JP2018193365A - Hemolysis suppressing freeze-preservation method and fresh fish freeze-preservation method, and freeze-device - Google Patents

Abstract

To provide: a blood freeze-preservation method in which hemolysis is suppressed without using additives; in addition, a fresh fish freeze-preservation method in which decrease in freshness is suppressed; and a vibration removal device used for transporting coolant or freeze-liquid used for these freeze-preservation methods.SOLUTION: In the method, a blood or fish meat is rapidly cooled to a first cooling temperature in the range of -4 to -6°C and slowly cooled to a second cooling temperature lower than the first temperature and in the range of -7 to -13°C which is the freeze-temperature, at a speed of -1.5°C/hour to -0.5°C/hour. In the vibration removal device, the transport pipe for the coolant or freeze-liquid supplied from a freeze-machine is branched and a device of half wavelength delay by multiple reflection is inserted on one side to remove the vibration of the freeze-machine by interference.SELECTED DRAWING: Figure 7A

Description

  The present invention relates to a method for cryopreserving whole blood, erythrocyte concentrate, and washed erythrocytes, and particularly to a cryopreservation method and a fresh fish cryopreservation method for suppressing hemolysis, and a refrigeration apparatus used for these methods.

  First, the background art regarding the cryopreservation method for suppressing hemolysis will be described in detail.

1) Prior art relating to a frozen storage method Although stored blood is limited to 4 weeks at most, frozen blood can be stored semipermanently. On the other hand, for human whole blood, red blood cell concentrate, and washed red blood cells provided by the Japanese Red Cross Society, the effective period is 3 weeks after blood collection at a storage temperature of 2 to 6 ° C. If the storage temperature is lowered and the erythrocytes are frozen for the purpose of extending the frozen storage period, there is a problem that the erythrocytes are destroyed due to the formation of ice crystals or the concentration of intracellular electrolytes. In short, a “hemolysis” phenomenon occurs. What solved this was glycene-added blood frozen storage by adding glycerin and removing glycerin with a 5% fructose solution at the time of use.

  As a specific example, a concentrated red blood cell solution is prepared by centrifugation of the collected blood, and an equal volume of a protective solution containing 79% glycerin is slowly mixed in an equal volume, and the rate is about 1 ° C. per minute in an ultra-low temperature bath. Cool down to below 80 ° C and store. In the case of quick freezing, it is stored at 196 ° C below zero in a liquid nitrogen tank. When returning from freezing, thaw at 40 ° C, mix with sugar solution to remove glycerin, and prepare and use a concentrated suspension of 70% red blood cells.

2) Problems of the prior art relating to the cryopreservation method In the above preservation method, since glycerin is an intracellular cryoprotectant for erythrocytes in the use of blood after thawing, a step for removing it is necessary. The problem is that management is cumbersome, takes a long time, is difficult to maintain under optimal conditions that do not cause hemolysis of the blood after thawing, and it is difficult to constantly control the hemolysis rate to a certain level. is there.

Therefore, a blood storage method that can use erythrocytes for blood transfusion without washing is desired.
In addition, there is a demand for blood storage that is not as long as cryogenic cryopreserved blood, and that it is desired to extend the storage period to several times the current period, or does not require washing of red blood cells. Therefore, a storage method that does not use an additive that can be used in such a case is desired.

In order to extend the storage period, it is necessary to suppress blood metabolism. For this purpose, low temperature and cryopreservation are considered effective. However, for example, cryopreservation has the following problems, which must be solved.
Hemolysis includes salt damage hemolysis, intracellular cause hemolysis, cell membrane cause hemolysis, and extracellular cause hemolysis.

  Salt-damaged hemolysis is a phenomenon in which the electrolyte in the cells of erythrocytes that occurs during freezing is concentrated, and as a result, the osmotic pressure from the outside of the cell rises and the cell membrane is destroyed. Red blood cells are 60% water, and the inside is filled with an electrolyte in which the solute is dissolved in water. When a cell freezes, water molecules in the cell form ice crystals by a bond chain by hydrogen bonds one after another. On the other hand, intracellular solutes do not participate in this chain of electrostatic binding. Therefore, ice crystals are composed of pure water molecules only.

  As a result, the solute protruding from the ice crystals is highly concentrated and collected in a small amount of cell fluid remaining in the ice crystal gap.

  This concentrated solution increases the osmotic pressure of the cell fluid. According to Non-Patent Documents (1) and (2), red blood cells cause hemolysis when 82% or more of the cell fluid is frozen. At this time, the intracellular electrolyte concentration is concentrated to a concentration of 0.8 M NaCl or more, and the osmotic pressure increases to 1,500 mOsm / L, which is more than 5 times the normal osmotic pressure. On the other hand, when water flows in from the outside of the red blood cell by diffusion, the red blood cell expands, and if the volume expands to about 1.25 times the normal volume, the membrane ruptures. It is a mechanism that causes hemolysis.

  Intracellular causal hemolysis means that red blood cells expand and the crystal growth is not uniform due to the freezing of the cell fluid, and the leading part grown from the surface of the crystal grows more than the other part. It is a theory that the constituting lipid bilayer membrane is destroyed.

  However, cells are not broken by simple volume expansion due to freezing of moisture in the cells. This is because 60% of the cells are water, and when this crystallizes in the cells, the volume expansion rate is 1.1 times, so that the whole cell becomes 1.06 times volume expansion. Because there is no.

  Therefore, there is a mechanism called cell membrane cause hemolysis. That is, according to Non-Patent Document (3), the lipid bilayer membrane is easily damaged by a local force, that is, a pinpoint force directed from the inside to the outside because the membrane is weakly coupled in the spreading direction. This is a mechanism in which the cell membrane of erythrocytes is destroyed by non-uniform crystal growth during freezing of the cell fluid during intracellular freezing. Furthermore, the lower the degree of supercooling at the start of nucleation, the larger the ice crystals formed during intracellular freezing, and the smaller the degree of ice crystals formed, the higher the degree of supercooling. Therefore, the lower the degree of supercooling, the easier it is to generate a pinpoint force from the inside to the outside, and hemolysis tends to occur.

  According to Non-Patent Document (4), extracellular cause hemolysis is a process in which plasma is frozen, and as the ice nuclei grow, concentrated plasma and cells are pushed into the gaps between the ice crystals, and the cells touch the ice crystals on the surface. This is a mechanism that is finally caused by destruction by the pressure of ice crystals. It has been reported that hemolysis occurs upon freezing and thawing at a relatively slow rate up to about −10 ° C.

  Next, a background technique related to a frozen storage method for fresh fish, particularly a storage method for maintaining freshness will be described in detail.

  As a freezing preservation method for maintaining the freshness of fresh fish, there is a freezing preservation method in which it is frozen to a temperature of -40 ° C. or less immediately after a net or after catching. However, in order to freeze at such a low temperature, it is necessary to provide an expensive refrigeration facility on the fishing boat, and this is not done except for high-quality tuna. Moreover, the use of such low-temperature equipment in the tuna distribution process is a major cause of raising the general consumer price of frozen fish.

  On the other hand, a freezing temperature of −18 ° C. is widely used as a standard in the cold chain during storage and distribution of fresh fish. This freezing temperature of −18 ° C. is widely used for freezing and storing fish caught especially in the near sea. However, this freezing temperature is insufficient to suppress the occurrence of drip in the fish meat after thawing, which is a measure of the freshness of the fish, especially the fish meat that has been cut, and browning during storage. These are mainly caused by brown degeneration due to destruction and methification of fish meat cell tissues, and a major factor in the deterioration of freshness of fresh fish.

  The occurrence of drip after thawing is not a problem in the thawing process, but it is caused by the growth of ice crystals during freezing, especially by the growth of ice crystal nuclei formed inside and outside the cell, and the size of the nuclei becoming larger. Arise. Specifically, ice nuclei grow inside the cell and the salinity of the ice crystal surface increases, and the intercellular fluid with relatively low salinity diffuses into the cell through the cell membrane, and the original cell Moisture enters the cells more than the water it has, and ice crystals grow, eventually destroying the cell membrane from the inside. On the other hand, the ice nucleus produced in the intercellular fluid freezes and grows on the surface of the surrounding intercellular fluid, and the size of the ice crystal increases. On the contrary, the cell membrane is destroyed from the outside. Whether the destruction of the cell membrane occurs from inside or outside the cell, or whether it occurs at different times inside and outside and damages the cell membrane, depends on the randomness of the initial ice crystal nucleus and the freezing process Depending on time.

  In the conventional freezing method, for example, even if it is frozen at -40 degrees, the cooling process is often slow, and the ice crystal nuclei are gradually scattered in the fish tissue as the temperature decreases in the freezing process. The energy difference of water molecules occurs between the surface and the surrounding area, and the electrolyte concentration gradient becomes inhomogeneous.As a result, the electrolyte is condensed in a part of the tissue, resulting in uneven tissue environment and damage to the tissue. give. This greatly affects the texture and taste of the fish meat after thawing, and the greater the effect, the more different the fish meat from fresh fish before freezing. And such a fish meat will be judged and judged by the consumer as a fish meat with a reduced freshness.

  Brown degeneration during cryopreservation is caused by the growth of ice crystals in which dissolved oxygen in the intracellular fluid is generated in the intracellular fluid. It is produced by binding to oxymyoglobin or deoxymyoglobin in the middle to form methotrexate. On the other hand, the ice nuclei generated in the intercellular fluid are frozen and grown on the surface of the surrounding intercellular fluid, oxygen molecules that cannot be dissolved in the intercellular fluid appearing on the surface of the ice crystal diffuse into the cell through the cell membrane, Similarly, it causes methation of intracellular myoglobin. This mechanism also causes browning. On the other hand, myoglobin obtains energy from the surroundings and is reduced by the action of metmyoglobin reductase. However, the effect of methoglobinization of myoglobin by the aforementioned mechanism is large.

  Dissolved oxygen in water is held in a group of water molecules, and when it grows from ice nuclei to ice crystals during freezing, this water molecule group receives the free energy of the surface of the ice crystals. A part of water molecules are released by releasing intermolecular bonds through hydrogen bonds with other water molecules, and exchange energy on the surface of ice crystals to refreeze on the ice crystal surface. As a result, dissolved oxygen is released from the group of water molecules and drifts and diffuses between the water molecule groups as free oxygen. As a result, free oxygen binds to myoglobin existing between cells, or diffuses into the cell through the cell membrane and binds to intracellular myoglobin to cause methation.

  Next, the refrigeration apparatus required when using the frozen storage method according to the present invention will be described in detail.

3) Prior art related to refrigeration equipment In general, a cooling device that is widely used cools an object with heat of evaporation when a refrigerant changes from a liquid phase to a gas phase in a thermal cycle involving a liquid phase and a gas phase. To do. A refrigerator using such a vapor compression refrigeration cycle is widely used from a small device to a large device.

  1A and 1B show the configuration and operation outline of a typical liquid cooling apparatus. The gas circuit that makes up the refrigeration cycle consists of a compressor, an expansion valve or capillary tube, a condenser and evaporator for moving heat, or a refrigeration pipe.

  In the compressor, a gas having a large latent heat of vaporization, such as ammonia gas, carbon dioxide gas, and chlorofluorocarbon, which is a refrigerant in the gas circuit, is compressed. After the compression, it becomes a high-temperature high-pressure gas, and the generated heat is transferred to low-temperature water or air by the condenser. As a result, the high-temperature high-pressure gas is reduced in temperature and condensed into a high-pressure liquid. On the other hand, the low-temperature water or air becomes high-temperature water or high-temperature air, and is returned to the reservoir or the atmosphere as exhaust heat. The high-pressure liquid in the gas circuit is opened to a low pressure by an expansion valve or a capillary tube to become a low-pressure liquid. Furthermore, the low-pressure liquid is vaporized and changed to low-pressure gas. At the time of the change, heat is absorbed and the temperature of the secondary refrigerant is lowered through the evaporator (FIG. 1A). Alternatively, in the refrigeration pipe, the primary refrigerant changes from low-pressure liquid to low-temperature low-pressure gas, and the temperature of the object to be frozen is lowered (FIG. 1B).

  The object to be frozen is the cooling liquid in the cooling tank in the present invention. When cooling a plurality of objects to be frozen, a cooling device (FIG. 1A) using a secondary refrigerant is used. This is because the cooling temperature of the object to be frozen can be changed independently.

  The compressor generates a large vibration from its operation. In particular, in a reciprocating pump, a compression operation is performed by a reciprocating motion, and the vibration is remarkable. Although it is possible to reduce the vibration of the compressor by using the rotary pump, since the structure is not a rigid structure, the reduction of the vibration is limited.

  The vibration generated in the compressor is transmitted to the blood pack through the refrigerant and the coolant. As long as refrigerants and coolants are used to freeze the blood pack, it is inevitable that vibrations are transmitted to the blood pack through these.

4) Problems with the prior art related to the refrigeration system As a method of reducing the transmission of vibrations through the refrigerant and the cooling liquid, the vibration transmitted through the refrigerant and the cooling liquid is attenuated by increasing the distance from the refrigerator to the object to be frozen. There is a method and a method of attenuating vibration by mixing bubbles in the middle of the transfer path in the refrigerant or coolant.

  However, increasing the distance from the refrigerator to the object to be frozen increases the transfer path in the refrigerant and coolant, and during that time, the low temperature of the refrigerant and coolant escapes to the outside world, resulting in comprehensive refrigeration. A decline in capacity is inevitable. On the other hand, even if a region in which bubbles are mixed is provided in the middle of the transfer path, a new vibration is generated due to the collapse of the bubbles, so that a low vibration environment cannot be created.

JP2014-214911

“Freezing of red blood cells”, Ken Miura, Blood and Blood Vessels, Vol. 1, No. 9, November 1970 "The Biophysical Society of Japan General Incorporated Association, NII-Electronic Library Service, Hemolytic properties of human erythrocytes under hydrostatic pressure, Takeo YAMAGUCHI, Department of Chemistry, Faculty of Science, Fukuoka University, Vol.37, No.2 (1997), "Hemolysis characteristics of human erythrocytes under pressure" "Control of ice crystal morphology in frozen pharmaceuticals-by controlling nucleation using ultrasound", (Hyogo Pref.) Nakagawa, Sakuya, Kitamura, Mitsutaka (Lyon Daiichi University) Hottot, S.H. Vessot, J. et al. Andrieu, SCEJ, 38th Automatic Meeting (Fukuoka, 2006), C202 “Mechanism of hemolysis by freezing at temperatures close to freezing point I: Morphological changes during freezing and thawing process”, Yoshio Nei, Cryogenic Science, Biology, December 25, 1967 “Study on recrystallization behavior of ice crystals in frozen foods”, Tomoaki Sugawara, Journal of Japan Society for Food Engineering, Vol.11, No.4, p169-175, December 2010 Morphological Anxiety in Ice Crystal Growth-ISS "Kibo" Experiment-Hokkaido University Yoshizumi Furukawa, Gakushuin University Yokoyama Goro, JAXA Izumi Yoshizaki, Yoda Junichi, Tanaka Tetsuo, JSF Taro Shimaoka, JAMSS Takehiko Sone, IHI Aerospace Toshiyuki Tomobe, Space Util. Res. , 26 (2010)

  First, the problem to be solved by the present invention regarding the frozen storage method will be described in detail.

  For long-term or semi-permanent storage of blood, cryopreservation of blood is performed. However, the problem is that hemolysis occurs after thawing. On the other hand, there is a method in which an additive is added to blood and stored frozen without freezing, but there is a problem that a blood washing step after storage is required.

Thus, a blood cryopreservation method that suppresses hemolysis without using additives is a main problem to be solved by the present invention. The mechanisms and causes of hemolysis associated with freezing of blood include salt damage hemolysis, intracellular cause hemolysis, cell membrane cause hemolysis, and extracellular cause hemolysis.
On the other hand, the main problems to be solved by the present invention in the method for freezing and storing fish meat while maintaining the freshness of fresh fish are that a large amount of drip comes out from the fish meat after thawing and that the fish meat undergoes browning during frozen storage. It is.
The present invention provides a cooling condition that does not cause any mechanism and a control method thereof. More specifically, there are the following problems and methods for solving them.

The blood cryopreservation method that suppresses hemolysis has the following problems and solutions.
(A) Prevention of salt-damaged hemolysis Prevents extracellular solutions from entering the cells by diffusion.
(I) Intracellular cause hemolysis Suppresses freezing of moisture in the cell, and suppresses cell expansion due to freezing to an allowable volume expansion of red blood cells.
(C) Cell membrane-causing hemolysis The ice crystal size is reduced to 3 to 4 microns by quick freezing and the cell membrane is not damaged.
(D) Prevention of extracellular cause hemolysis Causes ice crystals outside the red blood cells to freeze while maintaining the gap. For this purpose, ice crystals are recrystallized. This prevents red blood cells from being destroyed by the pressure of ice crystals. The recrystallization process is the result of a phenomenon that reduces the surface area of the crystal. For this purpose, the freezing start temperature is raised, the growth rate of the frozen surface is slowed, and the temperature gradient inside the ice crystal is reduced.

Regarding the fresh fish meat, there are the following solutions to the problem of a large amount of drip coming out of the fish after thawing and the problem of the fish meat browning during frozen storage.
(E) Drip after thawing When growing from ice crystal nuclei to ice crystals during freezing, keep the ice crystal size at the time of ice crystal nucleus generation without increasing the ice crystal size as much as possible.
(F) Brown degeneration Maintains the grouping of water molecules with dissolved oxygen in groups of water molecules, and recrystallizes on the ice crystal surface even when growing from ice nuclei to ice crystals during freezing. Suppress that. Suppression of this recrystallization suppresses the generation of free oxygen and prevents the formation of myoglycin.

  Next, problems to be solved by the invention related to the refrigeration apparatus will be described in detail.

  During the freezing process according to the present invention, it is necessary to suppress the generation of ice crystal nuclei generated in the blood pack or fresh fish and to prevent the ice crystal nuclei from growing as much as possible. One of the major causes of supercooled water icing is the vibration of the supercooled water. That is, since it is necessary to lower the temperature of the blood pack or the fresh fish while freezing as much as possible without freezing, in the present invention, the blood pack or the fresh fish is placed in an environment of low vibration or very low vibration during the cooling process. There is a need.

  The compressor generates a large vibration from its operation, and the vibration is transmitted to the blood pack or the fresh fish through the refrigerant. Since the compressor uses a pump with a high compression rate, there is a limit to reducing the vibration of the compressor itself. Therefore, it is necessary to insert a vibration transmission preventing device that does not transmit the vibration generated by the compressor into the path through which the cooling liquid for cooling the blood pack or fresh fish or the refrigerant at the end circulates.

  The vibration transmitted through the coolant or the refrigerant is a longitudinal wave. Therefore, as a vibration transmission prevention method, the vibration transmission path is divided into two, the length of one of the transmission paths is lengthened, and the difference between the two transmission path lengths is half of the period of vibration to prevent transmission or vibration. As a half of the wavelength (half wavelength), there is a method of smoothing vibrations or canceling peak and valley of vibrations by interference. Longitudinal waves in a liquid phase medium such as a cooling liquid or a refrigerant are difficult to attenuate and have a long coherence length. Therefore, after the longitudinal wave is divided into two, it is an extremely effective method to extinguish the longitudinal wave by interference so that one phase has a half-wave phase difference compared to the other phase. Here, for the sake of simplicity, the period of vibration and the smoothing of vibration are also represented by the terms of vibration wavelength and vibration interference.

  However, since the vibration transmission speed in the liquid is about 1500 m / second, if the vibration of the compressor is 60 Hz, for example, the half wavelength is 12.5 m, and a long path difference must be provided in the cooling circuit. There are challenges.

  First, means for solving the problems of the method for freezing and storing blood will be described in detail.

  The means used in the method for preventing hemolysis in the present invention rapidly cools the blood to a temperature (first cooling temperature) that does not cause salt damage hemolysis and intracellular hemolysis (hereinafter referred to as “first cooling process”). . Thereafter, cooling until reaching a second cooling temperature lower than the first cooling temperature (hereinafter referred to as “second cooling process”) is performed for a certain period of time (elapsed temperature) in which the temperature is maintained at a slow cooling or step cooling and the temperature continues for each step. It is performed by stage cooling that provides a maintenance period). Moreover, at least the second cooling process is performed in a low vibration environment.

The first cooling temperature is in the range of −4 ° C. to −6 ° C., which is lower than the temperature at which the solute of red blood cells begins to freeze, and the cooling rate is −10 ² ° C./min or higher. The second cooling temperature is −7 to −13 ° C. When the slow cooling is employed as the cooling from the first cooling temperature to the second cooling temperature, the cooling rate is -0.5 ° C / hour to -1.5 ° C / hour. On the other hand, when adopting step cooling as the cooling during this period. For each step cooling from -0.5 ° C to -1.5 ° C, select a longer time from 0.5 hours or a numerical value of Celsius temperature display of step cooling temperature x 0.5 hours, which is longer Is set as the elapsed temperature maintenance period. The second cooling process is performed by placing a container containing blood in a low vibration environment.

  Here, even if the blood is whole blood, RCC, RCC-LR, or RC-M. A. P. Concentrated erythrocytes abbreviated as “Red Cell Concentrates Mannitol Adenine Phosphate”, or simply referred to as “MAP” in the following).

  The first cooling process and the second cooling process will be described from the mechanism and effect of the solution provided in the present invention.

[First cooling process]
Red blood cells are about 6 to 8.5 microns in diameter. Ice crystal nuclei are formed from about -3 ° C which is the freezing temperature. The ice nuclei remain at most 3 to 4 microns in diameter. However, in the supercooled state, the temperature at which ice crystal nuclei are formed decreases. For example, when quick freezing is performed at a cooling rate of −10 ² ° C / min or higher, the ultimate temperature of the supercooling becomes the temperature at which ice crystal nuclei start to form. In a supercooled state, the ice crystal size decreases as the temperature at which the formation of ice crystal nuclei decreases (Non-patent Document 3). The first cooling temperature is in the range of -4 ° C to -6 ° C. Therefore, the ice crystal size in the cell is smaller than 3 microns. That is, the ice crystal size is considerably smaller than the size of red blood cells, so that hemolysis due to the cause of red blood cells does not occur.

  Since erythrocytes are rapidly frozen from the outside, the temperature inside the ice crystal nucleus is higher than the heat generated by ice crystal formation and the surface temperature of the supercooled ice crystal. For this reason, the water molecules present on the crystal surface maintain an energy balance with the molecules inside the crystal, so that the molecules remain in motion and the phenomenon of reducing the surface area of the crystal, that is, the growth of ice crystals, is unlikely to occur. Furthermore, a part of the water in the erythrocytes is ice crystals, and accordingly, the concentration of the solute in the erythrocyte increases and the freezing point of the solute decreases.

  The increase in concentration and the change in coagulation temperature are roughly proportional. For this reason, in the first cooling process, unless the first cooling temperature, which is the ultimate temperature, is extremely low, ice crystals hardly grow. That is, when the first cooling temperature is in the range of −4 ° C. to −6 ° C., ice crystal nuclei do not grow inside the erythrocytes even after a long time has passed. Therefore, the salt concentration of the solution in the erythrocyte does not increase extremely, and the water in the plasma outside does not diffuse into the erythrocyte by osmotic pressure, and the cell membrane of the erythrocyte is not destroyed. That is, no salt damage occurs.

  On the other hand, before reaching the first cooling temperature, the red blood cells are rapidly cooled from -1 ° C., which is a so-called maximum ice crystal formation zone, to the lower limit temperature. The lower limit temperature of the maximum ice crystal formation zone is said to be −5 ° C., which is close to that of pure water. In water containing an electrolyte such as red blood cells, −4 ° C. may be the lower limit temperature. It is a guideline. If the blood pack is small and the internal heat transfer is sufficiently ensured, the cooling time can be equivalently achieved by setting the maximum ice crystal formation zone to be shorter than 30 minutes. .

[Second cooling process]
Plasma occupies most of the outside of the red blood cells. Plasma has ice nuclei at about -3 ° C. At the first cooling temperature, ice crystal nuclei begin to grow in the plasma. However, since the cooling process in the second cooling process is slow and is in a low vibration environment, the growth of ice crystals is also unique. The feature is that the temperature difference between the inside and outside of the ice crystal is small, in other words, the temperature gradient inside the ice crystal is small, and further, the progress speed of the frozen surface of the ice crystal is low. Therefore, the ice crystal size L ^* depends on the growth rate R of the frozen surface and the temperature gradient G inside the ice crystal.
(Non-patent document (3)) a is a fitting parameter that matches the actual ice crystal size and the theoretical value of Formula 1. As this parameter value, for example, −0.2 at −3 ° C. At a cooling rate of ° C / min, L ^* = 160 microns, which is understood from Equation 1 that the ice crystal size is relatively large in such a characteristic cooling process as compared with other cooling processes. It is to become.

  In addition, since the vibration is low, collision of external water molecules on the ice crystal surface and subsequent freezing is less likely to occur. Therefore, in addition to the increase in the size of the ice crystal, a recrystallization process occurs at the interface between the ice crystal surface and the plasma, that is, the molecules present on the ice crystal surface are higher than the molecules inside the ice crystal. It is in an energy state.

  Thus, in order to reduce the number of molecules, the molecules move spontaneously and the process of reducing the surface area of ice crystals proceeds (Non-patent Document 5). These result in an increase in the average size of ice crystals, a decrease in the number of ice crystals, and smoothing of ice crystals. As a result, freezing outside the plasma, that is, erythrocytes, causes the ice crystal nuclei to grow slowly and large, and the void size between the ice crystals is larger than when no recrystallization process occurs. Between the first cooling temperature and the second cooling temperature, an ice crystal part growing in plasma and a solution part in a liquid state coexist.

  In such ice crystal growth, the ice crystal part is in a state of enclosing a void filled with the solution part. As ice crystals grow, the plasma solution concentration increases and the relative osmotic pressure on the blood cells decreases. Furthermore, with respect to the void size, the growth of ice crystals is slowed by the increase in the solution concentration, and the decrease in the solution portion (that is, the reduction in the size of the solution void) is suppressed. Thus, the red blood cells remain in the solution voids and do not cause crushing of the red blood cells due to ice crystals, so that hemolysis due to red blood cell extracellular causes hardly occurs.

  In step cooling, each time the cooling temperature is slightly lowered (steps from -0.5 ° C to -1.5 ° C), the cooling temperature is maintained for a long time (0.5 hours or the Celsius temperature of the step cooling temperature). An over-temperature maintaining period is provided, which is the longer of the displayed numerical value × 0.5 hour. During this overtemperature maintenance period, the ice crystal recrystallization process is maintained, and the ice crystal part maintains a state in which the void filled with the solution part is included even when the temperature becomes close to the second cooling temperature. That is, the above-mentioned recrystallization process is maintained quasi-statically, the reduction of the solution gap is suppressed, and the red blood cells stay in the solution gap and do not cause crushing of the red blood cells by ice crystals. Is less likely to occur.

  The above cooling process has been described for whole blood. However, in MAP, the coagulation point is almost the same as that of whole blood, and the osmotic pressure for red blood cells is not different from that of whole blood. Therefore, also in MAP, the phenomenon seen in the first cooling process and the second cooling process and the effect of preventing hemolysis can be explained as in the case of whole blood.

  Next, detailed description will be given of means for solving the problems of the method for freezing and preserving fish while maintaining the freshness of fresh fish.

  The means used in the method for suppressing the occurrence of drip at the time of thawing of the fish meat and the brown deterioration of the fish meat in the present invention is rapidly cooled to the first freezing temperature (first cooling temperature) (hereinafter referred to as “first” Cooling process). Thereafter, cooling until reaching a second cooling temperature lower than the first cooling temperature (hereinafter referred to as “second cooling process”) is slow cooling, or a fixed period (elapsed time) in which the temperature continues for each step and step cooling. (Temperature maintenance period) is performed by two-stage cooling.

[First cooling process]
Fish meat begins to freeze at around -1 ° C. Thereafter, for example, when rapid freezing is performed at a cooling rate of −10 ² ° C./min or higher, the ultimate temperature of the supercooling becomes the temperature at which ice nucleus formation starts. However, if the fish meat is left for a while without being vibrated after that, the whole temperature of the fish meat reaches its temperature without icing. The water in the fish meat, that is, the water in the intracellular or extracellular fluid, is dispersed in these liquids in order to maintain a thermal equilibrium state with cell proteins, lipids and cell interstitial fluid rather than forming ice crystal nuclei. Therefore, in the range of -4 ° C to -6 ° C which is the first cooling temperature, the fish meat is supercooled without icing.

  On the other hand, by the time the first cooling temperature is reached, the fish meat is rapidly cooled from -1 ° C, which is a so-called maximum ice crystal formation zone, to a minimum temperature of the electrolyte of the fish cells or intercellular fluid, -4 ° C. As for the cooling time, when the fish meat is small and the internal heat transfer is sufficiently ensured, the passage time of the maximum ice crystal formation zone can be easily performed in less than 30 minutes, and freezing can be avoided. On the other hand, in the case of large fish, internal heat transfer is not sufficiently ensured, and in order to shorten the cooling time, it may be necessary to cool by a contact method or the like.

[Second cooling process]
After the fish meat is left at the first cooling temperature of −4 ° C. to −6 ° C. to reach the same temperature as a whole, the second cooling process is started. The second cooling process is a slow cooling process toward the second cooling temperature lower than the first cooling temperature, and is in a low vibration environment, so that it is difficult to start freezing. That is, the moisture in the fish meat, that is, the moisture in the intracellular or extracellular fluid, maintains a thermal equilibrium state with the cell protein, lipid, and cell interstitial fluid, and therefore does not start freezing with these as nuclei. But I
Due to the 10 micrometer, moisture inside and outside the cells freezes at once from ice crystal nuclei randomly generated in the fish meat. Therefore, ice crystal nuclei are generated everywhere inside and outside the cell and maintain a thermal equilibrium state, so that ice crystal nuclei hardly actually grow.

In many cases, icing will occur immediately after cooling from the first cooling temperature to the second cooling temperature.
It becomes difficult. Since the diameter of the ice crystal grains is inversely proportional to the square root of the ice crystal growth rate (Non-Patent Document 3), the growth of ice crystal nuclei becomes more difficult as supercooling proceeds.

  As a result, the phenomenon that the expansion of the outer size due to the growth of ice crystals in the cell destroys the cell membrane is unlikely to occur. On the other hand, cell membranes are hardly broken from the outside due to freezing and growth of the intercellular fluid. The fact that cell destruction is less likely to occur is that after freezing the fish meat in such a freezing process and thawing, the ice crystal melts in the space occupied by the original ice crystal, forming a water space, The so-called drip-typical phenomenon of oozing out from the fish piece as a drip is unlikely to occur. This is because the ice crystals in the cells do not grow large and immediately return to the intracellular solution after thawing, and the ice crystals generated between the cells do not grow large and immediately return to the intercellular fluid after thawing. That is, the occurrence of drip can be suppressed.

  In the second cooling process in which the cooling is performed slowly toward the second cooling temperature lower than the first cooling temperature, when the supercooling temperature becomes sufficiently large and reaches the supercooling limit temperature, the fish meat randomly The water inside and outside the cell freezes from the ice crystal nuclei generated at once. At this time, ice crystals are generated everywhere inside and outside the cell and maintain a thermal equilibrium state, so that ice crystals hardly grow. Therefore, oxygen confined in the group of water molecules is hardly liberated, and is also suppressed from being combined with oxymyoglobin or deoxymyoglobin in fish meat to be converted into a methionate.

In this way, after supercooling has progressed as much as possible in a low-vibration environment, the moisture in the fish is frozen at once, thereby suppressing cell destruction and methation, and losing freshness even after thawing after frozen storage Fish meat can be stored frozen.
Conversely, when not in a low-vibration environment, before the supercooling temperature increases, that is, before the supercooling reaches the limit temperature, ice crystal nuclei are generated in the water in the fish by vibration, and ice crystal growth begins. . At this time, since the supercooling temperature is small, the growth rate of ice crystals is slow, as can be seen from Equation 2, resulting in cell destruction due to ice crystal growth and further methation due to free oxygen generated by breaking hydrogen bonds of grouped water molecules. .

  When step cooling is adopted in the second refrigeration process, the cooling temperature is maintained for a long time (0..0 ° C) every time the cooling temperature is slightly lowered (step of -0.5 ° C to -1.5 ° C). An overtemperature maintenance period of 5 hours or a numerical value of Celsius temperature display of step cooling temperature × 0.5 hour, whichever is longer) is provided. The rate of progress of the cooling temperature at each step is small, and the water in the fish meat, that is, the water in the intracellular or extracellular fluid, is in a thermal equilibrium state with cell proteins, lipids, and cell interstitial fluid, as in the continuous freezing process. In order to preserve, do not start freezing with these as nuclei. Therefore, supercooling can be performed. As a result, cell destruction and methotrelation are suppressed, and the fish meat can be stored frozen without losing freshness even if it is thawed after frozen storage.

  Freezing is likely to occur when the cooling step proceeds. This is because, at the moment when the step proceeds, the thermal equilibrium state between the water in the intracellular fluid or the extracellular fluid and the cellular protein, lipid, and cellular interstitial fluid tends to collapse.

  Next, means for solving the problems of the frozen storage method according to the present invention will be described in detail. Specifically, a vibration transmission preventing device will be described.

  In the cooling process according to the present invention, it is necessary to place the blood pack, fish meat or packaged fish meat in an environment of low vibration or very low vibration.

  The limit value necessary for the vibration to be low vibration or ultra-low vibration in the present invention will be described. The limit value is determined from the condition of continuing supercooling without starting freezing as much as possible after reaching the first cooling temperature (−4 ° C.) until reaching the second cooling temperature. The limit value is a phenomenologically limit value of vibration in which water molecules are not frozen or frozen by vibration. Water molecules freeze when hydrogen atoms constituting water molecules form a hexagonal system by hydrogen bonding with hydrogen atoms constituting other water molecules.

  The hydrogen bond is formed or broken when the water molecule has a transfer of energy corresponding to the heat of melting of ice when viewed macroscopically. Its energy is calculated from the generation of ice crystal nuclei when the water is in a supercooled state. The start of the generation of ice nuclei is considered to be when 10,000 ice nuclei having a diameter of 1 micron per cubic centimeter can be formed. The energy corresponding to the heat of fusion per gram of water is 333 Joules, and the crystal growth rate at the subcooling temperature of −4 ° C., which is the first cooling temperature, is 3.29 cm / sec from Equation 2, An energy transfer of 5.73 microwatts per square centimeter is required. This is 284 Pascals when converted to underwater sound pressure, and the sound pressure level is 169 dB. If vibration greater than this is applied, generation of ice crystal nuclei starts at the first cooling temperature, and refrigeration cannot be continued in a supercooled state. Therefore, the limit sound pressure level at the time of freezing of the present invention in a blood pack or fish meat or packaged fish meat is 169 dB.

  On the other hand, the specific gravity and sound speed of the refrigerant are almost the same as water. Therefore, the critical vibration sound pressure in the refrigerant is almost the same as the above-mentioned limit sound pressure level.

  On the other hand, in order to break the hydrogen atom bond in the water molecule group, the hydrogen bond can be broken by separating one of the two hydrogen atoms bonded by the van der Waals radius. Where the melting temperature of the energy ice is 6 kilojoules per mole, the hydrogen bonds in water are 10 to 40 kilojoules. This value is between 555 and 22220 joules per gram of water, which is greater than the melting temperature of ice. Therefore, as long as the above limit vibrational sound pressure is maintained, the water molecule group does not destroy the grouping, and therefore, no free oxygen is generated. As a result, fish cell methation does not occur.

  Also from this point, in order to maintain the freshness of fresh fish, it is necessary to place the fish meat in an environment of low vibration or ultra low vibration that maintains the above-mentioned limit sound pressure level in the freezing process.

  In the following description and examples, the freezing of the blood pack will be described, but the same applies to the freezing of fish meat and the like unless otherwise specified.

  The cooling device cools the object with the heat of evaporation when the refrigerant is changed from the liquid phase to the gas phase by applying a cycle of the liquid phase to the gas phase. 1A and 1B show the configuration and operation outline of a typical liquid cooling apparatus. The gas circuit that makes up the refrigeration cycle consists of a compressor, an expansion valve or capillary tube, a condenser and evaporator for moving heat, or a refrigeration pipe. In the compressor, a gas having a large latent heat of vaporization, such as ammonia gas, carbon dioxide gas, and chlorofluorocarbon, which is a refrigerant in the gas circuit, is compressed. After the compression, it becomes a high-temperature and high-pressure gas, and the generated heat is transferred to air or water by a condenser.

  As a result, the high-temperature high-pressure gas is reduced in temperature and condensed into a high-pressure liquid. The high-pressure liquid is opened to a low pressure by an expansion valve or a capillary tube to become a low-pressure liquid. Furthermore, the low pressure liquid is vaporized and replaced with the low pressure gas. At the time of the change, heat is absorbed and the temperature of the secondary refrigerant is lowered through the evaporator (FIG. 1A). Alternatively, in the refrigeration pipe, the primary refrigerant changes from low-pressure liquid to low-temperature low-pressure gas, and the temperature of the object to be frozen is lowered (FIG. 1B). The object to be frozen is the cooling liquid in the cooling tank in the present invention.

  The compressor generates a large vibration from its operation, and the vibration is transmitted to the blood pack through the refrigerant. Since the compressor uses a pump with a high compression rate, there is a limit to reducing the vibration of the compressor itself. Therefore, it is necessary to insert a vibration transmission preventing device that does not transmit the vibration generated by the compressor into the path through which the cooling liquid for cooling the blood pack or the refrigerant at the end circulates.

  The vibration transmitted through the coolant or the refrigerant is a longitudinal wave. Therefore, as a vibration transmission prevention method, the vibration transmission path is divided into two, the length of one transmission path is lengthened, and the difference between the two transmission paths is half the wavelength of the vibration to be blocked (half wavelength). ), There is a method of canceling out the peaks and valleys of vibration by interference.

  The reason why the vibration can be canceled in this way is that most of the vibration of the liquid refrigerator is generated from the refrigerant compressor. However, since the vibration transmission speed in the liquid is about 1500 m / second, if the vibration of the compressor is 60 Hz, for example, the half wavelength is 12.5 m. Must be provided in the circuit.

  Therefore, the vibration transmission preventing apparatus according to the present invention uses a delay device that substantially lengthens the vibration transmission distance by multiple reflections to increase the delay phase, and a rectifier that uses a diffraction hole that diffuses vibration, Therefore, vibration transmission can be prevented with a short length.

here,

The dynamic transmission speed is the same as the transmission speed of sound waves, about 1500 m / sec. When the vibration of the compressor is 60 Hz, the half-wave distance is about 12.5 m, and a long path difference is provided in the cooling circuit. Must be. Conversely, for example, for a 1 m path length, a phase shift of 0.04 radians

When devices using such multiple reflections are arranged in series in four stages, a delay amount of 0.72 radians × 4 is generated. This delay is 0.46 wavelength, and it is possible to divide the vibration wave source into two to generate two vibration waves, and to cancel the original vibration wave by interference between the two vibration waves. In order to correctly adjust the delay amount to the half wavelength, between the vibration reflector 1 and the vibration reflector 2

  As described above, the cooling device can be reduced in size by the delay device using the multiple reflection, and an extremely low vibration cooling device having a practical size can be realized.

  Even if the original vibration wave is canceled by interference between the two vibration waves, vibration may appear again depending on the propagation direction of the two vibration waves after the interference. This phenomenon is likely to appear in the harmonics of the vibration wave in question. In order to prevent this, after the interference between the two vibration waves, a vibration hole may be diffused by providing a diffraction hole in the propagation path of the vibration wave and passing through it. In practice, a rectifying device for diffusing this vibration is provided downstream of the delay device. As a result, the vibration is removed everywhere in the propagation path that hits the downstream side of the sizing device, and the other state is maintained.

  The blood frozen by the method of the present invention hardly undergoes hemolysis even when it is returned to body temperature. Since the freezing temperature is −10 ° C., it can be stored for a long period of time, and a person with a rare blood group can easily store his blood from normal times and be ready for an emergency blood transfusion.

  The fresh fish frozen and stored by the method of the present invention can be passed through a cold chain of −18 ° C. that is normally used without using an expensive freezing facility that is frozen and stored at −40 ° C. or lower that is used in high-grade fish. Freshness can be maintained. At that time, after reaching the second cooling temperature, the frozen fish meat is stored frozen at −18 ° C.

  Further, when the above frozen storage method is used, it is necessary to keep the blood pack in a low vibration environment, but the liquid refrigerator can be reduced in size by using the vibration transmission preventing device according to the present invention. The blood pack is small, and a large realistic effect that the entire apparatus can be miniaturized for using the present cryopreservation method and the benefit thereof are obtained.

FIG. 1A is a conceptual diagram showing a refrigeration cycle of a refrigerator using a secondary refrigerant. FIG. 1B is a conceptual diagram showing a refrigeration cycle of a refrigerator that uses only a primary refrigerant. FIG. 2A is a view showing a vibration isolating means comprising a horizontal bar or rail and a string to be hung on a blood pack. FIG. 2B is a view showing a vibration isolating means including a vibration isolator and a container for storing a blood pack, and a lid covering the upper part of the container. FIG. 3A is a photograph showing muscle fiber cells of non-frozen raw bluefin tuna meat. 3B is a photograph showing the muscle fiber cells of bluefin tuna meat that has been frozen and stored after undergoing the freezing process of Example 3. FIG. FIG. 3C is a photograph showing bluefin tuna muscle fiber cells that have been thawed after being frozen and stored through a normal slow freezing method. FIG. 4A is a photograph showing the appearance of non-frozen raw bluefin tuna meat. 4B is a photograph showing the appearance of bluefin tuna meat that has been frozen and stored after the freezing process of Example 3. FIG. FIG. 4C is a photograph showing the appearance of bluefin tuna meat that has been frozen and stored through a normal slow freezing method. FIG. 5 is a schematic diagram showing a state in which longitudinal waves are multiply reflected between the vibration reflector 1 and the vibration reflector 2. FIG. 6A is an external view of the vibration removing module. FIG. 6B is an internal cross-sectional view of the vibration removal module. FIG. 6C is an internal cross-sectional view of the vibration removal module when a baffle is used. FIG. 6D is an internal cross-sectional view of the vibration removing module when a vibration reflector is used. FIG. 7A is a diagram showing a main part of a phase difference generating circuit in which a vibration removing module is arranged in parallel with a direct transfer pipe. FIG. 7B is a diagram showing a main part of a phase difference generating circuit in which the vibration removing module is arranged perpendicular to the direct transfer pipe. FIG. 7C is an external view of a simple merger. FIG. 8A is an external view of another merger that merges the flow of refrigerant or coolant. FIG. 8B shows an internal cross-sectional view of another merger. FIG. 9 shows the pressure loss versus expansion angle in a gently expanding tube. FIG. 10A shows a schematic diagram of a refrigerator in which the vibration removing device is installed in a coolant circulation circuit. FIG. 10B shows a schematic diagram of a refrigerator in which the vibration removing device is used in the primary refrigerant circulation circuit.

  An example in which collected blood is frozen by the method of the present invention will be described below.

  This embodiment is an embodiment of a cooling process when slow cooling is employed.

  After collecting the blood, it is injected into a sterilized container (pack), and after filling the container with the blood, the injection port is sealed without containing an air phase. The material of the pack is TOTM (trimellitic acid tris − 2 − ethylhexyl) instead of polyvinyl chloride (PVC) or its plasticizer DEHP (diphthalic acid − 2 − ethylhexyl). ) Using a material made of polyvinyl chloride, polyolefin, polyethylene, polybutadiene or similar materials. Hereinafter, a container filled with blood in this way is called a blood pack.

  Alternatively, whole blood in a pack already sold as a blood pack may be used. These are all called blood packs.

  The blood pack may be placed in the following freezing step from room temperature. On the other hand, in order to shorten the refrigeration process time, it is desirable to perform preliminary refrigeration or cooling with a low-temperature liquid before placing the following refrigeration process.

  It is desirable that the liquid refrigerator be capable of cooling at a low temperature of about −18 ° C. as the cooling temperature. It may be a liquid refrigerator that can be cooled at a low temperature of −30 ° C., which is the cold resistance limit temperature or the embrittlement temperature of the blood pack. Furthermore, it is desirable to have a temperature control mechanism that can change the set value of the cooling temperature with time.

  Ethanol is circulated in the cooling tank of the liquid refrigerator as a secondary refrigerant for directly cooling the blood pack. The temperature setting of the liquid refrigerator is set to the first cooling temperature in the range of −4 ° C. to −6 ° C., and the solution is allowed to stand until the ethanol reaches the first cooling temperature. Thereafter, in order to cool in a low vibration state, the operation of compression of the liquid refrigerator is stopped or the minimum operation mode is set.

  When the blood pack is submerged in the cooling tank, the blood pack at room temperature temporarily raises the temperature of ethanol as a secondary refrigerant. In order to suppress this temperature rise, the volume of ethanol in the cooling tank needs to be sufficiently larger than the volume of the blood pack. Specifically, even when the temperature of ethanol rises temporarily, ethanol sufficient to maintain a sufficient heat capacity for the blood pack so that it does not exceed -4 ° C is stored and circulated in the cooling tank of the liquid refrigerator. Need to be. However, the blood after thawing that sets the first cooling temperature to a temperature lower than −6 ° C. in anticipation of the temperature rise causes hemolysis.

  Thereafter, the blood pack is left to stand in the cooling bath for a time longer than about 5 minutes. However, even if the time is shorter than this, the hemolysis of blood is hardly affected.

  After the elapse of the standing time, the cooling temperature of the liquid refrigerator is set to the second cooling temperature in the range of −7 to −13 ° C. The selection of the second cooling temperature affects the storage period of the blood in the blood pack. When the second cooling temperature is −7 ° C., the storage period is short, and when it is −13 ° C., the storage period is long. The cooling rate from the first cooling temperature to the second cooling temperature requires slow cooling in the range of -1.5 ° C / hour to -0.5 ° C / hour. For this reason, it is desirable that the liquid refrigerator has a temperature control mechanism, and the cooling temperature is set so that the temperature changes in any of the above ranges over time. During the slow cooling period, the cooling operation of the liquid refrigerator is set to the minimum operation mode in order to cool in a low vibration state. If it is still not possible to create a sufficiently low vibration state, the vibration isolation means described later is used.

  Slowly cool the blood pack and maintain that temperature after reaching the second cooling temperature. Since a low vibration state is not required to maintain the second cooling temperature, the blood pack is at or below its second cooling temperature, which is the cold limit temperature or embrittlement temperature of the blood pack material, -30 ° C. You may move to other refrigeration equipment, maintaining the temperature up to. This second cooling temperature or lower and the temperature up to −30 ° C., which is the cold resistance limit temperature or embrittlement temperature of the blood pack material, is called the storage temperature. In this series of processes, the blood pack freezing process is completed, and then the blood pack is simply stored while maintaining the storage temperature.

  As shown in FIG. 2A, the above-mentioned vibration isolating means includes a horizontal bar or rail 105 installed on the upper part of the cooling liquid circulation tank 101 that is a cooling tank of the liquid refrigerator, and a hanging string 104 that hangs on the blood pack 103. There is a means. By this means, the blood pack 103 can be cooled in a low vibration state without the vibration of the liquid refrigerator mainly generated by the compressor being transmitted to the blood pack 103. The hanging strap 104 may be a hole formed in the pouch bag joint at the top of the blood pack 103 or the whole blood pack 103 may be bound by the hanging strap 104.

  Further, as another vibration isolating means, as shown in FIG. 2B, the container 106 for putting the vibration isolator 108 and the blood pack 103 having the vibration isolating effect in the cooling liquid circulation tank 101 and the upper part of the container 106 are covered. There is a container lid 107. The anti-vibration table 108 makes it difficult for vibration generated by the compressor of the liquid refrigerator to be transmitted to the storage device 106 via the cooling liquid circulation tank 101 and is made of plastic such as vinyl chloride, silicone rubber or foamed plastic. The vessel 106 is prevented from coming into direct contact with the bottom surface which floats from the bottom surface of the cooling liquid circulation tank 101.

  On the other hand, the container 106 is intended to be isolated from the vibration of the compressor transmitted through the coolant 102. As the material of the container 106, the thermal conductivity is high in order to quickly transmit the temperature of the cooling liquid 102, and the acoustic impedance is greatly different from that of the cooling liquid 102 in order to reflect the vibration transmitted through the cooling liquid 102 and to have a blocking effect. is important. For this purpose, the material of the container 106 is preferably a metal such as aluminum. The material of the container lid 107 is also preferably a metal such as aluminum for the same reason.

  Even if the storage device lid 107 is attached to the storage device 106, the cooling effect on the blood pack 103 is reduced because the circulation of the cooling liquid 102 does not touch the blood pack 103 even if it has a vibration isolation effect. Therefore, it is not used except when it is necessary to place the blood pack 103 in an ultra-low vibration state.

  As a low vibration state, the sound pressure limit value is 284 Pascals. Therefore, it is desirable that the vibration pressure of the vibration transmitted to the blood pack is not more than this sound pressure limit value. If the vibration transmitted to the blood pack is larger than these values, collision of external water molecules on the ice crystal surface and subsequent freezing and fixation are likely to occur. As a result, a large ice crystal is formed and hemolysis occurs upon thawing. Loss of freshness in fish meat.

  This low vibration state is necessary at least in the cooling process from the first cooling temperature to the second cooling temperature, but the cooling is performed even in the cooling process to the first cooling temperature and after the second cooling temperature is achieved. It may be maintained in the process.

The present embodiment is an embodiment of a cooling process when step cooling is employed. Here, the production or origin of the blood pack 103 is the same as in the first embodiment.
The conditions and steps until the blood pack 103 is allowed to stand in the cooling liquid circulation tank 101, cooled to the first cooling temperature, and left for a time longer than about 5 minutes are the same as in the first embodiment.

  The second cooling temperature is set to -7 to -13 ° C. During the period from the first cooling temperature to the second cooling temperature, a temperature in the range of −0.5 ° C. to −1.5 ° C. is selected as the step temperature. After leaving for about 5 minutes or more, the cooling temperature of the liquid refrigerator is lowered by the selected step temperature. After that, the blood pack 103 is left in the liquid refrigerator for 0.5 hour or the time indicated by the step cooling temperature in degrees Celsius x 0.5 hour, whichever is longer.

  Thereafter, the cooling temperature of the liquid refrigerator is further lowered by the above-mentioned step temperature, and the blood pack 103 is placed in the liquid refrigerator for 0.5 hour or the time indicated in degrees Celsius of the step cooling temperature × 0.5 hour, whichever is longer Leave it alone. This is repeated to reach the second cooling temperature. If the temperature difference between the second cooling temperature and the temperature immediately before reaching the second cooling temperature is smaller than −0.5 ° C., the last step temperature is the temperature difference, and the second cooling temperature is further increased. After reaching the value, the blood pack 103 is left in the liquid refrigerator for a time longer than the numerical value of Celsius temperature display of the last step cooling temperature × 0.5 hours.

  When the cooling temperature of the liquid refrigerator is lowered for each step temperature, the compressor in the liquid refrigerator operates strongly and the blood pack 103 is left unattended for a while to lower the temperature of the cooling liquid circulation tank 101 by the step temperature. The vibration of the compressor is transmitted to the cooled cooling liquid circulation tank 101. Therefore, the blood pack 103 is not cooled in the low vibration state. Therefore, when it is expected that cooling in a low vibration state is difficult, when the blood pack 103 is put into the storage container shown in FIG. 2B and the temperature of the cooling liquid circulation tank 101 is lowered by the step temperature. Covers the upper part of the storage container with a storage cover 107.

  The container lid 107 is removed when it is left standing. The container lid 107 may be left in that state, but the leaving time is set to be two to three times longer than the above time so that the temperature of the coolant 102 is sufficiently transmitted to the blood pack 103. After the last standing time, the temperature of the liquid refrigerator is maintained as it is. Since the low vibration state is not necessary for maintaining the second cooling temperature, the blood pack is at a temperature equal to or lower than the second cooling temperature of the blood pack, which is −30 ° which is the cold limit temperature or the embrittlement temperature of the blood pack material. You may move to another freezing apparatus, maintaining the preservation | save temperature to C. In this series of processes, the blood pack cryopreservation step is completed, and then the blood pack is only stored while maintaining the storage temperature.

  The low vibration environment during freezing is preferably the same as in the first embodiment. Further, the application may be the same as in the first embodiment.

  In this embodiment, bluefin tuna meat is frozen instead of the blood pack. The bluefin tuna is cut into a fence shape, and the fillet is wrapped with a plastic film for packaging or a plastic bag for packaging without containing an air layer, and placed in the container 106 of the cooling liquid circulation tank 101 shown in FIG. 2A. The sample was frozen in the freezing process used in No. 2, and then stored frozen at -18 ° C. 3A, 3B, 3C and 4A, 4B, and 4C show the results of thawing after the same continuous cooling as in Example 1 and after storage at -18 ° C for 1 week. 3A, 3B, and 3C show bluefin tuna muscle fiber cells, which are unfrozen raw bluefin tuna meat, the above thawed bluefin tuna meat, and thawed bluefin tuna meat after undergoing a normal slow freezing method, respectively.

From this, the bluefin tuna meat of this example has almost no cell destruction similar to the raw meat not frozen. On the other hand, in the conventional slow freezing method (FIG. 3C), it can be seen that the destruction of myofiber cells occurs in various places and that water accumulates on the ice marks in the cells. 4A, 4B, and 4C show the overall appearance of bluefin tuna meat, which are unfrozen raw bluefin tuna meat, the above thawed bluefin tuna meat, and thawed bluefin tuna meat after undergoing a normal slow freezing method, respectively. Compared with the non-frozen raw bluefin tuna meat and the thawed bluefin tuna meat that has undergone the normal slow freezing method, the browned tuna meat of this example has a slight browning due to methification. However, raw bluefin tuna meat cannot be stored for one week, so we use bluefin tuna meat collected from the same part of the same kind of bluefin tuna.

  The plastic film for packaging and the plastic bag for packaging may use polyvinylidene chloride in addition to the above-described polyvinyl chloride, polyolefin, polyethylene, polybutadiene, etc., or may use a flexible and malleable material.

  From these results, it is shown that the bluefin tuna meat in this example is kept fresher than the bluefin tuna meat obtained by the conventional slow freezing method. This result is the same even when the step refrigeration method shown in Example 2 is used.

  In the temperature measurement, the temperature of the fish meat may be directly measured, or may be measured via a packaging plastic film or a packaging plastic bag.

In addition, it is preferable that the low vibration environment at the time of freezing is the same as Example 1. If the vibration is larger than this, collision of external water molecules on the surface of the ice crystal and subsequent freezing are likely to occur. As a result, a large ice crystal is formed, and the crystallization proceeds during freezing, and drip occurs during thawing.
Further, the application may be the same as in the first embodiment.

  In the first embodiment and the second embodiment, the two means and the usage method, that is, the blood pack 103 is suspended from above by the hanging strap 104 so that the vibration of the liquid cooling device is not applied to the cooling blood pack ( 2A), and a case in which the anti-vibration table 108 and the container 106 for storing the blood pack 103 and the container lid 107 covering the upper part of the container 106 are used in combination (in the case illustrated in FIG. 2B). ing. However, these are active in the mechanism that the vibration generated by the compressor, which is the main vibration source of the liquid cooling device, is transmitted to the fish meat in the blood pack or packaging plastic film or packaging plastic bag via the secondary refrigerant. It does not give a simple solution.

  In this embodiment, as an active vibration isolating means, the vibration removal function has a function of preventing vibration generated by the compressor from being transmitted to the primary refrigerant and further to the secondary tank such as ethanol or ethylene glycol in the cooling tank. Specific examples of the apparatus will be described. The vibration removing device is used in the middle of the primary refrigerant circuit. The vibration removing device blocks the vibration generated by the compressor, which is the main vibration source of the liquid cooling device, and does not transmit to the fish meat contained in the blood pack or the packaging plastic film. The vibration removing apparatus can be operated in cooperation with any liquid cooling apparatus using a compressor, thereby realizing a refrigeration apparatus in which vibration is hardly transmitted to the refrigerant.

  When the refrigerant that causes a phase change between gas and liquid cools the primary coolant such as ethanol or ethylene glycol in the cooling tank, the vibration transmission preventing device is inserted into the primary coolant circulation circuit.

6A and 6B show a vibration removal module 10 that forms part or all of the vibration removal apparatus according to the present embodiment. FIG. 6A shows the external appearance of the vibration removing module 10, and FIG. 6B shows its internal cross section. The vibration removing device element 10 includes an input tube 1, an output tube 3, and a multiple reflection tube 2. The input tube 1, the multiple reflection tube 2, and the output tube 3 of the vibration removal module 10 have a cylindrical shape, and their inner diameters are d1, d2, and d3. The transfer of the primary refrigerant or the secondary refrigerant is an order of movement in which these refrigerants enter from the input pipe 1 and exit from the output pipe 3 through the multiple reflection pipe 2. The longitudinal vibration wave in the refrigerant is transmitted from the input tube 1 to the multiple reflection tube 2, where the multiple anti-reverse wave is transmitted.

  Since the vibration removal module 10 does not actively control the flow rate of the refrigerant, d1 and d3 are generally equal. Multiple reflection of vibration waves occurs between two walls forming the cylindrical end face of the multiple reflection tube 2. Strictly speaking, the reflection coefficient and the transmission coefficient must take into account the refrigerant, the vibration removal module 10, and the acoustic impedance due to the density of the air outside the vibration removal module 10 and the sound transmission speed.

The vibration removing module 10 is made of metal or plastic. Therefore, there is a great difference in density and sound wave transmission speed from refrigerant and air. Therefore, even if the effect of the acoustic impedance is ignored and the reflection coefficient and the transmission coefficient of the vibration removal module 10 are obtained only from the shape, a large error does not occur. Therefore, the reflection coefficient and transmission coefficient are

  In the case where the internal structure of the vibration removal module 10 is as shown in FIG. 6B, a part of vibration transmitted from the input pipe 1 is directly transmitted from the output pipe 3 to the downstream refrigerant without causing the multiple reflection described above. . Therefore, it is difficult to give a half-wave phase difference of complete vibration to the downstream refrigerant.

  FIG. 6C shows the internal structure of the vibration removing module 10 that has improved the problem. That is, a baffle 31 made of a disk-shaped plate material is provided on the outlet side where the output pipe 3 looks into the input pipe 1 so that vibrations propagated from the input pipe 1 do not propagate directly to the output pipe 3. The baffle 31 functions to prevent transmission of vibration, and its diameter d4 is approximately the same as or slightly larger than the inner diameter d3 of the output tube 3. The baffle 31 is attached to the inside of the multiple reflection tube 2 by the attachment leg 32.

Because of the baffle 31, most of the vibration propagating from the input tube 1 undergoes the multiple reflection described above, and therefore, the multiple reflection tube 2 can give a half-wave phase difference of complete vibration.
Note that the baffle 31 may not be a disk shape but may be a quadrangle or a polygon having a shape that covers the inner diameter d3 of the output tube 3.

  FIG. 6D shows another internal structure of the vibration removing module 10. In the above-described embodiment, the vibration propagating from the input pipe 1 by the baffle 31 is not directly propagated to the output pipe 3. However, instead of the baffle 31, a vibration reflector 33 made of a disk-shaped plate material is provided. Yes. The diameter d5 of the vibration reflector 33 is slightly smaller than the inner diameter d2 of the multiple reflector 2 and larger than the inner diameter d3 of the output tube 3. The vibration reflection plate 33 almost reflects the vibration propagated from the input tube 1, and multiple reflection occurs between the vibration reflection plate 34 and the inner wall of the multiple reflection tube 2 on the input tube 1 side. The vibration after the multiple reflection is propagated in the refrigerant flowing out from the gap between the vibration reflection plate 33 and the multiple reflection tube 2 to the output tube 3. The vibration reflector 33 is attached to the inside of the multiple reflector 2 by the attachment leg 34.

  In FIG. 6D, the surface of the vibration reflection plate 33 is concave toward the terminal inner wall of the multiple reflection tube 2 on the input tube 1 side. This is to match the phase plane of vibration propagating from the input tube 1. However, although the spatial dispersion of the vibration waveform due to multiple reflection occurs, the surface shape of the vibration reflection plate 33 may be a flat plate parallel to the terminal inner wall of the multiple reflection tube 2 on the input tube 1 side.

The vibration reflecting plate 33 may not be a disk shape but may be a quadrangular shape or a polygon having a shape covering the inner diameter d3 of the output tube 3.
The baffle 31 works so as not to propagate vibration directly to the output tube 3, and the vibration reflector 33 works so as to generate multiple reflections with the terminal inner wall of the multiple reflection tube 2 on the input tube 1 side. The diameter d4 is smaller than the diameter d5 of the vibration reflector 33.

  In order to cancel out by the interference of vibration due to the half-wave phase difference of vibration, the circuit of primary refrigerant, secondary refrigerant or frozen liquid is divided into two, one of which is directly transferred (direct transfer), and the other is One or more vibration removal modules 10 are connected in series to generate a half-wave phase delay and transfer (phase delay transfer). 7A and 7B show the main part of an embodiment of a circuit (hereinafter referred to as “phase difference generation circuit”) that realizes these direct transfer and phase-delayed transfer.

  The piping 11 of the primary / secondary refrigerant / freezing liquid circuit is divided into two by a branch pipe 12. In the phase difference generation circuits 6 and 7, a straight pipe 4 extending substantially linearly is used for direct transfer in order to reduce the phase delay. In the phase difference generation circuit 6, the vibration removal module 10 is arranged in parallel to the direct transfer pipe 4 (FIG. 7A) for phase delay transfer, and in the phase difference generation circuit 7, the vibration removal module 10 is directly connected to the pipe for transfer. 4 is arranged at a right angle (FIG. 7B). In the phase difference generation circuit 7, the vibration removal module 10 is arranged at a right angle to the pipe 4 for direct transfer, and therefore the overall length thereof can be shortened compared to the phase difference generation circuit 6. be able to.

  A pipe 5 is used in a circuit that passes from the branch pipe 12 through the vibration removal module 10. The circuit of the direct transfer pipe 4 and the phase delay transfer circuit composed of the pipe 5 and the vibration removal module 10 have the same conductance, and the primary refrigerant supplied from the direct transfer circuit and the phase delay transfer circuit or The flow rate of the secondary refrigerant per unit time is the same. Thereby, it is possible to reliably cancel the vibration at the total flow rate due to the interference of vibration at the total flow rate after the merge.

  FIG. 7C shows a simple merger 17. That is, the primary refrigerant or secondary refrigerant supplied from the circuit of the direct transfer pipe 4 and the primary refrigerant or secondary refrigerant supplied from the pipe 5 via the phase delay transfer circuit are respectively input pipe 13. 14 and the pipe 16 are joined.

  FIGS. 8A and 8B show the external appearance and internal cross section of another merger 20 that merges the flow of refrigerant or coolant supplied from the direct transfer circuit and the phase delay transfer circuit. The circuit of the direct transfer pipe 4 and the circuit of the phase delay transfer flow composed of the vibration removal module 10 and the pipe 5 are connected to the input pipes 13 and 14 of the merger 20. The refrigerant flowing in from the input pipes 13 and 14 enters the entrance 25 of the expansion pipe 21 having a conical internal space to which the outlets of the two input pipes are connected, and is introduced into the interference chamber 26.

  The angle θ of the interference chamber 26 viewed from the input pipes 13 and 14 is a cone apex angle or a flow expansion angle, and a value that does not increase the flow pressure loss is desirable. In the expansion pipe, the flow is first released from the wall surface, goes downstream to some extent, and then contacts the pipe wall. The cause of the pressure loss occurs at the beginning of the expansion tube because the jets disturb the surrounding fluid and create vortices. From FIG. 9, the enlargement angle is preferably about 25 degrees, which is about half of the maximum pressure loss. Although the angle may be larger than this, the load on the circulation pump 52 (FIG. 10A) or the compressor 72 (FIG. 10B) that creates the flow increases. On the other hand, if the angle is less than this, the outer length of the merger 20 becomes large, and the vibration removing device for mounting it becomes large.

  The primary refrigerant or the secondary refrigerant supplied from the direct transfer circuit and the phase delay transfer circuit interfere with each other in the interference chamber 26, and the longitudinal wave vibration disappears in the interference chamber 26. However, the interference chamber 26 is just a space where two vibrations, a longitudinal wave vibration and a vibration delayed by a half wavelength, interfere with each other, so that the interference may be broken and the vibration may appear again in the refrigerant. In order to prevent this danger, a diffusion plate 23 having many diffraction holes 24 is provided in the downstream portion of the interference chamber 26. The vibration behaves using this diffraction hole 24 as a new vibration source.

  The diffusion plate 23 is preferably made of a material having a significantly different acoustic impedance than that of the coolant or the cooling liquid, for example, a plastic or metal plate having a cavity inside, in order not to transmit vibration. However, since the vibration disappears in the diffraction hole 24, a flow in which the vibration disappears gathers in the merge chamber 27 formed by the cylindrical tube 22 on the downstream side. Thereafter, the flow passes through the outlet 28 as an ultra-low vibration flow from the output pipe 15 through the cooling liquid supply pipe 54 (FIG. 10A) or the refrigerant supply pipe 84 (FIG. 10B) or the cooling liquid circulation tank 58 or cooling. It reaches a cooling pipe 75 for cooling the liquid circulation tank 78.

  The vibration removing device can be configured by connecting the phase difference generating circuit 6 or 7 to the merger 20. On the other hand, the pipes 4 and 5 of the phase difference generating circuit 6 or 7 may be connected to a merging pipe (not shown) that uses the branch pipe in the reverse direction without using the merging device 20, so that a vibration removing device is obtained. it can. In this case, if the pipes after merging are bent, the original vibration wave may appear, so that the usage is limited. On the other hand, when the merger 20 is used, since the original vibration wave does not appear even if the downstream pipe after the merger 20 is bent, the application is not limited.

  FIG. 10A shows an embodiment in which the vibration removing device 53 is used by installing a circulating circuit 65 for the coolant 55. The cooling liquid circulation tank 58 is filled with the cooling liquid 55. The coolant 55 is circulated through the circulation circuit 65 by the circulation pump 52. The circulation circuit 65 is provided with a primary cooler 51, and the coolant 55 is cooled by the primary cooler 51 and enters the vibration removing device 53 via the cooling pipe 66. The ultra-low vibration coolant 55 is supplied to the coolant circulation tank 58 via the coolant supply pipe 54. Since the coolant 55 does not transmit the vibration generated by the circulation pump 52 or the compression pump 42, the blood pack 57 is placed in a low vibration or very low vibration environment in the cooling process required in the first or second embodiment. be able to. The blood pack 57 may be placed on the anti-vibration table 56 or may be placed on the anti-vibration table 56 in a container (not shown).

  The primary refrigerant that cools the coolant 55 by the primary cooler 51 circulates in the cooling cycle in the circulation circuit 63. That is, the primary refrigerant circulates in the circulation circuit 63 due to the pressure gradient caused by the compression of the compression pump 42 and the low pressure in the expansion valve 43. After being compressed by the compression pump 42, the primary refrigerant is cooled by the secondary cooler 41 and enters the primary cooler 51 via the expansion valve 43. The secondary cooler 41 is supplied with low-temperature water or air 61 and takes the heat of the primary refrigerant, and then becomes hot water or hot air 62. In this embodiment, the primary refrigerant is compressed by the compression pump 42 and then cooled by the secondary cooler 41 and enters the expansion valve 43. However, instead of the expansion valve 43, a capillary may be used.

  The present embodiment can be applied not only to blood packs but also to fish meat placed in plastic films for packaging or plastic bags for packaging.

  FIG. 10B shows an embodiment in which the vibration removing device 74 is used in the primary refrigerant circulation circuit 83. The cooling liquid circulation tank 78 is filled with a cooling liquid 77. The primary coolant circulates in the circulation circuit 83 due to the pressure gradient caused by the compression of the compression pump 72 and the low pressure at the expansion valve 73. The primary refrigerant is compressed by the primary cooler 71 after being compressed by the compression pump 72 and enters the vibration removing device 74 via the expansion valve 73. The ultra-low vibration primary refrigerant is supplied to the cooling pipe 75 around the cooling liquid circulation tank 78 via the refrigerant supply pipe 84.

  The cooling liquid circulation tank 78 is cooled by the cooling pipe 75 and the cooling liquid 77 is cooled. Since the primary refrigerant does not transmit the vibration generated by the compression pump 72, the blood pack 79 can be placed in an environment of low vibration or very low vibration in the cooling process required in the first or second embodiment. The blood pack 57 may be placed on the anti-vibration table 76 or may be placed on the anti-vibration table 76 in a container (not shown).

  The primary cooler 71 is supplied with low-temperature water or air 81 and takes the heat of the primary refrigerant, and then becomes hot water or hot air 82. The primary refrigerant is compressed by the compression pump 72 and then cooled by the primary cooler 71 and enters the expansion valve 73. Instead of the expansion valve 73, a capillary may be used.

  The present embodiment can be applied not only to blood packs but also to fish meat placed in plastic films for packaging or plastic bags for packaging.

  Moreover, you may use together vibration isolation means, such as the hanging strap A shown to FIG. 2A, or the vibration isolation stand shown to FIG. 2B, with the freezing using the vibration removal module which concerns on this invention.

  In this specification, about hemolysis suppression, whole blood is explained as whole blood, and the present invention is explained based on whole human blood in Examples. However, the hemolysis-suppressed cryopreservation method and the refrigeration apparatus used in the method according to the present invention can be applied to blood of marine animals such as terrestrial animals or fish, or whole blood collected from these animals as long as they have red blood cells.

  In the present specification, the present invention has been described with an example using a container filled with blood, particularly a bag-like pack, at the time of freezing blood. However, as long as it can store blood and prevent external contamination, Even if it is not a pack, it may be a box shape, a spherical shape or the like. Any container that can freeze blood may be used.

  In addition, the present invention is applicable not only to blood transfusion, but also to cryopreservation of blood used for such purposes or applications as long as it is an object or use that requires prevention of hemolysis.

  On the other hand, with regard to the frozen storage method for maintaining the freshness of fresh fish, Example 3 describes bluefin tuna meat in this specification. However, this frozen storage method can also be applied to other tuna and other fish species such as yellowtail, mackerel, Thailand, skipjack, amberjack, saury and sardines.

  Further, for the bluefin tuna, other tuna and other fish species, in addition to the cooling process employing the slow cooling shown in the first embodiment, a cooling process employing the step cooling shown in the second embodiment may be used. Similarly, freshness can be maintained.

  When the fish body is relatively small, the frozen storage method of the present invention can be applied to the whole fish body by applying the same freezing process as in Example 3 not only to the fillet. The same applies to the refrigeration apparatus.

  The cooling process may include a cooling process that employs slow cooling and a cooling process that employs step cooling. For example, when shifting from the first cooling temperature to the second cooling temperature, a method in which the first half is cooled by a cooling process based on slow cooling and the second half is cooled by a cooling process based on step cooling, or vice versa, or a cooling process based on slow cooling. Alternatively, a cooling process based on step cooling and a cooling process in which the ratio of these processes is changed may be used.

  Moreover, the case where a liquid refrigerator is used as a refrigerator is demonstrated. However, an air blast refrigerator or the like can be used as long as the first freezing temperature and the second freezing temperature can be set and a rapid freezing process up to the first freezing temperature can be provided. Further, a contact freezer that directly contacts the refrigerant guide tube or the guide housing can be used as a refrigerator in which the blood pack or fish meat does not directly contact the coolant.

  When an air blast refrigerator is used, vibration transmitted through the refrigerant liquid is transmitted to the blood pack or fish meat, which is the object to be frozen, through the air. In the propagation of vibration energy in refrigerant or water and the propagation of energy in air, air is about 3400 times larger. However, the ratio of sound pressure energy at which reflection is greatly transmitted when sound waves are transmitted from the air to a blood pack or fish meat is 1/3700. Therefore, the sound pressure 294 Pascal becomes the limit sound pressure in the air. In this case, the sound pressure level is 143 dB. Therefore, it is necessary to suppress the air vibration of the air blast refrigerator to 143 dB or less.

  In the contact freezer, it is necessary to directly measure the vibration of the object to be frozen and suppress the sound pressure to 169 dB or less, which is the same as the sound pressure level in the refrigerant.

  In any case, the critical sound pressure inside the object to be frozen is the same when any of a liquid refrigerator, an air blast refrigerator, and a contact freezer is used.

  Cooled quickly to the first cooling temperature, and then applied to the storage of blood packs where hemolysis is unlikely to occur by a process consisting of a slow cooling process to the second cooling temperature in an environment where vibration is hardly transmitted from the outside to the blood pack. can do. Furthermore, this frozen storage method can also be applied to frozen storage that maintains the freshness of fish meat.

DESCRIPTION OF SYMBOLS 1 Input pipe 2 Multiple reflection pipe 3 Output pipe 4, 5, 16 Pipe 6, 7 Phase difference generation circuit 10 Vibration removal module 11 Pipe 12 Branch pipe 13, 14 Input pipe 15 Output pipe 17, 20 Merger 21 Expansion pipe 22 Cylindrical Tube 23 Diffusion plate 24 Diffraction hole 25 Entrance 26 Interference chamber 27 Merge chamber 28 Exit 31 Baffle 32, 34 Mounting leg 33 Vibration reflector 41, 71 Secondary cooler 42, 72 Compression pump 43, 73 Expansion valve 51 Primary cooler 52 Circulating pumps 53, 74 Vibration removing device 54 Cooling liquid supply pipes 55, 77, 102 Cooling liquids 56, 76 Anti-vibration tables 57, 79 Blood packs 58, 78, 101 Cooling liquid circulation tanks 65, 83 Circulating circuit 66 Cooling pipe 75 Cooling pipe 84 Refrigerant supply pipe 103 Blood pack 104 Suspension string 105 Rail 106 Storage unit 107 Storage unit cover 108 Vibration isolation table

Claims (33)

A method for freezing blood comprising the following steps:
Step 1 After injecting blood into a sterilized container (pack) and filling the container, the inlet is sealed in a state that does not include an air layer to form a blood pack.
Step 2 The blood pack is immediately cooled to the first cooling temperature (−4 to −6 ° C.).
Step 3 The blood in the blood pack is left at the first cooling temperature for 5 minutes or more after the first cooling temperature is reached.
Step 4 Slowly (−1.5 ° C./hour to −0.5 ° C./second) to the second cooling temperature (−7 to −13 ° C.) under an environment in which vibration is hardly transmitted from the outside to the blood pack. Cooling time),
Step 5 The blood pack is stored at a storage temperature that is equal to or lower than the second cooling temperature and is between the cold resistance limit temperature and the embrittlement temperature. A method for freezing blood comprising the following steps:
Step 1 After injecting blood into a sterilized container (pack) and filling the container, the inlet is sealed in a state that does not include an air layer to form a blood pack.
Step 2 The blood pack is immediately cooled to the first cooling temperature (−4 to −6 ° C.).
Step 3 The blood in the blood pack is left at the first cooling temperature for 5 minutes or more after the first cooling temperature is reached.
Process 4 Under the environment where vibration is hardly transmitted from the outside to the blood pack, the temperature selected in the range of -0.5 ° C to -1.5 ° C is set as the step temperature, and the temperature of the blood pack is set as the step temperature. Just lower,
Step 5 The blood pack is left at a temperature lower by the step temperature for a time longer than the numerical value of Celsius temperature display of the step temperature × 0.5 hours.
Step 6 Repeat Step 4 and Step 5 until the temperature of the blood pack reaches the second cooling temperature (−7 to −13 ° C.).
Step 7 After the temperature of the blood pack reaches the second cooling temperature, the blood pack is stored at a storage temperature that is equal to or lower than the second cooling temperature and is between the cold resistance limit temperature and the embrittlement temperature.   A method for cryopreserving blood comprising the freezing method according to claim 1.   A method for cryopreserving blood, comprising the freezing method according to claim 2.   The method for cryopreserving blood according to any one of claims 1 to 4, wherein the blood is any one of whole blood, red blood cell concentrate, and washed red blood cells.   The blood according to any one of claims 1 to 5, wherein a cooling liquid circulation tank is used as means for cooling the blood to a temperature up to the first cooling temperature and the second cooling temperature. Frozen storage method.   The cooling liquid circulation tank is used for the means which cools the said blood pack to the temperature until it reaches the 1st cooling temperature and the 2nd cooling temperature, The any one of Claim 1 to 6 characterized by the above-mentioned. How to store frozen blood packs.   The method of quickly cooling the blood pack to the first cooling temperature in the step 2 according to claim 1 or 2 is the cooling liquid stored in the cooling liquid circulation tank according to claim 7 and set to the first cooling temperature. The method for cryopreserving blood according to any one of claims 1 to 7, wherein a method of quickly sinking the blood pack is used.   In order to provide an environment in which vibration is hardly transmitted from the outside to the blood pack according to step 4 of claim 1 or 2, the horizontal bar installed on the upper part of the cooling liquid circulation tank, The method for cryopreserving blood according to any one of claims 6 to 8, wherein a string passed through the blood pack is used.   In order to provide an environment in which vibration is hardly transmitted to the blood pack from the outside according to step 4 of claim 1 or 2, the vibration isolator installed on the bottom surface of the cooling liquid circulation tank and the blood pack are stored. The method for cryopreserving blood according to any one of claims 6 to 8, wherein a container and a container lid that covers an upper part of the container are used. Method of freezing fish including the following steps:
Step 1 The whole fish body or the fillet, or those wrapped with a plastic film for packaging or a plastic bag for packaging without containing an air layer, is to be frozen.
Step 2 The object to be frozen is quickly cooled to the first cooling temperature (−4 to −6 ° C.).
Step 3 The object to be frozen is left at the first cooling temperature for 5 minutes or more after reaching the first cooling temperature.
Step 4 Slowly (−1.5 ° C./hour to −0.5 ° C.) to the second cooling temperature (−7 to −13 ° C.) under an environment where vibration is hardly transmitted from the outside to the object to be frozen. Cooling)
Step 5 The object to be frozen is stored at a storage temperature that is equal to or lower than the second cooling temperature and is between the cold resistance limit temperature and the embrittlement temperature.
Method for freezing fresh fish containing Method of freezing fish including the following steps:
Step 1 The whole fish body or the fillet, or those wrapped with a plastic film for packaging or a plastic bag for packaging without containing an air layer, is to be frozen.
Step 2 The object to be frozen is quickly cooled to the first cooling temperature (−4 to −6 ° C.).
Step 3 The object to be frozen is left at the first cooling temperature for 5 minutes or more after reaching the first cooling temperature.
Process 4 Under the environment where vibration is hardly transmitted from the outside to the object to be frozen, the temperature selected in the range of -0.5 ° C to -1.5 ° C is set as the step temperature, and the temperature of the blood pack is set as the step temperature. Just lower,
Step 5: The temperature of the object to be frozen is decreased by the step temperature, and is allowed to stand for a time longer than the value of the step cooling temperature displayed in degrees Celsius x 0.5 hours.
Step 6 Repeat Step 4 and Step 5 until the temperature of the object to be frozen reaches the second cooling temperature (−7 to −13 ° C.).
Step 7 After the temperature of the object to be frozen reaches the second cooling temperature, the object to be frozen is stored at a storage temperature that is equal to or lower than the second cooling temperature and between the cold resistance limit temperature and the embrittlement temperature. ,
Method for freezing fresh fish containing   A cooling liquid circulation tank is used as a means for cooling the whole fish body according to claim 11 or claim 12 or a fillet of the fish body to a temperature up to the first cooling temperature and the second cooling temperature. The method for cryopreserving fresh fish according to claim 11 or claim 12.   A means for cooling the whole fish body according to claim 11 or claim 12 or a fillet of the fish body wrapped in a plastic film for packaging or a plastic bag for packaging to a temperature up to the first cooling temperature and the second cooling temperature. The method for cryopreserving fresh fish according to claim 11 or 12, wherein a cooling liquid circulation tank is used.   In order to provide an environment in which vibration is hardly transmitted from the outside to the blood pack according to step 4 according to claim 1 or 2, the cooling liquid supplied to the cooling liquid circulation tank is a branch pipe. Branching into two paths, one becomes a cooling liquid via a linear tube extending almost linearly, and the other consists of a multiple reflection tube composed of tubes having an inner diameter larger than the inner diameter of the input tube and the output tube After the phase difference generation circuit in which one or more vibration removal modules are connected in series, a coolant having a phase delay of approximately half a wavelength of external vibration is generated, and after the one coolant and the other coolant are merged, The method for cryopreserving blood according to any one of claims 6 to 10, wherein the blood is supplied to the cooling liquid circulation tank.   In order to provide an environment in which vibration is hardly transmitted from the outside to the object to be frozen according to the step 4 according to claim 11 or 12, the cooling liquid supplied to the cooling liquid circulation tank is a branch pipe. Branching into two paths, one becomes a cooling liquid via a linear tube extending substantially linearly, and the other is from a multiple reflector tube composed of a tube having an inner diameter larger than the inner diameter of the input tube and the output tube After the phase difference generation circuit in which one or more vibration removal modules are connected in series, the coolant is delayed by approximately half a wavelength of the external vibration, and the one coolant and the other coolant are merged. The fresh fish frozen storage method according to claim 13 or 14, wherein the fresh fish is supplied to the cooling liquid circulation tank.   The cooling liquid supplied to the cooling pipe to surround the cooling liquid circulation tank in order to provide an environment in which vibration is hardly transmitted to the blood pack from the outside according to the step 4 according to claim 1 or 2. Is divided into two paths by a branch pipe, one becomes a cooling liquid via a straight pipe extending substantially linearly, and the other is constituted by a pipe having an inner diameter larger than the inner diameter of the input pipe and the output pipe. The phase difference generation circuit in which one or more vibration elimination modules composed of multiple reflection tubes are connected in series results in a cooling liquid having a phase delay of approximately half a wavelength of external vibration, and the one cooling liquid and the other cooling liquid. The method for cryopreserving blood according to any one of claims 6 to 10, wherein after the merging, the cooling liquid circulation tank is supplied to a cooling pipe.   The cooling supplied to the cooling pipe to surround the cooling liquid circulation tank in order to provide an environment in which vibration is hardly transmitted from the outside to the object to be frozen, according to the step 4 according to claim 11 or claim 12. The liquid is branched into two paths by a branch pipe, one becomes a cooling liquid via a straight pipe extending substantially linearly, and the other is constituted by a pipe having an inner diameter larger than the inner diameter of the input pipe and the output pipe. A cooling liquid having a phase lag corresponding to almost half wavelength of the external vibration is obtained through a phase difference generation circuit in which one or more vibration removing modules each composed of multiple reflection tubes are connected in series. 15. The method for cryopreserving fresh fish according to claim 13 or claim 14, wherein the liquids are supplied to a cooling pipe so as to surround the cooling liquid circulation tank after joining.   The vibration removal module according to any one of claims 15 to 18, wherein a baffle having a diameter equal to or larger than an inner diameter of the output tube is provided inside the multiple reflection tube and attached to the front of the output tube. The method for cryopreserving blood according to any one of claims 6 to 10, 15 or 17, or the fresh fish according to claim 16 or 18, wherein the blood is composed of a characteristic multiple reflection tube. Frozen storage method.   The vibration removal module according to any one of claims 15 to 18, wherein a vibration reflector having a diameter that is equal to or larger than the inner diameter of the output tube and smaller than the inner diameter of the multiple reflection tube is provided in the multiple reflection tube. The method for cryopreserving blood according to any one of claims 6 to 10, or 15 or 17, wherein the method is constituted by a multi-reflection tube, which is mounted in front of the output tube. Item 19. A method for cryopreserving fresh fish according to item 16 or claim 18.   The vibration removal module according to any one of claims 15 to 20, wherein the vibration removal module is disposed in parallel to the straight pipe, and the vibration removal module according to any one of claims 6 to 10, The method for cryopreserving blood according to claim 19 or 20, or the method for cryopreserving fresh fish according to claim 16, 18, 19 or 20.   The vibration removal module according to any one of claims 15 to 20, wherein the vibration removal module is disposed perpendicularly to the straight pipe. A method for cryopreserving blood according to claim 19 or claim 20 or a method for cryopreserving fresh fish according to claim 16, 18, 19 or 20.   The cooling liquid passing through the straight pipe according to claim 15 and the cooling liquid cooling liquid passing through the phase difference generation circuit according to claim 15 have an interference chamber composed of an enlarged pipe having a conical inner space. It joins with the merger which has, It supplies to the said cooling liquid circulation tank, The claim 1, The claim 15, The claim 15, or any one of the claims 19-22 23. A method for cryopreserving fresh blood, or a method for cryopreserving fresh fish according to any one of claims 16 and 19-22.   The cooling liquid passing through the straight pipe according to claim 17 or 18 and the cooling liquid cooling liquid passing through the phase difference generation circuit according to claim 18 are provided with an interference chamber composed of an enlarged pipe having a conical internal space. The merging device has a merging device and then is supplied to a cooling pipe so as to surround the cooling liquid circulation tank. 21. Any one of claims 17, 17, or 19 to 22. The method for cryopreserving blood according to claim 1, or the method for cryopreserving fresh fish according to any one of claims 18 or 19 to 22.   The merging device according to claim 23 or claim 24 has a diffusion plate in which a plurality of diffraction holes are formed inside, the merging device according to any one of claims 6 to 10, claim 15, claim 17, The method for cryopreserving blood according to any one of claims 19 or 20 to 24, or the cryopreservation of fresh fish according to any one of claims 16, 18 or 20 to 24. Method.   The material of the container according to step 1 according to claim 1 or claim 2 and the material of the plastic film for packaging or the plastic bag for packaging according to step 1 according to claim 11 or claim 12 are DEHP (phthalate). Polyvinyl chloride, polyolefin, polybutadiene, polyethylene, or polyvinylidene chloride using diacid − 2 − ethylhexyl) or TOTM (trimellitic acid tris − 2 − ethylhexyl) The method of cryopreserving blood according to any one of claims 6 to 10, claim 15, claim 17, or claim 19 to 25, or claim 11 to 13, The method for freezing and storing fresh fish according to any one of claims 1, 16, 18, or 19 to 25. A vibration removing device used in the method for cryopreserving blood according to claim 15 or the method for cryopreserving fresh fish according to claim 16, each of which comprises:
A phase difference generation circuit in which one or more vibration elimination modules according to claim 15 or claim 17 or claim 16 or claim 18 are coupled in series; and claim 15 or claim 17 or claim 16 or claim 18. The vibration removal apparatus which consists of a confluence | merging device which joins one cooling fluid and the other cooling fluid in the downstream of the said linear tube and a phase difference generation circuit.   The vibration removal module according to claim 15, further comprising a baffle having a diameter equal to or larger than the inner diameter of the output tube, and a multiple reflection tube attached to the front of the output tube inside the multiple reflection tube. The vibration removing device according to claim 27, wherein the vibration removing device is configured.   The vibration removing module according to claim 15, further comprising a vibration reflector having a diameter not less than an inner diameter of the output tube and smaller than an inner diameter of the multiple reflection tube, the output of the multiple reflection tube. 28. The vibration removing device according to claim 27, wherein the vibration removing device is constituted by a multiple reflection tube attached to the front of the tube.   The merger according to claim 27 is a merger having an interference chamber composed of an expansion pipe having a conical inner space to which two input pipes and outlets of the two input pipes are connected. 30. The vibration removing apparatus according to any one of claims 27 to 29.   The merging device according to any one of claims 27 to 30, wherein the merging device according to claim 30 is a merging device in which a diffusion plate having a plurality of diffraction holes is provided in a downstream portion of the interference chamber. The vibration removing apparatus according to item 1.   The vibration according to claim 1, 2, 9, 10, 15, or 17 is hardly transmitted to the blood pack or the vibration according to claim 11, 12, 16, or 18. The environment is characterized in that the sound pressure inside the blood pack or the frozen object caused by vibration applied to the blood pack or the frozen object placed under the environment is made smaller than 284 Pascals. Alternatively, the method for freezing blood according to claim 2, or the method for cryopreserving blood according to any one of claims 3 to 10, claim 15, claim 17, or claim 19 to 26. Or a frozen storage method for fresh fish according to any one of claims 11 to 14, claim 16, claim 18, or claim 19 to 26.   In the step 2 according to claim 1, claim 2, claim 8, claim 11 and claim 12, rapidly cooling to the first cooling temperature means that the temperature of the blood pack or the object to be frozen is -1. The method for freezing blood according to claim 1 or 2, or the method for freezing blood according to any one of claims 3 to 10, wherein the cooling is performed from ° C to -4 ° C in a time shorter than 30 minutes. 15. The method for cryopreserving blood according to any one of claims 15, 17 or 19 to 26, or any one of claims 11 to 14, claim 16, 18 or 19 to 26. The method for freezing and storing fresh fish according to any one of the above.