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Freeze-drying protective agent as well as preparation method and application method thereof

A technology of cryoprotectant and cryoprotectant, applied in the field of freeze protectant and its preparation, can solve the problem of increasing the complexity of finding cryoprotectant, heavy workload of exogenous cryoprotectant, screening of compound protectant Large volume and other issues, to achieve the effects of good cryoprotection effect, strong industrial implementation, and low cost

Active Publication Date: 2017-05-31
ZHENGZHOU UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, due to the specificity of different types of lactic acid bacteria cells, the specificity of the physical and chemical properties of different types of protective agents, and the complexity of the interaction between the two, a single protective agent alone cannot meet the requirements of bacteria to resist external harsh conditions, and the combination of protective agents Although the protective effect of cryoprotectant is obvious, the screening workload is huge, and the protective effect of the same compound protective agent on different species of lactic acid bacteria will be different, which increases the complexity of finding cryoprotectant on the other hand. The workload of exogenous cryoprotectants is heavy, the method is cumbersome, and the cost is high

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  • Freeze-drying protective agent as well as preparation method and application method thereof
  • Freeze-drying protective agent as well as preparation method and application method thereof
  • Freeze-drying protective agent as well as preparation method and application method thereof

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preparation example Construction

[0029] The invention provides a method for preparing and using such a cryoprotectant, comprising the following steps:

[0030] (a) Carry out freezing pretreatment to the lactic acid bacteria cultivated to the logarithmic phase; wherein said lactic acid bacteria include at least one selected from the group consisting of Lactococcus lactis, Pediococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium , Lactobacillus delbrueckii subsp. bulgaricus; culture conditions: temperature 31-37 ℃, time 12-36h. It should be understood that the culture method of the above-mentioned strains can adopt the existing culture technology, and the purpose is to cultivate the strains to the logarithmic phase.

[0031] The conditions for the freezing pretreatment of lactic acid bacteria are as follows: the gradient cooling treatment is 0-31° C., 0-45° C., and -45-80° C., and the freezing treatment is 0.5-5 hours under each temperature gradient.

[0032] (b) Collect the lactic aci...

Embodiment 1

[0045] Such as figure 1As shown, the intracellular substance of Lactococcus lactis was prepared as a protective agent according to the preparation process of the lactic acid bacteria freeze-drying protective agent. Specifically, after Lactococcus lactis was cultured at 31°C for 16 hours, freezing pretreatment was performed, and the gradient cooling conditions were 8°C, -16°C, and -67°C, and each treatment was performed for 5 hours in sequence; 5000rpm, centrifuged for 20min, and 4°C, collected the bacteria. Wash three times with Tris-HCl buffer solution in step (b) of the technical solution for body use, and the mass-volume ratio of bacteria and buffer solution is 1:50; then perform ultrasonic crushing, power 300w, ultrasonic 5s, stop 5s, ultrasonic 60min in total; centrifuge Collect the supernatant after ultrasonic crushing, centrifuge at 6000rpm, 20min, and 4°C; vacuum freeze-dry the supernatant, cold trap temperature -70°C, and vacuum 1Pa to obtain a cryoprotectant; then ve...

Embodiment 2

[0051] Such as figure 1 As shown, the intracellular substance of Lactococcus lactis was prepared as a protective agent according to the preparation process of the lactic acid bacteria freeze-drying protective agent. Specifically, after Lactococcus lactis was cultured at 31°C for 16 hours, freezing pretreatment was performed, and the gradient cooling conditions were 31°C, 0°C, and -45°C, and each treatment was performed for 2 hours in sequence; 5000rpm, centrifuged for 10min, and 6°C, collected the bacteria. Wash three times with Tris-HCl buffer solution in step (b) of the technical scheme, and the ratio of the mass volume of the bacteria to the buffer solution is 1:30; then perform ultrasonic crushing, power 350w, ultrasonic 5s, stop 5s, ultrasonic 30min in total; centrifuge and collect The supernatant after ultrasonic crushing was centrifuged at 16000rpm for 10min at a temperature of 4°C; the supernatant was vacuum freeze-dried at a cold trap temperature of -70°C and a vacuum...

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Abstract

The invention provides a freeze-drying protective agent as well as a preparation method and an application method thereof, in order to solve the problem on the preparation of the freeze-drying protective agent for different lactic acid bacteria strains in the prior art. According to the freeze-drying protective agent as well as the preparation method and the application method thereof disclosed by the invention, the freeze-drying protective agent is directly derived from corresponding lactic acid bacteria cells after freezing pretreatment at a low temperature, and is directly obtained by cell crushing treatment; and the freeze-drying protective agent disclosed by the invention has the advantages of easy availability for raw materials, simple preparation process, low cost, good freezing protection effect and stronger industrialized implementation.

Description

technical field [0001] The invention relates to the technical field of microorganism preservation, in particular to a freeze-drying protective agent and its preparation method and usage method. Background technique [0002] Lactic acid bacteria are an important microorganism in the food industry and are widely used in the fermentation of dairy products and meat products. In order to facilitate production, high-viability strains are often preserved by freeze-drying methods, and the development of freeze-drying technology and related cryoprotectants is very critical. [0003] 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. If lactic acid bacteria are directly freeze-dried, the mortality rate will reach more than 90%, so it is very important to choose a suitable freeze-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/04C12N1/20C12R1/46C12R1/25C12R1/245C12R1/225C12R1/01
CPCC12N1/04C12N1/20
Inventor 鲁吉珂林松洋刘鑫康巧珍郝利民鲁来政
Owner ZHENGZHOU UNIV
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