CN110819578A - Strain preservation medium of clostridium butyricum and culture method thereof - Google Patents

Strain preservation medium of clostridium butyricum and culture method thereof Download PDF

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CN110819578A
CN110819578A CN201911312445.2A CN201911312445A CN110819578A CN 110819578 A CN110819578 A CN 110819578A CN 201911312445 A CN201911312445 A CN 201911312445A CN 110819578 A CN110819578 A CN 110819578A
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clostridium butyricum
strain preservation
medium
peptone
sterilizing
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CN110819578B (en
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吴微
张茜茜
李洋
周樱
詹志春
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WUHAN SUNHY BIOLOGY CO Ltd
HUBEI HUAYANG TECHNOLOGY DEVELOPMENT Co Ltd
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HUBEI HUAYANG TECHNOLOGY DEVELOPMENT Co Ltd
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    • 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
    • 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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Abstract

The invention provides a strain preservation culture medium and a culture method of clostridium butyricum, wherein the strain preservation culture medium comprises the following components in percentage by mass: 0.5-7% of maltodextrin, 0.1-5% of peptone, 0.1-3% of corn steep liquor, 0.1-2% of anhydrous sodium acetate, 0.1-3% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1, adding 0.1-4% of calcium carbonate, and sterilizing at 115 ℃ for 30 min. According to the invention, the clostridium butyricum is converted into a stable spore dormancy form from a rod-shaped nutrient form by optimizing the components of the culture medium, so that the storage and preservation of strains are facilitated, and the quality of the storage and preservation of the strains is greatly improved.

Description

Strain preservation medium of clostridium butyricum and culture method thereof
Technical Field
The invention belongs to the technical field of microbial culture, and particularly relates to a strain preservation culture medium of clostridium butyricum and a culture method thereof.
Background
Clostridium butyricum, also known as butyric acid bacteria and clostridium butyricum, is an obligate anaerobic gram-positive bacterium which can produce short-chain fatty acid and has stronger resistance, heat resistance, acid resistance and antibiotic resistance, so clostridium butyricum can be approved as a feed additive for broilers and weaned pigs in 2003 by the european union, and is also brought into annex two of the Chinese feed additive variety catalog (2013) in 2013 in China.
At present, in the experimental and production processes, the storage and preservation medium of clostridium butyricum is generally RCM medium, i.e. reinforced clostridium culture medium, which comprises the following components: 0.3% of yeast powder, 1% of beef powder, 1% of peptone, 0.5% of glucose, 0.1% of soluble starch, 0.5% of sodium acetate trihydrate, 0.5% of sodium chloride, 0.05% of cysteamine hydrochloride, pH7.0 +/-0.1 and sterilizing at 115 ℃ for 30 min. However, the RCM medium is widely used for proliferation culture and counting of clostridium butyricum, but does not convert clostridium butyricum into spore form in large quantity, which is not favorable for storage and preservation.
Disclosure of Invention
The invention aims to solve the problem that the clostridium butyricum cultured by the conventional RCM culture medium cannot be converted into spore forms in a large amount and is not beneficial to storage and preservation.
Therefore, the invention provides a strain preservation medium of clostridium butyricum, which comprises the following components in percentage by mass: 0.5-7% of maltodextrin, 0.1-5% of peptone, 0.1-3% of corn steep liquor, 0.1-2% of anhydrous sodium acetate, 0.1-3% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1, adding 0.1-4% of calcium carbonate, and sterilizing at 115 ℃ for 30 min.
Specifically, the strain preservation medium of clostridium butyricum comprises the following components in percentage by mass: 0.5-5% of maltodextrin, 0.1-3% of peptone, 0.1-3% of corn steep liquor, 0.1-1% of anhydrous sodium acetate, 0.1-2% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1, adding 0.1-2.5% of calcium carbonate, and sterilizing at 115 ℃ for 30 min.
Specifically, the strain preservation medium of clostridium butyricum comprises the following components in percentage by mass: 1-4% of maltodextrin, 0.1-2% of peptone, 0.5-2% of corn steep liquor, 0.1-0.5% of anhydrous sodium acetate, 0.2-0.5% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1, adding 0.5-2% of calcium carbonate, and sterilizing at 115 ℃ for 30 min.
Specifically, the strain preservation medium of clostridium butyricum comprises the following components in percentage by mass: 1% of maltodextrin, 0.5% of peptone, 1% of corn steep liquor, 0.3% of anhydrous sodium acetate, 0.5% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1, adding 1% of calcium carbonate, and sterilizing at 115 ℃ for 30 min.
Specifically, the pH value is adjusted by using dilute hydrochloric acid.
In addition, the invention also provides a strain preservation culture method of clostridium butyricum, which comprises the following steps:
1) picking a single colony of clostridium butyricum in a sterile environment, and performing static culture for 24-36 h at 34-37 ℃ in a strain preservation culture medium of the clostridium butyricum;
2) mixing the culture solution cultured in the step 1) with glycerol, storing at-40 ℃ for 48-72 h, and storing at-80 ℃.
Specifically, the concentration of the glycerol in the step 2) is 40%, and the mass ratio of the glycerol to the culture solution is 1: 1.
Compared with the prior art, the invention has the beneficial effects that:
(1) the clostridium butyricum strain preservation culture medium provided by the invention has the advantages that the component is optimized, the growth of bacteria is effectively accelerated by utilizing a carbon source and a nitrogen source in maltodextrin, peptone and corn steep liquor, and calcium carbonate is added to neutralize acid produced in the metabolic process of bacteria growth and play a buffering role in the growth process, so that the clostridium butyricum is converted into a stable spore dormancy form from a rod-shaped nutrient form, the storage and preservation of strains are facilitated, and the quality of the strain storage and preservation is greatly improved.
(2) The clostridium butyricum cultured by the clostridium butyricum strain preservation culture medium obviously improves the strain quality and shortens the lag phase in the growth process in the later strain recovery and proliferation process, and the strain quantity and the growth speed of the clostridium butyricum are obviously superior to those of the clostridium butyricum cultured by an RCM culture medium.
The present invention will be described in further detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a microscopic image of Clostridium butyricum cultured in a conventional RCM medium;
FIG. 2 is a microscopic image of Clostridium butyricum cultured in the culture collection medium of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the implementation provides a strain preservation medium of clostridium butyricum, which comprises the following components in percentage by mass: 1% of maltodextrin, 0.5% of peptone, 1% of corn steep liquor, 0.3% of anhydrous sodium acetate, 0.5% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1 by using dilute hydrochloric acid, subpackaging the mixture into 200mL/250mL triangular bottles, adding 1% of calcium carbonate into the bottles, and sterilizing the bottles at 115 ℃ for 30 min.
The culture process of the clostridium butyricum by adopting the strain preservation culture medium of the clostridium butyricum in the embodiment is as follows:
placing the required inoculation tool (sterilized bamboo stick or inoculating loop) and the culture medium in a super clean bench or sterile room, irradiating with ultraviolet lamp for about 30min, turning off the ultraviolet lamp, and blowing for about 15 min; and then, sterilizing the skin exposed in the operation area, performing aseptic operation, picking a single colony in the strain preservation culture medium, sealing a bottle opening, and performing static culture in an incubator at 34-37 ℃ for 24-36 hours after the operation is finished.
Taking the culture solution after the culture and uniformly mixing with 40% of sterilized glycerol in a ratio of 1:1 in a sterile room or a super clean bench after the ultraviolet sterilization and disinfection; subpackaging, namely storing in a refrigerator at the temperature of-40 ℃ for 48-72 h, and then storing in a refrigerator at the temperature of-80 ℃.
Example 2:
the embodiment provides a strain preservation medium of clostridium butyricum, which comprises the following components in percentage by mass: 2% of maltodextrin, 2% of peptone, 0.5% of corn steep liquor, 0.5% of anhydrous sodium acetate, 0.3% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1 by using dilute hydrochloric acid, subpackaging the mixture into 200mL/250mL triangular bottles, adding 0.5% of calcium carbonate into the bottles, and sterilizing the bottles at 115 ℃ for 30 min.
The culture medium for the strain preservation of Clostridium butyricum of this example was the same as that of example 1.
Example 3:
the embodiment provides a strain preservation medium of clostridium butyricum, which comprises the following components in percentage by mass: 4% of maltodextrin, 0.1% of peptone, 2% of corn steep liquor, 0.1% of anhydrous sodium acetate, 0.2% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1 by using dilute hydrochloric acid, subpackaging the mixture into 200mL/250mL triangular bottles, adding 2% of calcium carbonate into the bottles, and sterilizing the bottles at 115 ℃ for 30 min.
The culture medium for the strain preservation of Clostridium butyricum of this example was the same as that of example 1.
Example 4:
the embodiment provides a strain preservation medium of clostridium butyricum, which comprises the following components in percentage by mass: 0.5% of maltodextrin, 5% of peptone, 0.8% of corn steep liquor, 1% of anhydrous sodium acetate, 2% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1 by using dilute hydrochloric acid, subpackaging the mixture into 200mL/250mL triangular bottles, adding 1% of calcium carbonate into the bottles, and sterilizing the mixture at 115 ℃ for 30 min.
The culture medium for the strain preservation of Clostridium butyricum of this example was the same as that of example 1.
Example 5:
the embodiment provides a strain preservation medium of clostridium butyricum, which comprises the following components in percentage by mass: 6% of maltodextrin, 3% of peptone, 0.1% of corn steep liquor, 2% of anhydrous sodium acetate, 0.1% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1 by using dilute hydrochloric acid, subpackaging the mixture into 200mL/250mL triangular bottles, adding 2% of calcium carbonate into the bottles, and sterilizing the mixture at 115 ℃ for 30 min.
The culture medium for the strain preservation of Clostridium butyricum of this example was the same as that of example 1.
Example 6:
the embodiment provides a strain preservation medium of clostridium butyricum, which comprises the following components in percentage by mass: 5% of maltodextrin, 2% of peptone, 1% of corn steep liquor, 1.5% of anhydrous sodium acetate, 3% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1 by using dilute hydrochloric acid, subpackaging the mixture into 200mL/250mL triangular bottles, adding 2.5% of calcium carbonate into the bottles, and sterilizing the mixture at 115 ℃ for 30min to obtain the calcium carbonate-containing biological protein feed.
The culture medium for the strain preservation of Clostridium butyricum of this example was the same as that of example 1.
Example 7:
the embodiment provides a strain preservation medium of clostridium butyricum, which comprises the following components in percentage by mass: 7% of maltodextrin, 0.1% of peptone, 3% of corn steep liquor, 0.2% of anhydrous sodium acetate, 1% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1 by using dilute hydrochloric acid, subpackaging the mixture into 200mL/250mL triangular bottles, adding 4% of calcium carbonate into the bottles, and sterilizing the mixture at 115 ℃ for 30min to obtain the calcium carbonate-containing biological feed.
The culture medium for the strain preservation of Clostridium butyricum of this example was the same as that of example 1.
Comparative example:
the present comparative example uses the existing RCM medium to culture clostridium butyricum, and the components of the RCM medium are as follows: 0.3% of yeast powder, 1% of beef powder, 1% of peptone, 0.5% of glucose, 0.1% of soluble starch, 0.5% of sodium acetate trihydrate, 0.5% of sodium chloride, 0.05% of cysteamine hydrochloride, pH7.0 +/-0.1 and sterilizing at 115 ℃ for 30 min.
The procedure of the present comparative example for culturing Clostridium butyricum using the above-mentioned RCM medium was the same as in example 1.
The strain cultured in the RCM medium of this comparative example and the strain cultured in the preservation medium of example 1 were examined under a microscope, and the results are shown in FIGS. 1 and 2, respectively.
As can be seen from the comparison between FIG. 1 and FIG. 2, the Clostridium butyricum cultured by the conventional RCM medium is in the form of a rod-shaped nutrient, while most of the Clostridium butyricum cultured by the strain preservation medium of the present embodiment is in the form of spore dormancy, and the amount of the strain is much larger than that cultured by the conventional RCM medium, thereby greatly improving the quality of strain storage and preservation.
The total bacterial count, the spore count and the lag phase duration of the clostridium butyricum cultured in the above examples 1 to 7 and comparative examples were measured, and the results are shown in table 1.
Detection of the culture medium: 0.3% of yeast powder, 1% of beef powder, 1% of peptone, 0.5% of glucose, 0.1% of soluble starch, 0.5% of sodium acetate trihydrate, 0.5% of sodium chloride, 0.05% of cysteamine hydrochloride, 2% of agar powder and pH7.0 +/-0.1, subpackaging into 18 x 180mm test tubes, adding 2mL of paraffin oil to a culture medium to form a liquid seal, isolating from air, plugging the test tubes with silica gel, and sterilizing for 30min at 115 ℃.
The total bacteria number detection process is as follows: gradually diluting a solution to be detected to a proper concentration, adding the sample into the detection culture medium, culturing for 18-36 h in an incubator at 34-37 ℃, counting the number of single colonies in a test tube, and multiplying the number by a dilution multiple to obtain the total number (including two types of nutrients and spores) of clostridium butyricum in the seed culture medium.
And in the spore number detection process, the diluted sample is placed in a water bath at 80 ℃ for 10min, and the rest operation process is consistent with the total bacteria number detection process.
Table 1:
total bacteria count (CFU/mL) Number of spores (CFU/mL) Lag phase (h)
Comparative example 1.2×108 ＜1×106 8
Example 1 2.3×108 1.4×108 1.5
Example 2 3.1×108 1.9×108 1.6
Example 3 1.9×108 1.2×108 1.5
Example 4 1.8×108 1.1×108 1.8
Example 5 2.2×108 1.6×108 2
Example 6 2.9×108 1.9×108 2
Example 7 3.4×108 2.0×108 2
As can be seen from Table 1, the clostridium butyricum cultured by the strain preservation medium obviously improves the strain quality and shortens the lag phase in the growth process in the recovery and proliferation process of strains used in the later period, and the strain quantity and the growth speed of the clostridium butyricum are obviously superior to those of the conventional RCM medium.
The above examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims and any design similar or equivalent to the scope of the invention.

Claims (7)

1. The strain preservation medium of clostridium butyricum is characterized by comprising the following components in percentage by mass: 0.5-7% of maltodextrin, 0.1-5% of peptone, 0.1-3% of corn steep liquor, 0.1-2% of anhydrous sodium acetate, 0.1-3% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1, adding 0.1-4% of calcium carbonate, and sterilizing at 115 ℃ for 30 min.
2. The strain preservation medium for clostridium butyricum according to claim 1, which comprises the following components in percentage by mass: 0.5-5% of maltodextrin, 0.1-3% of peptone, 0.1-3% of corn steep liquor, 0.1-1% of anhydrous sodium acetate, 0.1-2% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1, adding 0.1-2.5% of calcium carbonate, and sterilizing at 115 ℃ for 30 min.
3. The strain preservation medium for clostridium butyricum according to claim 1, which comprises the following components in percentage by mass: 1-4% of maltodextrin, 0.1-2% of peptone, 0.5-2% of corn steep liquor, 0.1-0.5% of anhydrous sodium acetate, 0.2-0.5% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1, adding 0.5-2% of calcium carbonate, and sterilizing at 115 ℃ for 30 min.
4. The strain preservation medium for clostridium butyricum according to claim 1, which comprises the following components in percentage by mass: 1% of maltodextrin, 0.5% of peptone, 1% of corn steep liquor, 0.3% of anhydrous sodium acetate, 0.5% of sodium chloride and the balance of water, adjusting the pH value to 7.0 +/-0.1, adding 1% of calcium carbonate, and sterilizing at 115 ℃ for 30 min.
5. The strain preservation medium for clostridium butyricum according to claim 1, wherein said pH value is adjusted with dilute hydrochloric acid.
6. The strain preservation culture method of clostridium butyricum is characterized by comprising the following steps:
1) picking a single colony of clostridium butyricum in a sterile environment, and carrying out static culture at 34-37 ℃ for 24-36 h in a strain preservation culture medium of clostridium butyricum of any one of claims 1-5;
2) mixing the culture solution cultured in the step 1) with glycerol, storing at-40 ℃ for 48-72 h, and storing at-80 ℃.
7. The method for preserving and culturing strains of Clostridium butyricum according to claim 6, wherein the concentration of glycerol in step 2) is 40% and the mass ratio of glycerol to the culture solution is 1: 1.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102363754A (en) * 2011-05-07 2012-02-29 华中农业大学 Clear liquid fermentation medium for clostridium butyricum and fermentation culture method thereof
CN105695370A (en) * 2016-04-13 2016-06-22 南京工业大学 Clostridium butyricum and culture method and application thereof
CN106479924A (en) * 2016-10-31 2017-03-08 湖北绿雪生物科技有限公司 The preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application
CN107988137A (en) * 2017-12-15 2018-05-04 武汉新华扬生物股份有限公司 A kind of highly concentrated clostridium butyricum gemma production method and highly concentrated clostridium butyricum gemma product
CN110151795A (en) * 2019-05-14 2019-08-23 广东大泽农生物科技股份有限公司 A kind of clostridium butyricum active bacteria preparation and its production technology

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102363754A (en) * 2011-05-07 2012-02-29 华中农业大学 Clear liquid fermentation medium for clostridium butyricum and fermentation culture method thereof
CN105695370A (en) * 2016-04-13 2016-06-22 南京工业大学 Clostridium butyricum and culture method and application thereof
CN106479924A (en) * 2016-10-31 2017-03-08 湖北绿雪生物科技有限公司 The preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application
CN107988137A (en) * 2017-12-15 2018-05-04 武汉新华扬生物股份有限公司 A kind of highly concentrated clostridium butyricum gemma production method and highly concentrated clostridium butyricum gemma product
CN110151795A (en) * 2019-05-14 2019-08-23 广东大泽农生物科技股份有限公司 A kind of clostridium butyricum active bacteria preparation and its production technology

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