CN103548563B - A kind of fragmentation processing method for germination bacterium liquid strain mycelium pellets - Google Patents
A kind of fragmentation processing method for germination bacterium liquid strain mycelium pellets Download PDFInfo
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- CN103548563B CN103548563B CN201310504715.6A CN201310504715A CN103548563B CN 103548563 B CN103548563 B CN 103548563B CN 201310504715 A CN201310504715 A CN 201310504715A CN 103548563 B CN103548563 B CN 103548563B
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Abstract
The open a kind of fragmentation processing method for germination bacterium liquid strain mycelium pellets of the present invention, process including preparing female kind, shaker flask stand cultivation, shaking flask concussion cultivation and mycelium pellet fragmentation, cost of material of the present invention is low, technique the most easily obtains, and Germination Strain mycelium pellet fragmentation is processed, fungus ball can be made to diminish and in the same size, improve the quality of mycelium pellet, solve the technique that Germination Strain mycelium pellet is difficult to fragmentation in shaking flask, make Germination Strain liquid spawn grow in accessing solid state cultivation kind rapidly, growth cycle significantly shortens, and improves production efficiency.
Description
Technical field
The invention belongs to Gastrodia planting technology field, particularly relate to a kind of Germination Strain liquid spawn
Mycelium pellet fragmentation processing method.
Technical background
Rhizoma Gastrodiae is a kind of orchid with mycorhiza, unrooted, without greenery, it is impossible to manufactures and is provided from
The nutrient substance of body growth, after gastrodia seed germination, relies primarily on decomposition and invades its internal honey
Ring mycelia and grow.The ripe bulb of Rhizoma Gastrodiae is rare Chinese medicine, have the title of " the second Radix Ginseng ",
It is the good medicine of China's successive dynasties treating disease and preserving health, is listed in top grade in successive dynasties bimillennium draft.My god
It is dizzy that fiber crops have wind-damp dispelling, convulsion, relieving fainting, effect of blood pressure lowering, especially in analgesia, calm
The effect of aspect is the most prominent, and has no side effect.At present, the most gradually Rhizoma Gastrodiae is used for
The aspects such as health care, medical treatment, food.
Entering long-term research to draw, Germination Strain is that the symbiosis of Rhizoma Gastrodiae rudiment, production and high yield is thin
Bacterium, Japanese wasteland person of outstanding talent helps and had once delivered " Rhizoma Gastrodiae and Armillaria mellea symbiosis " literary composition in 1911, you
Rear it is believed that Rhizoma Gastrodiae is in whole growth cycle, all it be unable to do without Armillaria mellea.Due to Gastrodia
The optimum temperature that grows with symbiosis Germination Strain of optimum temperature that son is sprouted does not matches, and this may be with
The biological characteristics of seeds of Gastrodia elata is closely related, in cultivation, on the one hand considers symbiotic germination
Condition needed for bacteria growing, meets the epidemic disaster that seeds of Gastrodia elata germinates again, could improve and plant
Sub-germination percentage and Output of Gastrodia elata, in order to solve above technical problem, research worker is first to Germination Strain
First cultivate, then when Rhizoma Gastrodiae is planted, Germination Strain be seeded in the afterbody of Rhizoma Gastrodiae, it is simple to
Rhizoma Gastrodiae is sprouted and growth.Known Germination Strain liquid spawn mycelium pellet is big, accesses solid state cultivation
Poor fluidity in kind, disperses uneven, easy bacteria infection, apparatus costliness during fragmentation
Deng；
Summary of the invention
For the problems referred to above, the present invention provides a kind of Germination Strain liquid spawn mycelium pellet fragmentation to process
Method has simple to operate, and fragmentation is convenient, and the cycle is short, and mycelium pellet density is high, it is little to infect probability,
Access good fluidity in solid state cultivation kind, be uniformly dispersed, send out bacterium fast.
The present invention is achieved by the following technical solutions:
A kind of fragmentation processing method for germination bacterium liquid strain mycelium pellets, the method includes preparing master clock, shaking
Bottle quiescent culture, shaking flask concussion are cultivated, fragmentation processes and inoculation six steps i.e. can obtain cultivation and use
Kind.
Further, described a kind of fragmentation processing method for germination bacterium liquid strain mycelium pellets, bag
Include following steps:
1) preparation mother plants: is inoculated into by strain block in test tube PDA culture medium, is placed in 20-30 DEG C
In environment, lucifuge is cultivated 6-16 days；
2) shaker flask stand is cultivated: by step 1) is cultivated female plant be cut into 3～5mm little
Block, provokes mycelia body with vaccinating lancet, is inoculated in shaking flask, and makes it be suspended on liquid level,
It is 5 10 pieces that every bottle graft enters amount, will connect the shaker flask stand planted at the constant temperature being placed on 20～30 DEG C
Case is cultivated 4-14 days；
3) shaking flask concussion is cultivated: by step 2) in shaker flask stand cultivate and be placed on frequency and be
Cultivate on the concussion shaking table of 200r/min, temperature 20～30 DEG C, make strain shake training in shaking flask
Support 2～12 days, obtain the mycelium pellet of a diameter of about 2cm.
4) fragmentation processes: shaking flask cultured in step 3) is transferred to frequency is 500r/min
Magnetic stirrer on make the stirring of 5～15 cycles, obtain the fragmentation mycelium of 40～60%.
5) inoculation: fragmentation mycelium is accessed solid state cultivation bag, at 20～30 DEG C of dark conditions
Lower cultivation 30～cultivation kind can be obtained for 35 days.
Further, described a kind of fragmentation processing method for germination bacterium liquid strain mycelium pellets,
It is characterized in that: comprise the following steps:
1) preparation mother plants: is inoculated into by strain block in test tube PDA culture medium, is placed in 24-27 DEG C
In environment, lucifuge is cultivated 8-12 days；
2) shaker flask stand is cultivated: the female kind cultivated in step 1) is cut into the fritter of 3～5mm,
Provoke mycelia body with vaccinating lancet, be inoculated in shaking flask, and make it be suspended on liquid level, every bottle
Access amount is 5～10 pieces, will connect the shaker flask stand planted in being placed on the calorstat of 22～27 DEG C
Cultivate 6-10 days；
3) shaking flask concussion is cultivated: by step 2) in shaker flask stand cultivate and be placed on frequency and be
Cultivate on the concussion shaking table of 200r/min, temperature 23～27 DEG C, make strain shake training in shaking flask
Support 6～10 days, obtain the mycelium pellet of a diameter of about 2cm；
4) fragmentation processes: shaking flask cultured in step 3) is transferred to frequency is 500r/min
Magnetic stirrer on make the stirring of 5～10 cycles, obtain the fragmentation mycelium of 40～60%；
5) inoculation: fragmentation mycelium is accessed solid state cultivation bag, at 20～26 DEG C of dark conditions
Lower cultivation 30～cultivation kind can be obtained for 35 days.
Further, described a kind of fragmentation processing method for germination bacterium liquid strain mycelium pellets,
It is characterized in that: comprise the following steps:
1) preparation mother plants: is inoculated into by strain block in test tube PDA culture medium, is placed in 25 DEG C
In environment, lucifuge is cultivated 11 days；
2) shaker flask stand is cultivated: the female kind cultivated in step 1) is cut into the fritter of 3～5mm,
Provoke mycelia body with vaccinating lancet, be inoculated in shaking flask, and make it be suspended on liquid level, every bottle
Access amount is 5～10 pieces, cultivates connecting the shaker flask stand planted in being placed on the calorstat of 27 DEG C
11 days；
3) shaking flask concussion is cultivated: by step 2) in shaker flask stand cultivate and be placed on frequency and be
200r/min, temperature 26 DEG C concussion shaking table on cultivate, make strain shake cultivation 8 in shaking flask
My god, obtain the mycelium pellet of a diameter of about 2cm；
4) fragmentation processes: shaking flask cultured in step 3) is transferred to frequency is 500r/min
Magnetic stirrer on make the stirring of 8 cycles, obtain the fragmentation mycelium of 40～60%；
5) inoculation: fragmentation mycelium is accessed solid state cultivation bag, trains under 26 DEG C of dark conditions
Support and cultivation kind within 34 days, can be obtained.
Wherein said PDA culture medium comprise Rhizoma Solani tuber osi, glucose, peptone, agar and
Water, is placed on PDA culture medium and carries out airtight sterilizing 30min under 1.3～1.7kPa, and will
Its pH value is arranged on 5.6～5.9.
Further, described PDA culture medium comprise Rhizoma Solani tuber osi 200 parts, glucose 30 parts,
Peptone 15 parts, agar 40 and 1000 parts of water.
Described shake flask culture is by peptone 0.2%, glucose 0.1%, potassium dihydrogen phosphate
0.01%, magnesium sulfate 0.01%, Carnis Bovis seu Bubali cream 0.01%, each 0.1 gram of vitamin B1, B2, add
20% potato juice composition, its pH value is arranged on 5.6～5.9.
The beneficial effects of the present invention is: cost of material is low, technique the most easily obtains, and to sprouting
Send out bacterium mycelium pellet fragmentation to process, fungus ball can be made to diminish and in the same size, improve the matter of mycelium pellet
Amount, solves Germination Strain mycelium pellet and is difficult to the technique of fragmentation in shaking flask, make Germination Strain liquid bacteria
Kind access in solid state cultivation kind growth rapidly, growth cycle significantly shorten, improve production efficiency.
Using shaking flask first to stand suspension culture in the present invention, bacterial strain draws nutrition in culture medium, from empty
Absorbing oxygen part in gas, mycelium, after surrounding growth to about 3cm bacterium colony, uses 200 turns
/ point frequency oscillation is cultivated, and obtains the mycelium pellet of diameter about 2cm；Then compact electromagnetic is used
Mycelium pellet is made fragmentation with the frequency of 500 revs/min and is processed by agitator.Obtained fragmentation mycelia
Account for the 60% of total liquid, relative to tradition Germination Strain liquid spawn operating procedure, simple to operate,
Fragmentation is convenient, and the cycle is short, and mycelium pellet density is high, it is little to infect probability, accesses in solid state cultivation kind
Good fluidity, is uniformly dispersed, and sends out bacterium fast.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described:
A kind of fragmentation processing method for germination bacterium liquid strain mycelium pellets, the method includes following step
Rapid:
A kind of fragmentation processing method for germination bacterium liquid strain mycelium pellets, comprises the following steps:
1) preparation mother plants: is inoculated into by strain block in test tube PDA culture medium, is placed in 20-30 DEG C
In environment, lucifuge is cultivated 6-16 days；
2) shaker flask stand is cultivated: by step 1) is cultivated female plant be cut into 3～5mm little
Block, provokes mycelia body with vaccinating lancet, is inoculated in shaking flask, and makes it be suspended on liquid level,
It is 5 10 pieces that every bottle graft enters amount, will connect the shaker flask stand planted at the constant temperature being placed on 20～30 DEG C
Case is cultivated 4-14 days；
3) shaking flask concussion is cultivated: by step 2) in shaker flask stand cultivate and be placed on frequency and be
Cultivate on the concussion shaking table of 200r/min, temperature 20～30 DEG C, make strain shake training in shaking flask
Support 2～12 days, obtain the mycelium pellet of a diameter of about 2cm.
4) fragmentation processes: shaking flask cultured in step 3) is transferred to frequency is 500r/min
Magnetic stirrer on make the stirring of 5～15 cycles, obtain the fragmentation mycelium of 40～60%.
5) inoculation: fragmentation mycelium is accessed solid state cultivation bag, at 20～30 DEG C of dark conditions
Lower cultivation 30～cultivation kind can be obtained for 35 days.
Wherein said PDA culture medium comprises Rhizoma Solani tuber osi 200 parts, glucose 30 parts, albumen
Peptone 15 parts, agar 40 and 1000 parts of water, its pH value is arranged on 5.6～5.9；Described shakes
Bottle culture fluid is by peptone 0.2%, glucose 0.1%, potassium dihydrogen phosphate 0.01%, magnesium sulfate
0.01%, Carnis Bovis seu Bubali cream 0.01%, each 0.1 gram of vitamin B1, B2, add 20% potato juice group
Becoming, its pH value is arranged on 5.6～5.9.
In order to preferably demonstrate that this Germination Strain is by liquid spawn mycelium pellet fragmentation processing method
The effect of the finished product obtained, obtains by changing step 1,2,3 and the temperature of 5 and incubation time
The Germination Strain that the fragmentation arrived processes, then carries out Rhizoma Gastrodiae plantation, by adding up its germination
Judge best cultivation temperature and incubation time.
Experimental group one
Using the temperature range of step 1 every 1 DEG C for gradient as experimental subject, step 1 time
Between and step 2, the temperature and time of 3,5 all take intermediate value, finally statistics Rhizoma Gastrodiae germination rate
Situation is as follows:
From experimental group one can draw step 1 when the temperature of 25 DEG C, the germination rate of Rhizoma Gastrodiae is
High.
Experimental group two
The temperature of fixing step 1 is 25 DEG C, with every 1 day of the time interval of step 1 as ladder
Degree is as experimental subject, and step 2, the temperature and time of 3,5 all take intermediate value, finally add up
The situation of Rhizoma Gastrodiae germination rate is as follows:
Can draw from experimental group two and cultivate 11 days in step 1, the germination rate of Rhizoma Gastrodiae is the highest,
Reach 67.2%.
Experimental group three
The temperature of step 1 is fixed on 25 DEG C, and incubation time is 11 days, with the temperature of step 2
Interval every 1 DEG C of degree is gradient as experimental subject, the time of step 2 and step 3,5
Temperature and time all takes intermediate value, and finally the situation of statistics Rhizoma Gastrodiae germination rate is as follows:
Can draw from upper table, when the cultivation temperature of step 2 is 27 DEG C, the rudiment of seed
Rate is the highest, reaches 67.3%.
Experimental group four
The temperature of step 1 is fixed on 25 DEG C, and incubation time is 11 days, with the temperature of step 2
Degree is set to 27 DEG C, and every 1 day of the time interval of step 2 is that gradient is as experimental subject, step
The rapid temperature and time of 3,5 all takes intermediate value, and finally the situation of statistics Rhizoma Gastrodiae germination rate is as follows:
Can draw from upper table, when the incubation time of step 2 is 11 days, Rhizoma Gastrodiae germination rate
The highest, reach 67.5%.
Experimental group five
The temperature of step 1 is fixed on 25 DEG C, and incubation time is 11 days, the temperature of step 2
Being fixed on 27 DEG C, incubation time is 11 days, with the temperature range of step 3 every 1 DEG C for ladder
Degree all takes intermediate value as experimental subject, the time of step 3 and the temperature and time of step 5,
Finally the situation of statistics Rhizoma Gastrodiae germination rate is as follows:
Can draw from upper table, when the cultivation temperature of step 2 is 26 DEG C, the rudiment of seed
Rate is the highest, reaches 67.4%.
Experimental group six
The temperature of step 1 is fixed on 25 DEG C, and incubation time is 11 days, the temperature of step 2
Being fixed on 27 DEG C, incubation time is 11 days, is fixed on 26 DEG C with the temperature of step 3, with step
Every 1 day of the time interval of rapid 3 is that gradient is as experimental subject, the temperature and time of step 5
All taking intermediate value, finally the situation of statistics Rhizoma Gastrodiae germination rate is as follows:
Can draw from upper table, when the incubation time of step 2 is 8 days, Rhizoma Gastrodiae germination rate is the highest,
Reach 67.4%.
Experimental group seven
The temperature of step 1 is fixed on 25 DEG C, and incubation time is 11 days, the temperature of step 2
Being fixed on 27 DEG C, incubation time is 11 days, and the temperature of step 3 is 26 DEG C, and the time is 8 days,
Take as experimental subject, the time of step 5 using every 1 DEG C of the temperature range of step 5 for gradient
Intermediate value, finally the situation of statistics Rhizoma Gastrodiae germination rate is as follows:
Can draw from upper table, when the cultivation temperature of step 2 is 26 DEG C, the rudiment of seed
Rate is the highest, reaches 67.7%.
Experimental group eight
The temperature of step 1 is fixed on 25 DEG C, and incubation time is 11 days, the temperature of step 2
Being fixed on 27 DEG C, incubation time is 11 days, and the temperature of step 3 is 26 DEG C, and the time is 8 days,
The temperature of step 5 is 26 DEG C, using every 1 day of the time interval of step 5 for gradient as reality
Testing object, finally the situation of statistics Rhizoma Gastrodiae germination rate is as follows:
Can draw from upper table, when the incubation time of step 2 is 35 days, Rhizoma Gastrodiae germination rate is
Height, reaches 67.8%.
To sum up eight embodiments are it follows that the temperature of step 1 is at 25 DEG C, and incubation time is
11 days；The temperature of step 2 is at 27 DEG C, and incubation time is 11 days, and the temperature of step 3 is 26 DEG C,
Time is 8 days；The temperature of step 5 at 26 DEG C, this liquid spawn mycelium pellet during 35 days time
The strain that fragmentation processing method obtains is best.
Claims (1)
1. a fragmentation processing method for germination bacterium liquid strain mycelium pellets, it is characterised in that: comprise the following steps:
1) preparation mother plants: be inoculated into by strain block in test tube PDA culture medium, is placed in lucifuge in 25 DEG C of environment and cultivates 11 days；Described PDA culture medium comprises Rhizoma Solani tuber osi 200 parts, glucose 30 parts, peptone 15 parts, 40 parts of agar and 1000 parts of water；PDA culture medium carries out airtight sterilizing 30min under 1.3～1.7kPa, and its pH value is arranged on 5.6～5.9；
2) shaker flask stand is cultivated: the female kind cultivated in step 1) is cut into the fritter of 3 ~ 5mm, provoke mycelia body with vaccinating lancet, be inoculated in shaking flask, and make it be suspended on liquid level, it is 5 ~ 10 pieces that every bottle graft enters amount, is placed in the calorstat of 27 DEG C cultivation 11 days by connecting the shaker flask stand planted；Described shake flask culture, by peptone 0.2%, glucose 0.1%, potassium dihydrogen phosphate 0.01%, magnesium sulfate 0.01%, Carnis Bovis seu Bubali cream 0.01%, each 0.1 gram of vitamin B1, B2, adds 20% potato juice composition；Its pH value is arranged on 5.6～5.9；
3) shaking flask concussion is cultivated: by step 2) in shaker flask stand cultivate and be placed on frequency and be 200r/min, cultivate on the concussion shaking table of temperature 26 DEG C, make strain shake in shaking flask and cultivate 8 days, obtain the mycelium pellet of a diameter of about 2cm；
4) fragmentation processes: transfers to shaking flask cultured in step 3) make 8 cycle stirrings on the magnetic stirrer that frequency is 500r/min, obtains the fragmentation mycelium of 40 ~ 60%；
5) inoculation: fragmentation mycelium is accessed solid state cultivation bag, cultivates under 26 DEG C of dark conditions and within 34 days, can obtain cultivation kind.
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| JPH06125647A (en) * | 1992-10-19 | 1994-05-10 | Katsunori Karasawa | Production of seed mycelium for mushroom cultivation |
| JP3430215B2 (en) * | 1998-04-30 | 2003-07-28 | 学校法人立命館 | Cultivation method of edible mushrooms using freshwater aquatic plants |
| JP2002051639A (en) * | 2000-08-11 | 2002-02-19 | World:Kk | Method for producing liquid spawn of mushroom and apparatus for inoculation of liquid spawn |
| JP2008079605A (en) * | 2006-09-01 | 2008-04-10 | Hiroichi Fujisawa | Culture liquid for liquid seed fungus, liquid seed fungus and method for producing liquid seed fungus |
| CN102550284B (en) * | 2011-12-31 | 2014-03-12 | 浙江大学 | Edible mushroom culturing method |
| JP5377804B1 (en) * | 2012-04-27 | 2013-12-25 | 共栄社化学株式会社 | Fungal culture method |
| CN103070012A (en) * | 2013-01-10 | 2013-05-01 | 关岭自治县丰硕食用菌农民专业合作社 | Culture method of halimasch liquid bacterial strains |
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