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Low-temperature microorganism preservation protectant

A technology for cryopreservation and microorganisms, which is applied in the fields of microorganisms, preserved microorganisms, and microorganism-based methods, and can solve the problems of slow recovery of freeze-dried strains.

Active Publication Date: 2017-05-31
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The invention provides a microbial low-temperature preservation protective agent, which can be used for but not limited to BCG live bacteria such as BCG strains, BCG live bacteria samples, or other bacteria and microorganisms in low-temperature refrigeration, so as to improve microorganisms, For example, the strains used for BCG production, the quality of the first recovery, and technical problems such as slow recovery of freeze-dried strains

Method used

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  • Low-temperature microorganism preservation protectant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1 Configure the stock solution of cryopreservation protection agent with different contents of trehalose, glycerin, polysorbate-80, potassium chloride and sodium glutamate

[0067] According to the content of the components in Table 1, take the corresponding substances, dissolve them in an appropriate amount of ultrapure water, set the volume to 100mL and adjust the pH value to 7.4 with hydrochloric acid and sodium hydroxide solution; The stock solution of the cryopreservation protective agent of the present invention.

[0068] Table 1 Proportion ratio table of cryopreservation protective agent stock solution (per 100ml)

[0069] Trehalose (g) Glycerin (ml) Polysorbate 80(ml) Potassium chloride (g) Sodium glutamate (g) Recipe 1 10 20 1 2 0.8 Recipe 2 10 10 2 2 0.8 Recipe 3 10 2 4 2 0.8 Recipe 4 20 20 1 2 0.8 Recipe 5 20 10 2 2 0.8 Recipe 6 20 2 4 2 0.8 Recipe 7 30 20 1 2 ...

Embodiment 2

[0072] The effect research of embodiment 2 cryopreservation protective agent

[0073] Harvest the BCG bacteria film grown on the surface of liquid Sutong medium, wash it with sterile physiological saline, press dry and weigh it, add physiological saline according to the weight of the wet bacteria, and grind it in a spherical bottle containing steel balls until the bacterial solution is uniform samples were taken for concentration determination. Dilute the bacterial solution with normal saline to a concentration of 0.2mg / ml, take the same volume of the bacterial solution and mix it with the low-temperature storage protection agent stock solution of formula 1-9 and control 1 in Example 1 and mix evenly at a ratio of 1:1 to obtain a semi-finished product, BCG The concentration is 0.1mg / ml, divided into 0.5ml per cryopreservation tube, pre-frozen at 4°C for more than 12 hours, pre-frozen at -20°C for more than 12 hours, and frozen at -70°C to obtain the finished product. In addit...

Embodiment 3

[0083] The long-term cryopreservation of embodiment 3 cryopreservation protective agent and the preservation effect of repeated freezing and thawing

[0084] According to the preparation method of Example 1, prepare the stock solution of the control frozen storage solution and the stock solution of the cryopreservation protection agent of the present invention with the composition content of Table 3:

[0085] Table 3 Ratio of original solution of cryopreservation protection agent (per 100ml)

[0086]

[0087] According to the preparation method of Example 2, take the same volume of BCG bacterial liquid and the low-temperature storage protection agent stock solution of formula 10-15 and control 2-4 and mix evenly at 1:1 to obtain a semi-finished product with a BCG concentration of 0.1 mg / ml, subpackage, 0.5ml per cryopreservation tube, pre-freeze at 4°C for more than 12 hours, pre-freeze at -20°C for more than 12 hours, and freeze at -70°C to obtain the finished product. T...

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Abstract

The invention relates to a low-temperature microorganism preservation protectant, as well as a preparation method and use thereof. The low-temperature microorganism preservation protectant is prepared from the following components: non-reducing saccharide, glycerin, a nonionic surfactant, a potassium salt, glutamate and water. The invention also discloses a technical method for preserving bacillus calmette-guerin at low temperature for long. A technical support is provided for deep development of related researches on the bacillus calmette-guerin.

Description

technical field [0001] The invention relates to a low-temperature storage protection agent for microorganisms, a preparation method and application thereof. Background technique [0002] Freeze-drying, a commonly used method to achieve long shelf-life of microorganisms (CarcobaR, 2000), is widely regarded as one of the most suitable dehydration processes for bacteria with the aim of obtaining stable final reagents (Carvalho AS, 2004), and is also used One of the most common methods for storing microbial cell cultures. Cell viability after freeze-drying reflects the resistance of cells to the effects of rapid freezing and drying (Miyamoto‐Shinohara Y, 2008). It is generally believed that most unprotected bacteria are killed after freeze-drying, while those that survive die rapidly when subjected to storage. In order to obtain ideal freezing and freeze-drying effects, in addition to the process, it is also very important to screen and use suitable cryoprotectants. Appropria...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/04C12R1/32
CPCC12N1/04C12N1/20
Inventor 赵爱华王国治付丽丽寇丽杰
Owner NAT INST FOR FOOD & DRUG CONTROL
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