CN105969653A - Bioreactor for standing and fermenting cordyceps militaris and fermenting method for cordyceps militaris - Google Patents
Bioreactor for standing and fermenting cordyceps militaris and fermenting method for cordyceps militaris Download PDFInfo
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Abstract
The invention discloses a bioreactor for standing and fermenting cordyceps militaris. The bioreactor comprises a wide-mouth glass container which is arranged in a magnetic stirring constant temperature water bath pot; an inverted stainless steel screen mesh is arranged at the bottom of the wide-mouth glass container; magnetic stirrers are arranged at the bottom of the wide-mouth glass container in the stainless steel screen mesh; a sterile agar gel layer is arranged on the stainless steel screen mesh; the top of the wide-mouth glass container is wrapped with gauze. The invention also discloses a method for standing and fermenting the cordyceps militaris by utilizing the bioreactor. According to the method, the limit to the height of a culture medium in existing standing and fermenting of the cordyceps militaris is overcome, and the practical fermenting volume is increased, so that cordycepin can be produced by performing industrial large-scale standing and fermenting on the cordyceps militaris, and the capacity of the cordycepin is greatly improved. Therefore, the method has very high practical application value.
Description
Technical field
The invention belongs to technical field of microbial fermentation, be specifically related to bioreactor and the fermentation process thereof of a kind of Cordyceps militaris (L.) Link. standing for fermentation.
Background technology
Cordycepin is one of active component main in Cordyceps.Cordycepin has various biological activity, such as antitumor, antiproliferative, metastasis, antibacterial, antiviral, immunomodulating and antiinflammatory etc..The preparation of cordycepin mainly has chemosynthesis and biosynthesis two ways.Owing to current chemosynthesis cordycepin production cost is high, synthesis technique is complicated, and yield is low, and product purification is more difficult, so cordycepin is mainly prepared by biological synthesis process.Biological synthesis process is prepared Cordyceps and is have two kinds of approach: one is that solid fermentation obtains Cordyceps sporophore, more therefrom extracts；Two is by Cordyceps liquid fermentation, extracting directly from fermentation liquid.Owing to liquid fermentation extracts, than solid fermentation advantage in fermentation-scale, biomass growth rate, stand density and controllability, Cordyceps liquid fermentation, the cordycepin preparation method that cordycepin becomes main.
Existing document report repeats interval surface liquid fermentation Cordyceps militaris (L.) Link. to be prepared cordycepin and can reach 15-17g/L, is the fermentation method best practice of preparing cordycepin.But this standing for fermentation method is owing to mass transfer rate is relatively slow, and culture medium height is less, greatly limit actual volume of culture and fermentation-scale.But there is presently no and strengthen the bioreactor of standing for fermentation mass transfer rate and corresponding fermentation process.
Summary of the invention
The technical problem to be solved is for the deficiencies in the prior art, it is provided that the new-type bioreactor of a kind of Cordyceps militaris (L.) Link. standing for fermentation.
The present invention also to solve the technical problem that and be to provide the method utilizing the new-type bioreactor of above-mentioned Cordyceps militaris (L.) Link. standing for fermentation to carry out fermenting and producing cordycepin.
For solving above-mentioned technical problem, the technical solution used in the present invention is as follows:
A kind of bioreactor of Cordyceps militaris (L.) Link. standing for fermentation, it includes the open mouth glass container being arranged in magnetic-mixing constant temperature water bath boiler, inverted stainless steel mesh it is provided with bottom open mouth glass container, magnetic stir bar it is provided with bottom open mouth glass container, inside stainless steel mesh, stainless steel sift arranges one layer of sterile letheen gel layer, open mouth glass container top parcel gauze on the net.
Wherein, the top planes of inverted stainless steel mesh has mesh；Described agar gel layer to cover all the top planes of inverted stainless steel mesh.
Wherein, described gauze is 6 layers.
Wherein, described open mouth glass container is 100-1000mL glass beaker, preferably 500mL glass beaker.
The length of magnetic stir bar is determined by the size of open mouth glass container.When described open mouth glass container is 100mL, support the use Φ 8mm × 30mm polytetrafluoro magnetic stir bar；When described open mouth glass container is 500mL, support the use Φ 8mm × 50mm polytetrafluoro magnetic stir bar；When described open mouth glass container is 1000mL, support the use Φ 9mm × 70mm polytetrafluoro magnetic stir bar.
The diameter of stainless steel mesh and height are determined by the size of open mouth glass container.When described open mouth glass container is 100mL, support the use diameter 5cm, highly 3cm, the stainless steel mesh of mesh 10 mesh；When described open mouth glass container is 500mL, support the use diameter 8cm, highly 4cm, the stainless steel mesh of mesh 10 mesh；When described open mouth glass container is 1000mL, support the use diameter 12cm, highly 5cm, the stainless steel mesh of mesh 10 mesh being inverted 2 layers.
Wherein, described sterile letheen gel layer by the 15-40g/L agar powder aqueous solution of the adenine containing 2-20g/L, is poured into cooling in Φ 6-12cm sterile petri dish and is prepared after high-temp steam sterilizing；Preferably by the 20g/L agar powder aqueous solution of the adenine containing 10g/L after high-temp steam sterilizing, pour cooling in Φ 9cm sterile petri dish into and prepare.
Utilizing the method that above-mentioned bioreactor carries out Cordyceps militaris (L.) Link. standing for fermentation, it comprises the steps:
1), in open mouth glass container, add magnetic stir bar, and be inverted stainless steel mesh, fermentation medium is poured in open mouth glass container and make liquid level exceed stainless steel mesh 1-3cm (preferably 2cm), system sterilizing；
2) Cordyceps militaris (L.) Link. is scraped from preservation inclined-plane 22-30 DEG C 180-300 rev/min concussion cultivation 3-5d in fermentation medium and obtains seed liquor；
3) by step 2) seed liquor for preparing accesses step 1 with the inoculum concentration of 5-20%v/v (preferably 10%v/v)) described in fermentation medium in, it is covered with sterile letheen gel layer, to cover the top planes of stainless steel mesh completely, open mouth glass container top sterile gauze seals；
4) by step 3) open mouth glass container be placed in stir culture in magnetic-mixing constant temperature water bath boiler, speed of agitator 60-120 rev/min, temperature is set to 25-27 DEG C, cultivate 21-34d.
Step 2) in, described Cordyceps militaris (L.) Link. is Cordyceps militaris CICC 14014.
Step 2) in, preferably Cordyceps militaris (L.) Link. is scraped from preservation inclined-plane 27 DEG C 250 revs/min concussions in fermentation medium and cultivates 4d.
Step 1) or 2) in, described fermentative medium formula is: glucose 30-50g/L, yeast extract 6-12g/L, peptone 10-30g/L, potassium dihydrogen phosphate 0.3-1.0g/L, dipotassium hydrogen phosphate 0.3-1.0g/L, magnesium sulfate 0.3-2.0g/L, solvent is water, with hydrochloric acid tune pH value to 5.0-7.0.Preferably fermentative medium formula is: glucose 42g/L, yeast extract 9g/L, peptone 17g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, and solvent is water, adjusts pH value to 5.8 with hydrochloric acid.
Step 4) in, preferred condition of culture is, speed of agitator 100 revs/min, and temperature is set to 27 DEG C, cultivates 21d.
Beneficial effect: the present invention has the advantage that relative to prior art
1) repeating intermittent liquid surface fermentation is a kind of standing for fermentation, is also the best approach of Cordyceps militaris (L.) Link. fermenting and producing cordycepin, and production concentration is high.But being as the needs that extensive cordycepin produces, in standing for fermentation, mass transfer rate becomes the maximum bottleneck of the method industrialized production slowly；And because the epontic reason of thalline liquid medium within, fermentation broth height is only 1-2cm.When fermentation broth height is more than more than 2cm, production concentration drastically declines.And this technology can solve this problem well, with stirrer when bottom is stirred, fermentation medium flows, and accelerates the mass transfer that agar gel is upper and lower, does not interferes with the most again the thalline growth at liquid surface.Therefore, use the method for the invention can be greatly increased the fermentation volume of Cordyceps militaris (L.) Link., increase fermentation-scale and obtain the production concentration identical with repeating intermittent liquid surface fermentation simultaneously.
2) use adenine immobilization technology to be embedded in agar gel by adenine, prevent adenine and cordycepin solids mixing.When cordycepin synthesizes in a large number, cordycepin concentration i.e. separates out formation precipitation more than 15g/L, so can be present in the most in solid form in fermentation liquid with unreacted adenine, affect later separation.When adenine is immobilized in agar gel, adenine solid is not present in fermentation liquid, reduces the difficulty of follow-up cordycepin purification.
Accompanying drawing explanation
Fig. 1 is the structural representation of the bioreactor of Cordyceps militaris (L.) Link. standing for fermentation in embodiment 1.
Detailed description of the invention
Following example further illustrate present disclosure, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment that the inventive method, step or condition are made or replacement, belong to the scope of the present invention.
If not specializing, the conventional means that technological means used in following example is well known to those skilled in the art.
Embodiment 1: the new-type bioreactor of a kind of Cordyceps militaris (L.) Link. standing for fermentation.
As shown in Figure 1, a kind of bioreactor of Cordyceps militaris (L.) Link. standing for fermentation, it includes the open mouth glass container 1 being arranged in magnetic-mixing constant temperature water bath boiler, inverted stainless steel mesh 2 it is provided with bottom open mouth glass container 1, the top planes of inverted stainless steel mesh 2 has mesh 6, bottom open mouth glass container 1, inside stainless steel mesh 2, it is provided with magnetic stir bar 3, one layer of sterile letheen gel layer 4 is set on stainless steel mesh 2, described agar gel layer 4 to cover all the top planes of inverted stainless steel mesh 2, open mouth glass container 1 top 6 layers of gauze 5 of parcel.
Wherein, described open mouth glass container 1 is 500mL glass beaker, supports the use Φ 8mm × 50mm polytetrafluoro magnetic stir bar and diameter 8cm, highly 4cm, the stainless steel mesh of mesh 10 mesh.
Wherein, described agar gel layer by the 20g/L agar powder aqueous solution of the adenine containing 10g/L, is poured into cooling in Φ 9cm sterile petri dish and is prepared after high-temp steam sterilizing.
Embodiment 2: the new-type bioreactor of a kind of Cordyceps militaris (L.) Link. standing for fermentation.
As shown in Figure 1, a kind of bioreactor of Cordyceps militaris (L.) Link. standing for fermentation, it includes the open mouth glass container 1 being arranged in magnetic-mixing constant temperature water bath boiler, inverted stainless steel mesh 2 it is provided with bottom open mouth glass container 1, the top planes of inverted stainless steel mesh 2 has mesh 6, bottom open mouth glass container 1, inside stainless steel mesh 2, it is provided with magnetic stir bar 3, one layer of sterile letheen gel layer 4 is set on stainless steel mesh 2, described agar gel layer 4 to cover all the top planes of inverted stainless steel mesh 2, open mouth glass container 1 top 6 layers of gauze 5 of parcel.
Wherein, described open mouth glass container 1 is 100mL glass beaker, supports the use Φ 8mm × 30mm polytetrafluoro magnetic stir bar and diameter 5cm, highly 3cm, the stainless steel mesh of mesh 10 mesh.
Wherein, described agar gel layer by the 15g/L agar powder aqueous solution of the adenine containing 2g/L, is poured into cooling in Φ 6cm sterile petri dish and is prepared after high-temp steam sterilizing.
Embodiment 3: the new-type bioreactor of a kind of Cordyceps militaris (L.) Link. standing for fermentation.
As shown in Figure 1, a kind of bioreactor of Cordyceps militaris (L.) Link. standing for fermentation, it includes the open mouth glass container 1 being arranged in magnetic-mixing constant temperature water bath boiler, inverted stainless steel mesh 2 it is provided with bottom open mouth glass container 1, the top planes of inverted stainless steel mesh 2 has mesh 6, bottom open mouth glass container 1, inside stainless steel mesh 2, it is provided with magnetic stir bar 3, one layer of sterile letheen gel layer 4 is set on stainless steel mesh 2, described agar gel layer 4 to cover all the top planes of inverted stainless steel mesh 2, open mouth glass container 1 top 6 layers of gauze 5 of parcel.
Wherein, described open mouth glass container 1 is 1000mL glass beaker, supports the use Φ 9mm × 70mm polytetrafluoro magnetic stir bar and is inverted diameter 12cm, highly 5cm, the stainless steel mesh of mesh 10 mesh of 2 layers.
Wherein, described agar gel layer by the 40g/L agar powder aqueous solution of the adenine containing 20g/L, is poured into cooling in Φ 12cm sterile petri dish and is prepared after high-temp steam sterilizing.
Embodiment 4:
Utilizing the method that the bioreactor of embodiment 1 carries out Cordyceps militaris (L.) Link. standing for fermentation, it comprises the steps:
1), in open mouth glass container 1, add magnetic stir bar 3, and be inverted stainless steel mesh 2, fermentation medium is poured into and open mouth glass container 1 makes liquid level 7 exceed stainless steel mesh 22cm, system sterilizing；
2) Cordyceps militaris (L.) Link. Cordyceps militaris CICC 14014 is scraped from preservation inclined-plane 27 DEG C 250 revs/min concussion cultivation 4d in fermentation medium and obtains seed liquor；
3) by step 2) seed liquor for preparing accesses step 1 with the inoculum concentration of 10%v/v) described in fermentation medium in, it is covered with sterile letheen gel layer 4, to cover the top planes of stainless steel mesh 2 completely, open mouth glass container 1 top sterile gauze seals；
4) by step 3) open mouth glass container 1 be placed in stir culture in magnetic-mixing constant temperature water bath boiler, speed of agitator 100 revs/min, temperature is set to 27 DEG C, cultivate 21d.Fermentation liquid is collected, filters, measure fermentating liquid volume with graduated cylinder, with the content of cordycepin in Fermentation Liquor by High Performance Liquid Chromatography, the results are shown in Table 1.
Step 1) or 2) in, described fermentative medium formula is: glucose 42g/L, yeast extract 9g/L, peptone 17g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, and solvent is water, adjusts pH value to 5.8 with hydrochloric acid.
Embodiment 5:
Utilizing the method that the bioreactor of embodiment 2 carries out Cordyceps militaris (L.) Link. standing for fermentation, it comprises the steps:
1), in open mouth glass container 1, add magnetic stir bar 3, and be inverted stainless steel mesh 2, fermentation medium is poured into and open mouth glass container 1 makes liquid level 7 exceed stainless steel mesh 21cm, system sterilizing；
2) Cordyceps militaris (L.) Link. Cordyceps militaris CICC 14014 is scraped from preservation inclined-plane 22 DEG C 300 revs/min concussion cultivation 5d in fermentation medium and obtains seed liquor；
3) by step 2) seed liquor for preparing accesses step 1 with the inoculum concentration of 5%v/v) described in fermentation medium in, it is covered with sterile letheen gel layer 4, to cover the top planes of stainless steel mesh 2 completely, open mouth glass container 1 top sterile gauze seals；
4) by step 3) open mouth glass container 1 be placed in stir culture in magnetic-mixing constant temperature water bath boiler, speed of agitator 60 revs/min, temperature is set to 25 DEG C, cultivate 34d.Fermentation liquid is collected, filters, measure fermentating liquid volume with graduated cylinder, with the content of cordycepin in Fermentation Liquor by High Performance Liquid Chromatography, the results are shown in Table 1.
Step 1) or 2) in, described fermentative medium formula is: glucose 30g/L, yeast extract 6g/L, peptone 10g/L, potassium dihydrogen phosphate 0.3g/L, dipotassium hydrogen phosphate 0.3g/L, magnesium sulfate 0.3g/L, and solvent is water, adjusts pH to 5.0 with hydrochloric acid.
Embodiment 6:
Utilizing the method that the bioreactor of embodiment 3 carries out Cordyceps militaris (L.) Link. standing for fermentation, it comprises the steps:
1), in open mouth glass container 1, add magnetic stir bar 3, and be inverted stainless steel mesh 2, fermentation medium is poured into and open mouth glass container 1 makes liquid level 7 exceed stainless steel mesh 23cm, system sterilizing；
2) Cordyceps militaris (L.) Link. Cordyceps militaris CICC 14014 is scraped from preservation inclined-plane 30 DEG C 180 revs/min concussion cultivation 3d in fermentation medium and obtains seed liquor；
3) by step 2) seed liquor for preparing accesses step 1 with the inoculum concentration of 20%v/v) described in fermentation medium in, it is covered with sterile letheen gel layer 4, to cover the top planes of stainless steel mesh 2 completely, open mouth glass container 1 top sterile gauze seals；
4) by step 3) open mouth glass container 1 be placed in stir culture in magnetic-mixing constant temperature water bath boiler, speed of agitator 120 revs/min, temperature is set to 27 DEG C, cultivate 21d.Fermentation liquid is collected, filters, measure fermentating liquid volume with graduated cylinder, with the content of cordycepin in Fermentation Liquor by High Performance Liquid Chromatography, the results are shown in Table 1.
Step 1) or 2) in, described fermentative medium formula is: glucose 50g/L, yeast extract 12g/L, peptone 30g/L, potassium dihydrogen phosphate 1.0g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate 2.0g/L, and solvent is water, adjusts pH to 7.0 with hydrochloric acid.
Cordycepin concentration in table 1 fermentating liquid volume and fermentation liquid:
| Fermentating liquid volume (mL) | Cordycepin concentration (g/L) in fermentation liquid | |
| Embodiment 4 | 350 | 21.7 |
| Embodiment 5 | 70 | 18.1 |
| Embodiment 6 | 900 | 19.2 |
Claims (10)
1. the bioreactor of a Cordyceps militaris (L.) Link. standing for fermentation, it is characterised in that it includes being arranged at magnetic agitation constant temperature
Open mouth glass container (1) in water-bath, open mouth glass container (1) bottom is provided with inverted stainless steel mesh (2),
Open mouth glass container (1) bottom, stainless steel mesh (2) inside are provided with magnetic stir bar (3), stainless steel mesh (2)
On one layer of sterile letheen gel layer (4) be set, open mouth glass container (1) top parcel gauze (5).
The bioreactor of Cordyceps militaris (L.) Link. standing for fermentation the most according to claim 1, it is characterised in that inverted not
The top planes of rust steel screen cloth (2) has mesh (6)；Described agar gel layer (4) to cover all inverted
The top planes of stainless steel mesh (2).
The bioreactor of Cordyceps militaris (L.) Link. standing for fermentation the most according to claim 1, it is characterised in that described yarn
Cloth (5) is 6 layers.
The bioreactor of Cordyceps militaris (L.) Link. standing for fermentation the most according to claim 1, it is characterised in that described is wide
Mouth glass container (1) is 100-1000mL glass beaker.
The bioreactor of Cordyceps militaris (L.) Link. standing for fermentation the most according to claim 4, it is characterised in that when described
When open mouth glass container (1) is 100mL, support the use Φ 8mm × 30mm polytetrafluoro magnetic stir bar；When described
Open mouth glass container (1) when being 500mL, support the use Φ 8mm × 50mm polytetrafluoro magnetic stir bar；Work as institute
When the open mouth glass container (1) stated is 1000mL, support the use Φ 9mm × 70mm polytetrafluoro magnetic stir bar.
The bioreactor of Cordyceps militaris (L.) Link. standing for fermentation the most according to claim 4, it is characterised in that when described
When open mouth glass container (1) is 100mL, support the use diameter 5cm, highly 3cm, the rustless steel of mesh 10 mesh
Screen cloth；When described open mouth glass container (1) is 500mL, support the use diameter 8cm, highly 4cm, mesh
The stainless steel mesh of 10 mesh；When described open mouth glass container (1) is 1000mL, supports the use and be inverted 2 layers
Diameter 12cm, highly 5cm, the stainless steel mesh of mesh 10 mesh.
The bioreactor of Cordyceps militaris (L.) Link. standing for fermentation the most according to claim 1, it is characterised in that described nothing
Bacterio-agar gel layer, is fallen by the 15-40g/L agar powder aqueous solution of the adenine containing 2-20g/L after high-temp steam sterilizing
Enter cooling in 6-12cm sterile petri dish to prepare.
8. utilize the method that the bioreactor described in claim 1 carries out Cordyceps militaris (L.) Link. standing for fermentation, it is characterised in that
It comprises the steps:
1), in open mouth glass container (1), add magnetic stir bar (3), and be inverted stainless steel mesh (2), will fermentation
Culture medium is poured into and is made liquid level (7) exceed stainless steel mesh (2) 1-3cm, system sterilizing in open mouth glass container (1)；
2) Cordyceps militaris (L.) Link. is scraped from preservation inclined-plane 22-30 DEG C 180-300 rev/min concussion in fermentation medium and cultivates 3-5d
Obtain seed liquor；
3) by step 2) seed liquor for preparing accesses step 1 with the inoculum concentration of 5-20%v/v) described in fermentation medium
In, it is covered with sterile letheen gel layer (4), to cover the top planes of stainless steel mesh (2) completely, open mouth glass holds
Device (1) top sterile gauze seals；
4) by step 3) open mouth glass container (1) be placed in stir culture in magnetic-mixing constant temperature water bath boiler, stirring turns
Speed 60-120 rev/min, temperature is set to 25-27 DEG C, cultivates 21-34d.
Method the most according to claim 8, it is characterised in that step 2) in, described Cordyceps militaris (L.) Link. is Cordyceps
militaris CICC 14014。
Method the most according to claim 8, it is characterised in that step 1) or 2) in, described fermentation training
Foster based formulas is: glucose 30-50g/L, yeast extract 6-12g/L, peptone 10-30g/L, potassium dihydrogen phosphate 0.3-1.0g/L,
Dipotassium hydrogen phosphate 0.3-1.0g/L, magnesium sulfate 0.3-2.0g/L, solvent is water, with hydrochloric acid tune pH value to 5.0-7.0.
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| CN110250496A (en) * | 2019-06-25 | 2019-09-20 | 泸州品创科技有限公司 | Cordyceps sinensis powder and preparation method thereof |
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| 汤佳鹏，柳依婷，赵强，董伟: "虫草菌素生物合成的研究进展", 《轻工科技》 * |
| 汤佳鹏，柳依婷: "蛹虫草静置发酵产虫草素的优化", 《食品工业科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110250496A (en) * | 2019-06-25 | 2019-09-20 | 泸州品创科技有限公司 | Cordyceps sinensis powder and preparation method thereof |
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| CN105969653B (en) | 2018-01-09 |
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