WO2012063345A1 - Immunopotentiating agent, immunopotentiating composition containing same, and immunopotentiating method - Google Patents

Abstract

The purpose of the invention is to provide an immunopotentiating agent, an immunopotentiating composition, a feed composition, and an immunopotentiating method, each of which has an excellent ability to potentiate the immunocompetence of a living body. The immunopotentiating agent according to the invention comprises a lactic acid bacterium derived from Lactobacillus sakei HS-1 (FERM BP-11312) strain or a processed substance thereof. The number of the lactic acid bacterial cells is preferably from 10⁶ to 10¹² cells/g. Further, the immunopotentiating composition according to the invention is characterized by containing the immunopotentiating agent so that the number of lactic acid bacterial cells derived from Lactobacillus sakei HS-1 (FERM BP-11312) strain is from 10³ to 10⁶ cells/g.

Description

Immunostimulating agent, immunostimulating composition containing the same, and immunostimulating method

The present invention relates to an immunostimulant, an immunostimulatory composition containing the same, and an immunostimulation method, and particularly to improvement of the immunostimulation.

Lactic acid bacteria have long been used for food preservation and food processing purposes and are recognized as safe and beneficial microorganisms for humans and animals. Many fermented foods such as cheese, yogurt, miso, soy sauce, and pickles are born from the use of lactic acid bacteria using milk, cereals and vegetables as raw materials.
In recent years, lactic acid bacteria are known not only to be useful for food production but also to have a positive effect on ingested humans, and many studies have been conducted on their probiotic functions. As the function of lactic acid bacteria, mainly the improvement of intestinal flora and cholesterol lowering have been reported, but recently, immunostimulatory action has attracted attention, and allergy reducing action and antiviral action are related to it. Infection protective effects have been studied.

Immunity is a function acquired for the purpose of eliminating heterologous organisms in multicellular organisms, and removes foreign substances by recognition of antigens different from self, inactivation by complements and antibodies, destruction by phagocytes, induction of apoptosis of infected cells, etc. Exclude. Immunity is broadly divided into innate immunity and adaptive immunity, but the latter is triggered when the pathogen is not eliminated by natural immunity such as complement or phagocytic cells, and an infected lesion is formed.
When microorganisms or viruses enter a living body, phagocytic cells such as macrophages and dendritic cells expressing receptors such as Toll-like receptor (TLR) first recognize it. TLR recognizes the unique RAMPs (pathogen-associated molecular patterns) of microorganisms and induces an immune response. Cell walls of Gram-positive bacteria such as lactic acid bacteria stimulate innate immunity, contain peptidoglucan and lipoteichoic acid as PAMPs, and are recognized by complexes of TLR2, TLR2, and TLR6, respectively. Stimulation of TLR activates NF-κB via MyD88 molecule and induces production of IL-12, interferon α, β and activates other immunocompetent cells such as NK cells. TLR4 recognizes lipopolysaccharide (LPS) of Gram-negative bacteria such as E. coli and activates NF-κB to produce TNF-α and IL-6.

Macrophages recognize and phagocytose microorganisms not only through TLRs but also through Fc receptors, complement receptors, mannose receptors, and scavenger receptors. The phagocytosed microorganism is transported to the phagosome, and is discarded by active oxygen, nitric oxide (NO), and degrading enzymes in the phagolysosome formed by fusing the phagosome with the lysosome. The phagocytosis is affected by the activated state of macrophages, and phagocytic ability and antigen presenting ability are low in non-activated macrophages. Macrophages are activated by various stimuli, and there are several stages in the activation. For example, in the case of inducing tumor cell destruction, macrophages are brought into a primed macrophage by stimulation with IFN-γ, which is one of cytokines. By administering LPS to macrophages that are ready for activation, tumor cell destruction is induced for the first time. That is, activated macrophages eliminate foreign substances and induce adaptive immunity through enhancement of phagocytosis, secretion of bactericidal components, production of cytokines, and the like.
Nitric oxide (NO) is an important bactericidal molecule and is temporarily produced through the expression of inducible NO synthase (iNOS) in macrophages. Therefore, it can be considered that the macrophages are activated by the production of NO.
In tests using macrophage-like cell lines, the NO produced is detected as nitrite ions in the medium. Tejada-Simon et al. Used RAW264.7, a macrophage-like cell line, and Lactobacillus sp. And Streptococcus sp. It is shown that heat-sterilized cells of lactic acid bacteria belonging to the above induce NO production. Regarding activation of macrophages by such lactic acid bacteria, for example, peptidoglycan of Bifidobacterium thermophilum, a kind of intestinal lactic acid bacteria derived from swine, also activates lower immune-competent cells such as mammals, birds and fish (Non-Patent Document 1). In this activation, the orally-administered peptidoglycan is taken up by M cells, and the information is transmitted to antigen-presenting cells such as macrophages. It has been suggested that this occurs because the system itself has been enhanced.

In addition, lactic acid bacteria used for the production of silage used for feed such as cattle are expected to be applied to the immunostimulatory action of livestock. Originally, silage uses fermentative ability of lactic acid bacteria adhering to grass as a means of preserving grass as feed for a long period of time, but in the livestock field where the immunity of livestock animals greatly affects productivity, Various feeds containing lactic acid bacteria have been developed based on the immunostimulatory action of lactic acid bacteria.
For example, feeds containing Lactobacillus plantarum (Patent Document 1), Lactobacillus gasseri (Patent Document 2), and Lactobacillus paracasei (Patent Document 3) have been reported as showing immunity promoting ability of specific lactic acid bacteria.

JP 2000-262247 A Japanese Patent No. 3648233 JP 2005-21156 A

However, the immunostimulatory effect exhibited by lactic acid bacteria varies in the action on the living body and the height of the stimulating effect depending on the bacterial species, and thus comprehensively excellent lactic acid bacteria have been demanded. In particular, a feed containing lactic acid bacteria has not yet achieved an immunostimulatory effect that brings about an increase in the survival rate of livestock, and there remains room for improvement in its composition and administration method.
This invention is made | formed in view of the said subject, and it aims at providing the immunostimulant excellent in the acceleration | stimulation of the immunity of a biological body, an immunostimulation composition, a feed composition, and an immunostimulation method.

In order to achieve the above object, the present inventors conducted extensive research and found that specific lactic acid bacteria belonging to Lactobacillus sakei have a very high immunostimulatory effect. Further, the present inventors have found that macrophages are ready for activation by administration of a small amount of the lactic acid bacteria, and are further activated by the action of dead cells of gram-negative bacteria or lipopolysaccharides, thereby completing the present invention.

That is, the immunostimulant according to the present invention is characterized by comprising a lactic acid bacterium derived from a Lactobacillus sakei HS-1 (FERM BP-11312) strain or a processed product thereof.
In the immunostimulant, the number of lactic acid bacteria derived from Lactobacillus sakei HS-1 (FERM BP-11312) strain is preferably 10 ⁶ to 10 ¹² / g.
Further, the immunostimulant composition according to the present invention provides the immunostimulant so that the number of lactic acid bacteria derived from Lactobacillus sakei HS-1 (FERM BP-11312) strain is 10 ³ to 10 ⁶ / g. It is characterized by blending.
Moreover, it is suitable that the said immunostimulant composition is a feed composition.

The immunostimulation method according to the present invention is characterized by administering a lactic acid bacterium derived from a Lactobacillus sakei HS-1 (FERM BP-11312) strain or a processed product thereof.
Furthermore, the immunostimulation method according to the present invention is characterized by administering a lactic acid bacterium derived from a Lactobacillus sakei HS-1 (FERM BP-11312) strain or a processed product thereof, and a dead cell of a Gram-negative bacterium. .

According to the present invention, an immunostimulant excellent in promoting immunity of a living body can be obtained. Furthermore, the feed composition containing the immunostimulant significantly enhances the target immunity, that is, resistance to pathogenic bacteria, so that productivity of livestock and the like can be improved.

It is a graph which shows the survival rate of the mouse | mouth which administered the immunostimulant of this invention. It is the graph which compared the activation of the macrophage by Lactobacillus sakei HS-1, microbial cell, and lipopolysaccharide. The upper graph is a graph showing the transition of the average body weight of each of the test groups A to C in each week in the combination test of the immunostimulant of the present invention in the feed composition for pigs. Further, the lower graph is a graph showing the average value of weight gain of each group during the period when the weight of the piglet is 10-30 kg, 30-70 kg, 10-70 kg. In the compounding test of the immunostimulant of the present invention in the feed composition for pigs, the feed requirement rate of the test groups A to C was shown in the period when the weight of the piglet was 10 to 30 kg, 30 to 70 kg, and 10 to 70 kg. It is a graph. FIG. 5 is a graph showing the average serum immunoglobulin (IgG) concentration in test groups A to C when the weight of a piglet is 30 kg and 70 kg in a combination test of the immunostimulant of the present invention in a feed composition for pigs. is there. 6 is a graph showing the average number of various intestinal bacteria in test groups A to C when the weight of a piglet is 30 kg in a combination test of the immunostimulant of the present invention in a feed composition for pigs. It is the graph which showed the transition of the average body weight of the test groups X and Y for every week in the combination test to the feed composition for pigs of the immunostimulant of this invention.

Hereinafter, the present invention will be described in detail.
Lactobacillus sakei HS-1 which is an immunostimulant according to the present invention has been isolated from kimchi. It is a lactic acid bacterium belonging to sakei. The lactic acid bacterium is deposited internationally as a microorganism having the deposit number FERM BP-11312 at the Patent Microorganism Consignment Center of the National Institute of Advanced Industrial Science and Technology. In addition, as a lactic acid bacterium, a pickle starter “lactic acid bacterium for pickles (Lactobacillus salmon HS-1)” sold by Nippon Green Packs Co., Ltd. may be used.

As a method for identifying and culturing Lactobacillus sakei HS-1, for example, a method disclosed in “Patent No. 3091196” relating to fermented pickles using the lactic acid bacteria can be applied.
First, the mycological properties of Lactobacillus sakei HS-1 are shown below.

A. Morphological properties Cell morphology Aspergillus Size 0.8 × 2-3μm
Gram staining positive

B. Physiological properties (+: positive,-: negative)
Growth at 15 ° C +
Growth at 45 ℃-
Growth in the presence of 6.5% salt +
Growth at pH 4-
Growth at pH 5 +
Fermentation type Homo lactic acid isomer type DL
Lactic acid isomer type L in the presence of acetic acid
Peptidoglycan Type Non-DAP

C. Sugar fermentability (+: positive,-: negative)
Arabinose +
Ribose +
Xylose-
Fructose +
Mannose +
Lactose +
Sucrose +
Trehalose +
Raffinose-
Mannitol-
Sorbitol-
Sodium gluconate +
Maltose +
Galactose +
Melibiose +

Lactobacillus sakei HS-1 can be cultured by a general lactic acid bacteria culture method or the method described in the aforementioned patent. As a general method for culturing the above-mentioned bacteria, for example, a synthetic medium such as MRS medium or GYP medium, or a semi-synthetic medium is used, and static culture is performed while maintaining the culture temperature at 10 to 15 ° C. and the pH at 5 to 7. To do. By such a culture method, Lactobacillus sakei HS-1 of about 10 ⁸ to 10 ¹¹ cells / g can be obtained in 2 to 3 days. Of course, the bacteria marketed as the above-mentioned pickle starter may be used as they are or after being cultured according to the usage.

The immunostimulant according to the present invention comprises the lactic acid bacterium Lactobacillus sakei HS-1 or a processed product thereof. The immunostimulatory ability of the lactic acid bacteria is not impaired even if the bacteria are inactivated by heating or the like. Therefore, in the present invention, either live or dead cells of the lactic acid bacteria can be used. In addition, the cells of the lactic acid bacterium may be in any form as long as it has the immunoreactivity intended in the present application. Therefore, the “bacteria” includes not only the form in which viable or dead bacteria cultured by the above method or the like are separated from the medium or the like, but also the form containing the medium components and products as the culture or culture residue. .

In addition, examples of the processed product of lactic acid bacteria include crushed bacteria, concentrates, pasted products, dried products, liquid products, diluted products, and sterilized products. In the present invention, a dried product is particularly preferable from the viewpoint of ease of processing and usability as a preparation. In addition, the use of live bacteria can be expected to produce an intestinal regulation effect, but live bacteria are more difficult to handle and store than dead bacteria. Regarding the immunostimulation aimed at by the present invention, the effect equivalent to that of live bacteria can be obtained even by using dried dead cells. The dried product of lactic acid bacteria can be obtained by, for example, drying treatment of bacteria by heat drying, spray drying, drum drying, microwave drying, vacuum tube layer, freeze drying, etc. A dry product is preferred.

The number of cells of Lactobacillus sakei HS-1 contained in the immunostimulant according to the present invention is preferably 10 ⁶ to 10 ¹² cells / g, more preferably 10 ⁶ to 10 ⁸ cells for both live and dead cells. / G.
The immunostimulant of the present invention may be used after diluting or concentrating. For example, an immunostimulant containing 10 ¹² bacteria / g can be diluted 10,000 times with other components and used as an immunostimulant composed of 10 ⁸ bacteria / g.

The immunostimulatory agent according to the present invention directly orally or parenterally administers macrophages widely distributed in the living body to enhance its non-specific phagocytic function, To improve. Lactobacillus sakei HS-1 constituting the immunostimulant of the present invention has an extremely high effect compared to other lactic acid bacteria known to have an immunostimulatory action.
Although the immunostimulant of the present invention depends on the administration target and means, if it is administered in an embodiment containing 10 ³ / g or more of the lactic acid bacteria, a sufficient activation effect is exhibited.

Further, the lactic acid bacterium Lactobacillus sakei HS-1 can be used as follows to obtain an immunostimulatory effect.
First, a small amount of the lactic acid bacteria as an immunostimulant or a processed product thereof is administered to a subject, and the inactive macrophages in the body are transitioned to an active preparation state. The dose of the lactic acid bacterium at this time is about 1/10 that of activating the macrophages with the lactic acid bacterium, and is usually 0.1 to 10 mg / kg as the dry weight of the microbial cell.

In the macrophages in the active ready state, the production of nitrous acid seen in the active state is hardly observed, and at first glance, there is no difference from the inactive state. However, if a small amount of dead Gram-negative bacteria is administered to the same subject at the same time as the administration of the lactic acid bacteria or 1 to 24 hours after the administration of the lactic acid bacteria, the macrophages that are in the active ready state rapidly and more than usual. Is also activated to a high level.

In the present application, the gram-negative bacterium preferably has a lipopolysaccharide on the outer membrane and is an intestinal resident bacterium. Examples As such bacteria, e.g. E. coli, E.I. aerogenes and the like.
These bacteria are used as dead cells that have been completely sterilized by heating or the like. The dead cells can be further subjected to treatment such as crushing, but it is necessary to leave the lipopolysaccharide related to the activity of macrophages in the treated product. The dose of dead cells of Gram-negative bacteria is about 1/10 to 1/10000 of the lactic acid bacteria, and is usually about 0.001 to 1 mg / kg as the dry weight of the bacteria. Further, instead of dead cells of Gram-negative bacteria, purified lipopolysaccharides in an amount corresponding to the dead cells can also be used.
The lactic acid bacteria and the dead cells or lipopolysaccharides of the gram-negative bacteria in this immunostimulation method may be administered either orally or parenterally, or both may be administered together or separately. Good.

The immunostimulant of the present invention based on the lactic acid bacterium Lactobacillus sakei HS-1 can be used alone as a preparation, or can be blended into foods, health foods, pharmaceuticals and the like as active ingredients and used as an immunostimulatory composition. In addition, Lactobacillus sakei HS-1 is a lactic acid bacterium that has been inhabited in pickles and used in the past, and therefore it is considered that there is no particular safety problem when taken orally in foods. Moreover, the immunostimulant of this invention can mix | blend an injection, an external preparation base, etc. suitably, and can also be used as a composition for parenteral use.

The compounding quantity of the said immunostimulant in an immunostimulatory composition can be determined according to the dosage of the immunostimulant mentioned above according to the kind, magnitude | size, age, etc. of administration object. In particular, when producing a composition comprising the immunostimulant according to the present invention as an active ingredient, the number of lactic acid bacteria Lactobacillus sakei HS-1 in the composition is 10 ³ to 10 ⁶ / g, preferably 10 ³ to 10 ^The immunostimulant of the present invention is preferably blended so as to be ⁴ / g.
As described above, the immunostimulant of the present invention can bring about a sufficient immunostimulatory effect in the composition with a very small amount of blending.
The immunostimulatory composition can be produced by appropriately blending various components used in foods, health foods, pharmaceuticals, etc. within the range not impairing the effects of the present invention in addition to the above active ingredients, and the dosage form of the composition Is not particularly limited.

In particular, the immunostimulatory composition according to the present invention is preferably used as a feed composition having an immunostimulatory activity by blending feed ingredients. The immunostimulant according to the present invention is effective not only for mammals such as cattle, pigs, horses, sheep, dogs, cats, rabbits, rats, mice, but also for fish, birds, insects, etc. By adjusting the ingredients, a feed composition effective for any living body can be obtained. Particularly preferred in the present invention is use as a feed composition for pigs, cows and chickens.

The feed component is not particularly limited. For example, as animal-derived components, livestock (for example, cattle, pigs, chickens, sheep, etc.) and seafood (for example, tuna, skipjack, horse mackerel, sardines, flounder, saury, shrimp, octopus, And meats, skins, organs, eggs, blood, milk, animal fats and other biological tissues obtained from scallops, etc.), dry powders thereof, or processed products thereof.
Examples of plant-derived components include vegetables such as carrots, pumpkins, cabbages and peppers, seeds such as sesame, almonds, poppy seeds, sunflower seeds, cereals such as rice, wheat, barley and corn, soybeans Beans such as green beans and broad beans, starches such as potatoes, sweet potatoes and corn starch, vegetable oils such as soybean oil and sesame oil, fruits such as mandarin oranges, apples and oysters, mycelium such as enoki, shiitake and shimeji, wakame , Plant tissues obtained from algae such as kombu and chlorella, grass, etc., powders thereof, processed products thereof, and the like.

In addition to the above components, the feed composition according to the present invention includes color formers, colorants, flavors, sweeteners, preservatives, emulsifiers, antioxidants, pH adjusters, seasonings, thickeners, and swelling agents. Additives usually used in feed compositions such as antifoaming agents, settling agents, and nutrient enhancers can be appropriately blended.
Furthermore, the feed composition is not particularly limited with respect to the dosage form, and any of a solid form, a pellet form, a liquid form, a paste form, a granule form, a powder form, a flake form and the like can be applied depending on the administration target. Good.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not restrict | limited to these. In addition, unless otherwise indicated, all compounding amounts are shown in wt%.
First, the infection resistance test with respect to colon_bacillus | E._coli of a mouse | mouth was done using the immunostimulant of this invention.
<Examples of bacterial preparation>
Lactobacillus sakei HS-1 strain is added to GYP medium (1 g glucose, 1 g yeast extract, 0.5 g peptone, 0.2 g sodium acetate, 0.5 mL salt solution, 1 mL Tween 80 solution in 100 mL distilled water and autoclaved) Inoculated and cultured at 30 ° C. for 2 days.
The proliferated bacterial cells were collected by centrifugation, and after further centrifuging by adding a phosphate buffer (PBS), the separated bacterial cells were washed twice with PBS. PBS containing 0.05% Tween 20 was added to the washed cells, heat-treated in boiling water for 15 minutes, allowed to cool, then washed 3 times with PBS and frozen. Then, it was freeze-dried (drying temperature 35 ° C.) using a freeze dryer to obtain dry cell powder. The number of Lactobacillus sakei HS-1 bacteria in the dry preparation was approximately 10 ¹² cells / g.

<Test method>
40 29-day-old male ICR mice that were preliminarily raised for 2 days (freezing temperature 24 ± 3 ° C., relative humidity 55 ± 5%) with free water and free feeding were divided into 4 groups of 10 mice each. The following samples were administered intraperitoneally to groups of mice.

Test group 1: 0.5 mL / mouse of a PBS suspension containing 2500 μg / mL of the above dry cell powder.
Test group 2: 0.5 mL / animal of PBS suspension containing 500 μg / mL of the above dry cell powder.
Test group 3: 0.5 mL / animal of PBS suspension containing 50 μg / mL of the above dry cell powder.
Control group: 0.5 mL / animal of the above-mentioned dry cell powder-free PBS.

Three days after the sample administration, the same sample as above was again administered intraperitoneally to each group. The day after this administration, 0.5 ml of PBS dilution solution (10 ⁸ CFU / mL) of E. coli (E.coli 72-5) was administered intraperitoneally to all groups, and 7 The animals were reared for days and observed for progress. Table 1 shows changes in the number of surviving mice in each group, and FIG. 1 shows the survival rate of each group after administration of E. coli.

As shown in Table 1 and FIG. 1, in the control group to which PBS was administered, 80% of the mice died one day after the administration of E. coli, but the test groups 1 to 3 to which Lactobacillus sakei HS-1 suspension was administered were administered. Each had a high survival rate, and the test groups 1 and 2 showed a survival rate of 100%. In addition, since all mice survived during the period from the sample administration to the E. coli administration on the third day (days 3 to 6), the safety of Lactobacillus sakei HS-1 administration to the living body is improved. confirmed.

From the above results, it can be seen that Lactobacillus sakei HS-1 significantly improved the resistance to infection of the subject Escherichia coli. That is, it was confirmed that Lactobacillus sakei HS-1 has an action that strongly activates the immune mechanism.
In addition, since such an action was observed in the killed bacteria of Lactobacillus sakei HS-1, components such as peptidoglycan constituting Lactobacillus sakei HS-1 were recognized as certain antigens in the living body, and E. coli infection was prevented. It is presumed that the production of immunoglobulins such as IgA to protect is promoted.

Next, the activation of macrophages by Lactobacillus sakei HS-1, microbial cells, and lipopolysaccharide was examined. The Lactobacillus sakei HS-1 used was prepared as a dry cell powder according to the above example. Gram-negative bacteria and lipopolysaccharide were prepared as follows.

Gram-negative bacteria and lipopolysaccharide (LPS)
Lactic acid bacteria were cultured for 2 days at 30 ° C. using GYP medium, and gram-negative bacteria (Escherichia Coli ATCC 11775, Enterobacter aerogenes ATCC 13048) were cultured for 2 days at 37 ° C. using LB medium. Microorganisms collected by centrifugation were sterilized by heating in boiling water for 30 minutes and then lyophilized.
As the lipopolysaccharide, Wako Pure Chemical / Escherichia coli LPS was used.

Macrophage culture and activation test Macrophage cell line J774.1 (RIKEN BRC Cell Bank RCB0434) is a PRMI1640 medium supplemented with 10% non-immobilized FBS (Sigma), 50 μg / ml streptomycin and penicillin at 37 ° C. The cells were cultured under 5% carbon dioxide gas-95% atmospheric conditions. The macrophage activation test was performed by adding 1 ml of 2 × 10 ⁶ / ml cells to each well of a 24-well culture plate (Falcon) and culturing for 2 days to confluence, then removing the old medium and adding new medium 0 9 ml and the following samples were suspended in PBS to make 0.1 ml. The nitrite concentration (μM) in the supernatant after 24 hours of culture was analyzed with a grease reagent.
Samples were dried bacterial powder 1 μg, 10 μg, 100 μg according to the above preparation example of Lactobacillus sakei HS-1, 0.1 μg, 1 μg of ATCC 11775 (E. Coli), 1 ng, 10 ng of LPS, and PBS alone A control example was used. The results are shown in FIG.

As shown in FIG. 2, in this test, macrophage activation was performed using 100 μg HS, 1 μg for E. coli and 10 ng for LPS. Example of adding 1 μg or 10 μg of HS-1, Macrophage activation was not observed in the case where 0.1 μg of E. coli was added and in the case where 10 ng of LPS was added. In each effective amount, HS-1 showed excellent macrophage activation ability.

Subsequently, the following test was conducted to examine the synergistic effect of the components that were found to have macrophage activation ability by the above test.
Activation of macrophages stimulated with lactic acid bacteria by low-level Escherichia coli, etc. 10 μg of Lactobacillus sakei HS-1 was added at the time of culturing the aforementioned macrophages J774.1 and cultured for 24 hours, then the medium was changed and ATCC 11775 (E. Coli) 1 μg, 1 μg ATCC 13048 (E. aerogenes), or 1 ng LPS was added. Thereafter, the cells were further cultured for 24 hours, and the nitrite concentration (μM) in the supernatant was analyzed with a grease reagent. As a control, an example in which PBS was added instead of HS-1 was also performed.

(Table 2)
Nitrite concentration (μM)
Pretreatment E. E. coli E. coli. aerogenes LPS
PBS (control example) 0.7 0.7 0.6
HS-1 10 μg 8.8 15 11.5

As shown in Table 2, by administering a combination of Lactobacillus sakei HS-1 that did not satisfy the effective amount of macrophage activation in FIG. 2 and Gram-negative bacteria (dead bacteria) or lipopolysaccharide (LPS) It was revealed that macrophages are remarkably activated. This is probably because the macrophage was stimulated by the small amount of HS-1 added earlier and became ready for activation, and therefore, the macrophage was markedly activated by the addition of a small amount of gram-negative bacteria or LPS.
Therefore, in the immunostimulation method according to the present invention, a lactic acid bacterium derived from Lactobacillus sakei HS-1 (FERM BP-11312) strain or a treated product thereof is administered at a low concentration, and at the same time or after the step, And a step of administering a dead cell of a negative bacterium or a lipopolysaccharide at a low concentration.

Furthermore, the phagocytic ability of macrophages activated by the present invention was evaluated by the following method.
Evaluation of phagocytic ability of activated macrophages In the above-described culture of macrophage cells, the number of E. coli trapped by macrophages when living E. coli was added to the medium was counted. Macrophages were cultured using a 24-well plate in the same manner as in the macrophage activation test. Control non-active macrophages were prepared by adding PBS, and activated macrophages were added with 10 μg HS-1 and 0.1 μg ATCC 11775 (E. coli) bactericidal cells and cultured for 1 day. After removal of the old medium, E. coli prepared in PBS in advance. E. coli viable bacteria were added to each well so as to be 6 × 10 ^6, and centrifuged (1000 rpm × 5 min) in order to increase contact between macrophages and E. coli. After standing in a carbon dioxide incubator for 30 minutes, the supernatant was discarded, 1 ml of PBS was added to the remaining macrophage cells, the cells were removed by pipetting, and the number of E. coli was measured by a conventional method to obtain the number of bound E. coli. The test was repeated twice with 12 holes for the control group and 12 holes for the test group. The results are shown in Table 3 below.

(Table 3)
Macrophage state Bound E. coli count (CFU / ml)
Deactivated 6.7 ± 1.7 × 10 ⁵
Activation 1.9 ± 0.3 × 10 ⁶
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Activation: Heat-sterilized HS-1 10 μg and E. coli. 1 day culture inactivated by adding 0.1 μg of E. coli: PBS is added instead of microbial cells and cultured for 1 day

As shown in Table 3, macrophages activated by the addition of lactic acid bacteria HS-1 and E. coli heated cells during culturing of macrophages J774.1 live almost three times as much as non-activated macrophages. E. coli bound. It is considered that the increase in the microorganism-bound receptor and the enhancement of the phagocytic ability were caused by the activation.

Furthermore, the compounding test to the feed composition for pigs of the immunostimulant of this invention was done.
<Test method>
Eighteen piglets (ternary crossbred (LWD)) were divided into three groups of 6 to 3 (3 males and 3 females), A to C, and each group was raised with the following feed for 84 days. In all groups, feed was freely consumed.
During breeding, for each group, measurement of body weight (weekly), calculation of feed request rate (when body weight is 10-30 kg, 30-70 kg), confirmation of diarrhea status (daily), general blood and biochemical tests and blood Immunoglobulin G (IgG) concentration was measured (at 30 kg body weight, at 70 kg), and intestinal bacterial tests with stool (at 30 kg) were performed.
Fig. 3 is a graph of the change in body weight and weight gain of each group, Fig. 4 is a graph of feed demand rate, Table 4 is the occurrence of diarrhea, Table 5 is a general blood test result, Table 6 is a blood biochemical test result, FIG. 5 is a graph of immunoglobulin G (IgG) concentration, FIG. 6 is a graph of intestinal bacteria count, and Table 7 is a comparison of intestinal bacteria count.

Test group A: Feed composition prepared by adding 0.02% of a dry preparation containing Lactobacillus sakei HS-1 at a concentration of 10 ⁸ cells / g to the following basic feed to contain 2 × 10 ⁴ bacteria / g I gave a thing.
Test group B: The following basic feed (without additives) was given.
Test group C: during a period of 10-30 kg of piglet weight, a feed composition containing 0.1% of an antibacterial feed additive consisting of a mixture of 5 g of enramycin and 30 g of morantel citrate was given, In the period of 30 to 70 kg, a feed composition was added to which 0.1% of an antibacterial feed additive consisting of a mixture of 10 g of enramycin, 20 g of colistin sulfate, and 30 g of morantel citrate was added.
(Basic feed)
Period of 10-30 kg of piglet weight: A specified mixed feed containing a crude protein (CP) content of 19.3% and a total digestible nutrient content (TDN) of 78.9%.
Period of piglet weight of 30 to 70 kg: feed for testing pork meat capacity containing crude protein (CP) content of 14.5% and total digestible nutrient (TDN) of 74.5%.
* Both basic feeds are drug-free samples that do not contain antibacterial feed additives.

(How to check the occurrence of diarrhea)
The breeding bed was observed every morning with + as soft stool, ++ as mud stool, and ++ as watery stool. When the solid could not be identified, the number of heads was judged from the situation, and when there were several, the number was added.

In Table 5, MCV represents average red blood cell volume, MCH represents average red blood cell hemoglobin content, MCHC represents average red blood cell hemoglobin concentration, and PLT represents platelets.

As shown in Tables 4 to 7 and FIGS. 3 to 6, the test group A fed with the diet administered with the specific lactic acid bacteria was compared with the test group B fed with the additive-free feed despite having a low concentration. In comparison, good results were shown in terms of weight gain, diarrhea, and immunoglobulin G concentration at 70 kg. In addition, in comparison with test group C fed with a feed supplemented with antibacterial sample additives normally added when raising pigs, test group A was equivalent in weight gain, but particularly with regard to immunoglobulin G concentration. Showed results exceeding test group C.
Moreover, the blood test showed no adverse effects due to the addition of specific lactic acid bacteria.
From the above results, it is clear that lactic acid bacteria derived from Lactobacillus sakei HS-1 (FERM BP-11312) strain show high activity as an immunostimulatory component of piglet feed compositions at a very low concentration. .

Furthermore, the compounding test to the feed composition for pigs of the immunostimulant of this invention was done by the following another systems.
<Test method>
Two abdominal ternary crossbred (LWD) pigs 12 and the like were divided into two groups of 6 (three males and three females), X and Y, and each group was bred with the following feed for 42 days. The test start date was 32.3 days for test group X and 33.0 days for test group Y. In all groups, feed was freely consumed.
For each group, body weight was measured weekly, and weight gain (kg), daily average weight gain (g), average feed intake (kg), and feed demand rate were calculated. These results are shown in Table 8, and the change in body weight for each week is shown in FIG.
Test group X: Milky piglet artificial milk made by Toyohashi feed containing new antibacterial feed additives (44 g titer / t of tylosin phosphate, 20 g titer / t of colistin sulfate, 30 g / t Morantel citrate) 0.02% of a dry preparation containing Lactobacillus sakei HS-1 at a concentration of 10 ⁸ cells / g was added to EX (CP 18.0%, TDN 81.0%)), and the bacteria were added at 2 × 10 ⁴ cells / g. A feed composition prepared for inclusion was given.
Test group Y: The same feed as in test group X was given without adding Lactobacillus sakei HS-1.

As shown in FIG. 7, the test group X fed with the feed to which the specific lactic acid bacterium was added increased the weight gain from the first week after the test compared to the test group Y fed with the feed to which the lactic acid bacterium was not added, and the test was started. There was a significant weight difference between the two groups at 6 weeks. From the above results, it is considered that the feed composition containing Lactobacillus sakei HS-1 improves the immune function of livestock and brings about weight gain. Further, by using the immunostimulant of the present invention in combination with a conventional antibacterial feed additive, a synergistic effect can be expected for the immunostimulation of livestock.

FERM BP-11312

Claims (6)

1. An immunostimulant comprising a lactic acid bacterium derived from Lactobacillus sakei HS-1 (FERM BP-11312) strain or a processed product thereof.
2. The immunostimulant according to claim 1, wherein the number of lactic acid bacteria derived from Lactobacillus sakei HS-1 (FERM BP-11312) strain is 10 ⁶ to 10 ¹² cells / g.
3. ^{3. The} immunostimulant according to claim 1 or 2 is blended so that the number of lactic acid bacteria derived from Lactobacillus sakei HS-1 (FERM BP-11312) strain is 10 ³ to 10 ⁶ / g. A characterized immunostimulant composition.
4. The immunostimulatory composition according to claim 3, which is a feed composition.
5. An immunostimulatory method comprising administering a lactic acid bacterium derived from a Lactobacillus sakei HS-1 (FERM BP-11312) strain or a processed product thereof.
6. A lactic acid bacterium derived from Lactobacillus sakei HS-1 (FERM BP-11312) strain or a processed product thereof,
   Dead cells of Gram-negative bacteria,
   An immunostimulation method comprising administering

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