JP6725031B2 - Culture device and culture method - Google Patents

Description

The present invention relates to a culture device and a culture method.

A sterile filling system (aseptic filling system) is known in which a sterilized container (PET bottle) is filled with sterilized contents in an aseptic environment, and then the container is closed with a cap. Specifically, in the aseptic filling system, the molded container is supplied to the aseptic filling system, and the container is sprayed with an aqueous solution of hydrogen peroxide as a bactericide in the aseptic filling system. It is then dried to sterilize the container and then the container is aseptically filled with the contents.

Before actually starting the filling of the container at the initial stage of such an aseptic filling system, it is necessary to confirm whether or not the sterility of the system is ensured. Therefore, various tests are performed to confirm the sterility of the system. After performing such various tests, in order to comprehensively evaluate the sterility of the aseptic filling system in the final stage, fill the medium into the container with the aseptic filling system and evaluate using the container containing this medium. Is being done.

In this case, it is general that the container filled with the medium is left to stand in a thermostatic chamber for culturing. Then, after a lapse of a predetermined period (for example, 7 days or more), the container filled with the medium is taken out from the thermostatic chamber, and it is verified whether or not the bacterium has survived or propagated in the medium in the container. However, conventionally, there is a problem that it is difficult to use the aseptic filling system during this period because it takes a long time to culture the container in a constant temperature oven.

JP, 2010-233563, A

For aerobic microbes, oxygen respiration is essential for energy harvesting. On the other hand, conventionally, as a culture device for aerobically culturing microorganisms, there are a culture device that agitates by applying force from above the culture container, and a culture device that rotates and agitates the culture container. There is known a culture device that supplies oxygen to microorganisms according to the purpose and conditions.

For example, a culture device is known in which an external force is applied from the upper surface of a culture container to stir a culture solution (see Patent Document 1). Such a culture device is designed so as not to interfere with the formation or decomposition of aggregates of microorganisms. However, in the culture device shown in Patent Document 1, since the culture container needs to be made of a soft material so that it can be stirred by an external force, when a culture container of a hard material that cannot be deformed by an external force is used, There are restrictions such as the inability to perform.

In another example, there is known a culture device in which a culture vessel is moved in the left-right direction (rotation, reciprocating movement, or the like) to stir the culture solution. Such a culture device is designed to improve the oxygen supply of microorganisms. However, since the culture container and the container for packing the culture container must be made of a strong material so as to withstand external force, if the culture container and the container are made of a weak material such as paper, the culture container and the container There is a risk that the container may be deformed by external force, or even damaged. Therefore, it is substantially difficult to use the above-mentioned culture device for verification of, for example, an aseptic filling system.

The present invention has been made in consideration of such points, by efficiently dissolving oxygen in the medium in the closed container, it is possible to rapidly culture the medium in the closed container, culture An object is to provide an apparatus and a culture method.

The present invention is a culturing device for culturing a medium filled in a closed container, wherein the container holding part for holding the closed container, and the closed container with respect to the closed container held in the container holding part. A culture device comprising: a motion imparting section that imparts physical motion from the outside.

The present invention is the culture device, wherein the container holding part is provided with a guide part for preventing or suppressing the fall of the closed container.

According to the present invention, the motion imparting unit is a vibration imparting unit that imparts motion to the sealed container vertically and horizontally, and when the motion is vibration, imparts vibration to the sealed container. It is a characteristic culture device.

The present invention is the culturing apparatus characterized in that the number of movements of the movement given to the closed container by the movement giving unit is 600 times/min or more.

The present invention is the culture apparatus, wherein the motion width (amplitude when the motion is vibration) that the motion imparting unit imparts to the closed container is 5 mm or less.

The present invention is the culture apparatus, wherein a plurality of the container holders are provided in the vertical direction, and each of the container holders holds the closed container.

The present invention is a culture method for culturing a medium filled in a closed container, the step of holding the closed container by a container holding part, and the closed to the closed container held in the container holding part And a step of imparting physical movement from the outside of the container.

The present invention is the culture method, wherein the step of imparting the physical movement is a step of imparting movement (vibration or the like) vertically and horizontally to the closed container.

The present invention is the culture method, wherein in the step of applying the movement (vibration or the like), the number of times of the movement given to the closed container is 600 times/min or more.

The present invention is the culturing method characterized in that, in the step of applying the motion (vibration, etc.), the motion width (amplitude in the case of vibration) of the motion applied to the closed container is 5 mm or less. ..

According to the present invention, the medium in the closed container can be rapidly cultured by efficiently dissolving oxygen in the medium in the closed container.

FIG. 1 is a schematic sectional view showing a culture device according to an embodiment of the present invention. FIG. 2 is a schematic plan view showing a culture device according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. 1 and 2 are diagrams showing an embodiment of the present invention.

(Cultivation device)
First, the culture apparatus according to the present embodiment will be described with reference to FIGS. 1 and 2.

The culture device 10 shown in FIGS. 1 and 2 is a device for culturing the medium M filled in the closed container 35. In particular, the culturing device 10 is for early checking whether or not the bacteria survive and reproduce in the medium M in the closed container 35. Such a culture device 10 applies vibration (movement) from the outside of the closed container 35 to the table (container holding portion) 12 holding the closed container 35 and the closed container 35 held on the table 12. A vibrator (motion imparting unit, vibration imparting unit) 15 is provided.

In this case, the closed container 35 includes a bottle (container) 30 and a cap 33 that closes the bottle 30. The bottle 30 is manufactured by subjecting a preform manufactured by injection molding of a synthetic resin material to biaxial stretch blow molding. As the material of the bottle 30, it is preferable to use a thermoplastic resin, particularly PE (polyethylene), PP (polypropylene), PET (polyethylene terephthalate), or PEN (polyethylene naphthalate). In addition, the closed container 35 may be glass, can, paper, pouch, or a composite container of these. In the present embodiment, a case where a bottle is used as the closed container 35 will be described as an example.

A plurality of closed containers 35 are housed in a housing container 37 such as a box in a lined state, and are placed on the table 12 in this state.

The table 12 is made of a flat plate-shaped member having a substantially rectangular shape. Further, a plurality of (in this case, two) tables 12 are provided separately from each other in the vertical direction. Each table 12 holds a closed container 35 housed in a container 37.

Further, the table 12 located below is provided with a guide portion 14 for preventing the closed container 35 and/or the storage container 37 from falling. Specifically, plate-shaped guide portions 14 are provided upright around the table 12, and each guide portion 14 extends in the vertical direction. Another table 12 is attached above the guide portion 14. The guide portions 14 are provided at both ends of the table 12, respectively. Further, the guide portion 14 is also provided at an end portion in the front direction and/or the rear direction of the paper surface of FIG. Further, the table 12 located above is also provided with a guide portion 14 for preventing or suppressing the closed container 35 and/or the container 37 from falling.

For example, the guide portion 14 may be detachably provided on the table 12, and the table 12 may be sequentially mounted on the guide portion 14. This makes it possible to arrange the tables 12 in multiple stages and arrange a large number of closed containers 35 and/or storage containers 37 in the culture device 10.

A vibrator 15 is attached below the table 12 via a connecting member 13. When the vibrator 15 applies vibration to the table 12, the table 12 vibrates and the closed container 35 on the table 12 also vibrates. As the vibrator 15, for example, an ultrasonic vibrator or the like may be used. In particular, by using the vibrator 15 as the motion imparting section, even if the culture container 35 and the storage container 37 are weak materials such as paper, the amplitude is small so that the culture container 35 and the storage container 37 are not deformed or damaged by an external force, and oxygen in the closed container 35 is used as a medium. The number of exercises can be increased in order to efficiently dissolve in M.

The number of motions of vibration applied to the table 12 and the closed container 35 by the vibrator 15 is 600 times/minute or more. By setting the number of motions of the vibration applied by the vibrator 15 within the above range, oxygen in the closed container 35 can be efficiently dissolved in the medium M, and the culture speed of the medium M can be improved.

Further, the amplitude of the vibration applied to the table 12 and the closed container 35 by the vibrator 15 is 5 mm or less. By setting the amplitude of the vibration applied by the vibrator 15 within the above range, oxygen in the closed container 35 can be efficiently dissolved in the medium M, and the closed container 35 and the storage container 37 are made of a weak material such as paper. Even in this case, it is possible to prevent the closed container 35 and the storage container 37 from being deformed by an external force, and it is possible to improve the speed of culturing the medium M.

The vibrator 15 is fixed to the mounting plate 17 using a vibrator fixing bolt 16. The mounting plate 17 is mounted on the base 11. The base 11 is placed, for example, on a flat horizontal surface in a thermostatic chamber (not shown).

(Culturing method)
Next, a culturing method for culturing the medium M in the closed container 35 using the culturing apparatus 10 described above will be described.

The culture method according to the present embodiment is performed, for example, to confirm whether or not the sterility of an aseptic filling system (aseptic filling system) not shown is secured. In the aseptic filling system, during normal production, a sterilized bottle (PET bottle) 30 is filled with sterilized contents under a sterile environment, and then the bottle 30 is closed with a cap 33. The culturing method according to the present embodiment is, for example, in the initial stage immediately after the above-mentioned aseptic filling system is completed, that is, before the actual filling of the bottle 30 with the aseptic filling system to start the production of the product bottle. It may be done. Alternatively, the present culturing method is performed when there is a risk of affecting sterility, such as when there is any change in the process or equipment in the aseptic filling system, or when the aseptic filling system is not used for a certain period of time. Is also good. Alternatively, the main culturing method may be carried out periodically every predetermined filling cycle, regardless of whether or not there is a risk of affecting sterility.

First, before carrying out the culture method according to the present embodiment, a test as to whether or not sterility is ensured is performed individually for each element of the aseptic filling system. Specifically, for example, a test as to whether or not the supply line for the contents is correctly heated (SIP temperature rise confirmation test), a test as to whether or not the bottle 30 and the cap 33 are properly sterilized (bottle sterilization test). , Cap sterilization test), and a test as to whether or not the aseptic chamber in which the aseptic filling system is arranged is sterilized (chamber sterilization test).

After performing such a test, the sterility of the aseptic filling system is verified in order to evaluate the sterility of the bottle 30. Specifically, a large number of bottles 30 are flown into the aseptic filling system, each bottle 30 is filled with a predetermined medium M instead of the contents to be actually filled, and the bottles are closed with a cap 33. After that, after putting a large number of the closed containers 35 thus produced in the storage container 37, the whole storage container 37 is placed on the culture device 10, and the culture medium 10 in the closed container 35 is cultured using the culture device 10. To do. Then, it is confirmed that the medium M in the closed container 35 does not decompose after a certain period of time.

Hereinafter, the culture method according to the present embodiment will be further described.

First, in the above-described aseptic filling system, a large number of sterilized bottles 30 are filled with the sterilized medium M and the cap 33 is used to close the closed container 35.
The number of bottles 30 is predetermined, and can be a predetermined number, for example, 100 or more and 300,000 or less (preferably 1,000 or more and 30,000 or less).

Next, the closed container 35 filled with the medium M is carried out of the aseptic filling system and packed in a container 37 such as a box. The storage container 37 is tilted (or inverted) manually or automatically on the conveyor so that the medium M is surely brought into contact with the inner surface of the closed container 35.

Next, the closed container 35 is conveyed to the culture device 10 while being housed in the container 37. The culturing apparatus 10 may be placed in a thermostatic chamber previously maintained at a predetermined temperature of 25° C. or higher and 40° C. or lower, for example. When the target product bottle is heated and sold by a hot bender or the like, it is preferable to check the sterility of thermophilic bacteria. In addition to the above culture conditions, the culture device 10 has a temperature of 40°C or higher and 65°C or lower. It may be placed in a constant temperature chamber.

Then, the closed container 35 contained in the container 37 is placed on each table 12 of the culture device 10 and held on the table 12.

Then, the power of the culture device 10 is turned on and the vibrator 15 is operated to vibrate the table 12 in the vertical and horizontal directions (directions of arrows in FIG. 1). As a result, vibration is applied to the closed container 35 held by the table 12 via the table 12. At this time, the number of times of vibration motion is 600 times/min or more, and the amplitude of vibration (motion width) is 5 mm or less. At this time, the oscillator 15 may constantly apply vibration to the closed container 35 with a constant number of movements and/or amplitudes, or vibrate the closed container 35 while periodically changing the number of movements and/or amplitudes. May be given. Alternatively, the vibrator 15 may intermittently apply vibration to the closed container 35.

After the lapse of a predetermined culturing period (for example, 3 days or more, preferably 7 days or more), the culturing apparatus 10 is stopped, all the closed vessels 35 are taken out from the culturing apparatus 10, and the culture medium M in the closed vessel 35 is cultivated with bacteria. Verify whether it is alive or breeding. As a result of this verification, if the number of closed containers 35 in which bacteria survive or propagated is less than a predetermined number (for example, zero), it is determined that the sterility of the aseptic filling system is secured. On the other hand, as a result of the verification, if there are a predetermined number or more (for example, one or more) of the closed containers 35 in which the bacteria survive or propagated, it is determined that the sterility of the aseptic filling system is insufficient, and measures are taken. For example, the bottle 30 may be transported and sterilized in the loading path, or the sterilization condition of the bottle 30 in the aseptic filling system may be adjusted (strengthened).

As described above, by vibrating the closed container 35 using the vibrator 15, the medium M in the closed container 35 is physically moved, and the closed container 35 is stored while the medium M is moving. It As a result, the period for culturing the medium M in the closed container 35 in the culture device 10 can be shortened. That is, by adding vibration to the medium M, the dissolution of oxygen into the medium M is promoted, so that the bacteria are cultivated aerobically, and thus the culture speed of the bacteria can be increased. As a result, the predetermined culture period necessary for the above culture (for example, 3 days or longer, preferably 7 days or longer) can be shortened, and it is possible to quickly determine whether or not bacteria are present in the closed container 35. ..

For example, according to the experiments conducted by the present inventors, when the closed container 35 filled with the medium M is vibrated by using the culture device 10 (Example A), the closed container 35 is not vibrated. It was found that the culture of bacteria can be promoted as compared with (Comparative Example B).

Specifically, a plurality of hermetically sealed containers 35 each filled with a liquid neutral medium were capped in a state in which their headspace contained falling bacteria floating in the laboratory and stored at 22°C to 27°C. During this period, the closed vessel 35 of one group (Example A) was constantly oscillated by the culture device 10 at a frequency of 3,000 movements/minute and with a vibration of 1 mm, and was added to the bottle 30 of another group (Comparative Example B). Was allowed to stand without vibration. As a result, as shown in the table below, the positive rate increased especially after 2 to 3 days of culture. This shows that the vibration can be applied to the closed container 35 to cultivate the bacteria in a shorter number of days than when the vibration is not applied. Here, the positive rate refers to the number of closed containers 35 that have survived or propagated (became positive) (total positive number) in the closed containers 35 that are positive at a predetermined time. It means the ratio of the number. Specifically, the positive rate is calculated based on the formula “positive rate (%)=(number of visually confirmed positives/total number of positives)×100”. The total number of positive cells was the number that was visually confirmed as positive after 13 days of culture.

As described above, according to the present embodiment, the vibrator 15 applies vibration to the closed container 35 held on the table 12 from the outside of the closed container 35. As a result, the period for culturing the medium M in the closed container 35 in the culture device 10 can be shortened. As a result, for example, it is possible to shorten the period during which the aseptic filling system, which is the subject of verifying sterility, cannot be used, and efficiently use the aseptic filling system.

Further, according to the present embodiment, vibration is applied from the outside of the closed container 35 by the vibrator 15, so that, for example, a culture device that applies force from above the culture container to stir the culture medium or a culture medium in the culture container is used. Unlike the culture device that directly stirs, it is not necessary to deform the closed container 35 or open the closed container 35. Further, as long as it can be held on the table 12, the medium M can be cultivated aerobically without being restricted by the material and packaging state of the closed container 35 and the storage container 37.

Further, according to the present embodiment, the table 12 is provided with the guide portion 14 that prevents or suppresses the fall of the closed container 35. As a result, it is possible to prevent the problem that the closed container 35 and the storage container 37 fall from the table 12 while applying vibration from the outside of the closed container 35.

Further, according to the present embodiment, since the vibrator 15 applies vibration to the closed container 35, the closed container 35 can be finely moved. As a result, it is possible to more effectively promote the dissolution of oxygen into the medium M in the closed container 35.

Further, according to the present embodiment, the number of times of vibrations given by the vibrator 15 is 600 times/min or more, and the amplitude of the vibration given by the vibrator 15 is 5 mm or less. As a result, the dissolution of oxygen into the medium M in the closed container 35 can be more effectively promoted.

Further, according to the present embodiment, a plurality of tables 12 are provided in the vertical direction, and each table 12 holds the sealed container 35. By thus arranging the tables 12 in the upper and lower stages in multiple stages, it is possible to collectively apply vibration to a large number of closed containers 35, so that oxygen can be efficiently supplied to the medium M in a large number of closed containers 35. The medium M can be efficiently cultured by allowing it to dissolve.

In addition to the above-mentioned vibration, the movement imparted to the closed container 35 may be a motion method used in a conventional culture device, for example, a motion such as rotation, reversal, and reciprocation. That is, instead of the vibrator 15 (vibration imparting unit), various motion imparting units (rotational motion imparting unit, which impart physical motions such as rotational motion, reversal motion, and reciprocating motion, from the outside of the closed container 35. A reversal motion imparting unit, a reciprocating motion imparting unit) may be used. In this case, by imparting physical motion such as rotation, reversal, and reciprocation to the medium M, the dissolution of oxygen into the medium M is promoted, so that the bacterium is cultivated aerobically, and thus the bacterium is cultured. It is possible to increase the culture rate of. Table 2 shows the motion method, the number of motions, and the like in the case of using the motion imparting unit including the oscillator 15 as well as the motion imparting unit that imparts physical motion such as reciprocating motion and rotary motion.

As described above, in the present embodiment, even if the closed container 35 and the storage container 37 are made of a weak material such as paper, the closed container 35 and the storage container 37 can be prevented from being deformed by an external force. On the other hand, in the culturing apparatus having a large amplitude like Comparative Examples 1, 2, and 3, the culturing vessel 35 and the container 37 for packing the culturing vessel 35 must be made of a strong material so as to withstand an external force. When the container 35 and the storage container 37 are made of a weak material such as paper, the culture container 35 and the storage container 37 may be deformed by an external force, and in some cases (for example, the value of the exercise width is changed). It is necessary to adjust the conditions.

Further, in the above description, the case where the culture apparatus 10 is one in which the closed container 35 filled with the medium M in the aseptic filling system is placed and the medium M in the closed container 35 is cultured is described as an example. However, the application of the culture device 10 is not limited to this, and the culture device 10 can be used for an arbitrary closed container 35 filled with the medium M. For example, the culture device 10 may be used for such purposes as obtaining a product produced in the process of microbial growth.

10 Culture Device 11 Base 12 Table 13 Connecting Member 14 Guide Part 15 Transducer 16 Transducer Fixing Bolt 17 Mounting Plate 30 Bottle 33 Cap 35 Sealed Container 37 Storage Container

Claims (5)

A culture method for culturing a medium filled in a closed container,
In the aseptic filling system, a process of filling a sterilized medium into a plurality of sterilized bottles and closing the cap with a cap to produce a closed container,
A step of carrying out the hermetically sealed container filled with the medium to the outside from the aseptic filling system and filling the container with a container,
A method of culturing, comprising the step of manually or automatically tilting or reversing the accommodation container on a conveyor so that the inner surface of the closed container is brought into contact with the medium. The culture method according to claim 1, further comprising a step of holding the storage container in a culture device and applying physical movement to the storage container from the outside of the storage container using the culture device. The culture method according to claim 2, wherein the step of imparting physical movement is a step of imparting movement vertically and horizontally to the closed container. The culturing method according to claim 3, wherein, in the step of applying the exercise, the number of times of the exercise applied to the closed container is 600 times/minute or more. The culturing method according to claim 3 or 4, wherein, in the step of applying the motion, the motion width of the motion applied to the closed container is 5 mm or less.

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