JPH0659225B2 - Extraction and separation method of 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 destroys the cell membrane of the microbial cells by a method that can easily recover the solvent in a short time, diffuses the components in the microbial cells to the outside of the cell membrane, and extracts the useful components therein. It is intended to provide a method of separation.

[Means for solving problems]

The present invention relates to the treatment of bacterial cells, which is obtained by mixing a culture solution of bacterial cells with a dissolving solvent in a supercritical state or a pseudocritical state, introducing the pressure into the first separation tank by instantaneously lowering the pressure, and taking out from the lower portion of the first separation tank. The liquid is introduced into the extraction tank and brought into countercurrent contact with the dissolution solvent in the supercritical state or pseudocritical state, and then the dissolution solvent phase in which the bacterial cell extract is dissolved is taken out from the upper portion of the extraction tank and is taken out as the second separation tank. Into the second separation tank, the microbial cell extract is recovered from the lower part of the second separation tank by gravity sedimentation separation under a pressure condition in which the dissolving solvent and the bacterial cell extract are substantially separated. This is a method for extracting and separating bacterial cells, characterized in that the dissolving solvent is recovered.

In the present invention, the temperature at which the mixture of the cell culture solution and the supercritical or pseudocritical dissolution solvent is introduced into the first separation tank by instantaneously lowering the pressure is maintained near the critical temperature of the dissolution solvent. Preferably.

First, the concept based on the present invention will be described.

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 critical temperature Tc of the dissolving solvent or lower and a countercritical temperature T _R = T / Tc (where 0.90 <T _R <1.
At the temperature T of 0), the pressure means a state equal to or higher than the saturated vapor pressure of the dissolving solvent at that 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 dissolving solvent near the critical point has the same density as the liquid, and by easily diffusing into the cell membrane,
When the pressure is suddenly lowered from this state, the high-density dissolved solvent that has diffused into the cell membrane expands rapidly, and the cell membrane cannot withstand this expansion force, so it can be broken into pieces.

Therefore, in the present invention, the culture solution of the bacterial cells is mixed with the solvent in the supercritical state or the pseudocritical state, and the dissolving solvent is diffused into the cell membrane of the bacterial cells, and then the pressure is instantly lowered, and the temperature is the lysis. The cell membrane of the microbial cells is crushed while being kept near the critical temperature of the solvent.

Since the temperature is close to the critical point even if the pressure is reduced here, the dissolution solvent can be separated from the crushed bacterial cells in an energy-saving manner without requiring the latent heat of vaporization of the dissolution solvent. The crushed bacterial cells are collected from the bottom of the tank, and the lysing solvent is collected from the top.

In addition, the inventors of the present invention have found that, when a cell membrane is present, a component in a bacterial cell cannot be extracted through the cell membrane even if it has a dissolving solvent in a supercritical state or a pseudocritical state. However, it has been found that when the cell membrane is destroyed, the component itself is easily dissolved in a dissolution solvent in a supercritical state or a pseudocritical state.

That is, among the various substances taken out of the cell membrane, valuable components such as fatty acids are dissolved in proportion to the density of the dissolving solvent.

Therefore, the valuable components are extracted into the dissolution solvent phase by countercurrently contacting the crushed cells recovered in the above step with the dissolution solvent in the supercritical state or pseudocritical state in the extraction tank. The dissolving solvent at this time may be the same as or different from the one used for disrupting the cell membrane of the bacterial cell. However, if the same dissolution solvent is used, particularly if the dissolution solvent recovered from the first separation tank after being used for cell membrane disruption is compressed to near the critical point and used in this extraction process, the process configuration can be simplified and By using the heat of compression for heating, it is possible to effectively use energy, and it is possible to extract a substance containing few impurities such as solid components.

Further, the present inventors introduce the dissolution solvent layer containing the valuable component into the second separation tank, and then reduce the density of the dissolution solvent in the supercritical state, that is, decrease the pressure or increase the temperature. By such means, it was found that the solubility of the extract such as valuable components is drastically lowered, and the solvent and the extract can be separated without the latent heat of vaporization of the solvent. The dissolved solvent and the extract, which are substantially insoluble to each other, are separated by gravity settling. The recovered dissolved solvent is recompressed and reused, and the compression heat at that time is used as a heating source to obtain an energy saving effect.

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 (℃) 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, its critical temperature is 31.1 ° C., and at a temperature of 35 ° C., a pressure of 100 kg / cm ² G and a density of about 0.1.
8 and at atmospheric pressure the density is of the order of 10 ⁻³ ,
Since the density difference of about 1000 times becomes the expansion force, it is understood 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. An example is Mortierella spp., Which is a type of filamentous fungus that selectively produces γ-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.

An embodiment of the present invention will be specifically described below 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 the bacterial cell culture liquid high-pressure feed pump 2, while 1 to 10 parts by weight of the dissolution solvent was supplied from the dissolution solvent supply line 3, For example, they 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. Next, the pressure is instantly reduced to near normal pressure by the pressure reducing valve 5 and the mixture is introduced into the first separation tank 6 where the temperature is near the critical point of the dissolving solvent, and the treated bacterial cell withdrawal port 8 at the bottom of the separation tank is introduced. The microbial cells whose cell membrane has been destroyed are collected, and the lysis solvent is collected from the lysis solvent extraction port 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, so that the crushing effect is large, but when re-compressing and reusing the dissolved solvent, energy is required correspondingly. It should preferably be in the range of 10 to 50 atm. On the other hand, if the pressure is higher than this, a large amount of the components in the bacterial cells start to dissolve in the dissolution solvent at the dissolution solvent outlet 7 in the upper part of the first separation tank, which is not preferable. As described above, in this step, the microbial cells are crushed and the dissolving solvent is separated without the need for latent heat of vaporization.

Next, the crushed microbial cell processed product is the microbial cell processed product take-out port 8
From the cell-treated product feed pump 11, the cell-treated product feed line 13 is supplied to the upper portion of the countercurrent contact extraction column 14 and the dissolution solvent supply line 18 is supplied to the lower portion of the countercurrent contact extraction column 14. After making countercurrent contact with the dissolved solvent in a supercritical state or a pseudocritical state of the dissolved solvent, the extraction residual liquid outlet 1
The residual liquid containing substantially no valuable components in the bacterial cells is taken out from 7 and the dissolving solvent phase containing the valuable components substantially not containing the cell membrane in the bacterial cells is taken out from the dissolving solvent phase extraction port 16. , Separate the two.

The structure of the gas-liquid contact portion 15 including, for example, a packed bed, a tray, etc. of the countercurrent contact extraction column 14 is not particularly limited, but a structure capable of preventing adhesion and deposition of solid components and the like is preferable.

The dissolution solvent supplied from line 18 does not necessarily have to be the same as the dissolution solvent used for cell disruption in line 3, and should be determined in consideration of the efficiency of each step. As mentioned above, preferably the same dissolving solvent is used.

Next, the dissolved solvent phase from the dissolved solvent phase outlet 16 passes through the pressure reducing valve 19 and the heat exchanger 20, and then the dissolved solvent phase supply line 2
1 is introduced into the second separation tank 23, and the solvent and the extract are substantially insoluble to each other and are maintained at the temperature and pressure necessary for gravity sedimentation separation. That is, by lowering the density in the supercritical state of the dissolved solvent, immediately lowering the pressure with the pressure reducing valve 19 or raising the temperature with the heater 29,
The dissolution solvent and the extract are separated by rapidly reducing the solubility of the extract in the dissolution solvent.

Thus, the extract from the bacterial cells can be taken out from the extract take-out line 24 and the dissolving solvent can be taken out from the dissolving solvent take-out line 25, so that both can be separated in an energy saving manner.

In addition, when the extract is also contained in the dissolving solvent at the dissolving solvent outlet 7, the pressure may be adjusted by the pressure reducing valve 10 and the extract may be supplied to the second separation tank 23 from the dissolving solvent supply line 22. it can.

The dissolution solvent from the take-out line 25 is recompressed by the compressor 28 or 12 via the dissolution solvent circulation line 27, and the compression heat obtained at this time is used as a heating source for the first separation tank 6 and the second separation tank 23. By recycling the dissolving solvent, the effective use of the dissolving solvent and the energy saving effect can be obtained. Reference numeral 26 is a dissolving solvent replenishing line.

〔Example〕

Example 1 1 part by weight of a cell culture solution having a water content of 50% by dehydrating a culture solution of Mortierella fungus, which is one of filamentous fungi, by a centrifuge, and 2 parts by weight of CO _{2 in} an autoclave having a volume of 1 And mixed at a temperature of 40 ° C. and a pressure of 105 kg / cm ² G for 5 minutes, and then rapidly opening the valve to introduce the mixture from the autoclave into a separation tank having a capacity of 10 and 20 kg / cm ² G, When CO ₂ was discharged from the upper part of the separation tank and the bacterial cell culture remaining in the lower part was observed with a microscope, the cell membranes of almost all bacterial cells 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 ℃ and a pressure of 20kg / cm
^When compressed from ² G to 110 kg / cm ² G, the temperature was about 85 ° C. Therefore, it was found that this heat can be used as a heating source.

Next, the treated cells were charged into the autoclave again,
After continuously flowing CO ₂ from the lower part at a temperature of 40 ° C. and a pressure of 140 kg / cm ² G for 5 hours, introduce a CO ₂ phase from the upper line into a separation tank having a pressure of 25 kg / cm ² G through a pressure control valve, The CO ₂ and extract were separated.

As a result, 98% by weight of the fatty acids in the cells were extracted from the extract.

On the other hand, almost no impurities were detected in the recovered CO ₂ . This CO ₂ is compressed by a compressor at 40 ℃, 20kg / cm ² G to 1
When compressed to 10 kg / cm ² G, the temperature becomes about 85 ℃,
It was discovered that this heat could be used as a heating source.

Comparative Example 1 Example 1 was charged cells that do not disrupt the cell membrane to the autoclave, the temperature 40 ° C. The CO ₂ from the bottom, but flowed continuously for 5 hours at a pressure 140 kg / cm ² G, into the CO ₂ Only 0.5% by weight of fatty acids in the cells were extracted.

(Effect of the present invention) As described in detail above, the present invention can easily destroy cell membranes of cells by using a dissolving solvent in a supercritical state or a pseudocritical state, and the solvent can be recovered. The effect is that energy can be saved easily.

[Brief description of drawings]

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

Claims (1)

[Claims] 1. A bacterium taken out from the lower part of the first separation tank after the culture solution of the microbial cells is mixed with a dissolution solvent in a supercritical state or a pseudocritical state, the pressure is instantly lowered and introduced into the first separation tank. The body treatment liquid is introduced into the extraction tank and brought into countercurrent contact with the supercritical or pseudocritical dissolution solvent, and then the dissolution solvent phase in which the bacterial cell extract is dissolved is taken out from the upper portion of the extraction tank It is introduced into a separation tank, and gravity sedimentation separation is carried out in the second separation tank under a pressure condition where the dissolution solvent and the bacterial cell extract are substantially separated, and the bacterial cell extract is recovered from the lower part of the second separation tank. A method for extracting and separating bacterial cells, characterized in that the dissolving solvent is recovered from the upper part.