A kind of lyoprotectant and its preparation method and use method
A cryoprotectant and cryoprotectant technology, which is applied in the field of freeze-dry protectant and its preparation, can solve the problems of exogenous cryoprotectant heavy workload, different protective effect, heavy screening of compound protective agent, etc.
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[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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