CN106416749A - Method for reducing operational pollution during cultivation process of liquid spawn - Google Patents
Method for reducing operational pollution during cultivation process of liquid spawn Download PDFInfo
- Publication number
- CN106416749A CN106416749A CN201610789477.1A CN201610789477A CN106416749A CN 106416749 A CN106416749 A CN 106416749A CN 201610789477 A CN201610789477 A CN 201610789477A CN 106416749 A CN106416749 A CN 106416749A
- Authority
- CN
- China
- Prior art keywords
- blake bottle
- culture
- bottle
- cultivation process
- tube
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention discloses a method for reducing operational contamination in the cultivation process of liquid spawn. The method comprises the steps of 1, preparing a culture medium, wherein packing the boiled culture medium raw materials into a culture tube; 2, sterilizing, wherein sterilizing the raw materials at a temperature of 120 DEG C under a pressure of 0.12MPa for 30-45min; 3, inoculating, wherein selecting spawn from the culture tube and placing the spawn into a culture bottle under the sterile condition, sealing the bottle opening, meanwhile, inserting a connecting pipe into the the culture bottle opening , sealing the bottle opening, and cultivating at a temperature of 18-20 DEG C for 3-5 days. The cultivation method of cordyceps militaris spawn can be applied to other liquid spawn. The culture bottle is opened once without being opened other times in the cultivation process, and the cultivation is conducted under an aseptic and enclosed environment, so that the problem of operational contamination in the cultivation process is effectively solved.
Description
Technical field
The present invention relates to field of microbial culture technology is and in particular to a kind of reduce the pollution of strain cultivation process operation
Method.
Background technology
Liquid spawn refers to use fluid nutrient medium, in biological fermentation tank, through the liquid shape of deep drainpipe technology production
The edible fungus species of state.
In liquid spawns such as cordyceps myceliums in incubation, it is readily incorporated operational pollution, ultimately cause culture
In cordyceps mycelium contain more miscellaneous bacteria, and existing incubation in, operational pollution uncontrollable it is impossible to be resolved.
And the method that introduces operational pollution is more, staff cannot accomplish to investigate one by one in operation, is difficult to carry
The purity of high final finished bacterial classification.
Based on this, study and develop a kind of method reducing the pollution of strain cultivation process operation of design.
Content of the invention
The technical problem to be solved is introducing dirt during the strain cultivation such as existing cordyceps mycelium
Dye ultimately results in more containing miscellaneous bacteria in finished product, impact product purity.It is an object of the invention to provide a kind of reduce liquid spawn
The method of incubation operational pollution, solves the technical problem of operational pollution in incubation.
The present invention is achieved through the following technical solutions：
A kind of method reducing the pollution of strain cultivation process operation, including following operating procedure：
) prepare culture medium
The culture medium preparing is dispensed in culture tube；
2) sterilization
To the medium sterilization in culture tube 30 45 minutes；
3) inoculate
Aseptically, the two ends of the first connecting tube are inserted into the first blake bottle respectively, in the second blake bottle, from training
Choose bacterial classification block in foster pipe to be placed in the bottle of the first blake bottle by the opening of the first blake bottle, sealing, the first blake bottle is built-in
There is solid medium, the second blake bottle is empty bottle, after the first inoculation in blake bottle, in the first blake bottle, solid medium passes through the
One connecting tube moves in the second blake bottle, cultivates at 18 20 DEG C, cultivates 35 days；
Fluid nutrient medium is loaded, the second blake bottle is connected by the second connecting tube with the 3rd blake bottle in 3rd blake bottle,
When having bacterium colony to occur in the first blake bottle, the second blake bottle, by the fluid nutrient medium in the 3rd blake bottle respectively back-pressure to the
Carry out deep drainpipe in one blake bottle, the second blake bottle, then cultured bacterial classification in the first blake bottle, the second blake bottle is led to
Cross closed conduit back-pressure to cultivate to fermentation tank.
In this incubation, only exist an opening, the bacterial classification block in culture tube is placed in the first blake bottle
In bottle in this operation, in post-treatment operations, the solid medium in the first blake bottle is passed through the second blake bottle, the 3rd
The fluid nutrient medium of blake bottle respectively back-pressure to the first blake bottle, the second blake bottle, operation all processed by sterilization
Connecting tube complete, opening manipulation mistake is not carried out to the first blake bottle, the second blake bottle, the 3rd blake bottle.Meanwhile, incubation
In the container that is applied to, connecting tube all thoroughly process it is ensured that being not introduced into polluting through sterilizing.Therefore, during whole operation, have
Effect avoids the problem that incubation introduces operational pollution, and during fundamentally solving strain cultivation, operational pollution draws
The path entering.
In technical solution of the present invention, the type of the 3rd blake bottle can be also fermentation tank, by postvaccinal first blake bottle,
Two blake bottles can be pressed directly in fermentation tank and carry out bacterium colony large-scale culture, realize the pollution-free big production of whole-process control.Whole behaviour
During work, the container being related to all is processed through sterilization, is not introduced into operational pollution, thus avoiding wherein operational pollution to ask
Topic.
In the technical program, in the first blake bottle, the solid medium of inoculation, also can be introduced in multiple blake bottles,
Inoculated, connecting tube after sterilization for the solid medium in such as the first blake bottle each leads into cultivates to first
Bottle, second culture, the 3rd blake bottle, the 4th blake bottle, to n-th blake bottle, then will be equipped with the training of fluid nutrient medium
Connecting tube after sterilization for the culture medium in foster bottle each leads into above-mentioned already equipped with solid medium first
Blake bottle, second blake bottle, the 3rd blake bottle carry out deep drainpipe to n-th blake bottle, after deep drainpipe, then convey
It is enlarged scale evaluation to fermentation tank.
It is related to deep drainpipe method in foregoing description to be inoculated in round for bacterial classification, so that bacterium solution cell free is suspended
In liquid medium within, and carry out a kind of method of grown cultures, the concrete operation step of the method and its principle are affiliated neck
The common knowledge in domain, no longer describes in detail.
Preferably, described bacterial classification block area is 4mmx5mm 6mmx6mm, in seeded process, the bacterium of selection from culture tube
Plant the area of block, can be adjusted as needed, as long as the requirement of inoculation can be met.
Bacterial classification block area is 4mmx5mm 6mmx6mm, is inoculated in the first blake bottle, and the inoculum concentration of selection is unsuitable excessive
Or too small, need to meet after being inoculated in the first blake bottle, can preferably sprout, promote the growth of bacterial classification.
Preferably, the culture volume chosen in described culture tube is the 1/5-1/4 of every culture pipe volume.
Preferably, described step 2) sterilization temperature be 121 DEG C, pressure be 0.12MPa.To the culture medium in culture tube
Carry out thorough disinfection sterilizing, it is to avoid introduce pollution in incubation.
Preferably, described step 2) in, the sterilized sterilizing of culture tube, culture tube is in 15 45 ° with horizontal line, at room temperature,
Start inoculate, in constant temperature oscillator at 23 24 DEG C quiescent culture.
Preferably, described inoculation is operation on superclean bench.Superclean bench is disappeared using uviol lamp before being operated
Poison sterilizing, avoids introducing the path of pollution from the container using.
Preferably, to superclean bench ultraviolet lamp disinfection 20 30min before described inoculation.
Preferably, described first blake bottle, the second blake bottle, the 3rd blake bottle, the second connecting tube, the second connecting tube, culture
Guan Jun is processed through sterilization.
The present invention compared with prior art, has such advantages as and beneficial effect：
(1) cultural method of the liquid spawn that the present invention adopts, whole incubation realizes no pollution, the pupa that culture obtains
The liquid spawns such as cordyceps mycelia are purity 100% finished product, no miscellaneous bacteria.
(2) method reducing operational pollution during strain cultivation of the present invention, can be applicable to extensive liquid
The culture of body bacterial classification, and in incubation, only have once opening during the culture medium inoculated in culture tube is operated in blake bottle
Mouthful, all there is not the operation of opening in other operations, and the equal specification of other associative operations, it is prevented effectively from and introduce in operation
The problem of operational pollution.
Brief description
Accompanying drawing described herein is used for providing the embodiment of the present invention is further understood, and constitutes of the application
Point, do not constitute the restriction to the embodiment of the present invention.In the accompanying drawings：
Fig. 1 is culture apparatus structural representation in the present invention；
Mark and corresponding parts title in accompanying drawing：
1- first blake bottle, 2- first connecting tube, 3- second blake bottle, 4- the 3rd blake bottle, 5- second connecting tube.
Specific embodiment
For making the object, technical solutions and advantages of the present invention become more apparent, with reference to embodiment and accompanying drawing, to this
Invention is described in further detail, and the exemplary embodiment of the present invention and its explanation are only used for explaining the present invention, do not make
For limitation of the invention.
Embodiment 1：
A kind of method reducing the pollution of strain cultivation process operation, including following operating procedure：
Taking cordyceps mycelium cultural method as a example：
1) prepare culture medium
Raw material in 1L culture medium：Potato 100-200g, glucose 20 30g, agar 20-30g, culture medium raw material is cooked
After be dispensed in culture tube；
The raw material of culture medium can be also raw material in 1L compost：Potato 200g, wheat bran 15g, glucose 25g, agar 15g,
Magnesium sulfate 0.4g, will be cooked for compost after be dispensed in culture tube, culture tube for specification be 15x20cm test tube.
2) sterilization
Sterilize 30 45 minutes under 120 DEG C of temperature, pressure 0.12MPa；
3) inoculate
As shown in figure 1, aseptically, the two ends of the first connecting tube 2 are inserted into the first blake bottle 1, second respectively
In blake bottle 3, choose bacterial classification block from culture tube and be placed in the bottle of the first blake bottle 1 by the opening of the first blake bottle 1, envelope
Mouthful, built with solid medium, the second blake bottle 3 is empty bottle to the first blake bottle 1, after inoculation in the first blake bottle 1, the first culture
In bottle 1, solid medium is passed through in the second blake bottle 3 by the first connecting tube 2, cultivates at 18 20 DEG C, and culture 35 days is
Can；
Fluid nutrient medium is loaded, the second blake bottle 3 and the 3rd blake bottle 4 pass through the second connecting tube 5 even in 3rd blake bottle 3
Connect, when having bacterium colony to occur in the first blake bottle 1, the second blake bottle 3, the fluid nutrient medium in the 3rd blake bottle 4 is anti-respectively
It is depressed into the first blake bottle 1, carries out deep drainpipe in the second blake bottle 3, then by culture in the first blake bottle 1, the second blake bottle 3
Good bacterial classification is cultivated to fermentation tank by closed conduit back-pressure；
Wherein, described bacterial classification block area is 4mmx5mm 6mmx6mm.
Wherein, described step 2) in sterilization temperature be 121 DEG C, pressure be 0.12MPa.
Wherein, the culture volume chosen in described culture tube is the 1/5-1/4 of every culture pipe volume.
Wherein, described step 2) in, after the sterilized sterilizing of culture tube, culture tube and horizontal line are in 15 45 °, tiltedly
Face, at room temperature, start inoculate, then in constant temperature oscillator at 23 24 DEG C quiescent culture.
Wherein, described inoculation is operation on superclean bench.
Wherein, described first blake bottle 1, the second blake bottle 3, the 3rd blake bottle 4, the second connecting tube 2, the second connecting tube 5,
Culture tube is all processed through sterilization.
Wherein, to superclean bench ultraviolet lamp disinfection 25 30min before described inoculation.
Wherein, described first blake bottle 1, the second blake bottle 3, the 3rd blake bottle 4, the second connecting tube 2, the second connecting tube 5,
Culture tube is all through disinfecting.
The cordyceps mycelium of culture is adopted conventional method to detect, such as the morphological feature according to cordyceps mycelium is aobvious
Whether micro- Microscopic observation, contain other impurities in finished product.
Except determining whether the product that final culture obtains has in addition to impurity using microscope according to the morphologic observation of bacterial classification, also
Can be detected by the physico-chemical process of the bacterial classification according to concrete culture, be determined its purity, if containing other impurities.
Using said method testing result：Do not contain and bacterial classification in the bacterial classification that cultural method described in the present embodiment 1 obtains
Unrelated miscellaneous bacteria.
By step 3) enter the use pupa polypide mycelium carrying out inoculated and cultured acquisition in fermentation tank, enter with using conventional method
The pupa polypide mycelium that row culture obtains, carries out pollution rate, goes out the isoparametric comparison of mycelia rate.
Wherein existing conventional method cultivates specific operation process：After preparing culture medium, sterilization, and it is dispensed into culture
Guan Zhong, cooling.Then choose the hypha body of a sesame size using inoculation hook from culture tube, be inoculated in blake bottle, connect
When entering to multiple blake bottle, all take the mode choosing hypha body from culture tube, inoculated, be required in this process
Open the opening of blake bottle, carry out inoculated and cultured.
The pupa polypide mycelial detection parameter that conventional practices are obtained with method of operating culture described in the present embodiment is such as
Shown in table 1 below,
| Bacterial classification parameter | Classical culture protocols | Cultural method of the present invention |
| Go out mycelia time (h) | 20 | 10 |
| Inoculum concentration | 30—40g | 20—25g |
| Go out mycelia temperature (DEG C) | 16—20 | 16—20 |
| Cultural hypha time (h) | 30 | 15 |
| Cultural hypha temperature (DEG C) | 20—25 | 23—24 |
| Pollution rate (%) | 10 | 0 |
Understand, the cordyceps mycelium being obtained using the cultural method described in the present embodiment is trained with traditional conventional practices
Support the finished product cordyceps mycelium obtaining to compare, using no miscellaneous bacteria in the present embodiment mycelium of preparing of culture, whole train
Foster process no pollution.
Above-described specific embodiment, has been carried out to the purpose of the present invention, technical scheme and beneficial effect further
Describe in detail, be should be understood that the specific embodiment that the foregoing is only the present invention, be not intended to limit the present invention
Protection domain, all any modification, equivalent substitution and improvement within the spirit and principles in the present invention, done etc., all should comprise
Within protection scope of the present invention.
Claims (8)
1. a kind of method reducing the pollution of strain cultivation process operation is it is characterised in that include following operating procedure：
1）Prepare culture medium
The culture medium preparing is dispensed in culture tube；
2）Sterilization
To the medium sterilization in culture tube 30 45 minutes；
3）Inoculation
Aseptically, by the first connecting tube（2）Two ends be inserted into the first blake bottle respectively（1）, the second blake bottle（3）
In, choose bacterial classification block from culture tube and pass through the first blake bottle（1）Opening be placed in the first blake bottle（1）Bottle in, sealing, the
One blake bottle（1）Built with solid medium, the second blake bottle（3）For empty bottle, the first blake bottle（1）After interior inoculation, the first training
Foster bottle（1）Interior solid medium passes through the first connecting tube（2）It is passed through the second blake bottle（3）In, cultivate at 18 20 DEG C, culture
35 days；
3rd blake bottle（3）Middle loading fluid nutrient medium, the second blake bottle（3）With the 3rd blake bottle（4）By the second connecting tube
（5）Connect, treat the first blake bottle（1）, the second blake bottle（3）When inside having bacterium colony to occur, by the 3rd blake bottle（4）In liquid training
Foster base difference back-pressure is to the first blake bottle（1）, the second blake bottle（3）Inside carry out deep drainpipe, then by the first blake bottle（1）、
Second blake bottle（3）Interior cultured bacterial classification is cultivated to fermentation tank by closed conduit back-pressure.
2. according to claim 1 a kind of reduce strain cultivation process operation pollution method it is characterised in that：Institute
Stating bacterial classification block area is 4mmx5mm 6mmx6mm.
3. according to claim 2 a kind of reduce strain cultivation process operation pollution method it is characterised in that：Institute
State step 2）The temperature of middle sterilization is 121 DEG C, pressure is 0.12MPa.
4. according to claim 3 a kind of reduce strain cultivation process operation pollution method it is characterised in that：Institute
State the 1/5-1/4 that the culture volume chosen in culture tube is every culture pipe volume.
5. according to claim 1 a kind of reduce strain cultivation process operation pollution method it is characterised in that：Institute
State step 2）In, after the sterilized sterilizing of culture tube, culture tube is in 15 45 ° with horizontal line, at room temperature, starts to inoculate, then
In constant temperature oscillator at 23 24 DEG C quiescent culture.
6. according to claim 1 a kind of reduce strain cultivation process operation pollution method it is characterised in that：Institute
Stating inoculation is operation on superclean bench.
7. according to claim 6 a kind of reduce strain cultivation process operation pollution method it is characterised in that：Institute
State before inoculating to superclean bench ultraviolet lamp disinfection 25 30min.
8. according to claim 7 a kind of reduce strain cultivation process operation pollution method it is characterised in that：Institute
State the first blake bottle（1）, the second blake bottle（3）, the 3rd blake bottle（4）, the second connecting tube（2）, the second connecting tube（5）, culture
Guan Jun is processed through sterilization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610789477.1A CN106416749B (en) | 2016-08-31 | 2016-08-31 | Method for reducing operation pollution in liquid strain culture process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610789477.1A CN106416749B (en) | 2016-08-31 | 2016-08-31 | Method for reducing operation pollution in liquid strain culture process |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106416749A true CN106416749A (en) | 2017-02-22 |
| CN106416749B CN106416749B (en) | 2020-02-07 |
Family
ID=58164321
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610789477.1A Active CN106416749B (en) | 2016-08-31 | 2016-08-31 | Method for reducing operation pollution in liquid strain culture process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106416749B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109089740A (en) * | 2018-08-06 | 2018-12-28 | 杨铁金 | A kind of method and device producing reduction liquid spawn |
| CN110628590A (en) * | 2018-06-22 | 2019-12-31 | 深圳市前海小藻科技有限公司 | Fermentation method for reducing risk of bacterial contamination |
| CN115926955A (en) * | 2022-12-30 | 2023-04-07 | 浙江安各洛生物技术有限公司 | Auxiliary mixing device for sterilization of multi-component culture medium |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1134780A (en) * | 1995-10-31 | 1996-11-06 | 远安县森源食用菌公司 | Process for mechanized prodn. of granular strains |
| JP2003250336A (en) * | 2002-02-15 | 2003-09-09 | Jae Mo Sung | Method for culturing liquid spawn for mass production of carpophore of vegetable wasp and plant work and apparatus for the same |
| CN1579124A (en) * | 2004-05-14 | 2005-02-16 | 活泼 | Method and apparatus for producing and inoculating liquid bacterial spawn in oxygenating mode |
| CN202406587U (en) * | 2012-01-07 | 2012-09-05 | 李好勇 | Edible fungus liquid strain culture device |
| CN102835254A (en) * | 2012-09-26 | 2012-12-26 | 烟台龙宇灵芝生物开发有限公司 | Ganoderma lucidum mycelia powder production process |
-
2016
- 2016-08-31 CN CN201610789477.1A patent/CN106416749B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1134780A (en) * | 1995-10-31 | 1996-11-06 | 远安县森源食用菌公司 | Process for mechanized prodn. of granular strains |
| JP2003250336A (en) * | 2002-02-15 | 2003-09-09 | Jae Mo Sung | Method for culturing liquid spawn for mass production of carpophore of vegetable wasp and plant work and apparatus for the same |
| CN1579124A (en) * | 2004-05-14 | 2005-02-16 | 活泼 | Method and apparatus for producing and inoculating liquid bacterial spawn in oxygenating mode |
| CN202406587U (en) * | 2012-01-07 | 2012-09-05 | 李好勇 | Edible fungus liquid strain culture device |
| CN102835254A (en) * | 2012-09-26 | 2012-12-26 | 烟台龙宇灵芝生物开发有限公司 | Ganoderma lucidum mycelia powder production process |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628590A (en) * | 2018-06-22 | 2019-12-31 | 深圳市前海小藻科技有限公司 | Fermentation method for reducing risk of bacterial contamination |
| CN109089740A (en) * | 2018-08-06 | 2018-12-28 | 杨铁金 | A kind of method and device producing reduction liquid spawn |
| CN115926955A (en) * | 2022-12-30 | 2023-04-07 | 浙江安各洛生物技术有限公司 | Auxiliary mixing device for sterilization of multi-component culture medium |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106416749B (en) | 2020-02-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20160369226A1 (en) | Solid-state biological reaction device and method for preparing filamentous organism spores by using the same | |
| CN106010975B (en) | Method for producing halimasch strains | |
| CN106416749A (en) | Method for reducing operational pollution during cultivation process of liquid spawn | |
| CN109609387A (en) | A kind of fast culture process of Bursaphelenchus xylophilus inner parasitic epiphyte Esteya vermicola | |
| CN101353618A (en) | Production method of edible fungus liquid spawn and fermentation plant thereof | |
| CN104557211B (en) | Mushroom liquefaction special bacteria culture medium and corresponding cultivation | |
| CN106676018A (en) | Agaricus bisporus strain breeding method applicable to standardized plant | |
| CN105602857B (en) | A kind of optimization method that wild cicada fungus liquid spawn is manually cultivated | |
| CN205408770U (en) | Hothouse plants cultivation device | |
| CN107118003A (en) | A kind of breeding method of Cordyceps militaris fermented bacterium and application | |
| CN104541983B (en) | Seafood mushroom liquefaction special bacteria culture medium and corresponding cultivation | |
| CN104988063B (en) | A kind of Tissue Culture Dish for preventing cell contamination and its application | |
| CN104557315A (en) | Strain culture medium for pleurotus geesteranus liquefaction and corresponding culture medium | |
| CN104620852A (en) | Mushroom liquefied strain cultivation method | |
| CN107815437A (en) | A kind of method of sweet potato black rot pathogen rapid, high volume production spore | |
| CN107580974A (en) | A kind of more batches of fruiting devices of flat mushroom and the fruiting method using the device | |
| CN103518534A (en) | Edible fungus liquid strain storing and inoculating integrated machine | |
| CN208586299U (en) | A kind of bacteria culture bottle convenient for sampling | |
| CN110484439A (en) | A method of the device and screening biocontrol microorganisms of screening biocontrol microorganisms | |
| CN106554934A (en) | The banana blight bacteria for polluting efficiently is avoided conidial to prepare cultural method | |
| CN104557209B (en) | Pleurotus eryngii liquefaction special bacteria culture medium and corresponding cultivation | |
| CN104745522B (en) | A kind of method for inducing silk spore fungi spore | |
| CN106867957A (en) | A kind of ustilaginoidea virens thin-walled of Pollution protection is conidial to prepare cultural method | |
| CN106754621A (en) | The efficient botrytis cinerea for preventing from polluting is conidial to prepare cultural method | |
| CN203537923U (en) | Edible mushroom liquid strain storing and inoculating all-in-one machine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |