CN103980027B - Bacterium culture medium of a kind of Volvariella brumalis and preparation method thereof and utilize its spawn culture method - Google Patents
Bacterium culture medium of a kind of Volvariella brumalis and preparation method thereof and utilize its spawn culture method Download PDFInfo
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- CN103980027B CN103980027B CN201410234837.2A CN201410234837A CN103980027B CN 103980027 B CN103980027 B CN 103980027B CN 201410234837 A CN201410234837 A CN 201410234837A CN 103980027 B CN103980027 B CN 103980027B
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Abstract
Bacterium culture medium of Volvariella brumalis and preparation method thereof and utilize its a spawn culture method, bacterium culture medium, corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%; Corn cob; Preparation method: corn cob adds boiling water makes it all flood raw material, soaks 0.8h ~ 1.2h, filters and to anhydrate point, add the rice bran of above-mentioned formula, calcium superphosphate, sucrose, stir, formation substratum; Check and adjust the moisture of described substratum, water content 50% ~ 55%; Bottling, sterilizing.Spawn culture method: strain separating, cultivates and transfers: again by same procedure, then carries out the switching of more than 2 times.The present invention adopts tissue isolation, and without Tube propagation, the directly corn cob+rice bran+calcium superphosphate+glucose feed culture medium culturing of access through preparing, by switching purifying, have successfully been obtained Volvariella brumalis Pure cultured spawn.The present invention has separation and substratum is easy to make, without test tube separation and Culture, shorten the spawn culture cycle.
Description
Technical field
The present invention relates to agricultural biological technical field, especially relate to bacterium culture medium of a kind of Volvariella brumalis and preparation method thereof and utilize its spawn culture method.
Background technology
Volvariella brumalis (VolvariellabrumalisHesp.), is commonly called as low temperature straw mushroom, paddy stake bacterium, shredded chicken bacterium.Wild Volvariella brumalis only Guizhou has distribution report, April in annual November to next year, plants in wheat, rape, broad bean, pea, potatoes and other crops ground after being grown on harvesting paddy rice.This bacterium is the unique low temperature modification kind found at present during straw mushroom belongs to, filled up existing straw mushroom belong to be in megathermal defect.This bacterium mushroom meat delicacy, delicious flavour, nutritious, there is very large value of exploiting and utilizing.But, about the strain separating of Volvariella brumalis and culture technique are but blank of the prior art, seriously govern the development of this industry.
Summary of the invention
The object of the invention is to bacterium culture medium designing a kind of Volvariella brumalis and preparation method thereof and utilize its spawn culture method.
To achieve these goals, the technical solution used in the present invention is as follows:
A bacterium culture medium for Volvariella brumalis, percent mass hundred: corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%;
Require:
Corn cob: without going mouldy, water content≤12%, pulverized particles≤5mm;
Rice bran: without going mouldy, water content≤13%;
Calcium superphosphate: containing effective P
2o
514% ~ 20%, wherein 80% ~ 95% is water-soluble;
Glucose: chemical pure;
The moisture of described substratum, water content reaches: 50% ~ 55%.
A preparation method for the bacterium culture medium of Volvariella brumalis, comprises step as follows:
The first step, prepares raw material: according to mass percent corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%;
Require:
Corn cob: without going mouldy, water content≤12%, pulverized particles≤5mm;
Rice bran: without going mouldy, water content≤13%;
Calcium superphosphate: containing effective P
2o
514% ~ 20%, wherein 80% ~ 95% is water-soluble;
Glucose: chemical pure;
Second step, substratum makes: the corn cob taking above-mentioned formula, in container, adds boiling water and makes it all flood raw material, soaks 0.8h ~ 1.2h, filters and to anhydrate point, add the rice bran of above-mentioned formula, calcium superphosphate, sucrose, stir, formation substratum; Check and adjust the moisture of described substratum, water content 50% ~ 55%.
Also comprise step as follows:
3rd step, bottling: the substratum prepared in second step is loaded in seed bottle, loading amount is to seed bottle shoulder, and compress the substratum of bottle internal surface, tightness is: seed bottle be inverted, substratum does not fall; Then, with outside clear water detergent bottle, then put in bottle with gauze, clean bottleneck shoulder, dry bottleneck moisture, beyond the Great Wall tampon, embrace bottleneck with kraft paper, tie up;
4th step, sterilizing: described seed bottle puts into high-pressure steam sterilizing pan, at 1.5kg/cm
2under pressure, keep 1h; Described seed bottle good for sterilizing is moved on in Bechtop, allows its naturally cooling, form finished product seed bottle.
Utilize a spawn culture method for the Volvariella brumalis of the bacterium culture medium of described Volvariella brumalis, comprise step as follows:
The first step, strain separating:
1) get the Volvariella brumalis sporophore of the fresh non-standard-sized sheet umbrella that the same day gathers, cut off stem base portion earth, wipe the foreign material on cap with aseptic wet absorbent cotton, load the plastics bag through sterilizing, be put in plastics casing and also take back laboratory in time;
2) with aseptic plastic bag outer wall described in 75% alcohol swab wiping, move on to Bechtop, under aseptic condition, scratch described aseptic plastic bag one osculum with scalper, cap top is just exposed, then to prune cap epidermis with scalper, expose the white tissues in cap, picking white tissues is about 5mm size as tissue block, in the middle part of described tissue block access seed bottle substratum, makes itself and substratum have better contact.
Second step, cultivate and transfer:
1) described finished product seed bottle is placed in 18 DEG C ~ 22 DEG C thermostat containers to cultivate.Observe the sprouting state of described tissue block of access every day: if described tissue block and pollution-free around, single bacterium colony, for normally, strain separating success; Otherwise for polluting, detect in time;
2) to above-mentioned normal sprouting, form the described seed bottle of single bacterium colony, the full bottle of Length discrepancy, when mycelia starts material feeding, when substratum is formed a loop diameter is 2.5cm ~ 3.5cm bacterium colony, transfers in time, make it purifying; The substratum that switching uses is identical with the substratum in the first step, and forwarding method is as follows:
S1, under Bechtop aseptic condition, with the edge mycelium of inoculating needle picking colony, is transferred on another seed bottle substratum, is placed in 18 DEG C ~ 22 DEG C thermostat containers and cultivates; Observe access thalli growth situation every day, mycelia material feeding, growth is neat, dense, healthy and strong, pollution-free, for normally, enters next step;
S2, then by same procedure, then carry out the switching of more than 2 times, be formed as Volvariella brumalis bacterial classification.
Bechtop (cleanbench) in the present invention refers to and to design to adapt to the demands of field to local working areas cleanliness factor such as modernization industry, opto-electronics, bio-pharmaceuticals and scientific research and testing.Bechtop principle is in specific space, room air is through prefilter initial filter, plenum chamber is pressed into by Small Centrifugal Fan, again through high-efficiency air filter cascade filtration, have certain for uniform sectional wind velocity from the clean gas flow of high-efficiency air filter outlet air surface blowout, the air that workspace is original can be got rid of, dust granules and biological particles are taken away, to form the Working environment of aseptic high-cleanness.The advantage of super clean bench is easy to operate freely, pleasant, working efficiency, and free time is short, starts shooting and can operate for more than 10 minutes, substantially can use at any time.In factorial praluction, inoculation workload is very large, and when needing often to work muchly, super clean bench is very desirable equipment.Super clean bench makes blowing power by three-phase machine, power about 145 ~ 260W, air " super purifier " after-blow by superimposed group of the microcellular foam lamella by spy is sent out, form the ultra-purify air laminar flow of continuously dust-free sterile, i.e. so-called " effective special air ", it eliminates the dust, fungus and bacterium spore etc. that are greater than 0.3 μm.The flow velocity of ultra-purify air is 24 ~ 30m/min, this pollution enough preventing neighbouring air from may harass and cause, and such flow velocity also can not hinder and adopt spirit lamp or Bunsen burner to sterilize to the calcination of apparatus etc.Staff just operates under such aseptic condition, keeps sterilizable material not contaminated in transfer is inoculated.But just in case operation midway runs into power failure, the material be exposed in non-filtrated air is just difficult to pollution of escaping by luck.At this moment should power cut-off rapidly, and mark is made on bottle, the material in interior as place's multiplicative stage, is then no longer used as propagation after and proceeds to root culture.As produced material for general, extremely enrich also can discard.As taken root in place, then plant use after can waiting until.
The present invention includes concrete steps as follows:
One, strain separating
(1) material
1, bacterium source
Volvariella brumalis sporophore, on April 6th, 2010 picks up from Longli County, Guizhou Province.
2, substratum and preparation
(1) culture medium prescription
Corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%.PH value nature.
(2) prepare
Corn cob: without going mouldy, water content≤12%, pulverized particles≤5mm.
Rice bran: without going mouldy, water content≤13%.
Calcium superphosphate: containing effective P
2o
514% ~ 20% (wherein 80% ~ 95% is water-soluble).
Glucose: chemical pure.
Substratum makes: take corn cob in container by above-mentioned formula, add boiling water, make it all flood raw material, soak about 1h, elimination excessive moisture, adds rice bran, calcium superphosphate, sucrose by formula, stir, then check the moisture of substratum, water content 50% ~ 55%.
Bottling: the substratum prepared is loaded in seed bottle, loading amount is to seed bottle shoulder, and compress the substratum of bottle internal surface, tightness is: seed bottle be inverted, substratum does not fall, with outside clear water detergent bottle, then put in bottle with gauze, clean bottleneck shoulder, dry bottleneck moisture, tampon beyond the Great Wall, embraces bottleneck with kraft paper, ties up.
Sterilizing: the seed bottle installed is put into high-pressure steam sterilizing pan, under 1.5kg/cm2 pressure, keeps 1h.The seed bottle that sterilizing is good is moved on in Bechtop, allows its naturally cooling.
(2) separation method
Get the Volvariella brumalis sporophore of the fresh non-standard-sized sheet umbrella that the same day gathers, cut off stem base portion earth, wipe the foreign material on cap with aseptic wet absorbent cotton, load the plastics bag through sterilizing, be put in plastics casing and take back laboratory in time.
With 75% alcohol swab wiping plastics bag outer wall, under moving on to Bechtop aseptic condition, plastics bag one osculum is scratched with scalper, cap top is just exposed, to prune cap epidermis with scalper again, expose the white tissues in cap, picking white tissues one fritter (about about 5mm), in the middle part of access seed bottle substratum, itself and substratum is made to have better contact.
Two, spawn culture
(1) observation is cultivated
Vaccinated above-mentioned seed bottle is placed in 18 DEG C ~ 22 DEG C thermostat containers to cultivate.Observe the tissue block sprouting state of access every day, tissue block and pollution-free around, single bacterium colony, is normal, illustrates that strain separating is successful.Otherwise for polluting, detect in time.
(2) purifying
To above-mentioned normal sprouting, form the seed bottle of single bacterium colony, can not wait and cover with bottle.Namely, when mycelia starts material feeding, when substratum is formed circle (diameter is a 2.5cm ~ 3.5cm) bacterium colony, purifying of transferring in time, the substratum that switching uses is identical with above-mentioned, at least will carry out 3 purifying.
Forwarding method: under Bechtop aseptic condition, with the edge mycelium of inoculating needle picking colony, is transferred on another seed bottle substratum, is placed in 18 DEG C ~ 22 DEG C thermostat containers and cultivates.Observe access thalli growth situation every day, mycelia material feeding, growth is neat, dense, healthy and strong, pollution-free, is normal.Again by same procedure, carry out second time, third time transfers cultivation, is Volvariella brumalis bacterial classification.
Beneficial effect of the present invention can be summarized as follows:
1, the present invention adopts tissue isolation, and without Tube propagation, the directly corn cob+rice bran+calcium superphosphate+glucose feed culture medium culturing of access through preparing, by switching purifying, have successfully been obtained Volvariella brumalis Pure cultured spawn.
2, the present invention have separation and substratum is easy to make, without test tube separation and Culture, shorten the spawn culture cycle.
Embodiment
In order to make technical problem solved by the invention, technical scheme and beneficial effect clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
A bacterium culture medium for Volvariella brumalis, percent mass hundred: corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%; Require:
Corn cob: without going mouldy, water content≤12%, pulverized particles≤5mm;
Rice bran: without going mouldy, water content≤13%;
Calcium superphosphate: containing effective P
2o
514%, wherein 80% is water-soluble;
Glucose: chemical pure;
The moisture of described substratum, water content reaches: 50%.
A preparation method for the bacterium culture medium of Volvariella brumalis, comprises step as follows:
The first step, prepares raw material: according to mass percent corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%; Require:
Corn cob: without going mouldy, water content≤12%, pulverized particles≤5mm;
Rice bran: without going mouldy, water content≤13%;
Calcium superphosphate: containing effective P
2o
514%, wherein 80% is water-soluble;
Glucose: chemical pure;
Second step, substratum makes: the corn cob taking above-mentioned formula, in container, adds boiling water and makes it all flood raw material, soaks 0.8h, filters and to anhydrate point, add the rice bran of above-mentioned formula, calcium superphosphate, sucrose, stir, formation substratum; Check and adjust the moisture of described substratum, water content 50%.
3rd step, bottling: the substratum prepared in second step is loaded in seed bottle, loading amount is to seed bottle shoulder, and compress the substratum of bottle internal surface, tightness is: seed bottle be inverted, substratum does not fall; Then, with outside clear water detergent bottle, then put in bottle with gauze, clean bottleneck shoulder, dry bottleneck moisture, beyond the Great Wall tampon, embrace bottleneck with kraft paper, tie up;
4th step, sterilizing: described seed bottle puts into high-pressure steam sterilizing pan, at 1.5kg/cm
2under pressure, keep 1h; Described seed bottle good for sterilizing is moved on in Bechtop, allows its naturally cooling, form finished product seed bottle.
Utilize a spawn culture method for the Volvariella brumalis of the bacterium culture medium of described Volvariella brumalis, comprise step as follows:
The first step, strain separating:
1) get the Volvariella brumalis sporophore of the fresh non-standard-sized sheet umbrella that the same day gathers, in the present embodiment, Volvariella brumalis sporophore picks up from Longli County, Guizhou Province on April 6th, 2010.
Cut off stem base portion earth, wipe the foreign material on cap with aseptic wet absorbent cotton, load the plastics bag through sterilizing, be put in plastics casing and also take back laboratory in time;
2) with aseptic plastic bag outer wall described in 75% alcohol swab wiping, move on to Bechtop, under aseptic condition, scratch described aseptic plastic bag one osculum with scalper, cap top is just exposed, then to prune cap epidermis with scalper, expose the white tissues in cap, picking white tissues is about 5mm size as tissue block, in the middle part of described tissue block access seed bottle substratum, makes itself and substratum have better contact.
Second step, cultivate and transfer:
1) described finished product seed bottle is placed in 18 DEG C of thermostat containers to cultivate.Observe the sprouting state of described tissue block of access every day: if described tissue block and pollution-free around, single bacterium colony, for normally, strain separating success; Otherwise for polluting, detect in time;
2) to above-mentioned normal sprouting, form the described seed bottle of single bacterium colony, the full bottle of Length discrepancy, when mycelia starts material feeding, when substratum is formed a loop diameter is 2.5cm ~ 3.5cm bacterium colony, transfers in time, make it purifying; The substratum that switching uses is identical with the substratum in the first step, and forwarding method is as follows:
S1, under Bechtop aseptic condition, with the edge mycelium of inoculating needle picking colony, is transferred on another seed bottle substratum, is placed in 18 DEG C of thermostat containers and cultivates; Observe access thalli growth situation every day, mycelia material feeding, growth is neat, dense, healthy and strong, pollution-free, for normally, enters next step;
S2, then by same procedure, then carry out the switching of more than 2 times, be formed as Volvariella brumalis bacterial classification.
The present invention adopts tissue isolation, and without Tube propagation, the directly corn cob+rice bran+calcium superphosphate+glucose feed culture medium culturing of access through preparing, by switching purifying, have successfully been obtained Volvariella brumalis Pure cultured spawn.The present invention has separation and substratum is easy to make, without test tube separation and Culture, shorten the spawn culture cycle---in the present embodiment, 45 days used times, and be usually separated through test tube in prior art, purifying, cultivate, obtain female kind, then original seed of transferring, cultivar, need 60-75 days, this technology 45-60 days, can shorten 15 days spawn culture cycles.
The qualification of Volvariella brumalis bacterial classification:
Colony characteristics: white, mycelia fine hair shape, sprawl growth, colony edge is neat, and edge and central part solid colour are homogeneous.
Mycelium feature: visual inspection white mycelium, under microscope, mycelia is transparent, branch, have every, primary mycelium is very thin, and secondary mycelium is more sturdy.
Prove through fruiting experiment, the entity for Volvariella brumalis bacterial classification obtained in the present embodiment.
Embodiment 2
A bacterium culture medium for Volvariella brumalis, percent mass hundred: corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%; Require:
Corn cob: without going mouldy, water content≤12%, pulverized particles≤5mm;
Rice bran: without going mouldy, water content≤13%;
Calcium superphosphate: containing effective P
2o
520%, wherein 95% is water-soluble;
Glucose: chemical pure;
The moisture of described substratum, water content reaches: 55%.
A preparation method for the bacterium culture medium of Volvariella brumalis, comprises step as follows:
The first step, prepares raw material: according to mass percent corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%; Require:
Corn cob: without going mouldy, water content≤12%, pulverized particles≤5mm;
Rice bran: without going mouldy, water content≤13%;
Calcium superphosphate: containing effective P
2o
520%, wherein 95% is water-soluble;
Glucose: chemical pure;
Second step, substratum makes: the corn cob taking above-mentioned formula, in container, adds boiling water and makes it all flood raw material, soaks 1.2h, filters and to anhydrate point, add the rice bran of above-mentioned formula, calcium superphosphate, sucrose, stir, formation substratum; Check and adjust the moisture of described substratum, water content 55%.
3rd step, bottling: the substratum prepared in second step is loaded in seed bottle, loading amount is to seed bottle shoulder, and compress the substratum of bottle internal surface, tightness is: seed bottle be inverted, substratum does not fall; Then, with outside clear water detergent bottle, then put in bottle with gauze, clean bottleneck shoulder, dry bottleneck moisture, beyond the Great Wall tampon, embrace bottleneck with kraft paper, tie up;
4th step, sterilizing: described seed bottle puts into high-pressure steam sterilizing pan, at 1.5kg/cm
2under pressure, keep 1h; Described seed bottle good for sterilizing is moved on in Bechtop, allows its naturally cooling, form finished product seed bottle.
Utilize a spawn culture method for the Volvariella brumalis of the bacterium culture medium of described Volvariella brumalis, comprise step as follows:
The first step, strain separating:
1) get the Volvariella brumalis sporophore of the fresh non-standard-sized sheet umbrella that the same day gathers, in the present embodiment, Volvariella brumalis sporophore picks up from Longli County, Guizhou Province on April 6th, 2010.
Cut off stem base portion earth, wipe the foreign material on cap with aseptic wet absorbent cotton, load the plastics bag through sterilizing, be put in plastics casing and also take back laboratory in time;
2) with aseptic plastic bag outer wall described in 75% alcohol swab wiping, move on to Bechtop, under aseptic condition, scratch described aseptic plastic bag one osculum with scalper, cap top is just exposed, then to prune cap epidermis with scalper, expose the white tissues in cap, picking white tissues is about 5mm size as tissue block, in the middle part of described tissue block access seed bottle substratum, makes itself and substratum have better contact.
Second step, cultivate and transfer:
1) described finished product seed bottle is placed in 22 DEG C of thermostat containers to cultivate.Observe the sprouting state of described tissue block of access every day: if described tissue block and pollution-free around, single bacterium colony, for normally, strain separating success; Otherwise for polluting, detect in time;
2) to above-mentioned normal sprouting, form the described seed bottle of single bacterium colony, the full bottle of Length discrepancy, when mycelia starts material feeding, when substratum is formed a loop diameter is 2.5cm ~ 3.5cm bacterium colony, transfers in time, make it purifying; The substratum that switching uses is identical with the substratum in the first step, and forwarding method is as follows:
S1, under Bechtop aseptic condition, with the edge mycelium of inoculating needle picking colony, is transferred on another seed bottle substratum, is placed in 22 DEG C of thermostat containers and cultivates; Observe access thalli growth situation every day, mycelia material feeding, growth is neat, dense, healthy and strong, pollution-free, for normally, enters next step;
S2, then by same procedure, then carry out the switching of more than 2 times, be formed as Volvariella brumalis bacterial classification.
The present invention adopts tissue isolation, and without Tube propagation, the directly corn cob+rice bran+calcium superphosphate+glucose feed culture medium culturing of access through preparing, by switching purifying, have successfully been obtained Volvariella brumalis Pure cultured spawn.The present invention has separation and substratum is easy to make, without test tube separation and Culture, shorten the spawn culture cycle.In the present embodiment, 68 days used times, and be usually separated through test tube in prior art, purifying, cultivate, obtain female kind then original seed of transferring, cultivar, need 60-75 days, this technology 45-60 days, 15 days spawn culture cycles can be shortened.
The qualification of Volvariella brumalis bacterial classification:
Colony characteristics: white, mycelia fine hair shape, sprawl growth, colony edge is neat, and edge and central part solid colour are homogeneous.
Mycelium feature: visual inspection white mycelium, under microscope, mycelia is transparent, branch, have every, primary mycelium is very thin, and secondary mycelium is more sturdy.
Prove through fruiting experiment, the entity for Volvariella brumalis bacterial classification obtained in the present embodiment.
Embodiment 3
A bacterium culture medium for Volvariella brumalis, percent mass hundred: corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%; Require:
Corn cob: without going mouldy, water content≤12%, pulverized particles≤5mm;
Rice bran: without going mouldy, water content≤13%;
Calcium superphosphate: containing effective P
2o
518%, wherein 87.5% is water-soluble;
Glucose: chemical pure;
The moisture of described substratum, water content reaches: 52.5%.
A preparation method for the bacterium culture medium of Volvariella brumalis, comprises step as follows:
The first step, prepares raw material: according to mass percent corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%; Require:
Corn cob: without going mouldy, water content≤12%, pulverized particles≤5mm;
Rice bran: without going mouldy, water content≤13%;
Calcium superphosphate: containing effective P
2o
518%, wherein 87.5% is water-soluble;
Glucose: chemical pure;
Second step, substratum makes: the corn cob taking above-mentioned formula, in container, adds boiling water and makes it all flood raw material, soaks 1h, filters and to anhydrate point, add the rice bran of above-mentioned formula, calcium superphosphate, sucrose, stir, formation substratum; Check and adjust the moisture of described substratum, water content 52.5%.
3rd step, bottling: the substratum prepared in second step is loaded in seed bottle, loading amount is to seed bottle shoulder, and compress the substratum of bottle internal surface, tightness is: seed bottle be inverted, substratum does not fall; Then, with outside clear water detergent bottle, then put in bottle with gauze, clean bottleneck shoulder, dry bottleneck moisture, beyond the Great Wall tampon, embrace bottleneck with kraft paper, tie up;
4th step, sterilizing: described seed bottle puts into high-pressure steam sterilizing pan, at 1.5kg/cm
2under pressure, keep 1h; Described seed bottle good for sterilizing is moved on in Bechtop, allows its naturally cooling, form finished product seed bottle.
Utilize a spawn culture method for the Volvariella brumalis of the bacterium culture medium of described Volvariella brumalis, comprise step as follows:
The first step, strain separating:
1) get the Volvariella brumalis sporophore of the fresh non-standard-sized sheet umbrella that the same day gathers, in the present embodiment, Volvariella brumalis sporophore picks up from Longli County, Guizhou Province on April 6th, 2010.
Cut off stem base portion earth, wipe the foreign material on cap with aseptic wet absorbent cotton, load the plastics bag through sterilizing, be put in plastics casing and also take back laboratory in time;
2) with aseptic plastic bag outer wall described in 75% alcohol swab wiping, move on to Bechtop, under aseptic condition, scratch described aseptic plastic bag one osculum with scalper, cap top is just exposed, then to prune cap epidermis with scalper, expose the white tissues in cap, picking white tissues is about 5mm size as tissue block, in the middle part of described tissue block access seed bottle substratum, makes itself and substratum have better contact.
Second step, cultivate and transfer:
1) described finished product seed bottle is placed in 20 DEG C of thermostat containers to cultivate.Observe the sprouting state of described tissue block of access every day: if described tissue block and pollution-free around, single bacterium colony, for normally, strain separating success; Otherwise for polluting, detect in time;
2) to above-mentioned normal sprouting, form the described seed bottle of single bacterium colony, the full bottle of Length discrepancy, when mycelia starts material feeding, when substratum is formed a loop diameter is 2.5cm ~ 3.5cm bacterium colony, transfers in time, make it purifying; The substratum that switching uses is identical with the substratum in the first step, and forwarding method is as follows:
S1, under Bechtop aseptic condition, with the edge mycelium of inoculating needle picking colony, is transferred on another seed bottle substratum, is placed in 20 DEG C of thermostat containers and cultivates; Observe access thalli growth situation every day, mycelia material feeding, growth is neat, dense, healthy and strong, pollution-free, for normally, enters next step;
S2, then by same procedure, then carry out the switching of more than 2 times, be formed as Volvariella brumalis bacterial classification.
The present invention adopts tissue isolation, and without Tube propagation, the directly corn cob+rice bran+calcium superphosphate+glucose feed culture medium culturing of access through preparing, by switching purifying, have successfully been obtained Volvariella brumalis Pure cultured spawn.The present invention has separation and substratum is easy to make, without test tube separation and Culture, shorten the spawn culture cycle.In the present embodiment, 75 days used times, and be usually separated through test tube in prior art, purifying, cultivate, obtain female kind then original seed of transferring, cultivar, need 60-75 days, this technology 45-60 days, 15 days spawn culture cycles can be shortened.
The qualification of Volvariella brumalis bacterial classification:
Colony characteristics: white, mycelia fine hair shape, sprawl growth, colony edge is neat, and edge and central part solid colour are homogeneous.
Mycelium feature: visual inspection white mycelium, under microscope, mycelia is transparent, branch, have every, primary mycelium is very thin, and secondary mycelium is more sturdy.
Prove through fruiting experiment, the entity for Volvariella brumalis bacterial classification obtained in the present embodiment.
Visible, be usually separated through test tube in prior art, purifying, cultivate, obtain female kind, then original seed of transferring, cultivar, need 60-75 days, this technology 45-60 days, 15 days spawn culture cycles can be shortened.
The present invention is described in detail in preferred embodiment above by concrete; but those skilled in the art should be understood that; the present invention is not limited to the above embodiment; within the spirit and principles in the present invention all; any amendment of doing, equivalent replacement etc., all should be included within protection scope of the present invention.
Claims (4)
1. a bacterium culture medium for Volvariella brumalis, is characterized in that:
Mass percent: corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%;
Require:
Corn cob: without going mouldy, water content≤12%, pulverized particles≤5mm;
Rice bran: without going mouldy, water content≤13%;
Calcium superphosphate: containing effective P
2o
514% ~ 20%, wherein 80% ~ 95% is water-soluble;
Glucose: chemical pure;
The moisture of described substratum, water content reaches: 50% ~ 55%.
2. a preparation method for the bacterium culture medium of Volvariella brumalis according to claim 1, is characterized in that, comprises step as follows:
The first step, prepares raw material: according to mass percent corn cob 80%, rice bran 18%, calcium superphosphate 1%, glucose 1%;
Require:
Corn cob: without going mouldy, water content≤12%, pulverized particles≤5mm;
Rice bran: without going mouldy, water content≤13%;
Calcium superphosphate: containing effective P
2o
514% ~ 20%, wherein 80% ~ 95% is water-soluble;
Glucose: chemical pure;
Second step, substratum makes: the corn cob taking above-mentioned formula, in container, adds boiling water and makes it all flood raw material, soaks 0.8h ~ 1.2h, filters and to anhydrate point, add the rice bran of above-mentioned formula, calcium superphosphate, sucrose, stir, formation substratum; Check and adjust the moisture of described substratum, water content 50% ~ 55%.
3. the preparation method of the bacterium culture medium of Volvariella brumalis according to claim 2, is characterized in that: also comprise step as follows:
3rd step, bottling: the substratum prepared in second step is loaded in seed bottle, loading amount is to seed bottle shoulder, and compress the substratum of bottle internal surface, tightness is: seed bottle be inverted, substratum does not fall; Then, with outside clear water detergent bottle, then put in bottle with gauze, clean bottleneck shoulder, dry bottleneck moisture, beyond the Great Wall tampon, encase bottleneck with kraft paper, tie up;
4th step, sterilizing: described seed bottle puts into high-pressure steam sterilizing pan, at 1.5kg/cm
2under pressure, keep 1h; Described seed bottle good for sterilizing is moved on in Bechtop, allows its naturally cooling, form finished product seed bottle.
4. utilize a spawn culture method for the Volvariella brumalis of the bacterium culture medium of the Volvariella brumalis described in claim 1, it is characterized in that, comprise step as follows:
The first step, strain separating:
1) get the Volvariella brumalis sporophore of the fresh non-standard-sized sheet umbrella that the same day gathers, cut off stem base portion earth, wipe the foreign material on cap with aseptic wet absorbent cotton, load the plastics bag through sterilizing, be put in plastics casing and also take back laboratory in time;
2) with aseptic plastic bag outer wall described in 75% alcohol swab wiping, move on to Bechtop, under aseptic condition, scratch described aseptic plastic bag one osculum with scalper, cap top is just exposed, then to prune cap epidermis with scalper, expose the white tissues in cap, picking white tissues is about 5mm size as tissue block, in the middle part of described tissue block access seed bottle substratum, makes itself and substratum have better contact;
Second step, cultivate and transfer:
1) described finished product seed bottle is placed in 18 DEG C ~ 22 DEG C thermostat containers to cultivate, observes the sprouting state of the described tissue block of access every day: if described tissue block and pollution-free around, single bacterium colony, for normally, strain separating success; Otherwise for polluting, detect in time;
2) to above-mentioned normal sprouting, form the described seed bottle of single bacterium colony, the full bottle of Length discrepancy, when mycelia starts material feeding, when substratum is formed a loop diameter is 2.5cm ~ 3.5cm bacterium colony, transfers in time, make it purifying; The substratum that switching uses is identical with the substratum in the first step, and forwarding method is as follows:
S1, under Bechtop aseptic condition, with the edge mycelium of inoculating needle picking colony, is transferred on another seed bottle substratum, is placed in 18 DEG C ~ 22 DEG C thermostat containers and cultivates; Observe access thalli growth situation every day, mycelia material feeding, growth is neat, dense, healthy and strong, pollution-free, for normally, enters next step;
S2, then press and S1 same procedure, then carry out the switching of more than 2 times, be formed as Volvariella brumalis bacterial classification.
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| CN108401786B (en) * | 2018-05-07 | 2019-12-31 | 贵州省山地资源研究所 | Dictyophora rubrovalvata cultivation raw material and Dictyophora rubrovalvata cultivation method |
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| JP2000287537A (en) * | 1999-04-02 | 2000-10-17 | Nobuo Inoue | Culture of spawn of volvariella speciosa sing. var. volvacea and production of mushroom bed for cultivating volvariella speciosa sing. var. volvacea |
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| JP2000287537A (en) * | 1999-04-02 | 2000-10-17 | Nobuo Inoue | Culture of spawn of volvariella speciosa sing. var. volvacea and production of mushroom bed for cultivating volvariella speciosa sing. var. volvacea |
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| CN102210233A (en) * | 2011-03-31 | 2011-10-12 | 上海高榕食品有限公司 | Production method of calcium-enriched edible fungi and formula of culture medium |
| CN102617240A (en) * | 2012-04-10 | 2012-08-01 | 绥化学院 | Novel edible fungus culture medium and preparation method thereof |
| CN103250552A (en) * | 2012-05-03 | 2013-08-21 | 广东海洋大学 | High-yield volvariella volvacea strain produced by banana leaves |
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