CN100425689C - Pupa cordyceps fruiting body cultured using large galleria waxmoth larva and its culturing method - Google Patents
Pupa cordyceps fruiting body cultured using large galleria waxmoth larva and its culturing method Download PDFInfo
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Abstract
This invention relates to a cordyceps militaris (L.) link encarpium which is raised by Galleria mellonella L larva and its cultural method. The characteristic of this invented Cordyceps encarpium is that Galleria mellonella L larva shows Buff, body surface has little orange yellow bacterial filament, encarpium is Club shape or cladodromous, orange yellow, one or more encarpium grow thickly from body surface of Galleria mellonella L larva pupa, and after Cordyceps militaris(L.)Link strain is cultivated by first class strain cultivation and second order strain cultivation, using second order liquid strain to infect Galleria mellonella L larva, and it is obtained by raising encarpium. This invented Cordyceps militaris(L.)Link encarpium is similar to wild Cordyceps militaris(L.)Link, nutritional ingredient of them is similar.
Description
Technical field
The present invention relates to a kind of Cordyccps-militaris-(L.)-link. Sporophore, relate to a kind of Cordyccps-militaris-(L.)-link. Sporophore specifically, also relate to the method for cultivation of this Cordyccps-militaris-(L.)-link. Sporophore with the greater wax moth larvae cultivation.
Background technology
Cordyceps militaris (L.) Link. (Cordyceps militaris (L.) Link) is called Cordyceps militaris again, belongs to Ascomycota Ascomycota, Ascomycetes Ascomycetes, Hypocreales Hyocreales, Clavicipitaceae Clavicipitaceae, Cordyceps Cordyceps.Cordyceps militaris (L.) Link. is at the pupa of nature main parasitic in lepidopterous insects.Cordyceps militaris (L.) Link. blazons all over the world, and all there is distribution China each province.
(C.sinensis (Berk) Sacc.) is traditional natural resources of Chinese medicinal materials with Cordyceps militaris (L.) Link. generic Cordyceps sinensis, has health care and pharmaceutical use, particularly antineoplastic effect.The nutritive ingredient of Cordyceps militaris (L.) Link. is similar to Cordyceps sinensis with pharmaceutical use, thereby also is subjected to human consumer's favor, and the market requirement is vigorous.Natural resources is difficult to satisfy the demands.The artificial culture of Cordyceps militaris (L.) Link. is the optimal selection that solves this demand.
At present except utilization contains the substratum cultivation Cordyccps-militaris-(L.)-link. Sporophore of rice, also by being that the host cultivates Cordyceps militaris (L.) Link. with the insect.Pan Zhonghua etc. (Jiangsu silkworm industry, 2002,24:21-24) and Wang Xiqiang etc. (the Anhui agricultural sciences, 2002,30:965-968) utilize silkworm pupa (Bombyx mori L.) and grasshopper cicada pupa (Cryptotympana pustulata F.) cultivation Cordyceps militaris (L.) Link. respectively.Li Ruchun etc. (the fungus journal, 2005,24:349-355) infect tenebrio molitor (Tenebrio molitor L.) and form shoestring with Taiwan Chinese caterpillar fungus (C.formosana Lobayasi et al.) bacterium.Li Taihui etc. (Chinese patent 200510101348.0) utilize the Tenebrio molitor of artificial culture successfully to nurture the Cordyceps militaris (L.) Link. stroma.Chinese patent 87106987 in addition, Chinese patent 00130381.3, and Chinese patent 01141386.7, Chinese patent 97113190.2 grades have also been described the method for utilizing silkworm chrysalis to cultivate Cordyceps militaris (L.) Link..
Greater wax moth (Galleria mellonella L.) belongs to Pyralidae Galleriinae insect, moves in naturally in the honeybee honeycomb.The greater wax moth larva contains nutritive substances such as rich in protein, fat, trace element and VITAMIN, and artificial already at present mass rearing as important experiment and mensuration insect, also is the good biological feed of freshwater fish, birds and amphibious reptile.But, up to the present all do not see and utilize the greater wax moth larva to cultivate the report of Cordyceps sporophore.
Summary of the invention
The objective of the invention is to utilize greater wax moth to turn out a kind of nutritive ingredient Cordyccps-militaris-(L.)-link. Sporophore close with wild cordyceps militaris nutrition, another purpose is to develop the method for cultivation of this Cordyceps militaris (L.) Link. entity.
The present invention is by infecting Cordyceps militaris spawn the greater wax moth larva, and the Cordyccps-militaris-(L.)-link. Sporophore that obtains is close with the nutritive ingredient of wild cordyceps militaris, thereby has realized purpose of the present invention.
Cordyccps-militaris-(L.)-link. Sporophore with the greater wax moth larvae cultivation of the present invention, it is characterized in that infecting the greater wax moth larvae cultivation by Cordyceps militaris (L.) Link. (Cordyceps militaris (L.) Link) bacterial classification obtains, described greater wax moth larva body is light yellow, the slightly orange-yellow mycelia of body surface, sporophore clavate or divide dendritic, orange-yellow, single or many grow thickly in greater wax moth larva pupal cell table.
Described greater wax moth larva is obtained by the indoors artificial culture medium culturing, and described Cordyceps militaris (L.) Link. (Cordyceps militaris (L.) Link) bacterial classification can obtain from Chinese common micro-organisms culture presevation administrative center, is numbered 3.4655.
The method of cultivation of the Cordyccps-militaris-(L.)-link. Sporophore with the greater wax moth larvae cultivation of the present invention, its feature comprises the steps:
(1) Cordyceps militaris (L.) Link. (Cordyceps militaris (L.) Link) bacterial classification is cultivated and the second class inoculum cultivation through first class inoculum, infected greater wax moth (Galleria mellonella L.) larva with the Cordyceps militaris (L.) Link. second-class liquid isolate;
(2) infected greater wax moth larva is placed in the aseptic tissue culture bottle, in relative air humidity 80%～100%, temperature is the cultivation of preserving moisture under 18～25 ℃ the condition, when appearring in greater wax moth larva body surface, white hypha carries out light induction, intensity of illumination is 200～2000Lux, to body surface mycelia yellowing, after the larva body surface produces cordycep militaris fruiting body entity primodium, continue to cultivate 50～60 days; The Cordyccps-militaris-(L.)-link. Sporophore of when the atrophy of larva body, gathering.
The described spawn culture of step (1) adopts existing method, first class inoculum cultivate be bacterial classification inoculation with separation and purification to the solid slant culture base, 20～25 ℃, lucifuge was cultivated 7～10 days, cover with the inclined-plane to white fine hair shape mycelia, described substratum can adopt conventional prescription; Described second class inoculum cultivate be with first class inoculum from board joint to through the liquid nutrient medium of the second class inoculum of sterilization, 25 ℃, 120r/min shaking culture 7～10 days, to bacterium ball filled with liquid medium, described substratum can adopt conventional prescription; The greater wax moth larva material that preferably adds massfraction 0.5%～5.0% in first class inoculum and second class inoculum helps the growth of bacterial classification and the vigor of maintenance bacterial classification, and described greater wax moth larva material is homogenate of greater wax moth larva or larva hemolymph etc.
Can be that 75% ethanol carries out the body surface sterilization with massfraction before the described greater wax moth larva of step (1) the inoculation Cordyceps militaris (L.) Link., the greater wax moth larva of raising for artificial cleaning can not carry out the body surface sterilization, described infection can be adopted usual method, for example 0.01～0.2mL bacterial classification is injected in the greater wax moth larva body with asepsis injector, perhaps bacterial classification is soaked the greater wax moth larva, soak time is 1～5 minute.
Analyze the aminoacids content of Cordyccps-militaris-(L.)-link. Sporophore of the present invention, the results are shown in Table 1.
The aminoacids content of table 1 Cordyccps-militaris-(L.)-link. Sporophore of the present invention
Analyze the protein of Cordyccps-militaris-(L.)-link. Sporophore of the present invention, fat and polysaccharide content the results are shown in Table 2.
The protein of table 2 Cordyccps-militaris-(L.)-link. Sporophore of the present invention, fat and polysaccharide content
Analyze the vitamin contents of Cordyccps-militaris-(L.)-link. Sporophore of the present invention, the results are shown in Table 3.
The vitamin contents of table 3 Cordyccps-militaris-(L.)-link. Sporophore of the present invention
Similar to wild cordyceps militaris from the sporophore part of the visible Cordyceps militaris (L.) Link. of the present invention of above-mentioned data, nutritive ingredient is close, therefore has wide commercial applications prospect.
Embodiment
Following examples are to further specify of the present invention, are not limitations of the present invention.
Embodiment 1:
With potato 20% (massfraction, down together), glucose 2%, KH
2PO
40.3%, MgSO
4.7H
2O0.15%, vitamins B
120mg/L, agar 2%, greater wax moth larva material 0.5% makes the one-level substratum, with glucose 2%, peptone 0.4%, KH
2PO
40.4%, MgSO
4.7H
2O 0.4%, vitamins B
120mg/L, greater wax moth larva material 0.5%, pH6.5 makes secondary medium.
The Cordyceps militaris spawn of separation and purification is seeded to solid inclined-plane one-level substratum, 20 ℃, lucifuge was cultivated 7 days, extremely white fine hair shape mycelia is covered with the inclined-plane, then with first class inoculum from board joint to through the liquid nutrient medium of second class inoculum of sterilization, 25 ℃, 120r/min shaking culture 7 days are to bacterium ball filled with liquid medium.With massfraction is that 75% ethanol carries out body surface sterilization greater wax moth larva, with asepsis injector the 0.01mL bacterial classification is injected in the greater wax moth larva body then.
Infected greater wax moth larva is placed in the aseptic tissue culture bottle, in relative air humidity 80%, temperature is the cultivation of preserving moisture under 18 ℃ the condition, when appearring in greater wax moth larva body surface, white hypha carries out light induction, intensity of illumination is 200Lux, to body surface mycelia yellowing, after the larva body surface produces cordycep militaris fruiting body entity primodium, continue to cultivate 50 days; The Cordyccps-militaris-(L.)-link. Sporophore of when the atrophy of larva body, gathering.By above-mentioned aminoacids content, protein content, lipid content, polysaccharide content and vitamin contents confirm that its nutritive ingredient and wild cordyceps militaris are close.
Embodiment 2:
With potato 20%, glucose 2%, KH
2PO
40.3%, MgSO
4.7H
2O0.15%, vitamins B
120mg/L, agar 2%, greater wax moth larva material 5.0% makes the one-level substratum, with glucose 2%, peptone 0.4%, KH
2PO
40.4%, MgSO
4.7H
2O0.4%, vitamins B
120mg/L, greater wax moth larva material 5.0%, pH6.5 makes secondary medium.
The Cordyceps militaris spawn of separation and purification is seeded to solid inclined-plane one-level substratum, 25 ℃, lucifuge was cultivated 10 days, extremely white fine hair shape mycelia is covered with the inclined-plane, then with first class inoculum from board joint to through the liquid nutrient medium of second class inoculum of sterilization, 25 ℃, 120r/min shaking culture 10 days to bacterium ball filled with liquid medium, are injected into the 0.2mL bacterial classification in the greater wax moth larva body that artificial cleaning raises with asepsis injector then.
Infected greater wax moth larva is placed in the aseptic tissue culture bottle, in relative air humidity 100%, temperature is the cultivation of preserving moisture under 25 ℃ the condition, when appearring in greater wax moth larva body surface, white hypha carries out light induction, intensity of illumination is 2000Lux, to body surface mycelia yellowing, after the larva body surface produces cordycep militaris fruiting body entity primodium, continue to cultivate 60 days; The Cordyccps-militaris-(L.)-link. Sporophore of when the atrophy of larva body, gathering.By above-mentioned aminoacids content, protein content, lipid content, polysaccharide content and vitamin contents confirm that its nutritive ingredient and wild cordyceps militaris are close.
Embodiment 3:
With potato 20%, glucose 2%, KH
2PO
40.3%, MgSO
4.7H
2O0.15%, vitamins B
120mg/L, agar 2%, greater wax moth larva material 3.0% makes the one-level substratum, with glucose 2%, peptone 0.4%, KH
2PO
40.4%, MgSO
4.7H
2O0.4%, vitamins B
120mg/L, greater wax moth larva material 3.0%, pH6.5 makes secondary medium.
The Cordyceps militaris spawn of separation and purification is seeded to solid inclined-plane one-level substratum, 23 ℃, lucifuge was cultivated 8 days, extremely white fine hair shape mycelia is covered with the inclined-plane, then with first class inoculum from board joint to through the liquid nutrient medium of second class inoculum of sterilization, 25 ℃, 120r/min shaking culture 8 days are to bacterium ball filled with liquid medium.With massfraction is that 75% ethanol carries out body surface sterilization greater wax moth larva, soaks the greater wax moth larva with bacterial classification then, and soak time is 1 minute.
Infected greater wax moth larva is placed in the aseptic tissue culture bottle, in relative air humidity 85%, temperature is the cultivation of preserving moisture under 20 ℃ the condition, when appearring in greater wax moth larva body surface, white hypha carries out light induction, intensity of illumination is 500Lux, to body surface mycelia yellowing, after the larva body surface produces cordycep militaris fruiting body entity primodium, continue to cultivate 55 days; The Cordyccps-militaris-(L.)-link. Sporophore of when the atrophy of larva body, gathering.By above-mentioned aminoacids content, protein content, lipid content, polysaccharide content and vitamin contents confirm that its nutritive ingredient and wild cordyceps militaris are close.
Embodiment 4:
With potato 20%, glucose 2%, KH
2PO
40.3%, MgSO
4.7H
2O0.15%, vitamins B
120mg/L, agar 2%, greater wax moth larva material 2.0% makes the one-level substratum, with glucose 2%, peptone 0.4%, KH
2PO
40.4%, MgSO
4.7H
2O0.4%, vitamins B
120mg/L, greater wax moth larva material 3.0%, pH6.5 makes secondary medium.
The Cordyceps militaris spawn of separation and purification is seeded to solid inclined-plane one-level substratum, 22 ℃, lucifuge was cultivated 9 days, extremely white fine hair shape mycelia is covered with the inclined-plane, then with first class inoculum from board joint to through the liquid nutrient medium of second class inoculum of sterilization, 25 ℃, 120r/min shaking culture 9 days are to bacterium ball filled with liquid medium.With massfraction is that 75% ethanol carries out body surface sterilization greater wax moth larva, soaks the greater wax moth larva with bacterial classification then, and soak time is 5 minutes.
Infected greater wax moth larva is placed in the aseptic tissue culture bottle, in relative air humidity 95%, temperature is the cultivation of preserving moisture under 23 ℃ the condition, when appearring in greater wax moth larva body surface, white hypha carries out light induction, intensity of illumination is 1000Lux, to body surface mycelia yellowing, after the larva body surface produces cordycep militaris fruiting body entity primodium, continue to cultivate the Cordyccps-militaris-(L.)-link. Sporophore of when the atrophy of larva body, gathering 58 days.By above-mentioned aminoacids content, protein content, lipid content, polysaccharide content and vitamin contents confirm that its nutritive ingredient and wild cordyceps militaris are close.
Claims (5)
1. Cordyccps-militaris-(L.)-link. Sporophore with the greater wax moth larvae cultivation, it is characterized in that obtaining: at first use Cordyceps militaris (L.) Link. (Cordyceps militaris (L.) Link) bacterial classification to infect greater wax moth (Galleria mellonella L.) larva by following method cultivation, then in relative air humidity 80%～100%, temperature is the cultivation of preserving moisture under 18～25 ℃ the condition, when appearring in greater wax moth larva body surface, white hypha carries out light induction, intensity of illumination is 200～2000Lux, to body surface mycelia yellowing, after the larva body surface produces cordycep militaris fruiting body entity primodium, continue to cultivate 50～60 days, the Cordyccps-militaris-(L.)-link. Sporophore of when the atrophy of larva body, gathering, described greater wax moth larva body is light yellow, the slightly orange-yellow mycelia of body surface, sporophore clavate or divide dendritic, orange-yellow, single or many grow thickly in greater wax moth larva pupal cell table.
2. method of cultivation with the Cordyccps-militaris-(L.)-link. Sporophore of greater wax moth larvae cultivation, its feature comprises the steps:
(1) Cordyceps militaris (L.) Link. (Cordyceps militaris (L.) Link) bacterial classification is cultivated and the second class inoculum cultivation through first class inoculum, infected greater wax moth (Galleria mellonella L.) larva with the Cordyceps militaris (L.) Link. second-class liquid isolate;
(2) infected greater wax moth larva is placed in the aseptic tissue culture bottle, in relative air humidity 80%～100%, temperature is the cultivation of preserving moisture under 18～25 ℃ the condition, when appearring in greater wax moth larva body surface, white hypha carries out light induction, intensity of illumination is 200～2000Lux, to body surface mycelia yellowing, after the larva body surface produces cordycep militaris fruiting body entity primodium, continue to cultivate 50～60 days; The Cordyccps-militaris-(L.)-link. Sporophore of when the atrophy of larva body, gathering.
3. according to the method for cultivation of a kind of Cordyccps-militaris-(L.)-link. Sporophore with the greater wax moth larvae cultivation of claim 2, it is characterized in that it is that first class inoculum with separation and purification is seeded to the solid slant culture base that the described first class inoculum of step (1) is cultivated, 20～25 ℃, lucifuge was cultivated 7～10 days, extremely white fine hair shape mycelia is covered with the inclined-plane, described second class inoculum cultivate be with first class inoculum from board joint to through the liquid nutrient medium of the second class inoculum of sterilization, 25 ℃, 120r/min shaking culture 7～10 days are to bacterium ball filled with liquid medium.
4. according to the method for cultivation of a kind of Cordyccps-militaris-(L.)-link. Sporophore with the greater wax moth larvae cultivation of claim 3, it is characterized in that containing in the substratum of described one-level of step (1) and two spawn culture the greater wax moth larva material of massfraction 0.5%～5.0%, described greater wax moth larva material is homogenate of greater wax moth larva or larva hemolymph.
5. according to the method for cultivation of a kind of Cordyccps-militaris-(L.)-link. Sporophore with the greater wax moth larvae cultivation of claim 2 or 3 or 4, it is characterized in that before the described greater wax moth larva of step (1) the inoculation Cordyceps militaris (L.) Link. with massfraction being that 75% ethanol carries out body surface and sterilizes, described infection is with asepsis injector 0.01～0.2mL bacterial classification to be injected in the greater wax moth larva body to infect, perhaps bacterial classification is soaked the greater wax moth larva and infect, soak time is 1～5 minute.
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| CN104430183B (en) * | 2013-09-13 | 2017-06-16 | 广西壮族自治区林业科学研究院 | Large-scale breeding and method for breeding in a kind of greater wax moth room |
| CN104584858A (en) * | 2015-01-05 | 2015-05-06 | 鲁东大学 | Method for planting cordyceps militaris by taking wooden shaving as carrier |
| CN105981583B (en) * | 2015-02-10 | 2019-11-19 | 保山学院 | Add the method that fresh insect protein promotes Cordyceps nutans growth |
| CN105493899B (en) * | 2016-01-13 | 2018-10-09 | 吉林大学 | A method of using yellow meal worm worm pupa as carrier culture worm grass product |
| CN105557312B (en) * | 2016-01-13 | 2018-10-09 | 吉林大学 | A method of using Yellow meal worm larva as carrier culture worm grass product |
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| CN1297997A (en) * | 1999-12-02 | 2001-06-06 | 上海国宝企业发展中心 | Culture process of Nortern cordyceps sporophore |
| JP2003116522A (en) * | 2001-09-28 | 2003-04-22 | Zuiei Chin | Medium for culturing cordyceps sinensis sacc. and method for culturing cordyceps sinensis sacc. |
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| CN1034390A (en) * | 1987-10-13 | 1989-08-02 | 吉林省蚕业科学研究所 | The artificial culturing method of silkworm chrysalis Cordyceps sinensis |
| CN1297997A (en) * | 1999-12-02 | 2001-06-06 | 上海国宝企业发展中心 | Culture process of Nortern cordyceps sporophore |
| JP2003116522A (en) * | 2001-09-28 | 2003-04-22 | Zuiei Chin | Medium for culturing cordyceps sinensis sacc. and method for culturing cordyceps sinensis sacc. |
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