JP3103615B2 - Preventive and therapeutic agent for pathogen infection added to formulated feed for cultured aquatic animals - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an agent for preventing and treating pathogenic bacterial infections which is added to a feed mixture for cultured aquatic animals.

[0002]

2. Description of the Related Art In recent years, demand for crustaceans such as prawns, lobsters, king crabs, snow crabs and crabs, and high-grade fishes such as red sea bream and flounder has dramatically increased in Japan as the eating habits have become more sophisticated and gourmet. They are,
Originally, it is a fishery resource with a small population and high rare value.
Furthermore, due to overfishing, the population density has been significantly reduced, and resource nationalism has risen, so countries close to fishing grounds claim ownership of marine resources inhabiting waters beyond 200 nautical miles and affected by their own country. As a result, it has become more and more difficult to secure quantitative quantities since other countries have restricted their unreasonable catch. On the other hand, the fisheries so far are based on catching fish that inhabit the natural world. Therefore, the fish catch greatly fluctuates due to natural conditions, and the management is unstable. It is natural for Japan to shift from conventional fisheries that rely on natural fishery resources to aquaculture fisheries, which are “creating fisheries,” in order to secure scarce fish resources and stabilize management. I can say that

[0003] The aquaculture and fishery industry in Japan begins with carp and eel, and has been in Japan since ancient times such as saltwater fish such as hamachi, flounder and red sea bream, freshwater fish such as ayu, char and rainbow trout, crustaceans such as prawns, oysters and scallops and shellfish. In addition to the fishery resources that inhabited it, recently aquaculture resources such as tilapia, pehelay, and piranha have come to be used for aquaculture.

[0004] Since aquaculture is a new industry, it has its own irrationality. In other words, most of the aquatic resources to be cultured are inhabited in wide waters, and so many ecology studies have not been thoroughly conducted. In the cultivation of aquatic resources, it is very important to study the feed efficiency and how much and when to feed the target animals. It is no exaggeration to say that little has been done. Also important is the study of what diseases are found in farmed animals and how to prevent and treat them. In particular, among the diseases, pathogenic bacterial infections, unlike the vast nature, in the case of aquaculture fisheries that are bred at high density in narrow water, once the pathogenic bacterial infection occurs, it can be fatal to the point where it is annihilated . Indeed, the death of cultured aquatic animals from pathogenic infections is the most serious problem in the aquaculture industry. In particular, juveniles are highly susceptible to infection with pathogenic bacteria, so that careful attention is required when rearing them. Moreover, in the case of aquaculture, no precise measures have been found for infectious diseases caused by pathogenic bacteria, except for a very small portion. Therefore, once an infectious disease of a pathogenic bacterium occurs, antibacterial substances,
For example, β-lactam, tetracycline or macrolide antibiotics are simply mixed into the feed and given.
These antibacterials and antibiotics are expensive, and in aquaculture, their use for infection prevention and treatment is enormous. Since aquaculture is an economic act, the development of a method for preventing and treating pathogenic infectious diseases that does not impair economic efficiency has been anticipated. Moreover, in Japan, antibiotics remaining in foods are strictly prohibited by the Food Sanitation Law, and their use is greatly restricted. Therefore, if an antibacterial substance is used, a certain drug holiday must be taken before shipment.

[0005]

SUMMARY OF THE INVENTION The present inventors have studied the pathogen infection of aquatic animals for many years, and as a result, found a means for effectively preventing and treating the pathogen infection of cultured aquatic animals, and completed the present invention. I have reached. That is, it is thought that all animals and plants have pathogens unique to their species, and pathogens are always present in their habitats, but in normal conditions, they have a mechanism to prevent infection and are thus free from onset. The body's ability to escape this infection is called the host defense mechanism, but despite the rise of immunology, the substance of the host defense mechanism is not always clear. However, it is believed that there is no doubt that aquatic animals also have biological defense mechanisms that protect their bodies from infection with pathogenic bacteria. Phagocytic cells that eat fast foreign bodies also in aquatic animals,
Because it exists in the blood. If the function of the biological defense mechanism is reduced, or if the amount of pathogenic bacteria is too large to be processed by the biological defense mechanism, an infectious disease is considered to occur. In aquaculture fisheries, once infected with pathogens, the disease spreads rapidly and causes large numbers of farmed animals to become infected and die. In the case of aquaculture fisheries bred at a higher density than in the natural state, it is easy for the environment to deteriorate and lead to a decrease in the biological defense ability, and the bred aquatic animals are in a state easily infected by pathogenic bacteria and in an easily infected state It is considered something.

Therefore, in order to prevent and treat infectious diseases in cultured aquatic animals, in addition to preventing the infection of pathogenic bacteria by antibacterial substances, the normal state of the biological defense ability under culture conditions is maintained, and moreover, the pathogens are protected from the normal state. Some action is needed to increase resistance.

[0007]

Means for Solving the Problems Bioactive milk proteins, lactoferrin and lactoperoxidase, which are contained in very small amounts in the milk of mammals, are responsible for the normal maintenance of the biological defense ability in the mammal and in the digestive tract. It is considered one of the biological defense factors. Earlier, the present inventors orally administered lactoferrin to mice that had inoculated pathogenic bacteria and caused experimental infection, β-lactam antibiotics,
Enhanced antimicrobial activity in in vivo bacteriostatic antibiotics such as tetracycline antibiotics and macrolide antibiotics, antibiotics to significantly reduce the half effective amount ED ₅₀ rescue mice from death due to sepsis I found it. In other words, lactoferrin has the function of enhancing the host defense ability in mammals in a host-dependent manner, exerting a synergistic action with antibacterial active substances in vivo, and nonspecifically protecting against pathogenic bacterial infections.

The present inventors are taxonomically far apart from mammals and are significantly more susceptible to pathogenic infections.
Using cultured aquatic animals cultivated in fish cages or ponds,
Examining the preventive and therapeutic effects of the above-mentioned mammalian host defense factors, lactoferrin and lactoperoxidase alone or in combination with antibiotics in a feed, and examining the effect of preventing and treating infectious diseases, significant by preventing and treating pathogenic bacterial infections The present inventors have found that the mortality rate is reduced, and that the yield of aquaculture is significantly increased by improving the yield of aquaculture, thereby completing the present invention. Hereinafter, details of the invention will be described.

[0009] Lactoferrin and lactoperoxidase used in the following examples are mainly isolated from milk, but those derived from humans, sheep, goats, horses, dogs and cats are also similar to bovines. It is valid.
Lactoferrin and lactoperoxidase are proteins that are ubiquitous across animal species, and their structures are very similar to each other. It is also known that their biological activities are the same and are compatible between animal species. For example, humans, cows, sheep, goats, horses, dogs,
Lactoferrin and lactoperoxidase purified from feline milk were subjected to receptor assay using normal cells such as MDCK cells (derived from rat normal kidney cells), and the receptor on the cell membrane was analyzed by Scatchard analysis. It has been confirmed that there is no difference in the receptor characteristics due to the difference.

[0010] These milk proteins can be separated by a known method. That is, native mammalian milk is passed through a column filled with a weakly acidic ion exchange resin (Na ⁺ type) to adsorb lactoferrin and lactoperoxidase. Next, the column is washed with pure water, and eluted by flowing down a diluted saline solution. The eluate is collected, the ultraviolet absorption of the protein is measured, and the fraction from which the protein has been eluted is collected. This protein solution can be treated with an ultrafiltration membrane having a molecular weight cut-off of 20,000 daltons to remove water, electrolytes and low molecular weight compounds, and to separate a concentrated solution consisting mainly of milk protein. The content of lactoferrin and lactoperoxidase in the milk protein mixture separated in this way is usually 50-70% by weight of the former and 10-20% by weight of the latter, which are the main components. Other milk components include proteins such as α-lactalbumin, β-lactoglobulin, and bovine serum albumin, and both are absorbed by adsorbing lactoferrin and lactoperoxidase by column chromatography packed with heparin-sepharose. To separate. Since these proteins are basic, they adsorb to heparin having a sulfonic acid group. Next, when the column is washed and then eluted with a concentration gradient of saline, the two are separately separated and eluted. The eluate from which both have been separated is ultrafiltered to remove the electrolyte and low molecular weight compounds, and then freeze-dried to powder.

Another protein is separated by repeating column chromatography using various carriers,
It is also known to purify lactoferrin and lactoperoxidase (JP-A-61-145200, JP-A-61-246).
No. 198, JP-A-62-19523 and International Publication WO 89/04608
No.).

However, mammalian milk is relatively expensive and is not suitable as a raw material for producing these bioactive milk proteins. In order to separate and purify lactoferrin and lactoperoxidase, it is very easy to use milk, which is the most ubiquitous mammalian milk, but it is important to separate cheese, butter and other milk components from milk. By-products, whey or skim milk, etc., produced at this time are suitable as raw materials. Among the dairy by-products including lactoferrin and lactoperoxidase, by-products are cheese whey, a by-product of producing cheese from milk. Therefore, cheese whey is the most suitable raw material for efficiently producing lactoferrin and lactoperoxidase. Similar to the method of separating lactoferrin and lactoperoxidase from mammalian milk, an appropriate adsorbent and elution conditions can be selected, and both can be separated from other milk components by column chromatography. That is, when whey is allowed to flow down to a weakly acidic ion exchange resin (Na ⁺ type) column near pH neutral, lactoferrin and lactoperoxidase in the whey are adsorbed to the ion exchange resin. After the ion exchange resin is saturated with these proteins, the column is washed with pure water and the adsorbed proteins are eluted with dilute saline. The eluate is collected, the ultraviolet absorption of the protein is measured, and the fraction from which the protein has been eluted is collected. The protein solution is treated with an ultrafiltration membrane having a molecular weight cut off of 20,000 daltons to remove water, electrolytes and low molecular weight compounds, and the concentrated protein solution is freeze-dried and powdered. The average composition of the protein powder thus prepared is 60-75% by weight of lactoferrin, 10-15% by weight of lactoperoxidase.
It is. Next, using a column filled with heparin-Sepharose, lactoferrin and lactoperoxidase are adsorbed to heparin having a sulfonic acid group, and after washing the column, elution is performed with a concentration gradient of saline. . The eluate from which both have been separated is ultrafiltered to remove the electrolyte and low molecular weight compounds, and then freeze-dried to powder.

[0013] Acid whey, which is a by-product of producing casein from skim milk, can also be suitably used in the same manner as cheese whey.

Lactoferrin and lactoperoxidase present in living organisms are iron-containing proteins,
Both have iron saturations of 20-30%. By adjusting the purified lactoferrin and lactoperoxidase to pH 3.0 or less, the apolactoferrin and apolactoperoxidase apoprotein devoid of iron ions can be obtained.
In addition, in the case of both apoprotein and 20-30% iron-saturated lactoferrin and lactoperoxidase, addition of a calculated amount of ferric ion (lactoferrin) or ferric ion (lactoperoxidase) in the presence of carbonate causes an increase in iron. Lactoferrin and lactoperoxidase with various iron saturations up to the holoprotein of hololactoferrin and hololactoperoxidase, which are ion-saturated, can be prepared. Lactoferrin and lactoperoxidase having these various iron saturations can be effectively used as agents for preventing and treating pathogenic infections of the present invention.

The isolated and purified lactoferrin and lactoperoxidase may be used alone or as a mixture, as long as the antibacterial activity is assayed in vitro at 1,000 μM.
High concentrations of g / ml do not inhibit the growth of pathogenic bacteria. However, when using eel, yellowtail, red sea bream, black porcupine, flounder, flounder, coho salmon, rainbow trout, prawns, prawns, prawns, and the like, bred in a fish cage or a culture pond using a compound feed for each fish species mixed with lactoferrin, Significant effects on viability and yield. Table 1 shows the survival rate and yield when the feed containing the agent for preventing and treating pathogenic bacteria of the present invention was added to cultured animals for two months before shipment. In these aquatic animal test plots, the environmental conditions were set as equal as possible, and the control plot was fed with normal feed, and the lactoferrin-treated plot was fed with the same feed as the control plot mixed with lactoferrin at 0.03% by weight. Was. As is clear from Table 1, the addition of lactoferrin has an effect of significantly increasing the survival rate and significantly increasing the yield.

Table 1 Effect of lactoferrin on survival rate of cultured aquatic animals Animal species Control feed Lactoferrin-containing feed Survival rate (%) Yield (Kg) Survival rate (%) Yield (Kg) Yellowtail 55.3 133.5 95.9 ^* 277.8 Red sea bream 43.8 107.1 87.0 ^* 246.8 flounder 47.2 566.4 82.8 ^* 1152.6 eel 25.6 87.5 77.4 ^* 317.4 prawn ^{25.6 22.5 97.4 * 85.6 * X 2} P in the assay <0.01

As is clear from Table 1, even when lactoferrin is added to feed alone and ingested by cultured fish, it significantly improves the yield in aquaculture and also increases the yield in comparison with the control group. is there.

Table 2 shows the results obtained by breeding yellowtail, red sea bream, and prawns for two months before shipment on a feed to which lactoferrin, a lactoperoxidase mixture and lactoperoxidase alone were added, and examining the effect on the survival rate. .

Table 2 Effect of lactoferrin (LF) and lactoperoxidase (LPO) on the survival rate and yield of cultured aquatic animals Animal species Survival rate (%) Control feed LF + LPO mixed feed LPO mixed feed Yellowtail 67.8 88.3 85.3 75.4 90.0 91.6 Kuruma shrimp 39.4 82.9 70.0 LF + LPO mixed feed: LF, 0.013% by weight +
LPO with 0.033% by weight LPO mixed feed: LPO with 0.015% by weight added to control feed

The lactoferrin, lactoperoxidase and a mixture of the two in the present invention can be added to an artificial compound feed and a compound feed prepared at a farm. For example, as artificial feed for fish farming, fish meal, casein, squid meal, powdered whey, other animal raw materials, soybean meal, flour, α-starch, dried yeast, other vegetable raw materials,
After mixing with raw materials such as animal fats and oils, vegetable fats and oils, vitamins, minerals and antioxidants, they are kneaded well with a small amount of water, then extruded and granulated, dried at low temperature and granulated. Can be The reason for avoiding drying due to high temperature is to prevent heat denaturation of lactoferrin and lactoperoxidase.

[0021] Further, a feed mixture containing an antibiotic or an antibacterial substance, which is currently used for the prevention and treatment of pathogenic bacterial infections in cultured aquatic animals, to which the agent for preventing and treating pathogenic bacterial infection of the present invention is added. Has been found to act synergistically with antibiotics or antibacterials when given to cultured aquatic animals and to exhibit a marked effect compared to when given antibiotics or antibacterials alone.

The agent for preventing and treating pathogenic bacterial infections of the present invention is usually used by adding 0.0001 to 1.0 weight of the mixed feed to the mixed feed, and exhibits excellent effects. Hereinafter, the present invention will be described in detail with reference to Examples.

[0023]Reference example Production of blended feed pellets  30% by weight of squid meal, 20% by weight of krill powder, 15 layers of fish meal
%, Dry yeast 10%, wheat gluten 10%, wheat
Powder 5% by weight, Mineral mixture 5% by weight, Vitamin mixture
Lactate with a purity of 85% by weight to 100kg of powder consisting of 5% by weight
Add 1kg of whey protein containing 1% by weight of ferrin powder
And mixed well. Add a small amount of water to this powder and knead
3mm diameter, 15-20 length
It was formed into cylindrical pellets of mm and provided for practical use.

[0024]Example 1  A treatment for tuberculosis infecting yellowtails raised in a culture cage
The effect of ctoferrin was examined. Common nodular disease occurs frequently
Three fish cages with a height of 1.4m and a depth of 1.5m
Each containing 600 yellowtails, milk-derived native
A mixture of lactoferrin of 0.01% by weight and 0.03% by weight
From June 1st to July 31st, 1988, with the formula feed
Bred for up to 2 months. During this time, every day
The number of dead tails due to tuberculosis was investigated, and the
3 is shown.

[0025] In Table 3 cages cumulative death tail number test section rearing Buri cumulative death tail number Shioritsu (%) control group 453 75.5 Lactoferrin 0.01 wt% addition group 121 ^* 20.2 Lactoferrin 0.03 wt% addition group 87 ^* 14.5 * X ² test P <0.01

As shown in Table 3, in the control group, the number of dead cats due to tuberculosis exceeded 75%, while in the test group, 0.01% by weight of lactoferrin was added to the same feed and 0.03%.
It can be seen that the number of dead tails decreased in a dose-dependent manner in the test group to which was added.

[0027]Example 2  For streptococcal disease infecting yellowtail bred in a culture cage
The effect of lactoferrin was examined. Streptococcal disease occurs frequently
Three fish cages 1.4 m in length and 1.5 m in depth at a fish farm
And house 300 yellowtails in each
Feeding lett feed from June 11, 1988 to July 30, 1988
For 50 days. Test plot 1 consisted of a group fed with a control diet.
Test plot 2 is the same dry pellet for yellowtail as test plot 1.
0.3% by weight of erythromycin was added to
Erythromycin on the same dry pellet for yellowtail as in Test plot 1
0.3% by weight of syn and milk-derived native lactoferrin (LF)
0.03% by weight was added to give. Every day, a chain ball per fish cage
The number of dead tails due to mycosis was investigated, and
It was shown to.

Table 4 Cumulative number of dead tails of caged yellowtail breeding test group Cumulative number of dead tail Dead rate (%) Control group 165 55 Erythromycin 0.3% by weight added section 89 ^** 30 Erythromycin 0.3% by weight, LF, 0.03% by weight added section 33 ^** 11 ** P <0.01 in X ² test

As shown in Table 4, in the control group, the death rate due to streptococci was 55%, while in the test group in which 0.3% by weight of erythromycin was added to the same feed, the death rate was 30%. Diminished. On the other hand, erythromycin 0.3% by weight
In test group 3 in which 0.03% by weight of lactoferrin was used in combination, the mortality was further reduced to 11%, and the difference between test group 2 and test group 3 was statistically highly significant. Thus, it is shown that lactoferrin acts synergistically with erythromycin to prevent streptococcus in yellowtail.

[0030]Example 3  About 500m^TwoAquaculture created by introducing seawater to the remains of salt fields
Divide the pond evenly with nylon net, and
The test area was used for shrimp. On April 10, juvenile shrimp about 4cm long
M^Two20 ponds per pond, and one pond is used as a control.
Give a mixed feed, and the other one is milk-derived on the same mixed feed as the control group
Unmodified lactoferrin was added to give 0.01% by weight. 6
The number and yield of surviving shrimp ceased after a month
Was measured.

[0031] Table 5 Effect test group of lactoferrin on the yield of prawn survival head count / m ² yield ^{(kg / m 2) P <} 0.01 in the control group 4.68 0.455 lactoferrin silage 17.30 ^* 1.56 ^* X ² test

As shown in Table 5, the number of surviving animals per unit area was 3.7 times and the yield was
It increased by 3.4 times, and the mortality prevention effect of lactoferrin addition is clear.

[0033]Example 4  Milk-derived native lactoferrin in powdered feed for eels
And lactoperoxidase mixture (lactoferrin 60
Weight%, lactoperoxidase 15 weight%, remaining 25 weight
(% Is other milk protein) 0.03% by weight and add water
The dough was prepared using a dough feeding machine. As a control diet
Uses the same eel powdered feed as the above
I got it.

For eel breeding, the control group and the test group used the same volume of water tank at a water temperature of 29-31 ° C., and housed 1,000 eels each having an average body weight of 105 g.
Performed for 40 days in the range of 2.0%. The results are as shown in Table 6.

Table 6 Effect of lactoferrin + lactoperoxysidase on eel culture yield and body weight Control plot Test plot Yield (%) 93.8 98.7 ^* Body weight (g) 157 ± 8.2 169 ± 6.4 ^** Total weight (kg) 147.3 166.8 * P <0.01 in Student's t-test ** P <0.01 in X ² test

[0036]

As shown in the above examples, the present invention activates the biological defense mechanisms of fish and crustaceans by adding unmodified lactoferrin and / or lactoperoxidase to feeds. In addition, by providing means for preventing and treating pathogenic bacterial infections in cultured aquatic animals, it can contribute to the development of aquaculture and fisheries. Lactoferrin and lactoperoxidase, or a mixture of both, can themselves prevent and treat pathogenic bacterial infections in cultured aquatic animals, but they do not contain antimicrobial actives, such as β
When lactam antibiotics and tetracycline antibiotics coexist, the effects of these antibacterial active substances are synergistically enhanced, which is more effective for the prophylactic treatment of pathogenic bacterial infections.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification code FI A61P 31/04 171 A61K 37/50 (56) References JP-A-61-83131 (JP, A) JP-A-3-220130 ( JP, A) JP-A-2-48534 (JP, A) JP-A-60-227641 (JP, A) JP-A-59-63147 (JP, A) JP-B-46-39046 (JP, B1) JP-B 47-20955 (JP, B1) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/00-38/58 A23K 1/00-1/175 A23K 1/20-3/04 CA (STN) MEDLINE (STN)

Claims (10)

(57) [Claims] 1. An agent for preventing and treating pathogenic bacterial infection of cultured aquatic animals, which comprises unmodified lactoferrin, lactoperoxidase or a mixture thereof and is added to a feed mixture for cultured aquatic animals. 2. The agent according to claim 1, wherein the lactoferrin and lactoperoxidase are lactoferrin and lactoperoxidase isolated from animal milk. 3. The milk of the animal is human, cow, sheep, goat, horse, dog or cat milk.
2. The agent for preventing and treating pathogenic bacterial infection according to item 1. 4. The method according to claim 1, wherein the lactoferrin and lactoperoxidase are lactoferrin and lactoperoxidase at various iron saturation stages from a holoprotein saturated with iron ions to an apoprotein completely removed from iron ions. The agent for preventing and treating pathogen infection according to any one of claims 1 to 3. 5. The pathogenic bacterial infection according to claim 1, wherein the compound feed contains an antibiotic or a synthetic antibacterial agent having antibacterial activity against pathogenic bacteria in cultured aquatic animals. Prophylactic and therapeutic agents. 6. The composition according to claim 1, comprising 0.0001 to 1.0% by weight, based on the weight of the formula feed, of said native lactoferrin, lactoperoxidase or a mixture thereof.
The agent for preventing and treating pathogenic bacterial infection according to any one of claims 1 to 4. 7. The agent for preventing and treating pathogenic bacterial infection according to claim 1, wherein the cultured aquatic animal is a fish cultured in a fish cage or a culture pond. 8. The agent according to claim 7, wherein the fish is eel, yellowtail, red sea bream, black porgy, flounder, coho salmon or rainbow trout. 9. The agent for preventing and treating pathogenic bacterial infection according to claim 1, wherein the cultured aquatic animal is a crustacean cultured in a culture pond. 10. The agent for preventing and treating pathogen infection according to claim 9, wherein the crustacean is a prawn, a prawn or a prawn.