CN103766138A - Phlebopus portentosus mother culture preserving method - Google Patents
Phlebopus portentosus mother culture preserving method Download PDFInfo
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- CN103766138A CN103766138A CN201310546293.9A CN201310546293A CN103766138A CN 103766138 A CN103766138 A CN 103766138A CN 201310546293 A CN201310546293 A CN 201310546293A CN 103766138 A CN103766138 A CN 103766138A
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- mycelia
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- solid culture
- boletus aereus
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- 241001600007 Phlebopus portentosus Species 0.000 title claims abstract description 7
- 238000004321 preservation Methods 0.000 claims abstract description 44
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- 241001063964 Boletus aereus Species 0.000 claims description 36
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- 229960003495 thiamine Drugs 0.000 claims description 8
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 8
- 235000010374 vitamin B1 Nutrition 0.000 claims description 8
- 239000011691 vitamin B1 Substances 0.000 claims description 8
- 241000973497 Siphonognathus argyrophanes Species 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 4
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- 239000011159 matrix material Substances 0.000 description 12
- 238000003860 storage Methods 0.000 description 10
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- 239000002932 luster Substances 0.000 description 4
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- 235000015097 nutrients Nutrition 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
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- 244000025352 Artocarpus heterophyllus Species 0.000 description 2
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Mushroom Cultivation (AREA)
Abstract
The invention relates to a phlebopus portentosus mother culture preserving method. The method comprises the steps that mycelium is cultivated on a solid medium, sclertium is extracted to be placed in a container filled with sterile purified water, the container is sealed, and the sclertium is preserved at the constant temperature ranging from 10 DEG C to 15 DEG C. The invention further relates to the culture medium for preservation of the phlebopus portentosus mother culture and the purpose of the culture medium in cultivating and/or preserving of the culture medium.
Description
Technical field
The present invention relates to a kind of black cattle liver starter kind method for preserving, belong to edible fungus culture technique field.
Background technology
Boletus aereus (
phlebopus portentosu), also name Phlebopus portentosus, be edible mushroom torrid areas preciousness, most favored by customers, be also current Boletaceae (
boletaceae) arteries and veins handle Boletus (
phlebopus) in the unique a kind of bolete that can cultivate with saprophytic cultivation method, be distributed in the ground such as Sri Lanka, Vietnam, Indonesia, Thailand, Brazil, Mexico, Australia, New Zealand and Yunnan Province of China, Guangxi and Hainan.Boletus aereus quality is fresh and tender, delicious flavour, nutritive value abundant, high protein, low fat, be rich in the necessary mineral elements of human body such as 17 kinds of essential amino acids and abundant phosphorus, potassium, calcium, magnesium, iron, zinc, be good healthy food and there is good medical value, liked by consumer.In natural environment, often grow in flame tree (
delonix regia), mango (
mangifern indica), coffee (
coffea arabica), shaddock (
citrus grandis) and jack fruit (
artocarpus heterophyllus) etc. near the lower and meadow of tree, and root mealybug in soil (
pseudococcus) also there is substantial connection, its special life style is just under study for action.At present can with cultivation saprophytic bacteria mode carry out artificial cultivation, and the host tree such as inoculation flame tree, coffee, shaddock, mango host tree is carried out bionic cultivation.
But the preservation of black cattle liver starter kind is a difficult problem always.Boletus aereus is a kind of high temperature edible mushroom, 28 ℃～30 ℃ of the optimum temperatures of suitable mycelial growth.Under existing technical conditions, its bacterial classification can not carry out preservation as saprophytic edible mushroom.Inventor shows the practical experience of Boletus aereus culture presevation for many years, all effective preservation Boletus aereus bacterial classifications of the conventional low temperature preservation using of general microorganism fungus kind and nitrogen ultra low temperature preserving process.4 ℃ of low temperature preservations, preservation is after 15 days, mycelia death; Nitrogen ultra low temperature preservation, tested in protectant, add 20% glycerine and add 20% glycerine, culture presevation is carried out in two kinds of processing of 5% extra large bath sugar, preservation activates for 6 months, result shows: in protectant, add the processing of 20% glycerine, bacterial classification is all dead; Protectant adds the processing of 20% glycerine, 5% trehalose, has 10% mycelia piece germination and growth, but the experiment in cultivation result in later stage show bacterial strain occurred serious degradation, mycelia weak, form that mycoderma is many, fruiting is irregular, fruit body weight saving and the underproduction serious.Therefore, there is the active demand of the method for the preservation black cattle liver starter kind to simple and effective in edible fungus culture technique field.
Summary of the invention
One of object of the present invention is to provide a kind of easy and simple to handle, black cattle liver starter kind method for preserving that can keep for a long time bacterial strain vigor, to solve the problem that in black cattle liver cultivation and scientific research, its bacterial classification is difficult to preservation, and offer reference for other boletes and the preservation of mycorhiza edible fungus species.Another object of the present invention is to provide a kind of black cattle liver starter kind preservation medium and the purposes of this medium in Boletus aereus cultivation and/or preservation.
One aspect of the present invention relate to a kind of Boletus aereus (
phlebopus portentosus) method for preserving, described method comprises:
1) on solid culture medium, cultivate mycelia,
Wherein said solid culture medium comprises:
Red soil 100～200 g
Glucose 10.0～20.0 g
Yeast diffusion juice 1.0～2.0 g
NH
4NO
3 1.0～2.0g
CaCl
2 0.5～1.0 g
ZnSO
4 0.5～1.0 g
FeSO
4 0.5～1.0 g
Vitamin B1 100～200ug
Agar powder 15.0～20.0 g
Water 1000ml
PH 4.5～6.5; And
2) get mycelia piece and put it in the container that sterile purified water is housed, airtight container, at 10 ℃～15 ℃ constant-temperature preservings.
In an embodiment, step 2) in mycelia block size be the mycelia piece of 0.5 ㎝ × 0.5, ㎝～1 ㎝～1 ㎝.
In an embodiment, step 2) in described container be selected from glass container and plastic containers.
In an embodiment, step 2) in described container be selected from preservation dedicated pipe, test tube and blake bottle.
In an embodiment, step 2) in constant-temperature preserving carry out having in the device of steady temperature ability and/or environment.
The present invention relates to a kind of method for preserving of Boletus aereus on the other hand, and described method comprises:
1) gather wild Boletus aereus fruit body, the tissue block of getting bolete with tissue isolation is inoculated in solid culture medium, in 28 ℃～30 ℃ constant temperature culture 25 days～30 days to obtain mycelia,
Wherein said solid culture medium comprises:
Red soil 100～200 g
Glucose 10.0～20.0 g
Yeast diffusion juice 1.0～2.0 g
NH
4NO
3 1.0～2.0g
CaCl
2 0.5～1.0 g
ZnSO
4 0.5～1.0 g
FeSO
4 0.5～1.0 g
Vitamin B1 100～200ug
Agar powder 15.0～20.0 g
Water 1000ml
pH 4.5～ 6.5；
2) the mycelia piece that the mycelia obtaining from step (1) cuts 0.5 ㎝ × 0.5, ㎝～1 ㎝～1 ㎝ is put in the test tube that sterile purified water is housed, and every test tube is put into 3～5 of mycelia pieces, with stopper stoppered test tube mouth; Preservation in the insulating box of 10 ℃～15 ℃.
The present invention relates to a kind of Boletus aereus culture collection process on the other hand, comprises the steps:
(1) cultivate black cattle liver starter kind with the culture medium prescription of improvement, Mother culture based formulas and compound method are as follows:
A: culture medium prescription
Red soil 100～200 g
Glucose 10.0～20.0 g
Yeast diffusion juice 1.0～2.0 g
NH
4NO
3 1.0～2.0g
CaCl
2 0.5～1.0 g
ZnSO
4 0.5～1.0 g
FeSO
4 0.5～1.0 g
Cobastab
1100～200ug
Agar powder 15.0～20.0 g
Water 1000ml
pH 4.5～ 6.5
B: compound method
Get 1000 ml water, put into red soil, boil 5 minutes, then filter, get filtrate, add water and be settled to 1000 ml, add all the other compositions in formula, pour in the test tube of specification 25 × 200, every 20 ml of the amount of pouring into, 121 ℃ of sterilizings 20 minutes, are then put into inclined-plane;
(2) Mother culture: gather wild Boletus aereus fruit body, the tissue block of getting Boletus aereus with tissue isolation is inoculated in the medium of step (1) preparation, in 28 ℃～30 ℃ constant temperature culture, within 25 days～30 days, mycelia is covered with medium, obtains black cattle liver starter kind;
(3) female preservation storage tube of planting is made: with the test tube of 18 × 180 specifications, and every cuvette cartridge distilled water 10 ml～15ml, then conventional sterilizing is stand-by; The black cattle liver starter kind that step (2) is obtained, the mycelia piece that cuts 0.5 ㎝ × 0.5, ㎝～1 ㎝～1 ㎝ is put in the distilled water that sterilizing test tubes has filled, and every test tube is put into 3～5, with silica gel plug stoppered test tube mouth, obtains female kind of storage tube;
(4) female preservation temperature of planting: the mother that step (3) obtains plants storage tube and is put in preservation in the insulating box of 10 ℃～15 ℃;
(5) female kind of vigor and cultivated character check: the black cattle liver starter kind that step (4) is preserved, activate bacterial strain respectively at after preservation half a year, 1 year, 3 years, 5 years, expand numerous bacterial classification, cultivate into again liquid spawn, the packed matrix artificial cultivation of liquid-spawn inoculation Boletus aereus, check spawn activity and cultivated character, result is as follows: it is fast that a, bacterial classification expand numerous mycelial growth, be inoculated in cuvette cartridge slant medium with activation mycelium, inoculate latter 25 days mycelia and cover with inclined-plane; B, use " a " expand numerous mycelium inoculation liquid medium culture liquid spawn, inoculates and within the 3rd day, start to form bacterium ball, and during to the 9th day, bacterium ball color is golden yellow, color and luster bright, and size is even, be distributed in liquid nutrient medium, are bacterium ball vigorous period; C, the liquid-spawn inoculation solid matrix artificial cultivation Boletus aereus that " b " cultivated; mycelia material feeding in the 3rd day after inoculation; within the 50th day, mycelia is covered with matrix; then earthing is cultivated fruiting; the 10th day fruiting and neat of earthing; average each bacterium bag produces fruit body 100g-120g, can realize Boletus aereus large-scale planting.
The present invention relates to a kind of solid culture medium on the other hand, and it comprises:
Red soil 100～200 g
Glucose 10.0～20.0 g
Yeast diffusion juice 1.0～2.0 g
NH
4NO
3 1.0～2.0g
CaCl
2 0.5～1.0 g
ZnSO
4 0.5～1.0 g
FeSO
4 0.5～1.0 g
Vitamin B1 100～200ug
Agar powder 15.0～20.0 g
Water 1000ml
pH 4.5～ 6.5。
The present invention relates to the purposes of a kind of solid culture medium in Boletus aereus cultivation and/or preservation on the other hand, described solid culture medium, and it comprises:
Red soil 100～200 g
Glucose 10.0～20.0 g
Yeast diffusion juice 1.0～2.0 g
NH
4NO
3 1.0～2.0g
CaCl
2 0.5～1.0 g
ZnSO
4 0.5～1.0 g
FeSO
4 0.5～1.0 g
Vitamin B1 100～200ug
Agar powder 15.0～20.0 g
Water 1000ml
pH 4.5～ 6.5。
The composition of the solid culture medium the present invention relates to includes but not limited to red soil, glucose, Yeast diffusion juice, NH
4nO
3, CaCl
2, ZnSO
4, FeSO
4, vitamin B1, agar powder and water.
As used herein, number range comprises any number between higher limit, lower limit and higher limit and lower limit.For example, " red soil 100～200 g " refer to that the content of red soil can be any number option between 100g, 200g and 100g and 200g, such as 101g, 125g, 150g, 175g, 199g etc.
Use method of the present invention and medium preservation black cattle liver starter kind, its mycelia vigor and cultivating bacterial spawn proterties are planted identical with a godmother.The present invention can preserve more than black cattle liver starter kind vigor and merit reach 5-10, has solved a difficult problem for Boletus aereus culture presevation difficulty, for large-scale planting provides powerful guarantee.
The invention has the advantages that: utilize the method preservation Boletus aereus bacterial classification method the present invention relates to simple and effective; equipment is simple; solve the difficult problem that high temperature edible fungus species is difficult to preservation; (5-10) preserves Boletus aereus bacterial strain vigor and merit over a long time; provide powerful guarantee for realizing Boletus aereus large-scale production, there is higher using value, economic implications and social effect.
Embodiment
In following examples, further describe the present invention, described embodiment shows and implements concise and to the point method of the present invention, but does not limit its likely modification of institute.
Embodiment 1
(1) cultivate black cattle liver starter kind with the culture medium prescription of improvement, Mother culture based formulas and compound method are as follows:
A: culture medium prescription
Red soil 100 g
Glucose 10.0 g
Yeast diffusion juice 1.0g
NH
4NO
3 1.0g
CaCl
2 0.5 g
ZnSO
4 0.5 g
FeSO
4 0.5g
Cobastab
1100.0ug
Agar powder 15.0 g
Water 1000ml
pH 4.5～ 6.5
B: compound method
Get 1000 ml water, put into red soil, boil 5 minutes, then filter, get filtrate, add water and be settled to 1000 ml, add all the other compositions in formula, pour in the test tube of specification 25 × 200, every 20 ml of the amount of pouring into, 121 ℃ of sterilizings 20 minutes, are then put into inclined-plane.
(2) Boletus aereus Mother culture: gather wild Boletus aereus fruit body, the tissue block of getting Boletus aereus with tissue isolation is inoculated in the medium of step (1) preparation, 28 ℃ of constant temperature culture, within 25 days, mycelia is covered with medium, obtains black cattle liver starter kind.
(3) female preservation storage tube of planting is made: with the test tube of 18 × 180 specifications, and every cuvette cartridge distilled water 10 ml, then conventional sterilizing is stand-by; The black cattle liver starter kind that step (2) is obtained, the mycelia piece that cuts 0.75 ㎝ × 0.75 ㎝ is put in the distilled water that sterilizing test tubes has filled, and every test tube is put into 3, with silica gel plug stoppered test tube mouth, obtains female kind of storage tube.
(4) female preservation temperature of planting: the mother that step (3) obtains plants storage tube and is put in preservation in the insulating box of 10 ℃.
(5) female kind of vigor and cultivated character check: the Boletus aereus bacterial classification that step (3) is preserved, after 2 half a year of preservation, activate bacterial strain, expand numerous bacterial classification, cultivate into again liquid spawn, the packed matrix artificial cultivation of liquid-spawn inoculation Boletus aereus, check spawn activity and cultivated character, it is fast that result: a, bacterial classification expand numerous mycelial growth, be inoculated in cuvette cartridge slant medium with activation mycelium, inoculate latter 25 days mycelia and cover with inclined-plane; B, use " a " expand numerous mycelium inoculation liquid medium culture liquid spawn, inoculates and within the 3rd day, start to form bacterium ball, and during to the 9th day, bacterium ball color is golden yellow, color and luster bright, and size is even, be distributed in liquid nutrient medium, are bacterium ball vigorous period; C, the liquid-spawn inoculation solid matrix artificial cultivation Boletus aereus that " b " cultivated, mycelia material feeding in the 3rd day after inoculation, within the 50th day, mycelia is covered with matrix, then earthing is cultivated fruiting, the 10th day fruiting and neat of earthing, average each bacterium bag produces fruit body 120.8g, and spawn activity and cultivated character are better.
Embodiment 2-3
As preferably, mother culture media preparation in embodiment 2-3, Mother culture, female plant that preservation storage tube is made and preservation temperature, female kind of vigor and cultivated character check identical with embodiment 1, difference is in table 1.
Table 1
Embodiment 4
As preferably, Mother culture based formulas and preparation, Mother culture, female preservation storage tube of planting are made identically with embodiment 2, and difference is and preservation temperature is 13 ℃.
Female kind vigor and cultivated character check: be the Boletus aereus actication of culture of 13 ℃ of 2 half a year of preservation by preservation temperature, expand numerous liquid spawn of cultivating into again, the packed matrix artificial cultivation of liquid-spawn inoculation Boletus aereus, check spawn activity and cultivated character, it is fast that result: a, bacterial classification expand numerous mycelial growth, be inoculated in cuvette cartridge slant medium with activation mycelium, inoculate latter 28 days mycelia and cover with inclined-plane; B, use " a " expand numerous mycelium inoculation liquid medium culture liquid spawn, inoculates and within the 3rd day, start to form bacterium ball, and during to the 9th day, bacterium ball color is golden yellow, color and luster bright, and size is even, be distributed in liquid nutrient medium, are bacterium ball vigorous period; C, the liquid-spawn inoculation solid matrix artificial cultivation Boletus aereus that " b " cultivated, mycelia material feeding in the 3rd day after inoculation, within the 50th day, mycelia is covered with matrix, then earthing is cultivated fruiting, the 10th day fruiting of earthing is neat and neat, average each bacterium bag produces fruit body 118g, and spawn activity and cultivated character are better.
Embodiment 5
As preferably, Mother culture based formulas and preparation, Mother culture, female preservation storage tube of planting are made identically with embodiment 2, and difference is and preservation temperature is 15 ℃.
Female kind vigor and cultivated character check: be the Boletus aereus actication of culture of 15 ℃ of 2 half a year of preservation by preservation temperature, expand numerous liquid spawn of cultivating into again, the packed matrix artificial cultivation of liquid-spawn inoculation Boletus aereus, check spawn activity and cultivated character, it is fast that result: a, bacterial classification expand numerous mycelial growth, be inoculated in cuvette cartridge slant medium with activation mycelium, inoculate latter 30 days mycelia and cover with inclined-plane; B, use " a " expand numerous mycelium inoculation liquid medium culture liquid spawn, inoculates and within the 3rd day, start to form bacterium ball, and during to the 10th day, bacterium ball color is golden yellow, color and luster bright, and size is even, be distributed in liquid nutrient medium, are bacterium ball vigorous period; C, the liquid-spawn inoculation solid matrix artificial cultivation Boletus aereus that " b " cultivated, mycelia material feeding in the 3rd day after inoculation, within the 50th day, mycelia is covered with matrix, then earthing is cultivated fruiting, the 10th day fruiting of earthing is neat and neat, average each bacterium bag produces fruit body 115g, and spawn activity and cultivated character are better.
Embodiment 6-9
As preferably, Mother culture based formulas is identical with embodiment 2, medium preparation, Mother culture, femalely plant that preservation storage tube is made, female preservation temperature of planting is identical with embodiment 1, difference is that female kind preservation half a year, 1 year, 3 years, 5 years stepmother plants vigor and cultivated character check situation in table 2.
Table 2
Finally, note also that, what more than enumerate is only several specific embodiments of the present invention.Objectively, the present invention is not merely only limited to above embodiment, can also have quite a few variation.Therefore, all changes that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, are all considered to be protection scope of the present invention.
Claims (8)
- A Boletus aereus ( phlebopus portentosus) method for preserving, described method comprises:1) on solid culture medium, cultivate mycelia,Wherein said solid culture medium comprises:Red soil 100～200 gGlucose 10.0～20.0 gYeast diffusion juice 1.0～2.0 gNH 4NO 3 1.0～2.0gCaCl 2 0.5～1.0 gZnSO 4 0.5～1.0 gFeSO 4 0.5～1.0 gVitamin B1 100～200ugAgar powder 15.0～20.0 gWater 1000mlPH 4.5～6.5; And2) get mycelia piece and put it in the container that sterile purified water is housed, airtight container, at 10 ℃～15 ℃ constant-temperature preservings.
- 2. according to the process of claim 1 wherein in step 2) in mycelia block size be the mycelia piece of 0.5 ㎝ × 0.5, ㎝～1 ㎝～1 ㎝.
- 3. according to the method for claim 1 or 2, wherein step 2) in described container be selected from glass container and plastic containers.
- 4. according to the method for claim 3, wherein step 2) in described container be selected from preservation dedicated pipe, test tube and blake bottle.
- 5. according to the method for any one in claim 1-4, wherein step 2) in constant-temperature preserving carry out having in the device of steady temperature ability and/or environment.
- 6. a method for preserving for Boletus aereus, described method comprises:1) gather wild Boletus aereus fruit body, the tissue block of getting bolete with tissue isolation is inoculated in solid culture medium, in 28 ℃～30 ℃ constant temperature culture 25 days～30 days to obtain mycelia,Wherein said solid culture medium comprises:Red soil 100～200 gGlucose 10.0～20.0 gYeast diffusion juice 1.0～2.0 gNH 4NO 3 1.0～2.0gCaCl 2 0.5～1.0 gZnSO 4 0.5～1.0 gFeSO 4 0.5～1.0 gVitamin B1 100～200ugAgar powder 15.0～20.0 gWater 1000mlpH 4.5～ 6.5；2) the mycelia piece that the mycelia obtaining from step (1) cuts 0.5 ㎝ × 0.5, ㎝～1 ㎝～1 ㎝ is put in the test tube that sterile purified water is housed, and every test tube is put into 3～5 of mycelia pieces, with stopper stoppered test tube mouth; Preservation in the insulating box of 10 ℃～15 ℃.
- 7. a solid culture medium, it comprises:Red soil 100～200 gGlucose 10.0～20.0 gYeast diffusion juice 1.0～2.0 gNH 4NO 3 1.0～2.0gCaCl 2 0.5～1.0 gZnSO 4 0.5～1.0 gFeSO 4 0.5～1.0 gVitamin B1 100～200ugAgar powder 15.0～20.0 gWater 1000mlpH 4.5～ 6.5。
- 8. the purposes of the solid culture medium of claim 7 in Boletus aereus cultivation and/or preservation.
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