CN1223671C - Method and device for seed culture of claviceps strains in liquid culture media - Google Patents
Method and device for seed culture of claviceps strains in liquid culture media Download PDFInfo
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- CN1223671C CN1223671C CN 02104627 CN02104627A CN1223671C CN 1223671 C CN1223671 C CN 1223671C CN 02104627 CN02104627 CN 02104627 CN 02104627 A CN02104627 A CN 02104627A CN 1223671 C CN1223671 C CN 1223671C
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Abstract
The present invention relates to a method and a device for culturing seeds for cordyceps sporophores strains by using a liquid culture medium; the seed culture is used for the large-scale production of seed entities. Original seeds of the cordyceps sporophores strains are cultured in a basin; the original seeds of the cordyceps sporophores strains are inoculated in an inoculating source culture medium which is composed of a carbon source, a nitrogen source and distilled water; an inoculating source is prepared; afterwards, glucose, yeast extract, peptone, distilled water and defoaming agents are used for preparing the liquid culture medium; an inoculating unit of the present invention carries out inoculation according to the ratio of 100: 1 of the liquid culture medium to the inoculating source culture medium. A culture device culturing the seeds by using liquid provides sterilizing air of which the speed rate is 0.3 vvm at 25 DEG C; the inoculating source is cultured for 5 days; thereby, cultured seeds by using the liquid are prepared. The liquid culturing seeds are inoculated to a rice culture medium which is composed of unpolished rice and silkworm granules. The cultured seeds are diverted to a culture box; a fluorescent lamp is arranged in the culture box; the cultured seeds are cultured for 10 to 15 days under the conditions of 70 to 80% of humidity and of the temperature of 24 DEG C; afterwards, the cultured seeds are cultured for 15 to 18 days in the culture box at 20 DEG C under the conditions of 80 to 90% of humidity, and 500 to 1000Lux of illumination; thereby, the seed entities are prepared.
Description
Invention field
The present invention relates to be used for the method and apparatus of the seed culture of scale operation Cordycepssphecocephala bacterial strain sporophore with liquid nutrient medium, be specifically related to cultivate the method for the seed that is used for scale operation Cordyceps sphecocephala bacterial strain sporophore with a kind of liquid nutrient medium, this liquid nutrient medium is by glucose, yeast extract, peptone is formed and is formed through sterilization, and this substratum can be used inoculation unit (10) and liquid culture seed culture device (20), in the liquid medium within, based on asepsis, realize simplification cultivation to the seed of Cordyceps sphecocephala bacterial strain.
Background of invention
Common mushroom class obtains the vegetalitas organic composition as nutrition source, can obtain insect from vegetalitas worm bacterial strain.Vegetalitas worm bacterial strain belongs to a kind of mushroom, and it is lived in the insect health in winter, resembles the plant in summer but look.Certain plants property worm bacterial strain enters the insect health, in the insect health, form sporophore as parasite, therefore vegetalitas worm bacterial strain just is used as a kind of alive for evermore and long-lived close pharmacy material in China a long time ago, and as a kind of therapeutical agent for the treatment of pulmonary tuberculosis, asthma, jaundice, a kind of toxinicide of meconism, a kind of recovery and healthy toughener, and a kind of immunologic function potentiator.Vegetalitas worm bacterial strain very expensive (Yiang, J., Mao, X., Ma.Q., Zong, Y. and Wen, H.1989, Icons of medicinal fungi fromChina.p575; Kobayasi, genus Cordyceps and its allies.Sci.Rept.Tokyo Bunrika Daikaku Y.1940.The, Sect.B., 5:53-260; Shimizu, D.194.Coloriconography of vegetable wasp and plant worms.Seibundo Shinkosha.Japan.p381).Cordyceps sp. bacterial strain is very rare at occurring in nature.Therefore, vegetalitas worm bacterial strain ordinary person worker cultivation.In the method for artificial culture vegetalitas worm bacterial strain, adopt and carry out seed culture based on the seed culture method of solid medium with based on the seed culture method of liquid nutrient medium.For the common seed culture method that adopts based on liquid nutrient medium of vegetalitas worm.Yet the seed culture method of liquid culture only limits to triangular flask and cultivates.Still untappedly go out industrial culture method.Korean Patent Publication No.: 97-68810 discloses a kind of method that silkworm chrysalis carries out seed culture and growth of using.Yet therefore the large-scale cultivation method of systematic study seed does not have a kind of clear and definite seed culture method that is applied to industry.
The present inventor has invented a kind of application inoculation unit (10) and liquid nutrient medium culture apparatus (20), carries out the method and apparatus of liquid culture seed easily based on asepsis, and therefore realizes the scale operation of Cordyceps sp. bacterial strain.
Summary of the invention
Therefore, one object of the present invention is to provide a kind of cultural method that is used for the liquid culture seed of scale operation Cordyceps sp. bacterial strain sporophore.
Another object of the present invention is to provide a kind of culture apparatus that is used for the liquid culture seed of scale operation Cordyceps sp. bacterial strain sporophore.
To achieve these goals, a kind of method and apparatus that Cordyceps sp. bacterial strain is carried out seed culture with liquid nutrient medium is provided, this seed culture is used for this bacterial strain sporophore of scale operation, wherein the primordial seed of Cordyceps sp. bacterial strain obtains in a basin and cultivates, the primordial seed of the Cordyceps sp. bacterial strain of cultivation is inoculated in the substratum, cultivated 5 days for 24 ℃, it is used to prepare inoculation source, use glucose then, yeast extract, peptone, distilled water and defoamer prepare liquid nutrient medium, cultivate in the following manner with inoculation of the present invention unit (10), both liquid nutrient medium was 100: 1 with the ratio of cultivating inoculation source.Under 25 ℃, inject sterile air with liquid culture seed culture device (20), cultivated inoculation source 5 days, prepare the liquid culture seed thus with the speed of 0.3vvm.Aforesaid liquid is cultivated seed and is inoculated into by in brown rice and the granuloplastic rice medium of silkworm chrysalis, cultivates, and preparation is used for the sporophore of the Cordyceps sp. bacterial strain of scale operation thus.
The accompanying drawing summary
The present invention will become and be easier to understand under situation with reference to the accompanying drawings, and these accompanying drawings only are explanation and do not lie in qualification the present invention, wherein:
Fig. 1 is the synoptic diagram of explanation inoculation unit (10), and it is used for the seed with the liquid nutrient medium inoculation Cordeceps sp. bacterial strain of asepsis of the present invention; With
Fig. 2 is the synoptic diagram of explanation liquid culture seed culture device (20), and it is used to realize easy cultivation, cultivates and discharge the seed of Cordeceps sp. bacterial strain of the present invention.
Detailed description of the preferred embodiments
Embodiments of the invention are described below with reference to the accompanying drawings.
The present invention relates to a kind of method of carrying out the seed culture of Cordeceps sp. bacterial strain with liquid nutrient medium, described seed culture is used for the sporophore of this bacterial strain of scale operation, this method comprises such step, the primordial seed that promptly prepares Cordeceps sp. bacterial strain, be inoculated on the solid medium of forming by glucose, yeast extract, peptone, agar and distilled water, cultivated 4～8 days at 22～30 ℃ then; Such step promptly prepares the inoculation medium of being made up of glucose, yeast extract, peptone and distilled water, is used for the inoculation culture of Cordeceps sp. bacterial strain; Such step, the primordial seed that is about to Cordeceps sp. bacterial strain is inoculated in the original substratum of inoculation, cultivates 5 days at 24 ℃ then; Such step promptly prepares the cultivation liquid nutrient medium of being made up of glucose, yeast extract, peptone, distilled water and defoamer, is used for the liquid culture seed culture; Such step is promptly cultivated 0.1L with inoculation unit (10) and is injected liquid culture seed culture device of the present invention (20) with inoculation source, and cultivates in the 10L nutrient solution; Such step promptly is injected into the sterile air of 0.3vvm to have inoculated under 25 ℃ and cultivates with in the nutrient solution of inoculation source, cultivates 5 days, obtains the liquid culture seed thus; And such step, being about to the liquid culture seed is inoculated in the substratum, this substratum adds the 1000ml translucent plastic container with 80g brown rice and 5g silkworm chrysalis particle and prepares, and transfers to then in the incubator that is equipped with luminescent lamp, and humidity 70～80% is set, about 24 ℃ of temperature, cultivated 10～15 days, and then the incubator temperature was adjusted to 20 ℃, humidity 80～90%, cultivated again under 500～1000Lux illumination condition 15～18 days, and prepared the sporophore of Cordeceps sp. bacterial strain thus.
Be used for cultivating the primordial seed process of the Cordeceps sp. bacterial strain of inoculation source in inoculation, inoculate the mycelium particulate by this way, so that about 70% the time when the seed mycelial growth, other thalline can not enter in the nutrient solution of culture vessel.At this moment, because the quantity of mycelium particulate increases, incubation time reduces, and because mycelium density to be inoculated increases, hyphal diameter reduces.Inoculation source was cultivated 5～8 days at 22～30 ℃, preferably cultivated 5 days at 24 ℃.For same Cordyceps sp. bacterial strain, because the temperature classes of nutrient solution is similar to mycelial Optimal Temperature condition, so incubation time reduces.When being inoculated into inoculation source in the nutrient solution, nutrient solution is 20～200: 1 to the ratio of cultivating inoculation source, preferred 100: 1.In the culturing process of liquid culture seed, injected sterile air 4～10 days at 22～28 ℃ with 0.1～1.0vvm, realize aerated culture thus, preferably injected 5 days with 0.3vvm at 25 ℃.The solid medium that is used to cultivate Cordyceps sp. bacterial strain primordial seed is made up of 10～80g glucose, 2～20g yeast extract, 2～20g peptone, 10～20g agar and 1000ml distilled water, preferably is made up of 40g glucose, 10g yeast extract, 10g peptone, 18g agar and 1000ml distilled water.In above-mentioned steps, the substratum of inoculation source substratum and liquid culture seed is made up of 10～80g glucose, 2～20g yeast extract, 2～20g peptone and 1000ml distilled water, preferably is made up of 40g glucose, 10g yeast extract, 10g peptone and 1000ml distilled water.
To explain below can easy application based on asepsis liquid culture seed culture device of the present invention (20).Liquid culture seed culture device (20) comprises the culture vessel (21) of a filling nutrient solution, a stopper (22) that covers culture vessel (21), one links to each other with stopper and to be used to the combination of injecting air and discharging nutrient solution with pipe (23), one is used for regulating the locking latches (24) of combination with the air-tight state of pipe (23), an air filter that is used to sterilize (25) that is installed in combination with pipe (23) end, air that is used for culture vessel is discharged to the vapor pipe (26) of outside and one and is installed in the strainer (27) that is used to filter various assorted bacterium in the vapor pipe (26).
To describe method of the present invention in detail below.This explanation does not limit the scope of the invention.
Embodiment
The separation of the primordial seed of embodiment 1:Cordyceps sp. bacterial strain and enlarged culturing prepare the primordial seed of Cordyceps sp. bacterial strain in the present invention.The primordial seed of Cordyceps sp. bacterial strain is cultivated in the plate, be inoculated into then by 40g glucose, 10g yeast extract, 10g peptone, 18g agar and 1000ml distilled water and form in the solid medium, 22～30 ℃ of enlarged culturing 4～8 days.
The cultivation of the inoculation source of embodiment 2:Cordyceps sp. bacterial strain
Step 1: the nutrient solution that cultivate in preparation Cordyceps sp. inoculation source
In order to cultivate the inoculation source of Cordyceps sp. bacterial strain, preparation nutrient solution as shown in table 1 was sterilized 20 minutes at 121 ℃ with high-pressure sterilizing pot.
The substratum proportion of composing of the liquid culture seed of table 1Cordyceps sp. bacterial strain sporophore
| Composition | Liquid nutrient medium | Nutrient solution |
| Glucose | 4 | ?400 |
| Yeast extract | 1 | ?100 |
| Peptone | 1 | ?100 |
| Distilled water | 0.1L | ?10L |
| [notes] unit: g | ||
The cultivation of the inoculation source of step 2:Cordyceps sp. bacterial strain
The primordial seed of cultivating in embodiment 2 steps 1 is inoculated in the nutrient solution of preparation in embodiment 2 steps 1.Inoculate when the mycelial growth 70% in the basin, the mycelium particulate is inoculated into the nutrient solution of culture vessel, prevent that simultaneously assorted bacterium from entering culture vessel, postvaccinal nutrient solution was cultivated 5 days at 24 ℃.
Embodiment 3: the cultivation of liquid culture seed
Step 1: the preparation of cultivating the substratum of liquid culture seed
As shown in table 1, the preparation nutrient solution is used to cultivate liquid culture seed of the present invention.In addition, can remove the foam that produces in the liquid culture process, use the combination oil of a kind of edible oil, oleum gossypii seminis, Viscotrol C, sweet oil and palm tree oil as a kind of.The addition of defoamer is 0.1～1.0% (w/v) based on nutrient solution, so that evenly spread to the nutrient solution upper strata.
Step 2: be used to cultivate the cultivation of the inoculation source of liquid culture seed of the present invention
The inoculation source of the Cordyceps sp. bacterial strain of cultivation in embodiment 2 steps 2 is inoculated in the nutrient solution of embodiment 3 steps 1 preparation.Inoculum size is 0.1L based on the 10L nutrient solution.At this moment, as shown in Figure 1, use inoculation unit (10), to prevent some pollution of assorted bacterium.Inoculation unit (10) is made of an inoculation container (11), a stopper (12), an air injection tube (13), an air filter (14) and a nutrient solution vent pipe (15).Air injection tube (13) links to each other with the stopper (12) of inoculation container (11) with nutrient solution vent pipe (15).Air sterilization is installed in the air injection tube (13) with air filter (14).Air injection tube (13) one ends are placed into the bottle end of inoculation container (11), and the other end is connected in compressor.One end of nutrient solution vent pipe (15) is placed into the bottom of inoculation container (11), and the other end is connected in the combination pipe (23) that is installed in the liquid culture seed culture device (20).Can connect nutrient solution vent pipe (15) by the size of differentially determining pipe, connect a rubber hose then, and with a paillon foil sealing, to prevent by various living contaminantses.Air injection tube (13) and nutrient solution vent pipe (15) are gone up at a distance of 1cm at stopper (12).Therefore, in the time of in inoculation source being inoculated into liquid culture seed culture device (20), with compressor air is injected in the air injection tube (13), nutrient solution vent pipe (15) is connected in the combination of liquid culture seed culture device (20) with managing (23), thereby, inoculation source is injected in the culture apparatus (20) of liquid culture seed owing to flow into the pressure of the air of air injection tube (13).
Step 3: cultivate liquid culture seed of the present invention
As shown in Figure 2, realize the cultivation of liquid culture seed with liquid culture device of the present invention (20).Liquid culture seed culture device comprises that a culture vessel (21), a stopper (22), a combination are with managing (23), a locking latches (24), an air filter (25), a vapor pipe (26) and a strainer (27).Culture vessel includes nutrient solution in (21).Stopper (22) links to each other with vapor pipe (26) with pipe (23) with combination, when injecting air, injects inoculation source, and finishes cultivation final vacuum pipe (26) and can discharge nutrient solution.Locking latches (24) and air sterilization are installed in combination with in the pipe (23) with air filter (25), so that regulate the tightness of pipe.Strainer (27) is installed in the vapor pipe (26), is discharged to the outside to prevent the assorted bacterium of the inner issuable poisonous and harmful of culture vessel.Make up the bottom that places culture vessel (21) with an end of pipe (23), the other end is connected in compressor, so that provide air to culture vessel (21) inside.One end of vapor pipe (26) is connected to the inside of culture vessel (21), and the other end is opened to the external world.Therefore, in the present invention,, cultivated 5 days, obtain the liquid culture seed thus at 25 ℃ of sterile airs to liquid culture seed culture device (20) injection 0.3vvm.
Embodiment 4: with its sporophore of liquid culture seed culture of Cordyceps sp. bacterial strain
In the present invention, 80g brown rice and 5g silkworm chrysalis particle are put into the 1000ml translucent plastic container prepare substratum, be used for producing its sporophore with the liquid culture seed of Cordyceps sp. bacterial strain.At this moment, when using brown rice, the ratio of brown rice and moisture is 1: 1.5, when using silkworm chrysalis, does not add moisture.The sporophore substratum was sterilized 20～30 minutes at 121 ℃ with high-pressure sterilizing pot.The liquid culture seed of embodiment 3 preparation is inoculated in wherein, the liquid culture seed is evenly spread on the rice medium in the aseptic sporophore substratum.In sterile cupboard, inoculate, prevent to enter various assorted bacterium.The media transfer of having inoculated the liquid culture seed is to incubator, and wherein humidity 70～80%, and temperature maintenance provides certain illumination at about 24 ℃ with luminescent lamp, cultivates therein 10～15 days.When mycelia is distributed on whole substratum, with the incubator of media transfer to 20 ℃, so that cultivate sporophore.At 20 ℃, the mycelia original state is an oyster white, all increases yl moiety gradually on the thalline.At this moment, the temperature of incubator is 20 ℃, and humidity is 80～90%, and intensity of illumination is 500～1000Lux, thereby the media surface mycelia becomes the cotton type.After 15～18 days, the silkworm chrysalis surface becomes deep yellow, and the piece of mycelia formation protrusion-shaped, forms stroma simultaneously.Stroma is grown in the depth direction of original state, stroma top thickening, thereby formation bag quilt wherein.When grow bag by the time, stop the growth of sporophore, degenerate gradually.The bag that bacterium gill fungus head forms is by less with comparing of nature discovery.The shape of ascus and ascus bag is the same with the nature discovery.In having the fungi of good mushroom shaped, for the fungi of the mycelia opposite with general feature at the media surface raised growth, the formation of sporophore reduces to some extent.
As mentioned above, in the method with the liquid culture seed production sporophore of Cordeceps sp. bacterial strain of the present invention, compare with conventional solid culture seed and may significantly reduce because the bad product that various living contaminantses produce.In addition, because the sporophore that produces has covered the substratum all surfaces, production efficiency has increased, thereby realizes scale operation.Especially,, therefore may prevent the pollution of various assorted bacterium because cultivate the liquid culture seed with inoculation unit and liquid culture device, thus the sporophore of effectively cultivating Cordyceps sp. bacterial strain.
Because can implement the present invention and not depart from inner characteristic of the present invention with several forms, it should also be understood that above-mentioned embodiment is not limit by previously described any details, except as otherwise noted, and should constitute essence of the present invention and the scope that claims limit more widely, so all in claim limits or the change and the modification that are equal to this qualification comprise within the scope of the appended claims.
Claims (5)
1. one kind is carried out the method for seed culture with liquid nutrient medium to Cordyceps sp. bacterial strain, and this seed culture is used for its sporophore of scale operation, and described method comprises:
Step (a), wherein the primordial seed of Cordyceps sp. bacterial strain is inoculated in the solid medium of being made up of 10～80g glucose, 2～20g yeast extract, 2～20g peptone, 10～20g agar and 1000ml distilled water, in couveuse 22～30 ℃ of enlarged culturing 4～8 days;
Step (b) wherein prepares the inoculation source substratum by mixing 10～80g glucose, 2～20g yeast extract, 2～20g peptone and 1000ml distilled water;
Step (c), wherein the substratum with step (b) preparation joins in the inoculation unit, the primordial seed of the Cordyceps sp. bacterial strain of enlarged culturing in the inoculation step (a), inoculation source was cultivated 4～8 days at 22～30 ℃ in couveuse;
Step (d) adds the nutrient solution that liquid seeds is cultivated in 10～80g glucose, 2～20g yeast extract, 2～20g peptone, 0.1～1.0% (w/v) defoamer and the preparation of 1000ml distilled water;
Step (e) wherein with respect to the cultivation inoculation source of step (c), is 20～200: 1 according to the ratio of nutrient solution and inoculation source, inoculates;
Step (f), wherein contain in steps (e) inoculation source nutrient solution by provide speed be 0.1～1.0vvm sterile air, 22～28 ℃, in the liquid culture device, cultivated 4～10 days, cultivate liquid seeds thus; With
Step (g), wherein the liquid seeds of cultivating in the step (e) is inoculated on the substratum that is prepared as follows, promptly brown rice particle and silkworm chrysalis are added the 1000ml translucent plastic container, transfer to humidity 70～80% then, about 24 ℃ of temperature according to asepsis, with keep with luminescent lamp in the incubator of certain illumination, cultivated therein 10～15 days, then at 20 ℃, humidity 80～90%, cultivated 15～18 days under luminosity 500～1000Lux condition, prepare the sporophore of Cordyceps sp. bacterial strain thus.
2. the process of claim 1 wherein as the foamy defoamer in the liquid seeds culturing process of removing step (d) Cordyceps sp. bacterial strain, use one or more to be selected from edible oil, oleum gossypii seminis, Viscotrol C, sweet oil and palmitic oil.
3. the process of claim 1 wherein that the inoculation unit of step (c) comprises an inoculation container; A stopper that is installed in the inoculation container top; An air injection tube, the one end connects stopper, and leads to the bottom of inoculation container, and the other end connects compressor, so that air is injected inoculation container; A sterile air strainer that is installed in the air injection tube; With a nutrient solution vent pipe, the one end leads to the inoculation container bottom, and the other end is connected on the cultivation unit that communicates with atmosphere.
4. the process of claim 1 wherein that the liquid seeds culture apparatus of step (f) comprises a culture vessel; A stopper that is installed in the culture vessel top; A combination is with managing; One is used to regulate the locking latches of combination with the pipe degree of opening; An air sterilization air filter; A vapor pipe and a filtration unit, wherein the inoculation source of Cordyceps sp. bacterial strain is injected with pipe by combination, sterile air injects culture vessel inside by air filter, and the air in the culture vessel is discharged to the outside by vapor pipe and filtration unit, realizes the cultivation of airiness thus.
5. one is carried out the device of Cordyceps sp. bacterial strain seed culture with liquid nutrient medium, and this seed culture is used for its sporophore of scale operation, and described device comprises:
A culture vessel that holds nutrient solution;
A stopper that is installed in the culture vessel top;
A combination is with managing, and the one end places the bottom of culture vessel, and the other end is connected on the compressor;
One is installed in the locking latches that makes up with in the pipe;
An air filter that is installed in combination with the pipe end; With
A vapor pipe, the one end is connected to culture vessel inside, and the other end is outwards open;
A filtration unit that is installed in the vapor pipe.
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| CN 02104627 CN1223671C (en) | 2002-02-19 | 2002-02-19 | Method and device for seed culture of claviceps strains in liquid culture media |
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| CN 02104627 CN1223671C (en) | 2002-02-19 | 2002-02-19 | Method and device for seed culture of claviceps strains in liquid culture media |
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Cited By (1)
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| CN104381010A (en) * | 2014-10-08 | 2015-03-04 | 生展生物科技股份有限公司 | Cultivation method of cordyceps sinensis sporocarps and composition and application of cordyceps sinensis sporocarps |
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| CN101649290B (en) * | 2008-08-14 | 2012-05-16 | 上海国宝企业发展中心 | Culture utensil for large-scale cultivation of cordyceps militaris |
| CN106636284B (en) * | 2016-12-22 | 2020-06-19 | 驻马店华中正大有限公司 | Aureomycin fermentation culture medium and aureomycin fermentation production method using same |
| CN107746805B (en) * | 2017-11-17 | 2019-04-23 | 江南大学 | A kind of method of Liquid static culture edible and medicinal fungi |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104381010A (en) * | 2014-10-08 | 2015-03-04 | 生展生物科技股份有限公司 | Cultivation method of cordyceps sinensis sporocarps and composition and application of cordyceps sinensis sporocarps |
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