CN101210224A - Liquid culture medium for culturing mematophagous fungi and method for preparing chlamydospore of the same - Google Patents

Liquid culture medium for culturing mematophagous fungi and method for preparing chlamydospore of the same Download PDF

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Publication number
CN101210224A
CN101210224A CNA2006100636864A CN200610063686A CN101210224A CN 101210224 A CN101210224 A CN 101210224A CN A2006100636864 A CNA2006100636864 A CN A2006100636864A CN 200610063686 A CN200610063686 A CN 200610063686A CN 101210224 A CN101210224 A CN 101210224A
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chlamydospore
culture
liking
preparation
nematode
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王治才
王琳
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王治才
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Abstract

A liquid culture medium for culturing Duddingtonia flagrans and a preparation method of chlamydospore thereof belongs to the field of prevention and treatment technology of gastrointestinal parasitic disease of livestock. The liquid culture medium mainly contains potato juice and trace amount of salt, and is filled in a 100 to 1,000 mL triangular flask or a 5 to 15 L fermentation tank by a volume of 1/3 to 1/5 of the container volume. The liquid culture medium can be used for preparing seeds of Duddingtonia flagrans and enlarging fermentation production after high-pressure sterilization. The technology has abundant and cost-effective raw materials, which facilitates the reduction of production cost, the alleviation of labor intensity, the improvement of a strain preparation as well as the improvement of scale production and industrialization level of the preparation. The technology can be used for fermentatively culturing Duddingtonia flagrans, which can provide seeds for batch production of chlamydospore of Duddingtonia flagrans and can be directly used for the production of chlamydospore.

Description

Have a liking for a kind of liquid nutrient medium and the chlamydospore preparation method thereof of nematode fungus culture
One, affiliated technical field
The present invention relates to a kind of animal gastrointestinal tract oxyuriasis biological control with having a liking for liquid nutrient medium and the culture technique that nematode fungal bacterial strain----Duddingtonia flagrans cultivates, belong to the sick Prevention Technique of domestic animal gastrointestinal parasite field, especially have a liking for a kind of liquid nutrient medium and the thick spore preparation method thereof of nematode fungus culture.
Two, background technology
At present, to the control of cattle and sheep gastrointestinal parasite disease, mainly be animal to be carried out regular expelling parasite with various chemicalses.Prolonged and repeated medication causes on the one hand the medicine savings in the carcass of being in, and makes the livestock product downgrade; Cause the polypide resistance to strengthen on the other hand, anti-system difficulty further strengthens.Drug resistance in parasite becomes one of threat of livestock industry maximum in the world.Thereby the biological control research of domestic animal livestock product parasitosis has begun to draw attention.In disclosed associated biomolecule Prevention Technique, develop comparatively fast with having a liking for the control that nematode fungi Duddingtonia flagrans carries out animal gastrointestinal tract oxyuriasis.
Have a liking for nematode fungi Duddingtonia flagrans and in classification, belong to imperfect fungi Du Dingtun genus.Have a liking for nematode fungi chlamydospore with this strain culture production, add feeding animals in the animal-feed to, chlamydospore can not only be by keeping active behind the animal digestive tract, but follower ight soil enters the grassland, utilize nutrition, the moisture content on ight soil and grassland to develop into mycelia with predation infective larvae, predation and kill worm's ovum in the ight soil of grassland and grow the gi tract nematode third phase infective larvae that, thus reach the biological control effect that purifies grassland control animal infection.
In the research and development of the cultivation of having a liking for nematode fungi Duddingtonia flagrans and biological control preparation, disclosed technology contents mainly comprises the agar plate of different components or is basic raw material with broomcorn millet grain and wheat, filter out the solid medium that carries out seed culture and chlamydospore production, carry out chlamydospore production by the mode of solid culture.At present, by solid culture preparation have a liking for nematode fungi chlamydospore preparation, the biological control that has been used to carry out cattle and sheep gi tract oxyuriasis is used, and obtains biotechnological formulation in some countries and use license licensed licenser licence.Yet find by experimental study, carry out the cultivation of Duddingtonia flagrans, the volume of employed culture vessel is had very big restriction with solid medium.The excessive diffusion that influences culture exchange gas and meta-bolites of container slows down mycelial growth rate, has reduced chlamydospore output; In addition, still all be to use solid medium to have a liking for the cultivation of nematode fungi Duddingtonia flagrans both at home and abroad at present, its characteristic has also just determined can only adopt the solid culture fermentation technique on cultural method.And solid culture does not also have the equipment and the device of special large-scale production.Use culture dish, culturing bottle, triangular flask and similar container to cultivate and seriously limited the scale and the level of this fungi being carried out mass production, increased production cost and technical difficulty.Solid culture occupies a large amount of spaces in sterilization, culturing process, culture cycle is long, the large-scale production cost height of preparation.
The relevant report that carries out this strain culturing with liquid nutrient medium seldom, only one piece of document is that the Vogels substratum that utilizes yeast extract-peptone-dextrose culture-medium (MYPG) and contain plant peptone, casein, sodium-acetate, glycerine carries out the test of liquid culture.And this liquid nutrient medium preparation cost height only can be used for the laboratory culture of this bacterial strain.
Two, summary of the invention
The objective of the invention is to, it is low to propose a kind of preparation cost, guarantees and promote to improve chlamydospore output by mycelial growth rate, be fit to the industrial nematode fungus culture technology of having a liking for, promptly have a liking for a kind of liquid nutrient medium and the chlamydospore preparation method thereof of nematode fungus culture.
Technical scheme of the present invention is:
1, the major ingredient of this liquid nutrient medium is potato juice and trace salt.Potato juice content is at 0.5-20%; Various inorganic salt, trace salt content are respectively: KH 2PO 40.50-2.0g/L; MgSO 4.7H 2O 0.3-1.5g/L; KCI 0.3-1.50g/L; FeSO 4.7H 2O0.005--0.02g/L; ZnSO 4.7H 2O 0.005--0.02g/L; CuSO 4.5H 2O0.003--0.008g/L.This medium pH is 5.5-7.5.
2, have a liking for the seed technology of preparing of nematode fungi Duddingtonia flagrans with this liquid nutrient medium: go bail for be stored in the agar culture plate have a liking for nematode fungi Duddingtonia flagrans culture, be inoculated in and be equipped with in above-mentioned substratum and the autoclaved container, put in the constant temperature jolting incubator, cultivate when finishing and take out, stand-by.
3, have a liking for nematode fungi Duddingtonia flagrans and enlarge production technology:
Adopt larger container, inoculate with the nematode fungi Duddingtonia flagrans seed of having a liking for of aforementioned preparation.Put in the constant temperature jolting incubator, cultivate when finishing and take out, stand-by.
The concrete cultivation time length is with chlamydospore number 〉=1 * 10 in the culture 5/ ml gets final product.
The having a liking for nematode fungi Duddingtonia flagrans culture and can pass through centrifugal or suction filtration of preparation, the gleanings on collecting precipitation or the filter, seasoning is pulverized, and counts the chlamydospore number, and packing is standby.
The invention has the beneficial effects as follows; this technology is that main raw material prepares liquid nutrient medium with the potato juice; and carry out the liquid fermenting production of this bacterium chlamydospore mycelia on this basis; for the large-scale production of having a liking for nematode fungi Duddingtonia flagrans provides a kind of new technological method; make and in this fungi preparation production, utilize large-scale liquid fermenting equipment to carry out cultivation and fermentation to become possibility; sufficient raw and cost are low; this helps to reduce production costs; reduce labor intensity; improve the production efficiency of bacterial strain preparation, improved the production and the industrialization level of said preparation mass-producing.Have a liking for the fermentation culture of nematode fungi Duddingtonia flagrans with this technology, both can also can be directly used in chlamydosporic production for the mass production of Duddingtonia flagrans chlamydospore provides seed.
Three, embodiment
The main raw material of embodiment 1:1, this liquid nutrient medium is potato and trace salt, and with peeling potatoes, fragmentation adds water cure filtering slag promptly, and potato juice content is at 0.5-20%; Various inorganic salt, trace salt content are respectively: KH 2PO 40.5.0-2.0g/L; MgSO 4.7H 2O0.3-1.5g/L; KCI 0.3-1.50g/L; FeSO 4.7H 2O 0.005--0.02g/L; ZnSO 4.7H 2O 0.005--0.02g/L; CuSO 4.5H 2O 0.003--0.008g/L.This medium pH is 5.5-7.5.
2, have a liking for the seed technology of preparing of nematode fungi Duddingtonia flagrans with this liquid nutrient medium: go bail for be stored in the agar culture plate have a liking for nematode fungi Duddingtonia flagrans culture, be inoculated in and be equipped with in above-mentioned substratum and the autoclaved 100-250ml triangular flask, put 25 ℃--in 30 ℃ the constant temperature jolting incubator, jolting every day was 30-60 minute in preceding 5 day, all the other times are left standstill cultivation, or directly jolting was cultivated 10-14 days.Take out when cultivate finishing, it is stand-by to put 4 ℃ or room temperature.
3, have a liking for nematode fungi Duddingtonia flagrans and enlarge production technology:
1), adopt the 500---1000ml triangular flask, aforementioned preparation have a liking for nematode fungi Duddingtoniaflagrans seed, with the amount inoculation of 2-5%.Put 25 ℃--in 30 ℃ the constant temperature jolting incubator, jolting every day was 30-60 minute in preceding 5 day, and all the other times are left standstill cultivation, or directly the jolting incubation time continues 3-4 week.Take out when cultivate finishing, it is stand-by to put 4 ℃ or room temperature.The concrete cultivation time length is with chlamydospore number 〉=1 * 10 in the culture 5/ ml gets final product.
2), adopt 5-15 to rise fermentor tank, aforementioned preparation have a liking for nematode fungi Duddingtoniaflagrans seed, inoculate with the amount of 2-5%.Put 25 ℃--in 30 ℃ the constant temperature jolting incubator, adopt preceding 5 day every day low speed (30-100rpm) to stir aerated culture 30-60 minute, all the other times are left standstill cultivation, or (30-100rpm) stirred aerated culture 10-14 days directly to adopt low speed.Take out when cultivate finishing, it is stand-by to put 4 ℃ or room temperature.The concrete cultivation time length is with chlamydospore number 〉=1 * 10 in the culture 5/ ml gets final product.
1) or 2) produce have a liking for centrifugal that nematode fungi Duddingtonia flagrans culture can be by 1000 rev/mins, or with G5 filter suction filtration, the gleanings on collecting precipitation or the filter, seasoning is pulverized, and counts the chlamydospore number, packing is standby.
This technology is that main raw material prepares liquid nutrient medium with the potato juice; and carry out chlamydosporic liquid production on this basis; for the large-scale production of having a liking for nematode fungi Duddingtonia flagrans provides a kind of new technological method; make and in this fungi preparation production, utilize large-scale liquid fermenting equipment to carry out cultivation and fermentation to become possibility; sufficient raw and cost are low; this helps to reduce production costs; reduce labor intensity; improve the production efficiency of bacterial strain preparation, improved the production and the industrialization level of said preparation mass-producing.Have a liking for the fermentation culture of nematode fungi Duddingtoniaflagrans with this technology, both can also can be directly used in chlamydosporic production for the mass production of Duddingtonia flagrans chlamydospore provides seed.
Embodiment 2: in embodiment 1:
1, takes by weighing the fresh potato 200g that cleans peeling, thinly slice, stir into pasty state.It is an amount of to add water, and low baking temperature boiled 30 minutes.Double gauze filters.Filtrate is supplied water to 1000ml, leaves standstill 12 hours.
2, preparation contains KH 2PO 41.0g, MgSO 4.7H 2O 0.5g, KCI 0.5g, FeSO 4.7H 2O0.01g, ZnSO 4.7H 2O 0.01g, CuSO 4.5H 2The trace salt solution 1000ml of O 0.005g.
3, get the potato juice supernatant 100ml that leaves standstill after 12 hours, above-mentioned trace salt solution 100ml supplies water to 1000ml, transfers PH to 6.5-7.5.
4, above-mentioned substratum is sub-packed in the 100---1000ml triangular flask or 5-15 rises fermentor tank.Loading amount is the 1/3-1/5 of container volume, preferred 1/4; Triangular flask clogs bottleneck with ventilative silica gel plug, encases bottleneck with kraft paper or flannelette again, and lace cord is tied up.15 pounds of 20 minutes autoclavings.
5, go bail for be stored in the agar culture plate have a liking for nematode fungi Duddingtonia flagrans culture, be inoculated in and be equipped with in above-mentioned substratum and the autoclaved 100-250ml triangular flask, put in 26 ℃ of constant temperature jolting incubators.At preceding 5 days that cultivate, every day, all the other times were left standstill cultivation with 100--110rpm jolting 60 minutes, or directly jolting was cultivated 10-14 days.Take out when cultivate finishing, it is stand-by to put 4 ℃ or room temperature.
6, enlarged culturing can rise fermentor tank with 500---1000ml triangular flask or 5-15 respectively and carries out.With the above-mentioned liquid culture seed of having a liking for nematode fungi Duddingtonia flagrans, with the amount inoculation of 2-5%.The cultivation of 500---1000ml triangular flask culture is with above-mentioned seed culture, but incubation time continues 3-4 week.The culture temperature of fermentor tank and time are with the triangular flask culture, but employing low speed (60-100rpm) stirs aerated culture.Concrete inspection of cultivating the time length basis to culture, chlamydospore number 〉=1 * 10 5/ ml gets final product.
6, culture centrifugal by 1000 rev/mins produced in the expansion that will have a liking for nematode fungi Duddingtonia flagrans, or with G5 filter suction filtration, the gleanings on collecting precipitation or the filter, and seasoning is pulverized, and counts behind the chlamydospore number standby.
Find that by test growing of this bacterial strain is lower to the requirement of nutrition, the trace element that adds certain kind and concentration in potato juice can adapt to and satisfy growing of this fungi different steps.

Claims (6)

1. have a liking for a kind of liquid nutrient medium and the chlamydospore preparation method thereof of nematode fungus culture, it is characterized in that, the liquid nutrient medium major ingredient is potato juice and trace salt, and potato juice content is at 0.5-20%; Various inorganic salt, trace salt content are respectively: KH 2PO 40.50-2.0g/L; MgSO 4.7H 2O 0.3-1.50g/L; KCI 0.3-1.50g/L; FeSO 4.7H 2O0.005--0.02g/L; ZnSO 4.7H 2O 0.005--0.02g/L; CuSO 4.5H 2O0.003--0.008g/L, this medium pH are 5.5-7.5.
2. a kind of liquid nutrient medium and the chlamydospore preparation method thereof who has a liking for the nematode fungus culture according to claim 1,, it is characterized in that liquid nutrient medium is sub-packed in the 100---1000ml triangular flask or 5-15 rises fermentor tank, loading amount is the 1/3-1/5 of container volume; Can be used for the seed preparation of fungi behind the autoclaving and enlarge producing fermentation.
3. a kind of liquid nutrient medium and the chlamydospore preparation method thereof who has a liking for the nematode fungus culture according to claim 1, it is characterized in that; go bail for be stored in the agar culture plate have a liking for nematode fungi Duddingtonia flagrans culture; be inoculated in and be equipped with in above-mentioned substratum and the autoclaved 100-250ml triangular flask; put 25 ℃--in 30 ℃ the constant temperature jolting incubator; jolting every day was 30-60 minute in preceding 5 day; all the other times are left standstill cultivation; or directly jolting was cultivated 10-14 days; cultivate when finishing and take out, it is stand-by to put 4 ℃ or room temperature.
4. a kind of liquid nutrient medium and the chlamydospore preparation method thereof who has a liking for the nematode fungus culture according to claim 2, it is characterized in that, enlarge and produce fermentation, adopt the 500---1000ml triangular flask, preparation have a liking for nematode fungi Duddingtonia flagrans seed, amount inoculation with 2-5%, put 25 ℃--in 30 ℃ the constant temperature jolting incubator, jolting every day was 30-60 minute in preceding 5 day, all the other times are left standstill cultivation, or directly the jolting incubation time continues 3-4 week, cultivates when finishing and takes out, and it is stand-by to put 4 ℃ or room temperature.The concrete cultivation time length is with chlamydospore number 〉=1 * 10 in the culture 5/ ml gets final product.In incubation time 3-4 week, specifically cultivate the time length according to the inspection to culture, chlamydospore number 〉=1 * 10 5/ ml gets final product.
5. a kind of liquid nutrient medium and the chlamydospore preparation method thereof who has a liking for the nematode fungus culture according to claim 2, it is characterized in that, enlarge and produce fermentation, adopt 5-15 to rise fermentor tank, aforementioned preparation have a liking for nematode fungi Duddingtonia flagrans seed, with the amount inoculation of 2-5%.Put 25 ℃--in 30 ℃ the constant temperature jolting incubator, adopt preceding 5 day every day low speed (30-100rpm) to stir aerated culture 30-60 minute, all the other times are left standstill cultivation, or (30-100rpm) stirred aerated culture 10-14 days directly to adopt low speed, take out when cultivate finishing, it is stand-by to put 4 ℃ or room temperature.The concrete cultivation time length is with chlamydospore number 〉=1 * 10 in the culture 5/ ml gets final product.
6. according to claim 4 and 5 described a kind of liquid nutrient medium and the chlamydospore preparation methods thereof that have a liking for the nematode fungus culture, it is characterized in that, enlarge centrifugal that nematode fungi Duddingtonia flagrans culture can be by 1000 rev/mins of having a liking for of production fermentative production, or with G5 filter suction filtration, the gleanings on collecting precipitation or the filter, seasoning, pulverize, counting chlamydospore number, packing, standby.
CNA2006100636864A 2006-12-28 2006-12-28 Liquid culture medium for culturing mematophagous fungi and method for preparing chlamydospore of the same Pending CN101210224A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102648714A (en) * 2011-02-24 2012-08-29 上海万力华生物科技有限公司 Method for preparing biopesticide through liquid fermentation
CN104004663A (en) * 2014-05-27 2014-08-27 内蒙古农业大学 Preparation method of Duddingtonia flagrans biological freeze-drying preparation
CN104560729A (en) * 2014-12-24 2015-04-29 西北民族大学 Liquid culture medium for culturing nematophagous fungi and preparation method for liquid culture medium
CN109843067A (en) * 2016-08-05 2019-06-04 国际动物保健品有限公司 Composition comprising Duddingtonia flagrans

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102648714A (en) * 2011-02-24 2012-08-29 上海万力华生物科技有限公司 Method for preparing biopesticide through liquid fermentation
CN102648714B (en) * 2011-02-24 2014-07-02 上海万力华生物科技有限公司 Method for preparing biopesticide through liquid fermentation
CN104004663A (en) * 2014-05-27 2014-08-27 内蒙古农业大学 Preparation method of Duddingtonia flagrans biological freeze-drying preparation
CN104560729A (en) * 2014-12-24 2015-04-29 西北民族大学 Liquid culture medium for culturing nematophagous fungi and preparation method for liquid culture medium
CN109843067A (en) * 2016-08-05 2019-06-04 国际动物保健品有限公司 Composition comprising Duddingtonia flagrans
CN109843067B (en) * 2016-08-05 2021-09-21 国际动物保健品有限公司 Compositions comprising Duddingtonia flagrans

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