JP2010077056A - Infectious disease prophylactic - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a preparation and a feed that simply prevent infectious diseases at a low cost and enhance immunoactivities without using an antibiotic and a vaccine. <P>SOLUTION: The infectious disease prophylactic comprises a component obtained by mixed culture of useful microorganisms. The useful microorganisms are lactic acid bacteria, yeast fungi and Bacillus natto. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an infectious disease preventive agent and a feed containing the same. More specifically, the present invention relates to an infectious disease preventive agent or immune activity enhancer containing a component obtained by mixing and culturing a plurality of useful bacteria, and a feed containing them.

Problems that fish farmers have in recent years include the occurrence of infectious diseases due to the breeding of large numbers and high density breeding, and the harmful effects of the use of antibiotics. Antibiotics are concerned about the establishment of resistant bacteria in the aquatic environment and the impact on the environment, while food safety is attracting attention, and their use is limited due to the higher cost.
As a typical disease, atypical Aeromonas salmonicida, which is called puncture disease (edible goi, Nishikigoi, goldfish, etc.), head ulcer disease (eel), ulcer disease (salmon, trout, etc.) is infected. Examples include bacterial infections that occur. For example, in the Japanese swordfish, the scales protrude and peel off due to the puncture disease, and the muscles of the fish are exposed to other illnesses, or the scales are red and ulcerated (perforated) after death, resulting in death. Measures are needed. At present, however, there are fish vaccines against the four types of diseases described above, but no effective vaccine against puncture disease has been developed.
On the other hand, by improving the balance of microorganisms in the intestines, the animals can ingest probiotics defined as microorganisms (live bacteria additives) that are beneficial to the host animal, and enhance the body's defenses. The method of making it hard to get sick has also been reported (nonpatent literatures 1-3). However, the method of ingesting propiotics requires viable bacteria, and in order to acquire an ecological defense effect, it requires ingestion of a large amount of viable bacteria.
On the other hand, there is a report that a component obtained by mixing and culturing a plurality of useful bacteria is used as an immune enhancer in animals such as dogs and cats (Patent Document 1, Non-Patent Documents 4 and 5). However, there is no known effective treatment for fish infections, particularly infections caused by atypical Aeromonas salmonicida.

Japanese Unexamined Patent Publication No. 2007-117083 Balcazar, JL, de Blas, I., Ruiz-Zarzuela, I., Vendrell, D., Girones, O. and Muzquiz, JL Enhancement of the immune response and protection induced by probiotic lactic acid bacteria against furunculosis in rainbow trout (Oncorhynchus mykiss). FEMS Immunol. Microbiol., 51: 185-193, 2007 Brunt, J. and Austin, B. Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish Dis., 28: 693-701, 2005 Nikoskelainen, S., Ouwehand, A., Salminen, S. and Bylund, G. Protection of rainbow trout (Oncorhynchus mykiss) from furunculosis by Lactobacillus rhamnosus. Aquaculture, 198: 229-236, 2001 Biagi, G., Cipollini, I., Pompei, A., Zaghini, G. and Matteuzzi, D. Effect of a Lactobacillus animals strain on composition and metabolism of the intestinal microflora in adult dogs.Vet. Microbiol., 124: 160 -165, 2008 Marshall-Jones, Z. V., Baillon, M. L. A., Croft, J. M. and Butterwick, R. F. Effects of Lactobacillus acidophilus DSM13241 as a probiotic in healthy adult cats. Am. J. Vet. Res., 67: 1005-1012, 2006

  An object of the present invention is to provide a preparation and a feed that can prevent infectious diseases easily and at low cost without using antibiotics or vaccines and enhance immune activity.

As a result of intensive studies to solve the above problems, the present inventors have found that components obtained by mixing and culturing useful bacteria enhance immune activity and prevent infectious diseases. As a result, the present invention has been completed.
That is, the present invention
[1] Infectious disease preventive agent containing a component obtained by mixing and culturing useful bacteria,
[2] The preventive agent according to [1], wherein the useful bacteria are lactic acid bacteria, yeasts, and natto bacteria,
[3] The preventive agent according to [2], wherein the lactic acid bacterium is Lactobacillus paracasei, the yeast is Saccharomyces cerevisiae and Pichia membranfaciens, and the natto bacterium is Bacillus pamilus,
[4] The preventive agent according to any one of [1] to [3], wherein the infectious disease is an infectious disease caused by Aeromonas salmonicida,
[5] The preventive agent according to [4], wherein Aeromonas salmonicida is atypical Aeromonas salmonicida,
[6] The preventive agent according to any one of [1] to [5], wherein the infectious disease is a fish infectious disease,
[7] The preventive agent according to [6], wherein the fish is a freshwater fish,
[8] Fish feed containing the preventive agent according to any one of [1] to [7],
[9] An immune activity enhancer containing a component obtained by mixing and culturing useful bacteria,
[10] A method for preventing infectious diseases comprising a step of administering an effective amount of a component obtained by mixing and culturing useful bacteria to an animal other than a human,
About.

According to the present invention, components obtained by mixing and culturing useful bacteria enhance the immune activity of animals, and effective against infectious diseases at a bacterial count of 1 / 10,000 to 1 / 100,000 of probiotics. Because it exerts, infectious diseases can be prevented with a small amount of ingestion. That is, since it is not necessary to consume a large amount, it is economical and advantageous for breeding and aquaculture. Furthermore, since the component does not require viable bacteria, it is easy to manage, the quality is kept constant, and it can be easily added to foods, animal feeds and the like. The above ingredients have a mouse LD ₅₀ of 2.0 g / Kg or more, and have no safety problems as supplements or feed additives for humans, pets, livestock, etc., and can be taken safely and for a long time. It is.

  The present invention relates to an infectious disease preventive agent containing a component obtained by mixing and culturing useful bacteria (also referred to as the preventive agent of the present invention).

  The useful bacteria in the present invention are not particularly limited as long as they do not adversely affect the living body, but mean healthy bacteria that have a favorable influence on the living body. Specific examples include lactic acid bacteria, yeasts, natto bacteria, and koji molds. Preferred are lactic acid bacteria, yeasts, and natto bacteria.

  The lactic acid bacteria in the present invention are not particularly limited as long as they are bacteria involved in lactic acid fermentation, but include Lactobacillus, Bifidobacterium, Enterococcus, and Lactococcus. The bacteria to which it belongs. Of these, the genus Lactobacillus is preferable, and Lactobacillus paracasei is more preferable.

  The yeast in the present invention is not particularly limited as long as it is a fungus that is mostly a single cell in the life cycle and proliferates mainly by budding, but includes bacteria belonging to the genus Saccharomyces and Pichia. Of these, Saccharomyces cerevisiae and Pichia membranifaciens are preferable.

  The Bacillus natto in the present invention is not particularly limited as long as it belongs to the genus Bacillus, but Bacillus pumilus is preferable.

  The lactic acid bacteria, yeasts, and natto bacteria of the present application are preferably obtained by screening a sample collected from soil near the hillside at an altitude of about 1500 m at the prefectural border between Tottori Prefecture and Hyogo Prefecture.

  The component obtained by mixing and culturing useful bacteria in the present invention (also referred to as component in the present invention) is a component obtained by mixing and culturing the useful bacteria, that is, a component obtained from a culture obtained by co-fermenting the useful bacteria. It is. That is, examples include live bacteria of the above-mentioned useful bacteria, dead bacteria after heating, secreted products secreted by useful bacteria into the culture medium during culture, mixtures thereof, and components obtained therefrom. Among them, a culture solution obtained by mixing and culturing useful bacteria (also referred to as a mixed culture solution in the present invention), and a solution obtained by removing dead bacteria from the mixed culture solution are preferable. The mixed culture solution preferably contains a soluble component obtained after heat treatment and killed bacteria.

Examples of the medium for mixed culture in the present invention include a medium containing rice bran, water, glucose, honey, mash, malt juice, meat extract and the like. A medium containing rice bran, water, and glucose is preferable.
Although the kind of rice bran which is a composition component of a culture medium is not specifically limited, For example, what generate | occur | produced when brown rice is polished can be used. The ratio of rice bran, water and glucose is usually 1: 10-40: 0.5-4, preferably 1: 10-30: 0.5-3, and more preferably 1: 20: 1. In addition, other components useful for culturing useful bacteria may be added to the medium.

An embodiment of a method for producing a component in the present invention will be described.
A medium for the mixed culture of the lactic acid bacteria, yeast and natto is prepared (manufactured) as follows. First, rice bran and water are mixed and the mixture is filtered. Glucose is added to the mixture (filtrate), the mixture is heated and the mixture is filtered. Further, water is added, sterilized and cooled, and the obtained medium is subjected to mixed culture.

Next, the above three types of bacteria are cultured in the obtained medium as follows to obtain a mixed culture solution.
Add bacteria to the medium. Primary culture is performed by aeration culture. The culture temperature is usually 32 to 40 ° C, preferably 35 to 37 ° C. The culture time is 36 to 60 hours, preferably 40 to 55 hours.

  The secondary culture is performed by cold aging. The culture temperature is usually 2 to 15 ° C, preferably 5 to 8 ° C. The culture period is 30 to 60 days, preferably 40 to 50 days.

  The medium obtained by the secondary culture is sterilized. The sterilization method is not particularly limited, but is usually performed by heat sterilization, electromagnetic wave sterilization, filter sterilization, or the like. Among them, heat sterilization is preferable. In the case of heat sterilization, the sterilization temperature is usually 110 to 130 ° C, preferably 120 to 121 ° C. The sterilization time is 10 to 60 minutes, preferably 16 to 17 minutes. The component obtained in the present invention is a culture solution obtained after sterilization or one obtained by filtering to remove cells (dead bacteria).

  The infectious disease in the present invention is an infectious disease caused by bacteria, such as atypical Aeromonas salmonicida, typical Aeromonas salmonicida (causal symptom causing fungus), Aeromonas hydrophila (causing fungus), Flavobacterium psychrophilum (causing bacterial mania) ) And Flavobacterium columnare (causal agent of columnaris disease). Among these, the preventive agent of the present invention is effective for infectious diseases caused by atypical Aeromonas salmonicida. Infectious diseases caused by atypical Aeromonas salmonicida have different names depending on the affected fish, and infectious diseases in the present invention include edible diseases such as edible goi, seigogoi, goldfish, eel head ulcer disease, salmon, trout, etc. Including ulcer disease.

  The subject of the preventive agent of the present invention is not particularly limited as long as it is an animal, but a subject suffering from an infection caused by atypical Aeromonas salmonicida is desirable. Usually, it is effective for fish and the like, preferably fish, and more preferably freshwater fish. Especially, it is effective for edible goi, Nishikigoi, goldfish, eel, salmon, trout, etc., and edible goi, Nishikigoi, goldfish are preferable.

  There is no particular limitation on the administration form (intake form) of the prophylactic agent of the present invention, and parenteral administration (intake) such as immersion, oral administration or injection administration (intraperitoneal administration, etc.) may be used. Is a component obtained by mixing and culturing useful bacteria, and is preferably administered orally. Oral agents include granules, fine granules, powders, coated tablets, tablets, suppositories, powders, (micro) capsules, chewables, syrups, juices, liquids, suspensions, emulsions, etc. As an injection, a general dosage form such as an intraperitoneal administration or subcutaneous injection can be employed, such as a form added to a water tank for fish.

  The preventive agent of this invention can be prepared by formulating by a conventional method. For example, the mixed culture solution can be processed into a powder and used, for example, as a skin external preparation for humans or pets. Alternatively, the powder can be processed without removing the cells and used as a supplement for pets such as humans, dogs and cats, and a supplement for livestock such as cows, pigs, horses and chickens. Furthermore, these can also be used as a liquid without powder processing. Moreover, the said external preparation and supplement can mix | blend various pharmacologically acceptable substances for a formulation (as adjuvants etc.) as needed on a formulation. The substance for the preparation can be appropriately selected depending on the dosage form of the preparation. For example, the excipient, the diluent, the additive, the disintegrant, the binder, the coating agent, the lubricant, the lubricant, the lubricant, and the flavoring agent. , Sweeteners, solubilizers and the like.

  In addition, when orally administering the prophylactic agent of the present invention to fish, a form usually contained in feed is desirable. However, in order to prevent the above-mentioned components from flowing out of the feed into the water, the above-mentioned components are coated with an ester compound, or prepared using a known formulation technique such as a water-in-oil emulsion that does not disperse in water or liposomes. May be. In addition, in order to make it difficult to dissolve in water, an adhesive (viscous agent) such as carboxymethylcellulose, sodium alginate, starch, and polyallyl agent is mixed together with fish moist pellets (raw fish fillet loosely hardened). The resulting floating bait can be mentioned.

In the preventive agent of the present invention, in the case of oral administration (ingestion), the dose varies depending on the subject to be administered, the symptom of the subject, the state of the subject, the administration method, etc. × 1 / 1,000,000 / day to body weight × 1/500 / day, preferably body weight × 1 / 100,000 / day to body weight × 1/1000 / day, more preferably body weight × 1/70000 / day to body weight × 1 / Administer about 2000 / day.
For example, in the case of fish, in order to obtain the above-mentioned infection prevention effect by oral administration, the dose, period, and interval vary depending on the season of administration, water temperature, type of fish, and age. (As dry weight) is usually about 0.1 mg / 100 g body weight / day to about 200 mg / 100 g body weight / day, preferably about 1 mg / 100 g body weight / day to about 100 mg / 100 g body weight / day, more preferably about 5 mg / 100 g body weight / day The dose is about 50 mg / 100 g body weight / day, particularly preferably about 5 mg / 100 g body weight / day to 30 mg / 100 g body weight / day. The administration period is not particularly limited, and the administration frequency is 1 to 5 times / day, preferably 1 to 2 times / day.

  Another aspect of the present invention is a fish feed containing the preventive agent of the present invention. Fish feed can be prepared by adding conventional additives to the preventive agent of the present invention. Specifically, dry pellets (dried solid type, feed mainly molded from fish meal), extruder pellets (feed formed by high-temperature and high-pressure processing using a granulator called an extruder) ), A feed for fish in which the preventive agent of the present invention is added to moist pellets (raw fish fillet loosely hardened).

  As another embodiment of the present invention, an immune activity enhancer containing components obtained by mixing and culturing useful bacteria can be mentioned. The conditions such as the components and dosage of the immune activity enhancer of the present invention are as described above. By ingesting the agent, lymphocytes are activated and immune activity is enhanced. As a result, the body defense power increases and it becomes difficult to suffer from various diseases. The administration target of the immune activity enhancer of the present invention is not particularly limited, and examples thereof include mammals including humans, birds, fishes / reptiles (turtles), and the like.

  The present invention provides a method for preventing infectious diseases comprising the step of administering an effective amount of a component obtained by mixing and culturing useful bacteria to an animal. An effective amount refers to an amount sufficient to provide the desired prophylactic effect and is approximately the same as the dose described above. According to the prevention method of the present invention, infectious diseases can be prevented without using antibiotics or vaccines by enhancing immunity.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these at all.

(Reference example)
[Production of medium]
In the production of the medium, 450 g of fresh rice bran and 9 L of water were first mixed and stirred for 1 to 1.5 hours to extract a 5% by weight aqueous solution of rice bran. Next, this rice bran mixture was filtered through a cotton cloth. In addition to the cotton cloth, for example, a suction machine or a filter press can be used for the filtration. Then, the filtered residue (slag) after filtration was removed and 450 g of glucose was added to the filtrate, and further heated at 100 ° C. for 10 minutes to prepare a mixed solution. Next, the heated mixed solution was filtered with a cotton cloth. In this case, a suction machine or a filter press can be used in addition to the cotton cloth. The filtration residue after filtration was removed, and the filtrate was supplemented with about 3 L of water to prepare a 9 L mixed solution. This solution was sterilized at 121 ° C. for 17 minutes, and then cooled to obtain a medium. As raw rice bran, what was generated when brown rice was polished was used. Furthermore, tap water was used as water.

[Manufacture of culture solution]
In the production of the culture solution, first, the medium obtained as described above was added to Lactobacillus paracasei, Saccharomyces cerevisiae, Pichia membranifaciens and Bacillus pamilus. Four types of bacteria (Bacillus pumilus) were added and mixed. Next, aeration culture was performed at 37 ° C. for 48 hours (primary culture). After the primary culture, cold aging was performed at 5 ° C. for 1.5 months (secondary culture). After this secondary culture, the mixture was sterilized at 121 ° C. for 17 minutes, and then filtered to remove the cells to obtain a mixed culture solution. This mixed culture was stored refrigerated at 5 ° C.

(Example)
material
LBS culture (trade name of Rtanial Biosciences Co., Ltd., fermentation broth containing components obtained by mixing and cultivating useful bacteria described in this specification) is a cell after heat treatment described in Reference Examples. What was not removed was used.
The carp feed used in Experiments 1 and 2 (Hikari Germ, Kyorin Co., Ltd.) was commercially available, and the LBS culture was added to the feed and molded.
In Experiment 3, the carp feed was mixed with wheat germ, wheat flour, shrimp meal, brewer's yeast, skim milk powder, soybean cake, roussan meal, various vitamins, various minerals, antioxidants, digestive enzymes, and kneaded with an extruder. What was molded and dried was used. For the experiment, a mixture of the above materials mixed with LBS culture was kneaded, processed by an extruder and dried.

Test method
The following experiment was performed under different conditions of LBS culture administration concentration and water temperature.

(Experiment 1) Experiment in which the physiological state of carp is slightly lower than the optimal condition and the growth of atypical A. salmonicida is below the optimal temperature
Carp feed supplemented with 5% LBS culture was fed twice daily for 32 days. One group of 10 carp (Nishigoi, male and female unselected) was used, the average body weight at the start of the experiment was 32 ± 5 g, and the daily feed dose was 0.3 g / animal. A feed supplemented with 5% of the medium used for LBS culture production shown in Reference Examples and a normal feed with no addition were used as negative controls.
The breeding water temperature was 18 ° C at the start of the experiment, 13 ° C at the time of A. salmonicida infection, and 10 ° C at the end of the experiment.
On the 32nd day after the start of the experiment, a highly poisonous atypical A. salmonicida T1031 strain (distributed from Niigata Prefectural Water Surface Laboratory) was immersed in water with a concentration of 6.7 x 10 ⁶ / ml for 1 hour. Infected with salmonicida.
After infection, the carp were observed for life and death and lesions on the body surface for 33 days, and the dead fish were isolated from the lesion and internal organs. After the experiment was completed, macroscopic lesions of the surviving fish were observed, and bacteria were also isolated. LBS culture supplemented feed and control feed were administered throughout the experimental period.

In control fish with medium or LBS culture non-added diets, localized swelling of the body surface, bleeding and skin erosion were observed 6 days after infection. Table 1 shows macroscopic lesions of carp 33 days after infection, and FIG. 1 shows changes in gross lesion scores. After 9 days from the infection, the bleeding lesion became extensive and severe, and the skin lesion showed a so-called 'perforated' state. The number of dead fish on day 33 of infection is 1 and 2 in each group due to the slow growth of the fungus due to the low water temperature. A. salmonicida was isolated from the surface bleeding lesions and ulcers of some fish.
In contrast, in carp fed with a diet containing 5% of LBS culture, no skin lesions were observed or slight and no fish died.
No bacteria were isolated from the skin and intra-abdominal organs of surviving fish across all groups.

(Experiment 2) Experiment on the physiological condition of carp and the growth of A. salmonicida in the optimal water temperature range
Feeds supplemented with 3%, 0.5%, or 0.1% LBS culture were fed twice daily for 25 days. The average body weight at the start of the experiment of 10 carp (Nishigoi, male and female unselected) per group was 30 ± 4 g, and the daily dose of feed was 0.3 g / animal. LBS culture-free feed was used as a negative control.
The breeding water temperature was 15 ° C. at the start of the experiment, 18 ° C. at the time of A. salmonicida infection, and 20 ° C. at the end of the experiment.
On the 25th day after the start of the experiment, atypical A. salmonicida T1031 strain (distributed from Niigata Prefectural Water Surface Laboratory) was immersed in water with a concentration of 4.1 x 10 ⁶ / ml for 1 hour. A. salmonicida Infected with.
After the infection, carp life and death and surface lesions were observed for 22 days, and bacteria were also isolated. LBS extract supplemented feed and control feed were administered throughout the experimental period.
Significant difference test was performed with χ ² test.

In control fish treated with LBS culture-free diet, localized swelling of the body surface, bleeding and skin erosion were observed 4 days after infection, after which the bleeding lesions were extensive and severe, and some were 'perforated' Was presented. Table 2 shows macroscopic lesions of carp 22 days after infection. The fish died 6 days after the infection, and the survival rate after 22 days was 10% (Fig. 2).
On the other hand, in carp fed with 3% LBS culture, only 2 animals died 10 days after infection and survived throughout the experiment (survival rate 80%) (P <0.01). The skin lesions were milder than the control fish.
On the other hand, 6 (P> 0.05) and 5 (P <0.05) carp fed with 0.5% or 0.1% LBS culture feed were killed, and gross lesions were observed. It was done.
Throughout the four groups, A. salmonicida was isolated from the surface bleeding lesions and ulcers of some fish and from dead fish.

(Experiment 3) Experiment at water temperature where the physiological condition of carp is at the optimum condition, and at a temperature where A. salmonicida grows slightly higher than the optimum range
An experiment similar to Experiment 2 was performed using the above-mentioned feed as an LBS culture-added feed.
Feeds supplemented with 3%, 0.5%, or 0.1% LBS culture were fed twice daily for 25 days. The average body weight of the group of 10 carp at the start of the experiment was 31 ± 6 g, and the daily feed dose was 0.3 g / animal. LBS culture-free feed was used as a negative control.
The breeding water temperature was 22 ° C at the start of the experiment, 23 ° C at the time of A. salmonicida infection, and 28 ° C at the end of the experiment.
On the 25th day after the start of the experiment, the carp was immersed for 1 hour in water at a concentration of 4.2 × 10 ⁶ / ml of A. salmonicida bacteria. After the infection, carp life and death and surface lesions were observed for 32 days, and bacteria were also isolated. LBS extract supplemented feed and control feed were administered throughout the experimental period.
Significant difference test was performed with χ ² test.

In control fish treated with dietary supplements without LBS culture, localized swelling of the body surface, bleeding and erosion of the skin were observed 3 days after infection, after which the bleeding lesions were extensive and severe, and some were 'perforated' Was presented. Table 3 shows macroscopic lesions of carp 32 days after infection, and FIG. 3 shows changes in gross lesion scores. The fish died 18 days after infection, and the survival rate after 32 days was 30% (Fig. 4).
On the other hand, carp fed with 3% LBS culture survived throughout the experimental period (P <0.01, one animal died from a jumping out of the aquarium and tested in 9 animals), and the degree of skin lesions was also control fish. It was mild compared to.
Only 1 carp (P> 0.01) died by the end of the experiment.
In contrast, carp fed with 0.1% LBS culture feed died by the end of the experiment.
Throughout the four groups, A. salmonicida was isolated from the surface bleeding lesions and ulcers of some fish and from dead fish.

  From the results of this experiment, it was clarified that feeding the carp with LBS culture supplements has a protective effect against atypical A. salmonicida infection. In particular, the formation of skin lesions could be suppressed almost completely at low water temperatures. The combined results of the two experiments showed that the administered LBS culture concentration was effective at 3% or more, and the protective effect was weak at 0.5% or less.

(Experiment 4)
LBS culture (20-fold to 2,560-fold) diluted 2-fold was added to BALB / c mice (10-15 weeks old) spleen lymphocyte culture (1 x 10 ⁵ cells / 0.1 ml, medium RPMI1640 plus 10 fetal calf serum. In addition, the cells were cultured at 37 ° C. for 72 hours. The negative control used the medium used for LBS culture manufacture, and the positive control used concanavalin A.
After culturing for 72 hours, a predetermined amount of Premix WST-1 Cell Proliferation Assay System reagent (Takara) was added, and after reacting at 37 ° C. for 4 hours, absorbance was measured at a wavelength of 450 nm.
The measurement results are shown in a graph with the difference in absorbance when cultured with the medium used for LBS culture production, and a significant difference test was performed by Student's t-test (in the case of Concanavalin A, only the cell culture solution was used) Comparison with culture).
Next, separation of components having lymphocyte activation ability was attempted by gel filtration.
The material used was a concentrated LBS culture. First, bacterial components were removed from the LBS culture by centrifugation, and the supernatant was lyophilized. After re-dissolving in a small amount of deionized water, it was dialyzed against phosphate buffered saline to obtain a concentrated solution finally concentrated to 1/8 of the starting material.
The concentrate was separated using Sephacryl S200. The absorbance of the protein was measured at a wavelength of 280 nm.
A mouse lymphocyte activation test was performed on each fraction.

The LBS culture significantly activated lymphocytes at a dilution of 40-fold to 2560-fold when compared to the negative control medium-only reaction (FIG. 5). The degree of activation was higher than the positive control concanavalin A, which is generally considered to have a superior immune response.
As a result of gel filtration, the component having the ability to activate lymphocytes was separated into two parts having a high molecular weight and a low molecular weight (FIG. 6).

From the above experimental results, it was shown that feeding the carp with an LBS culture-added feed has a protective effect against atypical A. salmonicida infection. That is, the appearance of bleeding lesions and the formation of ulcers were suppressed, and death was further suppressed.
In particular, the formation of skin lesions could be almost completely suppressed at low water temperatures where the bacterial growth was below the optimum water temperature (Experiment 1).
In experiments with water temperature suitable for or close to the growth of A. salmonicida, the survival rate of the carp fed with LBS culture (3% and 0.5%) was significantly higher than the carp fed with no added food ( Experiment 2, 3).

As one of the defense mechanisms, it is speculated that the basic immunity of carp is enhanced by LBS culture. Since there is no reliable carp lymphocyte activation test method at this time, a lymphocyte activation test was conducted using mice in this test. As a result, it was shown that LBS culture activates mouse lymphocytes (Experiment 4). It is effective in a wide range of 40 times to 2,560 times, and the degree of activation is higher than that of the positive control concanavalin A, indicating that the effect is strong.
As a result of gel filtration, the active ingredient was separated into two main parts, high molecular weight and low molecular weight. This experiment is the result of a test using a material from which bacterial components have been removed, which indicates that only the LBS culture soluble component has the ability to activate lymphocytes.
It was shown that the LBS culture administered as described above is absorbed from the intestinal tract or is present in the blood, so that some physiological change occurs in the body, resulting in a protective effect against infection. Since LBS culture activates mouse lymphocytes, it was suggested that basal immunity (lymphocytes and macrophages) can be enhanced in various animals other than carp to enhance immune activity and protect against infectious diseases.

It is a figure which shows the macroscopic lesion of a carp after atypical Aeromonas salmonicida infection. It is a figure which shows the survival rate of a carp after atypical Aeromonas salmonicida infection. It is a figure which shows the macroscopic lesion of a carp after atypical Aeromonas salmonicida infection. It is a figure which shows the survival rate of a carp after atypical Aeromonas salmonicida infection. It is a figure which shows the lymphocyte activation in a mouse | mouth spleen cell. It is a figure which shows isolation | separation of a lymphocyte activation component.

Claims (10)

  An infectious disease preventive agent containing a component obtained by mixing and culturing useful bacteria.   The preventive agent according to claim 1, wherein the useful bacteria are lactic acid bacteria, yeasts and natto bacteria.   The preventive agent according to claim 2, wherein the lactic acid bacterium is Lactobacillus paracasei, the yeast is Saccharomyces cerevisiae and Pichia membranifiens, and the Bacillus natto is Bacillus pamilus.   The preventive agent according to any one of claims 1 to 3, wherein the infectious disease is an infectious disease caused by Aeromonas salmonicida.   The preventive agent according to claim 4, wherein Aeromonas salmonicida is atypical Aeromonas salmonicida.   The preventive agent according to any one of claims 1 to 5, wherein the infectious disease is a fish infectious disease.   The preventive agent according to claim 6, wherein the fish is a freshwater fish.   Fish feed comprising the preventive agent according to any one of claims 1 to 7.   An immune activity enhancer comprising a component obtained by mixing and culturing useful bacteria.   A method for preventing infectious diseases, comprising a step of administering an effective amount of a component obtained by mixing and culturing useful bacteria to an animal other than a human.

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