Patent Application
20220120650
The present disclosure relates to a system for preparing a sample solution for testing and a method for preparing a sample solution for testing in order to obtain, from an undiluted sample solution, a sample solution for testing in a suitable state for testing for the purpose of testing of microorganisms or the like contained in a liquid.
In general, a microorganism test is conducted in order to control safety and quality of products while pharmaceutical products, and food and drink are manufactured. In the test, contamination with fungi such as yeast and mold, and bacteria such as colon bacillus and Legionella bacteria is checked. It is required to subject a large number of samples to testing for sufficient and proper product management.
Hitherto, a culture method has been used for the microorganism test. A liquid sample is used to perform culture with an agar medium, and the number of colonies on the culture medium is counted as the number of living bacteria. However, the culture method requires a long time period for culture, and hence rapid counting is difficult to achieve. In view of this, for the purpose of achieving rapid counting of living bacteria, various counting methods have been developed in recent years. As one of those methods, there has been proposed a method for counting the number of living bacteria by optically analyzing particles dispersed in the fluid (see Non-patent Literature 1 given below).
Further, in a case of liquid pharmaceutical products such as an injection solution, a test is conducted for foreign matters and fine particles contained in the liquid. Japanese Pharmacopeia specifies that a test is conducted in accordance with a light-shutoff particle counting method or a microscope coefficient method. In a case of an expensive liquid such as an injection solution, a product loss during the test is undesirable. Thus, it is conceived to recover a liquid in which particles that may be contained in a sample before testing are concentrated so as to suppress a product loss. However, during this operation, there may be caused a risk of damaging particles due to a centrifugal force or a shearing force applied to a sample and deteriorating components due to a turbulent flow or bubbles (see Non-patent Literature 2 given below).
As a literature with regard to separation of particles contained in a fluid, Patent Literature 1 is given below that describes a hydrodynamic separation device including a curved channel. In this device, a fluid containing particles is supplied to the curved channel, and the particles are separated through use of a force acting on the fluid flowing through the curved channel.
Patent Literature 1: Japanese Unexamined Patent Application Publication (Translation of PCT Application) No. 2016-526479
Non-patent Literature 1: Sasaki Yasuhiko, Takeuchi Ikuo, Watanabe Yusuke, and Inami Hisao, “Easy and Rapid Live Bacteria Counter by Flow Cytometry”, Japan Journal of Food Engineering, Vol. 14, 131-136, September 2013
Non-patent Literature 2: Yamaura, Kazuo, “Denaturation of Biopolymer by Flow and Shaking of Solution”, Journal of The Society of Rheology, Vol. 11, 107-116, (1983)
In the counting method as described in Non-patent Literature 1, for the purpose of removing contaminants being obstacles for measurement or concentrating bacterial bodies contained in a sample, preliminary processing is required. With this, efficient counting is achieved. For example, when solid particles and the like derived from raw materials are contained in a sample of food and drink, those substances need to be removed. During such preliminary processing, an impact on bacterial bodies is undesirable. Further, microorganism contamination during preliminary processing needs to be avoided. Therefore, it is desired that preliminary processing be performed in an aseptic state. When a sample for a foreign matter test is subjected to preliminary processing that damages particles or degrades components, correct testing results cannot be obtained, and there is a difficulty in recovering a liquid.
As described above, when pharmaceutical products, food and drink, and the like are manufactured, problems such as cost increase and efficiency reduction of a sample for a microorganism test or a foreign matter test need to be solved. Here, preliminary processing for a testing sample is a key factor. The separation technique described in Patent Literature 1 relates to separation of solid particles. Still, regarding the key factor, when a sample is obtained for the purpose of a microorganism test, an impact on microorganisms needs to be examined.
Therefore, it is important to prepare a sample that enables an efficient test. When the separation technique is applied to a test for management of product manufacturing, sufficient examination on whether a proper sample can be obtained is required.
It is an object of the present disclosure to provide a system for preparing a sample solution for testing and a method for preparing a sample solution for testing that enable an efficient test and are capable of supplying a sample solution for testing without affecting microorganisms.
In order to achieve the above-mentioned object, there has been examined an impact on microorganisms, which is caused at the time of performing separation of particle-like microorganisms or solid matters dispersed in an undiluted sample solution. As a result, it has been found that a suitable sample solution for testing can be obtained by separating and concentrating microorganisms or particle-like solid matters contained in an undiluted sample solution efficiently in accordance with a particle size through use of a hydrodynamic separation technique.
According to one aspect of the present disclosure, a system for preparing a sample solution for testing is configured to prepare a sample solution for testing from an undiluted sample solution, and includes a microorganism separation device. The microorganism separation device includes a hydrodynamic separation device and a liquid feeding unit. The hydrodynamic separation device includes a curved flow channel having a rectangular cross-section, and is configured to separate a liquid into a first segment and a second segment through use of a vortex flow generated in the liquid flowing through the curved flow channel. The liquid feeding unit is configured to supply the undiluted sample solution to the hydrodynamic separation device. With the hydrodynamic separation device, particles contained in the undiluted sample solution are concentrated in the first segment.
The undiluted sample solution may include a liquid raw material, a liquid intermediate, and a liquid final product in manufacturing food and drink or pharmaceutical products.
The liquid feeding unit may include a supply channel being branched from a manufacturing line and connected to the hydrodynamic separation device, and a pump configured to apply, to the undiluted sample solution, a hydrodynamic pressure for supplying the undiluted sample solution to the hydrodynamic separation device. It may be configured such that part of a liquid raw material, a liquid intermediate, or a liquid final product that flows through a manufacturing line is supplied as the undiluted sample solution to the hydrodynamic separation device. The liquid feeding unit may further include a return channel that returns one of the first segment and the second segment to the manufacturing line. Alternatively, the liquid feeding unit may include a container configured to store the undiluted sample solution, a supply channel configured to connect the container to the hydrodynamic separation device, and a pump configured to apply, to the undiluted sample solution, a hydrodynamic pressure for supplying the undiluted sample solution to the hydrodynamic separation device. Further, it is suitable that the liquid feeding unit further includes a return channel configured to return one of the first segment and the second segment to the container. The liquid feeding unit may further include a circulation channel configured to cause a part of the other one of the first segment and the second segment to circulate to the supply channel.
The system for preparing a sample solution for testing may further include an additional hydrodynamic separation device. It may be configured such that the liquid feeding unit further includes a flow channel for supplying, to the additional hydrodynamic separation device, a remaining part of the other one of the first segment and the second segment after separation in the hydrodynamic separation device. Further, it may be configured such that the system for preparing a sample solution for testing further includes a test device, a flow channel configured to connect the hydrodynamic separation device to the test device, and a flow channel configured to connect the test device to the manufacturing line. The other one of the first segment and the second segment may be returned to the manufacturing line via the test device.
Moreover, according to one aspect of the present disclosure, a method for preparing a sample solution for testing is performed to prepare a sample solution for testing from an undiluted sample solution, and includes microorganism separation processing. The microorganism separation processing includes a hydrodynamic separation step of separating a liquid into a first segment and a second segment through use of a vortex flow generated in the liquid flowing through a curved flow channel having a rectangular cross-section, and a liquid feeding step of feeding the undiluted sample solution to the hydrodynamic separation step. In the hydrodynamic separation step, particles contained in the undiluted sample solution are concentrated in the first segment.
The particles may be microorganisms of at least one type selected from a virus, a eubacterium, an archaebacterium, a fungus, a myxomycete, an alga, and a protist, the first segment may be supplied as a sample solution for testing, and a return channel may return the second segment from the hydrodynamic separation device. Further, the particles may be particle-like solid matters larger than microorganisms, and the second segment may be supplied as a sample solution for testing. In this case, the first segment may be returned through the return channel, or may be discharged to the outside of the system.
According to the present disclosure, the particle-like microorganisms or the particle-like solid matters dispersed in the liquid can be separated efficiently. At the same time, the liquid after separation can be used as the sample solution for testing, and a test for microorganisms that may be contained in the undiluted sample solution can be conducted efficiently. Further, a separation operation can be performed in a sequential and aseptic manner, and hence contamination can be prevented during preparation of the sample solution for testing. Therefore, there can be provided the system for preparing a sample solution for testing and the method for preparing a sample solution for testing that enable efficient preparation of the sample solution for testing, and efficient control for safety and quality of products can be performed.
Embodiments of the present disclosure are described below in detail with reference to the drawings. Note that dimensions, materials, and other specific numerals in the embodiments are given for easy understanding of the contents, and do not limit the present disclosure unless otherwise noted. Further, in the description and the drawings of the present application, elements having substantially the same function and configuration are denoted with the same reference symbol, and redundant description thereof is omitted. Elements that are not directly related to the present disclosure are omitted in illustration.
When food and drink or pharmaceutical products are produced, a microorganism test, a foreign matter test, or the like is conducted for the purpose of controlling safety and quality of the products. In a microorganism test, the number of microorganisms contained in a sample solution is counted, thereby confirming microorganism contamination. The number of microorganisms is counted by optically analyzing particles dispersed in a fluid. With this, the number of living bacteria can be counted rapidly. For such efficient counting, it is effective to prepare a sample solution in which microorganisms that may be contained in an undiluted sample solution are concentrated. Further, when particle-like solid matters larger than the microorganisms are contained in the undiluted sample solution, a sample solution for testing from which such matters are removed as contaminants is prepared, which is effective for precise counting. Further, in a foreign matter test, when a sample solution in which particles that may be contained in a liquid are concentrated is prepared, the remaining part of the undiluted sample solution can be recovered. Thus, when no abnormality is found during a foreign matter test, the recovered solution can be reused. Therefore, concentration of particles contained in a liquid and separation in accordance with a size are key factors in preparing a sample solution for testing.
One of the separation techniques for separating particles contained in a liquid uses an effect of a Dean vortex generated in the liquid (see Patent Literature 1 given above; hereinafter, the technique is referred to as hydrodynamic separation). This separation technique utilizes uneven distribution of the particles in the liquid in which a Dean vortex is generated. The liquid flows through a curved flow channel that has a rectangular cross-section perpendicular to a flow direction and is curved to one side. The particles flowing through the curved flow channel are distributed differently in the flow channel in accordance with a size (see Patent Literature 1 given above). Specifically, ring-like particle distribution is formed in the cross-section of the flow channel, and the particles spirally flow through the flow channel. In this state, a particle having a relatively large size is positioned on the outer side of the ring, and a particle having a relatively small size is positioned on the inner side of the ring. Further, when a predetermined separation condition is set, a particle distribution mode further changes. As a result, particles having a size exceeding a certain level converge on the outer circumferential side of the flow channel.
In the present disclosure, the above-mentioned hydrodynamic separation is applied to separation of particles, in other words, microorganisms or solid matters having a particle-like shape contained in the liquid, and is used in a separation mode in which microorganism particles or solid matter particles having a predetermined size or larger converge on the outer circumferential side of the curved flow channel. With this, a liquid raw material, a liquid intermediate, or a liquid final product in manufacturing products is used as an undiluted sample solution, and then a sample solution for testing is prepared. In the system for preparing a sample solution for testing according to the present disclosure, a liquid is supplied to a curved flow channel at a relatively high flow speed. As described above, while the liquid to be supplied flows through the curved flow channel, relatively small particles are distributed in a ring shape in the cross-section of the flow channel. Meanwhile, relatively large particles converge evenly on the outer side (outer circumferential side) of the curved flow channel. In other words, a liquid in which the relatively large particles are concentrated can be isolated as a first segment. Therefore, when the undiluted sample solution contains microorganism particles or solid matter particles, the undiluted sample solution is separated into the first segment in which the particles are concentrated and a second segment being the rest. With this, the first segment can be used as a sample solution for a microorganism test or a foreign matter test. When there is no abnormality found during the test, the second segment being the rest can be recovered and reused. With this, in a case of an undiluted sample solution in manufacturing an expensive pharmaceutical product such as an injection solution, a loss caused as a result of testing can be reduced. The microorganisms are separated in accordance with a particle size during hydrodynamic separation. Thus, the microorganisms are separated as particle-like organisms, and may be any one of microorganism single cells and particle-like aggregates of a plurality of cells. The solid matter particles may be any one of organic particles and inorganic particles, and the solid matter particles formed of, for example, plastic, elastomer, fibers, ceramics, and metal can be separated suitably.
An undiluted sample solution in manufacturing food and drink contains particle-like solid matters larger than microorganisms being a test target in some cases. Specifically, the particles contained in the undiluted sample solution may contain relatively large particle-like solid matters and relatively small microorganism particles. For example, when a seasoning liquid such as soy sauce is used as a liquid raw material, fine particles derived from the raw material or component aggregates may be contained. The same holds true for a liquid intermediate in the course of manufacture. Tea beverage, fruit beverage, or the like being a liquid final product contains particle-like solid matters such as tea leaf particles, fruit particles, or the like. In this case, during hydrodynamic separation, the particle-like solid matters being relatively large particles are concentrated in the first segment, and the second segment being the rest contains most of the relatively small particles. Therefore, the second segment obtained after removing the particle-like solid matters is used as a sample solution for testing in a microorganism test. With this, microorganism particles being relatively small particles can be detected. When there is no abnormality found during the test, the first segment may be recovered and reused. When the undiluted sample solution contains microorganisms, the second segment in this case may contain dead cell pieces (debris) and the like. In order to remove debris and the like from the sample solution for testing, a separation state may be adjusted, and hydrodynamic separation is further repeated. With this, microorganisms being relatively large particles and debris being relatively small particles can be separated. A size of the concentrated particles can be adjusted based on settings of a flow speed (flow rate) of the liquid supplied to hydrodynamic separation and a size of the cross-section of the curved flow channel.
A microorganism grows large in a highly active state. However, as activity lowers, a microorganism becomes dead and dissolved while having a relatively small size. Most of the relatively small microorganism particles are dead microorganisms or dead microorganism pieces, and the chances that active cells in the course of DNA synthesis are contained are low. A size of the particles to be separated through hydrodynamic separation can be adjusted by changing a separation condition (a supply flow rate of the liquid). Thus, by repeating hydrodynamic separation under different separation conditions, active microorganisms and debris and the like can be separated. Through multi-step hydrodynamic separation in which hydrodynamic separation is repeated while changing the separation condition, particles having different particle sizes can be isolated at the respective steps. Alternatively, when hydrodynamic separation is repeated under a constant separation condition, a concentration degree and separation accuracy can be improved.
In hydrodynamic separation, a flow in the flow channel is a laminar flow. Fracture of the particles contained in the liquid is relatively less likely to happen, which is significantly effective in preparing a sample solution for a foreign matter test. However, in consideration of maintaining an efficient microorganism test and an action performed when culture is required, presence or absence of damage on microorganisms during preparation of a sample solution needs to be examined. As a result of examination on this point, microorganisms such as a yeast and fungi have resistance under a relatively high pressure, and a survival rate thereof is maintained even when a pressure of, for example, approximately 1 MPa is applied. Moreover, the microorganisms have considerably high durability against pressure fluctuation. In a case where animal cells are cultured, the animal cells are affected by fluctuation of a pressure (pressure reduction) applied to the cell during separation, and a survival rate of the cells is lowered easily. Nevertheless, even when the microorganisms are under considerably large pressure fluctuation during hydrodynamic separation, a survival rate thereof can be maintained. Therefore, during hydrodynamic separation of microorganism particles, it is not particularly required to control a pressure environment. Thus, a hydrodynamic pressure can be changed by changing a driving force of a pump or the like that is used to supply a liquid to a hydrodynamic separation device, and hence a flow rate of the liquid can be adjusted based on a hydrodynamic pressure in a freely selective manner. In other words, settings for the separation condition can be changed easily through drive control of a pump, and hence the system configuration can be easily simplified. Further, hydrodynamic separation has advantages in that an aseptic state of the liquid can be easily maintained and that separation with high reproducibility can be achieved regardless of the skill level of an operator.
When an impact on microorganisms being a test target, which is caused by a pressure environment, is of concern, fluctuation of a pressure applied to microorganisms through hydrodynamic separation may be controlled. In this case, the separation condition is set so that fluctuation of a pressure (pressure reduction) applied to the cell during separation does not exceed a certain level. With this, a pressure environment in which the liquid is supplied to the curved flow channel is controlled. In other words, supply of the liquid is controlled so that fluctuation of a pressure (a difference between an inlet pressure and an outlet pressure) applied to the microorganisms during separation is equal to or lower than a predetermined value. Specifically, control is performed appropriately when pressure fluctuation (pressure difference) is set to be less than 0.60 MPa, preferably, equal to or less than 0.45 MPa, and more preferably, equal to or less than 0.40 MPa. Under the separation condition for controlling pressure fluctuation as described above, microorganisms are less damaged, and a survival rate of the microorganisms can be maintained at approximately 98% or higher. Therefore, large microorganism particles after concentration and separation can be isolated, and a proliferation function or the like thereof can be maintained. Under this state, the large microorganism particles can be supplied for measurement of particle diameter distribution, microorganism culture, testing, and the like.
With reference to the embodiments illustrated in the drawings, the configuration of the system for preparing a sample solution for testing is described below. A system 1 for preparing a sample solution for testing in
In the embodiment of
The hydrodynamic separation device 4 includes the curved flow channel inside, which has a constant rectangular cross-section perpendicular to the flow direction. At one end of the curved flow channel, a single inlet port 41 through which the undiluted sample solution L is taken in is formed. At the other end of the curved flow channel, at least two outlet ports 42 and 43 for separating and discharging the undiluted sample solution are formed. While the liquid flows through the curved flow channel, a vortex flow is generated in the liquid swirling in one direction. The hydrodynamic separation device 4 utilizes the vortex flow, and causes relatively large particles among the particles contained in the undiluted sample solution L to be evenly present on the outer side (outer circumferential side) of the flow channel. Therefore, the liquid discharged from the curved flow channel is divided into the outer-side segment and the inner-side segment, and hence the liquid in which the relatively large particles are concentrated can be separated as the outer-side segment. A liquid La (first segment) containing the concentrated particles having a relatively large size is discharged from the one outlet port 42 through a flow channel 7 connected to the outlet port 42. A liquid Lb (second segment) being the rest that contains relatively small microorganism particles is discharged through a flow channel 8 connected to the other outlet port 43.
Therefore, when the undiluted sample solution L does not contain particles that are larger than the microorganisms being a test target, the microorganism particles are concentrated in the first segment, and can be obtained as the liquid La from the outlet port 42. The liquid La can be used as a sample solution for testing in a microorganism test, and thus presence or absence of microorganisms can be tested. As required, counting of the number of microorganism particles, measurement of particle diameter distribution and a particle shape, and the like may be performed.
The curved shape of the curved flow channel may be a substantially circular shape, a substantially arc shape (partial circumference), a spiral shape, or the like, and may be selected freely from those shapes. The hydrodynamic separation device 4 may be designed with a flow channel unit including one curved flow channel as a flow channel unit. Specifically, the flow channel unit is configured in the following manner. A molded body having a plane layer is formed of plastic or the like, and one curved flow channel is formed inside the molded body. Then, the end surfaces of the molded body are opened at both the terminal ends of the curved flow channel, and hence one inlet port and at least two outlet ports are formed. The molded body as described above is used as the flow channel unit. The hydrodynamic separation device may consist of one flow channel unit or a combination of a plurality of flow channel units. When the plurality of flow channel units are layered to form the flow channel in a parallel state, a processing flow rate of the undiluted sample solution L can be increased.
Efficiency in separating the particles in the hydrodynamic separation device 4 is changed in accordance with a De number and a pressure of the liquid supplied to the curved flow channel. When the De number and the pressure fall within proper ranges, separation can be performed suitably. The Dean number is expressed in Equation of De=Re(D/2Rc)1/2 (Re denotes a Reynold's number (−), D denotes a representative length (m), and Rc denotes a turning radius of the flow channel (m)). The De number is proportional to a flow speed of the liquid. Thus, when a flow speed of the liquid supplied to the hydrodynamic separation device is controlled properly, the particles can be separated suitably. In general, the De number is preferably 30 or greater and 100 or less, and more preferably, approximately from 50 to 80. Therefore, a flow speed (flow rate) of the liquid is set so that the De number falls within this range. In accordance with a size of the particles to be separated, a flow rate of the liquid supplied to the hydrodynamic separation device 4 may be adjusted.
The liquid feeding unit 5 of the system 1 for preparing a sample solution for testing includes a pressurizing device that applies, to the undiluted sample solution L, a hydrodynamic pressure for supplying the undiluted sample solution L to the hydrodynamic separation device 4, namely, the pump 10. The hydrodynamic pressure applied by the pump 10 changes a flow rate and a flow pressure at which the undiluted sample solution L is supplied to the hydrodynamic separation device 4. A survival rate of the microorganisms is an important factor when a microorganism test is conducted. In this regard, the microorganisms have durability against separation performed by the hydrodynamic separation device 4, and a survival rate can be maintained. However, in a case where it is desired that an impact of pressure fluctuation be eliminated, a suitable pressure environment can be controlled through drive control of the pump 10, for example. When a pressure environment is controlled so that a pressure difference is less than 0.60 MPa, preferably, equal to or less than 0.45 MPa, and more preferably, equal to or less than 0.40 MPa, reduction of a survival rate and damage on the microorganisms during separation can be prevented. When a pressure control valve and a flow rate adjusting valve are provided, a supply pressure and a supply flow rate of the undiluted sample solution can be adjusted in a freely selective manner. Thus, a pressure and a flow rate may be monitored through use of a pressure gauge and a flowmeter.
In the system 1 for preparing a sample solution for testing in
When an impact on the microorganisms is to be eliminated as much as possible while preparing a sample solution for testing, a pump of such a type that does not apply a shearing force to the microorganisms may be used. Specifically, it is suitable to use a positive displacement pump that utilizes a volume change caused by reciprocating motion or rotating motion and pushes out a liquid having a predetermined volume. Examples of the positive displacement pump include a reciprocating pump such as a piston pump, a plunger pump, a diaphragm pump, and a wing pump, and a rotary pump such as a gear pump, a vane pump, and a screw pump. In one embodiment, for example, a pressure tank to which a compressor is attached may be used. In this embodiment, a liquid stored in the pressure tank can be pressurized by the compressor, and then the liquid can be forcibly fed from the pressure tank to the hydrodynamic separation device.
The liquid feeding unit of the system for preparing a sample solution for testing may further include a return channel that returns one of the first segment and the second segment to the manufacturing line. The system 1 for preparing a sample solution for testing in
In the system 1 for preparing a sample solution for testing, a method for preparing a sample solution for testing is performed to prepare a sample solution for testing from an undiluted sample solution. The preparation method involves microorganism separation processing. The microorganism separation processing includes a hydrodynamic separation step and a liquid feeding step of supplying the undiluted sample solution to the hydrodynamic separation step. In the hydrodynamic separation step, the liquid is separated into the first segment and the second segment through use of a vortex flow generated in the liquid flowing through the curved flow channel having a rectangular cross-section. With this, the particles contained in the undiluted sample solution are concentrated in the first segment. By using an undiluted sample solution not containing particles that are larger than the microorganisms being a test target, the liquid in which the microorganisms are concentrated can be obtained as the first segment. This liquid can be used as a sample solution for testing in a microorganism test, thereby confirming presence or absence of microorganisms. As required, counting of the number of microorganism particles, measurement of particle diameter distribution and a particle shape, and the like may be performed in combination.
In other words, the above-mentioned particles being the separation target during hydrodynamic separation are microorganism particles. The first segment is supplied as a sample solution for testing. The second segment is discharged from the hydrodynamic separation device to the outside of the system, or is returned to the manufacturing line PL through the return channel. The microorganism particles having a size of approximately 0.1 μm to a few millimeters may be suitably separated. Microorganism particles exceeding such a size can be separated in accordance with the separation condition. Microorganisms of at least one type selected from a virus, a eubacterium, an archaebacterium, a fungus, a myxomycete, an alga, and a protist may be used as a test target.
In the hydrodynamic separation step, the undiluted sample solution is introduced through a single inlet port into the curved flow channel having a rectangular cross-section, and is supplied to the curved flow channel under a uniform state. The curved flow channel of the hydrodynamic separation device is a flow channel having a rectangular cross-section perpendicular to the flow direction (radial cross-section). While the uniform liquid flows through the curved flow channel, distribution is changed to a ring-like shape in the rectangular cross-section with a Dean vortex. At the same time, a dynamic lifting force stagnating on the outer circumferential side of the flow channel acts on the particles, and distribution thereof is concentrated on the outer circumferential side. The terminal end outlet of the curved flow channel is divided into two ports, namely, the outlet port 42 positioned on the outer circumferential side and the outlet port 43 positioned on the inner circumferential side. The liquid containing the concentrated particles is discharged from the outlet port 42 on the outer circumferential side, and the liquid being the rest is discharged from the outlet port 43 on the inner circumferential side. The separation condition in the hydrodynamic separation step can be changed by adjusting a flow rate in the liquid feeding step. An appropriate flow rate may be set in accordance with a size of the particles being the separation target.
A system 1A for preparing a sample solution for testing in
In
A system 1B for preparing a sample solution for testing in
The liquid feeding unit 5 of the system 1B for preparing a sample solution for testing may further include a return channel that returns one of the first segment and the second segment to the container 2. In the embodiment in
The container 2 is a container capable of preventing microorganism contamination. A container equipped with a heater or a cooler and having a temperature adjusting function may be used as the container 2, as required. The liquid inside is maintained at a temperature suitable for storing the undiluted sample solution. The container 2 may include a stirring device for uniformizing the liquid. With this, stirring can be performed at such an appropriate speed that the microorganisms are not damaged. Alternatively, in order to perform adjustment to achieve an environment suitable for the microorganisms, a container having a function of adjusting an amount of oxygen/carbon dioxide/air, pH, conductivity, a light amount, or the like may be used as required.
The method for preparing a sample solution for testing that can be performed in the system 1B for preparing a sample solution for testing in
The configuration in which the undiluted sample solution L is supplied from the container 2 is suitable to a case where the liquid that is directly extracted from the manufacturing line is subjected to dilution or filtration/separation, or the like and is used. Further, the configuration may be used in case of conducting a test as a control test and the like using cultured microorganism, and a sample solution may be prepared.
A system 1C for preparing a sample solution for testing in
The method for preparing a sample solution for testing, which is performed in the system 1C for preparing a sample solution for testing in
In the system 1 for preparing a sample solution for testing in
A system 1E for preparing a sample solution for testing in
In the system 1E for preparing a sample solution for testing, a liquid Ld is discharged as the second segment through a flow channel 13 connected to an outlet port 43a of the additional hydrodynamic separation device 4a. Further, the configuration is made so that a return channel 13a that is branched from the flow channel 13 and is connected to the manufacturing line PL is provided. With this, the liquid Ld being the second segment can be returned to the manufacturing line PL. Similarly, a switching valve 9a is provided at a branching point of the return channel 13a, and is capable of selecting any one of discharging to the outside of the system and returning to the manufacturing line PL. The configuration of the system 1E for preparing a sample solution for testing in
In the system 1E for preparing a sample solution for testing in
Note that multi-step hydrodynamic separation as performed in the system 1E for preparing a sample solution for testing in
A system 1F for preparing a sample solution for testing in
Specifically, the system 1F for preparing a sample solution for testing includes a test device 14 and the flow channel 7 that connects the hydrodynamic separation device 4 to the test device 14. The flow channel 7 connects the outlet port 42 of the hydrodynamic separation device 4 and the test device 14 to each other, and the liquid discharged as the first segment is supplied to the test device 14 as a sample solution for testing. The system 1F for preparing a sample solution for testing further includes a flow channel that connects the test device 14 to the manufacturing line PL. Specifically, a flow channel 15 for discharging the sample solution after testing from the test device is branched, and a return channel 15a is provided. At the branching point, a switching valve 16 is provided. Therefore, the sample solution after testing can be discharged to the outside of the system or can be returned to the manufacturing line PL in a selective manner, and a change therefore can be made by the switching valve 16.
As illustrated in
Through use of the system in
As shown in the Equation given above, the De number is changed in accordance with the turning radius Rc of the curved flow channel and the dimension of the cross-section of the flow channel (the representative length D in the Equation given above can be regarded as the width of the curved flow channel). Therefore, adjustment can be performed based on the design of the curved flow channel so as to obtain a suitable value for the De number. With this, the hydrodynamic separation device can perform concentration and separation of particles with satisfactory separation efficiency. Further, the flow rate of the liquid can be adjusted in accordance with the settings of any one of the width (radial cross-section) and the height of the cross-section of the curved flow channel. Thus, the hydrodynamic separation device may be configured based on the design of the curved flow channel so that separation of the particles can be performed at a desired flow rate. Therefore, the design of the curved flow channel may be changed as appropriate so that suitable separation is achieved in accordance with conditions of the separation target (dimension distribution of microorganisms, viscosity of a liquid, and the like). From a viewpoint of efficiency in separating the microorganism particles, it is suitable that the curved flow channel is a flow channel with a rectangular cross-section having an aspect ratio (width/height) of 10 or greater. The liquid is supplied to the curved flow channel described above at a flow rate of approximately 100 mL to 500 mL per minute. With this, separation progresses satisfactorily, and the microorganism particles can be concentrated and separated at an efficiency of approximately five billion to twenty-five billion cells per minute. In this case, the microorganism particles having a particle diameter of approximately 10 μm or larger can be concentrated and isolated as the segment on the outer circumferential side. The relatively small microorganism particles having a particle diameter of approximately 5 μm or smaller are distributed in a ring-like shape, and the most part thereof is isolated as the segment on the inner circumferential side. With the design of the terminal end outlet of the curved flow channel (positions of the divided outlet ports), a size and separation accuracy of the particles contained in the segment on the outer circumferential side can be adjusted. Thus, the lower limit size of the microorganism particles concentrated in the segment on the outer circumferential side can be smaller than 10 μm. As illustrated in
A separation state of the particles in the hydrodynamic separation device (a size and an isolation ratio of the concentrated particles) differs depending on positions of the divided outlet ports. In general, a cross-sectional area ratio of the flow channel outlet is designed so that a division ratio (volume ratio) of the segment on the outer circumferential side/the segment on the inner circumferential side is approximately 10/90 to 70/30, which is suitable for concentration and separation for the particles as described above. In the system for preparing a sample solution for testing in
The system for preparing a sample solution for testing described above is used, and thus sample solutions for testing can be prepared from various types of undiluted sample solutions. Specific examples that can be used as the undiluted sample solution include liquids such as a liquid raw material, a liquid intermediate, a liquid final product in manufacturing pharmaceutical and medical supplies and chemical products, a culture solution for microorganisms or cells, drinking water, water for manufacture, and a food sample extracted from food. Examples of the water for manufacture include water for manufacturing medical and pharmaceutical products, water for manufacturing food and drink, and water for washing that is used in manufacturing industrial products. Examples of the liquid final product include a refreshing beverage, a lactic fermenting beverage, milk, beer, Japanese sake, and alcoholic beverages such as spirits. Examples of water for washing industrial products include water used in manufacturing process of an electronic substrate, a semiconductor, a precision apparatus, and medical equipment.
In the system for preparing a sample solution for testing according to the present disclosure, an impact on the components contained in the liquid is small during hydrodynamic separation. Thus, the system is applicable to a test for a substance that is easily deteriorated. For example, a glycoprotein such as an antibody contained in a liquid for culturing cells is a long chain polymer substance that is vulnerable. Even for such a substance, a sample solution for testing can be prepared through use of hydrodynamic separation. Specifically, through hydrodynamic separation, the cells contained in the culturing liquid are concentrated in the first segment, and are removed. Then the second segment can be subjected to testing and measurement. Therefore, the system for preparing a sample solution for testing in
Examples of the microorganism includes a procaryote (a eubacterium and an archaebacterium), a eucaryote (an alga, a protist, a fungus, and a myxomycete), and a virus. Specifically, examples of the procaryote include bacteria such as a colon bacillus, a hay bacillus, and Staphylococcus aureus, and hay bacilli such as actinomyces, cyanobacteria, and methanogenic bacteria. Examples of the eucaryote include algae such as spirogyra and wakame seaweed, protists such as a paramecium, and eumycetes such as mold, a yeast, and a mushroom. Further, examples of the protists include amebas. Trochelminths such as a rotifer and microalgae such as Botryococcus braunii are also included as microorganisms.
Hydrodynamic separation is applicable to a liquid culture medium for culturing microorganisms. The liquid culture medium may be any liquid culture medium such as a synthetic medium, a semi-synthetic medium, and a natural medium, and may contain a nutrient, a differentiating agent for the purpose of testing (such as a pH indicator, an enzyme substrate, and a sugar), a selective agent for suppressing growth of microorganisms other than target microorganisms, and the like. A liquid culture medium having low viscosity is preferably used so that hydrodynamic separation efficiently progresses. Further, in a case of a microorganism particle such as a virus having a small particle diameter, a flocculation agent may be added to a liquid culture medium so as to form a particle having a size that facilitates hydrodynamic separation. Examples of the flocculation agent include a polymer flocculation agent and an inorganic flocculation agent such as polyaluminum chloride, aluminum sulfate, and polysilica iron.
When the test device 14 is a test device for microorganisms, the test device 14 may include a means for measuring at least one of the number of microorganism particles, particle diameter distribution, and a survival rate of the microorganisms. With a test device performing an optical analysis, a sequential and efficient test can be conducted. Further, measurement performed by the test device 14 may involve operations such as culture, observation with a microscope, staining or light emission with a reagent, fluorescence detection, an electrophoresis, and absorption spectrum measurement through use of an electromagnetic wave such as an infrared spectrum. Further, an operation utilizing an indicator such as a marker selectively reacting to a specific component, structure, or gene may be performed. Measurement for microorganisms may be performed by an operation such as an optical analysis through flow cytometry. Examples of the devices capable of performing those operations include a flow cytometer (product name: Attune NxT) available from Thermo Fisher Scientific Inc., a cell analyzer (product name: Vi-Cell) available from Beckman Coulter Inc., and a plate reader (product name: ViewLux) available from PerkinElmer Inc.
The sample solution for testing may be used to culture the microorganisms contained in the liquid. The microorganisms may be cultured in accordance with a usual method under culture conditions that have been known to be suitable for the microorganisms. A method suitable for the microorganisms to be cultured may be selected and used as appropriate. In general, a selective enrichment medium or a selective separation medium prescribed for increasing a specific strain type is suitably used. The culture medium may be selected and used from commercially available liquid culture media as appropriate, or may be produced in accordance with a known prescription through use of a nutrient, purified water, and the like. A differentiating agent for the purpose of testing (such as a pH indicator, an enzyme substrate, and a sugar), a selective agent for suppressing growth of microorganisms other than target microorganisms, and the like may be added as required.
As described above, through use of the hydrodynamic separation technique, the particles contained in the undiluted sample solution can be concentrated and separated, and thus the sample solution for testing can be prepared. At the time of preparation, damage on the solid matter particles and an impact on the microorganisms can be prevented. Hydrodynamic separation can promote concentration and separation for particle components much more efficiently than centrifugal separation, and can perform concentration and separation with a higher survival rate without damaging microorganisms as compared to filter separation and the like. Further, an expensive component or a rare substance can be recovered. The liquid is supplied to the curved flow channel at a relatively high speed during hydrodynamic separation. Thus, the system for preparing a sample solution for testing according to the present disclosure has high processing capacity of performing concentration and separation for particles, and a sample solution for testing can be prepared efficiently. Further, the present disclosure is sufficiently applicable to quality control and safety control in manufacturing products.
With reference to a particle separator described in Patent Literature 1 given above, a resin molded body having a flat plate-like shape was produced. Inside the molded body, an arc-shaped curved flow channel (a turning diameter: approximately 115 mm, a radial cross-section of a flow channel: a rectangular shape, a flow channel width: approximately 3 mm) was formed. The molded body was used as a flow channel unit, and a hydrodynamic separation device was configured. A syringe pump (that pressurized and fed the liquid by pushing and pulling a plunger repeatedly) was used as the pump 10 for supplying the liquid, and the hydrodynamic separation device described above was used. In this manner, the system 1B for preparing a sample solution for testing in
Yeast were added and dispersed in water. A yeast aqueous liquid containing yeast particles was thus produced as an undiluted sample solution, and was stored in the container 2. Through use of the syringe pump, the yeast aqueous liquid in the container 2 was pressurized and fed to the curved flow channel of the hydrodynamic separation device at a constant flow rate of 110 mL/min. The first segment on the outer circumferential side and the second segment on the inner circumferential side that are discharged from the outlet ports 42 and 43 of the hydrodynamic separation device, respectively, were recovered in recovery containers. For each of the undiluted sample solution and the first segment, the number of yeast contained in the liquid and a survival rate (a rate of living yeast in the total number of yeast) were measured through use of an image type particle measurement device (available from Beckman Coulter Inc., product name: Vi-Cell). This operation and measurement were performed six times. The results obtained in each time are shown in Table 1. Note that a concentration rate in Table 1 is a yeast concentration in the first segment, and is expressed as a percentage (%). The concentration rate was obtained while regarding the number of yeast particles per unit volume of the liquid as a yeast concentration and regarding a yeast concentration in the undiluted sample solution as a reference.
As understood from Table 1, in any one of the six operations, the yeast were concentrated in the first segment. Significant reduction of a survival rate of the yeast was not confirmed before and after hydrodynamic separation. Thus, it is concluded that a separation operation caused little damage on the yeast. Further, a survival rate of the yeast in the first segment was significantly increased as compared to the yeast aqueous liquid before separation. The survival rate was increased because a rate of the relatively small particles contained in the first segment was reduced, and thus a rate of dead fungus bodies, debris, and impurities was reduced. Therefore, it is clear that the first segment in which the yeast were concentrated was able to be recovered through hydrodynamic separation without reducing a survival rate of the yeast and be supplied as a sample solution for testing.
A suitable amount of an amino acid (L-alanyl-L-glutamine) and a suitable amount of an antibiotic (penicillin, streptomycin, amphotericin B) were added to a liquid culture medium of 1.5 L (available from GE Healthcare, product name: SH30934.01 HyCell CHO Medium). CHO cell strains (ATCC CRL-12445 Cricetulus griseus/CHO DP-12 clone #1932) were cultured in the liquid culture medium while maintaining the temperature from 36.5 degrees Celsius to 37.0 degrees Celsius. During culture, a pH value of the liquid culture medium was controlled so that the pH value was not reduced under 6.70. The culture liquid thus obtained contained an IgG antibody secreted by the cells, and a concentration thereof was measured.
Similarly to Example 1, the system for preparing a sample solution for testing was configured, and the cell culture liquid obtained as described above was stored as the undiluted sample solution in the container 2. Through use of the syringe pump, the cell culture liquid in the container 2 was pressurized and fed to the curved flow channel of the hydrodynamic separation device at a constant flow rate. The first segment on the outer circumferential side and the second segment on the inner circumferential side that are discharged from the outlet ports 42 and 43 of the hydrodynamic separation device, respectively, were recovered in recovery containers. For the second segment, the number of cells contained in the liquid was counted through use of an image type particle measurement device (available from Beckman Coulter Inc., product name: Vi-Cell). Further, a concentration of the IgG antibody contained in the second segment was measured.
This operation and measurement were performed three times. The results obtained in each time are shown in Table 2. Note that the cell separation efficiency in Table 2 is a value obtained by calculating [1−(x/X)]×100 (%), where, in the expression, X denotes a cell concentration in the undiluted sample solution, and x denotes a cell concentration of the second segment (the segment on the inner circumferential side) after separation.
As understood from Table 2, in any one of the three operations, the separation efficiency was 90% or higher, and the cells were concentrated in the first segment. Further, a significant change in antibiotic concentration was not found before or after hydrodynamic separation. Based on this fact, even a vulnerable long chain protein such as an antibody protein can be supplied for testing without causing composition changes. In other words, an analysis on components in a liquid, a microorganism test, and the like can be conducted without changing the components in the liquid. With this, quality of the liquid can be confirmed. Therefore, by preparing a sample solution for testing through utilization of hydrodynamic separation, degradation of analysis quality, which is caused by contamination with solid components, can be prevented, and a highly accurate test can be conducted.
A sample solution for testing can be prepared efficiently by concentrating and isolating particles contained in a liquid without affecting components contained in the liquid. The sample solution for testing can be used for various tests in manufacturing medical and pharmaceutical products, or food and drink, and quality control and safety control of a manufactured product can be performed efficiently. This utilization contributes to economic efficiency and quality improvement.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-130254 | Jul 2019 | JP | national |
This application is a continuation application of International Application No. PCT/JP2020/026711, filed on Jul. 8, 2020, which claims priority to Japanese Patent Application No. 2019-130254, filed on Jul. 12, 2019, the entire contents of which are incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2020/026711 | Jul 2020 | US |
| Child | 17562242 | US |
