CN111363671B - Intestinal anaerobic microorganism culture bottle and preparation method thereof - Google Patents
Intestinal anaerobic microorganism culture bottle and preparation method thereof Download PDFInfo
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- CN111363671B CN111363671B CN202010121809.5A CN202010121809A CN111363671B CN 111363671 B CN111363671 B CN 111363671B CN 202010121809 A CN202010121809 A CN 202010121809A CN 111363671 B CN111363671 B CN 111363671B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/20—Material Coatings
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/08—Flask, bottle or test tube
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/38—Caps; Covers; Plugs; Pouring means
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses an intestinal anaerobic microorganism culture bottle and a preparation method thereof, wherein the culture bottle comprises a culture bottle which is sealed and packaged and a culture solution which is filled in the culture bottle; sequentially filling a basic culture solution obtained by mixing a basic culture medium solution, an L-cysteine salt aqueous solution and a resazurin aqueous solution in proportion into a culture bottle, and sealing and packaging; autoclaving the sealed flask; finally, 1640 culture medium liquid, BI serum, vitamin K and hemin are mixed according to a proportion to obtain an active additive mixed solution, and the active additive mixed solution is injected into a culture bottle which is sterilized under high pressure and cooled to room temperature to finish the preparation; the anaerobic microorganism culture bottle adopts a culture bottle body and a packaging piece, all components for preparing culture solution adopt commercial products, raw materials are easy to obtain, the threshold and the cost for anaerobic culture are reduced, the operations of anaerobic culture, storage and transportation are simpler, the preparation method is simple and feasible, the requirement of mass production is met, and the use requirements of hospitals and scientific research units are met.
Description
Technical Field
The invention relates to the technical field of microbial cultivation, in particular to an intestinal anaerobic microbial culture bottle and a preparation method thereof.
Background
At present, most microorganisms in the environment cannot be cultured, and the microorganisms which can be cultured account for about 1% of the total amount, so that the microorganisms cannot be cultured, and besides the low abundance, the culture conditions including temperature, pH, oxygen utilization rate, nutrition and the like are not optimized. Technically, the temperature and the pH can be adjusted, and the general difficulty is in anaerobic treatment and nutrition; thus, the emphasis of anaerobic microorganisms in the in vitro culture process is on the simulation of anaerobic environments and their nutritional environments.
At present, the anaerobic culture technology commonly used in research uses an anaerobic box, an anaerobic bag, an anaerobic tank, complex operation and higher cost, and can culture the solid culture medium in the anaerobic box, the anaerobic bag and the anaerobic tank after coating, but when the solid culture medium is used for proliferation, the open space is inconvenient to operate, and researchers solve the problem of anaerobic environment before, but the anaerobic bottle is insufficient in nutrition and the microorganism is not good in growth. The anaerobic culture bottle developed by the invention is added with active nutrient substances on the basis of deoxidization, ensures the growth of microorganisms, is convenient to operate and low in cost, and is suitable for being implemented by technicians for vast microorganism research.
In addition, the preservation of anaerobic bacteria has been a major problem, if the bacteria are frozen in a normal way, the bacteria must be contacted with oxygen unless the bacteria are operated in an anaerobic tank, but the anaerobic tank is troublesome to prepare an anaerobic environment, the operation with rubber gloves is inconvenient, and additionally, anaerobic frozen stock solution needs to be prepared. In addition, the transportation of anaerobic bacteria is currently carried out by preparing dry powder of bacteria under specific conditions, then filling the dry powder into ampoule bottles for deoxidization and sealing, and reactivating the ampoule bottles after reaching the destination. For long-distance transportation, the treatment mode of the dry powder is very suitable, but for short-distance transportation, the operation cost performance is not high, and the transportation of anaerobic bacteria can only depend on the transfer of anaerobic bags and anaerobic tanks from one laboratory to another laboratory, so that the method is inconvenient.
Disclosure of Invention
The invention aims to provide an anaerobic microorganism culture bottle which is convenient for culturing, storing and transporting intestinal anaerobic microorganisms.
Another object of the present invention is to provide a method for preparing the intestinal anaerobic microorganism culture flask
For this purpose, the technical scheme of the invention is as follows:
an intestinal anaerobic microorganism culture bottle comprises a culture bottle which is sealed and packaged and a culture solution filled in the culture bottle; the culture solution comprises 100 parts of basic culture medium aqueous solution, 4 parts of 2.5wt.% L-cysteine salt aqueous solution and 0.1 part of 0.1wt.% resazurin aqueous solution which are mixed in parts by volume, and an active additive mixed solution which is 4% of the basic culture solution; the active additive mixed solution is prepared by mixing 10 parts of 1640 culture medium solution, 1 part of BI serum, 1 part of vitamin K and 1 part of hemin in volume parts.
The intestinal anaerobic microorganism culture bottle is sealed and completely meets the anaerobic environment, and the culture solution is used for providing proper nutrition environment for anaerobic microorganisms; the L-cysteine in the culture solution has stronger reducibility, and the L-cysteine and oxygen in the culture bottle sealed and packaged are subjected to oxidation reaction under the action of high temperature to generate L-cystine, so that the L-cystine is deoxidized and amino acid meeting the requirement of microorganism culture is further provided; the resazurin is an oxygen indicator for indicating the oxygen content in the bottle, and if oxygen exists, the resazurin is pink or light blue, so that whether the internal state of the culture bottle meets the requirement of anaerobic culture environment or not is convenient to observe; the mixed liquid of the active additives supplements nutrition of the basic culture liquid, and experiments prove that the active additives can meet the growth of most anaerobic microorganisms.
Preferably, the filling volume of the culture solution is 90% -95% of the volume of the culture bottle.
Preferably, the basal medium is a powdered RAM basal medium, CDC basal medium, RCM basal medium, or MRS basal medium.
Preferably, the culture bottle consists of a penicillin bottle, a rubber plug inserted at the bottleneck of the penicillin bottle, a hollow aluminum cover sealed at the ends of the rubber plug and the bottleneck of the penicillin bottle, and a top cover covered on the hollow aluminum cover. The penicillin bottle is made of borosilicate glass, has a rigid structure, is resistant to high temperature and high pressure, is used for storing a liquid culture medium, and is suitable for sterilization operation and observation of bacterial growth states; the penicillin bottle is sealed by adopting the rubber plug, so that the outflow of culture medium in the bottle can be effectively prevented, the air tightness is better, the operation such as the passage and culture of anaerobic bacteria can be conveniently carried out by inserting the syringe, and the anaerobic environment in the bottle is ensured; the hollow aluminum cover has the function of reinforcing the rubber plug on one hand, and prevents the rubber plug from being ejected out under high temperature and high pressure, and on the other hand, the hollow structure can facilitate the subsequent anaerobic bacteria culturing operation.
Preferably, the penicillin bottle adopts a borosilicate glass bottle, and in the process of culturing anaerobic microorganisms, the total microorganism in the bottle can be measured by measuring the turbidity of the liquid in the bottle without opening the cover; the top cover adopts polytetrafluoroethylene top cover, is difficult for yielding under high temperature high pressure, effectively prevents the bottleneck pollution during the blake bottle stores and transportation.
The preparation method of the anaerobic microorganism culture bottle comprises the following steps:
s1, dissolving a powdery basic culture medium in sterile water according to a proportion to prepare a basic culture medium solution; dissolving powdered resazurin in sterile water to obtain 0.1wt.% resazurin aqueous solution; dissolving powdery L-cysteine salt in sterile water to obtain 2.5wt.% of L-cysteine salt aqueous solution;
s2, mixing a basic culture medium solution, an L-cysteine salt aqueous solution and a resazurin aqueous solution according to a volume ratio of 100:4:0.1 to obtain a basic culture solution, respectively filling the basic culture solution into a plurality of culture bottles, and sealing and packaging the filled culture bottles; wherein, the filling volume is 90% -95% of the volume of the culture flask;
s3, autoclaving the filled and sealed culture bottles obtained in the step S2;
s4, mixing 1640 culture medium liquid, BI serum, vitamin K and hemin according to the volume ratio of 10:1:1:1 to prepare an active additive mixed solution, and injecting the active additive mixed solution into the culture flask through an injector in a sterile operation table after the temperature of the culture flask subjected to high-pressure sterilization in the step S4 is reduced to room temperature; the volume ratio of the injection amount of the active additive mixed solution to the basic culture solution is 1:25.
Compared with the prior art, the anaerobic microorganism culture bottle is assembled from commercial products by adopting the culture bottle, is convenient to purchase and use, and the culture solution filled in the culture bottle is also prepared from commercial products, and the preparation and preparation methods are simple and feasible; according to the anaerobic microorganism culture bottle, the anaerobic microorganism culture bottle is assembled to obtain the anaerobic microorganism culture bottle, so that the anaerobic microorganism culture bottle is reduced in threshold and cost, operation of anaerobic culture, storage and transportation is simpler, the requirement of mass production is met, the use requirements of clinical laboratory and related scientific research institutions of hospitals can be met, work of scientific research personnel is greatly facilitated, and the anaerobic microorganism culture bottle has good commercial popularization prospect.
Drawings
FIG. 1 is a schematic diagram of the structure of an intestinal anaerobic microorganism culture flask according to the present invention;
fig. 2 is a statistical chart of the differences in MCF value production of the test results according to example 2 of the present invention.
Detailed Description
The invention will now be further described with reference to the accompanying drawings and specific examples, which are in no way limiting.
Example 1
The method for preparing a plurality of intestinal anaerobic microorganism culture bottles comprises the following steps:
a preparation method of an intestinal anaerobic microorganism culture bottle comprises the following steps:
s1, dissolving a powdery RAM basic culture medium (Haibo) in sterile water according to a ratio of 71:1000 to prepare a basic culture medium solution; dissolving powdered resazurin in sterile water to obtain 0.1wt.% resazurin aqueous solution; dissolving powdery L-cysteine salt in sterile water to obtain 2.5wt.% of L-cysteine salt aqueous solution;
s2, respectively filling mixed liquid obtained by mixing 100mL of basic culture medium solution, 4mL of L-cysteine salt aqueous solution and 0.1mL of resazurin aqueous solution into 10 penicillin bottles with the specification of 10mL, and sealing and packaging the penicillin bottles by inserting rubber plugs into the bottle mouths, packaging hollow aluminum covers at the rubber plugs and the ends of the penicillin bottle mouths and covering polytetrafluoroethylene top covers on the hollow aluminum covers; wherein, the filling volume of the mixed solution is 10mL, and the bottleneck part of the culture bottle is left empty at the moment;
s3, placing the 10 culture bottles subjected to filling and sealing packaging in the step S2 in a pressure cooker with the model of Zealway, setting the temperature to 121 ℃, and autoclaving the 10 culture bottles for 20min;
s4, mixing 10mL of 1640 culture medium solution, 1mL of BI serum, 1mL of vitamin K and 1mL of hemin to prepare an active additive mixed solution; after the temperature of the culture bottles subjected to the high-pressure sterilization in the step S4 is reduced to room temperature, 0.4mL of active additive mixed solution is injected into each culture bottle through a 1mL syringe in a sterile operation table;
s5, repeating the steps S1 to S4 until a sufficient number of anaerobic microorganism culture bottles are prepared.
Comparative example 1
A plurality of anaerobic microorganism culture flasks to which the active additive mixture was not added were prepared by the same method as in example 1, i.e., repeating steps S1 to S3 in example 1.
Example 2
The 40 strains of Table 1 below were anaerobically cultured using two intestinal anaerobic microorganism flasks prepared in example 1 and comparative example 1. The specific culture method comprises the following steps: the freeze-dried powder of 40 intestinal anaerobic microorganisms in table 1 was rapidly dissolved in 1mL of PBS according to serial numbers, and 500 μl of the dissolved bacterial liquid was sucked by a 1mL syringe, and injected into anaerobic microorganism culture flasks marked with the same serial numbers, which were prepared in example 1 and comparative example 1; culturing all culture flasks in a 37 ℃ incubator for 24 hours, and respectively measuring the turbidity of the cultured microorganisms, wherein the turbidity unit is MCF,1 MCF=3×10≡8CFU/mL, and the CFU is the unit of strain; all flasks had a turbidity of 0MCF before incubation.
Table 1:
as can be seen from the test results in Table 1, the culture flask prepared by the method of comparative example 1 was able to culture a strain, but the culture flask prepared by the method of example 1 was significantly better in strain culture. Fig. 2 is a statistical chart showing the differences in MCF values based on the test results of table 1. As can be seen from FIG. 2, the flask prepared by the method of example 1 has a remarkable strain culturing effect compared with the flask prepared by the method of comparative example 1, and the p value between the two is 0.0154 and less than 0.05, which is statistically significant.
Example 3
Taking out the 40 culture bottles prepared in the example 2 from the samples subjected to culture, placing the culture bottles in a refrigerator at the temperature of 4 ℃, taking out the culture bottles after the culture bottles are placed for 30 days, uniformly mixing the culture bottles, and standing the culture bottles for 1 hour to restore the room temperature; 500. Mu.l of each of the 40 cultures was aspirated with a syringe, injected into 40 new anaerobic microorganism flasks prepared using example 1, and the 40 new flasks were placed in a 37℃incubator for 24 hours.
After the cultivation, the turbidity of the microorganism after the cultivation in 40 new flasks was measured, respectively. The test results show that: except that four strains of bacteria of Bactoides PAC002443, lachnospiraceae PAC001085_s, lachnospiraceae PAC001459_s and Lachnospiraceae PAC001772_s are not grown, other 36 bacteria are activated, the activation rate reaches 90%, and the anaerobic microorganism culture flask can meet the requirement for short-term storage of anaerobic microorganisms.
In addition, because the liquid in the intestinal anaerobic microorganism culture bottle is always in the sealed and packaged environment, the liquid is not influenced by external environment basically, and the volume is small, the portable carrying or transporting requirements can be met, for example, other scientific research institutions need that certain anaerobic bacteria strains can be directly put in pockets to carry, or put in a refrigerating condition to transport scientific research institutions or hospitals in a short distance.
In conclusion, the intestinal anaerobic microorganism culture bottle disclosed by the application achieves the aim of simpler operation of anaerobic culture, storage and transportation, meets the requirement of mass production, and has good market popularization prospect.
Claims (5)
1. An intestinal anaerobic microorganism culture bottle is characterized by comprising a culture bottle which is sealed and packaged and a culture solution filled in the culture bottle; the culture solution comprises 100 parts of basic culture medium aqueous solution, 4 parts of 2.5wt.% L-cysteine salt aqueous solution and 0.1 part of 0.1wt.% resazurin aqueous solution which are mixed in parts by volume, and an active additive mixed solution which is 4% of the basic culture solution; the active additive mixed solution is prepared by mixing 10 parts of 1640 culture medium solution, 1 part of BI serum, 1 part of vitamin K and 1 part of hemin in parts by volume; wherein the basal medium is powdery RAM basal medium, CDC basal medium, RCM basal medium or MRS basal medium.
2. The intestinal anaerobic microorganism culture bottle according to claim 1, wherein the filling volume of the culture solution is 90% -95% of the volume of the culture bottle.
3. The intestinal anaerobic microorganism culture bottle according to claim 1, wherein the culture bottle comprises a penicillin bottle, a rubber plug inserted at the mouth of the penicillin bottle, a hollow aluminum cap sealed at the ends of the rubber plug and the mouth of the penicillin bottle, and a top cover covered on the hollow aluminum cap.
4. The intestinal anaerobic microorganism culture flask according to claim 1, wherein the penicillin flask is a borosilicate glass flask; the top cover adopts a polytetrafluoroethylene top cover.
5. The preparation method of the intestinal anaerobic microorganism culture bottle is characterized by comprising the following steps:
s1, dissolving a powdery basic culture medium in sterile water according to a proportion to prepare a basic culture medium solution; dissolving powdered resazurin in sterile water to obtain 0.1wt.% resazurin aqueous solution; dissolving powdery L-cysteine salt in sterile water to obtain 2.5wt.% of L-cysteine salt aqueous solution;
s2, mixing a basic culture medium solution, an L-cysteine salt aqueous solution and a resazurin aqueous solution according to a volume ratio of 1000:40:1 to obtain a basic culture solution, respectively filling the basic culture solution into a plurality of culture bottles, and sealing and packaging the filled culture bottles; wherein, the filling volume is 90% -95% of the volume of the culture flask;
s3, autoclaving the filled and sealed culture bottles obtained in the step S2;
s4, mixing 1640 culture medium liquid, BI serum, vitamin K and hemin according to the volume ratio of 10:1:1:1 to prepare an active additive mixed solution, and injecting the active additive mixed solution into the culture flask through an injector in a sterile operation table after the temperature of the culture flask subjected to high-pressure sterilization in the step S4 is reduced to room temperature;
the volume ratio of the injection amount of the active additive mixed solution to the basic culture solution is 1:25.
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Citations (5)
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| CN104164396A (en) * | 2014-08-15 | 2014-11-26 | 南昌大学 | Method for screening live pig intestinal micro-ecological preparation with high adhesive capacity |
| CN104507483A (en) * | 2012-04-13 | 2015-04-08 | 波士顿学院理事会 | Prebiotic compositions and methods of use |
| JP2016065795A (en) * | 2014-09-25 | 2016-04-28 | ヤマサ醤油株式会社 | Nonspecific reaction inhibitor, and immunoassay using it |
| CN108135944A (en) * | 2014-11-25 | 2018-06-08 | 伊夫罗生物科学公司 | Probiotics and prebiotic compositions and its method and purposes for adjusting microorganism group |
| CN109576218A (en) * | 2018-12-25 | 2019-04-05 | 苏州荣世医药科技有限公司 | A kind of Peripheral blood culture base and preparation method thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104507483A (en) * | 2012-04-13 | 2015-04-08 | 波士顿学院理事会 | Prebiotic compositions and methods of use |
| CN104164396A (en) * | 2014-08-15 | 2014-11-26 | 南昌大学 | Method for screening live pig intestinal micro-ecological preparation with high adhesive capacity |
| JP2016065795A (en) * | 2014-09-25 | 2016-04-28 | ヤマサ醤油株式会社 | Nonspecific reaction inhibitor, and immunoassay using it |
| CN108135944A (en) * | 2014-11-25 | 2018-06-08 | 伊夫罗生物科学公司 | Probiotics and prebiotic compositions and its method and purposes for adjusting microorganism group |
| CN109576218A (en) * | 2018-12-25 | 2019-04-05 | 苏州荣世医药科技有限公司 | A kind of Peripheral blood culture base and preparation method thereof |
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