CN116790380A - Bacterial preservation solution, preparation method and application thereof - Google Patents
Bacterial preservation solution, preparation method and application thereof Download PDFInfo
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- CN116790380A CN116790380A CN202310857348.1A CN202310857348A CN116790380A CN 116790380 A CN116790380 A CN 116790380A CN 202310857348 A CN202310857348 A CN 202310857348A CN 116790380 A CN116790380 A CN 116790380A
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- 230000001580 bacterial effect Effects 0.000 title claims abstract description 75
- 239000003761 preservation solution Substances 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 238000004321 preservation Methods 0.000 claims abstract description 31
- 241000894006 Bacteria Species 0.000 claims abstract description 22
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 63
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 claims description 34
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 32
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 32
- 239000003755 preservative agent Substances 0.000 claims description 21
- 230000002335 preservative effect Effects 0.000 claims description 21
- 239000001888 Peptone Substances 0.000 claims description 18
- 108010080698 Peptones Proteins 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- 235000019319 peptone Nutrition 0.000 claims description 18
- 239000001509 sodium citrate Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 17
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 17
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 17
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 17
- 229960004172 pyridoxine hydrochloride Drugs 0.000 claims description 17
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 17
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 17
- 239000011780 sodium chloride Substances 0.000 claims description 17
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 17
- 235000010265 sodium sulphite Nutrition 0.000 claims description 17
- 239000011668 ascorbic acid Substances 0.000 claims description 16
- 235000010323 ascorbic acid Nutrition 0.000 claims description 16
- 229960005070 ascorbic acid Drugs 0.000 claims description 16
- 239000008367 deionised water Substances 0.000 claims description 15
- 229910021641 deionized water Inorganic materials 0.000 claims description 15
- 239000012530 fluid Substances 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 235000011083 sodium citrates Nutrition 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 235000011187 glycerol Nutrition 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 235000002639 sodium chloride Nutrition 0.000 claims description 3
- 241000894007 species Species 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 241000192125 Firmicutes Species 0.000 claims description 2
- 239000003002 pH adjusting agent Substances 0.000 claims description 2
- 230000004083 survival effect Effects 0.000 abstract description 18
- 238000011084 recovery Methods 0.000 abstract description 7
- 230000007774 longterm Effects 0.000 abstract description 6
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 230000008859 change Effects 0.000 abstract description 3
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- 241000588724 Escherichia coli Species 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 10
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- 238000003860 storage Methods 0.000 description 4
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 2
- 240000006024 Lactobacillus plantarum Species 0.000 description 2
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- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940072205 lactobacillus plantarum Drugs 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
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- 239000001257 hydrogen Substances 0.000 description 1
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- 238000011534 incubation Methods 0.000 description 1
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- 210000002966 serum Anatomy 0.000 description 1
- PVGBHEUCHKGFQP-UHFFFAOYSA-N sodium;n-[5-amino-2-(4-aminophenyl)sulfonylphenyl]sulfonylacetamide Chemical compound [Na+].CC(=O)NS(=O)(=O)C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=C1 PVGBHEUCHKGFQP-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000000859 sublimation Methods 0.000 description 1
- 230000008022 sublimation Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
Classifications
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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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
- C12R2001/385—Pseudomonas aeruginosa
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/44—Staphylococcus
- C12R2001/445—Staphylococcus aureus
Abstract
The invention discloses a bacterial preservation solution, a preparation method and application thereof, and relates to the technical field of microbial engineering. The components of the preservation solution are simple to prepare, the bacterial strain preservation solution can maintain the microbial activity for a long time, the preservation time is greatly prolonged, the preserved bacterial strain has high recovery survival rate, the bacterial strain can be preserved for a long time, meanwhile, the change of the biological characteristics of the bacterial strain caused by the passage times of the bacterial strain and the possibility of contaminating other bacteria can be reduced, the survival rate of the bacterial strain is not influenced after the bacterial strain is preserved for a long time, and a way is provided for the long-term preservation of the bacterial strain.
Description
Technical Field
The invention relates to the technical field of microbial engineering, in particular to a bacterial preservation solution, a preparation method and application thereof.
Background
Bacteria are an important biological resource, and the preservation of bacteria is an important basic work for bacterial scientific research and clinical detection. The purpose of preserving the microorganism is to ensure that the microorganism strain maintains the original vitality, excellent production performance and morphological characteristics and also ensures the stable inheritance of the excellent shape, thereby better applying advanced technology to develop and use the microorganism for the latter.
During the preservation of a bacterial sample, the metabolism of the microorganism must be kept at a least active or relatively quiescent state so as to not change over a period of time while maintaining viability. The preservation methods adopted at present mainly comprise a glycerol preservation method, a liquid paraffin method, a semi-solid culture medium method, a vacuum freeze drying method, a refrigerator freezing method at 80 ℃, a liquid nitrogen ultralow temperature freezing method and the like, and the aim is to create an environment suitable for long-term dormancy for excellent strains, and the method of drying, low temperature, lack of oxygen, nutrients and the like is generally adopted to enable the metabolic activity of microorganisms to be in a minimum state but not to die, so that the preservation purpose is achieved. However, the semisolid culture medium preservation method cannot preserve strains for a long time; the simple glycerol preservation method is characterized in that the glycerol is only used for preventing freezing and preserving moisture, and certain special strains have low survival rate in glycerol stock solution, short preservation time and reduced toxicity; the bacteria are easy to damage by the classical freeze-vacuum drying method, the operation technology is difficult, the cost is high, and special equipment is needed; and some conventional methods are often disadvantageous in preservation due to the fact that the passaging times are large, the pollution and the mutation are easy to occur. Therefore, the method for searching the bacterial strain which is simple and convenient to operate and high in survival rate and can preserve the bacterial strain for a long time is of great significance to the subsequent research of the separated bacterial strain.
Therefore, it is a problem to be solved by those skilled in the art how to provide a bacterial preservative solution having a long shelf life and a high survival rate.
Disclosure of Invention
In view of the above, the invention provides a bacterial preservation solution, a preparation method and application thereof, and the preservation solution can prolong the preservation time of bacterial strains, keep the activity of the strains, improve the preservation efficiency and is convenient to operate.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a bacterial preservative solution, wherein 100mL of the bacterial preservative solution comprises: 10-30g of peptone, 5-25g of trehalose, 5-15g of sodium chloride, 5-10g of sodium citrate, 0.1-1g of sodium sulfite, 0.1-0.5g of pyridoxine hydrochloride, 0.1-0.7g of ascorbic acid and 5-15mL of glycerol, and the volume is fixed to 100mL by deionized water.
Still further, the 100mL of the bacterial preservation solution comprises: 16g of peptone, 11g of trehalose, 7g of sodium chloride, 8g of sodium citrate, 0.8g of sodium sulfite, 0.4g of pyridoxine hydrochloride, 0.6g of ascorbic acid and 7mL of glycerol, and the volume is fixed to 100mL by deionized water.
Still further, the 100mL of the bacterial preservation solution comprises: 21g of peptone, 13g of trehalose, 11g of sodium chloride, 5g of sodium citrate, 0.5g of sodium sulfite, 0.2g of pyridoxine hydrochloride, 0.4g of ascorbic acid and 13mL of glycerol, and the volume is fixed to 100mL by deionized water.
Further, the bacteria preservation solution also comprises a pH regulator.
Further, the pH regulator is added in an amount to regulate the pH of the bacterial preservation solution to 6.5-7.5.
Still further, the pH adjuster comprises dipotassium hydrogen phosphate or sodium hydroxide.
As the same inventive concept as the technical scheme, the invention also claims a preparation method of the bacteria preservation solution, which specifically comprises the following steps:
1) Weighing: weighing the raw materials according to the weight parts for standby;
2) Mixing peptone, trehalose, sodium chloride, sodium citrate, sodium sulfite, pyridoxine hydrochloride, ascorbic acid, glycerol and deionized water, and uniformly stirring to obtain a mixed solution;
3) And (3) regulating the pH value of the mixed solution in the step (2) to 6.5-7.5 by adopting a pH regulating reagent, and then sterilizing at 120-122 ℃ for 15-20min to obtain the bacteria preservation solution.
As the same invention conception as the technical scheme, the invention also claims the application of the bacterial preservation solution in bacterial strain culture and preservation.
Further, the bacterial species is one of gram-negative and gram-positive bacteria.
Compared with the prior art, the invention discloses a bacteria preservation solution, a preparation method and application thereof, and has the following beneficial effects: the bacterial preservation solution has simple preparation, and under the preparation of the specific components and the specific proportion, the peptone and the trehalose contain vitamins and saccharides, so that nutrients such as C sources, N sources, growth factors and the like can be provided for the growth and survival of bacteria, and the shape of cell membranes can be maintained; the glycerol can be added to stabilize the configuration of cell components through the affinity of hydrogen and ionic bonds to water and cells, so that the damage to the cells caused by freezing or continuous sublimation of the water is prevented; pyridoxine hydrochloride and sodium chloride provide energy for the bacteria and prevent their death. And the sodium citrate, sodium sulfite and ascorbic acid with proper concentrations are added into the protective solution, so that the components and the concentrations of the protective solution can be optimized, the activity of preserving bacterial strains can be effectively improved, and the oxidation resistance of the protective solution is improved, so that the bacterial strain preserving solution can maintain the microbial activity for a long time, the preserving time is greatly prolonged, meanwhile, the change of biological characteristics of the bacterial strains caused by the passage times of the bacterial strains and the possibility of polluting other bacteria can be reduced, the survival rate of the bacterial strains is not influenced after the long-term preservation, and a way is provided for the long-term preservation of the bacterial strains.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Experimental materials
The strains such as E.coli, staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, bifidobacterium, lactobacillus plantarum and the like used in the following examples were all commercial strains.
Example 1
A bacterial preservative fluid comprising the following components: 10g of peptone, 5g of trehalose, 5g of sodium chloride, 5g of sodium citrate, 0.1g of sodium sulfite, 0.1g of pyridoxine hydrochloride, 0.1g of ascorbic acid and 5mL of glycerol, and the volume is fixed to 100mL by deionized water, and the pH is adjusted to 6.7.
Example 2
A bacterial preservative fluid comprising the following components: 30g of peptone, 25g of trehalose, 15g of sodium chloride, 10g of sodium citrate, 0.8g of sodium sulfite, 0.5g of pyridoxine hydrochloride, 0.5g of ascorbic acid and 15mL of glycerol, and the volume is fixed to 100mL by deionized water, and the pH is adjusted to 7.0.
Example 3
A bacterial preservative fluid comprising the following components: 26g of peptone, 22g of trehalose, 13g of sodium chloride, 8g of sodium citrate, 0.6g of sodium sulfite, 0.4g of pyridoxine hydrochloride, 0.4g of ascorbic acid and 15mL of glycerol, and the volume is fixed to 100mL by deionized water, and the pH is adjusted to 7.3.
Example 4
A bacterial preservative fluid comprising the following components: 21g of peptone, 13g of trehalose, 11g of sodium chloride, 5g of sodium citrate, 0.5g of sodium sulfite, 0.2g of pyridoxine hydrochloride, 0.4g of ascorbic acid and 13mL of glycerol, and the volume is fixed to 100mL by deionized water, and the pH is adjusted to 6.9.
Example 5
A bacterial preservative fluid comprising the following components: 16g of peptone, 11g of trehalose, 7g of sodium chloride, 8g of sodium citrate, 0.8g of sodium sulfite, 0.4g of pyridoxine hydrochloride, 0.6g of ascorbic acid and 7mL of glycerol, and the volume is fixed to 100mL by deionized water, and the pH is adjusted to 7.1.
Example 6
A bacterial preservative fluid comprising the following components: 12g of peptone, 8g of trehalose, 9g of sodium chloride, 10g of sodium citrate, 0.6g of sodium sulfite, 0.4g of pyridoxine hydrochloride, 0.1g of ascorbic acid and 7mL of glycerol, and the volume is fixed to 100mL by deionized water, and the pH is adjusted to 7.5.
The preparation process of the bacterial preservative fluid is as follows:
1) Weighing: weighing the raw materials according to the weight parts for standby;
2) Mixing peptone, trehalose, sodium chloride, sodium citrate, sodium sulfite, pyridoxine hydrochloride, ascorbic acid, glycerol and deionized water, and uniformly stirring to obtain a mixed solution;
3) And (3) regulating the pH of the mixed solution in the step (2) to 6.5-7.5 by adopting a pH regulating reagent, and then sterilizing at 120-122 ℃ for 15-20min to obtain the bacteria preservation solution.
Example 7
Bacterial preservation solutions prepared by the methods of examples 1 to 6 and glycerol-physiological saline preservation solutions were used to determine the activity of E.coli strains at different preservation times, respectively.
Respectively picking fresh bacterial colonies of Escherichia coli, respectively dissolving in 5mL of the bacterial preservation solution and glycerol-physiological saline preservation solution prepared by the method to prepare 5×10 3 Sucking 100 mu L of the bacterial suspension to be coated on a beef extract peptone plate, and coating 10 plates in total; the remaining 4mL of the bacterial suspension was packed in 8 centrifuge tubes, each tube was packed with 500. Mu.L, sealed and stored in a-4℃refrigerator, and after 0, 3, 5, 10, 15, 20, 25 and 30 days of storage, the bacterial suspension was taken out and plated in the same manner. All plates were counted after incubation in a constant temperature phase at 37℃for 24h and mean and standard deviation were calculated. The results are shown in Table 1.
TABLE 1 preservation test results of examples 1-6 and glycerol-physiological saline preservation solution on E.coli
As shown in Table 1, the survival rate of the escherichia coli prepared by the invention after being preserved for one month is obviously higher than that of glycerol-physiological saline, and the survival rate is as high as 95.7%.
Example 8
E.coli preserved by the bacterial preservation solution prepared in the examples 1-6 and the glycerol-normal saline preservation solution are randomly selected for 20 pipes for resuscitation respectively in 6 months, 12 months, 18 months, 24 months, 30 months and 36 months after freezing, the resuscitation method is to quickly defrost the frozen stock in 37 ℃ water bath and then put the frozen stock into serum broth for culturing for 24 hours, then the resuscitated bacterial solution is picked by an inoculating loop and evenly coated on beef extract peptone culture medium, culturing is carried out for 24 hours at 37 ℃, whether colony generation exists is observed, and the survival rate is calculated. The calculation formula of the survival rate is as follows: survival = number of surviving tubes/number of tubes selected, test results are shown in table 2.
TABLE 2 results of recovery test of E.coli with examples 1-6 and glycerol-saline stock solution
As shown in table 2, the recovery survival rate of the escherichia coli strain is significantly reduced with the prolongation of the preservation time in the glycerol-physiological saline preservation group when the storage time is 6 months, 12 months, 18 months and 24 months, and the recovery survival rate of the escherichia coli strain is 0 when the storage time is 30 months and 36 months; when the preservation time is 36 months, the bacteria preservation solution prepared by the method can preserve escherichia coli, and the recovery survival rate can reach 94%. Compared with the glycerol-normal saline preservation group, the bacterial preservation solution is more beneficial to long-term preservation of bacterial strains and can also ensure the recovery survival rate of the bacterial strains.
Example 9 determination of the preservation Effect of bacterial preservative fluid on different strains at different preservation temperatures
The bacterial preservation solution and the glycerol-normal saline preservation solution prepared by the method of the embodiment 4 are used for preserving strains such as staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, bifidobacterium, lactobacillus plantarum and the like, and the viability of the different strains is measured after the strains are preserved for 20 days at 10 ℃, 4 ℃ below zero and 20 ℃ below zero respectively, and the test results are shown in table 3.
TABLE 3 preservation Effect of bacterial preservation solutions on different strains at different preservation times
As shown in Table 3, the bacterial preservation solution of the present invention and the liquid bacterial strain products obtained by mixing with different bacterial strains are preserved for 20 days at-20-10 ℃ respectively, the viable cell rate can reach more than 90%, and the viable cell rate is reduced only slightly; the liquid bacterial strains obtained by mixing glycerol-physiological saline and different bacterial strains are respectively preserved for 20 days at the temperature of-20 ℃ to 10 ℃, the living cell rate is only 36 percent, and the living cell rate is obviously reduced by 60 percent compared with the preservation solution for the bacteria. Therefore, the bacterial preservative fluid provided by the invention cannot be suitable for various bacterial strains, can prolong the preservation time of the bacterial strain at low temperature, and is convenient for long-time cold chain transportation and product storage.
Example 10 demonstrates that the components have a synergistic effect
Comparative example 1: compared with the example 4, the trehalose is removed, and the rest components and the proportion are unchanged;
comparative example 2: in comparison to example 4, sodium citrate was replaced with sodium citrate;
comparative example 3: compared with example 4, pyridoxine hydrochloride is removed, and the rest components and the proportion are unchanged;
comparative example 4: compared with the example 4, the sodium sulfite is removed, and the rest components and the proportion are unchanged;
comparative example 5: compared with example 4, the glycerol is removed, and the rest components and the proportion are unchanged.
Recovery survival rates of E.coli at various shelf lives were determined as in example 8 and are shown in Table 4;
TABLE 4 Table 4
| 6 months of | For 12 months | 18 months of | 24 months of | For 30 months | For 36 months | |
| Comparative example 1 | 82％ | 71％ | 57％ | 42％ | 38％ | 33％ |
| Comparative example 2 | 86％ | 75％ | 62％ | 53％ | 47％ | 39％ |
| Comparative example 3 | 72％ | 63％ | 51％ | 44％ | 40％ | 37％ |
| Comparative example 4 | 66％ | 60％ | 49％ | 41％ | 35％ | 29％ |
| Comparative example 5 | 63％ | 56％ | 50％ | 46％ | 42％ | 38％ |
As can be seen from Table 4, the survival rate of the prepared bacterial preservation solution after resuscitating is obviously reduced under different preservation time, and the bacterial preservation solution is not suitable for long-term preservation due to the removal of any one of the components or the replacement of the components with the same functions. It will be appreciated that the components of the present invention work together and are not a superposition of the effects of the individual components.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A bacterial preservative solution, wherein 100mL of the bacterial preservative solution comprises: 10-30g of peptone, 5-25g of trehalose, 5-15g of sodium chloride, 5-10g of sodium citrate, 0.1-1g of sodium sulfite, 0.1-0.5g of pyridoxine hydrochloride, 0.1-0.7g of ascorbic acid and 5-15mL of glycerol, and the volume is fixed to 100mL by deionized water.
2. The bacteria preservative fluid of claim 1, wherein the 100mL of bacteria preservative fluid comprises: 16g of peptone, 11g of trehalose, 7g of sodium chloride, 8g of sodium citrate, 0.8g of sodium sulfite, 0.4g of pyridoxine hydrochloride, 0.6g of ascorbic acid and 7mL of glycerol, and the volume is fixed to 100mL by deionized water.
3. The bacteria preservative fluid of claim 1, wherein the 100mL of bacteria preservative fluid comprises: 21g of peptone, 13g of trehalose, 11g of sodium chloride, 5g of sodium citrate, 0.5g of sodium sulfite, 0.2g of pyridoxine hydrochloride, 0.4g of ascorbic acid and 13mL of glycerol, and the volume is fixed to 100mL by deionized water.
4. A bacteria preservation solution according to claim 1 or 3, further comprising a pH adjuster.
5. The bacterial preservative solution according to claim 4, wherein the pH adjustor is added in an amount to adjust the pH of the bacterial preservative solution to 6.5 to 7.5.
6. The preparation method of the bacterial preservation solution is characterized by comprising the following steps of:
1) Weighing: weighing raw materials according to any of the weight parts of claims 1-3 for later use;
2) Mixing peptone, trehalose, sodium chloride, sodium citrate, sodium sulfite, pyridoxine hydrochloride, ascorbic acid, glycerol and deionized water to obtain a mixed solution;
3) And regulating the pH value of the mixed solution to 6.5-7.5 by adopting a pH regulating reagent, and sterilizing at 120-122 ℃ for 15-20min to obtain the bacteria preservation solution.
7. Use of a bacterial preservative fluid obtained by the method according to claim 6 for the cultivation and preservation of bacterial species.
8. The use of the bacterial preservation solution according to claim 7 for culturing and preserving bacterial species, wherein the bacteria are gram-negative bacteria or gram-positive bacteria.
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