CN107513501A - The long spore bacterium Spawn incubation of disk and technical process - Google Patents
The long spore bacterium Spawn incubation of disk and technical process Download PDFInfo
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- CN107513501A CN107513501A CN201710888557.7A CN201710888557A CN107513501A CN 107513501 A CN107513501 A CN 107513501A CN 201710888557 A CN201710888557 A CN 201710888557A CN 107513501 A CN107513501 A CN 107513501A
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Abstract
The invention discloses the long spore bacterium Spawn incubation of disk and technical process, by the purchase from strain, the preparation of culture medium, the preparation of seed, fermenting and producing finished product, sterile mill bacterium, strain counting, a set of flow of sterile filling, obtains the long spore bacteria liquid of disk；The present invention improves the vigor of strain, making liquid spawn activity can ensure, together with bacterium and culture medium that strain can also grow in transportation, and the postoperative infection sowed is easy, and the effect for killing Semen Cuscutae is more preferable by the way that the long spore bacterium of disk is made into liquid.
Description
Technical field
The present invention relates to the long spore bacterium Spawn incubation of disk and technical process, and parasitics weeds Semen Cuscutae is killed suitable for production
Fungal spore.
Background technology
The long spore bacterium of disk, depositary institution：China is commonly biological DSMZ；Address：Chaoyang District, Beijing City North Star west
The institute 3 of road 1 Institute of Microorganism, Academia Sinica (CGMCC)；Numbering：CGMCC NO.3.1620；Latin title：
Gloeosporium sp.
A kind of long fungi of spore bacterium of disk, can infect Soybean Dodder makes its death of falling ill, and then prevents and treats this weeds
Purpose, it is that Institute of Plant Protection of academy of agricultural sciences of Shandong Province of China province finds and applied first.Soybean Dodder is endanger soybean a kind of pernicious
Parasitic Weeds, are widely distributed in Chinese main soybean producing region, general harm can the 10-20 of the underproduction percent, serious particle in blocks
Without receipts.It is all pulvis that No.1 is protected in Shandong in the past, and easily failure, the quantity of strain can not reach the concentration of needs, efficiency of infection
It is low, just it is unfavorable for thoroughly killing Semen Cuscutae.
The content of the invention
For problem present in background technology, the invention provides the long spore bacterium Spawn incubation of a discharge plate and technical process,
It is intended to improve the vigor of strain, making liquid spawn activity can ensure, together with bacterium and culture medium that strain is in transportation
In can also grow, the postoperative infection sowed is easy, and the effect for killing Semen Cuscutae is more preferable.
To achieve the above object, the present invention provides following technical scheme：The long spore bacterium Spawn incubation of disk and technical process, including
Following steps,
S10：Obtain the long spore bacterium of disk.
S20：The preparation of culture medium, including the preparation of PDA solid mediums, the preparation of YPD culture mediums and final resuspension
The preparation of the culture medium of strain is wherein：The process of the preparation of PDA solid mediums is as follows：Peeled potatoes are weighed, potato is cut into small
Block is put into pot, adds water 1000ml, is heated to seething with excitement on the heaters, 20-30 minutes is maintained, with two layers of gauze while hot in measuring cup
Upper filtering, filter residue are abandoned or adopted.Filtrate keeps the skin wet to 1000ml.Filtrate after keeping the skin wet is put into pot, adds glucose
20g, broken agar is done in advance and adds 15~20g, is then placed on asbestos gauge, small fire heating, and be stirred continuously with glass rod, treat fine jade
After fat is completely dissolved, moisture is supplemented, in 121 DEG C of autoclaving 15min.Packing is into 18mm × 180mm test tube, Mei Geshi
Pipe 10ml, tilt bevel.
The preparation of YPD culture mediums：Compound method：By 10g yeast extracts, 20g peptones, it is dissolved in 900ml water, 121 DEG C
Autoclaving, add the glucose of 100ml 20%.
Finally the preparation process of the culture medium of resuspension strain is：20g potatoes, 500ml is added after being blended using agitator and is steamed
In distilled water, then add 20g white sugar, 200g wheat brans and 200g and grind broken activated carbon, be finally settled to 1000ml, 121 degree, go out
Bacterium 15min
S30：The preparation of seed, including the preparation of the preparation of first order seed and the preparation of secondary seed, wherein first order seed
Process is that separation strain is seeded in PDA culture medium inclined-plane, cultivates 3-5 days under the conditions of 28 DEG C, is protected afterwards under the conditions of 4-10 DEG C
Deposit；
The preparation process of secondary seed is the YPD culture mediums that a certain amount of bacterium of picking is seeded to 10ml from PDA culture medium
In, at 28 DEG C and using rotating speed per minute as 220 under conditions of carry out shaking bacterium two days, then 10ml bacterium solution is all seeded to
In 1L YPD culture mediums, bacterium is shaken to saturation.
S40：Fermenting and producing finished product：According to 1% inoculum concentration, secondary seed is inoculated into 100L YPD culture mediums,
At 28 DEG C and using rotating speed per minute as 220rpm under conditions of shake bacterium two days to three days, until saturation.
S50：Sterile mill bacterium：Sterile mill bacterium refers under aseptic condition, is rotated with 2500rpm per minute rotating speed and centrifuges 10
Minute, thalline is then collected, bacterium ball is ground in the mortar of sterilizing, breaks up thalline, weight in the culture medium of strain is finally being resuspended
The broken strain of outstanding grinding.
S60：Strain counts：By gradient dilution colony counting method, the spore strain quantity in fermentation end products is calculated, most
Ensure 10,000,000,000/ml eventually.
S70：Sterile filling：By bacterium and culture medium together, dispensed according to every bottle of 500ml amount, into final products.
Compared with prior art, the beneficial effects of the invention are as follows：The present invention improves bacterium by the way that the long spore bacterium of disk is made into liquid
The vigor of kind, making liquid spawn activity can ensure, together with bacterium and culture medium that strain can also give birth in transportation
Long, the postoperative infection sowed is easy, and the effect for killing Semen Cuscutae is more preferable.
Embodiment
With reference to the embodiment in the present invention, the technical scheme in the embodiment of the present invention is clearly and completely retouched
State, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.Based on the present invention
In embodiment, the every other implementation that those of ordinary skill in the art are obtained under the premise of creative work is not made
Example, belongs to the scope of protection of the invention.
Embodiment
S10：Obtain the long spore bacterium of disk.
S20：The preparation of culture medium, including the preparation of PDA solid mediums, the preparation of YPD culture mediums and final resuspension
The preparation of the culture medium of strain is wherein：The process of the preparation of PDA solid mediums is as follows：Peeled potatoes are weighed, potato is cut into small
Block is put into pot, adds water 1000ml, is heated to seething with excitement on the heaters, 20-30 minutes is maintained, with two layers of gauze while hot in measuring cup
Upper filtering, filter residue are abandoned or adopted.Filtrate keeps the skin wet to 1000ml.Filtrate after keeping the skin wet is put into pot, adds glucose
20g, broken agar is done in advance and adds 15~20g, is then placed on asbestos gauge, small fire heating, and be stirred continuously with glass rod, treat fine jade
After fat is completely dissolved, moisture is supplemented, in 121 DEG C of autoclaving 15min.Packing is into 18mm × 180mm test tube, Mei Geshi
Pipe 10ml, tilt bevel.
The preparation of YPD culture mediums：Compound method：By 10g yeast extracts, 20g peptones, it is dissolved in 900ml water, 121 DEG C
Autoclaving, add the glucose of 100ml 20%.
Finally the preparation process of the culture medium of resuspension strain is：20g potatoes, 500ml is added after being blended using agitator and is steamed
In distilled water, then add 20g white sugar, 200g wheat brans and 200g and grind broken activated carbon, be finally settled to 1000ml, 121 degree, go out
Bacterium 15min.This culture medium advantage：Nutrition can be provided for the strain in transport, ensure spawn activity.Be adsorbed with strain wheat bran and
Activated carbon can be adsorbed on Semen Cuscutae blade and vines after splashing, difficult for drop-off, make contact of more strains with Semen Cuscutae, promoted
Enter infection.
S30：The preparation of seed, including the preparation of the preparation of first order seed and the preparation of secondary seed, wherein first order seed
Process is that separation strain is seeded in PDA culture medium inclined-plane, cultivates 3-5 days under the conditions of 28 DEG C, is protected afterwards under the conditions of 4-10 DEG C
Deposit；
The preparation process of secondary seed is the YPD culture mediums that a certain amount of bacterium of picking is seeded to 10ml from PDA culture medium
In, at 28 DEG C and using rotating speed per minute as 220 under conditions of carry out shaking bacterium two days, then 10ml bacterium solution is all seeded to
In 1L YPD culture mediums, bacterium is shaken to saturation.
S40：Fermenting and producing finished product：According to 1% inoculum concentration, secondary seed is inoculated into 100L YPD culture mediums,
At 28 DEG C and using rotating speed per minute as 220rpm under conditions of shake bacterium two days to three days, until saturation.
S50：Sterile mill bacterium：Sterile mill bacterium refers under aseptic condition, is rotated with 2500rpm per minute rotating speed and centrifuges 10
Minute, thalline is then collected, bacterium ball is ground in the mortar of sterilizing, breaks up thalline.Weight in the culture medium of strain is finally being resuspended
The broken strain of outstanding grinding.
S60：Strain counts：By gradient dilution colony counting method, the spore strain quantity in fermentation end products is calculated, most
Ensure 10,000,000,000/ml eventually.
S70：Sterile filling：By bacterium and culture medium together, dispensed according to every bottle of 500ml amount, into final products.
Shelf life of products simulated experiment：
25 DEG C of laboratory room temperature：Experimental result such as table 1：
25 DEG C of room temperatures are put, and product viable count in 28 days gradually increases, and viable count tails off within the 56th day.Illustrate the guarantor of room temperature
The matter phase is 56 days or so.
The placement of 30 DEG C of laboratory：Experimental result such as table 2：
30 DEG C of placements, viable count has increase in product 1 week, then gradually decreases, viable count maintains an equal level with most starting within 28 days.Say
Bright 30 DEG C of placements shelf life of products is shorter, between 2-3 weeks.
4 DEG C of laboratory condition is placed：Experimental result such as table 3：
4 DEG C of placements, product viable count are held essentially constant in 8 time-of-weeks.Illustrate that 4 DEG C of placement shelf life of products are longer,
Therefore product is relatively adapted to 4 DEG C of transports and 4 DEG C of preservations.
Application method：
Directly poured into after opening in 50 jin of water, the inside Archon is pulverized, it is standby after mixing.First manually break apart by chopping Semen Cuscutae stem
It is climing, then directly it is sprinkled upon in the dusk or cloudy day on Semen Cuscutae.One mu of 1 bottle of use, it can be taken the circumstances into consideration to increase usage amount according to Semen Cuscutae.Often
Week is using once.
Based on above-mentioned, present invention has the advantage that：The present invention improves strain by the way that the long spore bacterium of disk is made into liquid
Vigor, making liquid spawn activity can ensure, together with bacterium and culture medium that strain can also grow in transportation, broadcast
The postoperative infection spread is easy, and the effect for killing Semen Cuscutae is more preferable.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (10)
1. the long spore bacterium Spawn incubation of disk and technical process, it is characterised in that：Comprise the steps of,
S10：Obtain strain；
S20：The preparation of culture medium；
S30：The preparation of seed；
S40：Fermenting and producing finished product；
S50：Sterile mill bacterium；
S60：Strain counts：
S70：Sterile filling.
2. the long spore bacterium Spawn incubation of disk and technical process according to claim 1, it is characterised in that：The S20：Culture medium
Prepare, specifically include the system of the preparation of PDA solid mediums, the preparation of YPD culture mediums and the final culture medium that strain is resuspended
It is standby.
3. the long spore bacterium Spawn incubation of disk and technical process according to claim 2, it is characterised in that：The PDA solid cultures
The preparation process of base is specially：Peeled potatoes are weighed, potato, which is cut into small pieces, to be put into pot, adds water 1000ml, is added on the heaters
Heat maintains 20-30 minutes, filtered while hot on measuring cup with two layers of gauze, filter residue is abandoned or adopted to seething with excitement.Filtrate keep the skin wet to
1000ml.Filtrate after keeping the skin wet is put into pot, adds glucose 20g, broken agar is done in advance and adds 15~20g,
Then it is placed on asbestos gauge, small fire heating, and is stirred continuously with glass rod, after agar is completely dissolved, moisture is supplemented, 121
DEG C autoclaving 15min.Packing is into 18mm × 180mm test tube, each test tube 10ml, tilts bevel.
4. the long spore bacterium Spawn incubation of disk and technical process according to claim 2, it is characterised in that：The YPD culture mediums
Prepare, specific compound method：By 10g yeast extracts, 20g peptones, it is dissolved in 900ml water, 121 DEG C of autoclavings, added
The glucose of 100ml 20% of filter sterilization.
5. the long spore bacterium Spawn incubation of disk and technical process according to claim 2, it is characterised in that：The final resuspension strain
The preparation process of culture medium be：20g potatoes, added after being blended using agitator in 500ml distilled water, it is white then to add 20g
Sugar, 200g wheat brans and 200g grind broken activated carbon, are finally settled to 1000ml, 121 degree, sterilize 15min.
6. the long spore bacterium Spawn incubation of disk and technical process according to claim 1, it is characterised in that：The system of the S30 seeds
It is standby, including the preparation of first order seed and the preparation of secondary seed.
7. the long spore bacterium Spawn incubation of disk and technical process according to claim 6, it is characterised in that：The system of the first order seed
Standby process is that separation strain is seeded in PDA culture medium inclined-plane, cultivates 3-5 days under the conditions of 28 DEG C, afterwards in 4-10 DEG C of temperature
Under the conditions of preserve.
8. the long spore bacterium Spawn incubation of disk and technical process according to claim 6, it is characterised in that：The system of the secondary seed
Standby process is that a certain amount of bacterium of picking is seeded in 10ml YPD culture mediums from PDA culture medium, at 28 DEG C and with per minute
Rotating speed is carried out shaking bacterium two days under conditions of being 220rpm, and then 10ml bacterium solution is all seeded in 1L YPD culture mediums, shaken
Bacterium is to saturation.
9. the long spore bacterium Spawn incubation of disk and technical process according to claim 1, it is characterised in that：The S40：Fermenting and producing
The detailed process of finished product is：According to 1% inoculum concentration, secondary seed is inoculated into 100L YPD culture mediums, at 28 DEG C simultaneously
And using rotating speed per minute as 220rpm under conditions of shake bacterium two days to three days, until saturation.
10. the long spore bacterium Spawn incubation of disk and technical process according to claim 1, it is characterised in that：The S50：Sterile mill
Bacterium refers under aseptic condition, is rotated and centrifuged 10 minutes with 2500rpm per minute rotating speed, thalline is then collected, in sterilizing
Bacterium ball is ground in mortar, breaks up thalline, the strain that resuspension grinding is broken in the culture medium of strain is finally finally being resuspended.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112063537A (en) * | 2020-09-24 | 2020-12-11 | 瑞楚生物科技(江苏)有限公司 | Method capable of maintaining spore viability in discothrix bacterium liquid |
| CN112075424A (en) * | 2020-09-24 | 2020-12-15 | 瑞楚生物科技(江苏)有限公司 | Solution formula capable of promoting adherence of onchocercaria and infecting dodder leaves |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1651568A (en) * | 2004-02-03 | 2005-08-10 | 李勇 | Edible fungus liquid culture submerged fermentation technology |
| CN101914455A (en) * | 2010-08-13 | 2010-12-15 | 易丛琴 | Process for producing gloeosporium bacteria |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112063537A (en) * | 2020-09-24 | 2020-12-11 | 瑞楚生物科技(江苏)有限公司 | Method capable of maintaining spore viability in discothrix bacterium liquid |
| CN112075424A (en) * | 2020-09-24 | 2020-12-15 | 瑞楚生物科技(江苏)有限公司 | Solution formula capable of promoting adherence of onchocercaria and infecting dodder leaves |
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Application publication date: 20171226 |
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