CN103667084B - Method for co-culturing amanita subfrostiana mycelium by using mycorrhizal fungus and saprobic fungus - Google Patents
Method for co-culturing amanita subfrostiana mycelium by using mycorrhizal fungus and saprobic fungus Download PDFInfo
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- CN103667084B CN103667084B CN201310701996.4A CN201310701996A CN103667084B CN 103667084 B CN103667084 B CN 103667084B CN 201310701996 A CN201310701996 A CN 201310701996A CN 103667084 B CN103667084 B CN 103667084B
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Abstract
The invention relates to a method for co-culturing an amanita subfrostiana mycelium by using a mycorrhizal fungus and a saprobic fungus, and belongs to the technical field of macro fungus culture. The method is characterized in that the mycorrhizal fungus lactarius piperatus and the artificially cultured saprobic fungus lentinus edodes are added directly in multiplication media, so that the growth rate of the amanita subfrostiana mycelium can be increased by 9-12 times, and the growth is accelerated obviously. According to the method, a tedious and complex process is not required, the reproduction speed of the mycelium can be increased only by adding a mixture of the two fungi into the media, the effect is obvious, the culture is simple and easy to implement, and the production cost is low. Amanita toxins have potential application in the field of development of novel specific medicines such as anti-tumor medicines, anti-microbial and antiviral medicines, sedative or anaesthetic medicines and like, while are difficult to develop and apply due to bottleneck problems that resources are rare and valuable, artificial acclimation is difficult and the like. The method performs innovation research aiming at pure culture restraining problems such as slower growth of the amanita subfrostiana mycelium and the like, and provides a basis for large-scale culture, artificial acclimation and culture in the future and the like of the mycelium.
Description
Technical field
The present invention relates to a kind of method using VA Mycorrhizal Fungi and saprophytic microorganism co-cultivation yellow squama Amanita fuliginea filament, belong to biological technical field, specifically belong to poisonous macro fungi indoor cultivation category.
Background technology
Amanita (
amanita) be under the jurisdiction of Basidiomycotina, Hymenomycetes, Agaricales, Amanitaceae (
amanitaceae), be in poisonous macro fungi more special, more valuable one worldwidely blazon large genus, its species diversity is enriched very much.The whole world has reported nearly 400 kinds, nearly 100 kinds (containing subspecies, mutation and modification) that China has recorded.According to textual criticism, current China still has many kinds not yet study and name.But nowadays along with the appearance of global ecological crisis, many Macro-Fungi Resources of China accuse danger, are in slump of disastrous proportions and Critical Condition, and part plant be faced with may also not by human knowledge or find that it is worth time, with regard to the risk of having become extinct.
Major part kind in Amanita belongs to famous hypertoxic bacterium, it is reported, is because of caused by Amanita fuliginea because eating the wild gill fungus bacterium person of being poisoned to death 95% by mistake.Scientists is really interested to Amanita fuliginea is its toxicity, and therefore, greatly before 140 years, people just start the research of In The Studies On Toxins of Amanita.The applied research of amatoxin is mainly reflected in following several respects: 1. can be used for the expression of research eukaryotic gene, regulation and control and cellular localization; 2. antibacterial, antiviral special efficacy new drug can be developed; 3. can screening antineoplastic drugs; 4. calmness, anesthesia and other specifics can be developed; 5. can be used for biological anti-smelting etc.
The Main Bottleneck problem causing goose cream to be difficult to exploitation and Substantial evaluation at present has: 1. goose cream resource is rare, an its kind of group of mean people, individual few, yield poorly, distributed quantity is extremely limited, in addition responsive to habitat, once habitat is damaged, very easily disappear or extinction, belong to special and cause danger biological group, which increase its studied, difficulty of utilizing further; 2. Amanita, belongs to Applying Ectomycorrhizal Fungi mostly, at present still can not artificial culture.Therefore, also do not have both at home and abroad so far a kind of can the toxin product of mass-producing exploitation, the existing Peptide toxin as biochemical reagents is expensive (before more than 10 year every gram at about 100,000 dollars-Chen Zuohong etc., 1999) still, is difficult to meet needed for scientific research and application.
The pure culture of goose cream is the prerequisite or key that ensure that goose cream sustainability of natural resources utilizes; but this still belongs to the problem being difficult to capture at present; there is many blind spots, wherein mycelia is difficult to induction, mycelial growth is slowly the important step or the factor that restrict and affect pure culture.
Yellow squama goose cream (
a.subfrostiana), belong to goose cream subgenus (Amanita subgen. Amanita) goose cream group (sect. Amanita), its Species structure is less, successfully be separated to the mycelia of this kind at present, but the growth of mycelia is still very slow for early stage, is difficult to expansion numerous, the present invention levies this problem, add VA Mycorrhizal Fungi and saprophytic microorganism in the medium, substantially increase the speed of growth of mycelia, for important foundation has been established in the research and development utilization etc. of amatoxin.
Summary of the invention
The object of the invention is to, provide a kind of simple, easily realize, production cost is not high, the cultural method that Amanita fuliginea silk grows fast can be made.
Technical assignment of the present invention is, in proliferated culture medium, directly add the saprophytic microorganism of mycorrhizal fungi beyond Amanita and artificial culture simultaneously, and the speed of growth of yellow squama Amanita fuliginea filament can be made to improve 9 ~ 12 times;
Described multiplication culture based component is: potato 200g/L, fructose 9 ~ 11g/L, albumen freeze 1.00 ~ 1.25g/L, ZnSO
40.40 ~ 0.60g/L, MgSO
4habitat leaf-humidified soil 25 ~ 35g/L, the agar 10.00g/L of the mistake 80 order sub-sieve of 0.40 ~ 0.60g/L, zeatin ZT 0.80 ~ 1.00mg/L, pulverizing, controlling substratum pH value is 5.6 ~ 6.0;
Described mycorrhizal fungi be pick up from raised growth in goose cream habitat lime bacterium (
lactarius piperatus), saprophytic microorganism be can indoor scale produce mushroom (
lentinus edodes); The method of preparation and use of lime bacterium and mushroom is as follows: lime bacterium, mushroom are dried respectively, pulverize, the two mixes in 1:4 ratio, by mixed powder, water is added by the weightmeasurement ratio of 1:7 ~ 1:8, stir evenly, with ultrasonication 30 ~ 40 min, then add 15 ~ 20 times of water, regulate pH value to be 5.4 ~ 5.6, in 45 ~ 50 DEG C of water-baths, add cellulase by the weightmeasurement ratio of 2.5 ~ 3.0%, after enzymolysis 70 ~ 100 min, bath temperature is risen to 90 ~ 93 DEG C, move into during evaporate to dryness basic to moisture in 65 ~ 70 DEG C of baking ovens, dry 25 ~ 30hr; The dry powder of enzymolysis is joined in proliferated culture medium by 25 ~ 35g/L;
Described yellow squama Amanita fuliginea filament be the present invention early stage induction and cultivate, induction and cultural method as follows: field pluck well-grown, non-parachute-opening healthy sporophore; The internal aseptic tissue block of getting cap and stem junction is about 0.35cm
2size, embeds inducing culture gently, and described medium component is: potato 200g/L, fructose 9 ~ 11g/L, albumen freeze 1.00 ~ 1.25g/L, MgSO
41.00g/L, CaCl
21.00g/L, KH
2pO
4wort 100 ~ the 125ml of 0.50g/L, 16.0 Baumes, agar 10.00g/L, controlling substratum pH value is 5.6 ~ 6.0, light culture 50 ~ 60 days at 22 ~ 23 DEG C, and tissue block surface grows white fluffy mycelia; By mycelia succeeding transfer culture in above-mentioned inducing culture, obtain the present invention and use mycelia.
The invention has the beneficial effects as follows: directly in proliferated culture medium, add particular matter, the mycelial speed of growth can be made greatly to improve, and its feature is as follows:
(1) successful: add VA Mycorrhizal Fungi and saprophytic microorganism after mixture, the speed of growth is 9 ~ 12 times before not adding, and growth is obviously accelerated;
(2) cultivation is simple and easy to realize: the flow process not needing very complicated, as long as the thalline mixture adding through enzymolysis processing in the medium;
(3) production cost is not high: two kinds of thalline materials that the present invention adopts, and one picks up from field, and increment is large, is easy to get; Another kind of market can be bought everywhere, and low price, the two neither can cause increasing of toxigenic capacity.
Embodiment
Following examples of implementation further illustrate of the present invention, is not limitation of the present invention.
example one:
The healthy sporophore of well-grown, non-parachute-opening is plucked in field; The internal aseptic tissue block of getting cap and stem junction is about 0.35cm
2size, embeds inducing culture gently, and described medium component is: potato 200g/L, fructose 9g/L, albumen freeze 1.00g/L, MgSO
41.00g/L, CaCl
21.00g/L, KH
2pO
4the wort 100ml of 0.50g/L, 16.0 Baumes, agar 10.00g/L, controlling substratum pH value is 5.6, light culture 50 days at 22 ~ 23 DEG C, and tissue block surface grows white fluffy mycelia; By mycelia succeeding transfer culture in above-mentioned inducing culture, obtain the present invention with mycelia;
Mycelia is proceeded to proliferated culture medium, and described multiplication culture based component is: potato 200g/L, fructose 9g/L, albumen freeze 1.00g/L, ZnSO
40.40g/L, MgSO
4enzymolysis dry powder 25g/L, the agar 10.00g/L of the habitat leaf-humidified soil 25g/L of the mistake 80 order sub-sieve of 0.40g/L, zeatin ZT 0.80mg/L, pulverizing, lime bacterium and mushroom, controlling substratum pH value is 5.6;
The preparation method of described lime bacterium and mushroom is as follows: lime bacterium, mushroom are dried respectively, pulverize, the two mixes in 1:4 ratio, by mixed powder, add water by the weightmeasurement ratio of 1:7, stir evenly, use ultrasonication 30min, then add 15 times of water, regulate pH value to be 5.4, in 45 DEG C of water-baths, add cellulase by the weightmeasurement ratio of 2.5%, after enzymolysis 70min, bath temperature is risen to 90 DEG C, move in 65 DEG C of baking ovens during evaporate to dryness basic to moisture, dry 25hr.
example two:
The healthy sporophore of well-grown, non-parachute-opening is plucked in field; The internal aseptic tissue block of getting cap and stem junction is about 0.35cm
2size, embeds inducing culture gently, and described medium component is: potato 200g/L, fructose 11g/L, albumen freeze 1.25g/L, MgSO
41.00g/L, CaCl
21.00g/L, KH
2pO
4the wort 125ml of 0.50g/L, 16.0 Baumes, agar 10.00g/L, controlling substratum pH value is 6.0, light culture 60 days at 22 ~ 23 DEG C, and tissue block surface grows white fluffy mycelia; By mycelia succeeding transfer culture in above-mentioned inducing culture, obtain the present invention with mycelia;
Mycelia is proceeded to proliferated culture medium, and described multiplication culture based component is: potato 200g/L, fructose 11g/L, albumen freeze 1.25g/L, ZnSO
40.60g/L, MgSO
4enzymolysis dry powder 35g/L, the agar 10.00g/L of the habitat leaf-humidified soil 35g/L of the mistake 80 order sub-sieve of 0.60g/L, zeatin ZT 1.00mg/L, pulverizing, lime bacterium and mushroom, controlling substratum pH value is 6.0;
The preparation method of described lime bacterium and mushroom is as follows: lime bacterium, mushroom are dried respectively, pulverize, the two mixes in 1:4 ratio, by mixed powder, add water by the weightmeasurement ratio of 1:8, stir evenly, with ultrasonication 40 min, then add 20 times of water, regulate pH value to be 5.6, in 50 DEG C of water-baths, add cellulase by the weightmeasurement ratio of 3.0%, after enzymolysis 100 min, bath temperature is risen to 93 DEG C, move in 70 DEG C of baking ovens during evaporate to dryness basic to moisture, dry 30hr.
example three:
The healthy sporophore of well-grown, non-parachute-opening is plucked in field; The internal aseptic tissue block of getting cap and stem junction is about 0.35cm
2size, embeds inducing culture gently, and described medium component is: potato 200g/L, fructose 10g/L, albumen freeze 1.10g/L, MgSO
41.00g/L, CaCl
21.00g/L, KH
2pO
4the wort 110ml of 0.50g/L, 16.0 Baumes, agar 10.00g/L, controlling substratum pH value is 5.6, light culture 55 days at 22 ~ 23 DEG C, and tissue block surface grows white fluffy mycelia; By mycelia succeeding transfer culture in above-mentioned inducing culture, obtain the present invention with mycelia;
Mycelia is proceeded to proliferated culture medium, and described multiplication culture based component is: potato 200g/L, fructose 10g/L, albumen freeze 1.10g/L, ZnSO
40.50g/L, MgSO
4enzymolysis dry powder 30g/L, the agar 10.00g/L of the habitat leaf-humidified soil 30g/L of the mistake 80 order sub-sieve of 0.50g/L, zeatin ZT 0.90mg/L, pulverizing, lime bacterium and mushroom, controlling substratum pH value is 5.6;
The preparation method of described lime bacterium and mushroom is as follows: lime bacterium, mushroom are dried respectively, pulverize, the two mixes in 1:4 ratio, by mixed powder, add water by the weightmeasurement ratio of 1:7, stir evenly, with ultrasonication 35 min, then add 18 times of water, regulate pH value to be 5.4, in 48 DEG C of water-baths, add cellulase by the weightmeasurement ratio of 2.8%, after enzymolysis 90 min, bath temperature is risen to 91 DEG C, move in 68 DEG C of baking ovens during evaporate to dryness basic to moisture, dry 28hr.
example four:
The healthy sporophore of well-grown, non-parachute-opening is plucked in field; The internal aseptic tissue block of getting cap and stem junction is about 0.35cm
2size, embeds inducing culture gently, and described medium component is: potato 200g/L, fructose 10g/L, albumen freeze 1.20g/L, MgSO
41.00g/L, CaCl
21.00g/L, KH
2pO
4the wort 120ml of 0.50g/L, 16.0 Baumes, agar 10.00g/L, controlling substratum pH value is 6.0, light culture 58 days at 22 ~ 23 DEG C, and tissue block surface grows white fluffy mycelia; By mycelia succeeding transfer culture in above-mentioned inducing culture, obtain the present invention with mycelia;
Mycelia is proceeded to proliferated culture medium, and described multiplication culture based component is: potato 200g/L, fructose 10g/L, albumen freeze 1.20g/L, ZnSO
40.55g/L, MgSO
4enzymolysis dry powder 28g/L, the agar 10.00g/L of the habitat leaf-humidified soil 28g/L of the mistake 80 order sub-sieve of 0.55g/L, zeatin ZT 0.85mg/L, pulverizing, lime bacterium and mushroom, controlling substratum pH value is 6.0;
The preparation method of described lime bacterium and mushroom is as follows: lime bacterium, mushroom are dried respectively, pulverize, the two mixes in 1:4 ratio, by mixed powder, add water by the weightmeasurement ratio of 1:8, stir evenly, with ultrasonication 38 min, then add 17 times of water, regulate pH value to be 5.6, in 47 DEG C of water-baths, add cellulase by the weightmeasurement ratio of 2.6%, after enzymolysis 80 min, bath temperature is risen to 92 DEG C, move in 67 DEG C of baking ovens during evaporate to dryness basic to moisture, dry 26hr.
Claims (1)
1. use a method for VA Mycorrhizal Fungi and saprophytic microorganism co-cultivation yellow squama Amanita fuliginea filament, it is characterized by, add in proliferated culture medium mycorrhizal fungi and saprophytic microorganism mixed enzymolysis dry powder, the speed of growth of yellow squama Amanita fuliginea filament can be made to improve 9 ~ 12 times, described mycorrhizal fungi be pick up from raised growth in goose cream habitat lime bacterium (
lactarius piperatus), saprophytic microorganism be indoor scale produce mushroom (
lentinus edodes), the method of preparation and use of lime bacterium and mushroom is as follows: respectively by lime bacterium, mushroom is dried, pulverize, the two mixes in 1:4 ratio, by mixed powder, water is added by the weightmeasurement ratio of 1:7 ~ 1:8, stir evenly, with ultrasonication 30 ~ 40 min, add 15 ~ 20 times of water again, pH value is regulated to be 5.4 ~ 5.6, in 45 ~ 50 DEG C of water-baths, cellulase is added by the weightmeasurement ratio of 2.5 ~ 3.0%, after enzymolysis 70 ~ 100 min, bath temperature is risen to 90 ~ 93 DEG C, to moving in 65 ~ 70 DEG C of baking ovens during moisture evaporate to dryness, dry 25 ~ 30hr, the dry powder of enzymolysis is joined in proliferated culture medium by 25 ~ 35g/L, described multiplication culture based component is: potato 200g/L, fructose 9 ~ 11g/L, peptone 1.00 ~ 1.25g/L, ZnSO
40.40 ~ 0.60g/L, MgSO
4habitat leaf-humidified soil 25 ~ 35g/L, the agar 10.00g/L of the mistake 80 order sub-sieve of 0.40 ~ 0.60g/L, zeatin ZT 0.80 ~ 1.00mg/L, pulverizing, controlling substratum pH value is 5.6 ~ 6.0, concrete steps are: yellow squama goose ointment-like medicine for oral or plastering use entity is carried out mycelium induction, then proceeds to above proliferated culture medium, mycelium induction and cultural method as follows: field pluck well-grown, non-parachute-opening healthy sporophore, get the internal aseptic tissue block 0.35cm of cap and stem junction
2size, embeds inducing culture gently, and described medium component is: potato 200g/L, fructose 9 ~ 11g/L, peptone 1.00 ~ 1.25g/L, MgSO
41.00g/L, CaCl
21.00g/L, KH
2pO
4wort 100 ~ the 125ml of 0.50g/L, 16.0 Baumes, agar 10.00g/L, controlling substratum pH value is 5.6 ~ 6.0, and light culture 50 ~ 60 days at 22 ~ 23 DEG C, tissue block surface grows white fluffy mycelia.
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Citations (3)
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|---|---|---|---|---|
| CN102715089A (en) * | 2012-07-10 | 2012-10-10 | 云南大学 | Establishing method for indoor symbiotic relationship of amanita flavipes and cyclobalanopsis glaucoides as symbiotic plant |
| CN102726207A (en) * | 2011-09-28 | 2012-10-17 | 云南大学 | Method for rapidly propagating small leopard amanita mycelium |
| CN102754596A (en) * | 2012-06-21 | 2012-10-31 | 云南大学 | Establishing method of symbiont of cyclobalanopsis glaucoides and bolete |
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| CN102726207A (en) * | 2011-09-28 | 2012-10-17 | 云南大学 | Method for rapidly propagating small leopard amanita mycelium |
| CN102754596A (en) * | 2012-06-21 | 2012-10-31 | 云南大学 | Establishing method of symbiont of cyclobalanopsis glaucoides and bolete |
| CN102715089A (en) * | 2012-07-10 | 2012-10-10 | 云南大学 | Establishing method for indoor symbiotic relationship of amanita flavipes and cyclobalanopsis glaucoides as symbiotic plant |
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