JP5481702B2 - Novel lactic acid bacteria having immunostimulatory action and gastric juice resistance - Google Patents

Description

The present invention has an immunostimulatory activity, have a gastric fluid resistant and high adhesion to intestinal milk acid bacteria Pediococcus (Pediococcus) sp. The present invention relates to KB1 (NITE P-755), an immunostimulatory composition containing the lactic acid bacterium, a food and drink containing the immunostimulatory composition, and a pharmaceutical product.

  Lactic acid bacteria are microorganisms that have been used as a starter since ancient times for producing dairy products such as cheese, yogurt, and fermented butter, and livestock meat products such as fermented sausages and fermented salami. It has attracted attention because it has physiological activities that are extremely beneficial for health maintenance and promotion. Recent studies have revealed that 50-60% of all peripheral lymphocytes are concentrated in the intestinal tract, and it is considered that intestinal immune activation is extremely important for the control of the immune response in the whole body. It has been. In the intestine, it is said that lactic acid bacteria must be resistant to digestive juices such as gastric acid and bile acids in order to exert their physiological activity (Non-patent Document 1). In addition, it is known that lactic acid bacteria having high adhesion to the intestinal tract can establish in the intestinal tract and exert their effects sufficiently, and further prevent harmful microorganisms from attaching to the intestinal tract. Therefore, when lactic acid bacteria are ingested orally, it is desirable that the lactic acid bacteria can reach the intestinal tract and stay in a living state while being high in functionality.

  Many of such lactic acid bacteria having beneficial physiological activity are derived from animal materials isolated from fermented dairy products and intestinal tracts, but strains that also have an immunostimulatory effect in plant lactic acid bacteria from which plant materials are separated Have been found (Patent Document 1, Non-Patent Documents 2 and 3). In addition, as lactic acid bacteria that can reach the intestines alive, bifidobacteria (lactic acid bacteria belonging to the genus Bifidobacterium) and lactic acid bacteria belonging to the genus Lactobacillus are known, and by combining a plurality of these useful lactic acid bacteria, It is expected that the following effects will be obtained (Patent Documents 2 to 4). In addition, to date, Lactobacillus brevis (Patent Document 5), Pediococcus pentosaceus (Patent Document 6), and Leuconostoc gallicum are known as lactic acid bacteria having both immunostimulatory action and gastric juice resistance. (Patent Document 6) and the like are known.

Japanese translation of PCT publication 2008-501013 JP 2006-298778 A JP-A-2005-97280 JP 2007-259729 A JP 2009-112232 A JP 2008-54556 A

Society Publishing Center, Intestinal Flora Symposium 3, Intestinal Flora and Probiotics, p. Kishi Kyoko, Kokubo Aoi, Akaya Satoshi, Ougiya Eriko, Fujita Kaya, Kishida Tsunataro, Pasken Journal 15. 21-26, (2002) Atsushi Akaya, Eiko Kishi, Eriko Ougiya, Aoi Kokubo, Junya Fujita, Tsunataro Kishida, 6th Annual Meeting of the Intestinal Bacteriological Society

  An object of the present invention is to provide a novel lactic acid strain having an immunostimulatory action and gastric juice resistance, an immunostimulatory composition containing the novel lactic acid strain, a food and drink containing the immunostimulatory composition, and a pharmaceutical product.

  In order to solve the above-mentioned problems, the present inventors screened several thousand or more strains from soil, fermented food, and intestines, and intensively studied their properties. As a result, the lactic acid bacterium Pediococcus sp., Which has an excellent immunostimulatory action and gastric juice resistance, and has high adhesion to the intestinal tract. KB1 (NITE P-755) was successfully isolated, and the lactic acid bacteria Pediococcus sp. From the analysis result of the base sequence of 16S rRNA of KB1, this bacterium was found to be a new species not corresponding to any of the known lactic acid bacteria, and the present invention was completed.

That is, the present invention
(1) having an immunopotentiating effect, it has a gastric fluid resistant and high adhesion to intestinal milk acid bacteria Pediococcus (Pediococcus) sp. KB1 (NITE P-755),
(2) Lactic acid bacteria Pediococcus sp. An immunostimulatory composition containing KB1 (NITE P-755) as an active ingredient,
(3) Food / beverage products containing the immunostimulatory composition as described in (2) above, and (4) Pharmaceuticals containing the immunostimulatory composition as described in (2) above.

  The novel lactic acid bacteria Pediococcus sp. KB1 (NITE P-755) was found to be able to reach the intestine alive by oral ingestion since a high survival rate was observed even under conditions of strong acidity and protease. The lactic acid bacterium of the present invention exhibits a high immunostimulatory activity even if it is a dead cell, and can be expected to show a further sustained immunostimulatory effect by reaching the intestine as a living bacterium. From the above, by using the lactic acid bacteria of the present invention as an immunostimulatory composition, foods and beverages and pharmaceuticals having high immunostimulatory activity can be provided.

Lactic acid bacteria Pediococcus sp. It is a photograph which shows the form of KB1 (NITE P-755) stock | strain. Lactic acid bacteria Pediococcus sp. It is a figure which shows the result of having examined comparatively the influence which KB1 (NITE P-755) and a known lactic acid bacterium have on interferon (IFN) -γ production of immune cells. In the figure, KB1 represents Pediococcus sp. KB1 (NITE P-755), A for L. casei, B for P. pentosaceus, C for L. paracasei, D represents Lactobacillus brevis (L. brevis), and E represents Bifidobacterium longum (B. longum). Lactic acid bacteria Pediococcus sp. It is a figure which shows the result of having examined comparatively the influence which KB1 (NITE P-755) and known lactic acid bacteria have on interleukin (IL) -12 production of an immune cell. In the figure, KB1 represents Pediococcus sp. KB1 (NITE P-755), A for L. casei, B for P. pentosaceus, C for L. paracasei, D is L. brevis, E is B. longum, F is L. gasseri, G is Enterococcus faecalis Respectively. Lactic acid bacteria Pediococcus sp. It is a figure which shows the result of having processed KB1 (NITE P-755) and standard strain Pediococcus pentosaceus (JCM 5890) with the artificial gastric juice, and comparing the change of the survival rate (%) of each strain with time. is there. In the figure, KB1 represents Pediococcus sp. KB1 (NITE P-755) is indicated, and Z is Pediococcus pentosaceus (JCM 5890).

  The lactic acid bacterium of the present invention is a novel lactic acid bacterium Pediococcus sp. Having the 16S rDNA base sequence represented by SEQ ID NO: 1. It is KB1 and was deposited on May 14, 2009 as the accession number NITE P-755 at the National Institute of Technology and Evaluation, the National Institute of Technology and Evaluation, 2-5-8, Kazusa-Kamashita, Kisarazu City, Chiba Prefecture. ing. The lactic acid bacteria of the present invention include Pediococcus sp. Cultures containing live or killed KB1 are also included for convenience.

Lactic acid bacteria Pediococcus sp. KB1 (NITE P-755) can be cultured under normal conditions for culturing lactic acid bacteria. The medium used for the culture is not particularly limited as long as it can grow. However, as a liquid medium for culturing lactic acid bacteria, for example, MRS medium (manufactured by Merck), whey medium using milk components, skim milk medium And the like. The culture may be stationary culture or, if the culture solution is liquid, stirring culture by stirring. Further, the culture system may be anaerobic or aerobic.
FIG. 1 shows the lactic acid bacterium Pediococcus sp. The form of KB1 (NITE P-755) is shown. Details of mycological properties are shown below.
Cell morphology Tetraphytic spore Not formed Gram stain Positive motility Non-motile catalase reaction Negative growth at 15 ° C Growth at good growth at 45 ° C Gas production from glucose without growth Negative initial pH Can be propagated at pH 6

Assimilation of sugar (positive: +, negative:-) (30 ° C, 3 days culture)
mannitol (−) mannose (+)
lactose (+) fructose (+)
cellobiose (+) melibiose (+)
melezitose (−) raffinose (+)
maltose (+) starch (-)
sorbitol (−) D-ribose (+)
trehalose (+) D-xylose (-)
galactose (+) rhamnose (+)
salicin (+) L-arabinose (+)
sucrose (−)

  Lactic acid bacteria Pediococcus sp. KB1 (NITE P-755) is a lactic acid bacterium that is resistant to gastric juice and highly adherent to the intestinal tract. Furthermore, as shown in the following examples, the lactic acid bacterium Pediococcus sp. KB1 (NITE P-755) has the effect of strongly promoting interferon (IFN) -γ and interleukin (IL) -12 production in mouse spleen cells. Since IFN-γ is a cytokine that activates the immune system and exhibits antiviral activity, the immune system is activated when the production of INF-γ is promoted. INF-γ is also involved in differentiation into Th1 cells that activate cellular immunity. On the other hand, IL-12 is a cytokine that activates both non-specific immunity and antigen-specific cellular immunity through activation of natural killer cells (NK cells) and cellular immunity, When IL-12 production is promoted, cellular immunity is activated, and both non-specific immunity and antigen-specific cellular immunity are activated, so that Th1 / Th2 balance is made Th1 dominant. Is considered to be possible. From the above, the lactic acid bacterium Pediococcus sp. KB1 (NITE P-755) is a strain having excellent immunostimulatory activity and can be used as an active ingredient of an immunostimulatory composition.

  Examples of the immunostimulatory composition of the present invention include lactic acid bacteria Pediococcus sp. It is not particularly limited as long as it contains KB1 (NITE P-755) cells as an active ingredient, and the cells may be live cells or dead cells, Further, it may be a part of a cell body such as a cell membrane or cytoplasm. In the case where a sustained effect in the intestine is expected, viable cells are preferable. The immunostimulatory composition may further contain one or more different types of lactic acid bacteria. Furthermore, as an embodiment of the present invention, lactic acid bacteria Pediococcus sp. Use of KB1 (NITE P-755) for the preparation of an immunostimulatory composition, lactic acid bacteria Pediococcus sp. An immunostimulation method of orally administering KB1 (NITE P-755) is included.

The immunostimulatory composition of this invention can provide immunostimulatory activity to food-drinks by mix | blending and using for food-drinks. In order to mix and use the immunostimulatory composition of the present invention in a food or drink, an effective amount of the active ingredient is added and blended at the raw material production stage of the food or drink or the stage of the manufactured product. Here, the “effective amount of active ingredient” means a content such that the active ingredient is ingested when the amount normally consumed in each food or drink is ingested. It can be determined depending on body weight, symptoms, administration time, dosage form, administration method, combination of drugs and the like. When the effective dose or the effective intake amount of the active ingredient in the present invention is expressed by the number of lactic acid bacteria, the intake amount per day is preferably 1 × 10 ⁹ or more, more preferably 1 × per day. 10 ¹⁰ or more, further preferably 5 × ^{10 10} or more.

  The food / beverage product of the present invention is not particularly limited as long as it contains the immunostimulatory composition of the present invention. Specifically, the immunostimulatory composition of the present invention was directly prepared as a food / beverage product or a supplement. Examples include those that are further blended with foods, various proteins, sugars, fats, trace elements, vitamins, etc., those that are liquid, semi-liquid or solid, and those that are added to and blended with general foods and drinks. be able to. In addition, the food and drink of the present invention may be a food or drink produced using general lactic acid bacteria, such as dairy products, meat, breads, beverages, and vegetables. The immunostimulatory composition of the present invention can be used as a lactic acid bacterium in the production or a part thereof, or as an additive of the produced food or drink. In addition, when adding lactic acid bacteria, which are the active ingredients of the immunostimulatory composition of the present invention, to a food or drink, the fermentation of the lactic acid bacteria itself is not required, and if the flavor of the food or drink itself is to be preserved, For example, it is preferable to use after sterilization by a treatment such as heat sterilization.

  Furthermore, the food and drink of the present invention may be a health food, a functional food, a food for specified health use, or a food for a sick person, and for its production, in addition to commonly used food materials and food additives, Food and beverage preparations using adjuvants such as excipients, extenders, binders, disintegrants, lubricants, dispersants, preservatives, wetting agents, solubilizers, preservatives, stabilizers, capsule bases, etc. It can be used in the form. Examples of the adjuvant include lactose, fructose, glucose, starch, gelatin, magnesium carbonate, synthetic magnesium silicate, talc, magnesium stearate, calcium carbonate, methylcellulose, carboxymethylcellulose, or a salt thereof, gum arabic, polyethylene glycol Syrup, petrolatum, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium sulfite, sodium phosphate, pullulan, carrageenan, dextrin, reduced palatinose, sorbitol, xylitol, stevia, synthetic sweetener, citric acid, ascorbic acid, Examples include acidulants, baking soda, sucrose esters, plant oils and fats, potassium chloride, safflower oil, beeswax, soybean lecithin, and fragrances.

  In addition, the immunostimulatory composition of the present invention can be used as a pharmaceutical having immunostimulatory activity by mixing with a pharmaceutically acceptable carrier, excipient, binder, diluent or the like. The pharmaceutical product is not particularly limited as long as it contains the immunostimulatory composition of the present invention, and can be administered orally or parenterally. Examples of oral agents include granules, Powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, suspensions and the like can be mentioned. As parenterals, injections (for example, subcutaneous injections, intravenous injections, Intramuscular injections, intraperitoneal injections), drops, external preparations (eg, nasal preparations, transdermal preparations, ointments), suppositories (eg, rectal suppositories, vaginal suppositories) and the like. it can. Examples of pharmaceutically acceptable excipients and additives include carriers, binders, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, and the like. Specific examples of the carrier include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting wax, cocoa butter, etc. As an example.

  In the following examples, the present invention will be described specifically and in more detail. The following examples are for illustrative purposes only and are not intended to limit the technical scope of the present invention.

[Pediococcus sp. Immunostimulatory effect of KB1 (NITE P-755)]
The novel lactic acid bacteria Pediococcus sp. For the purpose of confirming the immunostimulatory effect of KB1 (NITE P-755), IFN-γ and IL- of immune cells of the novel lactic acid bacteria of the present invention and several known lactic acid bacteria having a high immunostimulatory effect. The effect on 12 production was compared in vitro. In the test, various cultured lactic acid bacteria dead cells were used. In a 96-well plate for cell culture, 5 × 10 ⁵ DBA / 2 mouse spleen cells / hole and dead cells of each lactic acid bacterium are adjusted to a concentration of 0.01 μg / mL, 0.1 μg / mL, and 1 μg / mL. Added. The total amount of cell culture fluid was 250 μL / well. Two days after the start of cell culture, the culture supernatant was collected, and the concentrations of IFN-γ and IL-12 in the supernatant were measured by ELISA.
FIG. 2 shows the measurement result of INF-γ. Pediococcus sp. In the system to which KB1 (NITE P-755) was added, IFN-γ production was significantly promoted as compared to the control (no additive system). This promoting action was confirmed to be equivalent to or better than the known lactic acid bacteria compared. Among them, a significant increase in the amount of IFN-γ production was confirmed when compared with Lactobacillus cathay, Pediococcus pentosaceus, Lactobacillus paracathesia, and Lactobacillus brevis.
FIG. 3 shows the measurement results of IL-12. Pediococcus sp. In the system to which KB1 (NITE P-755) was added, IL-12 production was significantly promoted as compared to the control (no additive system). This promoting action was confirmed to be equivalent to or better than the known lactic acid bacteria compared. Especially, when compared with Lactobacillus gasseri, a significant increase in IL-12 production was confirmed.

[Pediococcus sp. Artificial gastric juice resistance of KB1 (NITE P-755)]
Pediococcus sp. The gastric juice resistance of KB1 (NITE P-755) was evaluated using artificial gastric juice by the following method.
An artificial gastric juice was prepared by adding 0.04% pepsin to 5.2% MRS medium and adjusting the pH to 2.5 with 6M HCl. The KB1 strain was added to this artificial gastric juice so as to be about 2 × 10 ⁹ / ml, and cultured at 37 ° C. for 1 to 3 hours. A part of the culture solution was appropriately diluted at 1 hour, 2 hours and 3 hours of culture, applied to MRS medium, and anaerobically cultured at 37 ° C. for 48 hours. The number of colonies of the grown KB1 strain was counted by the colony count method, and the change in survival rate (%) was measured. A similar experiment was performed using the standard strain Pediococcus pentosaceus (JCM 5890) instead of the KB1 strain.
The results are shown in FIG. When the survival rate before the artificial gastric juice treatment is 100%, Pediococcus sp. The survival rate of KB1 (NITE P-755) was 90%, which was found to be significantly higher than that of the standard strain Pediococcus pentosaceus (JCM 5890). From this result, Pediococcus sp. It was suggested that KB1 (NITE P-755) has excellent artificial gastric juice resistance.

[Pediococcus sp. Adhesion of KB1 (NITE P-755) to intestinal tract]
Pediococcus sp. The ability of KB1 to adhere to the intestinal mucosa was evaluated by the following test method using Caco-2 cells.
10% inactivated FBS, penicillin (50 U / ml) and streptomycin (50 μg / ml) were added to a DMEM medium containing 25 mM glucose (GIBCO DMEM 11971) to prepare a Caco-2 cell culture medium. 2 ml of this medium was dispensed into a 6-well plate, and Caco-2 cells were added to a concentration of 10 ⁴ cells / ml.
The medium was changed every 2 days at 5% CO 2 and 37 ° C. and cultured for 2 weeks (2 × 10 ⁶ cells / well). After the culture, the culture solution was removed, and 2 ml of a lactic acid bacterium solution suspended in a Caco-2 cell culture medium to which no antibiotic was added was added thereto. As a lactic acid bacterium, Pediococcus sp. KB1 and L. have the ability to adhere to the intestinal mucosa. acidophilus JCM1132 was used. After addition of the lactic acid bacteria solution, incubation was performed at 5% CO 2 and 37 ° C. for 1 hour. The culture solution was removed, washed 3 times with PBS (+) buffer (manufactured by Sigma), Caco-2 cells were peeled off by trypsin treatment, and suspended and collected in PBS (-) (manufactured by Sigma). A 1/10 dilution series of the recovered solution was prepared, 1 ml of this diluted solution was added to the petri dish, and 20 ml of MRS agar medium was added to the petri dish and mixed. Anaerobic culture was carried out at 37 ° C. for 2 days, the number of colonies of the appeared lactic acid bacteria was measured, and the number of attached lactic acid bacteria was calculated.
The adhesion rate of lactic acid bacteria to Caco-2 cells was calculated according to the following formula. The results are shown in Table 1.
Lactic acid bacteria adhesion rate (%) = (number of lactic acid bacteria attached / number of lactic acid bacteria added) × 100

  Pediococcus sp. KB1 is L. It showed Caco-2 cell adhesion rate equal to or better than acidophilus JCM1132, and was found to be able to adhere to the human intestinal tract.

[Identification of bacteria by 16S rDNA base sequence]
Pediococcus sp. The analysis was performed by requesting Techno Suruga Lab Co., Ltd. for 16S rDNA-Full molecular phylogenetic analysis of KB1 (NITE P-755) strain. The base sequence obtained as a result of the analysis is shown in SEQ ID NO: 1. The analysis result of the base sequence of 16S rRNA revealed that this bacterium is a new species that does not fall under any of the known lactic acid bacteria.

NITE P-755

Claims (4)

Have immunostimulatory activity, it has a gastric fluid resistant and high adhesion to intestinal milk acid bacteria Pediococcus (Pediococcus) sp. KB1 (NITE P-755). Lactic acid bacteria Pediococcus sp. An immunostimulatory composition containing KB1 (NITE P-755) as an active ingredient. Food / beverage products containing the immunostimulatory composition of Claim 2. A pharmaceutical comprising the immunostimulatory composition according to claim 2.