CN103766138B - Phlebopus portentosus mother culture preserving method - Google Patents
Phlebopus portentosus mother culture preserving method Download PDFInfo
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- CN103766138B CN103766138B CN201310546293.9A CN201310546293A CN103766138B CN 103766138 B CN103766138 B CN 103766138B CN 201310546293 A CN201310546293 A CN 201310546293A CN 103766138 B CN103766138 B CN 103766138B
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 241001600007 Phlebopus portentosus Species 0.000 title claims abstract description 7
- 238000004321 preservation Methods 0.000 claims abstract description 44
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- 235000010374 vitamin B1 Nutrition 0.000 claims description 8
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- 244000002639 Delonix regia Species 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 244000025352 Artocarpus heterophyllus Species 0.000 description 2
- 235000008725 Artocarpus heterophyllus Nutrition 0.000 description 2
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- 235000014826 Mangifera indica Nutrition 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 technical field of cultivation.
Background technology
Boletus aereus (
phlebopus portentosu), also name Phlebopus portentosus is torrid areas preciousness, edible mushroom most favored by customers, be also current Boletaceae (
boletaceae) arteries and veins handle Boletus (
phlebopus) in unique a kind of bolete can cultivated 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, rich in nutritive value, high protein, low fat, be rich in the mineral element that the human bodies such as 17 kinds of essential amino acids and abundant phosphorus, potassium, calcium, magnesium, iron, zinc are necessary, be good healthy food and there is good medical value, deeply liking by consumer.Often be grown in natural environment flame tree (
delonix regia), mango (
mangifern indica), coffee (
coffea arabica), shaddock (
citrus grandis) and jack fruit (
artocarpus heterophyllus) etc. on the lower and neighbouring 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 carries 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, the optimum temperature 28 DEG C ~ 30 DEG C 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, and the low temperature preservation that general microorganism fungus kind routine uses and nitrogen ultra low temperature preserving process all can not effective preservation Boletus aereus bacterial classifications.4 DEG C of low temperature preservations, preservation is after 15 days, and mycelia is dead; Nitrogen ultra low temperature preservation, test in protectant, to add 20% glycerine and add 20% glycerine, culture presevation is carried out in the two kinds of process of 5% extra large bath sugar, preservation activates for 6 months, and result shows: the process adding 20% glycerine in protectant, and bacterial classification is all dead; Protectant adds the process of 20% glycerine, 5% trehalose, has 10% mycelia block germination and growth, but the experiment in cultivation result in later stage show bacterial strain there occurs 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 to the method for the preservation black cattle liver starter kind of simple and effective in edible fungus technical field of cultivation.
Summary of the invention
An 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 bacterial strain vigor for a long time, be difficult to the problem of preservation to solve its bacterial classification in black cattle liver cultivation and scientific research, 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, mycelia is cultivated,
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
31.0～2.0g
CaCl
20.5～1.0 g
ZnSO
40.5～1.0 g
FeSO
40.5～1.0 g
Vitamin B1 100 ~ 200ug
Agar powder 15.0 ~ 20.0 g
Water 1000ml
PH 4.5 ~ 6.5; And
2) get mycelia block to put it into and be equipped with in the container of sterile purified water, airtight container, at 10 DEG C ~ 15 DEG C constant-temperature preservings.
In an embodiment, step 2) in mycelia block size be the mycelia block 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 in the device with 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 getting bolete with tissue isolation is inoculated in solid culture medium, in 28 DEG C ~ 30 DEG C 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
31.0～2.0g
CaCl
20.5～1.0 g
ZnSO
40.5～1.0 g
FeSO
40.5～1.0 g
Vitamin B1 100 ~ 200ug
Agar powder 15.0 ~ 20.0 g
Water 1000ml
pH 4.5～ 6.5；
2) the mycelia block that the mycelia obtained from step (1) cuts 0.5 ㎝ × 0.5, ㎝ ~ 1 ㎝ ~ 1 ㎝ is put in and is equipped with the test tube of sterile purified water, often props up test tube and puts into mycelia block 3 pieces ~ 5 pieces, with stopper stoppered test tube mouth; Preservation in the insulating box of 10 DEG C ~ 15 DEG C.
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 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
31.0～2.0g
CaCl
20.5～1.0 g
ZnSO
40.5～1.0 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, the amount of pouring into often props up 20 ml, and 121 DEG C of sterilizings 20 minutes, are then put into inclined-plane;
(2) Mother culture: gather wild Boletus aereus fruit body, be inoculated in the medium that step (1) prepares with the tissue block that tissue isolation gets Boletus aereus, in 28 DEG C ~ 30 DEG C constant temperature culture, within 25 days ~ 30 days, mycelia covers with medium, obtains black cattle liver starter kind;
(3) female preservation storage tube of planting makes: with the test tube of 18 × 180 specifications, often prop up cuvette cartridge distilled water 10 ml ~ 15ml, then conventional sterilant is stand-by; The black cattle liver starter kind that step (2) is obtained, the mycelia block cutting 0.5 ㎝ × 0.5, ㎝ ~ 1 ㎝ ~ 1 ㎝ is put in the distilled water of sterilizing test tubes dress, often props up test tube and puts into 3 pieces ~ 5 pieces, with silica gel plug stoppered test tube mouth, obtain female kind of storage tube;
(4) female kind preservation temperature: the mother that step (3) obtains plants storage tube and is put in preservation in the insulating box of 10 DEG C ~ 15 DEG C;
(5) female kind of vigor and cultivated character inspection: the black cattle liver starter kind that step (4) is preserved, respectively at activated strains after preservation half a year, 1 year, 3 years, 5 years, expand numerous bacterial classification, be trained liquid spawn again, liquid-spawn inoculation packed matrix artificial cultivation Boletus aereus, inspection 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 start to form bacterium ball for the 3rd day, bright color golden yellow to bacterium ball color when the 9th day, and size is even, be distributed in liquid nutrient medium, is bacterium ball vigorous period; C, the liquid-spawn inoculation solid matrix artificial cultivation Boletus aereus that " b " is cultivated; mycelia material feeding in 3rd day after inoculation; within 50th day, mycelia covers with matrix; then earthing cultivates fruiting; earthing the 10th day fruiting and neat; 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
31.0～2.0g
CaCl
20.5～1.0 g
ZnSO
40.5～1.0 g
FeSO
40.5～1.0 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
31.0～2.0g
CaCl
20.5～1.0 g
ZnSO
40.5～1.0 g
FeSO
40.5～1.0 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 that 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 higher limit, lower limit and any number between higher limit and lower limit.Such as, " red soil 100 ~ 200 g " refers to that the content of red soil can for 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 black cattle liver starter kind vigor and merit reaches more than 5-10, solves 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 that 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) Boletus aereus bacterial strain vigor and merit can be preserved 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
Further describe the present invention in the examples below, described embodiment shows implements shorthand method of the present invention, but does not limit its institute's likely modification.
Embodiment 1
(1) cultivate black cattle liver starter kind with the culture medium prescription of improvement, Mother culture based formulas and compound method as follows:
A: culture medium prescription
Red soil 100 g
Glucose 10.0 g
Yeast diffusion juice 1.0g
NH
4NO
31.0g
CaCl
20.5 g
ZnSO
40.5 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, the amount of pouring into often props up 20 ml, and 121 DEG C of sterilizings 20 minutes, are then put into inclined-plane.
(2) Boletus aereus Mother culture: gather wild Boletus aereus fruit body, be inoculated in the medium that step (1) prepares with the tissue block that tissue isolation gets Boletus aereus, 28 DEG C of constant temperature culture, within 25 days, mycelia covers with medium, obtains black cattle liver starter kind.
(3) female preservation storage tube of planting makes: with the test tube of 18 × 180 specifications, often prop up cuvette cartridge distilled water 10 ml, then conventional sterilant is stand-by; Black cattle liver starter kind step (2) obtained, the mycelia block cutting 0.75 ㎝ × 0.75 ㎝ is put in the distilled water of sterilizing test tubes dress, often props up test tube and puts into 3 pieces, with silica gel plug stoppered test tube mouth, obtain female kind of storage tube.
(4) female kind preservation temperature: the mother that step (3) obtains plants storage tube and is put in preservation in the insulating box of 10 DEG C.
(5) female kind of vigor and cultivated character inspection: the Boletus aereus bacterial classification that step (3) is preserved, activated strains after preservation 2 half a year, expand numerous bacterial classification, be trained liquid spawn again, liquid-spawn inoculation packed matrix artificial cultivation Boletus aereus, inspection 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 start to form bacterium ball for the 3rd day, bright color golden yellow to bacterium ball color when the 9th day, and size is even, be distributed in liquid nutrient medium, is bacterium ball vigorous period; C, the liquid-spawn inoculation solid matrix artificial cultivation Boletus aereus that " b " is cultivated, mycelia material feeding in 3rd day after inoculation, within 50th day, mycelia covers with matrix, then earthing cultivates fruiting, earthing the 10th day fruiting and neat, average each bacterium bag produces fruit body 120.8g, spawn activity and cultivated character better.
Embodiment 2-3
As preferably, mother culture media preparation in embodiment 2-3, Mother culture, female plant preservation storage tube make and the inspection of preservation temperature, female kind of vigor and cultivated character 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 make identical with embodiment 2, and difference is and preservation temperature is 13 DEG C.
Female kind vigor and cultivated character inspection: the Boletus aereus actication of culture by preservation temperature being 13 DEG C of preservation 2 half a year, expand and be numerously trained liquid spawn again, liquid-spawn inoculation packed matrix artificial cultivation Boletus aereus, inspection 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 start to form bacterium ball for the 3rd day, bright color golden yellow to bacterium ball color when the 9th day, and size is even, be distributed in liquid nutrient medium, is bacterium ball vigorous period; C, the liquid-spawn inoculation solid matrix artificial cultivation Boletus aereus that " b " is cultivated, mycelia material feeding in 3rd day after inoculation, within 50th day, mycelia covers with matrix, then earthing cultivates fruiting, earthing the 10th day fruiting is neat and neat, average each bacterium bag produces fruit body 118g, spawn activity and cultivated character better.
Embodiment 5
As preferably, Mother culture based formulas and preparation, Mother culture, female preservation storage tube of planting make identical with embodiment 2, and difference is and preservation temperature is 15 DEG C.
Female kind vigor and cultivated character inspection: the Boletus aereus actication of culture by preservation temperature being 15 DEG C of preservation 2 half a year, expand and be numerously trained liquid spawn again, liquid-spawn inoculation packed matrix artificial cultivation Boletus aereus, inspection 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 start to form bacterium ball for the 3rd day, bright color golden yellow to bacterium ball color when the 10th day, and size is even, be distributed in liquid nutrient medium, is bacterium ball vigorous period; C, the liquid-spawn inoculation solid matrix artificial cultivation Boletus aereus that " b " is cultivated, mycelia material feeding in 3rd day after inoculation, within 50th day, mycelia covers with matrix, then earthing cultivates fruiting, earthing the 10th day fruiting is neat and neat, average each bacterium bag produces fruit body 115g, spawn activity and cultivated character better.
Embodiment 6-9
As preferably, Mother culture based formulas is identical with embodiment 2, medium preparation, Mother culture, female plant that preservation storage tube makes, female preservation temperature of planting is identical with embodiment 1, difference is femalely to plant preservation half a year, 1 year, 3 years, 5 years stepmother and plant vigor and cultivated character checks 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, and quite a few can also be had to change.Therefore, all changes that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, are all considered to be protection scope of the present invention.
Claims (8)
1. a Boletus aereus (
phlebopus portentosus) method for preserving, described method comprises:
1) on solid culture medium, mycelia is cultivated,
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
31.0～2.0g
CaCl
20.5～1.0 g
ZnSO
40.5～1.0 g
FeSO
40.5～1.0 g
Vitamin B1 100 ~ 200ug
Agar powder 15.0 ~ 20.0 g
Water 1000ml
PH 4.5 ~ 6.5; And
2) get mycelia block to put it into and be equipped with in the container of sterile purified water, airtight container, at 10 DEG C ~ 15 DEG C constant-temperature preservings.
2. method according to claim 1, wherein in step 2) in mycelia block size be the mycelia block 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. method according to 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 claim 1 or 2, wherein step 2) in constant-temperature preserving carry out in the device with 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 getting bolete with tissue isolation is inoculated in solid culture medium, in 28 DEG C ~ 30 DEG C 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
31.0～2.0g
CaCl
20.5～1.0 g
ZnSO
40.5～1.0 g
FeSO
40.5～1.0 g
Vitamin B1 100 ~ 200ug
Agar powder 15.0 ~ 20.0 g
Water 1000ml
pH 4.5～ 6.5；
2) the mycelia block that the mycelia obtained from step (1) cuts 0.5 ㎝ × 0.5, ㎝ ~ 1 ㎝ ~ 1 ㎝ is put in and is equipped with the test tube of sterile purified water, often props up test tube and puts into mycelia block 3 pieces ~ 5 pieces, with stopper stoppered test tube mouth; Preservation in the insulating box of 10 DEG C ~ 15 DEG C.
7. a solid culture medium, it comprises:
Red soil 100 ~ 200 g
Glucose 10.0 ~ 20.0 g
Yeast diffusion juice 1.0 ~ 2.0 g
NH
4NO
31.0～2.0g
CaCl
20.5～1.0 g
ZnSO
40.5～1.0 g
FeSO
40.5～1.0 g
Vitamin B1 100 ~ 200ug
Agar powder 15.0 ~ 20.0 g
Water 1000ml
pH 4.5～ 6.5。
8. the purposes of solid culture medium in Boletus aereus cultivation and/or preservation of claim 7.
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| CN104663251A (en) * | 2015-03-18 | 2015-06-03 | 景洪宏臻农业科技有限公司 | Boletus aereus mother strain long-time preservation method |
| CN106105783A (en) * | 2016-08-01 | 2016-11-16 | 景洪宏臻农业科技有限公司 | A kind of batch production Boletus aereus cultural method |
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Effective date of registration: 20180605 Address after: 562200 Guizhou Zhenfeng County, Southwest Guizhou Province, Buyi and Miao Autonomous Prefecture, min Valley Street, the two floor of landscape gold city. Patentee after: Guizhou Hong Zhen fungus industry investment development Co., Ltd. Address before: 200042 block A, 18 floor, Zhaofeng building, 369 Jiangsu Road, Shanghai. Patentee before: Shi Quan |
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Denomination of invention: A method for preservation of mother species of Boletus nigrum Effective date of registration: 20200929 Granted publication date: 20150617 Pledgee: ZHENFENG sub branch of Guiyang Bank Co., Ltd Pledgor: Guizhou Hong Zhen fungus industry investment development Co.,Ltd. Registration number: Y2020980006513 |
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