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A high-efficiency wall-breaking method for single-cell microorganisms

A microbial and single-cell technology, applied in the biological field, can solve the problems of large differences in the composition and thickness of microbial cell walls, high cost, and easily damaged products, and achieve immeasurable economic benefits, reduce production costs, and improve work efficiency.

Inactive Publication Date: 2011-12-28
CHINA AGRI UNIV
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  • Description
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Problems solved by technology

Due to the wide variation in microbial cell wall composition and thickness, there is no universal method for cell disruption for bacteria and fungi
Generally, the use of acid and alkali to break the wall will cause pollution, and it is easy to destroy the product to be extracted. The chemical method of breaking the wall is not friendly to the environment. The existing pressure breaker (FrenchPress) has extremely low efficiency and is only used in laboratories and is not suitable for use. break down of yeast
However, some yeasts need to use helicase or cellulase to assist in breaking the wall, which has no significant effect on some yeasts with thick walls. It is often necessary to mix different enzyme solutions according to the situation and adjust the enzymatic hydrolysis conditions, which is costly and inefficient, and is not suitable for mass production use

Method used

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  • A high-efficiency wall-breaking method for single-cell microorganisms

Examples

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Effect test

Embodiment 1

[0021] The cell disruption of embodiment 1 prokaryotic unicellular organism

[0022] In this example, the single-cell model microorganism Magnetotactic spirilla (Magnetospirillum gryphiswaldense) is used as a representative strain, and the fermented magnetotactic bacteria are directly passed into the high-pressure homogenizer through a pipeline from the fermenter, and the pressure is set. The flow rate is 7L / h. After 2 cycles, the cells were all crushed, and the magnetosomes were extracted. Observation by an electron microscope showed that the particles of the magnetosomes were uniform, the outer membrane was complete, and the quality was intact. Table 1 shows the wall breaking effect of Magnetospira under different conditions. figure 1 It is the result of 20% cell fluid, broken under 60Mpa pressure, and stained with crystal violet. The cell disruption rate reaches 90%.

[0023] Table 1 The wall-breaking effect of Magnetospira under different conditions

[0024] Ce...

Embodiment 2

[0025] The cell disruption of embodiment 2 eukaryotic unicellular organisms

[0026] In this example, the unicellular microorganism Phaffia rhodozyma was used as a representative strain, and 10%, 20% and 30% cell suspensions were prepared respectively, and passed into a high-pressure homogenizer to break the wall under different conditions. The results are shown in Table 2.

[0027] Table 2 The wall breaking effect of Phaffia yeast under different conditions

[0028] Cell concentration (W / V)

[0029] It can be seen from the above table that the wall breaking rate of 10% cell suspension can reach 87.7% under the pressure of 90Mpa, and the treatment of 120Mpa and 150Mpa can completely break the cell wall, and the pigment extraction rate is nearly 100%. Therefore, we choose 120Mpa as the optimum pressure for Phaffia yeast wall breaking, and apply it in large-scale production. figure 2 It is a 30% cell suspension, a 400-fold optical microscope photo of the cells after...

Embodiment 3

[0030] Example 3 Large-flow high-pressure homogenizer to the wall-breaking effect of bacteria and yeast

[0031] Table 3 The effect of breaking the wall under different conditions

[0032] Cell concentration (W / V)

[0033] Table 3 shows the wall-breaking effect of large-flow high-pressure homogenizers on Magnetospira and Phaffia yeast. Using a working pressure of 80-140Mpa, the flow rate is 75 liters / h, and the circulation is 2-4 times, all of which can reach 98%- 100% wall breaking effect.

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Abstract

The invention provides an efficient and fast method for breaking the wall of single-cell microorganisms. In the method, the cells are first prepared into a 10-30% cell suspension and passed into a high-pressure homogenizer. For prokaryotic single-cell microorganisms, the pressure is set to For eukaryotic single-cell microorganisms, set the pressure at 90-150Mpa, flow rate 5-80 liters / hour, and cycle 1-4 times to achieve 100% wall breaking of bacteria or yeast. The method of the invention can be effectively applied to the extraction process of fermentation engineering products, saves time, improves production efficiency, and does not require any chemical drug assistance, and is especially suitable for the fermentation industry.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cell breaking method for single-cell microorganisms. Background technique [0002] Microbial fermentation plays a huge role in industry. Many antibiotics, enzymes, organic acids, pigments, etc. are obtained through microbial fermentation. In particular, the construction of genetically engineered strains and the efficient expression of target products have become a way for people to obtain a large number of beneficial products. Due to the wide variation in microbial cell wall composition and thickness, there is no general method for cell disruption for bacteria and fungi. Generally, the use of acid and alkali to break the wall will cause pollution, and it is easy to destroy the product to be extracted. The chemical method of breaking the wall is not friendly to the environment. The existing pressure breaker (FrenchPress) has extremely low efficiency and is only used in laboratories...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12R1/01C12N1/20C12R1/85C12N1/16
Inventor 李颖唐涛苗莉莉姜伟田杰生王珍芳李季伦
Owner CHINA AGRI UNIV
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