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Method for improving survival ratio of lactobacillus after freeze drying by liquid nitrogen prefreezing

A technology of lactic acid bacteria and survival rate, applied in the field of pre-freezing to improve the survival rate of lactic acid bacteria after freeze-drying, can solve the problems of cell membrane and organelle damage, solute damage, high solute concentration, etc., achieve strong industrial implementation, prevent material foaming, pre-freeze Freezing Method Simple Effects

Inactive Publication Date: 2009-08-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, if the freezing speed is too slow, it will also cause partial freezing of the extracellular solution, so that the concentration of solute in the unfrozen solution outside the cell is too high, resulting in solute damage; when the freezing speed is too fast, the water in the cell has no time to leak out. Larger ice crystals will be formed, which will damage the cell membrane and organelles and cause mechanical damage to the ice crystals in the cells.
There are also research reports that when the freezing rate is between 5°C / min and 180°C / min, the intracellular water will completely leak out of the cells during the freezing process, no crystallization will occur in the cells, and the cell survival rate is high; when the freezing rate is greater than At 5000°C / min, the intracellular water quickly forms crystals, no extravasation occurs, and the cell survival rate is relatively high; but if the freezing speed is between 180°C / min and 5000°C / min, the intracellular water is easy to flow during the extravasation process. mechanical damage to cells

Method used

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  • Method for improving survival ratio of lactobacillus after freeze drying by liquid nitrogen prefreezing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Centrifuge 1000ml of Lactobacillus acidophilus bacteria solution at a speed of 6000r / min and a temperature of 4°C for 20min, discard the supernatant, wash with physiological saline and collect the bacteria sludge. In the bacteria sludge obtained by centrifugation, add the compound protective agent of twice the volume of the bacteria sludge, in the protective agent, skim milk: trehalose: vitamin C: sodium glutamate (mass ratio) = 15:4:1:1.5, and mix uniform.

[0026] Take a 500ml beaker, fill it with 15ml of bacteria slime, put it into a vacuum freeze dryer for pre-freezing, the pre-freezing temperature is -80°C, and the time is 2h.

[0027] Take four 500ml beakers, fill them with liquid nitrogen, quickly use 5ml, 1ml, 200μl, and 100μl pipette guns to drop 15ml of bacteria sludge into the four beakers respectively, and place them in the liquid nitrogen environment for 2 hours, then put Freeze dry in a freeze dryer.

[0028] To measure the number of viable bacteria in t...

Embodiment 2

[0030] Centrifuge 1000ml of Lactobacillus bulgaricus liquid at a speed of 4000r / min for 20min, discard the supernatant, wash with normal saline and collect the sludge. Add the same volume of the compound protective agent of Example 1 to the bacteria sludge obtained by centrifugation, and mix well.

[0031] Take a 500ml beaker, fill it with 15ml mixed sludge, put it into a vacuum freeze dryer for pre-freezing, the pre-freezing temperature is -80°C, and the time is 2h.

[0032] Take a 500ml beaker, fill it with liquid nitrogen, quickly drop 15ml of bacterial solution into the beaker with a 200μl pipette gun, place it in the liquid nitrogen environment for 1 hour, and then freeze it in a freeze dryer.

[0033] To measure the number of viable bacteria in the freeze-dried bacterial powder, add 10ml of normal saline to the bacterial powder, dilute it step by step, and then spread it on a flat plate to count the number of viable bacteria. After ordinary freeze-drying, the number of ...

Embodiment 3

[0035] Centrifuge 1000ml of Streptococcus thermophilus bacterial liquid at a speed of 6000r / min for 20min, discard the supernatant, wash with normal saline and collect the sludge. Add the same volume of the compound protective agent of Example 1 to the bacteria sludge obtained by centrifugation, and mix well.

[0036] Take a 500ml beaker, fill it with 15ml mixed sludge, put it into a vacuum freeze dryer for pre-freezing, the pre-freezing temperature is -80°C, and the time is 2h.

[0037] Take a 500ml beaker, fill it with liquid nitrogen, quickly drop 15ml of bacterial solution into the beaker with a pipette gun with a 100μl tip, place it in the liquid nitrogen environment for 1 hour, and then freeze it in a freeze dryer.

[0038] To measure the number of viable bacteria in the freeze-dried bacterial powder, add 10ml of normal saline to the bacterial powder, dilute it step by step, and then spread it on a flat plate to count the number of viable bacteria. After ordinary freeze...

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PUM

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Abstract

The invention discloses a method for improving the survival rate of lactobacilli after freeze drying by using prefreezing with liquid nitrogen, which comprises the following steps: (1) collecting well-cultivated bacterial sludge of lactobacilli and adding a protective agent according to a certain mixing ratio; (2) dripping the bacterial sludge into liquid nitrogen for prefreezing; (3) freeze drying the pre-frozen bacterial sludge; and (4) collecting product for storage under vacuum. The method of the invention effectively increases the number of live bacteria after freeze drying by replacing the conventional prefreezing method with prefreezing with the liquid nitrogen, and adopting the certain compound protective agent at the same time, is simple in related materials and convenient operation, and has high industrial implementation performance.

Description

technical field [0001] The invention relates to a freeze-drying pre-freezing method, in particular to a pre-freezing method for improving the survival rate of lactic acid bacteria after freeze-drying. Background technique [0002] With the improvement of people's quality of life requirements, freeze-dried products are becoming more and more popular. However, due to the fact that the technology of vacuum freeze-drying microorganisms in China is not perfect enough, the number of viable bacteria in freeze-dried products is not high enough, and the freeze-drying technology is still in the development stage. [0003] The direct-throwing freeze-dried starter has a high content of viable bacteria, is convenient for storage and transportation, has a high degree of mechanization, and is convenient for industrialized mass production. It is the main development direction of high-efficiency concentrated starter in the future. The main method of preparing direct-throwing freeze-dried st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12R1/225C12R1/23C12R1/46
Inventor 朱东升何国庆李青青
Owner ZHEJIANG UNIV
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