JP2012115250A - Method for transporting live fish by use of nitrification bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for stably maintaining oxygen and water quality in a sealed container over a long time in transportation of live fish.SOLUTION: Fish is contained in fresh water or salt water filled with micro-nano bubbles, and oxygen is filled in the sealed container. In addition, carbon dioxide remover is used to maintain a high oxygen state, so that respiration of fish is enabled over a long period. By further inserting foam resin where nitrification bacteria is implanted, and adding copper, iron, phosphoric acid, oil and fat, or aliphatic acid, that is necessary for its activation, nitrification action is accelerated, thereby water quality is stably maintained.

Description

  TECHNICAL FIELD The present invention relates to a technique for improving the quality of water contaminated by oxygen supply, removal of carbon dioxide, and excretion in live fish transportation.

  The demand for live fish transport is increasing worldwide with the rise of the sushi boom. In order to provide fresh fish meat, 1) live tightening, 2) chilled storage, 3) rapid freezing storage, 4) cryogenic storage, and 5) live fish transportation are used as the main methods. Live tightening kills fish by severing the nervous system and maintains freshness. Chilled storage is a technique for storing freshness at a temperature just before freezing. Recently, there are things that are always available in refrigerators, and they are well established as general technology. Rapid freezing is a technique that suppresses the formation of crystals that destroy cells by rapidly passing through a temperature zone in which needle-like ice crystals are formed during freezing. For this reason, there are few drips which exude from a cell when thawing freezing, and freshness is maintained. The low freezing temperature is a technique for storing at a low temperature far exceeding -20 ° C, which has been widely used so far, that is, at -40 ° C to -60 ° C. Each is beyond the conventional technology, you can feel the freshness.

Compared to preservation, live fish transportation is utilized until just before, so it can provide the freshest fish meat. However, sealing a fish in a container for transport creates many problems and has not been fully solved so far.
Live fish transportation is mainly applied to cultured fish, but to date there are established forms of transportation from farms to urban areas. The technology is improving day by day, but the main means of transportation is a live fish cart or a live fish boat. However, both have no septic tank. Live fish boats are structured to allow seawater to enter and irrigate during movement, but live fish carts usually have only a ventilation device.
It is difficult to install a septic tank in a live fish cart because of the following reasons: 1) The area occupied by the septic tank is large, and 2) The transportation density of fish is high and considerable purification capacity is required to increase economy. .

  In terms of distribution, live fish transportation means other than live fish carts or live fish boats are not well established. That is, in the case of ornamental fish, the fish is put in a plastic bag or the like, and the bag is filled with air or oxygen and transported. It is also used for transporting some live fish for edible use, lowering the water temperature to reduce fish metabolism and avoiding water quality deterioration. However, in the case of a short time, it is possible to transport by this method, but when it is several days at a long distance, the water quality will deteriorate unless there is a septic tank, and the fish will be killed. Have difficulty.

In the closed state, fish excrement causes water quality deterioration, among which ammonia affects fish survival. Since ammonia is highly toxic, it is immediately converted into harmless urea and uric acid in the body of terrestrial animals, which are excreted in the urine. However, fishes release most of the ammonia produced in the body from the salmon and the rest as urine components into the water. In general, about 16% of the protein weight constituting the bait is nitrogen atoms, and it is said that about half of the weight is released as ammonia.
Ammonia exists in a different manner depending on pH and temperature. If it is acidic, that is, in a state where the hydrogen ion concentration is high, most of it is converted to ammonium ion, which is not as toxic as ammonia. However, it is extremely toxic because it exists as ammonia in basic form.
If this ammonia can be removed, the fish can survive, but since the fish is constantly excreted, the ammonia is constantly increasing. Although there is a method of trying to remove ammonia that is increasing with an adsorbent such as activated carbon, an overwhelmingly large amount of adsorbent must be used for ammonia that is increasing, which is not practical. Only biological purification methods can cope with this, but this is an activity that can be found only in a septic tank that stands up naturally in a general water tank and is stable.

There are various forms of septic tanks for water tanks, but basically water is circulated with a pump and purified through porous materials or fibers. Nitrifying bacteria are found in the porous material or fiber, and ammonia is purified by the action of these bacteria. The nitrifying bacteria responsible for nitrification are ammonia-oxidizing bacteria (or nitrite bacteria) that oxidize ammonia nitrogen to nitrite, and nitrite-oxidizing bacteria (or nitrate bacteria that oxidize nitrite to nitrate). 2) bacteria group. These bacteria are autotrophic bacteria that use carbon dioxide (CO ₂ ) as their only carbon source, and oxidize ammonia or nitrite to acquire energy and perform metabolism.
Most of the nitrifying bacteria have settled on the surface of the individual, and about 15% of the dividing bacteria have detached from the colonization site, but the colonization is almost proportional to the height of activity. Therefore, nitrifying bacteria that are fixed on a carrier are effective. Objects with a larger surface area of the carrier can attach more bacteria.

  The present inventors previously described a “fish and aquatic animal tank purification filter, a method for producing the purification filter, a purification filter storage method, and a fish and aquatic animal tank purification method” (Patent Document 1), and In “Purification Filter and its Manufacturing Method and Preservation Method of the Filter” (Patent Document 2), carbonized cotton (carbonized cotton) was proposed based on this principle. However, in live fish transportation, the carbonized cotton powder gradually flows out into the water, clogging the fish traps, and hindering breathing. It is hydrophilic and porous, and requires a stable material to absorb bacteria well.

In the aquarium where fish are raised, nitrifying bacteria grow and stabilize naturally over time. In the water tank in which ammonia nitrogen has accumulated, ammonia-oxidizing bacteria will start up first, and ammonia nitrogen will be changed to nitrite nitrogen, but after sufficient nitrite nitrogen has accumulated, nitrite-oxidizing bacteria will start up. Thus, it usually takes several months for nitrifying bacteria to have stable activity.
In the case of ammonia-oxidizing bacteria, if the elements necessary for growth are sufficient, the cells divide in a minimum of 24 hours. In the case of nitrite oxidizing bacteria, it divides in a minimum of 48 hours. The energy obtained when oxidizing 1 mol of ammonia nitrogen is 39.5 kcal, and the energy obtained by oxidizing 1 mol of nitrite nitrogen is only 21.6 kcal. Very little compared to 686 kcal, the energy obtained by respiration from a mole (180 g). This growth rate is extremely slow compared to the rate at which bacteria such as E. coli divide within one hour. This slow growth has become a problem in all situations where nitrifying bacteria are used.

  Ammonia-oxidizing bacteria include the genus Nitrosomonas, the genus Nitrosolobus, the genus Nitrosococcus, the genus Nitrosospira, and the genus Nitrospira. . In addition to the genus Nitrobacter, nitrite-oxidizing bacteria are also known in the genus Nitrococcus and Nitrospina.

The fact that fish metabolizes is to breathe oxygen primarily, decompose nutrients by oxidation, gain energy, and biosynthesize new substances by catabolism. As a result, it emits carbon dioxide. On the other hand, the above-mentioned nitrifying bacteria for water purification are the same, and consume a great deal of oxygen.
The major problem in live fish transportation is this oxygen supply and carbon dioxide treatment. In a closed space, there is only limited oxygen, so not only is oxygen consumed and reduced by fish and nitrifying bacteria, but carbon dioxide produced by respiration significantly lowers the residual oxygen partial pressure. .

JP-A-2005-117925 JP-A-2005-87083 Japanese Patent Application No. 2010-159127

  The objectives of the present invention are: 1) a method in which the fish can breathe with limited oxygen for a long time in a container for transporting live fish; It is to provide a method for detoxifying activated oxygen conditions and 4) a large amount of ammonia that is constantly excreted.

The inventors of the present invention previously proposed “Activating substances for nitrification and denitrification” (Patent Document 1). The present invention is intended to devise a use for live fish transport, focusing on the activation of nitrifying bacteria of the previous invention. However, it is necessary to add more conditions to achieve the above objectives. As a result of extensive research, the combined use of micro-nano bubble water and pure oxygen, the addition of a minimum substance for nitrifying bacteria activation, and stable nitrifying implantation The knowledge that the above problem can be solved by using the material was obtained.
The present invention is based on this finding.
1. Under high oxygen concentration, in the presence of nitrifying bacteria activating substance, in the presence of nitrifying bacteria-implanted material, under proper pH and water temperature, ensure fish respiration and immediately develop nitrifying action of nitrifying bacteria A live fish transportation method characterized by maintaining water quality by allowing the fish to survive in a closed container for a long period of time,
2. In the water phase where the transported fish enters, high-oxygen concentration uses fresh water or seawater filled with micro-nano bubbles of oxygen, and in the gas phase, pure oxygen is enclosed in a sealed container, and the carbon dioxide generated by the respiration of the fish is 2. The live fish transportation method according to 1, characterized in that the high oxygen partial pressure is maintained by removing with an absorbent filled in a polyethylene bag,
3. The composition of the nitrifying bacteria activating substance is 1) a phosphate buffer (pH 7.0 for fresh water, pH 8.2 for seawater), and its final concentration is 0.1 to 5 mM, preferably 1 mM, 2) It is an iron salt (II) and its final concentration is 150 to 500 μg / L, preferably 200 to 400 μg / L, 3) It is a copper salt (II), its final concentration 20 to 200 μg / L, Preferably 40 to 80 μg / L, and 4) Fats and oils or their constituent fatty acids are long chains, preferably have 12 to 24 carbon atoms, more preferably 18 carbon atoms, and preferably not. Fats and oils composed of saturated fatty acids or unsaturated fatty acids, final addition amount is 0.1 to 50 μL / L, preferably 10 μL / L, 5) Carbon source indicates carbonate hardness (KH) To 8 or more, preferably 12 or more, and a live fish transport method according to each of 1-2,
4). The nitrifying bacteria implantation material is a foam of a hydrophilic resin, preferably a foam of melamine resin (melamine foam), or a fiber, preferably a carbon fiber, according to each of 1 to 3 Live fish transport method,
5. The appropriate pH is 5.0 to 9.2, preferably 7.0 to 8.0 for fresh water, 7.5 to 9.0 for seawater, preferably 8.0 to 8.5. The live fish transportation method according to each of 1 to 4, characterized in that:
6). The live water transportation method according to each of 1 to 5, characterized in that an appropriate water temperature is 4 ° C to 30 ° C, preferably 10 ° C to 25 ° C,
I will provide a.

The present inventor has shown that nitrifying bacteria can be activated by the prior application (Reference 1). However, in a closed container, there is limited oxygen, and conditions that allow oxygen to be sufficiently supplied in an environment where carbon dioxide is constantly produced by respiration and ammonia is excreted, and nitrifying bacteria are further reduced. It is necessary to obtain activated conditions. For this purpose, first, conditions under which oxygen can be sufficiently supplied were obtained.
The fish transport container is sealed so that the enclosed water (fresh water or seawater) does not leak during transport. Thus, oxygen for fish respiration fills the container. When a container is filled with 1 atmosphere of oxygen, the amount of oxygen dissolved in water is determined by temperature. For example, the dissolved oxygen amount is about 8 mg / L at 1 atmosphere and room temperature (25 ° C.), and about 6.5 mg / L in the case of seawater.

In order to maintain a high oxygen state, it was examined whether micro-nano bubble water is effective.
It is known that micro-nano bubble water forcibly supplies oxygen gas into the water in the form of bubbles, and its size is micro to nano level, so it does not expand and remains in the water for a long time with small bubbles. . Eventually, it shrinks and dissolves in water to become dissolved oxygen, but it can exist as foam for several weeks. The amount of dissolved oxygen in micro-nano bubble water varies greatly depending on the measurement method, and it is difficult to determine how much actual dissolved oxygen (DO) is. When measured with a DO meter (diaphragm electrode method), it was about 32 mg / L, but with the modified azide Winkler method, it was about 20 mg / L. Both are official methods of measuring environmental standards.

(Experiment 1)
[Effect of micro / nano bubble water]
Commercial mineral water and oxygen were converted into micro / nano bubble water by gas-liquid mixing shearing method, and artificial sea salt was dissolved therein. When fish (Cuffoonfish, about 30 g) and micro / nano bubble seawater were placed in a sealed container and kept at 25 ° C. without air, they became difficult to breathe in 45 minutes and tumbled. When DO at this time was measured by the modified azide Winkler method, it was 20.2 mg / L at the beginning, and 4.64 mg / L after the fish was recovered. On the other hand, using the same weight fish, it became difficult to breathe in 7 minutes compared to artificial seawater, and it overturned. The DO at this time was 6.7 mg / L at the beginning and 4.50 mg / L after the fish was recovered. When comparing oxygen consumption per hour,
Micro-nano bubble artificial seawater: 20.2-4.64 mg / L / 45 min = 0.346 mg / L / min
Artificial seawater: 6.7-4.5 mg / L / 7 min = 0.314 mg / L / min
It became. Therefore, it was found that the oxygen consumption per minute of this fish was about 0.3 mg / L, and there was almost no difference. In other words, the DO of the micro / nano bubble artificial seawater means that the numerical value measured by the azide winkler is good. Micro-nano bubble water does not support the respiration of fish for a long time, but it is not necessary to urgently supply oxygen after fish entrapment during the operation of commercial live fish transportation, which is advantageous in that many simultaneous operations are possible. There is.

(Experiment 2)
[Effect of micro / nano bubble water on nitrifying bacteria]
Next, the effect of micro-nano bubble water on aerobic nitrifying bacteria was verified. This is because the present inventor has found that the nitrification action rises quickly when the oxygen supply of nitrifying bacteria is sufficiently performed. The activator for nitrification was according to the method described in Patent Document 1. That is, 100 mL of artificial seawater made with micro-nano bubble water and 100 mL of artificial seawater made with ion-exchanged water were put into a 150 mL beaker, and 5 g / L chelated iron (FeSO ₄₎ was added to each.
0.5 g, H ₂ SO ₄ 50 μL, and EDTA 0.14 g dissolved in 100 mL of ion-exchanged water) 5.6 μL, 1 g / L chelating copper (CuSO ₄ .5H ₂ O 0.5 g, H ₂ SO ₄ 50 μL , 0.14 g of EDTA dissolved in 100 mL of ion-exchange water) 5.6 μL, 0.15 mL of 0.2 M phosphate buffer (pH 7.0), 1 mL of 50 g / L sodium bicarbonate, 5 μL of olive oil added did. To this, 5 mL of nitrifying bacteria stock, melanin foam block (1 cm × 1 cm × 2 cm) as a material for implantation were added, and ammonium sulfate was added as ammonia to a final concentration of 5 mg / L. It stored at room temperature (17 degreeC), ventilating in a beaker with an air pump.
The results are shown in Table 1. When ammonia nitrogen was measured and the water quality of both was compared, the nitrification reaction started more quickly with micro / nano bubble water than with pure water.

(Experiment 3)
[Cultivation method of nitrifying bacteria]
Nitrifying bacteria must be cultured separately. As the nitrifying bacteria, a mixed bacterium of nitrosomonas and nitrobacter collected from the natural world by the present inventor was used. The culture was performed as described in (Reference 1). That is, for the medium, NaCl 35 g, K ₂ HPO ₄ 0.5 g, MgSO ₄ .7H ₂ O 0.5 g, 0.2 g / L MnCl ₂ 2.3 mL, 10 mg / L with respect to 1 L of ion-exchanged water. CaCl ₂ 0.5 mL, 0.05 mg / L Na ₂ MoO ₄ .2H ₂ O 0.5 mL, 0.05 mg / LZnSO ₄ .7H ₂ O 0.5 mL, 0.001 mg / L CoCl ₂ .6H ₂ O 0. 5 mL, 5 g / L chelated iron 280 μL, and 1 g / L chelated copper 280 μL were dissolved, 50 g / L NaHCO ₃ 2 mL was added, and the pH was adjusted to 8.2 with hydrochloric acid. Nitrifying bacteria are planted in 6 L of this medium, (NH ₄ ) ₂ SO ₄ is added so that the final concentration is 0.2 g / L, and it is circulated with a circulation pump at 30 ° C. until ammonia, nitrous acid and nitric acid are not detected. Cultured.
(NH ₄ ) ₂ SO ₄ was added again, and the culture was continued until it was completely nitrified. This was repeated four times to obtain stock bacteria.
The melamine foam was placed in a circulation pump with a sheet having a thickness of 2 cm, a length of 16 cm, and a width of 8 cm, and nitrifying bacteria were implanted. While circulating, nitrifying bacteria gradually begin to implant into melamine foam, and the activity rises. The melamine foam on the floor was cut into blocks with a cutter knife and used.

Examples and Comparative Examples

  Next, examples and comparative examples of the present invention will be described. In addition, the Example shown below is for making an understanding of this invention easy, and this invention is not restrict | limited to these Examples. That is, other examples or modifications based on the technical idea of the present invention are naturally included in the present invention.

[Examination of carbon dioxide removal agent]
When Takifugu niphobles (weight approximately 30g) is put together with artificial seawater or micro-nanobubble artificial seawater in a plastic bag, oxygen is sealed, and when stored at room temperature, with or without carbon dioxide removal agent Then, we verified the difference in survival time. Add iron, copper, and phosphate buffer solution (pH 7.0) to 400 mL of artificial seawater or micro-nanobubble artificial seawater as described in Experiment 2, and then add about 1 mL of NaHCO ₃ so that KH = 10. Was adjusted to pH 8.2 with hydrochloric acid.
Nitric acid bacteria are placed on melamine foam as described in Experiment 3, and 2 μL of olive oil is infiltrated into this block (2 cm × 2 cm × 2 cm), and then put in each plastic bag, one as it is (A), and one is carbon dioxide. 10 g of carbon removing agent (soda lime (water: about 20%, calcium hydroxide: about 75%, sodium hydroxide: about 3%, potassium hydroxide: about 1%)) was put in a polyethylene bag, heat sealed and sealed. (B). The polyethylene bag used is low density polyethylene, and the density is 0.90 to 0.925. Oxygen gas, carbon dioxide and other inorganic gases pass well, but water molecules hardly pass. Then, oxygen was enclosed, and each was about 800 mL. The pressure was approximately 140%. A control (C) in which seawater and nitrifying melamine foam were added was also prepared for comparison.

Stored at room temperature (25 ° C.) and compared for survival. The results are shown in Table 2, and (B) survived up to 67.5 hours. The concentration of ammonia nitrogen in the artificial seawater after breeding was almost 0, and it was found that among the nitrifying bacteria, ammonia oxidizing bacteria were very well activated. On the other hand, nitrite nitrogen was slightly higher but not at a concentration that would endanger survival, and nitrite-oxidizing bacteria were also activated. The difference between (A) and (B) was whether carbon dioxide was absorbed or not, so in (A) where carbon dioxide accumulated, the oxygen partial pressure was lowered by carbon dioxide, and oxygen supply to fish was sufficient It was not performed, and the nitrifying bacteria were not fully activated. The pH also decreased in the order of death, and was proportional to the deterioration of water quality. In the control using only seawater, since there was no activator, the activity of nitrifying bacteria was not observed, high ammonia nitrogen was accumulated, and neither nitrite nitrogen nor nitrate nitrogen was detected.

[Experiment at room temperature using ezomeval]
Since the storage conditions were prepared in Example 1, the effect was confirmed with another fish. The fish used was Sebastes taczanowski and weighed about 60 g. All were added and filled with oxygen in the manner of Example 1 (B) to 800 mL of nanobubble artificial seawater. Three sets of the same thing were prepared. Each oxygen amount was 2L. It was stored at room temperature and examined for its survival. The results are shown in Table 3. All survived in the bag for 70 hours and survived after collection. Ammonia nitrogen also showed the same tendency as in Example 1, with an average of 0.3 mg / L, nitrite nitrogen with an average of 4.3 mg / L, and nitrate nitrogen with an average of 7.1 mg / L. It was.

[Low-temperature storage experiment by ezomebaru]
In the case of actual transportation, it is difficult to maintain room temperature (usually 25 ° C.). In the summer season as well as the season, the temperature of the truck bed may exceed 40 ° C. on a day with sunshine. On the other hand, in winter, the temperature may be below freezing in cold regions, which is not a preferable temperature for live fish. However, transportation at low temperatures has been established through the use of refrigerated vehicles. In view of this situation, a low temperature storage experiment was conducted. The same fish and procedure as in Example 2 were used. However, storage was performed in a refrigerator (average 4 ° C.). The results are shown in Table 4. All survived for 170 hours, and one of the three died, because of continued storage for an additional 3 hours, resulting in a lack of oxygen. However, it was found that the water quality was maintained very well.

The invention's effect

The method for transporting live fish of the present invention aims at sufficient oxygen supply and activation of nitrifying bacteria, and can quickly purify water during the transport period and stably transport fish for a long period of time. For this reason, individual fish transportation becomes possible, and not only it becomes possible to use a courier nationwide, but also global transportation is possible for the world. It can be transported for 3 days at room temperature, and can be transported for about 1 week in refrigeration. Since it can be transported and stored for a long time, it can be widely used for transporting live fish.
[Brief description of chart]

[Table 1]
Effect of Micro-Nano Bubble Water on Fish Tables comparing the activity of nitrifying bacteria between artificial seawater prepared with oxygen micro-nano bubble water and artificial seawater prepared with ion-exchanged water.
[Table 2]
Examination of carbon dioxide removal agent by trough It is a table comparing the survival of fish with and without carbon dioxide removal agent. [Table 3]
It is the table | surface showing the result of having performed the storage experiment at room temperature using the Esomeval.
[Table 3]
It is a table showing the results of a low temperature storage experiment using ezomebaru.

Claims (6)

  Under high oxygen concentration, in the presence of nitrifying bacteria activating substance, in the presence of nitrifying bacteria-implanted material, under proper pH and water temperature, ensure fish respiration and immediately develop nitrifying action of nitrifying bacteria A live fish transport method characterized by maintaining water quality by allowing the fish to survive in a closed container for a long period of time.   In the water phase where the transported fish enters, high-oxygen concentration uses fresh water or seawater filled with micro-nano bubbles of oxygen, and in the gas phase, pure oxygen is enclosed in a sealed container, and the carbon dioxide generated by the respiration of the fish is 2. The live fish transportation method according to claim 1, wherein a high oxygen partial pressure is maintained except for an absorbent filled in a polyethylene bag.   The composition of the nitrifying bacteria activating substance is 1) a phosphate buffer (pH 7.0 for fresh water, pH 8.2 for seawater), and its final concentration is 0.1 to 5 mM, preferably 1 mM, 2) It is an iron salt (II) and its final concentration is 150 to 500 μg / L, preferably 200 to 400 μg / L, 3) It is a copper salt (II), its final concentration 20 to 200 μg / L, Preferably 40 to 80 μg / L, and 4) Fats and oils or their constituent fatty acids are long chains, preferably have 12 to 24 carbon atoms, more preferably 18 carbon atoms, and preferably not. Fats and oils composed of saturated fatty acids or unsaturated fatty acids, final addition amount is 0.1 to 50 μL / L, preferably 10 μL / L, 5) Carbon source indicates carbonate hardness (KH) To 8 or more, preferably 12 or more, and a live fish transport method according to each of claims 1-2.   Each of claims 1 to 3, characterized in that the nitrifying bacteria implantation material is a hydrophilic resin foam, preferably a melamine resin foam (melamine foam), or a fiber, preferably a carbon fiber. The live fish transportation method described.   The appropriate pH is 5.0 to 9.2, preferably 7.0 to 8.0 for fresh water, 7.5 to 9.0 for seawater, preferably 8.0 to 8.5. The live fish transport method according to claim 1, wherein the live fish transport method is provided.   The method for transporting live fish according to each of claims 1 to 5, wherein an appropriate water temperature is 4 ° C to 30 ° C, preferably 10 ° C to 25 ° C.