JPH069502B2 - Method of crushing bacterial cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for pulverizing bacterial cells, which enables efficient extraction of a useful substance produced in the cell membrane by the bacterial cell to the outside of the cell membrane.

[Conventional technology]

Recently, a large amount of raw materials for pharmaceuticals and the like are being produced by growing new strains of bacteria and bacteria by gene recombination technology.

In these manufacturing processes, it is necessary to extract specific components from the cells through the cell membrane.

As a conventional extraction method, after preliminarily shattering the bacterial cells with a ball mill, a method of extracting the specific component with a solvent, or by dissolving the bacterial cell with the cell membrane using a strong solvent such as chloroform to extract the specific component There is a method of extracting.

[Problems to be solved by the invention]

However, the former method among the above-mentioned conventional methods has a microbial cell size of a few microns, and when treated with a ball mill, the microbial cells slide into each other and enter the gap, so it takes a long time to shatter. It is time consuming and unsuitable for mass production.

On the other hand, in the latter method, a solvent such as chloroform is harmful and it is necessary to remove it in a post-treatment step.

In view of such a situation, the present invention intends to provide a method capable of destroying the cell membrane of a bacterial cell in a short time and by a method that is also easy to recover a solvent, and extracting a useful component in the bacterial cell out of the system. To do.

[Means for solving problems]

The present invention is a method for powdering bacterial cells, which comprises mixing a culture solution of bacterial cells with a dissolving solvent in a supercritical state or a pseudocritical state, and then immediately lowering the pressure.

The term "supercritical state" as used in the present invention means a state under a temperature or pressure condition that is a critical temperature or higher and a critical pressure or higher of a dissolving solvent,
The pseudocritical state is a temperature Tc below the critical temperature Tc of the dissolving solvent, a temperature T of the counter-critical temperature T _R = T / Tc (where 0.90 <T _R <1.0), and the pressure is the dissolving solvent at that temperature. Means a state above the saturated vapor pressure.

It is known that the density of the dissolved solvent changes rapidly with pressure near the critical point, and the present invention utilizes this. That is, the dissolution solvent near the critical point has a density comparable to that of a liquid, and when it is easily diffused into the cell membrane, if the pressure is suddenly lowered from this state, the high-density dissolution solvent diffused into the cell membrane expands rapidly. However, the cell membrane cannot be resisted by this expansion force and is broken into pieces.

In the supercritical state of a dissolving solvent, its density is similar to that of a liquid, but its diffusion coefficient is several tens of times larger than that of a liquid, and it easily passes through cell membranes, so it is preferable to make it a supercritical state rather than a pseudocritical state. .

The dissolution solvent used in the present invention is preferably a solvent that diffuses the cell membrane of bacterial cells, such as carbon dioxide gas and carbon number 2-4.
In addition to the hydrocarbons such as C ₂ H ₄ , C ₂ H _{6 and the} like and mixtures thereof, an inorganic or organic solvent whose critical temperature is below the heat distortion temperature of the bacterial cells or a mixed solvent thereof can be used. is there.

The following table illustrates the main solvents used in the present invention.

Table Solvent name Critical temperature (℃) CO ₂ 31.1 C ₂ H ₄ 9.7 C ₂ H ₆ 32.4 N ₂ O 36.5 C ₃ H ₆ 92.3 C ₃ H ₈ 96.8 H ₂ S 100.4 C ₄ H ₁₀ 152.0 In the present invention, it is particularly preferable to use CO ₂ which has a critical temperature near room temperature and is harmless and easy to collect.

The addition amount of the dissolution solvent in the present invention is preferably in the range of 1 to 10 parts by weight with respect to 1 part by weight of the bacterial cell culture solution.

When carbon dioxide is used, the pressure is 10 at a temperature of 35 ° C.
The density is about 0.8 at 0 kg / cm ² G and 10 at normal pressure.
It is on the order of ^-3 , and it is understood that the density difference of about 1000 times becomes expansion force, and the force is very large.

There is no particular limitation on the bacterial cell culture medium that can be the subject of the present invention. An example is Mortiereilan, which is a type of filamentous fungus that selectively produces γ-linolenic acid, an unsaturated fatty acid.

The culture broth should preferably be preliminarily freed of water by a centrifuge or the like. The presence of such water is not preferable because it causes an increase in the diffusion resistance of the dissolving solvent.

An embodiment of the present invention will be described below with reference to FIG.

In the configuration of FIG. 1, 1 part by weight of the bacterial cell culture liquid is pumped from the bacterial cell culture supply line 1 by a high-pressure pump 2 of the bacterial cell culture liquid, while 1 to 10 parts by weight of the dissolving solvent is supplied to the dissolving solvent supply line 3 And the two are mixed by a mixer 4 such as a static mixer.

In the mixer 4, the pressure and temperature are controlled so that the dissolved solvent is in a supercritical state or a pseudocritical state.

Next, the pressure is rapidly reduced to near normal pressure by the pressure reducing valve 5, the mixed solution is introduced into the separation tank 6, and the cell body whose cell membrane has been destroyed from the cell-treated product outlet 8 at the bottom of the separation vessel 6. Is collected, and the dissolving solvent is collected from the dissolving solvent outlet 7 on the upper side. In addition, in FIG. 1, 9 is a heater.

〔Example〕

Hereinafter, the present invention will be described in detail with reference to Examples of the present invention.

Example 1 An autoclave was charged with 1 part by weight of a culture solution of Mortierella fungus, which is a type of filamentous fungus, dehydrated by centrifugation and 1 part by weight of a cell culture solution having a water content of 50% by weight, and 2 parts by weight of CO ₂ .
The mixture was mixed at a temperature of 35 ° C. and a pressure of 105 kg / cm ² G for 5 minutes, and the mixture was introduced from the autoclave into 10 atmospheric pressure separation tanks by rapidly opening the valve to lower the pressure,
When CO ₂ was discharged from the upper part of the separation tank and the treated bacterial cell culture solution remaining in the lower part was taken out and observed under a microscope, the cell membranes of almost all bacterial cells were broken, and fatty acids were dispersed outside.

In addition, in the above-mentioned Examples, almost the same results were obtained even in the supercritical state although the supercritical state was performed.

〔The invention's effect〕

INDUSTRIAL APPLICABILITY As described in detail above, the present invention, by using a dissolution solvent in a supercritical state or a pseudocritical state, can easily and quickly destroy cell membranes of cells, and can also easily recover the dissolution solvent. That is the effect.

[Brief description of drawings]

FIG. 1 is a flow sheet for explaining an embodiment of the present invention.

Claims (1)

[Claims] 1. A method for crushing bacterial cells, which comprises mixing a culture solution of bacterial cells with a dissolving solvent in a supercritical state or a pseudocritical state and then immediately reducing the pressure.