃, mixing speed is 200 rev/mins, ventilation be 1: 0.4 volume/volume/minute, tank pressure 0.05 MPa is cultivated after 5 days, promptly obtain Brazilian Cordyceps mycelium, drying gets pure Brazilian mycelium powder of sinensis, yield 1.76%.
The main component of embodiment 2 mycelium powder of sinensis and natural cs relatively
Table 1, table 2, table 3 are respectively main effective constituent, the trace element of mycelium powder of sinensis and natural cs, the comparison of aminoacids content.
The effective component content that mainly contains of table 1 mycelium powder of sinensis and natural cs compares
| Project | Mycelium powder of sinensis | Natural cordyceps | Analytical procedure |
| D-N.F,USP MANNITOL (%) | 9.54％ | 7.46％ | " Chinese pharmacopoeia (1990) version |
| Ergosterol | 0.19％ | 0.13％ | High pressure liquid chromatography |
| Polysaccharide | 13.68％ | 5% (literature value) | Sulfuric acid-phynol variable color method |
| Adenosine | 0.15×10 -3 | / |
The micronutrient levels of table 2 mycelium powder of sinensis and natural cs relatively
| Element | Mycelium powder of sinensis (ppm) | Natural cordyceps (ppm) | Element | Mycelium powder of sinensis (ppm) | Natural cordyceps (ppm) |
| Al | 990.4 | 1637 | Mg | 2717.8 | 1177.3 |
| Ca | 1155.6 | 552 | Mn | 13.2 | 233.1 |
| Cr | 8.4 | Do not detect | Ni | 5.4 | Do not detect |
| Cu | 12.3 | 10.04 | Sr | 2.4 | 1.2 |
| Ag | Do not detect | Do not detect | Zn | 93.4 | 66.0 |
| Au | Do not detect | Do not detect | Ge | 0.1 | / |
| Fe | 116.1 | 5584.6 | Se | 0.1 | / |
The aminoacids content of table 3 mycelium powder of sinensis and natural cs relatively
| Amino acid | Mycelium powder of sinensis (ppm) | Natural cordyceps (ppm) | Amino acid | Mycelium powder of sinensis (ppm) | Natural cordyceps (ppm) |
| Aspartic Acid | 3.178 | 2.167 | Isoleucine | 0.789 | 0.985 |
| Threonine | 1.153 | 1.190 | Leucine | 1.070 | 1.580 |
| Serine | 0.787 | 0.984 | Tyrosine | 0.540 | 1.008 |
| L-glutamic acid | 2.650 | 3.719 | Phenylalanine | 0.956 | 1.174 |
| Proline(Pro) | 1.115 | 1.397 | Methionin | 0.503 | 1.686 |
| Glycine | 1.330 | 1.210 | Ammonia | 0.440 | 0.880 |
| L-Ala | 1.281 | 1.348 | Histidine | 0.417 | 1.318 |
| Gelucystine | 0.104 | 0.406 | Tryptophane | / | / |
| Xie Ansuan | 1.230 | 1.466 | Arginine | 0.301 | 2.235 |
| Methionine(Met) | 0.434 | 0.438 | Total amount | 18.28 | 24.30 |
From top table 1,2,3 result, as main effectively components D-N.F,USP MANNITOL (cordycepic acid), ergosterol (cordycepin), the Crude polysaccharides of Cordyceps sinensis, its content mycelium powder of sinensis is a little more than natural cordyceps.Both basically identicals of aminoacids content, micronutrient levels differ also little, and both contained chemical ingredients basically identicals are described, lay a good foundation for mycelium powder of sinensis replaces natural cs.
The pharmacological action of embodiment 3 mycelium powder of sinensis and natural cs relatively
(1) to immune organ weight and leukocytic influence
The NIH that gets body weight 12 ± 2g is 80 of mouse, divides equally 8 groups, 10 every group, and by the various dose gastric infusion, qd * 7d, capacity 0.2ml/10g, control group wait capacity distilled water.Immunocompromised 5-8 group is the 4th day intraperitoneal injection of cyclophosphamide 50mg/kg after administration then, last administration 1h posterior orbit is got blood numeration white corpuscle, and dislocation of cervical vertebra puts to death animal and cut open and get thymus gland, and spleen claims weight in wet base with torsion(type)balance, calculate immune organ index (mg/10g body weight), the results are shown in Table 4.
Table 4 pair immune organ weight and leukocytic influence are relatively
| Group | Number of animals | Dosage (g/kg) | E ± SD (mg/10g body weight) | White corpuscle * 10 9/L | |
| Thymus gland | Spleen | ||||
| Mycelium powder of sinensis | 10 | 4 | 33.5±5.0 | 42.2±5.4 | 53.2±10.8 |
| 10 | 8 | 36.2±4.8 | 46.8±8.0 | 55.2±12.2 | |
| Cordyceps sinensis | 10 | 8 | 37.8±5.2 | 47.8±6.6 | 57.7±12.0 |
| Distilled water | 10 | 30.4±6.8 | 38.5±7.8 | 50.8±8.2 | |
| Mycelium powder of sinensis+endoxan | 10 | 4+50mg | 28.6±6.0 | 29.3±8.0 | 31.8±7.2 |
| 10 | 8+50mg | 33.0±4.8 Δ | 33.4±5.8 Δ | 33.4±7.6Δ | |
| Cordyceps sinensis+endoxan | 10 | 8+50mg | 35.0±4.0 ΔΔ | 36.0±5.4 ΔΔ | 37.0±8.0ΔΔ |
| Distilled water+endoxan | 10 | 50mg | 22.0±4.5 | 25.0±4.8 | 26.3±6.6 |
Compare Δ P＜0.05 Δ Δ P＜0.01 (down together) with distilled water+endoxan group
The result shows, mycelium powder of sinensis and Cordyceps sinensis have the trend of increase to normal immune organ thymus gland, spleen and white corpuscle number average, but statistics does not have significant meaning.The thymus gland of immunocompromised group, spleen then have obvious effect of gain, and leukocytic reduction has the rising effect to caused by cyclophosphamide, with control group than (P＜0.05 and 0.01).Its effect is strengthened with dosage within the specific limits.
(2) mouse carbon is cleaned up the influence of speed
Get 40 of body weight 18 ± 2g mouse and divide four groups at random, 10 every group, by the various dose gastric infusion, qd * 7d.Control group give to wait capacity distilled water, and 1h intravenous injection india ink 0.1ml/20g after the last administration injects and got blood 20 microlitres from eye socket respectively in back 2 minutes and 20 minutes and put into 0.1%Na
2CO
3Shake up in the 2ml solution, 670nm surveys the OD value with RA-50 automatic chemical analyzer wavelength, calculates carbon and cleans up index K value, the results are shown in Table 5.
Table 5 pair mouse carbon is cleaned up the comparison of the influence of speed
| Group | Number of animals | Dosage (g/kg) | Carbon is cleaned up index (K value) (E ± SD) |
| Mycelium powder of sinensis | 10 | 4 | 0.044±0.024 |
| 10 | 8 | 0.051±0.022* | |
| Cordyceps sinensis | 10 | 8 | 0.055±0.019** |
| Control group | 10 | 0.036±0.020 |
With control group than * P＜0.05 * * P＜0.01 (down with)
The result shows that each group of mycelium powder of sinensis all can increase carbon and cleans up index, and wherein 8g/kg group and control group point out this product can significantly improve the phagocytic function of body to reticuloendothelial system than difference remarkable (P＜0.05).
(3) to the influence of mouse body endolymph cell transformation function
Get 40 of mouse, divide four groups at random, 10 every group, by the various dose gastric infusion, qd * 7d.Each organizes the 3rd day intramuscular injection PHA6mg/kg of administration, injects continuously 3 days.Except that the distilled water group, each is organized in 50mg/kg of injection the 2nd day equal intraperitoneal injection of cyclophosphamide of PHA, cuts tail after the last administration and gets the blood push jack.Rui Shi-Jim Albert'stain Albert calculates in 100 lymphocytes and transforms percentage of cells, and the difference between comparative group the results are shown in Table 6.
The comparison of the influence of table 6 pair mouse body endolymph cell transformation function
| Group | Number of animals | Dosage (g/kg) | Lymphocyte transformation rate (the % of E ± SD) |
| Distilled water | 10 | 26.5±5.4 | |
| Mycelium powder of sinensis+endoxan | 10 | 4+50mg | 22.4±5.4Δ |
| 10 | 8+50mg | 24.4±5.0Δ | |
| Cordyceps sinensis+endoxan | 10 | 8+50mg | 26.0±6.8ΔΔ |
| Distilled water+endoxan | 10 | 14.6±6.8 |
The result shows that mycelium powder of sinensis 4 and 8g/kg and Cordyceps sinensis all can improve mouse body endolymph cell transformation rate significantly, the antagonism endoxan suppress lymphocytic transformation, with control group than difference remarkable (P＜0.05 and 0.01).Show that this product can strengthen the cellular immune function of mouse.
(4) influence that the mice serum hemolytic antibody is generated
Get 80 of body weight 18 ± 2g mouse, divide 8 groups at random, 10 every group, by the various dose gastric infusion, qd * 7d.Administration was only respectively organized equal abdominal injection 5% chicken red blood corpuscle 0.2mg/ on the 1st day, sensitization, the immunocompromised group is in 50mg/kg of the 4th day intraperitoneal injection of cyclophosphamide of administration, and last administration 1h broken end is got blood and measured hemolysin O D value with RA-50 automatic chemical analyzer wavelength 540nm, calculates half hemolysis value (HC
50), the significance of difference between comparative group the results are shown in Table 7.
The comparison of the influence that table 7 pair mice serum hemolytic antibody generates
| Group | Number of animals | Dosage (g/kg) | HC50( E±SD) |
| Mycelium powder of sinensis | 10 | 4 | 72.3±29.0 |
| 10 | 8 | 77.2±35.0 | |
| Cordyceps sinensis | 10 | 8 | 78.0±26.0 |
| Distilled water | 10 | 67.5±30.6 | |
| Mycelium powder of sinensis+endoxan | 10 | 4+50mg | 40.7±15.2Δ |
| 10 | 8+50mg | 44.5±20.0Δ | |
| Cordyceps sinensis+endoxan | 10 | 8+50mg | 46.8±21.6ΔΔ |
| Distilled water+endoxan | 10 | 50mg | 28.0±18.5 |
The result shows, each group of mycelium powder of sinensis has the trend that promotes that serum hemolysin antibody generates to normal mouse, but statistics does not have significant difference, the effect that endoxan is suppressed the hemolysin generation then has tangible antagonistic action, with control group comparing difference remarkable (P＜0.05), show that mycelium powder of sinensis has enhancement to humoral immunization.
(5) to the influence of male mice testicular weight childhood
Get 40 of body weight 12 ± 2g mouse, divide 4 groups at random, 10 every group, by the various dose gastric infusion, qd * 7d.Control group is given distilled water such as the capacity of grade, and the execution animal is cutd open and gets testis behind the last administration 1h, weighs with torsion(type)balance, and the weight mg/10g of computation organization body weight the results are shown in Table 8.
The influence of table 8 male mice testicular weight pair childhood relatively
| Group | Number of animals | Dosage (g/kg) | Testis (E ± SD mg/10g body weight) |
| Mycelium powder of sinensis | 10 | 4 | 53.4±11.5 |
| 10 | 8 | 63.8±13.2* | |
| Cordyceps sinensis | 10 | 8 | 68.0±15.0* |
| Control group | 10 | 47.0±12.0 |
The result shows that mycelium powder of sinensis and Cordyceps sinensis have obvious effect of gain to the testis of male mice, with control group than difference remarkable (P＜0.05).
(6) to castration mouse sexual accessory gland organ weight's influence
Get 40 of male mices, extract testis down in etherization, postoperative the 5th day, by the various dose gastric infusion, qd * 7d.Control group is given distilled water such as the capacity of grade, and dislocation of cervical vertebra execution animal is cutd open respectively and gets seminal vesicle behind the last administration 1h, preputial glands is weighed, and the results are shown in Table 9.
Table 9 couple castration mouse sexual accessory gland organ weight's influence relatively
| Group | Number of animals | Dosage (g/kg) | (E ± SDmg/10g body weight) | |
| Preputial glands | Seminal vesicle | |||
| Mycelium powder of sinensis | 10 | 4 | 17.0±3.4* | 32.5±17.0* |
| 10 | 8 | 18.6±3.2* | 35.2±21.0* | |
| Cordyceps sinensis | 10 | 8 | 19.5±2.0** | 36.8±24.0** |
| Control group | 10 | 10.0±2.8 | 18.5±28.0 | |
The result shows that each group of mycelium powder of sinensis and Cordyceps sinensis have tangible effect of gain to preputial glands, the seminal vesicle of castration mouse, point out this product to have the effect of male sex hormone sample.
(7) mycelium powder of sinensis is to CCL
4Bring out the influence of mouse liver injury
40 of mouse are divided into 4 groups at random, and 10 every group, by the various dose gastric infusion, qd * 7d.In administration the 7th day, except that the normal control group, all the other 3 groups equal subcutaneous injection 0.1%CCL
4Peanut oil 10ml/kg, injection back fasting 16h broken end is got blood and is surveyed gpt (SGPT) content results such as table 10.
Table 10 mycelium powder of sinensis is to CCL
4Bring out the influence of mouse liver injury
| Group | Number of animals | Dosage (g/kg) | SGPT(μ/L)( E±SD) |
| Mycelium powder of sinensis | 10 | 4 | 70.2±37.2* |
| 10 | 8 | 61.8±28.0** | |
| CCL 4 | 10 | 123.0±56.8 | |
| Control group | 10 | 34.8±15.0 |
With CCL
4Group is than * P＜0.05 * * P＜0.01
Claims (3)
1, a kind of Deel-layer fermentation process for culturing Brazilian cordyceps mycelia comprises shake-flask culture, seed tank culture, fermentor cultivation, it is characterized in that:
(1) shake-flask culture: insert Brazilian cordyceps species,, under shaking speed 120-300 rev/min the condition, cultivate after 3-6 days, insert seed tank culture at 22-32 ℃;
(2) seed tank culture: the shake-flask seed that will shake inserts in the seeding tank, inoculum size 1-15%, at 22-32 ℃, stirring velocity 150-500 rev/min, tank pressure 0.03-0.12 MPa, air quantity 1: 0.3-1: 1.2 volume/volume/minute condition under, cultivated 1-4 days;
(3) fermentor cultivation: cultured seed inserts fermentor tank, inoculum size is 5-20%, at 22-32 ℃, stirring velocity 150-350 rev/min, tank pressure 0.02-0.12 MPa, air quantity 1: 0.3-1: 1.2 volume/volume/minute, under the condition of pH 6.5-8.0, cultivated 3-6 days, and got Cordyceps mycelium, the dry mycelium powder of sinensis that gets; Wherein used nutrient media components and weight percentage are: starch 0.5-4%, glucose 0.75-2%, pupa casein 0.5-4%, inorganic salt 0.01-0.2%, pH 6.5-8.0, solid medium add 1-2% agar; Described inorganic salt are MgSO
4And K
2HPO.
2, Deel-layer fermentation process for culturing Brazilian cordyceps mycelia according to claim 1 is characterized in that used nutrient media components and weight percentage are: starch 0.8-3.5%, glucose 0.8-1.8%, pupa casein 0.8-3%, MgSO
4And K
2HPO
40.03-0.1%, pH 6.5-8.0, solid medium add 1-2% agar.
3, Deel-layer fermentation process for culturing Brazilian cordyceps mycelia according to claim 2 is characterized in that used nutrient media components and weight percentage are: starch 1%, glucose 1%, pupa casein 1%, MgSO
40.05%, K
2HPO
40.05%, pH6.8.
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|---|---|---|---|
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|---|---|---|---|
| CNB021345732A CN1181186C (en) | 2002-08-19 | 2002-08-19 | Deel-layer fermentation process for culturing Brazilian cordyceps mycelia |
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| CN103130907B (en) * | 2013-02-16 | 2014-12-03 | 浙江省林业科学研究院 | Chinese cordyceps sinensis protein polysaccharide and preparation and application thereof |
| CN103211212B (en) * | 2013-04-11 | 2016-01-20 | 天津天狮生物发展有限公司 | A kind of Cordyceps mycelium and preparation method |
| CN103548571B (en) * | 2013-10-29 | 2017-01-04 | 中国海洋大学 | The cultural method of the Cordyceps mycelium of a kind of high yield acidic polysaccharose and acidic polysaccharose |
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