JP5867893B2 - Freezing method - Google Patents

Description

  The present invention relates to a method for freezing foods, organs, tissue pieces, animal cells, microorganisms and the like.

  When frozen objects with low water content such as animal cells are frozen, ice crystal nuclei (ice seeds) generated inside the frozen object grow into large size ice crystals and damage the cell membrane of the frozen object. Causes water to flow out of the damaged cells upon thawing. This outflow moisture (drip) contains intracellular fluid, and the amount of drip generated at the time of thawing greatly affects the freezing quality of the object to be frozen.

  The faster the freezing rate of the object to be frozen, the more the growth of ice crystal size can be suppressed. For this reason, the object to be frozen is rapidly cooled in a low temperature zone of −50 ° C. or less, which has a large temperature difference from the environmental temperature. However, since a large amount of electric power is required for cooling to a low temperature zone, it is not preferable from the viewpoint of energy saving.

  Therefore, in Patent Document 1, after the food is pre-cooled in a storage at −7 ° C. to be in a supercooled state, cold air is allowed to flow into the storage to rapidly cool the food. A technique for transitioning to freezing is disclosed.

  Patent Document 2 discloses a technique for freezing food stored in a warehouse by ejecting liquefied carbon dioxide into a warehouse such as a freezer and rapidly cooling the interior of the warehouse without temperature change by the latent heat of vaporization. Is disclosed.

Japanese Patent No. 4253775 Japanese Utility Model Publication No. 51-50444

  However, in the freezing method disclosed in Patent Document 1, only the surface layer of the food is partially frozen, and it has been difficult to preserve the freshness for a long time.

  In view of the above, an object of the present invention is to provide a freezing method capable of reducing the amount of drip generated when the object to be frozen is thawed after being frozen.

The sealed container of the present invention is a sealed container for storing an object to be frozen in an internal space, a diaphragm that divides and isolates the internal space into a first space and a second space, and a multicomponent composed of three or more atoms. A first gas supply capable of supplying the first space with an atomic molecular gas or a gas containing the polyatomic molecular gas so that the heat capacity of the gas in the first space is larger than the heat capacity of the dry air having the same number of molecules. A mechanism and a polyatomic molecular gas having 3 or more constituent molecules, or a gas containing the polyatomic molecular gas so that the heat capacity of the gas in the second space is larger than the heat capacity of dry air of the same number of molecules. A second gas supply mechanism capable of supplying to the second space, a first exhaust mechanism for exhausting the first space, a second exhaust mechanism for exhausting the second space, and an object to be frozen inside the first space. And an object-to-be-frozen installation mechanism that holds the object away from the wall surface. It is characterized in.
Moreover, the freezing method of the present invention is a freezing method using the above-described sealed container, wherein the frozen object placement step of sealing the sealed container after placing the frozen object in the frozen object installation mechanism, The first gas that exhausts the gas existing in the first space by the exhaust mechanism and fills the second space with the gas containing the polyatomic molecule gas or the polyatomic molecule gas by the second gas supply mechanism. The supply step and the gas containing the polyatomic molecule gas or the polyatomic molecule gas filled in the second space are exhausted until the diaphragm comes into close contact with the wall surface by the second exhaust mechanism, and the first gas supply mechanism A second gas supply step of filling a space containing a polyatomic molecular gas or a gas containing a polyatomic molecular gas, and cooling the sealed container to lower the temperature in the first space, Cool in natural convection at 16 ° C or less, and supercool After over, characterized in that it comprises a freezing step of freezing.

  In this case, the gas covering the object to be frozen is a polyatomic molecule gas having 3 or more constituent molecules or the heat capacity of the gas in the container is larger than the heat capacity of dry air having the same number of molecules. These gases have a larger heat capacity (molar specific heat) per mole than nitrogen molecules and oxygen molecules composed of two atoms, which are the main components of air.

  If the temperature difference and the initial pressure are the same, the heat transfer coefficient is determined by the specific heat capacity of the fluid in contact with the object to be frozen in natural convection, and the polyatomic gas has a higher heat transfer coefficient than air. Therefore, after canceling the supercooled state, the heat transfer coefficient is increased and the freezing speed of the object to be frozen can be increased compared to the case where the atmospheric gas is air.

  And the latent heat release time is shortened by increasing the freezing speed, and the time for exposure to a freezing temperature near 0 ° C. due to solidification latent heat is shortened. Therefore, it is possible to reduce the degree to which ice crystal nuclei generated inside the object to be frozen grow into large-sized ice crystals and damage the cell membrane of the object to be frozen. Therefore, it is possible to reduce the amount of drip generated during thawing.

  In addition, it is necessary to make the volume in an airtight container large enough compared with the volume of to-be-frozen material. Therefore, the object to be frozen is preferably a small-volume tissue sample, animal cell, microorganism or other research sample. However, in a freezer warehouse or the like that can secure a sufficient volume in the freezer compartment, a large volume of food etc. Can also be frozen.

  In order to bring the object to be frozen into a supercooled state with a deep depth to the center, the initial cooling rate is preferably a slow rate, but after the supercooling is released, it is cooled as quickly as possible to reduce the latent heat of solidification in a short time. Good-quality freezing becomes possible by absorbing heat.

The graph which shows the drip rate when the sample is thawed after freezing. The graph which shows the relationship between the drip rate when the sample is thawed after freezing and the constant pressure molar specific heat of the atmospheric gas. The graph which shows the drip rate when the sample is thawed after freezing. The graph which shows the drip rate when the sample is thawed after freezing. The graph which shows the drip rate when the sample is thawed after freezing. Explanatory drawing of the freezing instrument which concerns on this invention. Explanatory drawing of another freezing instrument which concerns on this invention.

[Freezing principle]
Damage to the object to be frozen at the time of freezing includes damage due to cracks that increase in proportion to the endothermic rate and damage due to ice crystal size growth that decreases in inverse proportion to the endothermic rate. When considering only the freezing without considering the storage and thawing of the object to be frozen, the damage during freezing can be considered as the sum of the two damages.

  The crack at the time of freezing in a to-be-frozen thing is damage to which the crack crack generate | occur | produces or deform | transforms in the outer layer part of a to-be-frozen object. The crack is caused by the outer layer portion that has been first frozen due to heat absorption from the surface layer expanding during freezing inside. The damage due to the ice crystal size growth in the object to be frozen is caused by the growth of ice crystal nuclei generated inside the object to be frozen, and the size of the ice crystal generated in the object to be frozen increases.

  If the object to be frozen has a low moisture content of 65% to 85%, such as animal cells, the damage due to cracking is small, and damage due to ice crystal size growth in the object to be frozen is the main cause of damage to the object to be frozen. . On the other hand, when the object to be frozen has a high water content of more than 90% such as single-cell microorganisms, damage due to cracking becomes a main factor of damage to the object to be frozen.

  The growth of ice crystal size can be suppressed by rapidly cooling the object to be frozen and reducing the time of exposure to a freezing temperature near 0 ° C. due to latent heat of solidification. For that purpose, it is important to freeze at a high freezing rate. However, cooling in a low temperature zone is not preferable from the viewpoint of energy saving because a large amount of electric power is required.

  In addition, when freezing via supercooling, increasing the degree of supercooling to the deep part of the object to be frozen can shorten the solidification latent heat release time after canceling the supercooling, and high-quality freezing becomes possible. In order to shorten the solidification latent heat release time, it is preferable that the heat capacity of the contact refrigerant is large because the heat transfer coefficient is large.

  In particular, when a small amount of an object to be frozen is frozen via supercooling, the heat absorption rate of the solidification latent heat can be increased by increasing the heat capacity of the atmospheric gas. The reason is that if the temperature difference and initial pressure are the same, the heat transfer coefficient is determined by the specific heat capacity of the fluid in contact with the object to be frozen in natural convection. This is because the freezing speed of the object can be increased.

  Specifically, for example, such an atmosphere can be formed by suspending an object to be frozen and placing it on a net-like shelf, and covering almost the entire periphery of the object to be frozen with such a gas.

  If the constituent molecule is a polyatomic gas composed of 3 atoms or more, the molar specific heat is larger than that of air. Examples of such a gas include carbon dioxide, water vapor, chlorofluorocarbon, methane, ethane, propane, and butane.

  In this way, the object to be frozen is mixed and covered with a gas having a molar specific heat larger than that of air, and the object to be frozen is frozen in the supercooling temperature region while allowing natural convection. In addition, the temperature range in which freezing via supercooling is possible is about −10 ° C. to −40 ° C. Therefore, the object to be frozen is suspended in the sealed container so as not to contact the wall surface, and a gas having a large specific heat capacity such as carbon dioxide gas is mixed and sealed, and then cooled in the freezer compartment in the temperature zone. In addition, the heat capacity of the atmosphere gas in the sealed container is the product of the temperature difference between the object to be frozen and the atmosphere gas and the molar specific heat and the number of moles of the atmosphere gas. growing. However, if the temperature difference becomes too large, the initial cooling rate increases and the degree of supercooling decreases. Therefore, an appropriate temperature range exists. If the temperature in the freezer is −10 ° C. or higher, the temperature difference is small, the heat capacity is insufficient, and drip increases. On the other hand, at -30 ° C. or lower, the initial cooling rate is too high, the degree of supercooling decreases, and the drip increases. The optimal temperature range is about -15 ° C to -25 ° C.

  Therefore, it is preferable to naturally release the supercooling under natural convection under a low temperature condition of −15 ° C. to −25 ° C., so that the object to be frozen does not crack and the solidification latent heat release time can be shortened.

〔Evaluation method〕
Conventionally, the quality of an object to be frozen by freezing or thawing has been evaluated by a method of measuring the drip amount at the time of freezing, the elastic value after freezing, the ice crystal size at the time of freezing, or a sensory test. However, these have different evaluation values depending on the part of the object to be frozen, and there are problems in the accuracy and reproducibility of the evaluation. Therefore, if there is not much difference in the quality of the object to be frozen due to freezing, the superiority or inferiority of freezing cannot be distinguished, which has been an obstacle for developing a new freezing method.

  Therefore, the inventors of the present application first developed a method for evaluating the quality of freezing by using Takano tofu as a model for low moisture content such as animal cells and measuring the amount of drip at the time of thawing.

  As a model for a low moisture content frozen object, a sample formed by mixing a powder of Takano tofu with a 1.5 wt% agar aqueous solution was used. Takano tofu is also called frozen tofu and is a preserved food that is freeze-dried tofu. The moisture content of this sample is about 80%, which is close to the moisture content of meat such as seafood and animal meat, which is 65% to 85%.

  Specifically, Koya tofu was crushed with a grater and powdered. Then, 7% by weight of Takano tofu powder with respect to the weight of water was mixed with the boiled 1.5% by weight agar aqueous solution, stirred for 5 minutes, placed in the container, and cooled with ice water to solidify the contents. . And after removing the condensed water droplets in the container, die-cutting was performed using a cylindrical mold having an inner diameter of 12 mm to obtain a cylindrical sample having a diameter of 12 mm and a height of 10 mm. Further, the molded sample was stored in a plastic bag with a chuck and refrigerated at 4 ° C. for 1 day.

  Using this sample, the drip rate was determined as follows.

  First, after measuring the weight Wep of the sample before freezing, the sample was frozen under various conditions without being put in a container or bag. Then, the sample was put in a centrifuge tube (Spitz tube) provided with a filter and a drip container in a frozen state, and naturally thawed by centrifuging at 220 G for 40 minutes using a swing rotor centrifuge. Then, the sample was taken out from the centrifuge tube, the weight Wer was measured, and the freezing drip rate Rer was obtained by the formula (1) based on the weight difference between the samples before and after freezing.

  Rer = 100 × (Wep−Wer) / Wep (1)

  On the other hand, after measuring the weight Wer of the sample before freezing, the unfrozen sample was centrifuged at 220 G for 40 minutes using a swing rotor centrifuge. Then, the weight Wc of the sample after centrifugation was measured, and the centrifugal drip rate Rc was determined by the formula (2) based on the difference in weight of the sample before and after centrifugation.

  Rc = 100 × (Wc−Wep) / Wep (2)

  Finally, from the difference between the frozen drip rate Rer and the centrifugal drip rate Rc, the drip rate Re was determined by Equation (3), and the difference in the drip rate Re depending on the freezing conditions was compared.

  Re = Rer-Rc (3)

  However, since there was variation in sample production, the drip rate Re was compared using samples of the same production lot.

  Since the sample was refrigerated and stored in a sealed bag for 1 day after molding, water droplets on the surface adhered to the bag and a certain amount of water evaporated in the bag, so that the error could be reduced. Furthermore, since it thawed during centrifugation, the drip generated during the thawing could be separated without being reabsorbed by agar or Takano tofu. In addition, since a swing rotor centrifuge is used, the sample can be squeezed in the vertical direction, and the error can be reduced.

〔Evaluation results〕
The sample is suspended in a sealed container so as not to contact the wall, and atmospheric gas in the sealed container having a volume 500 times larger than the volume of the sample is mixed with 20% by volume of carbon dioxide gas (Air + CO ₂ ) and water vapor. Saturated air (Air + H ₂ O), air dried with a desiccant (Air), air mixed with 20% by volume of argon gas (Air + Ar), and put the sealed container in a freezer at −16 ° C. Samples were frozen by natural convection. The respective drip rates Re at this time are shown in FIG.

FIG. 2 shows the relationship between the constant pressure molar specific heat (constant pressure molar heat capacity) of these atmospheric gases and the drip rate. From FIG. 2, it can be seen that the drip rate Re increases as the specific pressure molar specific heat increases in the order of Air + CO ₂ , Air + H ₂ O, Air, Air + Ar. This is considered to be because the freezing rate of the object to be frozen increases as the specific heat capacity of the atmospheric gas increases, the growth of ice crystals is suppressed, and damage to the object to be frozen due to the ice crystal size growth is reduced.

  Furthermore, FIG. 3 is a graph showing the drip rate when two samples are stored and four samples are stored in a closed container of the same volume. From FIG. 3, it can be seen that the smaller the number of storages, the smaller the drip amount. This indicates that the drip amount is smaller as the heat capacity of the atmospheric gas per object to be frozen is larger.

  The reason why the difference in drip rate between the atmospheric gases in FIGS. 1 and 2 is small is considered to be because the mixing ratio of the gas into the sealed container was low. However, the measurement variation of the drip rate is small and strongly suggests the above tendency.

  Therefore, when freezing a small amount of an object to be frozen in a large-capacity sealed container via supercooling, if the gas having a larger specific heat capacity than air is used as the atmosphere gas, the object to be frozen is more effective than the case where air is used as the atmosphere gas. It can be seen that the amount of drip generated at the time of thawing is reduced and the freezing quality is improved.

  Next, the sample was suspended in the sealed container so as not to contact the wall, the atmosphere in the sealed container was set to air, and the sealed container was placed in a freezer at −20 ° C. to freeze the sample by natural convection. At this time, the airtight container and the air inside thereof were pre-cooled air set to -20 ° C. and room temperature air kept at room temperature. These drip rates Re are shown in FIG.

  FIG. 4 shows that the drip rate Re is greater in the air precooled to −20 ° C. than in the case of cooling from room temperature (+ 20 ° C.). Therefore, it can be seen that it is preferable to keep the atmospheric gas at room temperature when a small amount of the object to be frozen is frozen in a large-capacity sealed container. This indicates that the drip amount is small when the difference between the sample temperature and the ambient temperature is small. The room temperature is preferably + 10 ° C. or higher.

  FIG. 5 is a graph showing the drip rate when the sample is suspended from the wall surface of the sealed container and frozen in the sealed container, and the drip rate when the sample is brought into contact with the floor surface of the sealed container and frozen. It is. FIG. 5 shows that the amount of drip is smaller when the sample is cooled away from the wall surface of the sealed container. This indicates that there is less drip when the sample is surrounded by ambient gas, and the gas has much higher heat insulation than the solid, and a better initial refrigeration can be achieved with a slower initial cooling rate.

  4 and 5, it can be seen that the initial cooling rate is slow and a high cooling rate is preferable after the supercooling is released.

[Freezing equipment]
As shown in FIG. 6, the freezing instrument 1 according to the present invention is provided with a hanging rod 4 for hanging a suspended body 3 that accommodates an object to be frozen inside a sealed container 2. Here, the suspended body 3 is a net-like bag or net made of resin. The hermetic container 2 is installed and cooled in a freezer (not shown) at −10 ° C. to −20 ° C.

  And the airtight container 2 has an internal space divided into two vertically by a diaphragm 5, and these spaces are isolated. The suspended body 3 is disposed in the upper space.

  An upper air supply pipe 6 is connected to the upper space, and a lower air supply pipe 7 is connected to the lower space. A gas cylinder 8 filled with liquefied carbon dioxide gas is connected to the upper air supply pipe 6 and the lower air supply pipe 7 via regulators 9 and 10, respectively. The upper air supply pipe 6 and the lower air supply pipe 7 are provided with air supply valves 11 and 12, respectively.

  Further, an upper exhaust pipe 13 is connected to the upper space, and a lower exhaust pipe 14 is connected to the lower space. Exhaust valves 15 and 16 are interposed in the upper exhaust pipe 13 and the lower exhaust pipe 14, respectively.

  Hereinafter, a freezing method using the freezing instrument 1 will be described.

  First, the suspended body 3 that accommodates an object to be frozen such as cells and microorganisms is suspended from the suspension rod 4 in the sealed container 2 through a lid (not shown). And the said lid | cover is closed and the airtight container 2 is sealed. At this stage, all valves 11, 12, 15, 16 are closed.

  Then, the upper exhaust valve 15 is opened to exhaust the air in the upper space through the upper exhaust pipe 13, and the lower air supply valve 12 is opened to supply carbon dioxide gas into the lower space through the lower air supply pipe 7. . After the upper space is sufficiently exhausted and the lower space is filled with carbon dioxide, the upper exhaust valve 15 and the lower air supply valve 12 are closed.

  Next, the lower exhaust valve 16 is opened to exhaust carbon dioxide in the upper space through the lower exhaust pipe 14, and the upper air supply valve 11 is opened to supply carbon dioxide into the upper space through the upper air supply pipe 6. Supply. After the lower space is sufficiently exhausted and the upper space is filled with carbon dioxide, and the diaphragm 5 is in close contact with the lower surface of the container, the lower exhaust valve 16 and the upper air supply valve 11 are closed. And in this state, the airtight container 2 is installed in the said freezer compartment of -10 degreeC--20 degreeC, and is cooled.

  By using such a freezing instrument 1, the carbon dioxide concentration in the internal space can be greatly increased. Therefore, the heat capacity of the atmospheric gas in the sealed container 2 can be increased, the heat transfer rate can be increased, and the amount of drip generated during thawing can be reduced. Note that instead of carbon dioxide, water at room temperature may be saturated with water vapor.

  Moreover, as shown in FIG. 7, an airtight bag 21 may be used as a freezing instrument according to the present invention. The airtight bag 21 is provided with an airtight chuck 22, and the inside of the airtight bag 21 is kept airtight by closing the airtight chuck 22.

  Furthermore, a suspended body 23 that accommodates an object to be frozen is provided inside the airtight bag 21. The suspended body 23 is a net-like bag or net made of resin. In addition, the airtight bag 21 is provided with a valved opening 24 for exhausting the gas inside and for filling the inside with carbon dioxide gas.

  If such an airtight bag 21 is used, since the amount of air initially remaining in the interior is small, the carbon dioxide concentration can be increased by filling the interior with carbon dioxide. Can keep. Therefore, the heat capacity of the atmospheric gas in the airtight bag 21 can be increased, the object to be frozen can be frozen via supercooling, and the amount of drip generated during thawing can be reduced.

Claims (2)

An airtight container for storing an object to be frozen in an internal space surrounded by wall surfaces,
A diaphragm that is divided into a first space and a second space and is isolated, and is deformable so as to be in close contact with the wall surface;
A polyatomic molecular gas comprising three or more constituent molecules, or a gas containing the polyatomic molecular gas so that the heat capacity of the gas in the first space is larger than the heat capacity of dry air having the same number of molecules, A first gas supply mechanism capable of supplying the first space;
A polyatomic molecular gas comprising three or more constituent molecules, or a gas containing the polyatomic molecular gas so that the heat capacity of the gas in the second space is larger than the heat capacity of dry air having the same number of molecules, A second gas supply mechanism capable of supplying the second space;
A first exhaust mechanism for exhausting the first space;
A second exhaust mechanism for exhausting the second space;
An airtight container comprising: an object to be frozen installation mechanism for holding the object to be frozen apart from the wall surface inside the first space. A freezing method using the sealed container according to claim 1,
After placing the object to be frozen in the object to be frozen installation mechanism, the object to be frozen placing step for sealing the sealed container;
The first exhaust mechanism exhausts the gas existing in the first space, and the second gas supply mechanism contains the polyatomic molecular gas or the polyatomic molecular gas in the second space. A first gas supply step for filling the gas to be
The polyatomic gas filled in the second space or the gas containing the polyatomic molecular gas is evacuated until the diaphragm is in close contact with the wall surface by the second exhaust mechanism, and the first gas supply A second gas supply step of filling the first space with a gas containing the polyatomic molecular gas or the polyatomic molecular gas by a mechanism;
A freezing step in which the temperature in the first space is lowered by cooling the sealed container, the object to be frozen is cooled in natural convection of -16 ° C. or lower, and after passing through supercooling, is frozen. And a freezing method characterized by comprising:

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