JPH0697988B2 - Sampling device for culture tank - Google Patents

Description

Detailed Description of the Invention [Industrial applications]

The present invention relates to a sampling device for collecting a culture solution from a culture tank, and more specifically, it is possible to aseptically collect a sample that can be directly separated from cells and applied to an analyzer such as a chromatograph, directly from the culture tank. The present invention relates to a sampling device that can be used. In this specification, the term "culture" is to be understood in the broadest sense including "fermentation" which means the culture of microorganisms. Cell culture in which cells of plants or animals are grown in a container is also included in the "culture" in the present specification. Therefore, in the present specification, a culture tank and a fermentation tank, a culture solution and a fermentation solution, and the like are used with the same meaning. Further, in the present specification, "culture liquid"
The terms "medium" and "medium" are treated as synonyms without distinction.

[Prior art]

In order to understand the composition of the culture solution (medium) such as metabolites and inhibitors in the culture tank, and to judge the culture status and control the culture, it is often necessary to collect and analyze the culture solution during the culture. Occurs. Although various methods are used as analysis means, when using a biosensor or liquid chromatography capable of quantitatively analyzing multiple components at one time, gas chromatography, supercritical chromatography, etc., a solid-liquid culture solution is used. Need to be separated. As a conventional sampling device for a culture tank capable of collecting a sample which can be applied to these analyzers, for example, a device as shown in FIGS. 10 to 12 has been proposed. In the sampling device of FIG. 10, the pipe 53 and the pipe 54 are arranged downstream of the pipe 51 via the valve 52, the pipe 56 is arranged downstream of the pipe 54 via the valve 55, and the pipe 53 and the pipe 54 are connected. It has a piping system in which a pipe 58 having a valve 59 is joined at a connection point. This piping
The upstream end of 51 is inserted into the culture tank 50, and a filtration filter 60 having a pore diameter of 0.2 μ or less that does not allow miscellaneous bacteria to pass through is attached. The downstream of the pipe 56 is opened to the sample tank 57, and the pipe 58 is connected to a sterile air source 61 via a valve 59. An extraction pump 62 is provided in the pipe 56 if necessary. In such an apparatus, first, before the start of culture, steam is supplied from a steam pipe (not shown) into the culture tank 50 to sterilize the culture tank itself, the culture medium and the filtration filter 60 with heating steam. When sampling, close all valves from the closed state, close valve 59, open valves 52, 55, and sample the culture solution in the culture tank through the piping 51, 53, 54, 56 by the tank internal pressure. Collect in tank 57. After collection is completed, valves 52 and 55 are closed. If the filtration filter 60 is clogged during sampling, open the valve 59 with the valve 55 closed and connect the sterile air to the pipe.
8, it is supplied into the culture tank 50 through the pipe 53, the valve 52, the pipe 51, and the filtration filter 60, and the filtration filter 60 is backwashed. Sterile air is used because the culture solution in the culture tank contains bacterial cells, enzymes, etc., and therefore cannot be backwashed with steam. According to the sampling device as shown in FIG. 10, since the sample is sampled through the filtration filter 60 having a pore diameter of 0.2 μ or less provided in the culture tank, it is possible to collect the culture solution without mixing various bacteria in the culture layer. Moreover, since large solids and bacterial cells in the culture solution have been removed from the collected sample by a filtration filter, this sample can be directly subjected to an analyzer. On the other hand, the sampling device shown in FIGS. 11 and 12 (Japanese Patent Laid-Open No. 61-5774) uses a filtration filter instead of the direct flow type filtration filter installed in the culture tank as used in FIG. Is placed outside the culture tank, and a parallel flow type filtration filter that is relatively unlikely to cause clogging is adopted. That is, the sampling device in FIG. 11 is provided with a culture solution circulation system consisting of a pipe 71, a circulation pump 72, and a pipe 73 outside the culture tank 70, and the pipe 73 has a pore diameter of 0.2 that does not allow bacteria to pass.
A tubular parallel-flow type filtration filter 74 having a size of μ or less is interposed, and the culture solution flows through the inside of the filtration filter 74 so that the filtration solution comes out to the outer surface of the filtration filter. In such a device, first, before the start of culturing, the whole device is placed in an autoclave, and the culture tank itself, the culture medium and the culture solution circulation system outside the culture tank are sterilized by heating with steam. At the time of sampling, the circulation pump 72 is started and the culture solution in the culture tank 70 is piped 71, the circulation pump 72, the pipe 73,
The filtration liquid is circulated through the filtration filter 74 and the pipe 73 to allow the filtration liquid to permeate the outer surface of the filtration filter 74, and the filtration liquid is collected in the sample tank 75. The sampling device of FIG. 12 employs a filtration filter module 74 ′ such as a ceramics filter module in place of the simple cylindrical filtration filter 74 of FIG. 11, and when the filtration filter is clogged, it is filtered. Liquid or sterile air is supplied into the culture tank through the filtration filter module 74 'so that the filtration filter module 74' can be backwashed. Also in this case, the back culture with steam cannot be performed because the culture solution in the culture tank contains bacterial cells, enzymes and the like. The above-mentioned filtration filter module 74 'has a structure in which one or more cylindrical filtration filters are loaded in the storage container, the culture fluid circulates inside or outside the filtration filter cylinder, and inside the filtration filter cylinder. In the type in which the culture fluid flows, the filtrate permeates to the outer surface of the cylinder, and in the type in which the culture fluid flows outside the cylinder of the filtration filter, the filtrate permeates to the inner surface of the cylinder, and the filtrate is connected to the housing. It flows through a pipe 76 and a valve 77 into a sample tank 78. When backwashing the filtration filter module 74 ',
The filtrate of the sample tank 78 is supplied to the filtration filter module 74 'by the pump 79, or aseptic air (not shown) is supplied to the filtration filter module 74' through the pipe 76, and the filtrate or sterile air is incubated in the filtration filter. Permeate to the tank side. Also in the device shown in FIGS. 11 and 12 described above, the hole diameter is 0.
Sampling is performed through a filtration filter 74 having a size of 2 μ or less or a filtration filter module 74 ′ having a pore size of 0.2 μ or less, so that the culture solution can be collected without contamination of various bacteria in the culture tank. Further, the collected culture solution has a large solid content removed by a filtration filter, and this sample can be directly applied to an analyzer.

[Problems to be Solved by the Invention]

The conventional device shown in FIGS. 10 to 12 is a sampling device capable of collecting a sample that can be directly subjected to an analyzer such as chromatography, but when backwashing the filtration filter, the filtration device used for backwashing is used. Since liquid and aseptic air flow into the culture tank, if various bacteria propagate in the downstream piping of the sampling device, these bacteria will be entrained in the culture tank together with the above-mentioned backwashing filtrate and sterile air. Causes nation. In order to prevent this, there is a restriction that a filtration filter having a pore diameter of 0.2 μ or less that does not allow miscellaneous bacteria to be used. Conversely due to this constraint,
When sampling a culture solution that contains a large amount of solids or large cells, cells with complex shapes, or adherent cells, clogging of the filtration filter occurs immediately.
Fig. 11 and Fig. 11 which adopted a parallel flow type filtration filter
Even with the device shown in Fig. 12, the filter is immediately clogged. Therefore, there has been a problem that backwashing with a filtered liquid or sterile air becomes frequent, and it is difficult to completely recover the clogging even when backwashed, and the flow rate of the filtered liquid passing through the filtration filter is not stable. As a result, there is also a problem that it is difficult to connect the sampling device and an analyzer such as chromatography to automate sampling and analysis. Therefore, an object of the present invention is to provide a new and improved sampling device that does not have a restriction on the pore size of a filtration filter and has no problem of contamination that tends to occur during sampling and can collect a sample that can be directly applied to an analyzer. It is to be. Another object of the present invention is to easily and sufficiently recover the clogging of the filtration filter by backwashing even if the clogging of the filtration filter occurs. By connecting a sampling device and an analyzer such as chromatography, It is to provide a new and improved sampling device that is easy to automate sampling and analysis.

[Means for solving problems]

That is, the sampling device of the present invention, a culture solution introduction pipe for introducing the culture solution from the culture tank into one space of the filtration filter storage container partitioned by the filtration filter, the filtrate that has passed through the filtration filter in the other space Of the above-mentioned one space (introduction of the culture solution of the filtration filter), while connecting the filtrate extraction pipe for extracting the Side) is connected to a steam discharge pipe for discharging the steam or condensed water passing through the filtration filter, and the culture solution from the culture tank is introduced into the one space (culture solution introduction side) and the other space (filtration). The step of extracting the filtrate that has permeated the liquid permeation side), and introducing the steam for heat sterilization into the other space (filtrate permeation side) and permeating into one space (culture liquid introduction side) The serial vapor or valve for switching the step of discharging the condensed water is characterized in that provided on the respective pipes. The filtration filter may be the above-mentioned direct flow type filtration filter or parallel flow type filtration filter. In the direct flow method, the culture solution introduction pipe and the steam discharge pipe are separated by the filtration filter of the filter storage container in one space [culture solution introduction side], and the filtrate extraction pipe and the steam introduction pipe are stored in the filter. In most cases, it is connected to the other space [the filtrate permeation side] defined by the filtration filter of the container. On the other hand, in the parallel flow method,
For example, in the type in which the culture solution flows through the inside of the filtration filter cylinder, the culture solution introduction pipe and the steam discharge pipe are connected to the inside of the filtration filter cylinder (that is, one space partitioned by the filtration filter of the filter storage container). [Communication liquid introduction side], and the filtrate extraction pipe and the steam introduction pipe are connected to the filtration filter storage container (that is, the other space partitioned by the filtration filter of the filter storage container [filtrate permeation side] ], And in the type in which the culture fluid flows outside the cylinder of the filtration filter, the culture fluid introduction pipe and the vapor discharge pipe are connected to the filtration filter storage container (that is, the filtration filter of the filter storage container). Is connected to one of the spaces [the culture solution introduction side], and the filtrate extraction pipe and the steam introduction pipe are inside the cylinder of the filtration filter. Is circuited (i.e., it is contacted to the other space defined by the filtration filter of the filter container [filtrate permeate side]) Ru. As described above, there are various types of connection of the filter storage container of each pipe to the filtration filter cylinder depending on the type of the filtration filter. Further, the culture solution introducing pipe, the filtrate extracting pipe, the steam introducing pipe and the steam discharging pipe may be independently connected to the predetermined space of the filtration filter storage container, and further, the culture solution introducing pipe. And the steam discharge pipe are partially configured as common pipes, or the vapor filtrate extraction pipe and the steam introduction pipe are configured as partially common pipes, and this common pipe is used for the filtration filter storage container. Connect to the designated space,
The above culture solution introduction pipe, filtrate extraction pipe, steam introduction pipe,
As a result, each of the vapor discharge pipes may communicate with a predetermined space of the filtration filter storage container. Further, the step of introducing the culture solution from the culture tank into the one space and extracting the filtrate that has permeated into the other space, and the steam for heating sterilization introduced into the other space and the vapor permeated into the one space or When a valve for switching the process of discharging the condensed water is provided in each of the pipes, specifically, for example, a two-way valve is a pipe for each of the culture solution introducing pipe, the filtrate extracting pipe, the steam introducing pipe, and the steam exhausting pipe. Three-way at the connection point between the culture solution introduction pipe, the steam discharge pipe and the common pipes, and the connection point between the filtered liquid discharge pipe, the steam introduction pipe and the common pipes. It can be realized by providing a valve. That is, a valve such as a two-way valve or a three-way valve may be provided in or at each end of the culture solution introducing pipe, the filtrate extracting pipe, the steam introducing pipe, and the steam discharging pipe. Examples of the material for the filtration filter include inorganic materials such as glass, ceramics and silicon, or organic polymer materials such as Teflon, nylon, polypropylene, polyvinyl alcohol, and cellulosic resins, and metal materials such as porous stainless steel. Other materials can be used as long as they can be sterilized by steam heating. As a filtration filter that can be preferably used, a pore size of several tens of μ ~ 0.1 μ
The ceramic filter and the sintered metal filter described above can be used, and a microporous membrane formed in a film shape and having a similar pore size or an ultrafiltration membrane having a molecular weight cutoff of several thousand to several hundred thousand can also be used. The pore size of the filtration filter may be appropriately selected depending on the size of cells in the culture solution, the components to be analyzed, and the like. It is preferable to select a cell having a pore size that is as large as possible without allowing the cells in the culture solution to permeate. As a material for each of the above-mentioned pipes, a stainless steel pipe can be preferably used, and a flexible tube made of an organic polymer material such as silicon, Teflon, or polypropylene can be used. Further, it is preferable to make these pipes as short as possible to facilitate steam heat sterilization. Stainless steel is preferably used as the material of the container for the filtration filter, and heat-resistant glass or the above-mentioned heat-resistant organic polymer or inorganic material can also be used.

[Work]

According to the sampling device of the present invention having the above-mentioned configuration, the step of introducing the culture solution from the culture tank to the culture solution introduction side of the filtration filter and extracting the permeated filtrate to the filtrate permeation side, that is, the sampling step, The step of introducing steam for heat sterilization into the filtrate permeation side of the filtration filter and discharging the vapor permeated into the culture solution introduction side or its condensed water, that is, the backwash step, is switched by the switching means.
Even if the steam for heat sterilization is used in the backwashing step, the steam for heat sterilization is not introduced into the culture tank, and thus does not affect the bacterial cells, enzymes or the like in the culture tank. As described above, in the apparatus of the present invention, since steam for heat sterilization can be used in the backwashing step, it is possible to remove the filter clogging much more easily and effectively than the conventional backwashing of the filter with sterile air or filtrate. In addition to being able to perform, sterilization by steam of the filter can be performed at the same time. Further, in the conventional backwashing using sterile air or filtrate, when germs propagate in the downstream pipe of the sampling device, these germs are entrained in the culture tank along with germicidal air for washing and filtrate. However, because of the risk of contamination, there is a restriction that a filtration filter with a pore size of 0.2μ or less that does not allow bacteria to pass must be used. However, in the device of the present invention, there is no risk of germs growing in the pipe because steam for heat sterilization can be used for backwashing,
Therefore, there is no longer a restriction that a filtration filter having a pore size of 0.2 μ or less, which does not allow various bacteria to pass, must be used. As a result, since a filtration filter having a larger pore size can be used, it is possible to sample a culture solution that cannot be sampled due to immediate clogging with a conventional filtration filter. As described above, in the present invention, it is possible to use a filtration filter that does not allow the cells in the culture tank to permeate and has a pore size as large as possible. As a result, some solids may be present in the filtrate, but it can be measured with an analyzer that uses a biosensor without any problems, and clogging of the prefilter or precolumn frequently occurs even in high performance liquid chromatography. Therefore, it is possible to carry out the measurement without any problem, and thus it is possible to collect a sample which can be directly applied to various analyzers.

【Example】

FIG. 1 is an embodiment showing the basic configuration of a sampling device according to the present invention. This device is connected in this order to a storage container 35 that stores the valve 12, the pipe 13, the pipe 14 and the filtration filter 15 in the downstream side of the pipe 11 that communicates with the inside of the culture tank 10, and from the filtrate permeation side of the storage container 35. Pipe 16, pipe 17 and valve 18 are connected in this order, and pipe 19 branches from the connection point between pipes 13 and 14, and the pipe is connected at the connection point between pipes 16 and 17.
21 joins. The pipe 19 has a drain valve 20, and the upstream side of the pipe 21 is connected to a vapor supply source 23 via a valve 22. The operation procedure of the sampling device of FIG. 1 will be described below. Sterilization of filtration filter: Before starting the culture, the valves 20 and 22 are opened, and the steam for heat sterilization is passed from the steam supply source 23 through the pipe 21, the pipe 16, the filtration filter 15, the pipe 14, and the pipe 19 to obtain the filtration filter 15. Sterilize. When sterilization of the filtration filter 15 is completed, open the valve 12,
The valve 20 is closed and the steam for heat sterilization is passed through the pipes 13 and 11 to sterilize the pipe system. After the sterilization is completed, the valve 12 is closed, and the valves 22 and 20 are slightly opened to allow the steam for heating sterilization to be released into the valve 20.
Continue to discharge little by little. In the present specification,
The steam for heat sterilization means steam having a temperature used for heat sterilization in ordinary culture, and if it has a temperature that can be sterilized under conditions of pressure resistance and heat resistance of the device, especially temperature, pressure, etc. There is no limitation to the above, and in some cases, some condensed water may be mixed. In normal culture, steam for heat sterilization is used at a temperature of 125-138 ° C and a pressure of 0.147-0.245.
Saturated steam of about MPa [gauge] is often used. Less than,
In the present specification, “steam for heat sterilization” is simply abbreviated as “steam”. Then, steam is supplied into the culture tank 10 from a steam pipe (not shown) to sterilize the culture tank itself and the culture solution charged therein with heating steam. Sampling: When sampling the culture solution in the culture tank 10 during culturing, close the valves 22 and 20 in the slightly opened state, then open the valves 12 and 18, and set the pressure in the culture tank 10 to the desired value. The culture solution sent out from the pipe 11 is filtered by the filtration filter 15, and the filtrate is collected as a sample through the pipes 16 and 17.
When sampling is complete, close valves 12 and 18, then valve 22 again.
The valve 20 is slightly opened, and the steam is continuously discharged little by little from the valve 20, and the steam is sealed while backwashing the filtration filter 15. In this way, since sampling is performed through the filtration filter 15, large solids and cultured bacterial cells in the culture solution have been removed from the collected sample, and this sample is directly used in an analyzer (not shown). You can call. Further, for example, even when using a filtration filter having a pore diameter of more than 0.2μ, the sampling device including the filtration filter 15 on the upstream side of the valve 18 has already been sterilized, and the filtration filter 15 acts as a kind of check valve. Therefore, the filtrate (sample) does not flow backward, and the culture solution can be collected without contamination of the culture tank 10 with bacteria. Backwashing of the filtration filter: As mentioned above, since the steam sealing is performed after the sampling operation is completed, the steam sealing also doubles as the backwashing of the filtration filter. Therefore, the backwashing of the filtration filter is usually performed again. No need. After the sampling operation is completed, if the valve 22 and the valve 20 are completely closed and the steam seal is not performed, when the clogging of the filtration filter 15 occurs, or after the completion of the sampling operation, the valve 12, if necessary, With the valve 18 closed, the valves 22 and 20 are opened, and steam is discharged from the valve 20 through the filter 15 to backwash the filter. By backwashing with steam seals and steam as described above,
The clogging of the filtration filter can be removed more easily and sufficiently than the conventional backwashing with sterile air or a filtrate, and furthermore, the filtration filter and the piping system connected thereto can be sterilized at the same time. In addition, backwashing with steam
It is considered that the gel-like substance formed on the surface of the filtration filter undergoes heat denaturation due to steam to improve the peeling property, so that a far superior cleaning effect is produced as compared with the conventional backwashing in which steam cannot be used. Therefore, the backwashing with steam, which is made possible by the present invention, can recover the clogging of the filter and sterilize the filter at the same time, and has a synergistic effect. Further, in the device of the present invention, the filtration filter is
It is also possible to exchange aseptically easily during the culture without affecting the culture system. That is, in FIG. 1, with the valve 12 closed, the filtration filter 15 is removed from the storage container 35 or the storage container 35 is removed from the pipes 14 and 16 and replaced. After that, the valves 22 and 20 may be opened, and steam may be passed through the newly replaced filtration filter for heat sterilization.
If the replacement of the filtration filter is not considered during culture, the storage container 35 may be integrated with the pipes 14 and 16, or the storage container 35 itself may be formed of pipes. Furthermore, in the device of the present invention, since a sample containing bacterial cells and SS (suspended solid substance) can be collected from the drain port downstream of the valve 20, various samples can be collected and can be widely used. That is, the valves 22 and 20 in FIG. 1 are opened and steam is passed through the pipes 14, 19 and 13 to heat and sterilize these pipe systems. Next, the valve 12 is slightly opened, and the piping systems are cooled while washing the pipings 13, 19 and 14 with the culture solution. Then close valve 22,
Collect the culture solution from the drain port downstream of the valve 20. When the sampling is finished, the valve 22 is opened while the valve 12 is closed, the sampling device is re-sterilized with steam, and then the valves 20 and 22 are closed to restore the original state. By this operation, aseptic sampling can be performed. As shown in FIG. 1, an extraction pump 25 is provided in the downstream pipe 24 of the valve 18, and the suction force of the pump 25 and the culture tank are used.
It is also possible to carry out filtration sampling with the tank internal pressure of 10. By providing the extraction pump 25, the sample can be easily extracted from the filtration filter 15, and the collected sample can be further transported to an analytical instrument. In the embodiment of FIG. 1, the valves 12, 18, 20, 22 are all two-way valves, but when steam sealing is not performed, instead of these, the connection points of the pipes 13, 14, 19 and the pipes are replaced. The number of valves may be reduced by using a T-port type three-way valve at the connection points of 16, 17, and 21. Further, in the embodiment shown in FIG. 1, the pipe 14 is used as a common pipe for introducing the culture solution into the filtration filter and discharging steam from the filtration filter. Similarly, the pipe 16 is used as a common pipe for introducing steam into the filtration filter and for withdrawing the filtrate from the filtration filter. However, as shown in FIG. 2, the culture solution introduction pipe 13 and the steam discharge pipe 19 are directly connected to the culture solution introduction side of the storage container 35 (filtration filter 15), and the steam introduction pipe
21 and filtrate extraction pipe 17 are stored in container 35 (filtration filter 1
It may be directly connected to the filtrate permeation side of 5). The filtration filter 15 in the embodiment of FIG. 1 can be used in either a direct flow system or a parallel flow system, but when a parallel flow system filtration filter is adopted,
It is preferable to provide a piping system shown by the broken line in FIG. This pipe system is configured by connecting a pipe 26 connected to the culture solution introduction side of the filtration filter 15, a circulation pump (rotary pump in this example) 27, a valve 28, and a pipe 29 communicating with the culture tank 10 in this order. ing. A branch pipe 31 having a drain valve 30 is connected between the circulation pump 27 and the valve 28. This allows piping 11, valve 12, piping 13, piping
14, filtration filter 15, piping 26, circulation pump 27, valve 28,
A culture solution circulation system consisting of the pipe 29 will be formed,
This circulation system can prevent clogging of the filtration filter 15 more effectively. The operation of the apparatus shown in FIG. 1 equipped with the above-mentioned culture solution circulation system will be described below. First, before the start of the culture, the valves 12 and 28 are closed and steam is supplied into the culture tank 10 from a steam pipe (not shown) to heat the culture tank itself and the culture medium charged therein, as well as the pipe 11 and the pipe 29. Sterilize. On the other hand, with the valve 18 closed, the valves 20, 30, 22 are opened to supply steam from the steam supply source 23, and the pipe 21, the pipe 16, the filtration filter 15, the pipe 14, and the pipe
13, the pipe 19, the pipe 26, the circulation pump 27, and the branch pipe 31 are sterilized by heating steam. After the sterilization is completed, the valves 22, 20 and 30 are slightly opened, and steam sealing is performed as described above. When sampling, the valves 22, 20 and 30 are closed, the valves 12 and 28 are opened, and the circulation pump 27 is activated to start the culture tank 10.
The culture solution inside is flowed into the culture solution circulation system, and the valve 18 is opened to collect the permeated filtrate from the filtration filter 15. When the sampling is completed, the circulation pump 27 is stopped, the valves 12, 28 and 18 are closed, and then the valves 22, 20 and 30 are slightly opened to perform the above-described vapor sealing. This allows sterilization and backwashing to be performed simultaneously. If steam sealing is not performed, the valves 12, 28, 18 are closed and the sampling operation is completed, and then the valves 20, 30,
22 is opened to filter steam from the steam supply source 23 Filter 15
And the filtration filter is backwashed. Thereby, also in this case, backwashing and sterilization can be performed simultaneously. As a result of making the pore size of the filtration filter as large as possible without allowing the cells in the culture tank to permeate, even when some solid content is present in the filtrate (sample), for example, in high performance liquid chromatography. Since the analyzer has a pre-filter and a pre-column, such a filtrate can be directly applied to the analyzer. However, when it is desired to prolong the replacement time of the prefilter or precolumn or the cleaning interval, or to completely eliminate the prefilter or precolumn, the filtration filters may be arranged in multiple stages as shown in the embodiment of FIG. Good. In this case, a coarse filter 15a is used on the culture tank side, and a fine filter 15b is used in the subsequent stage.
In the case of arranging the filtration filters in multiple stages, it is necessary to install the extraction pump 25 after the filtration filter 15b in the latter stage and perform suction filtration. The backwashing operation in the case of adopting the multistage filtration filter as shown in FIG. 3 can be carried out by the following procedure. For example, when the sampling operation is completed and all the valves in FIG. 3 are closed, first, the drain valve 20b and the valve 22 are opened to backwash the filtration filter 15b in the subsequent stage, and the steam supply is performed. Source 2
The steam from 3 is passed through the post-filtration filter 15b. Next, in order to backwash the filtration filter 15a in the previous stage, the valve 18a and the drain valve 20a are opened while the valve 22 is kept open, and the drain valve 20b is opened.
Is closed. As a result, the steam from the steam supply source 23 is introduced into the front stage filtration filter 15a through the rear stage filtration filter 15b. After these backwashing operations are completed, the valve 22 and the drain valve 20a are slightly opened to perform vapor sealing, or all the valves are closed. FIG. 4 shows an example in which the sampling device of the present invention is directly connected to an analysis device to fully automate the analysis. In the figure, the parts incorporating the sampling device of the present invention shown in FIG. 1 have the same reference numerals (12,15,18,20,22,23,25,2 as in FIG.
7, 28, 30, 35) will be omitted. The sample liquid (filtrated liquid) from the filtration filter 15 is sent by the pump 25 through the check valve 32 to the air bubble removal sample pot, where the air bubbles in the liquid are removed. A standard solution for calibration of the analyzer is sent to the bubble removal sample pot by the pump 41 via the check valve 33, or washing water for washing the piping system and each device is checked by the pump 42. It is sent through valve 34 and likewise eliminates air bubbles. The bubble-free sample pot has a volume of about 3 ml,
It consists of a cylindrical outer cylinder sealed by the top and bottom walls and a small-diameter inner cylinder with the upper end open, and an overflow pipe is connected to the top wall of the outer cylinder. Is a structure in which the inner cylinder into which is introduced is inserted through the bottom wall of the outer cylinder. The liquid overflows from the upper end of the inner cylinder, the gas in the liquid is separated from the liquid, and the separated liquid stays down between the outer circumference of the inner cylinder and the inner circumference of the inner cylinder. It is sucked and transported by the pump 43. Therefore, in the bubble removal sample pot, the flow rates of the pumps 25, 41 and 42 and the pump 43 are set so that the inflow amount> the outflow amount, and the liquid in the pot is exhausted so that the pump 43 does not suck air. ing. The air (air bubbles) separated from the liquid in the pot is sent from the upper part of the pot to the drain 1 through the overflow pipe and is discharged to the outside. The sample liquid from which air bubbles have been removed by the air bubble removal sample pot is sent by the pump 43 through the sampling injector, the three-way valve 45, and the prefilter to the online automatic analyzer for analysis. The sampling injector collects each predetermined volume of the sample solution, the standard solution, or the wash water sent from the pump 43, and the predetermined amount of each liquid collected in the injector is pumped by the eluent from the pump 44. It is a device that extrudes and sends it to the analyzer and discharges an excess amount of each liquid to the drain 2. As shown in FIGS. 5A and 5B, this sampling injector is composed of a hexagonal valve and a sampling loop (capillary tube), and the sampling loop has a predetermined volume of each liquid to be sent to the analyzer. Explaining an example of the operation of the sampling injector, in the state of FIG. 5A, the sample solution, the washing water or the standard solution sent from the pump 43 passes through the sampling loop and is discharged to the drain 2 as it is. On the other hand, the eluent from the pump 44 passes through the inside of the hexagonal valve and is flowed to the analyzer. Next, when the hexagonal valve is rotated to the state shown in FIG. 5B, a predetermined amount of sample liquid, washing water or standard liquid in the sampling loop is extruded by the eluent from the pump 44 and sent to the analyzer. Meanwhile, the pump 43
The sample liquid, the washing water or the standard liquid continuously sent from is passed through the inside of the hexagonal valve and is discharged to the drain 2 as it is. The sample liquid from the sampling injector is sent to the online automatic analyzer through the prefilter via the three-way valve 45. The pre-filter is provided to remove dust in the sample solution and prolong the replacement period of the pre-column. The three-way valve 45 receives the instruction of the process controller (for example, depending on the time) and pre-filters the sample solution flow. Switch from A to B or from B to A. The online automatic analyzer consists of a HPLC (High Performance Liquid Chromatography) device, an analyzer and a data analysis computer. The HPLC device consists of a pre-column and an analytical column, and the analyzer is equipped with UV (ultraviolet-visible spectroscopic detector) and RI (differential refractometer), respectively, to perform analysis of multiple items. The data analysis computer receives the signal from the process controller that the sample liquid flows in when the sampling injector is in the state shown in FIG. 5B, controls the HPLC device to start the analysis, and sends the analysis result to the computer. It is something to send. In the example of FIG. 4, the sampling / analyzing system for the gas in the culture tank is incorporated like the sampling / analyzing system for the culture solution (medium), and various gas concentrations in the culture tank are measured by the gas mass spectrometer. , Mainly measures oxygen concentration, carbon dioxide gas concentration, etc. and sends the measurement results to a computer. The computer receives signals from various sensors and analyzers, judges the culture state, and instructs the process controller to take necessary measures. The process controller receives the instruction from the computer, controls the culture, and controls the sampling device and the analyzer of the present invention. Therefore, the process controller controls the supply of acid, alkali, and sterile air to the culture medium in the culture tank via a computer based on the information from the pH sensor, DO sensor, etc., and the nutrient source such as sugar is the sample obtained by the HPLC device, etc. Is controlled by a process controller via a computer based on the analysis result of An example of the operation of the sampling device of the present invention will be described below with reference to the time chart of the sampling device shown in FIG. First, prior to the first sampling on that day, the analyzer is calibrated with the standard solution. For that purpose, the pumps 41 and 43 are operated to send bubbles to the standard solution to the sample pot and further to the sampling injector. Initially, this standard solution is discharged to the drain 2 through the sampling loop in the sampling injector, but by operating the six-way valve of the sampling injector to bring it to the state of FIG. The standard solution of a volume is extruded by the eluent sent by the pump 44 which is always operating, and is sent to the automatic analyzer through the prefilter A by the selection of the three-way valve 45, where the calibration is performed. Adjust the analyzer. After the calibration of the analyzer with the standard solution is completed, the pump 41 is stopped and the pump 42 is operated. As a result, the washing water is sent to the air bubble removal sample pot and further to the sampling injector by the pump 43. By operating the six-way valve in the sampling injector, a predetermined amount of washing water was sent to the analyzer through the pre-filter A in the same manner as above, and the standard solution residue was washed without being detected by the analyzer. To confirm. Such calibration with the standard solution and washing with washing water may be performed about once a day. During transport of the above standards and wash water to the analyzer, the valve
30, 20 and 22 are maintained in a slightly opened state, and the filtration filter 15 and the piping system connected thereto are steam-sealed. Then, in order to perform the first sampling of the sample solution of the day, the valves 30, 20 and 22 described above are closed, while
Open valves 28, 12 and 18. As a result, the culture solution in the culture tank is filtered by the filtration filter 15, the filtrate (sample solution) is sent by the pump 25 to the air bubble removal sample pot via the check valve 32, and further by the pump 43 to the sampling injector. Sent. By operating the six-way valve in the sampling injector, a predetermined amount of sample liquid is sent to the analyzer through the pre-filter A in the same manner as above, and a predetermined analysis is performed. The surplus of the culture liquid introduced from the culture tank to the filtration filter 15 is pumped.
It is circulated to the culture tank through the valve 28 by 27. After sampling, stop pumps 27, 25 and 43,
Valves 28, 12 and 18 are closed and valves 30, 20 and 22 are opened again to filter with steam from steam source 23.
Backwash 15. After the end of the first sampling, the valves 30, 20 and 22 are opened slightly, and the filtration filter 15 and the piping system connected thereto are steam-sealed. Also, during this period, the three-way valve 45 is operated to switch from the pre-filter A to the B, and the pre-filter A is replaced if necessary. For the second and subsequent samplings, the valve
28, 12 and 18 are opened and valves 30, 20 and 22 are closed, and sampling is performed in the same manner as the first time.
Backwashing may be performed as in the first time. Experimental Example 1 An experiment for confirming the backwashing effect of a filtration filter with steam was conducted using the sampling apparatus of the present invention shown in FIG. 1 equipped with a culture solution circulation system. Pore size for filtration filter
A 0.2 μ ceramics filter was used. While culturing baker's yeast in a medium containing molasses as a carbon source, the medium is continuously circulated through a filtration filter (membrane surface flow rate).
The filtrate was sampled at a sampling timing of every 20 minutes for 5 minutes (5 minutes after 15 minutes) from 0.16 m / sec), and the permeation amount of the culture solution was measured. When the permeation rate becomes 20 ml / min or less, saturated steam at 130 ° C (approx. 0.176 MPa [gaug
The filtration filter according to e]) was backwashed, and the recovery state of the permeation amount of the culture solution after the backwashing was examined. The results of plotting the measured values of the permeation amount of the culture solution and displaying them as continuous lines are shown in FIG. As a comparative experiment, an experiment was conducted using a conventional apparatus as shown in FIG. 12 and using a filtrate instead of steam for backwashing the filtration filter. The specifications of the filtration filter and other experimental conditions are the same as in the above experiment. The results of the comparative experiment are shown in FIG. From these experimental results, the permeation amount of the filtration filter recovers to the same level as in the initial stage by performing backwashing with steam, but in the case of backwashing with the filtrate, the permeation amount does not recover much and the sample The result was that it could not be sufficiently collected for a long time. Therefore, it can be seen that the backwashing with steam is more effective. In the comparative experiment, sterile air was used in place of the filtrate, and other experiments were conducted under the same experimental conditions. However, the same effect as that using the filtrate of FIG. 8 was obtained, Backwashing with steam was more noticeable. Experimental Example 2 Using the sampling apparatus of the present invention of FIG. 1 equipped with a culture solution circulation system, Aspergillu in a medium containing a large amount of soybean meal.
The s niger [Aspergillus niger (filamentous fungus)] was cultured, and a sampling experiment of a medium containing a large amount of cells and solids was performed. For this experiment, the circulation pump was started near the end of culture, the culture solution was circulated through the filtration filter at a membrane surface flow rate of 0.16 m / sec, and the permeation amount of the culture solution from the filtration filter was measured. As the filtration filter, a ceramics filtration filter having a pore size of 0.2 μ was used initially, but the filtration filter was clogged in 10 minutes, and it became impossible to collect a sample. After backwashing with steam, sampling was carried out again, but it became impossible to take a sample immediately. Therefore, sampling was stopped, the ceramic filter with a pore size of 0.2μ was replaced with a ceramic filter with a pore size of 2.0μ, and after the sterilization operation with steam, the circulation pump was started again and the culture solution was circulated through the filter at the same membrane surface velocity. Then, the permeation amount of the culture solution was measured. The above results are No. 9
Shown in the figure. Incidentally, the contamination of the culture tank due to the sampling operation including the filter replacement operation did not occur. From the results shown in FIG. 9, it is possible to perform stable sampling with a filtration filter having a pore size of 2.0 μ, and by using a backwashing operation with steam in combination, more stable and aseptic continuous sampling becomes possible. It was also found to be effective for sampling the culture broth.

【The invention's effect】

According to the sampling device of the present invention having the above-mentioned configuration, not only can the sample be separated from the culture solution and directly applied to the analysis device such as chromatography, but also the heating of the filtration filter by using steam. Sterilization and backwash can be performed simultaneously. As a result, there is no problem of contamination that is likely to occur during sampling, therefore there is no restriction on the pore size of the filtration filter, and even if the filtration filter is clogged, it can be easily back-flushed with conventional sterile air and filtration. It can be removed much more effectively than a backwash with liquid to restore the filtration effect. In this way, backwashing of the filtration filter with steam and heat sterilization can be combined with the fact that a sample that can be directly applied to the analyzer can be collected, and the sampling device and the analyzer such as chromatography can be connected to perform sampling. Facilitates the formation of automated analysis systems. Further, if necessary, it is possible to collect a sample containing bacterial cells and SS (suspended solid substance) from the drain port, so that various samples can be collected and widely used.

[Brief description of drawings]

FIG. 1 is an explanatory view of an embodiment showing a basic configuration of a sampling device of the present invention; FIGS. 2 and 3 are explanatory views showing different embodiments of the sampling device of the present invention; FIG. 4 is this Explanatory diagram showing an embodiment of a fully automated analysis system incorporating the sampling device of the invention; FIGS. 5A and 5B are explanatory diagrams showing the operation of the sampling injector used in the system of FIG. 4; FIG. 7 is a time chart of the sampling device of the present invention in the system shown in FIG. 7; FIG. 7 is a graph showing the effect of backwashing by vapor using the sampling device of the present invention; FIG. 8 is the conventional sampling device shown in FIG. FIG. 9 is a graph showing the backwashing effect by the filtered liquid which was used; FIG. 9 is a backwashing effect by the steam when using the sampling device of the present invention and using the filter having different pore sizes. Graph showing the difference; Fig. 10, 11
FIG. 12 and FIG. 12 are explanatory views showing conventional sampling devices having different configurations. 10 …… Incubator, 12,18,20,21 …… Valve (switching means), 13…
… Culture fluid introduction pipe, 15 …… Filtration filter, 17 …… Filtrate extraction pipe, 19 …… Steam discharge pipe, 21 …… Steam introduction pipe, 35 …… Storage container.

Claims (4)

[Claims] 1. A culture solution introducing pipe for introducing a culture solution from a culture tank into one space of a filtration filter storage container partitioned by a filtration filter, and a filtrate which has passed through the filtration filter into the other space. A vapor introducing pipe for connecting the filtered liquid extracting pipe and introducing a steam for heat sterilization into the other space, and a vapor discharging pipe for discharging the vapor having passed through the filter or the condensed water thereof into the one space. The step of introducing the culture solution from the culture tank into the one space and extracting the filtrate that has permeated into the other space, and introducing heating sterilization steam into the other space and permeating into the one space. A culture medium sampling device, wherein a valve for switching between the step of discharging the steam or condensed water thereof is provided in each of the pipes. 2. The sampling device according to claim 1, wherein the culture solution introducing pipe, the filtrate extracting pipe, the steam introducing pipe, and the steam discharging pipe are provided independently of each other. 3. The sampling device according to claim 1, wherein the culture solution introducing pipe and the steam exhausting pipe are partially constructed in common. 4. The sampling device according to claim 1, wherein the filtered liquid withdrawing pipe and the steam introducing pipe are partially constructed in common.