JP2010227043A - Infection protective agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism enhancing the effect of vaccine through humoral immunity and simultaneously preventing virus infection through cell-mediated immunity; and to provide uses thereof. <P>SOLUTION: There are disclosed Lactobacillus pentosus YM2-2 strain, and a mucosal immunostimulator, an IgA production promoting agent, a virus (especially rotavirus) infection protective agent, and foods and drinks, wherein these comprise the strain as an active ingredient. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a novel lactic acid strain and an infection protective agent using the same.

  Infectious diseases caused by pathogenic viruses occur every year in developing countries and developed countries, and are the main cause of death in infants and children. For example, rotavirus diarrhea is a disease that occurs frequently in infants aged 6 months to 2 years, which causes 600,000 deaths every year in developing countries. In addition, this disease occurs not only in humans but also in domestic animals such as pigs, and is a major economic hit.

  In the immune response, lymphocytes that have received an antigen make an antigen-specific IgA antibody, and the antibody is injected into the antigen like a missile to inactivate (neutralize) the pathogenicity, and macrophages As represented by lymphocyte killer cells, there are roughly two methods of cell-mediated immunity that visits and kills foreign microorganisms, tumor cells formed in the body, and virus-infected cells.

  In recent years, research and development aimed at preventing infection through the mucosal immune mechanism has been actively conducted. Mucosal immunity is an infection defense mechanism that is first performed when a pathogen adheres to the mucous membrane (see Non-Patent Document 1). Secretory IgA (S-IgA) in mucus exhibits a protective action against pathogens such as bacteria and viruses (see Non-Patent Document 2) and also plays a role in neutralizing toxins produced by microorganisms (Non-patent) Reference 3).

  Conventionally, vaccines using viral antigens have been developed as treatment methods for infectious diseases. This treatment involves intestinal IgA antibodies. Attenuated virus is immunized as a vaccine, which produces virus-specific IgA antibodies. IgA antibody works to prevent pathogenic microorganisms from entering the mucous membrane, neutralize viruses and toxins, prevent food allergens from entering, etc. It is important for the defense of living organisms to increase such IgA antibodies. is there.

  An adjuvant that promotes the production of IgA antibody is called an adjuvant, but an adjuvant using lactic acid bacteria has been reported. For example, Yasui et al. Conducted a mouse infection experiment with rotavirus, the main causative virus of infant diarrhea, ingested Bifidobakuterium breve YIT4064 strain in mother mice, and then used mother mice's breast milk As a result of ingestion to mice, it has been reported that the development of diarrhea was suppressed in pup mice infected with rotavirus (see Non-Patent Document 4 and Patent Document 1). In addition, there are some reports on strains with high IgA production promoting ability (see Patent Documents 2 and 3). However, it is not known that Lactobacillus pentosus has an IgA antibody production promoting action or a virus infection protecting action.

Japanese Patent No. 3269890 Japanese Patent No. 3818319 JP 2007-308419 A

Brandtzaeg, P. Curr. Top. Microbiol. Immunol. 146: 13 1989. Czinn, S, J, .et al Vaccine 11: 637 1993; Renegar, K. et al J. Immunol. 146: 1972 1991. Brandtzaeg, P APMIS 103: 1 1995; Kilian, M. et al Microbiol. Rev. 52: 296 1988. H. Yasui et al J. Infect. Dis. 172: 403.1995.

Depending on the type of virus, the development of appropriate vaccines has not yet been successful. Moreover, since the antibody produced by a vaccine is antigen-specific, there exists a subject that an effect is not anticipated with respect to the virus which is easy to mutate. Furthermore, although an IgA production promoter has an application as an adjuvant, there is a problem that it does not guarantee immune activation via cellular immunity. Thus, there is a strong craving for non-antigen-specific infection protection agents.
The present invention relates to providing a microorganism having the ability to enhance the effect of a vaccine via humoral immunity and at the same time protect against viral infection via cellular immunity, and uses thereof.

In order to solve the above-mentioned problems, the present inventors diligently searched for food-derived microorganisms. As a result, a novel lactic acid bacterium Lactobacillus pentosus strain YM2-2 (FERM AP-) isolated from Awa Bancha 21778) was found to have an adjuvant effect when used in combination with a vaccine and to protect against rotavirus infection to the same extent as vaccines even when administered alone (see Example 2).

That is, the present invention relates to the following 1) to 6).
1) Lactobacillus pentosus YM2-2 strain deposited as FERM AP-21778 at the National Institute of Advanced Industrial Science and Technology (AIST).
2) A mucosal immunostimulant comprising the bacterial cell of 1) above or a culture thereof as an active ingredient.
3) An IgA production promoter comprising the bacterial cell of 1) above or a culture thereof as an active ingredient.
4) A virus infection protective agent comprising as an active ingredient the bacterial cell of 1) above or a culture thereof.
5) The virus infection protective agent of said 4) whose virus is rotavirus.
6) A composition selected from foods and drinks, pharmaceuticals, external preparations and feed, which contains the bacterial cells of the strain 1) or a culture thereof.

  Since the lactic acid bacterium of the present invention has an IgA production promoting effect, administration of the lactic acid bacterium together with the vaccine enhances the production of antibodies against antigens including the vaccine, improves the induction of protective immunity, and enhances the effect of the vaccine. At the same time, cellular immunity can also be activated, so that virus infection can be prevented to the same extent as vaccines by administration of lactic acid bacteria alone, without relying on immunization with vaccines. Therefore, it can exert an effect on a virus that is difficult to act on a vaccine and is easily mutated, and is very useful. In addition, it is particularly useful as a food and drink and a pharmaceutical because it can exhibit the original effects of lactic acid bacteria, such as an intestinal regulating action, an intestinal bacterial flora improving action and the like.

It is the figure which showed the IgA production promotion ability of the lactic acid strain isolate | separated from Awaban tea. It is the figure which showed the experiment schedule.

[Lactic acid bacteria of the present invention]
The lactic acid bacterium Lactobacillus pentosus strain YM2-2 of the present invention was separated from Awa bancha tea using mouse Peyer's plate cell culture system as an index and induced by IgA production. A new lactic acid strain collected (screened in Example 1 below). On March 4, 2009, the National Institute of Advanced Industrial Science and Technology (AIST, Tsukuba City, Ibaraki, Japan) 1-1, center 6) and deposited as FERM AP-21778. The main mycological properties of the YM2-2 strain are shown below.

(A) Macroscopic characteristics (a-1) MRS agar medium Circularly slightly irregular, hemispherical, smooth, milky white (a-2) BL agar medium Circularly slightly irregular, hemispherical, smooth, white brown (b) Microscopic features It is a koji mold and has no motility. Spores do not form.
(C) Growth temperature It grows well at 30 to 37 ° C.
(D) Microbial taxonomic position The base sequence of 16S ribosomal DNA of this strain (GenBank / EMB / DDBJ accession number AB488810) shows 100.0% homology with the Lactobacillus pentosus strain and further targets the recA gene as a target. From the determined PCR, the strain was identified as belonging to Lactobacillus pentosus.

[Mucosal immunity, rotavirus infection protection]
As shown in Examples 1 and 2 below, the lactic acid bacterium of the present invention enhances the effect of the vaccine via humoral immunity, and at the same time causes rotavirus infection to the same extent as the vaccine through single cell administration. Has the ability to defend. Here, “protecting rotavirus infection” specifically means suppressing the onset of diarrhea caused by virus infection.

  The lactic acid bacterium of the present invention has an IgA antibody production promoting action, and has an action of reducing the incidence of diarrhea due to rotavirus infection as compared with a vaccine alone. That is, an adjuvant effect is exhibited when used in combination with a vaccine.

  The mechanism of action contributing to this humoral immunity is considered as follows. That is, first, the Peyer's patch M cells constituting the intestinal tract immune system take up the antigen in the lumen. The antigen is presented to Th cells by antigen presenting cells such as dendritic cells. Due to the action of antigen-specific Th2 cells, immature B cells mature and move to the lamina propria and finally differentiate into IgA antibody-producing cells. It is still unclear how the lactic acid bacteria of the present invention are involved in this IgA production promoting mechanism, but at least humoral immunity (IgA production) is promoted by the presence of the lactic acid bacteria of the present invention. Since cells need to take up an antigen, the lactic acid bacteria of the present invention are considered to function as this antigen.

  In addition, the lactic acid bacterium of the present invention can reduce the incidence of diarrhea due to rotavirus infection to the same level as that of a vaccine by the activation effect of cellular immunity. That is, even when administered alone, it has the effect of protecting against rotavirus infection to the same extent as vaccines.

  The mechanism of action that contributes to this cellular immunity is considered as follows. That is, the antigen taken into the M cell differentiates the helper T (Th) cell into a type I helper T (Th1) cell via the antigen presenting cell, thereby activating the T cell into a killer T cell. Since the production of interleukin 12 and interferon γ from Th1 cells and natural killer (NK) cells is promoted by the presence of the lactic acid bacterium of the present invention, the lactic acid bacterium of the present invention functions as an antigen that activates cellular immunity. it is conceivable that.

It is presumed that the difference in recognition of lactic acid bacteria by receptors expressed on antigen-presenting cells (Toll-like receptor) is involved in the control of humoral immunity and cellular immunity.
That is, it is considered that the lactic acid bacteria of the present invention have a mucosal immunity activation effect of activating humoral immunity in the presence of a vaccine and activating cellular immunity in the absence of a vaccine.

  Therefore, the lactic acid bacteria of the present invention or a culture thereof can be a mucosal immunity activator, IgA production promoter, virus infection protective agent for administration or ingestion to animals including humans, and is caused by virus infection. Food, food, medicine, feed, topical preparation, etc. that exerts an adjuvant effect when used in combination with a vaccine against the onset of diseases, particularly diarrhea caused by rotavirus infection, and can protect against the virus infection when administered alone It can be used as various compositions.

  Here, as an infectious disease to be protected against infection, IgA produced by the lactic acid bacterium of the present invention is considered to exert systemic functions, and thus is not necessarily limited to the intestinal tract, but is secreted IgA. Progenitor cells are present in Peyer's patches, and when sensitized, they circulate in the body, differentiate into IgA-producing cells, and return to the mucosal tissue. From the mucous membranes of the oral mucosa, respiratory mucosa, digestive mucosa, etc. It is particularly effective for invading mucosal infectious diseases and is considered to exhibit an excellent vaccine enhancement effect. Specific examples of such infectious diseases include rotavirus, poliovirus, influenza virus, AIDS virus, hepatitis A and hepatitis B viruses.

  In addition, vaccines against infectious diseases include transdermal inoculations, subcutaneous injections, oral administrations, and the like. In consideration of direct stimulation of Peyer's patches, ease of administration, etc., the lactic acid bacteria of the present invention Is preferably administered together with an orally administered vaccine. In addition, as the vaccine, any of vaccines using dead bacteria (inactivated vaccines), vaccines using attenuated strains (live vaccines), and those using a partial structure of bacteria (component vaccines) can be used. Is possible.

[Various compositions]
When the lactic acid bacteria of the present invention are used in the form of various compositions such as foods and drinks, pharmaceuticals, external preparations (external medicines, cosmetics, etc.), feeds, etc., the lactic acid bacteria cells are obtained by culturing according to conventional methods of lactic acid bacteria culture. In addition to using the product isolated from the cultured product by means of collection of bacteria such as centrifugation, the culture / fermentation liquid (culture supernatant), the crude or purified product of the culture, and lyophilization thereof A cytoplasm or cell wall fraction obtained by treating a product or bacterial cells with an enzyme or physical means can also be used.
Further, the microbial cells may be sterilized not only by viable cells but also by ordinary general heat sterilization operations. Even sterilized cells can not only be expected to maintain health and longevity due to intestinal regulation, intestinal microbiota improvement, etc., but in the case of live bacteria, it is in the form at the time of delivery and display after product manufacture Since there is a possibility of causing a change, dead cells that do not cause any further morphological change can be preferably used.

  For example, as shown in Example 3, the culture solution can be obtained by culturing at 30 to 37 ° C. for about 16 to 28 hours using a medium suitable for the lactic acid bacteria of the present invention, such as MRS medium. The cultured cells can be obtained after culturing, for example, by centrifuging the culture solution at 3,000 rpm for 10 minutes at 4 ° C. for collection. These can be purified according to conventional methods. Furthermore, the cells can be freeze-dried or spray-dried. The bacterial cells thus obtained can be used as an active ingredient of the composition of the present invention.

  In the composition of the present invention, the lactic acid bacterium of the present invention can be used as it is, but a suitable edible carrier (food material) and a pharmaceutically acceptable carrier are appropriately blended, and food and drink, pharmaceuticals, and external preparations as described later. It is preferably prepared in the form of a feed or the like.

In addition, the composition of the present invention can further contain an appropriate amount of nutritional components suitable for maintenance, growth, etc. of the lactic acid bacteria of the present invention, as necessary.
Specific examples of the nutritional component include carbon sources such as glucose, starch, sucrose, lactose, dextrin, sorbitol, and fructose, and nitrogen sources such as yeast extract and peptone that are used in a culture medium for culturing microorganisms. , Vitamins, minerals, trace metal elements, and other nutritional components. Examples of vitamins include vitamin B, vitamin D, vitamin C, vitamin E, vitamin K, and the like. Examples of trace metal elements include zinc and selenium. Examples of other nutritional components include various oligosaccharides such as dairy oligosaccharide, soybean oligosaccharide, lactulose, lactitol, fructooligosaccharide, and galactooligosaccharide. The blending amount of these oligosaccharides is not particularly limited, but it is preferably selected from an amount range which is usually about 1 to 30% by weight in the composition of the present invention.

  In addition, when the composition of the present invention contains lactic acid bacteria as dead cells, conditions such as heating and pressurization may be employed for commercialization of the composition.

The blending amount of lactic acid bacteria in the composition of the present invention is generally about 10 ⁸ to 10 ¹¹ bacteria in 100 g of the composition of the present invention (it is not necessary to be the number of living bacteria. If it is included, it is counted as the number of viable bacteria before sterilization. The number of viable bacteria is calculated by applying a diluted sample to an agar medium for culturing bacteria, culturing at 37 ° C., and counting the number of grown colonies. Since the viable cell count and turbidity are correlated, if the correlation between the viable cell count and turbidity is obtained in advance, the viable cell count is counted by measuring the turbidity instead of measuring the viable cell count. it can. The blending amount of the lactic acid bacterium can be appropriately changed according to the form of the composition of the present invention to be prepared, the type of lactic acid bacterium to be used, and the like, using the above amount as a guide.

The composition of the present invention is used with a vaccine or the composition alone. When used with a vaccine, the composition of the present invention can be administered before and after vaccine administration, and can also be used as a vaccine effect enhancer that enhances the effect.
The amount of the composition used varies depending on the type and quality of the vaccine used, age, symptoms, etc. For example, for use for prevention, it is about 0.01 to 10 g in terms of solid content per adult. It is desirable to take three times a day about 30 minutes before meals. In addition, when used as a health food, it is appropriate to use it in an amount that does not adversely affect the taste and appearance of the food, for example, in the range of about 0.1 to 100 g in terms of solid content with respect to 1 kg of the target food. .

1) Food / beverage products When the composition of the present invention is used as a food / beverage product, for example, fermented milk, lactic acid bacteria beverages, fermented vegetable beverages, fermented fruit beverages, fermented soy milk beverages, etc. Can be mentioned. In the present specification, the terms “fermented milk” and “lactic acid bacteria beverage” shall conform to the definitions of the former Ministry of Health and Welfare “Ministerial Ordinance on Components of Milk and Dairy Products”, Article 2 37 “Fermented Milk” and 38 “Lactic Acid Beverages”. To do. That is, “fermented milk” refers to milk or dairy products made into a paste or liquid obtained by fermenting with lactic acid bacteria or yeast. Accordingly, the fermented milk includes a yogurt form as well as a beverage form. The “lactic acid bacteria beverage” refers to a beverage obtained by diluting milk or a dairy product with a paste or liquid obtained by fermentation with lactic acid bacteria or yeast as a main ingredient.

  Examples of other food and drink products include fermented foods such as pickles, miso, fermented tea, ban, baby food such as baby food, powdered milk, baby food, foamed preparations, confectionery such as gum, gummi, pudding, noodles, Examples include dietary supplements such as capsules, granules, powders, tablets, and dairy products other than the fermented milk and lactic acid bacteria beverages.

  In addition, the food and drink of the present invention includes functional foods such as foods for specified health use and health foods, which are based on the concept of infection prevention, prevention of diarrhea, and the like as necessary. Health food means a food that is more active than ordinary food, and is intended for health, health maintenance and promotion, for example, liquid or semi-solid, solid products such as cookies, Examples include confectionery such as rice crackers, jelly, yokan, yogurt and manju, soft drinks, nutritional drinks, soups and the like.

In the production of such foods and drinks, various components can be added and blended to increase the flavor or make the necessary shape, and further flavor can be added to obtain a final product.
Examples of such addition and mixing components include various sugars, emulsifiers, sweeteners, acidulants, fruit juices, and the like. More specifically, sugars such as glucose, sucrose, fructose, and honey, sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, and palatinit, emulsifiers such as sucrose fatty acid ester, glycerin sugar fatty acid ester, and lecithin are included. . In addition to this, the composition of the present invention has excellent flavor even when various vitamins such as vitamin A, vitamin B, vitamin C and vitamin E, herbal extracts, grain ingredients, vegetable ingredients, milk ingredients, etc. are blended. Obtainable.

  In addition, flavors include yogurt, berry, orange, quince, perilla, citrus, apple, mint, grape, pair, custard cream, peach, melon, banana, tropical, herbal, tea And coffee-based flavors, which can be used alone or in combination of two or more. The addition amount of the flavor is not particularly limited, but is preferably 0.05 to 0.5% by mass, particularly preferably about 0.1 to 0.3% by mass in terms of the flavor.

Hereinafter, preparation of fermented vegetable drinks, fermented fruit drinks and fermented soymilk drinks will be described in detail.
Preparation into each of these forms is performed by culturing lactic acid bacteria in a liquid such as vegetables, fruits and soy milk (soy emulsified liquid) containing a nutrient source for lactic acid bacteria to ferment the raw material. Can be done.
Vegetables and fruits as raw materials for fermentation include cuts, crushed products, ground products, juices, enzyme-treated products, diluted products and concentrates of various vegetables and fruits. Vegetables include pumpkins, carrots, tomatoes, peppers, celery, spinach, colored sweet potatoes, corn, beet, kale, parsley, cabbage, broccoli. Fruits include apples, peaches, bananas, strawberries, grapes, watermelons, oranges, tangerines and the like. The raw material for fermentation may be a non-edible part residue of vegetables and fruits, a food residue such as okara and shochu.

Cut and crushed vegetables and fruits, grinds, etc., for example, after washing the vegetables or fruits, blanching treatment such as putting in hot water as necessary, crusher, mixer, food processor, pulper fisher It can be obtained by cutting, crushing, and grinding using Mycolloider (Mycolloider ^™ , manufactured by Tokushu Kika Kogyo Co., Ltd.).

  Juice can be prepared using a filter press, a juicer mixer, etc., for example. The juice can also be prepared by filtering the ground product using a filter cloth or the like. The enzyme-treated product can be prepared by allowing cellulase, pectinase, protopectin-degrading enzyme, etc. to act on the cut product, crushed product, ground product, and juice. Dilutions include those diluted 1-50 times with water. Concentrates include, for example, those concentrated 1 to 100 times by means such as freeze concentration and vacuum concentration.

  Soymilk, which is another specific example of the raw material for fermentation, can be prepared from a soybean raw material according to a conventional method. In the soy milk, for example, moistened soybeans are immersed in water, wet milled using a suitable mill such as a colloid mill, and then homogenized and homogenized according to a conventional method, water-soluble soybean protein is dissolved in water A solution and the like are also included.

Fermentation using lactic acid bacteria is preferably performed by preparing a starter in advance, inoculating the raw material for fermentation and fermenting it. The starter is typically inoculated with the lactic acid bacteria of the present invention into a fermentation raw material that has been subjected to normal sterilization treatment at 90-121 ° C. for 5-20 minutes in advance, 10% nonfat dry milk added with yeast extract, and the like. Can be mentioned. The starter thus obtained usually contains about 10 ⁷ -10 ⁹ cells / g culture of the lactic acid bacterium of the present invention.

  The fermentation raw material used for the starter includes, if necessary, fermentation promoting substances for good growth of the lactic acid bacteria of the present invention, for example, carbon sources such as glucose, starch, sucrose, lactose, dextrin, sorbitol, fructose, yeast extract, Nitrogen sources such as peptone, vitamins and minerals can be added.

Inoculum of lactic acid bacteria are generally bacterial cells fermentation material containing solution 1mL of about 1 × 10 ⁶ or more is suitable be selected from amounts that preferably is intended to include 1 × 10 ⁷ cells before and after . The culture conditions are generally selected from a fermentation temperature of about 20-42 ° C, preferably about 25-37 ° C, and a fermentation time of about 5-72 hours.

The lactic acid fermented product obtained as described above may have a card-like form (yoghurt-like or pudding form), which can be taken as it is as a solid food. The carded lactic acid fermented product can be made into a desired beverage form by further homogenizing it. This homogenization can be carried out using a general emulsifier (homogenizer). Specifically, the homogeneous structure formation, for example using a Gaulin (GAULIN) manufactured by a high pressure homogenizer (LAB40), about 200-1000kgf / cm ^2, preferably about 300-800kgf / cm ² condition, or Sanwa It can be carried out under a condition of 150 kg / cm ² or more using a machine industry homogenizer (product number: HA × 4571, H20-A2, etc.). By this homogenization, a beverage having an excellent texture, particularly smoothness, can be obtained. In addition, in this homogenization, appropriately diluted as necessary, organic acids for pH adjustment are added, and production of beverages such as sugars, fruit juices, thickeners, surfactants, flavorings, etc. Various additives that are usually used can be added as appropriate.

  Specific examples of preferable additives and the amount thereof added (% by weight based on the weight of the carded fermented product) include, for example, glucose 8% (% by weight, the same applies hereinafter), sugar 8%, dextrin 8%, and citric acid 0.1%. Glycerol fatty acid ester 0.2% and fragrance 0.1%.

  The beverage of the present invention thus obtained can be aseptically filled into a suitable container to obtain a final product. The product has a smooth texture and flavor.

The administration (intake) amount is appropriately determined according to the age, sex, body weight, degree of disease, etc. of the living body ingesting it, and is not particularly limited. In general, the amount of lactic acid bacteria is preferably selected from a range where the amount is about 10 ^{6 to} 10 ⁹ cells / mL. In general, about 50 to 1,000 mL of the product may be taken and taken per person per day.

2) Pharmaceuticals When the composition of the present invention is used as a pharmaceutical, it is prepared and used in the form of a general pharmaceutical composition using an appropriate pharmaceutical carrier that is pharmaceutically acceptable together with the lactic acid bacteria of the present invention. As the pharmaceutical carrier, diluents or excipients such as fillers, extenders, binders, moisturizers, disintegrants, surfactants, lubricants and the like that are usually known in this field are used. An agent can be illustrated. These are appropriately selected and used depending on the dosage unit form of the preparation to be obtained.

Various dosage forms can be selected as the dosage unit form of the pharmaceutical composition, and preferred examples include preparations for oral administration and preparations for external use.
Representative examples of oral preparations include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules and the like.

  In the case of forming into a tablet form, as the above-mentioned preparation carrier, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate and other excipients; water, ethanol , Propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone, and other binders; sodium carboxymethylcellulose, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, dry starch Disintegrating agents such as sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate; polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate Surfactants such as stearic acid monoglyceride; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil; absorption promoters such as quaternary ammonium base and sodium lauryl sulfate; humectants such as glycerin and starch; Adsorbents such as lactose, kaolin, bentonite and colloidal silicic acid; lubricants such as purified talc, stearate, boric acid powder and polyethylene glycol can be used. Further, the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets.

  When forming into a pill form, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc and the like as a formulation carrier; binders such as gum arabic powder, tragacanth powder, gelatin, ethanol; Disintegrants such as laminaran and agar can be used.

  Furthermore, in the pharmaceutical composition, a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals may be contained as necessary.

The amount of the lactic acid bacterium of the present invention to be contained in the pharmaceutical composition is not particularly limited and is appropriately selected from a wide range. Usually, about 10 < ⁷ > -10 < ¹² > pieces / dosage unit form should be contained in the composition.

  The administration method of the pharmaceutical composition is not particularly limited, and is determined according to various preparation forms, patient age, sex and other conditions, the degree of disease, and the like. The dose is appropriately selected depending on the usage, patient age, sex and other conditions, disease severity, etc. The amount of the lactic acid bacterium of the present invention, which is usually an active ingredient, is about 0.5-kg / kg body weight per day. The dosage is preferably about 20 mg, and the preparation can be administered to humans in 1 to 4 divided doses per day.

3) External preparation When the composition of the present invention is used as a composition for external use such as cosmetics, external medicines, quasi drugs, etc., it is generally used together with the lactic acid bacteria of the present invention using an appropriate pharmaceutical carrier that is pharmaceutically acceptable. Prepared in the form of a typical external preparation composition.
Such pharmaceutical carriers include, for example, humectants such as glycerin, petrolatum, urea, hyaluronic acid, heparin; PABA derivatives (paraaminobenzoic acid, Escalol 507, etc.), cinnamic acid derivatives (neoheliopan, Pulsol MCX, Sungard B, etc.) , Salicylic acid derivatives (octyl salicylate, etc.), benzophenone derivatives (ASL-24, ASL-24S, etc.), dibenzoylmethane derivatives (Parsol A, Parsole DAM, etc.), heterocyclic derivatives (tinuvin type, etc.), titanium oxide, etc. UV absorbers / scattering agents; metal sequestering agents such as edetate disodium, edetate trisodium, citric acid, sodium citrate, tartaric acid, sodium tartrate, lactic acid, malic acid, sodium polyphosphate, sodium metaphosphate, gluconic acid; Salicylic acid, sulfur, caffeine, Sebum inhibitors such as ninnin; bactericides and disinfectants such as benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate; Anti-inflammatory agents such as acids; vitamin A, vitamin B group (B1, B2, B6, B12, B15), folic acid, nicotinic acid, pantothenic acid, biotin, vitamin C, vitamin D group (D2, D3), vitamin E, Vitamins such as ubiquinones and vitamin K (K1, K2, K3, K4); amino acids such as aspartic acid, glutamic acid, alanine, lysine, glycine, glutamine, serine, cysteine, cystine, tyrosine, proline, arginine, pyrrolidone carboxylic acid And Derivatives; whitening agents such as retinol, tocopherol acetate, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, kojic acid, ellagic acid, placenta extract; antioxidants such as butylhydroxytoluene, butylhydroxyanisole, propyl gallate; Astringents such as zinc chloride, zinc sulfate, zinc carbonate, zinc oxide, aluminum potassium sulfate; sugars such as glucose, fructose, maltose, sucrose, trehalose, erythritol, mannitol, xylitol, lactitol; licorice, chamomile, maronier, yukinoshita, In addition to various plant extracts such as glaze, karin, ougon, abalone, auren, jujube, ginkgo biloba, etc., oily components, surfactants, thickeners, alcohols, powder components, pigments and the like can be mentioned.

  Specific examples of external preparation compositions include cosmetic creams, emulsions, lotions, packs, skin milk (emulsions), gels, powders, lip balms, lipsticks, under-makeups, foundations, sun cares, bath preparations, Examples include body shampoos, body rinses, soaps, cleansing foams, ointments, patches, jellies, and aerosols.

4) Feed When the composition of the present invention is used as a feed, for example, a preparation form for oral administration (aqueous solution, emulsion, granule) for the prevention of infectious diseases in the weaning period of pigs, cattle, etc. , Powder, capsule, tablet, etc.).

  EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all by these Examples.

Example 1
This example uses the Peyer's plate cell culture system according to the method described by Yasui et al. (Yasui, H., et al., Microbial Ecology in Health and Disease, 5,155, 1992.) This example was tested in vitro and was performed as follows.

(1) Test animals Inbred female mice SPF / VAF BALB / c AnNCrj were used.
The test mice were quarantined for 1 week after arrival. During the quarantine period, MF solid feed (Oriental Yeast Co., Ltd.) and tap water were freely ingested.

(2) Peyer's plate cell culture method After completion of the quarantine, 80 mice were divided into 10 groups so that the weight of each group was equal. After grouping, 10 mice were sacrificed every day, the small intestine was removed, Peyer's patch on the outer surface of the small intestine was cut out, MEM medium [Eagle's MEM (NISSUI), 2 mM glutamine (GIBCO), 1 mM sodium pyruvate ( GIBCO) and MEM nonessential amino acids (GIBCO)] were added in a centrifuge tube to which ice cooling was performed. A single cell suspension was prepared using a mesh and washed well with 5 mL of MEM medium. The cell suspension was filtered and centrifuged at 1,000 rpm at 4 ° C. for 10 minutes. After centrifugation, the culture supernatant was removed by suction, and the precipitate was suspended in 5 mL of MEM medium. After the same operation was repeated twice, the precipitate was suspended in 10 mL of MEM medium containing 10% FBS (GIBCO), the number of living Peyer's plate cells was counted, and the cell suspension was 96-well cell culture. A cell culture plate was prepared by plating on a plate for cell culture.

(3) Preparation of test bacterial cells 25 lactic acid bacteria isolated from Awa Bancha were used. Each bacterium was cultured in an MRS (GIBCO) medium until stationary phase, and then centrifuged to collect microbial cells (2,000 rpm × 20 minutes, 4 ° C.). After washing 3 times with PBS (-), it was lyophilized. At the time of use, various freeze-dried cells were suspended in PBS (−) to 1 mg / mL, and sterilized by heating at 121 ° C. for 15 minutes in an autoclave. Thereafter, the resultant was diluted to 200 μg / mL with RPMI 1640 medium (Sigma).

(4) Measurement of IgA concentration in culture supernatant Peyer's patch cells prepared in (1) above are suspended in MEM medium containing 10% FBS, adjusted to 5 × 10 ⁶ cells / mL, Placed in a well cell culture plate. To each well of this plate, 100 μL of the test cell suspension prepared at 200 μg / mL (prepared in the above (3)) was added and cultured at 37 ° C. under 5% CO ₂ for 7 days.
Next, the total IgA concentration of each culture supernatant obtained was measured by ELISA using a commercially available kit.

(5) IgA production promoting activity of the lactic acid bacterium of the present invention The total amount of IgA in the lactic acid bacterium measured according to (4) above was added with 100 μL of RPMI 1640 medium containing no microbial cells as a control (no microbial cells added), and cultured for 7 days. The measured value of the culture supernatant obtained in this manner is used as a reference (1.0), and the relative ratio (Stimulation Index; SI) is shown in FIG.

(6) Results As shown in FIG. 1, the IgA production inducing ability has the highest IgA production inducing ability of the lactic acid bacteria of the present invention (YM2-2 strain), which is 25.8, among the 25 test strains. I found out.
Furthermore, even when compared with the same kind of Lactobacillus pentosus strain and its related species Lactobacillus plantarum strain, according to the specification of Patent Document 2, in the case of Lactobacillus pentosus strain, NRIC0391 (1.0), NRIC0392 (1.0 ), NRIC0393 (1.2), NRIC0394 (1.2), and the related species Lactobacillus plantarum strains, NRIC1919 (1.3), NRIC1920 (1.1), NRIC1921 (1.1), NRIC1923 (1.0), NRIC1957 (1.0), and NRIC1958 (1.3) were remarkably low, indicating that the strain difference in IgA antibody production was significant.
Therefore,

Example 2
This test reveals that the intake of the lactic acid bacteria of the present invention is effective in protecting against rotavirus diarrhea.
[Anti-rotavirus infection effect]
The test method was based on the method described in Patent Document 1. As a test model, diarrhea due to rotavirus occurs only in the first few days after birth, when immunity is not established. Therefore, it is not possible to establish a model that first immunizes a newborn mouse before subjecting it to an infection test. Therefore, by first immunizing mother mice with rotavirus as a vaccine and transmitting the immunity substance to the pups through breast milk to the pups born to the mother, there was an effect equivalent to vaccine immunization to the pups. I saw it. That is, a system that protects rotavirus infection in suckling pups by activating the immune mechanism of the mother mouse with the lactic acid bacteria of the present invention was adopted.

  Specifically, the infection protection test by ingestion of the lactic acid bacteria of the present invention was examined using the incidence of diarrhea after rotavirus infection in pups as an index. This test was conducted as follows.

(1) Test animals Inbred female SPF / VA / VAF mice (strain name: BALB / c AnNCrj) (7 weeks old) received from SLC were quarantined for 4 days under the following conditions, and then grouped according to body weight. Went. In addition, 11-week-old male mice were used for mating, and 5-day-old mice were used for testing.
Feeding / feeding method: MM-3 solid feed (Funabashi Farm) / free intake water / water supply method: tap water / free intake with water bottle Environment: temperature: 23 ± 2 ° C, humidity: 60 ± 10%
Illumination time: light period 7: 00-19: 00, dark period 19: 00-7: 00

(2) Test rotavirus (RV)
The monkey-derived SA-11 strain (group A, type III; virus titer 10 ^8.6 TCID ₅₀ / mL) was used as a live vaccine.

(3) Test Lactic Acid Bacteria The heat-dead cells of the lactic acid bacteria YM2-2 strain of the present invention were added to the bait (MM-3) at 0.1%.

(4) Test Method FIG. 2 is an explanatory diagram showing a test schedule of this example. As shown in the figure, a specific schedule for feeding, mating, immunization, delivery, RV infection in suckling pups and diarrhea observation is shown. Specifically, 7-week-old female mice were divided into a YM2-2-added feed group (YM2-2 administration group) and an additive-free feed group (control group), and were freely ingested until the end of the experiment. Four weeks after the start of the experiment (11 weeks of age), males were allowed to coexist and mated. One week later, female mice were orally immunized with rotavirus (RV) (RV immunized group). PBS was orally administered to the non-immunized group. Finally, it was divided into 4 groups as shown in Table 1. For oral immunization of mother mice, 0.5 mL of virus solution diluted 10-fold with PBS was orally administered to each mouse. Also, 20 μL of rotavirus stock solution was orally administered to pup mice at 5 days of age. Thereafter, the abdomen of each pup was pressed daily for 7 days to observe whether diarrhea had developed. Diarrhea if watery stool appears, soft stool if non-watery but soft-shaped stool appears, no symptom if stool does not appear even after pressing hard stiff or abdomen, and more than soft stool was determined to have diarrhea . Differences in the incidence of diarrhea between pups in each group were compared using Fisher's exact calculation method. In both cases, when p <0.05, it was determined that there was a significant difference. The results are shown in Table 1.

(5) Results and discussion As shown in Table 1, when the non-immunized group (control group) and the RV immunized group were compared in the YM2-2-free diet group, the incidence of diarrhea in the non-immunized group (control group) was There was a statistically significant (p <0.05) decrease in 92% and 53% in the RV immunized group. This is presumably due to the increase in anti-IgA antibody in breast milk due to RV immunization.

  In addition, in the RV immunization group, the YM2-2-free diet group (RV immunization group) and the YM2-2-containing diet group (M2-2-administered RV immunization group) were compared. The incidence of diarrhea in the immunized group was 53%, and that in the YM2-2 containing diet group was 4%, indicating a statistically significant (p <0.001) decrease. This is presumably due to the increase of anti-IgA antibody in breast milk by administration of YM2-2.

  Furthermore, in the non-RV immunity group, when the YM2-2-free diet group (control group) and the YM2-2-containing diet group (M2-2 administration group) were compared, the YM2-2-free diet group (control group) The incidence of diarrhea was 92%, and that of the YM2-2-containing diet group was 50%, indicating a statistically significant (p <0.05) decrease. This is presumably because the administration of YM2-2 enhanced the cellular immunity of the mother mice, the cytokines produced by these transferred into the breast milk, and the intestinal immunity of the pups that drank it was activated.

  When the RV immunized group and the YM2-2 administration group were compared, the incidence of diarrhea was 53% and 50%, and it was revealed that the efficacy of the anti-rotavirus infection action was almost the same.

  As shown in Table 2, the anti-rotavirus infection action via cellular immunity by the single administration of YM2-2 is statistically different from that of Bifidobacterium breve YIT4064 strain described in Patent Document 1. A clinically significant (p <0.01) decrease in the incidence of diarrhea was observed.

  As described above, the pups that sucked the milk of the mother mouse fed the lactic acid bacterium YM2-2 of the present invention are more resistant to rotavirus infection than the pups that sucked the milk of the mother mouse that did not feed it. It was clarified in both RV immunity and non-RV immunity groups.

  The effect of suppressing the onset of diarrhea via cellular immunity that does not depend on vaccine immunity can be said to be immensely significant, considering that viruses tend to mutate and the vaccine is ineffective.

Example 3
Next, an Example is given about the food / beverage products which use the strain of this invention lactic acid bacteria YM2-2 as an active ingredient.
[Cultivation and collection of Lactobacillus pentosus YM2-2 strain, production method of dead cells]
A medium mainly composed of lactose (5.5%), yeast extract (1.0%) and skim milk powder (1.0%) is inoculated with the lactic acid bacterium YM2-2 strain of the present invention, and pH 5.5-6. While preparing at 0, the cells were cultured at 37 ° C. for 18 hours, washed and collected by centrifugation. The collected cells were washed, heated at 100 ° C. for 30 minutes, and lyophilized to obtain YM2-2 dead cell powder.

[1: Bread]
300 g of wheat flour, 4.5 g of salt, 3 g of sugar, 3 g of lard, 9 g of baker's yeast, 10 g of the above-mentioned YM2-2 dead cell powder, and 180 g of water are mixed well, put into a mold and baked to make bread containing YM2-2 cells Manufactured.

[2: Biscuits]
100% wheat flour, 10g sugar, 18g shortening, 1g salt, 1.2g baking powder, 5g invert sugar, and 5g YM2-2 dead cell powder are mixed well, removed from the mold, and baked in an oven. Body biscuits were produced.

[3: Chocolate]
180M of chocolate block, 165g of cacao butter, 430g of powdered sugar, 220g of whole milk powder, 5g of lentin, a little fragrance and 15g of YM2-2 dead cell powder are mixed well in a hot water bath, and then solidified by cooling. Body chocolate was produced.

[4: Drop]
YM2-2 cells by mixing 180 g of sugar, 120 g of starch syrup, organic acid 3 g, 10 g of YM2-2 dead cell powder, a little fragrance, and a little pigment while boiling at about 150 ° C., then pouring into a mold and solidifying by cooling. Incoming drop was manufactured.

[5: Prepared milk powder]
After skim milk is added to the raw material milk and standardized, sugars, minerals and vitamins are added as appropriate, sterilized and filtered, and then powdered with a spray dryer. To this, 5% amount of YM2-2 dead cell powder was added to prepare YM2-2 cell-containing prepared milk powder.

[6: Fermented milk]
A heated milk medium (fat dry milk 15%, yeast extract 0.1%) is inoculated with the YM2-2 strain, cultured at 35 ° C. until the medium pH becomes 4.6, cooled, and then homogenized with a homogenizer. . On the other hand, Streptococcus thermophilus was inoculated into a heat-sterilized milk medium (fat dry milk 12%), cultured at 37 ° C. until the medium pH reached 4.3, and then ice-cooled and homogenized with a homogenizer. Both were mixed with sucrose syrup in a ratio of 1: 3: 1 to produce a drink yogurt.

Since the Lactobacillus pentosas YM2-2 strain of the present invention has an IgA production promoting effect, administration of the vaccine together with the vaccine enhances the production of antibodies against the antigens contained in the vaccine, improves the induction of protective immunity, and improves the vaccine effect. Strengthen. At the same time, since cellular immunity can also be activated, viral infection can be prevented to the same extent as vaccines only by administration of the lactic acid bacteria of the present invention, without relying on immune enhancement by vaccines.
Thus, it can be said that Lactobacillus pentosasus YM2-2 strain is very useful for exhibiting the efficacy of infection protection against viruses that are easily mutated and difficult to act on vaccines.

Claims (6)

  Lactobacillus pentosus YM2-2 strain deposited as FERM AP-21778 at the National Institute of Advanced Industrial Science and Technology (AIST).   2. A mucosal immunostimulant comprising the bacterial cell of the strain according to claim 1 or a culture thereof as an active ingredient.   The IgA production promoter which uses the microbial cell of the strain of Claim 1, or its culture as an active ingredient.   A virus infection protective agent comprising the bacterial cell of the strain according to claim 1 or a culture thereof as an active ingredient.   The virus infection protective agent according to claim 4, wherein the virus is rotavirus.   A composition selected from foods and drinks, pharmaceuticals, external preparations, and feeds, comprising the bacterial cells of the strain according to claim 1 or a culture thereof.

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