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Method of conservation and recovery of haematococcus pluvialis cell

A technology of Haematococcus pluvialis and a preservation method is applied in the field of preservation and recovery of Haematococcus pluvialis cells, and can solve the problems such as the recovery survival rate of only 66.13%, the complicated operation technology preservation equipment, and the limitation of the efficiency and scale of preservation. , to achieve the effect of inhibiting the proliferation of bacteria and miscellaneous algae, high recovery survival rate, and simple method

Active Publication Date: 2017-08-18
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, Haematococcus pluvialis can be preserved for a long time by protecting it with glycerol at low temperature in liquid nitrogen (CN105779292A-a method for cryopreservation of aseptically treated Haematococcus pluvialis), but the recovery survival rate is only 66.13% , and requires complex operating techniques and expensive preservation equipment
Due to the limitations of the above methods in terms of cell survival rate, storage time, labor intensity, and equipment requirements, the efficiency and scale of storage are limited, which brings many difficulties to actual production.

Method used

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  • Method of conservation and recovery of haematococcus pluvialis cell

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Haematococcus pluvialis NIES-144 strain (commercially available), respectively: 1) in total nitrogen BBM medium (commercially available) at 50 μE / m 2 The green zoospores obtained by cultivating algal strains for 5 days under / s light;

[0025] 2) In nitrogen-free BBM medium at 300μE / m 2 The chlamydospores obtained by cultivating algal strains for 10 days under / s light;

[0026] The green zoospores and thick-walled spores obtained above were left to settle and concentrate, and then placed in the fresh-keeping room of the refrigerator at 5 degrees Celsius for cold acclimation for 24 hours; then transferred to the refrigerator at -20 degrees Celsius to freeze into ice and continue to freeze for 60 days; After storage, take it out and let it thaw at room temperature.

[0027] Afterwards, dilute the above-mentioned concentrated algae liquid with the BBM medium containing 4mmol / L sodium nitrate to a cell density of 0.449g / L, and place it at 200μE / m 2 / s light, and pass 1%...

Embodiment 2

[0042] Haematococcus pluvialis SCCAP K-0084 algal strain (commercially available), in nitrogen-free BBM medium at 300 μE / m 2 Cultivate the algal strain under the light of / s for 10 days to obtain chlamydospores. After standing and settling, the concentrated algae liquid with a cell density of 60g / L was obtained. The concentrated algae liquid was placed in the fresh-keeping room of the refrigerator at 5 degrees Celsius for 24 hours of cold acclimation; after that, it was packed in In multiple sample bags, after subpackaging, place them in refrigerators at -5, -20, and -80 degrees Celsius to freeze into ice, and continue to freeze for 60 days. Then take it out and let it melt at room temperature, dilute and melt it with BBM medium containing 4mmol / L sodium nitrate, concentrate the algae liquid to a cell density of 0.5g / L, and place it at 200μE / m 2 / s light, and pass 1% CO 2 air culture. After culturing under light for 48 hours, use a fluorescent microscope to observe and count...

Embodiment 3

[0044] In nitrogen-free BBM medium at 300 μE / m 2 Cultivate the algae strain under the light of / s for 10 days to obtain the thick-walled algae liquid. After standing and settling, the concentrated algae liquid with a cell density of 40g / L was obtained, which was placed in the fresh-keeping room of the refrigerator at 5 degrees Celsius for 24 hours of cold acclimation; transfer to a freezer at -5°C to freeze into ice. After 3, 38 and 75 days of frozen storage, take out a bag of frozen algae and thaw at room temperature; then dilute the thawed and concentrated algae liquid with BBM medium containing 4mmol / L sodium nitrate to a cell density of 0.5g / L , placed in 200μE / m2 / s light, and passed through 1% CO 2 air culture. Sampling at regular intervals to monitor cell growth. The experiment in which only cold-acclimated cells were used as inoculation materials without freezing was used as a control. The results showed that the survival rates of recovered cells after 3, 38 and 75 ...

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Abstract

The invention belongs to the field of algae biotechnology, in particular relates to a method of conservation and recovery of haematococcus pluvialis cells. After the cold acclimation of a concentrate solution of haematococcus pluvialis cells under 0-10 centigrade, the solution is subject to frosting and preserving in shading conditions and below the freezing point. By the method of conservation and recovery of haematococcus pluvialis cells, the seaweed concentrate solution after the short-time cold acclimation is subject to frosting and preserving, the middle-long term preservation method of high survival rate of the haematococcus pluvialis cells is achieved; at the same time the method of recovery of the preserved cells is achieved; the survival rate of cell recovery is high, after the preservation of 75 days, the survival rate of recovered cells is up to 95% or above, the growth after the recovery is fast; the propagation of bacteria and miscellaneous algae is effectively prevented, at the long-term preservation stage no obvious bacteria and miscellaneous algae are bred, the cells after the recovery are grown rapidly, and the proliferation of bacteria and miscellaneous algae is inhibited. The method is simple, does not require the assistance of specialized equipment; the method is tended to the preservation of a large number of algae species, and is easy to carry out in production and in practice.

Description

technical field [0001] The invention belongs to the field of algae biotechnology, and in particular relates to a method for preserving and recovering Haematococcus pluvialis cells. Background technique [0002] Haematococcus pluvialis is a green alga that produces natural astaxanthin, belonging to the family Chlorophyta Chlorophyta Volvoceae Haematococcus. Its cells produce a large amount of astaxanthin in a specific environment and store it in the cell, and the content of astaxanthin is as high as 7%. Astaxanthin in Haematococcus pluvialis is mainly an esterified form of astaxanthin, with a molecular structure of 3S, 3S' configuration, so it is considered to be the best source of natural astaxanthin, used in health food, coloring agent, personal care Products and high-end feeds are widely used, and have become a hot spot in the development and utilization of algae resources in recent years. [0003] In the large-scale production process of Haematococcus pluvialis, especia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/04C12N1/12C12R1/89
CPCC12N1/04C12N1/12
Inventor 陈林刘天中程文涛张维王俊峰
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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