JP6731972B2 - Frozen storage method for suppressing hemolysis, frozen storage method for fresh fish, and freezer - Google Patents

Description

The present invention relates to a method for cryopreserving whole blood, a concentrated red blood cell solution, and washed red blood cells, particularly a cryopreservation method for suppressing hemolysis and a fresh fish cryopreservation method, and a refrigerating apparatus used for these methods.

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

1) Conventional technology related to frozen storage method Although the 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. When the storage temperature is lowered and the red blood cells are frozen for the purpose of extending the frozen storage period, there are problems that the red blood cells are destroyed due to the formation of ice crystals or the concentration of intracellular electrolytes. In short, "hemolysis" phenomenon occurs. The solution to this problem is the addition of glycerin and glycene-added blood frozen storage by removing glycerin with a 5% fructose solution at the time of use.

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

2) Problems of the prior art relating to the cryopreservation method In the above preservation method, in the use of blood after thawing, glycerin is an intracellular cryoprotective material for erythrocytes, so a step for removing it is required. There is a problem that the management is complicated, it takes a long time, it is difficult to maintain the blood after thawing under optimal conditions that do not cause hemolysis, and it is difficult to constantly suppress the hemolysis rate to a constant level. is there.

Therefore, there is a demand for a blood preservation method in which red blood cells can be used for transfusion without washing.
There is also a demand for blood preservation that is not as long as cryogenic cryopreserved blood and that the preservation period is several times longer than that of the current situation or that washing of red blood cells is not required. Therefore, a storage method that does not use additives that can be used in such cases is desired.

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

Salt-induced hemolysis is a phenomenon that occurs during freezing, in which the electrolyte in the cells of red blood cells is concentrated, and as a result, the osmotic pressure from the outside rises and cell membrane destruction occurs. 60% of the red blood cells are water, and the solute is filled with electrolyte in which the solute is dissolved in water. When a cell freezes, water molecules in the cell successively form hydrogen crystals by hydrogen bonding to form ice crystals. On the other hand, intracellular solutes do not participate in such electrostatic binding chains. So ice crystals are composed of pure water molecules.

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

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

Intracellular cause hemolysis is the erythrocyte cell swelling due to freezing of the cell fluid and the crystal growth is not uniform. The theory is that the constituent lipid bilayer membranes are destroyed.

However, the cells are not destroyed by simple volume expansion due to freezing of water in the cells. Because 60% of the inside of the cell is water, and when it crystallizes inside the cell, its volume expansion rate is 1.1 times, so the volume expansion of the whole cell is 1.06 times, and at this level, the cell breaks down. Because there is no.

Therefore, there is a mechanism called cell membrane-caused hemolysis. That is, according to Non-Patent Document (3), since the lipid bilayer membrane has weak bonding in the spreading direction of the membrane, it is easily damaged by a local force, that is, a pinpoint force from the inside to the outside. It is a mechanism that, during intracellular freezing, the cell membrane of erythrocytes is destroyed by non-uniform crystal growth during freezing of the cell liquid. Further, during intracellular freezing, the smaller the degree of supercooling at the start of nucleation, the larger the ice crystals formed, and the higher the degree of supercooling, the smaller the ice crystals formed. Therefore, as the degree of supercooling is lower, pinpoint force from the inside to the outside is more likely to occur, and hemolysis is more likely to occur.

According to Non-Patent Document (4), extracellular-caused hemolysis is that in the process of freezing of plasma, concentrated plasma and cells are pushed into the gaps of ice crystals as ice nuclei grow, and the cells come into contact with ice crystals on the surface. , It is a mechanism that eventually occurs when the ice crystals are destroyed by the pressure of ice crystals. It has been reported that freezing and thawing at relatively slow rates up to about -10°C causes hemolysis.

Next, the background art relating to a method for freezing and storing fresh fish, particularly a method for keeping freshness will be described in detail.

As a frozen storage method for maintaining the freshness of fresh fish, there is a frozen storage method in which the fresh fish is frozen at a temperature of −40° C. or lower immediately after the net is caught or after it is captured. However, in order to freeze at such a low temperature, it is necessary to install expensive refrigeration equipment on the fishing boat, and it is not performed except for high-quality tuna. Moreover, the use of such low-temperature equipment in the distribution process of tuna is a major cause of raising the general consumer price of frozen fish.

On the other hand, in the cold chain during storage and distribution of fresh fish, a freezing temperature of -18°C is widely used as a standard. This freezing temperature of -18°C is widely used for freezing and preserving fish caught especially in the near sea. However, this freezing temperature is insufficient to suppress the occurrence of drip in fish meat after thawing, which is a measure of the freshness of fish, especially fish meat after thawing and browning during storage. These are mainly caused by the brown color change caused by the destruction of cellular structure of fish meat and the formation of meteorite, and are also a major factor in the deterioration of the freshness of fresh fish.

The occurrence of drip after thawing does not mean that there is a problem in the thawing process, but the growth of ice crystals at the time of freezing, especially because ice crystal nuclei formed inside and outside the cells grow and their size increases and the cell membrane is destroyed. Occurs. Specifically, the ice crystal nuclei grow inside the cell, the saltiness of the ice crystal surface rises, and the intercellular fluid with a relatively low saltiness diffuses into the cell through the cell membrane, and the original cell Water enters the cells more than it had and ice crystals grow and finally the cell membrane is destroyed from the inside. On the other hand, in the ice crystal nuclei generated in the intercellular fluid, the surrounding intercellular fluid freezes and grows on its surface, and the size of the ice crystal increases, and conversely the cell membrane is destroyed from the outside. Whether the destruction of the cell membrane occurs inside or outside the cell, or at different times inside and outside to break the cell membrane, depends on the randomness of the initial ice crystal nucleus generation and the freezing process. It depends on the time.

In conventional refrigeration methods, for example - 40 ° often the cooling process be frozen is slow, in the tissues of the fish, in the refrigeration process dotted ice nuclei gradually decreasing temperature ice crystals An energy difference between water molecules is generated on the surface and around it, and the concentration gradient of the electrolyte becomes inhomogeneous.As a result, the electrolyte is condensed in a part of the tissue, causing nonuniformity of the tissue environment and damage to the tissue. give. This has a great influence on the texture and taste of the fish meat after thawing, and the greater the influence, the more different from the fresh fish meat before freezing. Then, such fish meat will be evaluated and judged by the consumer as fish meat with a low freshness.

Brown degeneration during frozen storage is due to the fact that dissolved oxygen in the intracellular fluid grows in ice crystals formed in the intracellular fluid, resulting in an increase in free energy on the surface and excessive free oxygen molecules that cannot be dissolved. It is formed by binding with oxymyoglobin or deoxymyoglobin in the medium to form a methate. On the other hand, in the ice crystal nuclei generated in the intercellular fluid, oxygen molecules that cannot be dissolved in the intercellular fluid appearing on the ice crystal surface and the intercellular fluid surrounding the ice cell diffuses into the cell through the cell membrane, In the same way, it causes methification of intracellular myoglobin. This mechanism also causes browning. On the other hand, myoglobin receives energy from the surroundings and is reduced by the action of metmyoglobin reductase. However, the action of myoglobin methation by the above-mentioned mechanism is large.

Dissolved oxygen in water is held in multiple water molecule groups, and when it grows from an ice crystal nucleus to an ice crystal during freezing, this water molecule group receives the free energy of the surface of the ice crystal. Some of the water molecules release the intermolecular bonds through hydrogen bonds with other water molecules, and are released, and energy is exchanged on the surface of the ice crystal to refreeze on the ice crystal surface. As a result, dissolved oxygen is released from the water molecule groups and drifts and diffuses as free oxygen between the water molecule groups. As a result, free oxygen binds to myoglobin existing between cells, or diffuses into the cell through the cell membrane and binds to myoglobin in the cell to cause metation.

Detailed description will next be made about the refrigeration device that is required when using the cryopreservation method of the present invention.

3) Prior art related to refrigeration system Generally, a widely used cooling system cools an object by heat of vaporization when a refrigerant changes from a liquid phase to a gas phase in a heat cycle involving a liquid phase and a gas phase. To do. Refrigerators using such a vapor compression refrigeration cycle are widely used from small devices to large devices.

1A and 1B show the configuration and operation outline of a typical liquid cooling device. The gas circuit forming the refrigeration cycle comprises a compressor, an expansion valve or a capillary tube, a condenser for transferring heat and an evaporator or a refrigeration pipe.

The compressor compresses a gas having a large latent heat of vaporization such as ammonia gas, carbon dioxide gas, and chlorofluorocarbon, which are refrigerants in the gas circuit. After the compression, it becomes high-temperature high-pressure gas, and the heat generated in the condenser is transferred to low-temperature water or air. As a result, the high-temperature high-pressure gas is cooled to a low temperature and condensed into a high-pressure liquid. On the other hand, low-temperature water or air becomes high-temperature water or high-temperature air, and is returned to a reservoir or the atmosphere as waste heat. The high pressure liquid in the gas circuit is opened to a low pressure by the expansion valve or the capillary tube to become a low pressure liquid. Furthermore, the low-pressure liquid is vaporized and converted into low-pressure gas. When it changes, heat is absorbed and the temperature of the secondary refrigerant is lowered via the evaporator (FIG. 1A). Alternatively, in the freezing pipe, the primary refrigerant changes from low-pressure liquid to low-temperature low-pressure gas to lower the temperature of the object to be frozen (FIG. 1B).

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

The compressor generates large vibrations from its operation. Especially, in the reciprocating pump, the vibration is remarkable because the reciprocating motion causes the compression operation. Although it is possible to reduce the vibration of the compressor by using the rotary pump, the vibration reduction of the compressor is limited because it is not a structurally rigid mechanism.

The vibration generated in the compressor is transmitted to the blood pack via the refrigerant and the cooling liquid. As long as the refrigerant or the cooling liquid is used to freeze the blood pack, it is inevitable that vibration is transmitted to the blood pack through these.

4) Problems of the related art related to refrigeration equipment As a method of reducing the transmission of vibrations via the refrigerant or the cooling liquid, the distance from the refrigerator to the object to be frozen is increased to attenuate the vibrations transmitted through the refrigerant or the cooling liquid. There is a method, and a method in which bubbles are mixed in the middle of the transfer path in the refrigerant or the cooling liquid to damp the vibration.

However, increasing the distance from the refrigerator to the object to be refrigerated lengthens the transfer path in the refrigerant or cooling liquid, during which the low temperature of the refrigerant or cooling liquid escapes to the outside world and A decline in ability 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.

JP, 2014-214911, A

"Freezing Red Blood Cells," Takeshi Miura, Blood and Blood Vessels, Vol. 1, No. 9, November 1972 "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 red blood cells under pressure" "Control of ice crystal morphology in frozen pharmaceuticals -by controlling nucleation using ultrasonic waves-" (Hyogo Prefectural Carpenter) T. Nakagawa, Mitsutaka Kitamura (Lyon Daiichi University) A. Hottot, S.H. Vessot, J. et al. Andrieu, SCEJ, 38th Autumn Meeting (Fukuoka, 2006), C202 "Mechanism of Hemolysis by Freezing at Temperatures Near Freezing I: Morphological Changes during Freezing and Thawing", Yoshio Nei, Low Temperature Science, Biology, December 25, 1967. "Study on the recrystallization behavior of ice crystals in frozen foods", Tomoaki Ogiwara, Journal of the Food Engineering Society of Japan, Vol. 11, No. 4, p169-175, December 2010. "Anxiety of morphology in ice crystal growth-ISS "Kibo" experiment-", Hokkaido University Yoshizumi Furukawa, Gakushuin University Etsuro Yokoyama, JAXA Izumi Yoshizaki, Shinichi Yoda, Tetsuo Tanaka, JSF Shimataro, JAMSS Takehiko Sone, IHI Aerospace. Tomoyuki Tomobe, Space Utility. Res. , 26 (2010)

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

Freezing preservation of blood is performed for long-term preservation or semi-permanent preservation of blood. However, the problem is that hemolysis occurs after thawing. On the other hand, there is a method in which additives are added to blood and frozen storage is performed without freezing, but there is a problem that a blood washing step after storage is required.

Therefore, a blood cryopreservation method that suppresses hemolysis without using additives is the main problem to be solved by the present invention. Mechanisms and causes of hemolysis that accompany freezing of blood include salt-induced hemolysis, intracellular-induced hemolysis, cell-membrane-induced hemolysis, and extracellular-induced hemolysis.
On the other hand, the main problem to be solved by the present invention in the method for freezing and preserving the freshness of fresh fish is to cause a large amount of drip from the fish meat after thawing and to cause brown discoloration of the fish meat during frozen storage. 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.

There are the following problems and solutions in the blood cryopreservation method that suppresses hemolysis.
(A) Prevention of salt-damaged hemolysis Prevent the extracellular solution from entering from the outside by diffusion.
(A) Intracellular hemolysis Controls freezing of water in cells and suppresses cell expansion due to freezing to the allowable volume expansion of red blood cells.
(C) Hemolysis caused by cell membranes Do not damage the cell membranes by keeping the ice crystal size to 3 to 4 microns by rapid freezing.
(D) Prevention of extracellular cause hemolysis Ice crystals outside the red blood cells cause freezing that maintains the gaps. Therefore, ice crystals are recrystallized. This prevents the red blood cells from being destroyed by the pressure of the ice crystals. The recrystallization process is the result of the phenomenon of reducing the surface area of crystals. For that purpose, the freezing start temperature is raised, the growth rate of the frozen surface is slowed, and the temperature gradient inside the ice crystals is made small.

Regarding fresh fish meat, there are the following solutions to the problem that a large amount of drip is generated from the meat after thawing and the problem that the fish meat turns brown 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 nucleation generation as small as possible.
(F) Brown alteration Maintaining the grouping of water molecules while retaining dissolved oxygen among multiple water molecule groups, and recrystallizing on the ice crystal surface even when growing from ice crystal nuclei to ice crystals during freezing Suppress it. The suppression of this recrystallization suppresses the generation of free oxygen and prevents the myoglybin from becoming meth.

Next, the problems to be solved by the invention relating to the refrigeration system 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 the fresh fish and to prevent the growth of ice crystal nuclei even if they occur. One of the main causes of the freezing of supercooled water is that vibration is added to the supercooled water. That is, since it is necessary to lower the temperature of the blood pack or the fresh fish without superfreezing 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 ultra-low vibration in 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 in which the cooling liquid for cooling the blood pack or the fresh fish or the refrigerant at the end circulates.

Vibrations transmitted through the cooling liquid or the refrigerant are longitudinal waves. Therefore, as a method for preventing vibration transmission, the vibration transmission path is divided into two, the length of one transmission path is increased, and the difference between the two transmission path lengths is set to half the cycle of the vibration or vibration As a half of the wavelength (half wavelength), there is a method of smoothing the vibration or canceling the peaks and troughs of the vibration by interference. Longitudinal waves in a liquid medium such as a cooling liquid or a refrigerant are difficult to attenuate and have a long coherence length. Therefore, it is a very effective method to divide the longitudinal wave into two and to eliminate the longitudinal wave by interference so that one phase has a half-wavelength phase difference with respect to the other phase. Here, for simplification, the period of vibration and the smoothing of vibration are also represented by the terms of wavelength of vibration and interference of vibration.

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

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

The means used in the method for preventing hemolysis in the present invention rapidly cools blood to a temperature (first cooling temperature) at which a cause of salt-damaged hemolysis and intracellular hemolysis is not generated (hereinafter referred to as "first cooling process"). .. After that, 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 (elapsed temperature) that is maintained at the temperature which is slow cooling or step cooling and continues step by step. (Maintenance period) Provided by stage cooling. 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 more. The second cooling temperature is -7 to -13°C. When slow cooling is adopted 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 cooling during this period. For each step cooling from -0.5°C to -1.5°C, select 0.5 hour or 0.5 hour of step cooling temperature x 0.5 hour, whichever is longer, and select a period longer than that. Is set as the elapsed temperature maintenance period. The second cooling process is performed by placing the container containing blood in a low vibration environment.

Here, the blood may be whole blood, RCC, RCC-LR, or RC-M. A. P. Concentrated red blood cells such as (Red Cell Concentrates Mannitol Adenine Photoshop, or simply referred to as “MAP” hereinafter) may be used.

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 on the order of 6 to 8.5 microns in diameter. Ice crystal nuclei form at about -3°C, which is the freezing temperature. The ice nuclei remain at most 3 to 4 microns in diameter. However, when supercooled, the temperature at which ice crystal nuclei are formed decreases. For example, when rapid freezing is performed at a cooling rate of -10 ² °C/min or more, the temperature reached by the supercooling becomes the temperature at which the formation of ice crystal nuclei starts. In the supercooled state, the smaller the temperature at which the formation of ice crystal nuclei starts is, the smaller the ice crystal size becomes (Non-Patent Document 3). The first cooling temperature is in the range of -4°C to -6°C. Therefore, the intracellular ice crystal size is smaller than 3 microns. That is, the ice crystal size is much smaller than the size of red blood cells, so that hemolysis due to intracellular causes of red blood cells does not occur.

Since erythrocytes are rapidly frozen from the outside, the temperature inside the ice crystal nuclei is higher than the heat generated by ice crystal formation and the surface temperature of the supercooled ice crystals. Therefore, the water molecules existing on the crystal surface maintain energy equilibrium with the molecules inside the crystal, so that the molecule keeps a moving state and the phenomenon of reducing the surface area of the crystal, that is, the growth of ice crystals is difficult to occur. Further, a part of the water content in the red blood cells becomes ice crystals, and the concentration of the solute in the red blood cells rises and the freezing point of the solute decreases accordingly.

The increase in the concentration and the change in the solidification temperature are roughly proportional. Therefore, in the first cooling process, unless the first cooling temperature, which is the ultimate temperature, is extremely low, ice crystals are hard to 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 red blood cells even after a long time has passed. Therefore, the salt concentration of the solution in the red blood cells does not rise extremely, and the water content of the external plasma does not diffuse to the inside of the red blood cells due to the osmotic pressure and destroys the cell membrane of the red blood cells. That is, salt damage does not occur.

On the other hand, the red blood cells are rapidly cooled from the so-called maximum ice crystal production zone of -1°C to the lower limit temperature thereof until the first cooling temperature is reached. The lower limit temperature of the maximum ice crystal production zone is said to be -5°C, but this is the case when it is close to pure water, and in the case of water containing an electrolyte such as red blood cells, the lower limit temperature may be -4°C. That is a guide. As for the cooling time, when the blood pack is small and the internal heat transfer is sufficiently ensured, a considerable effect can be obtained by making the passage time of the maximum ice crystal production zone shorter than 30 minutes. .. On the other hand, even with a transit time of more than 1 hour or more, the hemolysis-suppressing cryopreservation method which is the object of the present invention is possible unless it is an extremely long time.

の関係がある（非特許文献（３）。ａは、実際の氷晶サイズと数式１の理論値を合わせるフィティングパラメータである。このパラメータ値としては、例えば−３°Ｃで−０．２°Ｃ／分の冷却速度では、Ｌ^＊＝１６０ミクロンとなる。この数式１から分かることは、このような特徴的な冷却過程では、他の冷却過程に比べて相対的に氷晶サイズは大きくなることである。 [Second cooling process]
The outside of the red blood cells is dominated by plasma. Ice crystal nuclei are formed in plasma at about -3°C. At the first cooling temperature, ice crystal nuclei start to grow in plasma. However, since the cooling process is slow in the second cooling process and the environment is low in vibration, the growth of ice crystals is also specific. The characteristics are 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 ^* is, with respect to 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 for matching the actual ice crystal size with the theoretical value of Equation 1. The parameter value is, for example, −0.2 at −3° C. At a cooling rate of °C/min, L ^* = 160 microns, which can be seen from Equation 1 that in such a characteristic cooling process, the ice crystal size is relatively large compared to other cooling processes. Is to be.

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

Thus, when the number of molecules is reduced, the molecules move spontaneously and a 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 a smoothing of ice crystals. As a result, in the freezing of blood plasma, that is, outside of red blood cells, ice crystal nuclei grow slowly and large, and the void size between ice crystals becomes larger than that in the case where the recrystallization process does not occur. Between the first cooling temperature and the second cooling temperature, the ice crystal part growing in the plasma and the solution part in the liquid state coexist.

In such ice crystal growth, the ice crystal part is in a state of containing voids filled with the solution part. When ice crystals grow, the solution concentration of plasma increases and the relative osmotic pressure on blood cells weakens. Further, with respect to the void size, the growth of ice crystals slows down due to the increase of the solution concentration, and the decrease of the solution portion (that is, the reduction of the size of the solution void) is suppressed. Thus, erythrocytes remain in the solution voids and do not cause crushing of erythrocytes by ice crystals, so that hemolysis due to erythrocyte extracellular causes is less likely to occur.

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

The above cooling process has been described for whole blood. However, in MAP, the freezing point is almost the same as that of whole blood, and the osmotic pressure on red blood cells is no different from that of whole blood. Therefore, in MAP as well, the phenomenon observed 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, the means for solving the problems will be described in detail for the method of frozen storage of fish while maintaining the freshness of fresh fish.

In the means used in the method of the present invention for suppressing the occurrence of drip during thawing of fish meat and suppressing browning of fish meat, rapid cooling (hereinafter referred to as “first cooling temperature”) to a first freezing temperature (first cooling temperature) is used. "Cooling process"). After that, cooling until reaching a second cooling temperature lower than the first cooling temperature (hereinafter, referred to as “second cooling process”) is slowly cooled, or step cooling and a certain period of time during which the temperature is maintained following each step (elapsed time). The temperature is maintained during two-stage cooling.

[First cooling process]
Fish meat begins to freeze at around -1°C. After that, for example, when it is rapidly frozen at a cooling rate of -10 ² °C/min or more, the reached temperature of the supercooling becomes the temperature at which formation of ice crystal nuclei starts. However, after that, if the fish meat is left for a while without being vibrated, the entire temperature of the fish meat will reach its ultimate temperature without starting to freeze. The water in fish meat, that is, the water in the intracellular fluid or the extracellular fluid, is dispersed in these fluids in order to maintain a thermal equilibrium state with cellular proteins, lipids and interstitial fluid rather than forming ice crystal nuclei. Therefore, in the first cooling temperature range of -4°C to -6°C, the fish meat is supercooled without freezing.

On the other hand, before reaching the first cooling temperature, the fish meat is rapidly cooled from the so-called maximum ice crystal formation zone of -1°C to the lower limit temperature of the electrolyte of the fish meat cells or the intercellular fluid of -4°C. As for the cooling time, when the fish meat is small and internal heat transfer is sufficiently ensured, the passage time of the maximum ice crystal production zone can be easily shorter than 30 minutes, and freezing can be avoided. On the other hand, in the case of a large fish, internal heat transfer is not sufficiently ensured, and therefore it may be necessary to cool by a contact method or the like in order to shorten the cooling passage time. However, in the case of small or large fish meat, even if the passage time exceeds 30 minutes, the fresh fish frozen storage method which is the object of the present invention is possible unless the time is extremely long.

１０ミクロンメータであることより、魚肉内にランダムに発生した氷晶核から細胞内外の水分は一気に氷結する。そのため、氷晶核は細胞内外にいたるところに発生し、かつ熱平衡状態を保つため、氷晶核は実際には成長することはほとんどない。 [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, the second cooling process is started. The second cooling process is a cooling process that is slower toward the second cooling temperature lower than the first cooling temperature and is in an environment of low vibration, so it is difficult to start freezing. That is, water in fish meat, that is, water in intracellular fluid or extracellular fluid, maintains thermal equilibrium with cellular proteins, lipids, and interstitial fluid, and does not start freezing with these as nuclei. But I

Since it is 10 micrometers, the water inside and outside the cells freezes at once from the ice crystal nuclei randomly generated in the fish meat. Therefore, ice crystal nuclei are generated everywhere inside and outside the cell, and the thermal equilibrium state is maintained, so that the ice crystal nuclei hardly actually grow.

にくくなる。氷晶粒の直径が氷晶成長速度の平方根に反比例すること（非特許文献３）から過冷却が進むに連れて氷晶核の成長はますます生じにくくなる。 In many cases, icing will occur immediately even after cooling from the first cooling temperature to the second cooling temperature.

It gets harder. 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 less likely to occur as supercooling proceeds.

As a result, the phenomenon that the expansion of the outer size due to the growth of ice crystals in the cells destroys the cell membrane is unlikely to occur. On the other hand, breakage of the cell membrane from the outside due to freezing and growth of the intercellular fluid is also unlikely to occur. The fact that cell destruction is unlikely to occur is that even if fish meat is frozen in this freezing process and then thawed, the ice crystals melt in the space occupied by the original ice crystals, forming a space for water. A so-called typical phenomenon of drip, which is a drip that exudes from a piece of fish meat, is unlikely to occur. This is because intracellular ice crystals do not grow much and return to intracellular fluid immediately after thawing, and ice crystals generated between cells also do not grow greatly and return to intercellular fluid immediately after thawing. That is, the occurrence of drip is 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 high and the limit temperature of the supercooling is reached, the fish meat is randomly distributed in the fish meat. The water inside and outside the cell freezes at once from the ice crystal nuclei generated in the. At this time, ice crystals are generated everywhere inside and outside the cells, and a thermal equilibrium state is maintained, so that the ice crystals hardly grow. Therefore, the oxygen trapped in the group of water molecules is hardly released and is also prevented from binding to oxymyoglobin or deoxymyoglobin in fish meat to be met.

In this way, in a low-vibration environment, after supercooling has proceeded as much as possible, the water content in the fish meat is frozen at a stretch to suppress cell destruction and methemolysis, and freshness is not lost even when thawed after frozen storage. It enables frozen storage of fish meat.
On the other hand, when the environment is not low vibration, before the supercooling temperature rises, that is, before the supercooling reaches the limit temperature, vibration causes ice crystal nuclei to form in the water in the fish meat and ice crystal growth starts. .. At this time, since the supercooling temperature is small, the growth rate of the ice crystals is slow, as can be seen from the mathematical formula 2, and cell destruction due to ice crystal growth and further metation due to free oxygen generated by the breaking of hydrogen bonds of grouped water molecules occur. ..

When step cooling is adopted in the second freezing process, the cooling temperature is maintained for a long time (0..C) every time the cooling temperature is slightly lowered (step from -0.5°C to -1.5°C). An over temperature maintaining period of 5 hours or a value expressed in degrees Celsius of the step cooling temperature×0.5 hour, whichever is longer, is provided. The rate of progress of the cooling temperature in each step is small, and like in the continuous freezing process, the water in fish meat, that is, the water in the intracellular fluid or extracellular fluid, is in thermal equilibrium with the cellular proteins, lipids and interstitial fluid. In order to keep it, these do not start freezing with these as nuclei. Therefore, supercooling can be performed. As a result, it is possible to prevent cell destruction and methoteration, and to freeze-preserve fish meat without losing freshness even after thawing after frozen storage.

Freezing is likely to occur when the cooling step progresses. This is because the thermal equilibrium state between the water content of the intracellular fluid or the extracellular fluid and the cellular proteins, lipids and interstitial fluid is likely to collapse at the moment when the step advances.

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

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

The limit value required 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 that after the first cooling temperature (−4° C.) is reached and before the second cooling temperature is reached, freezing is not started as much as possible and supercooling is continued. The limit value is phenomenologically a limit value of vibration in which water molecules do not freeze or freeze due to vibration. Water molecules freeze when hydrogen atoms forming water molecules form hydrogen bonds with hydrogen atoms forming other water molecules to form a hexagonal crystal system.

The hydrogen bond is formed or broken when the water molecule has energy transfer corresponding to the heat of melting of ice in macroscopic view. Its energy is calculated from the formation of ice nuclei when the water is supercooled. It is considered that the generation of ice crystal nuclei starts when 10,000 ice crystal nuclei having a diameter of 1 micron are formed per cubic centimeter. The energy corresponding to the heat of fusion per gram of water is 333 Joules, and the crystal growth rate at the supercooling temperature of −4° C., which is the first cooling temperature, is 3.29 cm/sec from Equation 2, Energy transfer of 5.73 microwatts per square centimeter is required. This is 284 Pascal when converted into underwater sound pressure, and the sound pressure level is 169 dB. When a vibration larger than this is applied, generation of ice crystal nuclei starts at the first cooling temperature, and it becomes impossible to continue freezing in the supercooled state. Therefore, the limit sound pressure level of the blood pack or fish meat or packed fish meat during freezing of the present invention is 169 dB.

On the other hand, the specific gravity and sonic velocity of the refrigerant are almost the same as those of water. Therefore, the limit vibration sound pressure in the refrigerant is almost the same as the above limit sound pressure level.

On the other hand, in order to break the bond of the hydrogen atom in the water molecule group, the hydrogen bond can be broken by separating one of the two bonded hydrogen atoms by the van der Waals radius. Where the melting temperature of the energetic ice is 6 kilojoules per mole, the hydrogen bonds of water are 10 to 40 kilojoules. This value is 555 to 22220 Joules per gram of water, which is higher than the melting temperature of ice. Therefore, as long as the above-mentioned limit vibration sound pressure is maintained, the water molecule group does not destroy its grouping, and thus, free oxygen is not generated. As a result, the methation of fish meat cells does not occur.

From this point as well, in order to maintain the freshness of the 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, freezing of blood packs will be described, but the same applies to freezing of fish meat and the like unless otherwise specified.

The cooling device cools the object with the heat of vaporization when the refrigerant is cycled between the liquid phase and the gas phase to change from the liquid phase to the gas phase. 1A and 1B show the configuration and operation outline of a typical liquid cooling device. The gas circuit forming the refrigeration cycle comprises a compressor, an expansion valve or a capillary tube, a condenser for transferring heat and an evaporator or a refrigeration pipe. The compressor compresses a gas having a large latent heat of vaporization such as ammonia gas, carbon dioxide gas, and chlorofluorocarbon, which are refrigerants in the gas circuit. After the compression, it becomes high-temperature high-pressure gas, and the heat generated in the condenser is transferred to air or water.

As a result, the high-temperature high-pressure gas is cooled to a low temperature and condensed into a high-pressure liquid. The high pressure liquid is released to a low pressure by an expansion valve or a capillary tube to become a low pressure liquid. Furthermore, the low-pressure liquid vaporizes and replaces the low-pressure gas. When it changes, heat is absorbed and the temperature of the secondary refrigerant is lowered via the evaporator (FIG. 1A). Alternatively, in the freezing pipe, the primary refrigerant changes from low-pressure liquid to low-temperature low-pressure gas to lower the temperature of the object to be frozen (FIG. 1B). In the present invention, the object to be frozen is the cooling liquid in the cooling tank.

The compressor generates a large vibration from its operation, and the vibration is transmitted to the blood pack via 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 in which the cooling liquid for cooling the blood pack or the refrigerant at the end circulates.

Vibrations transmitted through the cooling liquid or the refrigerant are longitudinal waves. Therefore, as a vibration transmission prevention method, the vibration transmission path is divided into two, one of the transmission paths is lengthened, and the difference between the two transmission paths is determined by half the wavelength of the vibration (half wavelength ), there is a method of canceling the peaks and troughs of vibration by interference.

The reason why the vibration can be canceled by such a method 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/sec, if the vibration of the compressor is, for example, 60 Hertz, the half-wavelength is 12.5 m, and in order to eliminate the vibration due to interference, cool the long path difference. Must be provided in the circuit.

Therefore, in the vibration transmission preventing device according to the present invention, a physical delay unit is used that substantially lengthens the vibration transmission distance by multiple reflection to increase the delay phase, and a rectifying device that uses a diffraction hole for diffusing the vibration. It is possible to prevent vibration transmission with an extremely short length.

ここで、

here,

動伝達速度は音波の伝達速度と同じであって約１５００ｍ／秒、圧縮機の振動が例えば６０ヘルツの場合、半波長となる距離は約１２．５ｍとなり、長い経路差を冷却回路内に設けなければならい。逆に言うなら、たとえば１ｍの経路長では、０．０４ラジアンの移相

The dynamic transmission speed is the same as the transmission speed of sound waves and is about 1500 m/sec. When the vibration of the compressor is, for example, 60 Hertz, the half wavelength distance is about 12.5 m, and a long path difference is provided in the cooling circuit. I have to. Conversely, for example, with a path length of 1 m, a phase shift of 0.04 radians

When four stages of devices using such multiple reflection are connected in series, a delay amount of 0.72 radian×4 occurs. This delay is 0.46 wavelength, and it is possible to divide the vibration wave source into two, generate two vibration waves, and cancel the original vibration wave by mutual interference of these two vibration waves. In order to properly adjust the delay amount to the half-wavelength, between the vibration reflection plate 1 and the vibration reflection plate 2,

In this way, the cooling device can be downsized by the delay device using the multiple reflection, and a cooling device with an ultra-low vibration of a practical size can be realized.

Even if the original vibration wave is canceled by the interference between the two vibration waves, the vibration may appear again depending on the propagation directions of the two vibration waves after the interference. This phenomenon is likely to appear at higher harmonics of the vibration wave in question. In order to prevent this, after 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 it through the diffraction hole. In practice, a rectifying device for diffusing this vibration is provided downstream of the delay device. As a result, the vibration is eliminated and the other state is maintained anywhere in the propagation path downstream of the particle size control device.

Blood cryopreserved by the method of the present invention hardly causes 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 rare blood group person can easily carry out autologous blood storage during normal times to prepare 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. which is usually used without using expensive refrigeration equipment that is frozen and stored at −40° C. or less, which is used for high-grade fish. The freshness can be maintained. At that time, after reaching the second cooling temperature, the frozen fish meat is frozen and stored at -18°C.

Further, when the above-mentioned frozen storage method is used, it is necessary to put the blood pack in a low vibration environment, but by using the vibration transmission preventing device according to the present invention, the liquid refrigerator can be downsized. Since the blood pack is small, the use of the present cryopreservation method has a great practical effect that the entire apparatus can be downsized, and a benefit thereof.

FIG. 1A is a conceptual diagram showing a refrigeration cycle of a refrigerator that uses 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 diagram showing a vibration isolator including a horizontal bar or a rail and a string attached to a blood pack. FIG. 2B is a diagram showing a vibration isolator including a vibration isolation table, 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 unfrozen raw bluefin tuna meat. FIG. 3B is a photograph showing muscle fiber cells of bluefin tuna meat that was thawed after being frozen and stored through the freezing process of Example 3. FIG. 3C is a photograph showing the muscle fiber cells of bluefin tuna meat that has been thawed after being cryopreserved through a normal slow freezing method. FIG. 4A is a photograph showing the appearance of unfrozen raw bluefin tuna meat. FIG. 4B is a photograph showing the appearance of bluefin tuna meat that was thawed after being frozen and stored through the freezing process of Example 3. FIG. 4C is a photograph showing the appearance of bluefin tuna meat that has been thawed after being 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 reflection plate 1 and the vibration reflection plate 2. FIG. 6A is an external view of the vibration removing module. FIG. 6B is an internal cross-sectional view of the vibration removing module. FIG. 6C is an internal cross-sectional view of the vibration removing module when the baffle is used. FIG. 6D is an internal cross-sectional view of the vibration removing module when the vibration reflecting plate is used. FIG. 7A is a diagram showing a main part of a phase difference generating circuit in which the vibration removing module is directly arranged in parallel with the transfer pipe. FIG. 7B is a diagram showing a main part of the phase difference generating circuit in which the vibration removing module is directly arranged vertically in the transfer pipe. FIG. 7C is an external view of a simple merger. FIG. 8A is an external view of another combiner that joins the flows of the refrigerant or the cooling liquid. FIG. 8B shows an internal cross-sectional view of another merger. FIG. 9 shows the pressure drop for the angle of expansion in a gently expanding tube. FIG. 10A is a schematic view of a refrigerator in which a vibration removing device is installed in a circulation circuit of a cooling liquid for use. FIG. 10B is a schematic view of a refrigerator in which the vibration elimination device is installed in the circulation circuit of the primary refrigerant and used.

An example of freezing the collected blood by the method of the present invention will be described below.

The present embodiment is an embodiment of the cooling process when slow cooling is adopted.

After collecting blood, the blood is injected into a sterilized container (pack), the blood is filled in the container, and then the injection port is sealed in a state where the air phase is not included. The material of the pack is polyvinyl chloride (PVC) or polyvinyl chloride using polyolefin (TO-2-(tris - 2 - ethylhexyl trimellitate)) instead of DEHP (di - 2 - ethylhexyl phthalate) which is a plasticizer thereof, and polyolefin. Use polyethylene, polybutadiene, or a material similar to these. Hereinafter, such a container filled with blood will be referred to as a blood pack.

Alternatively, whole blood in a pack already sold as a blood pack may be used. All of these are called blood packs.

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

It is desirable that the liquid refrigerator be capable of cooling at a low temperature of about -18°C as a 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 or embrittlement temperature of the blood pack. Further, it is desirable to have a temperature control mechanism that can change the set value of the cooling temperature with time.

In the cooling tank of the liquid refrigerator, ethanol is circulated as a secondary refrigerant that directly cools the blood pack. The temperature of the liquid refrigerator is set to the first cooling temperature in the range of -4°C to -6°C, and the liquid refrigerator is left to stand until the ethanol reaches the first cooling temperature. Then, in order to perform cooling in a low vibration state, the compression operation 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 the 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 if the temperature of ethanol temporarily rises, enough ethanol is stored in the cooling tank of the liquid refrigerator and circulated so that it maintains a sufficient heat capacity for the blood pack so that it does not exceed -4°C. Need to be However, the blood after thawing, which sets the first cooling temperature to a temperature lower than −6° C. in anticipation of the temperature increase, causes hemolysis.

After that, the blood pack is allowed to stand in the cooling tank for about 5 minutes or longer. 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 choice of the second cooling temperature affects the shelf life 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 as to temporally have a temperature change in any of the above ranges. During the slow cooling period, cooling is performed in a low vibration state, so that the compression operation of the liquid refrigerator is set to the minimum operation mode. If it is still not possible to create a sufficiently low vibration state, use the anti-vibration means described later.

The blood pack is slowly cooled and maintained at 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 endurance temperature or embrittlement temperature of the material of the blood pack, -30°C. The temperature may be maintained and the temperature may be moved to another refrigeration system. The second cooling temperature or lower temperature and the temperature up to −30° C. which is the cold resistance limit temperature or the embrittlement temperature of the material of the blood pack are called the storage temperature. In this series of processes, the cryopreservation step of the blood pack is completed, and the remaining storage temperature is maintained and the blood pack is simply stored.

As shown in FIG. 2A, the above-mentioned vibration isolator comprises a horizontal bar or rail 105 installed above the cooling liquid circulation tank 101, which is a cooling tank of the liquid refrigerator, and a hanging string 104 hung on the blood pack 103. There is a means. By this means, the vibration of the liquid refrigerator mainly generated by the compressor is not transmitted to the blood pack 103, and the blood pack 103 can be cooled in a low vibration state. The hanging strap 104 may be a hole formed in the mating portion of the bag of the upper part of the blood pack 103, or the entire blood pack 103 may be bound by the hanging strap 104.

Further, as another vibration isolating means, as shown in FIG. 2B, a container 106 for putting a vibration isolation table 108 and a blood pack 103 having a vibration isolation effect in the cooling liquid circulation tank 101, and an upper part of the container 106 are covered. There is a container lid 107. The vibration-proof table 108 is for making it difficult for the vibration generated by the compressor of the liquid refrigerator to be transmitted to the container 106 through the cooling liquid circulation tank 101, and is made of plastic such as vinyl chloride, silicone rubber or foamed plastic. The container 106 is not floated from the bottom surface of the cooling liquid circulation tank 101 and is not in direct contact with the bottom surface.

On the other hand, the container 106 is intended to isolate the vibration of the compressor transmitted through the cooling liquid 102. The material of the container 106 has high thermal conductivity for quickly transmitting the temperature of the cooling liquid 102, and has a large acoustic impedance different from that of the cooling liquid 102 in order to reflect and block vibrations transmitted through the cooling liquid 102. is important. For that purpose, a metal such as aluminum is preferable as the material of the container 106. The material of the container lid 107 is preferably metal such as aluminum for the same reason.

Although providing the container lid 107 on the container 106 has a vibration-proof effect, 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. Therefore, it is not used except when the blood pack 103 needs to be placed in an ultra-low vibration state.

In the low vibration state, the sound pressure limit value is 284 Pascal. Therefore, it is desirable that the oscillating pressure of the vibration transmitted to the blood pack be equal to or lower than the sound pressure limit value. If the vibration transmitted to the blood pack is larger than these values, collision of external water molecules on the surface of the ice crystal and subsequent freeze fixation are likely to occur, resulting in the formation of large ice crystals, which causes hemolysis during thawing. Loss 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 in the cooling process up to the first cooling temperature and even after the second cooling temperature is reached, cooling is performed. May be maintained in the process.

This embodiment is an embodiment of a cooling process when step cooling is adopted. Here, the manufacture 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 allowed to stand for a time longer than about 5 minutes are the same as those 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 value of the step cooling temperature displayed in degrees Celsius×0.5 hour, whichever is longer.

Thereafter, the cooling temperature of the liquid refrigerator is further lowered by the above step temperature, and the blood pack 103 is kept in the liquid refrigerator for 0.5 hour or 0.5 hour of the step cooling temperature indicated by 0.5 degree, whichever is longer. Leave it on. By repeating this, the second cooling temperature is reached. When the absolute value of 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 final step temperature is defined as the temperature difference and the second cooling temperature. After reaching, the blood pack 103 is left in the liquid refrigerator for a time longer than the value of the last step cooling temperature displayed in degrees Celsius×0.5 hours.

When the cooling temperature of the liquid refrigerator is lowered for each step temperature, the temperature of the cooling liquid circulation tank 101 is lowered by the step temperature for a while, so that the compressor in the liquid refrigerator operates strongly and the blood pack 103 is left unattended. The vibration of the compressor is transmitted to the cooled 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 placed in 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 container with the container lid 107.

When it is left as it is, the container lid 107 is removed. While the container lid 107 may be left in the left-standing state, the left-standing time is made 2 to 3 times longer than the above-mentioned time so that the temperature of the cooling liquid 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 required to maintain the second cooling temperature, the blood pack is at the second cooling temperature or lower, and the cold resistance limit temperature or the embrittlement temperature of the material of the blood pack is -30°. It may be moved to another refrigeration system while maintaining the storage temperature up to C. In this series of steps, the cryopreservation process of the blood pack is completed, and the rest of the process is simply maintained while maintaining the storage temperature.

The low vibration environment during freezing is preferably the same as that in the first embodiment. Also, when applied, it 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 an air layer, and placed in the container 106 of the cooling liquid circulation tank 101 shown in FIG. 2A. It was frozen in the freezing process used in 2 and then stored frozen at -18°C. After carrying out the same continuous cooling as in Example 1, it was stored at -18°C for 1 week and then thawed. The results are shown in FIGS. 3A, 3B, 3C and 4A, 4B, 4C. 3A, 3B, and 3C show muscle fiber cells of bluefin tuna meat, which are unfrozen raw bluefin tuna meat, the above-mentioned thawed bluefin tuna meat, and thawed bluefin tuna meat that has undergone the normal slow freezing method, respectively.

As a result, the bluefin tuna meat of this example has almost no cell destruction similar to that of raw meat that is not frozen. On the other hand, it can be seen that in the conventional slow freezing method (FIG. 3C), muscle fiber cells are destroyed in various places and water is accumulated in the intracellular ice marks. FIG. 4A, FIG. 4B, and FIG. 4C show the overall appearance of bluefin tuna meat, that is, unfrozen raw bluefin tuna meat, the above-mentioned thawed bluefin tuna meat, and the thawed bluefin tuna meat that has undergone the usual slow freezing method. Compared with the non-frozen raw bluefin tuna meat and the thawed bluefin tuna meat that has undergone the usual slow freezing method, the thawed bluefin tuna meat of this example has a slight browning change 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 species of bluefin tuna.

In addition to the above-mentioned polyvinyl chloride, polyolefin, polyethylene, polybutadiene, etc., polyvinylidene chloride may be used for the plastic film for packaging and the plastic bag for packaging, or a flexible and malleable material may be used.

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

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

The low vibration environment during freezing is preferably the same as that in the first embodiment. If the vibration is larger than this, collision of external water molecules on the surface of the ice crystal and subsequent freeze fixation are likely to occur, and as a result, large ice crystals are formed, which promotes met formation during freezing and drip occurs during thawing.
Also, when applied, it may be the same as in the first embodiment.

In the above-mentioned first and second embodiments, in order to prevent the vibration of the liquid cooling device from being applied to the blood pack being cooled, two means and the use thereof, that is, the case where the blood pack 103 is hung from above by the hanging string 104 ( 2A) and a case in which the vibration isolation table 108, the container 106 into which the blood pack 103 is inserted, and the container lid 107 that covers the upper part of the container 106 are used in combination (the case illustrated in FIG. 2B). ing. However, these are positive to the mechanism in which the vibration generated by the compressor, which is the main vibration source of the liquid cooling device, is transmitted to the fish meat contained in the blood pack, the plastic film for packaging, or the plastic bag for packaging through the secondary refrigerant. Does not give a good solution.

In this embodiment, as positive vibration damping means, vibration removal having a function of preventing vibration generated by the compressor from being transmitted to the primary refrigerant and further to ethanol or ethylene glycol in the cooling tank which is the secondary refrigerant. Specific examples of the apparatus will be described. The vibration eliminator is installed and used in the middle of the circuit of the primary refrigerant. By the vibration removing device, the vibration generated by the compressor, which is the main vibration source of the liquid cooling device, is blocked and is not transmitted to the fish meat contained in the blood pack or the plastic film for packaging. By operating this vibration removing device in cooperation with any liquid cooling device using a compressor, it is possible to realize a refrigeration system in which vibration is hardly transmitted to the refrigerant.

When the refrigerant that causes a phase change between gas and liquid cools the primary cooling liquid such as ethanol or ethylene glycol in the cooling tank, this vibration transmission prevention device is installed in the circulation circuit of the primary cooling liquid.

6A and 6B show a vibration eliminating module 10 which constitutes a part or the whole of the vibration eliminating apparatus according to the present embodiment. FIG. 6A shows the external appearance of the vibration removal module 10, and FIG. 6B shows its internal cross section. The vibration eliminator element 10 comprises 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 removing module 10 have a cylindrical shape, and their inner diameters are d1, d2 and d3, respectively. The transfer of the primary refrigerant or the secondary refrigerant is an order of movement in which these refrigerants enter from the input tube 1 and go out from the output tube 3 via the multiple reflection tube 2. The longitudinal oscillating wave in the refrigerant is transmitted from the input tube 1 to the multiple reflection tube 2, where multiple reflections occur.

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

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

When the internal structure of the vibration removing module 10 is that shown in FIG. 6B, a part of the vibration transmitted from the input pipe 1 is directly transmitted from the output pipe 3 to the refrigerant on the downstream side without causing the above-mentioned multiple reflection. .. Therefore, it is difficult to give a half-wave phase difference of complete vibration to the refrigerant on the downstream side.

FIG. 6C shows the internal structure of the vibration elimination module 10 that has solved the problem. That is, the output tube 3 is provided with a baffle 31 formed of a disk-shaped plate material on the outlet side of the input tube 1 so that the vibration propagating from the input tube 1 does not directly propagate to the output tube 3. The baffle 31 functions to prevent the transmission of vibration, and its diameter d4 is about the same as or slightly larger than the inner diameter d3 of the output tube 3. The baffle 31 is mounted inside the multiple reflection tube 2 by mounting legs 32.

Because of the baffle 31, most of the vibration propagating from the input tube 1 undergoes the above-described multiple reflection, so that the multiple reflection tube 2 can give a half-wave phase difference of the complete vibration.
The baffle 31 may have a rectangular shape or a polygonal shape as long as it has a shape that covers the inner diameter d3 of the output tube 3 instead of the disk shape.

FIG. 6D shows another internal structure of the vibration removing module 10. Although the baffle 31 prevents the vibration propagating from the input tube 1 from directly propagating to the output tube 3 in the above-described embodiment, the baffle 31 is replaced by a vibration reflection plate 33 formed of a disk-shaped plate material. There is. The diameter d5 of the vibration reflection plate 33 is slightly smaller than the inner diameter d2 of the multiple reflection tube 2 and larger than the inner diameter d3 of the output tube 3. The vibration reflector 33 almost reflects the vibration propagating from the input tube 1, and causes multiple reflection between the vibration reflector 34 and the inner end 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 to the output tube 3 through the gap between the vibration reflection plate 33 and the multiple reflection tube 2. The vibration reflection plate 33 is attached inside the multiple reflection tube 2 by the attachment legs 34.

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

The vibration reflection plate 33 may have a quadrangular shape or a polygonal shape as long as it has a shape that covers the inner diameter d3 of the output tube 3 instead of the disk shape.
The baffle 31 works so that the vibration does not directly propagate to the output tube 3, and the vibration reflection plate 33 works so as to cause multiple reflection with the inner wall of the terminal end of the multiple reflection tube 2 on the input tube 1 side. The diameter d4 is smaller than the diameter d5 of the vibration reflection plate 33.

In order to cancel by the interference of vibration due to the phase difference of half wavelength of vibration, the circuit of the primary refrigerant, the secondary refrigerant or the frozen liquid is divided into two, one of them is directly transferred as it is (direct transfer) and the other is transferred to the other. One or more vibration removing modules 10 are connected in series to generate a phase delay of half a wavelength and transfer (phase delay transfer). 7A and 7B show essential parts of an embodiment of a circuit (hereinafter, referred to as "phase difference generating circuit") for realizing the direct transfer and the phase delay transfer.

The pipe 11 of the circuit for the primary and secondary refrigerants or the frozen liquid is divided into two by a branch pipe 12. In the phase difference generating circuits 6 and 7, the linear 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 elimination module 10 is arranged parallel to the direct transfer pipe 4 (FIG. 7A) for the phase delay transfer, and in the phase difference generation circuit 7, the vibration elimination module 10 is directly transferred pipe. 4 at right angles (Fig. 7B). In the phase difference generating circuit 7, since the vibration removing module 10 is arranged at right angles to the pipe 4 for direct transfer, the total length thereof can be made shorter than that of the phase difference generating circuit 6, so that the vibration removing device can be made compact. be able to.

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

FIG. 7C shows a simple combiner 17. That is, the primary refrigerant or the secondary refrigerant supplied from the circuit of the direct transfer pipe 4 and the primary refrigerant or the secondary refrigerant supplied from the pipe 5 via the circuit of the phase delay transfer are respectively input pipes 13. And 14 to join them by the pipe 16.

FIG. 8A and FIG. 8B show the external appearance and the internal cross-section of another combiner 20 that joins the flows of the refrigerant or the cooling liquid 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 elimination module 10 and the pipe 5 are connected to the input pipes 13 and 14 of the combiner 20. The refrigerant flowing from the input pipes 13 and 14 is introduced into the interference chamber 26 through the inlet 25 of the expansion pipe 21 having a conical internal space to which the outlets of the two input pipes are connected.

The angle θ of the interference chamber 26 seen from the input pipes 13 and 14 is the apex angle of the cone or the expansion angle of the flow, and it is desirable that the value be in a range that does not increase the pressure loss of the flow. In the expansion tube, the flow is first separated from the wall surface, goes to some extent downstream, and then contacts the tube wall. The cause of the pressure loss occurs at the beginning of this expansion tube, where the jet disturbs the surrounding fluid to create a vortex. From FIG. 9, the expansion angle is preferably about 25 degrees, which is about half the maximum pressure loss. The angle may be larger than this, but the load on the circulation pump 52 (FIG. 10A) and the compressor 72 (FIG. 10B) that creates the flow becomes large. On the contrary, if the angle is less than this, the outer length of the merger 20 becomes large, and the vibration eliminating device equipped with this 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, since the interference chamber 26 is only a space where two vibrations of a longitudinal wave and a vibration delayed by a half wavelength from each other interfere with each other, the interference may be broken and the vibration may appear again in the refrigerant. In order to prevent this danger, a diffuser plate 23 having a large number of diffraction holes 24 is provided downstream of the interference chamber 26. The vibration acts as a new vibration source using the diffraction hole 24.

Since the diffusion plate 23 does not transmit vibration, it is desirable that the diffusion plate 23 is made of a material whose acoustic impedance is significantly different from that of the refrigerant or the cooling liquid, for example, a plastic or metal plate having a cavity inside. However, since the vibration has disappeared in the diffraction hole 24, the flow in which the vibration has disappeared gathers in the confluence chamber 27 constituted by the cylindrical tube 22 on the downstream side. After that, 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) and the cooling liquid circulation tank 58 or cooling. The cooling pipe 75 for cooling the liquid circulation tank 78 is reached.

The vibration eliminator 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 opposite direction without using the merging device 20 to form a vibration eliminating device. it can. In this case, the original vibration wave may appear when the pipes after merging are bent or the like, so that the use is limited. On the other hand, when the combiner 20 is used, the original vibration wave does not appear even if the downstream pipe after the combiner 20 is bent, and therefore the application is not limited.

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

The primary refrigerant that cools the cooling liquid 55 in the primary cooler 51 circulates in the cooling circuit 63 in the cooling cycle. 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 of 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. Low-temperature water or air 61 is supplied to the secondary cooler 41, and heat of the primary refrigerant is removed to become hot water or high-temperature air 62. In the present embodiment, the primary refrigerant is compressed by the compression pump 42 and then cooled by the secondary cooler 41 to enter the expansion valve 43, but a capillary may be used instead of the expansion valve 43.

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

FIG. 10B shows an embodiment in which the vibration eliminator 74 is installed and used in the circulation circuit 83 for the primary refrigerant. The cooling liquid circulating tank 78 is filled with the cooling liquid 77. The primary cooling liquid circulates in the circulation circuit 83 due to the pressure gradient caused by the compression of the compression pump 72 and the low pressure of the expansion valve 73. The primary refrigerant is cooled by the primary cooler 71 after being compressed by the compression pump 72, and enters the vibration elimination 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.

By this cooling pipe 75, the cooling liquid circulation tank 78 is cooled 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 ultra-low vibration in the cooling process required in the first or second embodiment. The blood pack 57 may be placed on the vibration isolation table 76, or may be placed in a container (not shown) and placed on the vibration isolation table 76.

Low temperature water or air 81 is supplied to the primary cooler 71, and heat of the primary refrigerant is removed to become hot water or high temperature air 82. After the primary refrigerant is compressed by the compression pump 72 and cooled by the primary cooler 71 and enters the expansion valve 73, a capillary may be used instead of the expansion valve 73.

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

In addition, the vibration isolation means such as the hanging strap A shown in FIG. 2A or the vibration isolation table shown in FIG. 2B may be used together with refrigeration using the vibration removal module according to the present invention.

In the present specification, for hemolysis suppression, whole human blood is described as whole blood, and the present invention is also described in Examples based on whole human blood. However, the hemolysis-suppressing cryopreservation method according to the present invention and the refrigerating apparatus used for the method can be applied to blood of land animals, marine animals such as fish, or whole blood collected from these animals as long as they have red blood cells.

In the present specification, the present invention is described with reference to an example of using a container filled with blood, particularly a bag-shaped pack, when freezing blood, but it is possible to store blood and prevent contamination from the outside. The shape does not have to be a pack, but may be box-shaped, spherical, or the like. Any container that can freeze blood may be used.

The blood is not limited to blood transfusion, and the present invention can be applied to cryopreservation of blood used for such purpose or use as long as the purpose or use is required to prevent hemolysis.

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

Furthermore, for the above-mentioned bluefin tuna, other tuna, and other fish species, in addition to the cooling process adopting the slow cooling shown in Example 1, a cooling process adopting the step cooling shown in Example 2 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 not only the fillet but also the whole fish body by applying the same freezing process as in Example 3. The same applies to the refrigerator.

The cooling process may include a cooling process that employs slow cooling and a cooling process that employs step cooling. For example, when transitioning from the first cooling temperature to the second cooling temperature, a cooling method based on slow cooling in the first half and a cooling step based on step cooling in the latter half, or vice versa, or a cooling step based on slow cooling Alternatively, a cooling process based on step cooling and a cooling process based on step cooling may be mixed to change the ratio of these processes.

Further, the case where a liquid refrigerator is used as the refrigerator has been described. However, as long as the first freezing temperature and the second freezing temperature can be set and a rapid freezing process can be provided up to the first freezing temperature, an air blast freezer or the like can be used instead of the liquid freezer. Further, as a refrigerator in which the blood pack or the fish meat does not come into direct contact with the cooling liquid, a contact freezer which is in direct contact with the refrigerant guide tube or the guide housing can be used.

When the air blast refrigerator is used, the vibration transmitted through the refrigerant liquid is transmitted through the air to the blood pack or the fish meat which is the object to be frozen. Regarding the propagation of vibrational energy in the refrigerant or water and the propagation of energy in air, air is about 3400 times larger. However, the ratio of the sound pressure energy that is largely transmitted through the reflection when the sound wave is transmitted from the air to the blood pack or the fish meat is 1/3700. Therefore, in the air, the sound pressure of 294 Pascal is the limit sound pressure. 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 limit sound pressure inside the object to be frozen is the same regardless of whether the liquid refrigerator, the air blast refrigerator, or the contact freezer is used.

Applicable to the preservation of blood packs where hemolysis is unlikely to occur by the process of rapidly cooling to the first cooling temperature and then slowly cooling to the second cooling temperature in an environment in which vibration from the outside is hardly transmitted to the blood pack. can do. Furthermore, the present frozen storage method can also be applied to frozen storage for maintaining the freshness of fish meat.

1 Input pipe 2 Multiple reflection pipe 3 Output pipe 4, 5, 16 Piping 6, 7 Phase difference generation circuit 10 Vibration elimination module 11 Piping 12 Branch pipe 13, 14 Input pipe 15 Output pipe 17, 20 Combiner 21 Expansion pipe 22 Cylinder Tube 23 Diffusion plate 24 Diffraction hole 25 Inlet 26 Interference chamber 27 Merging chamber 28 Outlet 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 circulation pumps 53, 74 vibration elimination device 54 cooling liquid supply pipes 55, 77, 102 cooling liquid 56, 76 anti-vibration table 57, 79 blood packs 58, 78, 101 cooling liquid circulation tanks 65, 83 circulation circuit 66 cooling pipe 75 Cooling pipe 84 Refrigerant supply pipe 103 Blood pack 104 Hanging string 105 Rail 106 Storage device 107 Storage device lid 108 Vibration isolation table

Claims (33)

Blood freezing method including the following steps:
Step 1 Inject blood into a sterilized container (pack), fill the container, and then seal the inlet with no air layer to obtain a blood pack,
Step 2 is cooled to changes in the blood pack from at least -1 ° C to -4 ° C in the first cooling temperature in a shorter transit times than 30 minutes (-4~-6 ° C),
Step 3 After the blood in the blood pack reaches the first cooling temperature, it is left at the first cooling temperature for 5 minutes or more.
Step 4 Under the environment in which the sound pressure inside the blood pack is smaller than 284 Pascal , slowly move the blood pack to the second cooling temperature (-7 to -13°C) (-1.5°C/hour- -0.5°C/hour) cool,
Step 5 The blood pack is stored at a storage temperature which is equal to or lower than the second cooling temperature and up to the cold resistance limit temperature or the embrittlement temperature. Blood freezing method including the following steps:
Step 1 Inject blood into a sterilized container (pack), fill the container, and then seal the inlet with no air layer to obtain a blood pack,
Step 2 is cooled to changes in the blood pack from at least -1 ° C to -4 ° C in the first cooling temperature in a shorter transit times than 30 minutes (-4~-6 ° C),
Step 3 After the blood in the blood pack reaches the first cooling temperature, it is left at the first cooling temperature for 5 minutes or more.
Step 4 Under the environment in which the sound pressure inside the blood pack is smaller than 284 Pascal , the temperature of the blood pack is set as a step temperature which is selected in the range of −0.5° C. to −1.5° C. Lower the temperature of the stage,
Step 5 The blood pack is left at a temperature lowered by the stage temperature for a time longer than the value of the stage temperature in degrees Celsius×0.5 hours,
Step 6 Steps 4 and 5 are repeated 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 which is a temperature equal to or lower than the second cooling temperature and up to the cold resistance limit temperature or the embrittlement temperature. A method for cryopreserving blood, including the method for freezing according to claim 1. A method for cryopreserving blood, including the method for freezing according to claim 2. The frozen storage method of blood according to any one of claims 1 to 4, wherein the blood is any one of whole blood, concentrated red blood cells, 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 a means for cooling the blood to a temperature up to the first cooling temperature and the second cooling temperature. Frozen storage method. 7. The cooling liquid circulation tank is used as a means for cooling the blood pack to a temperature up to the first cooling temperature and the second cooling temperature, according to any one of claims 1 to 6. How to store blood packs frozen. 7. The temperature of the refrigerant does not exceed −4° C. even when the blood pack is submerged after the refrigerant in the cooling tank of the cooling liquid circulation tank is set to the first cooling temperature. Or the method for cryopreserving blood according to any one of 7 or 7 . In order to provide an environment in which the sound pressure inside the blood pack is smaller than 284 Pascal according to step 4 of claim 1 or 2, a horizontal bar installed on the upper part of the cooling liquid circulation tank and the horizontal bar are provided. The method for cryopreserving blood according to any one of claims 6 to 8, characterized in that a string which is suspended and passed through the blood pack is used. The anti-vibration table installed on the bottom surface of the cooling liquid circulation tank in order to provide an environment in which the sound pressure inside the blood pack is smaller than 284 Pascals according to step 4 of claim 1 or 2, and the blood pack. 9. The method of cryopreserving blood according to claim 6, wherein a container for storing the container and a container lid for covering the upper part of the container are used. Method of freezing fish meat 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 in a state not containing an air layer is a frozen body,
Step 2 is cooled the the first cooling temperature in a shorter transit times than 30 minutes changes to -4 ° C to be frozen bodies from at least -1 ° C (-4~-6 ° C),
Step 3 After the object to be frozen reaches the first cooling temperature, it is left at the first cooling temperature for 5 minutes or more.
Step 4 Under an environment in which the sound pressure inside the frozen object is smaller than 284 Pascal , slowly cool the frozen object to the second cooling temperature (-7 to -13°C) (-1.5°C/ Time to −0.5° C./hour),
Step 5 The object to be frozen is stored at a storage temperature which is equal to or lower than the second cooling temperature and up to the cold resistance limit temperature or the embrittlement temperature,
Method for frozen storage of fresh fish containing. Method of freezing fish meat 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 in a state not containing an air layer is a frozen body,
Step 2 is cooled the the first cooling temperature in a shorter transit times than 30 minutes changes to -4 ° C to be frozen bodies from at least -1 ° C (-4~-6 ° C),
Step 3 After the object to be frozen reaches the first cooling temperature, it is left at the first cooling temperature for 5 minutes or more.
Step 4 Under the environment in which the sound pressure inside the object to be frozen is smaller than 284 Pascal , 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 object to be frozen is set . Lower the temperature by the relevant stage temperature,
Step 5 The object to be frozen is left at a temperature lowered by the step temperature for a time longer than the value of the step cooling temperature displayed in degrees Celsius×0.5 hours,
Step 6 Steps 4 and 5 are repeated 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 which is a temperature equal to or lower than the second cooling temperature and up to the cold resistance limit temperature or the embrittlement temperature. ,
Method for frozen storage of fresh fish containing. A cooling liquid circulation tank is used as a means for cooling the entire fish body according to claim 11 or 12 or the fillet of the fish body to a temperature up to the first cooling temperature and the second cooling temperature. The method for frozen storage of fresh fish according to claim 11 or 12. A means for cooling the whole fish body according to claim 11 or 12 or the fish body fillet wrapped with 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 freezing and preserving fresh fish according to claim 11 or 12, wherein a cooling liquid circulation tank is used. The cooling liquid supplied to the cooling liquid circulation tank in order to provide the environment in which the sound pressure inside the blood pack is smaller than 284 Pascal according to the step 4 of claim 1 or 2, Multiple reflections composed of a pipe branched into two paths, one of which serves as a cooling liquid through a straight pipe that extends almost linearly, and the other of which has an inner diameter larger than the inner diameters of the input and output pipes. A cooling liquid having a phase delay of approximately half a wavelength of the external vibration is generated via a phase difference generation circuit in which one or more vibration elimination modules made up of pipes are connected in series, and the one cooling liquid and the other cooling liquid merge. The frozen storage method for blood according to any one of claims 6 to 10, wherein the blood is supplied to the cooling liquid circulation tank after being processed. The cooling liquid supplied to the cooling liquid circulation tank in order to provide an environment in which the sound pressure inside the object to be frozen is smaller than 284 Pascal according to the step 4 of claim 11 or 12. , A branch pipe is branched into two paths, one of which is a cooling liquid passing through a substantially straight linear pipe, and the other is a multiplex pipe composed of pipes having an inner diameter larger than that of the input pipe and the output pipe. A cooling liquid having a phase delay of about half a wavelength of external vibration is generated via a phase difference generating circuit in which one or more vibration elimination modules each including a reflection tube are coupled in series, and the one cooling liquid and the other cooling liquid are 15. The method of frozen storage of fresh fish according to claim 13 or 14, wherein the fresh liquid is supplied to the cooling liquid circulation tank after being merged. In order to provide an environment in which the sound pressure inside the blood pack is smaller than 284 Pascal according to the step 4 of claim 1 or 2, it is supplied to a cooling pipe surrounding the cooling liquid circulation tank. The cooling liquid is branched into two paths with a branch pipe, one of which is a cooling liquid that passes through a substantially straight linear pipe, and the other is a pipe having an inner diameter larger than the inner diameters of the input pipe and the output pipe. One or more cooling liquids, which are phase-delayed by about half a wavelength of the external vibration, are generated through a phase difference generation circuit in which one or more vibration elimination modules composed of multiple reflection tubes are connected in series. The cryopreservation method for blood according to any one of claims 6 to 10, wherein the cooling liquid of (1) is joined and then supplied to a cooling pipe surrounding the cooling liquid circulation tank. In order to provide an environment in which the sound pressure inside the object to be frozen is smaller than 284 Pascal according to the step 4 of claim 11 or 12, the cooling liquid is supplied to a cooling pipe surrounding the circulation tank. The coolant to be branched is branched into two paths by a branch pipe, one of which is a coolant through a linear pipe that extends substantially linearly, and the other is a pipe having an inner diameter larger than the inner diameters of the input pipe and the output pipe. Via a phase difference generation circuit in which one or more vibration removal modules each consisting of a multiple reflection tube are connected in series to form a cooling liquid with a phase delay of approximately half a wavelength of the external vibration, and one of the cooling liquids The method for refrigerating preservation of fresh fish according to claim 13 or 14, wherein the other cooling liquid is supplied to a cooling pipe surrounding the cooling liquid circulation tank after being merged. The vibration elimination module according to any one of claims 15 to 18, wherein a baffle having a diameter equal to or larger than the inner diameter of the output tube is installed inside the multiple reflection tube and in front of the output tube. The method for cryopreserving blood according to any one of claims 6 to 10, claim 15 or claim 17, or the fresh fish according to claim 16 or claim 18, which is constituted by a multi-reflecting tube. Frozen storage method. The vibration elimination 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 inside the multiple reflection tube. The blood cryopreservation method or claim according to any one of claims 6 to 10, which is configured by a multiple reflection tube, characterized in that the method is attached to the front of the output tube. Item 16. A method for frozen storage of fresh fish according to Item 16 or 18. The vibration elimination module according to any one of claims 15 to 20 is arranged in parallel to the linear pipe, and the vibration elimination module according to any one of claims 6 to 10, claim 15 and claim 17 , cryopreservation method or claim 16 of the blood according to claim 19 or claim 20, claim 18, fresh fish cryopreservation method according to claim 19 or claim 20. The vibration eliminating module according to any one of claims 15 to 20 is arranged perpendicularly to the straight pipe, and the vibration removing module according to any one of claims 6 to 10, claim 15 and claim 17. 21. The frozen storage method for blood according to claim 19 or 20, or the frozen storage method for fresh fish according to claim 16, 18, 19, or 20. The cooling liquid passing through the linear pipe according to claim 15 or 16 and the cooling liquid cooling liquid passing through the phase difference generating circuit according to the same claim form an interference chamber including an expansion pipe having a conical inner space. The flow is supplied to the cooling liquid circulation tank after being combined by a combiner that has the structure, according to any one of claims 6 to 10, claim 15 or claim 19 to 22. 23. The frozen storage method for blood, or the frozen storage method for fresh fish according to claim 16 or any one of claims 19 to 22. The cooling liquid passing through the linear pipe according to claim 17 or 18 and the cooling liquid cooling liquid passing through the phase difference generating circuit according to the same claim form an interference chamber including an expansion pipe having a conical inner space. Any one of claims 6 to 10, claim 17 or claims 19 to 22 which is supplied to a cooling pipe surrounding the cooling liquid circulation tank after merging with a confluence device. The method for cryopreserving blood according to claim 1, or the method for cryopreserving fresh fish according to claim 18 or any one of claims 19 to 22. The confluencer according to claim 23 or claim 24 has a diffusion plate having a plurality of diffraction holes formed therein, any one of claims 6 to 10, claim 15, claim 17, and 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 of claim 1 or claim 2 and the material of the plastic film for packaging or the plastic bag for packaging according to step 1 of claim 11 or claim 12 are DEHP (phthalate). Acid (2-ethylhexyl acid) or TOTM (tris-2-ethylhexyl trimellitate), which is any one of polyvinyl chloride, polyolefin, polybutadiene, polyethylene or polyvinylidene chloride. 16. The method for cryopreserving blood according to claim 1, claim 15, claim 17, or any one of claims 19 to 25, or any one of claims 11 to 13, claim 16, and claim Item 18. The method for frozen storage of fresh fish according to any one of Items 18 and 19 to 25. A vibration eliminating 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 generating circuit in which one or more vibration elimination modules according to claim 15 or 17 or 16 or 18 are coupled in series, and claim 15 or 17 or claim 16 or 18. A vibration eliminator comprising one cooling liquid and the other cooling liquid, which joins the straight pipe and the phase difference generating circuit on the downstream side. The vibration elimination module according to any one of claims 15 to 18, further comprising a baffle having a diameter equal to or larger than an inner diameter of the output tube, the baffle being provided inside the multi-reflection tube and attached in front of the output tube. 28. The vibration elimination device according to claim 27, wherein the vibration elimination device is configured. The vibration eliminating module according to any one of claims 15 to 18, further comprising a vibration reflection plate 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 inside the multiple reflection tube. 28. The vibration eliminator according to claim 27, wherein the vibration eliminator comprises a multiple reflection tube attached to the front of the tube. The confluencer according to claim 27 is a confluencer having an interference chamber composed of two input pipes and an expansion pipe having a conical internal space to which outlets of the two input pipes are connected. The vibration elimination device according to any one of claims 27 to 29. The confluence device according to claim 30 is a confluence device in which a diffusion plate having a plurality of diffraction holes is further provided in a downstream portion of the interference chamber. The vibration removing device according to item 1. In the case where the cooling of the blood pack in steps 2 to 5 in claim 1 or the cooling of the blood pack in steps 2 to 7 in claim 2 is performed by an air blast refrigerator,
The cryopreservation method for blood according to any one of claims 1 to 5, wherein the air vibration generated by the air blast refrigerator and transmitted to the blood pack is 143 dB or less. In the case where the cooling of the object to be frozen 2 to step 5 in claim 11 or the cooling of the object 2 to step 7 in claim 12 is performed by an air blast refrigerator,
13. The method for freezing and preserving fresh fish according to claim 11, wherein the air vibration generated by the air blast refrigerator and transmitted to the object to be frozen is 143 dB or less.

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