JPH0661260B2 - Method of crushing bacterial cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to a method for crushing bacterial cells, which enables efficient and energy-saving extraction of useful substances produced by bacterial cells inside the cell membrane to the outside of the cell membrane. Regarding

[Conventional technology]

Recently, a large amount of raw materials for pharmaceuticals 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, a method of extracting specific components after preliminarily shattering the bacterial cells with a ball mill, or a method of dissolving the bacterial cells together with the cell membrane using a strong solvent such as chloroform and extracting the specific components Etc.

[Problems to be solved by the invention]

However, the former method out of 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 is long 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 this case, if the evaporation separation by heating which is usually performed for the separation of the solvent is performed, the useful component is modified by the heating and the energy cost is increased due to the load of the latent heat of evaporation, which is not preferable. In particular, when the amount of solvent is large, the energy cost increases remarkably in proportion to it.

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 directed to a critical temperature T _R = T / T of a culture of a microbial cell at a temperature above the critical temperature and above the critical pressure, under pressure conditions (hereinafter, this state is referred to as the supercritical state) or below the critical temperature T _c.
C (where 0.90 <T _R <1.0), the pressure is higher than the saturated vapor pressure of the dissolving solvent at that temperature, and the condition is under pressure (hereinafter, this state is referred to as pseudocritical state). After mixing with a dissolving solvent that diffuses inside the cell membrane of the body, the pressure is instantly lowered and introduced into the separation tank at the same temperature, and the crushed bacterial cells are collected from the lower part of the separation tank and from the upper part of the separation tank. The solvent is recovered, the recovered solvent is then compressed, and the heat of compression generated thereby is used as a heating source for the separation tank.
The compressed solvent is a method for pulverizing bacterial cells, which is characterized by being mixed with the above culture solution and reused.

First, the concept underlying the present invention will be described.

As mentioned above, the term "supercritical state" as used in the present invention means a state under a temperature or pressure condition that is not lower than the critical temperature of the dissolving solvent and not lower than the critical pressure, and the pseudocritical state is the critical temperature T of the dissolving solvent.
c below, versus the critical temperature _T R = _T / Tc (where 0.90
At a temperature T of _<T R <1.0), pressure means a state on the saturated vapor pressure of the dissolved solvent in the temperature.

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

Therefore, in the present invention, the culture solution of the bacterial cells is mixed with a solvent in a supercritical state or a pseudocritical state, and after the dissolving solvent is diffused into the cell membrane of the bacterial cells, the pressure is instantly lowered to the temperature as it is. While retaining, the cell membrane of the microbial cells is crushed, and then the crushed bacterial cell product and the dissolving solvent are separately recovered.

Now the pressure is reduced and the temperature is near its critical point,
The dissolution solvent can be separated from the crushed bacterial cells in an energy-saving manner without the need for latent heat of vaporization of the dissolution solvent. The crushed bacterial cells are recovered from the lower part of the separation tank and the dissolved solvent is recovered from the upper part. . The recovered dissolved solvent is compressed in a compressor and used again for cell crushing in a super or pseudocritical state, and the compression heat generated in the compressor is used as a heat source for the separation process, so the entire process is extremely short. In addition, the solvent recovery is simple and can be reused, and the effective components in the bacterial cells can be taken out of the system by effectively utilizing energy.

The present invention will be described in more detail below.

While the dissolution solvent has a density similar to that of a liquid in the supercritical state, its diffusion coefficient is several tens of times larger than that of a liquid, and it easily passes through the cell membranes of bacterial cells. It is preferable to use a dissolving solvent.

The dissolution solvent used in the present invention is preferably a solvent that diffuses in the cell membrane of the bacterial cells, such as carbon dioxide CO ₂ , hydrocarbons having 2 to 4 carbon atoms such as C ₂ H ₄ , C ₂ H _{6 and the} like. In addition to the mixture and the like, an inorganic or organic solvent whose critical temperature is lower than the heat denaturation temperature of the bacterial cells or a mixed solvent thereof can be used. The following table illustrates the main solvents used in the present invention.

Table Solvent name Critical temperature (° C.) 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 As the dissolving solvent in the present invention, carbon dioxide gas, which has a critical temperature near room temperature and is harmless and easy to recover, is particularly preferable.
In the case of carbon dioxide gas, its critical temperature is 31.1 ° C., and at a temperature of 35 ° C., the density is about 0.8 at a pressure of 100 kg / cm ² G, and at normal pressure the density is on the order of 10 ⁻³ , This density difference of about 1000 times becomes the expansion force, so it can be seen that the force is very large.

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.

There is no particular limitation on the bacterial cell culture medium that can be the subject of the present invention. One example is Mortierella spp., Which is a type of filamentous fungus that selectively produces r-linolenic acid, an unsaturated fatty acid.

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

Hereinafter, an embodiment of the present invention will be described in detail with reference to the flow sheet shown in FIG. 1 part by weight of the bacterial cell culture liquid was pumped from the bacterial cell culture liquid supply line 1 by a high pressure bacterial cell culture liquid pump 2.
On the other hand, 1 to 10 parts by weight of the dissolving solvent is supplied from the dissolving solvent supply line 3, and both are mixed by a mixer 4 such as a static mixer. The pressure and temperature are controlled so that the dissolved solvent in the mixer 4 is in a supercritical state or a pseudocritical state. 9 is a heater. Then, the pressure is instantly reduced to near normal pressure by the pressure reducing valve 5, the mixture is introduced into the first separation tank 6, and the cell body whose cell membrane has been destroyed is discharged from the cell-treated product outlet 8 at the bottom of the separation vessel. Is collected, and the dissolving solvent is collected from the dissolving solvent outlet 7 on the upper side.

The lower the pressure in the first separation tank 6 is, the larger the differential pressure before and after the pressure reducing valve 5 is, and the greater the crushing effect is, but since more energy is required when re-compressing and reusing the dissolved solvent, The pressure in the first separation tank 6 is preferably in the range of 10 to 50 atm. When the pressure exceeds 50 atm, a large amount of components in the bacterial cells start to dissolve in the dissolving solvent in the upper part of the separation tank 7, which is not preferable.

The inventors of the present invention set the temperature of the first separation tank 6 near the critical point of the dissolution solvent and drastically lowered the pressure to accelerate the crushing of the microbial cells, and at the same time, to separate the microbial cells and the dissolution solvent from each other. It has been found that this method is very advantageous from the viewpoint of energy saving as it can be carried out without requiring latent heat of vaporization of the solvent.

On the other hand, the bacterial cells are introduced into the second separation tank 15 from the bacterial cell treated product outlet 8 by the pressure reducing valve 14. The purpose of installing the second separation tank 15 is to collect the dissolved solvent dissolved in the treated bacterial cell product and prevent its loss. Therefore, the second
It is preferable that the pressure in the separation tank 15 be near atmospheric pressure,
The temperature is preferably 20 to 50 ° C. in order to increase the vapor pressure of the dissolving solvent. It is not preferable to set the temperature over 50 ° C. because of energy loss.

According to the research conducted by the present inventors, it was found that the dissolved solvent is easily recovered at a temperature of 20 to 50 ° C.

The microbial cells are collected from the treated microbial cell withdrawal port 17, and the lysing solvent collected from the lysing solvent withdrawing ports 16 and 7 are compressed by the compressors 12 and 18, respectively, and then transferred to the mixer 4 by the lysing solvent supply line 3. Supplied and reused. The inventors can effectively use energy by using the compression heat of the compressors 12 and 18 as a heating source of the first separation tank 6 and / or the second separation tank 15 as shown in FIG. I also found In FIG. 1, 13 is a heater,
10 represents a pressure reducing valve.

〔Example〕

And which is one type Moruteiera centrifuge at dehydrated moisture content 50% by weight and the cell culture solution 1 part by weight of culture of bacteria Example 1 filamentous fungi, the CO ₂ 2 parts by weight, volume 1 of the autoclave The mixture was charged into the autoclave and mixed for 5 minutes at a temperature of 40 ° C. and a pressure of 105 kg / cm ² G, and then the mixture was introduced from the autoclave into a separation tank having a capacity of 10 and 20 kg / cm ² G by rapidly opening the valve. Then, CO ₂ was discharged from the upper part of the separation tank, and the cell culture remaining in the lower part was observed under a microscope. As a result, the cell membranes of almost all the cell bodies were broken, and fatty acids were dispersed outside. .

On the other hand, almost no impurities were detected in the recovered CO ₂ . This CO ₂ is compressed by a compressor at a temperature of 40 ° C and a pressure of 20 kg / c.
When compressed from m ² G to 110 kg / cm ² G, the temperature was about 85 ° C. It was found that this heat can be used as a heating source.

Although the above example was performed in the supercritical state, almost the same result was obtained even in the pseudocritical state.

〔The invention's effect〕

The present invention, as described in detail above, by using a dissolution solvent in the supercritical state or pseudocritical state, it is possible to easily and quickly destroy the cell membrane of the bacterial cells, and the recovery of the dissolution solvent is also easy, Since the recovered dissolved solvent and the recycled solvent is reused as a super or pseudocritical state and the compression heat generated at that time is used as a heat source, it is possible to mass-produce useful components from bacterial cells by saving resources and energy. This is a very advantageous industrial method.

[Brief description of drawings]

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

Claims (1)

[Claims] 1. A culture solution of a microbial cell at a temperature above the critical temperature and above the critical pressure, under pressure conditions or below the critical temperature T _C to a critical temperature T _R = T / T _C (where 0.90 <T _R <1.0)
At that temperature, the pressure is equal to or higher than the saturated vapor pressure of the dissolving solvent at that temperature, and after mixing with the dissolving solvent that diffuses inside the cell membrane of the cells under pressure conditions, the pressure is instantly lowered and the separation tank is maintained at that temperature. And recovering the crushed bacterial cells from the lower part of the separation tank and recovering the solvent from the upper part of the separation tank, and then compressing the recovered solvent, and the heat of compression generated thereby is applied to the heating source of the separation tank. The method for crushing bacterial cells, characterized in that the compressed solvent is reused after being mixed with the above culture solution.