JPH1192390A - Agent for improving stressed state - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject improving agent capable of improving the stressed state of livestock, poultry or the like, capable of exhibiting an excellent effect on the prevention of the deterioration of immunological activity, capable of being produced at a low cost and useful for the livestock, the poultry or the like by using capsule-free bacterial cells as an active ingredient. SOLUTION: This agent for improving a stress state contains (A) capsule- removed bacterial cells (e.g. capsule-free cells obtained by removing capsules from the cells of a bacterium belonging to the genus Bifidobacterium) as an active ingredient. This improving agent is obtained e.g. by suspending 50-100 g of wet bacterial cells, such as wet cells obtained by centrifuging the cells at 3000-8000 rpm for 10-60 min, in 1-10 fold-volume of a 0.05-5 vol.% surfactant solution at room temperature to 60 deg.C for 1-24 hr, washing out the surfactant adhered to the cells with water or a buffer solution by a centrifugal washing method or by an ultrafiltration washing method and subsequently using the obtained cells A as an active ingredient. The improving agent is preferably administered at a daily dose of 10-1000 μg/kg body as the cells A.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a stress condition improving agent for improving the stress condition of livestock and poultry.

[0002]

2. Description of the Related Art At present, the stress of livestock and poultry in animal husbandry is various, such as stress caused by grouping of piglets, transport stress of calves, heat stress, poaching stress, stress due to moulting, birth / spawning, blood sampling, etc. There is stress. It is known that these stress loads make the immune system susceptible to illness and disease and decrease productivity. Therefore, a method for improving stress is desired. As a method for improving the breeding environment, breeding environment, breeding management, and improvement of feed materials are being devised. Academic support of these improvement measures is desired, but it is difficult to objectively introduce stress stimuli by changing the feeding management method into the experimental system, and it is not easy to conduct tests.

On the other hand, under such experimental restrictions,
There are reports on stress caused by environmental temperature and long-term social psychological stress (stress due to psychological suppression based on social rank of strong and weak people in a group) using mice and rats. Regarding the effects of environmental temperature, changes in the percentage of leukocytes, reduction in the thymus / spleen weight ratio, reduction in lymphocyte blastogenesis, reduction in NK activity, and reduction in the proportion of T lymphocytes have been reported. Regarding the effects of social psychological stress, there are reports that the stronger have lower antibody production and the weaker have higher antibody production than normal mice. It has been reported that the embryonic response, NK activity, and IL-2 production are reduced.

Conventionally, L-tryptophan (L-tryptophan literature collection, No. 2 (1987), Showa Denko), vitamin B ₂ (Seii) has been used as an agent for improving the stress state of livestock and poultry.
chiARAKI et al., J. Vet.Med.Sci. 57 (4): 599-602,
1995), tranquilizers and antibiotics (Journal of the Study on Chicken Disease, Vol. 7, 1971). However, the effects of these stress condition improving agents are not always satisfactory and are relatively expensive.

[0005]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a stress condition improving agent which has an excellent stress condition improving effect and can be supplied at low cost.

[0006]

Means for Solving the Problems As a result of intensive studies, the inventors of the present application have found that the bacterial cells from which the capsular membrane has been removed are effective for improving the stress state, and have completed the present invention.

[0007] That is, the present invention provides an agent for improving a stress state, comprising a bacterial cell from which a capsule has been removed as an active ingredient.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, the stress condition improving agent of the present invention comprises, as an active ingredient, a substance obtained by removing a capsular film from cells. The cells may be any of bacteria and yeasts. Bacteria such as Bifidobacterium thermophilum, Bacillus subtilis (Baci subtilis), and the like can be used.
Bacillus bacteria such as Corynebacterium ovis; Corynebacterium bacteria such as Corynebacterium ovis; Actinomyces bacteria such as Actinomyces pyogenes; Lactobacillus acidophilus. (Lactobacillu
Lactobacillus bacteria such as S. acidophilus, Streptococcus faecali
s) and bacteria belonging to the genus Staphylococcus such as Staphylococcus cohnii, and yeasts such as Kluyveromyces lactis and Saccharomyces cerevisiae. Preferred and particularly preferred are bacteria of the genus Bifidobacterium.

The cells may be dead cells or live cells if they are not pathogenic.

The stress condition improving agent of the present invention can be produced by treating cells with a surfactant, and does not require another enzyme treatment or the like. The treatment with a surfactant can be performed by suspending the cells in a surfactant solution, or by suspending the cells in water or a buffer in advance and adding a surfactant thereto. be able to.

As the surfactant, any type of surfactant can be used, but a nonionic surfactant which can be relatively easily removed in a later step is preferable. Commercially available nonionic surfactants can be used, and examples thereof include Tween 20 (trade name) and Triton X-100 (trade name).

When the cells are treated with a surfactant solution, the concentration of the surfactant used is 0.05% to 5% (v / v).
v) is preferred, and more preferably 0.1% to 1.5% (v / v)
It is. The amount of the surfactant solution to be used is determined by the wet cell weight (3000-8000 rpm, centrifuged cells for 10-60 minutes).
The amount is preferably 1 to 10 times the amount (50 to 100 times the amount of dried cells) with respect to 50 to 100 g. When the suspension is previously suspended in water or a buffer, a surfactant can be added at the above ratio after the suspension.

The surfactant treatment can be carried out at room temperature or at a temperature of 30 ° C. to 60 ° C. The processing time is preferably 1 hour to 24 hours. Preferably, the suspension is stirred during the treatment.

After the treatment, it is preferable to carry out centrifugal washing with water or a buffer or washing by ultrafiltration in order to remove the surfactant adhering to the cells.

Bacteria have their own autolytic enzymes and are known to lyse the cell walls of the cells (Katsu Funazu and Daisuke Tsuru, "Lytic Enzymes", page 14, Kodansha, 197).
7 years). For this reason, it is preferable to inactivate the autolytic enzyme and to protect the cell walls of the cells before performing the surfactant treatment. This can be performed by heat-treating the cells. The conditions of the heat treatment are, for example, to completely inactivate the autolytic enzyme and to prevent heat denaturation of the protein of the bacterial cells at 56 ° C to 75 ° C for 15 minutes to 60 minutes, or about 10 minutes in boiling water. Short-time processing is desirable.

The cells from which the capsular layer has been removed by the treatment with a surfactant can be used as they are, or can be used after freeze-drying.

The stress condition improving agent of the present invention can be used for livestock such as pigs, cows, horses, goats, dogs, cats, and rodents, which are raised in a stressed state or are expected to be in a stressed state. , Poultry such as chickens, and freshwater fish such as carp, rainbow trout, sweetfish, eel, tilapia, squid, salmon and trout, and seawater fish such as Thailand, yellowtail, flounder, tiger pufferfish and shrimp. be able to. The stress includes, but is not limited to, stress caused by piglet grouping, calf transport stress, heat stress, poaching stress, stress caused by forced moulting, birth / spawning, blood sampling, etc. is not.

The agent for improving a stress state of the present invention may be added to feed of livestock or the like as it is, or a suspension in water may be orally administered to livestock or the like, or mixed with other excipients and dried. These may be mixed with the feed and administered.

The dose for livestock and the like is appropriately selected depending on the degree of stress, the age and the type of livestock, and the like.
Usually, the body weight is about 10 μg to 1 mg per kg per day.

[0020]

EXAMPLES The present invention will be described below more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1 Preparation of Stress State Improving Agent Bifidobacterium thermophilum
ium thermophilum PNA1-24, distributed by RIKEN
Was cultured in a conventional manner, suspended in 1 liter of 0.05 M phosphate buffer (pH 7.0) per 100 g of the obtained wet cells, and heated at 75 ° C. for 30 minutes to remove the autolyzing enzyme possessed by the bacteria itself. Deactivated. Thereafter, a surfactant (Tween 20) was added at a ratio of 0.5% (v / v), and the mixture was stirred at room temperature for 12 to 15 hours, and then centrifuged at 8000 rpm for 20 minutes. The cells were collected and subjected to centrifugal washing three times to obtain bacterial cells. The stress condition improving agent of the present invention obtained by freeze-drying the cells was used as a feed material in the following Examples.

Example 2 Dexamethasone (hereinafter referred to as "De
x ") in the immunosuppressive situation, the effect of the stress condition-improving agent of the present invention in improving the stress condition. In a stress condition, secretion of adrenocortical hormone is enhanced,
It is known that the immune system is suppressed. In general, an immunosuppressive state can be created by exogenously administering a high-dose corticosteroid preparation, and many immunosuppressive tests have been performed using this experimental system (edited by Akihide Eda: Toxicology Test Lecture (10 ) Immunotoxicity, 70-94, Jinjinshokan, 199
1). In this example, the stress-ameliorating agent of the present invention (hereinafter, sometimes referred to as “PG”) produced in Example 1 was fed to an immunosuppressed animal produced by Dex, The improvement effect was examined.

The immunosuppressive conditions were as follows: BALB / c mice,
It was produced by subcutaneous injection of 200 μg / 0.4 ml of Dex at the age of 7 weeks, and the immunoreactivity was measured 18 hours later.

The PG group (5 animals per group) was fed a feed supplemented with 20 ppm of PG, and the control group (5 animals per group) was fed a non-added feed for 3 weeks and then subjected to immunosuppression treatment under the above conditions. The ratio decreased (FIG. 1), peripheral lymphocyte reduction / granulocyte increase (FIG. 2), and a stress state was confirmed.
Furthermore, lymphocyte blastogenesis (Fig. 3, SASAKI T. et al., En
hanced Resistance of Mice to Escherichia coli Infe
ction Induced by Administration of Peptideglycan d
erived from Bifidobacterium thermophilum, J. Vet.M
ed. Sci. 56 (3) 433-437, measured by the method described in 1994), phagocytic activity of peritoneal macrophages (FIG. 4, SASAKI).
T. et al., Phagocytosis of Splenetic Neutrophils of Mi
ce Enhanced by Orally Administered Peptideglycan f
rom Bifidobacterium thermophilum, J. Vet. Med. Sc
i. 58 (1) 85-86, measured by the method described in 1996), and phagocytic activity of spleen neutrophils (FIG. 5, SASAKI T. et al., Phagocyt.
osisof Splenetic Neutrophils of Mice Enhanced by O
rally Administered Peptideglycan from Bifidobacter
ium thermophilum, J. Vet.Med.Sci. 58 (1) 85-86, 1
(Measured by the method described in 996) was suppressed by Dex injection, but the degree was reduced by feeding PG-added feed. In addition, NK activity was also suppressed by Dex injection, but was reduced by feeding PG-added feed (FIG. 6, SASA
KI T. et al., Enhancement of Cytotoxic Activity of Lym
phocytes in Mice by Oral Administration of Peptide
glycan (PG) Derived from Bifidobacterium thermophi
lum, J. Vet. Med. Sci. 56 (6) 1129-1133, 1994)). As described above, when the PG-added feed was fed for a long time, the immunosuppressive state due to Dex was reduced.

Example 3 Effect of Improving PG under Closely Keeping Conditions At a livestock production site, multi-headed breeding is performed, and animals are often kept close together. Under such circumstances, animals are under stress due to poaching. Therefore, as an experimental model of stress, a stress condition was set by BALB / c mice, 4 weeks old, and males kept close to each other, and the effect of PG on improving the stress was examined. A cage having a floor area of about 432 cm ² was used, and a densely cultivated area (20 animals / cage) and a normal density breeding area (5 animals / cage) were used.

Three weeks after the close keeping, 500 μg of PG was orally administered by gavage once. As a result, a significant increase in the spleen weight ratio was observed in the normal density breeding and densely bred groups by PG administration (FIG. 7). Percentage of peripheral blood cells showed a tendency to decrease lymphocytes and increase granulocytes in a close breeding, but PG did not show a clear improvement effect (FIG. 8). Lymphocyte transformation (Fig. 9), phagocytic activity of peritoneal macrophages (Fig. 1)
0) was suppressed by poaching, but was improved by a single oral administration of PG, and the PG-administered closely-kept group was significantly higher than the non-PG-administered normal density group. The phagocytic activity of spleen neutrophils (FIG. 11) was also suppressed by close keeping, but improved by PG administration. NK activity tended to decrease with close keeping, but no apparent difference was observed (FIG. 12).

From the above, it is confirmed that the immune activity is reduced by the close breeding, but the immunological activity is increased by the single oral administration of PG to a level equal to or higher than that of the normal breeding, and the stress of the close breeding is reduced. Was done.

Example 4 Effect of Long-Term Feeding of PG in Closely Closed Feeding Next, in the method of Example 3, a test was conducted in which the feed supplemented with 20 ppm of PG was fed throughout the period of close keeping.
As a result, there was no effect on the spleen weight ratio due to close keeping (FIG. 13). Peripheral blood cell percentages tended to decrease lymphocytes and increase granulocytes in close breeding (FIG. 14). Although the lymphocyte weakening response tended to be reduced by poaching, it was increased by PG administration.
The results were equal to or higher than those of the non-administered normal density group (FIG. 15). The phagocytic activity of peritoneal macrophages (FIG. 16) and the phagocytic activity of spleen neutrophils (FIG. 17) are reduced by poaching, but are improved by PG administration, and are higher in the PG-administered group than in the non-PG-administered normal density group. The value was significantly higher.
In addition, NK activity was increased by feeding PG-added feed (FIG. 18).

From the above, it has been confirmed that the immune activity is reduced by close breeding, but the feeding of PG-added feed is increased to or higher than that of normal breeding, and the stress of close breeding is reduced.

[0030]

EFFECT OF THE INVENTION The agent for improving a stress state of the present invention comprises livestock,
It has an excellent effect in improving the stress state of poultry and the like and preventing a decrease in immune activity. Moreover, since the stress condition improving agent of the present invention can be produced simply by culturing bacteria and treating it with a surfactant, it can be produced at lower cost than conventional stress improving agents.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the spleen weight ratio when a diet supplemented with a stress condition-improving agent according to one embodiment of the present invention is fed for 3 weeks and then dexamethasone is injected subcutaneously into an immunosuppressed state, in comparison with a control. It is.

FIG. 2 is a graph showing the percentage of peripheral blood cells in a case where dexamethasone was subcutaneously injected into an immunosuppressed state after feeding a feed supplemented with a stress condition improving agent according to one embodiment of the present invention for 3 weeks, in comparison with a control. It is.

FIG. 3 shows a lymphocyte blastogenesis reaction when mice were fed a diet supplemented with a stress condition-improving agent according to one embodiment of the present invention for 3 weeks and then subcutaneously injected with dexamethasone to make them immunosuppressed. It is a figure shown in comparison.

FIG. 4 shows the phagocytic activity of peritoneal macrophages when mice were fed a diet supplemented with a stress condition improver according to one embodiment of the present invention for 3 weeks and then subcutaneously injected with dexamethasone to be in an immunosuppressed state. FIG.

FIG. 5: Control of the phagocytic activity of spleen neutrophils when mice were fed a diet supplemented with a stress condition improver according to one embodiment of the present invention for 3 weeks and then subcutaneously injected with dexamethasone to be in an immunosuppressed state. It is a figure shown in comparison with.

FIG. 6 shows NK activity in a case where a diet supplemented with a stress condition-improving agent according to one embodiment of the present invention was fed to mice for 3 weeks and then dexamethasone was injected subcutaneously into an immunosuppressed state, as compared with a control. FIG.

FIG. 7 is a graph showing the spleen weight ratio when a stress state improving agent according to one example of the present invention is orally administered by gavage once to mice which are closely housed for 3 weeks.

FIG. 8 is a graph showing the percentage of peripheral blood cells when mice are closely gavaged for three weeks and administered once by gavage with the stress condition-improving agent of one example of the present invention, as compared with the control.

FIG. 9 is a graph showing a lymphocyte blastogenesis reaction when a stress condition-improving agent according to one embodiment of the present invention is orally administered once to mice that are closely housed for 3 weeks, in comparison with a control. .

FIG. 10 is a graph showing the phagocytic activity of peritoneal macrophages when a stress state-improving agent according to one example of the present invention is orally administered to a mouse that has been closely housed for 3 weeks, as compared with a control.

FIG. 11 is a diagram showing the phagocytic activity of spleen neutrophils when a stress state improving agent according to one embodiment of the present invention is gavaged once orally to mice strictly housed for 3 weeks, as compared with a control. is there.

FIG. 12 is a graph showing, in comparison with a control, NK activity when a stress state-improving agent, which is one example of the present invention, is forcibly administered orally once to mice that have been closely housed for 3 weeks.

FIG. 13 is a diagram showing the spleen weight ratio when mice are closely bred for 3 weeks and the stress condition improving agent of one example of the present invention is supplied throughout the strictly bred period, as compared with a control.

FIG. 14 is a graph showing the percentage of peripheral blood cells when mice are closely bred for 3 weeks and the stress condition improving agent of one example of the present invention is supplied throughout the strictly bred period, as compared with a control.

FIG. 15 is a graph showing the lymphocyte blastogenesis reaction when mice given close-contact for 3 weeks are fed the stress condition-improving agent of one example of the present invention throughout the period of close-feeding, as compared with controls. It is.

FIG. 16 is a graph showing the phagocytic activity of peritoneal macrophages when mice are closely bred for 3 weeks and a stress condition-improving agent according to one embodiment of the present invention is fed throughout the strictly bred period, as compared with a control. is there.

FIG. 17 compares the phagocytic activity of spleen neutrophils to the control when a stress condition-improving agent according to one embodiment of the present invention is fed to a mouse strictly bred for 3 weeks throughout the strictly bred period. FIG.

FIG. 18: Comparison of the phagocytic activity of spleen neutrophils in NK activity when mice were closely bred for 3 weeks and the stress condition-improving agent of one embodiment of the present invention was fed throughout the bred period. FIG.

Claims (2)

[Claims] An agent for improving a stress state, comprising a bacterial cell from which a capsular membrane has been removed as an active ingredient. 2. The agent for improving a stress state according to claim 1, wherein the cells are cells of a genus Bifidobacterium.