HK1093355B - Lactic acid bacteria capable of stimulating mucosal immunity - Google Patents

Description

Lactic acid bacteria capable of stimulating mucosal immunity

Technical Field

The present invention relates to lactic acid bacteria and compositions comprising these bacteria, more particularly lactic acid bacteria capable of stimulating mucosal immunity, and food and beverages comprising these bacteria.

Technical Field

Various lactic acid bacteria have been detected in vegetable foods such as kimchi, kimchi (kimchi), bread, sake (japanese liquor), japanese soybean paste, and soy sauce. Professor Sanae Okada of Tokyo agricultural university names Lactic Acid Bacteria detected in vegetable food as "vegetable Lactic Acid Bacteria" and suggests to distinguish them from Lactic Acid Bacteria derived from animal food such as fermented milk and cheese (Japanese Journal of Lactic Acid Bacteria, Vol.13, No.1, pp.23-36 (2002)). This is because plant lactic acid bacteria can utilize a wider variety of sugars in their growth environment, unlike animal lactic acid bacteria, and adapt themselves to more severe environments in terms of resistance to bacterial substances, enzyme resistance, oxygen resistance, and the like.

The present inventors studied these plant lactic acid bacteria, and have reported that fermented milk prepared using Lactobacillus plantarum ONC141 as a starting strain has the following properties: improving the human gastrointestinal microflora (Megumi Kumemura, Masamichi Toba, Yoshiro Sogawa, Seiichi Shimizu, Shinzo Kawaguchi, "journal of intestinal bacteriology (Enterobacteriology Magazine)" 15, 15, (2001)); increasing the frequency of defecation in constipated adults (Masamichi Toba, Megusi Kumemura, Satoshi Muneyuki, Yoshiro Sogawa, Hisao Yoshizawa, Yoichi Yajima, Yutaka Matsuda, Hajimieijima "journal of Enterobacteriaceae" 15, 21, (2001)); and increased host resistance to oral infection by pathogenic Salmonella typhimurium (increased IgA production, irritation of gastrointestinal mucosa) (Takeshi Ikenaga, Satoko Yamahara, Hideki Nachi, Masamichi Toba, Hiroshi Okamatsu, "Dairy Science (Milk Science)", Vol.51, No.1, pp.27-32 (2002)).

Lactobacillus plantarum ONC141 (fermented milk) has the highest effect of enhancing host resistance to salmonella infection among known Lactobacillus plantarum and animal lactic acid bacteria, and thus is considered to be capable of enhancing mucosal immune function and very useful for human host defense.

Disclosure of Invention

It is an object of the present invention to provide lactic acid bacteria which have higher mucosal immunostimulating ability and ability to enhance host defense mechanism than those lactic acid bacteria which the present inventors have studied and developed, and can be used as a microecological modulator, and further to provide end products (foods and beverages, such as fermented milk and lactic acid bacteria beverages) containing these lactic acid bacteria.

The present inventors have recently acquired a number of different microorganisms and tested their ability to induce IgA production using a mouse Peyer's Patch cell culture system. As a result, the present inventors have found that two lactic acid bacteria have particularly excellent ability to induce IgA production. The present inventors have further studied on the basis of this finding, and have completed the present invention.

The present invention provides the inventions summarized in the following items 1 to 13.

The first, a composition comprising a lactic acid bacterium selected from the group consisting of Lactobacillus (Lactobacillus) onrichb 0239(FERM BP-10064) and Lactobacillus (Lactobacillus) ONRIC b0240(FERM BP-10065), and an edible carrier, said composition being capable of stimulating mucosal immunity and being in the form of a food or beverage.

The composition of the second and first strips is fermented milk, lactic acid bacteria beverage, fermented vegetable beverage, fermented fruit beverage or fermented soy milk beverage.

The composition of the third strip, the first strip, is in the form of granules, powders, tablets, effervescent products or puddings.

Use of the composition of any of the fourth, first to third for the preparation of a food or beverage composition for stimulating mucosal immunity in a human subject in need thereof.

Use of the composition of any of the fifth, first to third strips for the preparation of a food or beverage composition for promoting IgA production in a human subject in need of such treatment.

Use of a lactic acid bacterium selected from the group consisting of lactobacillus ONRIC b0239(FERM BP-10064) and lactobacillus ONRIC b0240(FERM BP-10065) for the manufacture of a food or beverage composition for stimulating mucosal immunity in a human subject in need thereof.

The use of a lactic acid bacterium selected from the group consisting of Lactobacillus onRIC b0239(FERM BP-10064) and Lactobacillus onRIC b0240(FERM BP-10065) in the preparation of a food or beverage composition for promoting IgA production in a human subject in need of a treatment for promoting IgA production.

The eighth item, a pharmaceutical composition for human mucosal immunostimulation, comprising a lactic acid bacterium selected from the group consisting of Lactobacillus onRIC b0239(FERM BP-10064) and Lactobacillus onRIC b0240(FERM BP-10065), and a pharmaceutically acceptable excipient or diluent.

The ninth item, a pharmaceutical composition for promoting IgA production in humans, comprising a lactic acid bacterium selected from the group consisting of lactobacillus ONRIC b0239(FERM BP-10064) and lactobacillus ONRIC b0240(FERM BP-10065), and a pharmaceutically acceptable excipient or diluent.

Use of the composition of the tenth and eighth aspects in the manufacture of a medicament for stimulating mucosal immunity in a human subject in need thereof.

Use of the composition of the tenth and ninth aspects in the manufacture of a medicament for promoting IgA production in a human subject in need of such treatment.

Use of a lactic acid bacterium selected from the group consisting of lactobacillus ONRIC b0239(FERM BP-10064) and lactobacillus ONRIC b0240(FERM BP-10065) for the manufacture of a medicament for stimulating mucosal immunity in a human subject in need thereof.

The use of a lactic acid bacterium selected from the group consisting of Lactobacillus onRIC b0239(FERM BP-10064) and Lactobacillus onRIC b0240(FERM BP-10065) for the manufacture of a medicament for promoting IgA production in a human subject in need of a treatment for promoting IgA production.

The lactic acid bacteria strain of the present invention and the composition of the present invention containing the bacteria will be described below.

Lactic acid bacterium strain of the present invention

The lactic acid bacteria strains of the present invention are named Lactobacillus (Lactobacillus) ONRICb0239(FERM BP-10064) and Lactobacillus (Lactobacillus) ONRIC b0240(FERM BP-10065).

(1) Screening

(1-1) Source microorganism

The source microorganism used is lactic acid bacteria isolated from human intestinal contents, vegetable foods and animal foods and preserved in Otsuka Nutraceuticals institute of Otsuka pharmaceuticals, Inc.

(1-2) screening method

The target strain was screened using a mouse peyer's patch cell culture system using the IgA production-inducing ability as an index. The detailed screening method is described in example 2 below.

(2) Microorganisms obtained by screening

(2-1) Lactobacillus (Lactobacillus) ONRIC b0239

(a) Macroscopic features

(a-1) MRS agar Medium

Round to slightly irregular, hemispherical, smooth, milky white

(a-2) BL agar Medium

Round to slightly irregular, hemispherical, smooth, brown-white

(b) Microscopic features

Bacillus, no motility and no spore

(c) Optimum growth temperature

30 to 33 DEG C

(d) Physiological and biochemical characteristics

Gram staining capacity: positive for

Utilization of saccharides

Glycerin-

Erythritol-

D-arabinose-

L-arabinose-

Ribose ± +/-

D-xylose ± -s

L-xylose-

Ribitol-

beta-methyl-D-xyloside-

Galactose +

D-glucose +

D-fructose +

D-mannose +

L-sorbose-

Rhamnose-

Dulcitol-

Inositol-

Mannitol-

Sorbitol acid calcium chloride

alpha-methyl-D-mannoside +

alpha-methyl-D-glucoside ± -alpha-methyl-D-glucoside

N-ethyl-glucosamine +

Almond glycoside +

Hydroquinone glucoside +

Esculin +

Water lily glycoside +

Cellobiose C

Maltose syrup

Lactose

Honey disaccharide C

Sucrose +

Trehalose +

Inulin-

Melezitose-

D-raffinose +

Amidon -

Glycogen-

Xylitol-

Gentiobiose

D-turanose-

D-lyxose-

D-tagatose-

D-fucose-

L-fucose-

D-arabitol ± -

L-arabitol-

Gluconic acid-

2-keto-gluconic acid-

5-keto-gluconic acid-

The obtained isolate was identified as a Lactobacillus plantarum (Lactobacillus plantarum) strain according to the above characteristics according to the criteria shown in Bergey's systematic bacteriology Manual, and named Lactobacillus (Lactobacillus) ONRIC b0239, which was deposited at 8.6.2003 at the International patent organism depositary of national institute of Industrial science and technology, AIST Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-keb, Japan, accession number FERM P19469. It is then transferred to the international deposit under the Budapest convention and received as accession number FERM BP-10064.

(2-2) Lactobacillus (Lactobacillus) ONRIC b0240

(a) Macroscopic features

(a-1) MRS agar Medium

Round to slightly irregular, hemispherical, smooth, milky white

(a-2) BL agar Medium

Round to slightly irregular, hemispherical, smooth, brown-white

(b) Microscopic features

Bacillus, no motility and no spore

(c) Optimum growth temperature

30 to 33 DEG C

(d) Physiological and biochemical characteristics

Gram staining capacity: positive for

Utilization of saccharides

Glycerin-

Erythritol-

D-arabinose-

L-arabinose-

Ribose ± +/-

D-xylose-

L-xylose-

Ribitol-

beta-methyl-D-xyloside-

Galactose +

D-glucose +

D-fructose +

D-mannose +

L-sorbose-

Rhamnose-

Galactitol ± -r

Inositol-

Mannitol

Sorbitol acid calcium chloride

alpha-methyl-D-mannoside-

alpha-methyl-D-glucoside-

N-ethyl-glucosamine +

Almond glycoside +

Hydroquinone glucoside +

Esculin +

Water lily glycoside +

Cellobiose C

Maltose syrup

Lactose

Honey disaccharide C

Sucrose +

Trehalose-

Inulin-

Melezitose-

D-raffinose +

Amidon -

Glycogen-

Xylitol-

Gentiobiose

D-turanose-

D-lyxose-

D-tagatose-

D-fucose-

L-fucose-

D-arabitol-

L-arabitol-

Gluconic acid-

2-keto-gluconic acid-

5-keto-gluconic acid-

The obtained isolate was identified as a Lactobacillus plantarum (Lactobacillus plantarum) strain according to the above characteristics according to the criteria shown in Bergey's systematic bacteriology Manual, and named Lactobacillus (Lactobacillus) ONRIC b0240, which was deposited at the International patent organism depositary, national institute of advanced Industrial science and technology, AIST Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-keb, Japan, accession number FERM P-19470, on 8/6.2003. It is then transferred to the international deposit under the Budapest convention and received as accession number FERM BP-10065.

Compositions of the invention

The composition of the present invention mainly contains the lactic acid bacterium strain of the present invention as an active ingredient. The composition can be prepared in the form of a food, beverage or pharmaceutical product by using a suitable edible carrier (food material). The composition may also be prepared in the form of a pharmaceutical product by using suitable pharmaceutically acceptable excipients or diluents.

The significant mucosal immune stimulation and enhanced IgA production obtained with the compositions of the present invention is believed to result from: peyer's lymph node M cells, which are part of the intestinal immune system, receive antigen in the cell lumen. The antigen is presented by antigen presenting cells, such as dendritic cells, to CD 4T cells. When immature B cells mature into IgA antibody-producing cells through an antigen-specific reaction of T cells, the B cells move to the lamina propria mucosa and finally differentiate into IgA antibody-secreting cells. Although it is not clear how the lactic acid bacteria of the present invention participate in the mechanism of enhancement of IgA production, the uptake of antigen by at least peyer's patch M cells is essential for the enhancement of IgA production caused by the presence of the bacteria of the present invention. Therefore, the lactic acid bacterium of the present invention is presumed to function as such an antigen. In order to function as an antigen, the lactic acid bacteria of the present invention need not be living cells. The bacteria may be sterilized by conventional heat sterilization methods. However, as is well known, ingestion of live lactic acid bacteria in fermented milk and the like is effective for health maintenance and persistence due to the regulation in the intestinal tract and the balanced effect of the microbial community in the intestinal tract, and ingestion of live lactic acid bacteria of the present invention is also expected to have these effects, and therefore live lactic acid bacteria are preferably included in the composition of the present invention.

These lactic acid bacteria (living cells) may be contained in the composition of the present invention in the form of, for example, cultures, crude or purified products of these cultures, and lyophilized powders thereof.

In general, the culture can be obtained by various methods, including culturing at 30 ℃ for about 16 hours in a medium suitable for each strain, such as MRS medium.

After the culture, the cells can be recovered by, for example, centrifuging the culture solution at 4 ℃ at a speed of 3000 rpm for about 10 minutes. They may be purified in a conventional manner or lyophilized. The lyophilized powder thus obtained may also be used as an active ingredient of the composition of the present invention.

If desired, the composition may be supplemented with appropriate amounts of nutrients suitable for the maintenance and growth of the microorganisms of the present invention. Specific examples include nutrients used for culturing microorganisms in a medium, such as various carbon sources such as glucose, starch, sucrose, lactose, dextrin, sorbitol, fructose, etc., nitrogen sources such as yeast extract, peptone, etc., vitamins, minerals, trace metal elements, and other nutrients. Examples of such vitamins include vitamin B, vitamin D, vitamin C, vitamin E and vitamin K. Examples of such trace metal elements include zinc, selenium, and the like. Examples of other nutrients include various oligosaccharides such as lactosucrose, soy oligosaccharides, lactulose, lactitol, fructooligosaccharides and galactooligosaccharides. The amount of these oligosaccharides incorporated is not particularly limited, but it is preferable that the concentration thereof in the composition of the present invention is in the range of about 1 to 3% by weight.

Specific food and beverage forms of the composition of the present invention include fermented milk, lactic acid bacteria beverages, fermented vegetable beverages, fermented fruit beverages, and fermented soy milk beverages. The terms "fermented milk" and "lactic acid bacteria beverage" as used in the present specification and claims are in accordance with the definitions in clauses 2-37 "fermented milk" and clauses 2-38 "lactic acid bacteria beverage" of the ministry of health and welfare of the previous date, "ministry of treaty relating to ingredients and the like of milk and milk products". That is, "fermented milk" refers to a paste-like or liquid preparation prepared by fermenting milk or a milk product with lactic acid bacteria or yeast. Thus, "fermented milk" includes not only products in the form of beverages but also products in the form of fermented milk. "lactic acid bacteria beverage" means a beverage prepared by diluting with water using a paste-like or liquid preparation prepared by fermenting milk or a dairy product with lactic acid bacteria or yeast as a main raw material.

Fermented vegetable beverages, fermented fruit beverages, and fermented soy milk beverages are described later herein.

Other food forms of the composition of the invention include cell-containing microencapsulated forms, solid food forms (e.g., granules, powders (including lyophilized powders of fermented milk, etc.), tablets, effervescent products, chewing gums, goumi and puddings), and dairy products other than the fermented milks and lactic acid bacteria beverages described above.

Examples of pharmaceutical forms include forms for oral administration, such as solutions, emulsions, granules, powders, capsules, tablets and the like.

The processing of these food or beverage forms and pharmaceutical forms can be carried out in a conventional manner. The carrier for processing of these forms may be any edible carrier, pharmaceutically acceptable excipients and diluents. Details regarding edible carriers and processes that can be used in food and beverage forms are described in the "food and beverage forms of the compositions" section below. Particularly preferred carriers in the preparation of the food form are those having good mouthfeel and flavour enhancing effect. The processing of the drug form and the pharmaceutical excipients and diluents that can be used are described in the "drug form of composition" section below.

The amount of lactic acid bacteria incorporated in the composition of the invention can be suitably selected so as to obtain about 10 per 100 g of the composition⁸To 10¹¹The concentration of cells (cell count does not have to be a viable cell count; when the number of dead cells is included, it should be calculated as the number of viable bacteria before sterilization; the same is true hereinafter). The number of living cells was determined in the following manner. The diluted samples were added to agar bacterial medium, incubated anaerobically at 37 ℃ and the colonies formed were counted. Since the number of living cells and the turbidity are correlated with each other, if such correlation between the number of living cells and the turbidity is determined in advanceThe number of viable cells can be calculated using the measured turbidity instead of the viable cell count. The amount of incorporated lactic acid bacteria can be suitably adjusted according to the form of the composition of the present invention to be prepared, the kind of lactic acid bacteria used, and the like, using the above-mentioned range as a guideline.

Because the compositions of the present invention are designed to contain lactic acid bacteria (primarily living cells), the use of conditions such as heat and pressure during processing of the composition into the final product is not recommended. Thus, for example, in processing the composition of the invention into a solid food form, the lactic acid bacteria are preferably formulated directly in the form of freeze-dried cells, or the freeze-dried cells are treated with a suitable encapsulating agent and the encapsulated cells are used.

Food and beverage forms of the composition

Representative preferred food and beverage forms of the compositions of the present invention include fermented milk, lactic acid bacteria beverages, fermented vegetable beverages, fermented fruit beverages, fermented soy milk beverages, and the like. The fermented vegetable beverage, the fermented fruit beverage, and the fermented soybean milk beverage are described in detail later. This form of processing can be carried out by a procedure comprising culturing lactic acid bacteria in a suitable fermentation raw material containing a nutrient of lactic acid bacteria such as juice from vegetables or fruits, soybean milk (soybean milk), etc., to thereby cause fermentation of these raw materials. Vegetables and fruits used as fermentation materials include cuttings, press chips, mill dust, pressed juice, enzyme-treated products and dilutions or concentrates thereof. Useful vegetables include, for example, pumpkin, carrot, tomato, bell pepper, celery, spinach, colored sweet potato, corn, tables, cabbage, parsley, cabbage and cauliflower. Useful fruits include, for example, apples, peaches, bananas, strawberries, grapes, watermelons, oranges and tangerines.

The vegetable and fruit shavings, scraps and shavings may be obtained, for example, by washing at least one of the vegetables and fruits and optionally blanching them, for example, in hot water, and then passing them through a crusher, blender, or food processorMachine for processing articles, grinder, Mycolloider^TM(product of Tokushu Kika Kogyo Co., Ltd.) and the like by chopping, pulverizing or grinding. The press juice may be prepared by using a filtering press juice blender or the like. Pressed juices can also be prepared by filtering the ground material through a filter cloth. The enzyme-treated product can be prepared by acting cellulase, pectinase, protopectinase, etc. on cuttings, press chips, mill dust or press juice. The dilution comprises 1 to 50 times the dilution with water. The concentrated solution includes juice concentrated 1 to 100 times by a method such as freeze concentration, concentration under reduced pressure, etc.

Soymilk is another example of a particular fermentation material, which may be prepared in a conventional manner from soy materials. Examples of such soy milk include a homogenate prepared by immersing the skinned soybeans in water, wet milling the soybeans with a suitable mill such as a colloid mill, homogenizing the mill grind in a conventional manner, and then dissolving the water-soluble soy protein in water.

For fermentation using lactic acid bacteria, it is preferable to prepare a starting material in advance and inoculate the fermented material with the starting material. A representative example of such a starting material is a culture obtained by inoculating the lactic acid bacterium of the present invention to 10% skim milk powder or a fermented material supplemented with a yeast extract, which was previously sterilized at 90 to 121 ℃ for 5 to 20 minutes in a conventional manner, and then culturing the lactic acid bacterium of the present invention. The starter thus prepared often contains about 10 per gram of culture⁷To about 10⁹And (3) lactic acid bacteria cells.

The fermentation material for the starting material may also be supplemented with fermentation-promoting substances to ensure good growth of the lactic acid bacteria of the present invention, such as various carbon sources such as glucose, starch, sucrose, lactose, dextrin, sorbitol, fructose, etc., nitrogen sources such as yeast extract, peptone, etc., vitamins and minerals.

The lactic acid bacteria inoculum should generally be equivalent to no less than about 1X10 per cubic centimeter of fermentation broth⁶Number of living cells, preferably about 1X10⁷Number of living cells. As for the culture conditions, the fermentation temperature is generally aboutIn the range of 20 to about 45℃, preferably in the range of about 25 to about 37℃, and the fermentation time in the range of about 5 to 72 hours.

The lactic acid fermentation product thus obtained may be in the form of a curd (in the form of a fermented milk or pudding), and such product may be directly digested as a solid food. The lactic acid fermentation product in the form of a curd can thus be further homogenized to prepare the desired beverage form. Homogenization can be carried out using a conventional homogenizer. In particular, Gaulin high pressure homogenizer (LAB 40) at about 200 to about 1000kgf/cm may be used²Preferably from about 300 to about 800kgf/cm²Or a homogenizer (product No. HA x 4571, H20-A2, etc.) of Sanwa machine industry Co., Ltd. at not less than 150kg/cm²Under pressure of (c). By such a homogenate, a beverage product having excellent palatability, in particular a smooth mouthfeel, can be obtained. During the homogenization, it is also possible, if necessary, to carry out appropriate dilution, to adjust the pH by adding organic acids, and/or to add appropriate amounts of various other additives commonly used in the manufacture of beverages, such as oligosaccharides, juices, thickeners, surfactants and flavorings. Preferred additives and their amounts (weight percentage based on the fermentation product in the form of a curd) are, for example, 8% glucose (weight percentage, see below), 8% sucrose, 8% dextrin, 0.1% citric acid, 0.2% fatty acid glycerides and 0.1% flavourings.

The beverage of the invention thus obtained can be aseptically dispensed into suitable containers to provide the final product. The product has good palatability, allows smooth swallowing, and has pleasant aroma.

The amount of the product to be used (intake amount) may be appropriately selected depending on the age, sex, body weight, severity of disease and the like of the recipient, and is not particularly limited. Generally, the viable count is about 10 per ml⁶-10⁹The cellular product may be administered to a human at a rate of about 50-1000 ml per day.

Another specific example of a food form of the composition of the invention is a composition in the form of an effervescent product. The product is prepared by formulating 10 to 35 wt% of sodium carbonate and/or sodium bicarbonate and 20 to 70 wt% of a neutralizing agent as an effervescent ingredient, and 0.01 to 50 wt% of the lactic acid bacteria (lyophilized cells) of the present invention. The neutralizing agent used is an acidic compound capable of neutralizing sodium carbonate and/or sodium bicarbonate to produce carbon dioxide gas. Representative examples of such compounds are organic acids such as L-tartaric acid, citric acid, fumaric acid and ascorbic acid.

The amount of effervescent ingredients in the effervescent product of the invention is such that when the product of the invention is dissolved in water, the solution is acidic, in particular the acidity is approximately ph 3.5-4.6. More specifically, the content may be selected within the range of 10-35% sodium carbonate and/or sodium bicarbonate and 20-70% neutralizing agent. In particular, the amount of sodium carbonate can be chosen in the range 11-31%, preferably 22-26%; and/or sodium bicarbonate in the range of 10-35%, preferably 20-30%. Most preferred is the use of sodium bicarbonate alone, in the range of 20-25%. The amount of neutralizing agent is chosen from the range of 20-70%, preferably 30-40%. In particular, it is most preferred to use L-tartaric acid in the range of 20-25% and ascorbic acid in the range of 8-15%.

The effervescent product of the present invention contains the lactic acid bacterium of the present invention and an effervescent ingredient as main ingredients, and may be supplemented with appropriate amounts of various known additives, such as excipients, binders, disintegrants, lubricants, thickeners, surfactants, osmotic pressure regulators, electrolytes, sweeteners, flavoring agents, coloring agents, pH regulators, and the like. Examples of such additives include starches such as wheat starch, potato starch, corn starch, dextrin, and the like; sugars such as sucrose, glucose, fructose, maltose, xylose, lactose, and the like; sugar alcohols such as sorbitol, mannitol, xylitol, and the like; glycosides such as ligating sugar (sugar), palatinose and the like; excipients such as calcium phosphate, calcium sulfate, and the like; binders and thickeners such as starches, sugars, gelatin, gum arabic, dextrin, methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl cellulose, xanthan gum, pectin, tragacanth gum, casein, alginic acid, and the like; lubricants such as leucine, isoleucine, L-valine, sugar esters, hydrogenated oil, stearic acid, magnesium stearate, talc, polyethylene glycol and the like; disintegrants such as crystalline cellulose (trade name "Avicel", product of Asahi chemical industries, Ltd.), carboxymethyl cellulose (CMC), sodium carboxymethyl cellulose (CMC-Na), calcium carboxymethyl cellulose (CMC-Ca), etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters of fatty acids (polyoxyethylene sorbitol fatty acid esters), lecithin, and the like; dipeptides such as aspartame, alitame, and the like; and sweeteners such as steviol (stevia), saccharin and the like. These additives may be appropriately selected with reference to the relationship of each additive to the main ingredient, the properties of the formulation, the method of producing the formulation, and other factors, and may be used in appropriate amounts.

In addition, vitamins, particularly cyanocobalamin and ascorbic acid (vitamin C), may be added in suitable amounts to the effervescent product of the invention. The amount is not particularly limited, but for example, vitamin C is usually added up to 30%, preferably in the range of about 5% to about 25%.

The process for producing the effervescent product of the present invention may be substantially the same as the conventional process for producing such effervescent tablets. Thus, the products of the invention in the form of effervescent tablets can be prepared by weighing predetermined amounts of the respective ingredients, mixing them and then processing them, for example, by direct powder compression or wet or dry granulation compression.

The product of the invention thus obtained can be converted into a form suitable for oral administration, simply by placing in water, and administered orally.

The dose (intake amount) is not particularly limited, and may be appropriately determined depending on the age, sex, body weight, disease severity, and other variables of the recipient. Generally, 1-2 tablets of the effervescent tablet of the present invention weighing about 1.5 to 6.0 grams per tablet are dissolved in 100ml of water and 300 ml of water to produce a single dose for human recipients.

Pharmaceutical forms of the composition

The composition of the present invention can be manufactured into general pharmaceutical products using an appropriate pharmaceutically acceptable carrier and the lactic acid bacteria of the present invention as a main ingredient, and put into practical use. Examples of pharmaceutically acceptable carriers that may be used include various diluents and excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, and the like, which are well known in the art. These carriers may be used selectively according to the unit dosage form of the resulting pharmaceutical preparation.

The unit dosage form of the pharmaceutical product may be selected from a variety of dosage forms. Representative forms are tablets, pills, powders, solutions, suspensions, emulsions, granules and capsules.

Tablets may be prepared using, as pharmaceutical carriers, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc.; binders such as water, ethanol, propanol, neat syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, polyvinylpyrrolidone, etc.; disintegrants such as sodium carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose, dry starch, sodium alginate, agar powder, laminarin powder, sodium hydrogen carbonate, calcium carbonate, and the like; surfactants such as polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate, and the like; disintegration-inhibiting compositions such as sucrose, stearin, cacao butter, hydrogenated oil and the like; adsorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, and the like; moisturizers such as glycerin, starch, etc.; adsorbents such as starch, lactose, kaolin, bentonite, silica gel, etc.; and lubricants such as purified talc, stearate, boric acid powder, polyethylene glycol, and the like.

Further, if desired, the tablets may be coated with a standard coating material to provide sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, etc., or processed into multi-layered tablets such as bilayer tablets.

Pills can be prepared using pharmaceutical carriers such as excipients for example glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, kaolin, talc and the like; binders such as gum arabic powder, tragacanth powder, gelatin, ethanol, etc.; and disintegrating agents such as laminarin, agar, and the like.

In addition, color enhancers, preservatives, flavor compounds, flavoring agents, sweeteners, and other pharmaceutical substances may also be incorporated into the pharmaceutical products of the present invention, if desired.

The number of the lactic acid bacteria of the present invention to be incorporated into the pharmaceutical product of the present invention is not particularly limited and may be appropriately selected from a wide range. Generally, a recommended ratio is about 10 per unit dosage form of the pharmaceutical product⁷-10¹²A cell.

The method of administering the pharmaceutical product is not particularly limited, and may be suitably determined depending on the form of the pharmaceutical product, the age, sex and other variables of the patient, the severity of the disease, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules can be administered orally.

The dose of the pharmaceutical product may be appropriately selected depending on the method of administration, age, sex and other variables of the patient, severity of the disease, etc., but is preferably about 0.5 to 20 mg of the lactic acid bacterium of the present invention, i.e., the active ingredient, per kg of body weight per day. The pharmaceutical product may be administered in 1-4 divided doses a day.

The modification of the composition of the present invention allows the lactic acid bacteria in the composition to colonize the lower digestive tract as part of the gut microflora once ingested (administered), where the desired effects of the lactic acid bacteria, such as gut modulation and gut microflora improvement, can be obtained. Thus, a particularly preferred form of pharmaceutical product is an enteric tablet, through which the lactic acid bacteria can be transported to the intestine avoiding attack by gastric acid.

The lactic acid bacterial strains and the compositions containing the bacteria of the present invention, once ingested or administered, are capable of stimulating mucosal immunity and promoting IgA production in humans. The present invention therefore provides a method of stimulating mucosal immunity in a human subject in need thereof, comprising administering to the human subject a lactic acid bacterium of the present invention; a method of stimulating mucosal immunity in a human subject in need of such stimulation, comprising administering to the human subject a composition of the invention; a method for promoting IgA production in a human subject in need of treatment for promoting IgA production, comprising administering to the human subject a lactic acid bacterium of the present invention; and a method of promoting IgA production in a human subject in need of such treatment comprising administering to the human subject a composition of the invention.

The invention also provides the application of the lactobacillus in the immunostimulation of human mucosa; the use of the composition of the invention for mucosal immunostimulation in humans; the application of the lactobacillus in promoting the production of human IgA; use of a composition of the invention for promoting human IgA production.

Furthermore, the present invention provides the use of the lactic acid bacteria of the present invention for the preparation of the composition of the present invention.

Effects of the invention

The present invention provides lactic acid bacteria which have excellent IgA production-inducing ability, are effective in providing improved immunostimulation of human mucosa, particularly intestinal immunostimulation, and enhance host defense system, and provides a composition containing the bacteria. More specifically, compositions in the form of food or pharmaceutical products are provided.

Brief Description of Drawings

FIG. 1 shows the effect of administration of lactic acid bacteria of the present invention on IgA production by Peyer's patch cells.

FIG. 2 shows the effect of administration of lactic acid bacteria of the present invention on IgG production.

Best Mode for Carrying Out The Invention

The following examples and experimental examples are provided to further describe the present invention in detail.

Example 1

Examples of formulations for the compositions of the present invention are shown below.

(1) Preparation of fermented soymilk beverage

The ingredients were weighed according to the following formulation and then mixed to prepare the composition of the present invention in the form of a beverage.

100mL of soymilk fermented by Lactobacillus (Lactobacillus) ONRIC b0239

Milk cane sugar (content 55%) 10.0g

Appropriate amount of vitamins and minerals

Proper amount of flavoring agent

Proper amount of water

Total 150mL

Lactobacillus (Lactobacillus) ONRIC b0239 fermented soymilk is prepared by adding 10 liters of soymilk (protein content about 5g/100mL) to 1 liter of soymilk⁸Lactobacillus (Lactobacillus)us) ONRICb0239(FERM BP-10064) cells, and then fermented at 37 ℃ for 48 hours. The bacterial cell content of the fermented milk is 1x10⁹cells/mL.

(2) Preparation of fermented milk

The ingredients were weighed according to the following formulation and then mixed to prepare the composition of the present invention in the form of fermented milk.

Milk cane sugar (content 55%) 10.0g

Lactobacillus (Lactobacillus) ONRIC b0240 fermented milk 100mL

Appropriate amount of vitamins and minerals

Proper amount of flavoring agent

Proper amount of water

Total 150mL

Lactobacillus (Lactobacillus) ONRIC b0240 fermented milk is prepared by adding 10L milk into 1L milk⁸Individual cells of Lactobacillus (Lactobacillus) ONRIC b0240(FERM BP-10065) and then fermented at 37 ℃ for 24 hours. The bacterial cell content of milk is 1x10⁸cells/mL.

(3) Preparation of freeze-dried fermented milk powder

Use about 10⁷Lactobacillus (Lactobacillus) ONRIC b0239(FERM BP-10064) cells were subjected to lactic acid fermentation of 100 g of milk at 37 ℃ for 24 hours, and then the fermented product (including bacteria) was freeze-dried to prepare powder.

The resulting powder and various other ingredients were weighed according to the following formulation and then mixed to prepare the composition of the present invention in the form of fermented milk lyophilized powder. The bacterial cell content of the powder was 1x10⁹cells/mL.

Lactobacillus (Lactobacillus) ONRIC b0239 fermentation

2.2g of freeze-dried powder of cow milk

Proper amount of excipient

Appropriate amount of vitamins and minerals

Proper amount of flavoring agent

20g in total

Corn starch is used as excipient.

(4) Preparation of the powder

The compositions of the present invention were prepared in powder form by weighing the ingredients according to the following formulation and then mixing.

Casein 4.5g

Milk cane sugar (content 55%) 10.0g

Lactobacillus (Lactobacillus) ONRIC b0240 lyophilized powder 1.0g

Appropriate amount of vitamins and minerals

Proper amount of flavoring agent

20g in total

Lactobacillus (Lactobacillus) ONRIC b0240 lyophilized powder is obtained by culturing Lactobacillus (Lactobacillus) ONRIC b0240(FERM BP-10065) in 10% skimmed milk powder water solution, i.e. fermentation raw material of Lactobacillus growth, at 37 deg.C for 24-48 hr, and freeze-drying. The bacterial cell content of the powder was 10⁹-10¹⁰cells/mL.

(5) Preparation of granules

The compositions of the present invention in the form of granules were prepared by weighing the ingredients according to the following formulation and then mixing.

Milk cane sugar (content 55%) 10.0g

Lactobacillus (Lactobacillus) ONRIC b0240 lyophilized powder 1.0g

Appropriate amount of sorbitol

Appropriate amount of vitamins and minerals

Proper amount of flavoring agent

20g in total

The lyophilized powder of Lactobacillus (Lactobacillus) ONRIC b0240 used was the same as that used in example 1- (4).

(6) Microcapsule containing Lactobacillus

Lactobacillus (Lactobacillus) ONRIC b0239(FERM BP-10064) was freeze-dried in the same manner as in example 1- (4). Lyophilized powder obtained per gram contains 6 x10¹⁰The individual cells were dispersed in melted hydrogenated coconut oil (melting point 34 ℃) together with milk sucrose to prepare a mixed melt containing lactic acid bacteria (25%), oil (70%) and oligosaccharides (5%). The resulting melt was added dropwise to the flowing cooling oil through the innermost nozzle of the triple concentric nozzle at an average flow rate of 0.3 m/s; the mixed melt of hydrogenated coconut oil (melting point 34 ℃) and hydrogenated soybean oil was added dropwise through an intermediate nozzle near the inner nozzle at an average flow rate of 0.3 m/sec; the gelatin/pectin solution (volume ratio 85/15) used to form the capsule shell was added dropwise through the outermost nozzle at an average flow rate of 0.3 m/s, which resulted in three layers of seamless capsules (1.4X 10 per gram of capsule)⁹Individual cells) with a diameter of 2.5 mm.

The weight ratio of the contents, intermediate coating and outer capsule shell was 35:35: 30.

The capsules are air-dried and then vacuum-dried or vacuum-lyophilized to reduce the water activity of the capsules to an Aw value of 0.20 or less and a thermal conductivity of 0.16kcal/mh ℃ or less. The Aw value WAs measured using an electrical impedance water activity meter (Aw meter, WA-360, Shibaura electronics Co., Ltd.). The thermal conductivity was measured using the catch method.

Example 2

In this example, the capacity of the lactic acid bacteria of the present invention to induce IgA production was tested in vitro using the Peyer's lymph node cell culture system according to the method described in Yasui et al and Ikenaga et al (Yasui, H. et al, microbiological Ecology in health and Disease, 5, 155 (1992); Ikenaga, T. et al, Milk Science, 51, 27 (2002)). The test is as follows.

(1) Laboratory animal

Pure line SPF/VAF BALB/cAnNCrj female mice were used.

The resulting test mice were quarantined for one week. During quarantine, solid diet (MF, a product of organic Yeast Co., Ltd.) and tap water were supplied ad libitum.

(2) Peyer's lymph node cell culture method

After the quarantine period, 80 mice were divided into 8 groups of 10 mice each so that the average body weight of each group was substantially the same. After grouping, 10 mice were sacrificed each day, the small intestine was removed, and the peyer's patches were isolated from the small intestine. Peyer's lymph nodes were placed in a centrifuge tube containing MEM [ Eagle's MEM (NISSUI product), 2mM glutamine (GIBCO product), 1mM sodium pyruvate (GIBCO product), and MEM non-essential amino acid (GIBCO product) ], and cooled with ice. Single cell suspensions were prepared by passing the cells through a sieve and washing the wells with 5 mM MEM. The cell suspension was filtered and centrifuged at 1000 rpm for 10 minutes at 4 ℃. After centrifugation, the culture supernatant was aspirated, and the pellet was suspended in 5mL of MEM. After repeating this procedure twice, the pellet was suspended in 10mL of MEM containing 5% FBS (GIBCO product) and viable peyer's lymph node cells were counted. The cell suspension was seeded into a 96-well plate to prepare a cell culture plate.

(3) Preparation of test cells

Lactobacillus (Lactobacillus) ONRIC b0239(FERM BP-10064) and Lactobacillus (Lactobacillus) ONRIC b0240(FERM BP-10065) were used as the lactic acid bacteria of the present invention. These bacteria were cultured in a medium suitable for their culture until a stationary growth phase was reached, and then the resulting culture broth was centrifuged at 7000g for 10 minutes (4 ℃). Cells were washed three times with PBS (-) and suspended in 5mL of physiological saline. To determine the number of cells, the turbidity was measured at 660 nm. The cells were then autoclaved at 100 ℃ for 30 minutes. It was determined that a haze of 1.0 at 660nm is equivalent to 2.0 x10⁹Cells/ml.

(4) Determination of IgA concentration in culture supernatant

The peyer's lymph node cells prepared in the above (2) were suspended in MEM containing 5% FBS and adjusted to 2.5X 10⁶Cells/ml, 200 μ L of suspension was seeded into 96-well cell culture plates. 20 μ L of the concentration prepared in (3) above was 2.0X 10⁹A test cell suspension of cells/ml is added to each well of the plate in the presence of 5% CO₂In the case of (3), the culture was carried out at 37 ℃ for 7 days.

20 μ L of LPS (lipopolysaccharide) at a concentration of 50 μ g/mL was used as a positive control instead of 20 μ L of the above cells.

Subsequently, the total IgA concentration of the obtained culture supernatant was determined by ELISA using a commercial kit.

(5) IgA production enhancing Activity of lactic acid bacterium of the present invention

Table 1 below shows the IgA production enhancing activity of the lactic acid bacterium of the present invention as an index of stimulation index (S.I.), a value of the total IgA concentration of the supernatant containing the lactic acid bacterium of the present invention measured in the above (4) with respect to the total IgA concentration in the control culture supernatant prepared by adding 10. mu.L of PBS (-) to MEM and then culturing the cell-free medium in the same manner for 7 days as a reference (1.0).

The test results obtained using various known lactic acid bacteria are shown in tables 1 to 4. The test result of the positive control (LPS 50. mu.g/mL) was designated as "positive control (LPS)". In the table, "strain number" the abbreviations shown below represent the following collections of microorganisms:

ATCC: american type culture Collection; manassas, VA, u.s.a.

JCM: RIKEN, the microbiological occlusion center of the Japan institute for physical and chemical research

NRIC: NODAI culture collection, tokyo university of agriculture; setagaya-ku, Tokyo, Japan.

TABLE 1

Line number	Belong to	Seed of a plant	Subspecies of the species	IgAS.I.
						Control (PBS)			1
	Positive control (LPS)			13.1
					ONRIC b0239	Lactobacillus	plantarum		5.61
ONRIC b0240	Lactobacillus	Dlantarum		6.31
					JCM 1132	Lactobacillus	acidophilus		1.15
ATCC 43121	Lactobacillus	acidoDhilus		1.1
					JCM 1059	Lactobacillus	brevis		1.2
JCM 1115	Lactobacillus	buchneri		1.17
					JCM 1134	Lactobacillus	casei	casei	1.03
JCM 1096	Lactobacillus	curvatus		1.63
					JCM 1002	Lactobacillus	delbrueckii	bulgaricus	1.23
JCM 1012	Lactobacillus	delbrueckii	delbrueckii	1.41
					JCM 1248	Lactobacillus	delbrueckii	lactis	1.31
JCM 1173	Lactobacillus	fermentum		1.08
					JCM 1131	Lactobacillus	gasseri		1.15
JCM 1155	Lactobacillus	hilgardii		1.11
					JCM 2012	Lactobacillus	iohnsonii		1.11
JCM 8572	Lactobacillus	kefirgranum		1.08
					JCM 5818	Lactobacillus	kefiri		1.21
JCM 8130	Lactobacillus	paracasei	Daracasei	1.11
					JCM 1171	Lactobacillus	Daracasei	tolerans	1.11
JCM 1149	Lactobacillus	plantarum		1.66
					JCM 1551	Lactobacillus	plantarum		1.14
JCM 8341	Lactobacillus	plantarum		1.18
					JCM 1112	Lactobacillus	reuteri		1.15
ATCC 7469	Lactobacillus	rhamnosus		1.05
					JCM 1157	Lactobacillus	sakei	sakei	1.52
JCM 1150	Lactobacillus	salivarius	salicinius	1.06
					JCM 1231	Lactobacillus	salivarius	salivarius	1.14
JCM 9504	Lactobacillus	suebicus		1.28
					JCM 5885	Pediococcus	acidilactici	(pentosaceus)	1.51
JCM 5890	Pediococcus	pentosaceus		1.44
					JCM 6124	Leuconostoc	mesenteroides	mesenteroides	1
NRIC 0103	Enterococcus	faecalis		1.06
					NRIC 0110	Enterococcus	faecalis		1.08
NRIC 0134	Lactobacillus	brevis		1.07
					NRIC 0137	Lactobacillus	brevis		1.13
NRIC 1713	Lactobacillus	brevis		1.08
					NRIC 1950	Lactobacillus	brevis		1.12
NRIC 1964	Lactobacillus	brevis		1.07
					NRIC 1965	Lactobacillus	brevis		1.07

TABLE 2

Line number	Belong to	Seed of a plant	Subspecies of the species	IgAS.I.
					NRIC 1042	Lactobacillus	casei	casei	1.00
NRIC 1597	Lactobacillus	casei	casei	0.96
					NRIC 1917	Lactobacillus	casei	casei	1.01
NRIC 1941	Lactobacillus	casei	casei	1.02
					NRIC 1962	Lactobacillus	casei	casei	1.00
NRIC 1963	Lactobacillus	casei	casei	1.05
					NRIC 1968	Lactobacillus	casei	casei	1.07
NRIC 1975	Lactobacillus	curvatus		1.02
					NRIC 1976	Lactobacillus	curvatus		1.14
NRIC 1977	Lactobacillus	curvatus		1.04
					NRIC 1978	Lactobacillus	curvatus		1.11
NRIC 1979	Lactobacillus	curvatus		0.99
					NRIC 0191	Lactobacillus	delbrueckii	bulgaricus	1.07
NRIC 1682	Lactobacillus	delbrueckii	lactis	1.12
					NRIC 0129	Lactobacillus	fermentum		1.00
NRIC 0131	Lactobacillus	fermentum		1.19
					NRIC 0132	Lactobacillus	fermentum		1.03
NRIC 0135	Lactobacillus	fermentum		1.02
					NRIC 0139	Lactobacillus	fermentum		1.14
NRIC 0141	Lactobacillus	fermentum		1.08
					NRIC 0142	Lactobacillus	fermentum		0.94
NRIC 0143	Lactobacillus	fermentum		1.04
					NRIC 0144	Lactobacillus	fermentum		0.97
NRIC 0145	Lactobacillus	fermentum		1.09
					NRIC 0146	Lactobacillus	fermentum		1.05
NRIC 0147	Lactobacillus	fermentum		1.05
					NRIC 1949	Lactobacillus	fermentum		1.09
NRIC 1952	Lactobacillus	fermentum		1.06
					NRIC 1955	Lactobacillus	fermentum		1.12
NRIC 1966	Lactobacillus	hilgardii		0.94
					NRIC 1967	Lactobacillus	hilgardii		1.06
NRIC 1936	Lactobacillus	paracasei	paracasei	0.96
					NRIC 1937	Lactobacillus	paracasei	paracasei	0.94
NRIC 1942	Lactobacillus	paracasei	paracasei	0.93
					NRIC 1944	Lactobacillus	paracasei	paracasei	1.00
NRIC 1945	Lactobacillus	paracasei	paracasei	0.98
					NRIC 1946	Lactobacillus	paracasei	paracasei	1.01
NRIC 1934	Lactobacillus	paracasei	tolerans	1.09
					NRIC 1935	Lactobacillus	paracasei	tolerans	1.03
NRIC 1938	Lactobacillus	paracasei	tolerans	1.03

TABLE 3

Line number	Belong to	Seed of a plant	Subspecies of the species	IgAS.I.
					NRIC 1939	Lactobacillus	paracasei	tolerans	1.0
NRIC 1940	Lactobacillus	paracasei	tolerans	1.0
					NRIC 1943	Lactobacillus	paracasei	tolerans	0.99
NRIC 1947	Lactobacillus	paracasei	tolerans	0.98
					NRIC 0391	Lactobacillus	pentosus		1.00
NRIC 0392	Lactobacillus	pentosus		1.04
					NRIC 0393	Lactobacillus	pentosus		1.19
NRIC 0394	Lactobacillus	pentosus		1.15
					NRIC 1919	Lactobacillus	plantarum		1.32
NRIC 1920	Lactobacillus	plantarum		1.08
					NRIC 1921	Lactobacillus	plantarum		1.14
NRIC 1922	Lactobacillus	plantarum		1.37
					NRIC 1923	Lactobacillus	plantarum		0.96
NRIC 1957	Lactobacillus	plantarum		1.01
					NRIC 1958	Lactobacillus	plantarum		1.31
NRIC 1715	Lactobacillus	reuteri		0.95
					NRIC 1974	Lactobacillus	reuteri		1.16
NRIC 1980	Lactobacillus	reuteri		1.31
					NRIC 1599	Lactobacillus	sakei		0.97
NRIC 1600	Lactobacillus	sakei		1.52
					NRIC 1601	Lactobacillus	sakei		1.07
NRIC 1602	Lactobacillus	sakei		1.37
					NRIC 1603	Lactobacillus	sakei		1.03
NRIC 1575	Leuconostoc	lactis		0.85
					NRIC 1576	Leuconostoc	lactis		0.92
NRIC 1578	Leuconostoc	lactis		1.00
					NRIC 1580	Leuconostoc	lactis		1.03
NRIC 1582	Leuconostoc	lactis		0.93
					NRIC 1750	Leuconostoc	lactis		1.03
NRIC 1087	Leuconostoc	mesenteroides	mesenteroides	1.33
					NRIC 1507	Leuconostoc	mesenteroides	mesenteroides	1.02
NRIC 1541	Leuconostoc	mesenteroides	mesenteroides	0.90
					NRIC 0124	Pediococcus	acidilactici		0.93
NRIC 0122	Peoiococcus	pentosaceus		1.03
					NRIC 0123	Pediococcus	pentosaceus		0.96
NRIC 1913	Pediococcus	pentosaceus		1.62
					NRIC 1914	Pediococcus	pentosaceus		1.05
NRIC 1915	Pediococcus	pentosaceus		1.28
					NRIC 0001	Saccharomvces	cerevisiae		1.04
NRIC 0002	Saccharomvces	cerevisiae		1.02
					NRIC 0004	Saccharomvces	Cerevisiae		1.12

TABLE 4

Line number	Belong to	Seed of a plant	Subspecies of the species	IgAS.I.
					NRIC 0005	Saccharomvces	cerevisiae		1.00
NRIC 0006	Saccharomvces	cerevisiae		1.01
					NRIC 0007	Saccharomvces	cerevisiae		0.98
NRIC 0008	Saccharomvces	cerevisiae		0.97
					NRIC 0009	Saccharomvces	cerevisiae		0.98
NRIC 0011	Saccharomvces	cerevisiae		1.03
					NRIC 0013	Saccharomvces	cerevisiae		0.95
NRIC 0014	Saccharomvces	cerevisiae		0.94
					NRIC 0015	Saccharomvces	cerevisiae		1.04
NRIC 0016	Saccharomvces	cerevisiae		0.88
					NRIC 0059	Saccharomvces	cerevisiae		1.12
NRIC 0060	Saccharomvces	cerevisiae		1.11
					NRIC 1412	Saccharomvces	cerevisiae		1.00
NRIC 1414	Saccharomvces	cerevisiae		1.03
					NRIC 1415	Saccharomvces	cerevisiae		0.85
NRIC 1417	Saccharomvces	cerevisiae		0.97
					NRIC 1461	Saccharomvces	cerevisiae		0.92
NRIC 1465	Saccharomvces	cerevisiae		1.00
					NRIC 1466	Saccharomvces	cerevisia		1.07
NRIC 1624	Saccharomvces	cerevisiae		0.91
					NRIC 1478	Saccharomvces	cerevisiae		0.91
NRIC 1482	Saccharomvces	cerevisiae		0.94
					NRIC 1483	Saccharomvces	cerevisiae		1.24
NRIC 1484	Saccharomvces	cerevisiae		0.87
					NRIC 1485	Saccharomvces	cerevisiae		0.95
NRIC 1486	Saccharomvces	cerevisiae		1.04
					NRIC 1487	Saccharomvces	cerevisiae		0.91
NRIC 1488	Saccharomvces	cerevisiae		0.91
					NRIC 1489	Saccharomvces	cerevisiae		0.84
NRIC 1490	Saccharomvces	cerevisiae		0.88
					NRIC 1811	Saccharomvces	cerevisiae		1.03

As shown in tables 1-4, the IgA production of the PBS control was taken as 1 and the average s.i. of the positive control was 13.1, indicating a strong enhancement of IgA production. Thus, it was confirmed that the culture system can be used for evaluating IgA production by Peyer's patch cells.

Comparison of various lactic acid bacteria, based on their ability to induce IgA production, showed that the lactic acid bacteria ONRIC b0239 and ONRIC b0240 of the present invention have s.i. values of 5.61 and 6.31, respectively, and thus have significantly higher ability to induce IgA production than other strains, which have s.i. values of 0.8 to 1.4.

IgA inhibits pathogenic bacteria invasion, neutralizes viruses and toxins, and inhibits dietary allergen invasion. These increases in IgA are important for host defense.

Example 3

In this example, the ability of the lactic acid bacterium of the present invention to induce IgA production was tested in vivo in the following manner.

(1) Laboratory animal and breeding thereof

50 male 8-week-old BALB/c mice were purchased and quarantined for one week. MF solid diet (product of OrientalYeast) and tap water were supplied ad libitum during quarantine and during subsequent trials.

After the quarantine period, the mice were divided into 3 groups, i.e., a physiological saline administration group (15 mice), a lactic acid bacterium (living cell) administration group of the present invention (15 mice), and a lactic acid bacterium (non-living cell) administration group of the present invention (15 mice)

(2) Preparation of lactic acid bacteria of the present invention for oral administration

The lactic acid bacteria (live and non-live cells) of the present invention for oral administration were prepared by the following method.

Live cells:

lactobacillus plantarum b0240(FERM BP-10065; hereinafter simply referred to as "b 0240") was cultured in MRS medium until a stationary growth phase was reached, and the resultant culture was centrifuged at 3500rpm for 10 minutes (4 ℃). The cells were washed twice with physiological saline and suspended in physiological saline to a concentration of 4X 10⁹CFU/mL。

Non-viable cells:

the obtained living cells were autoclaved (heated at 121 ℃ for 15 minutes) and then sonicated for 45 minutes using a sonication washer (brasson).

(3) Test method

The lactic acid bacteria (live cells) of the present invention prepared in (2) were orally administered to 15 mice (5+5+5 ═ 15 mice) of the group to which the lactic acid bacteria (live cells) of the present invention were administered, and were orally administered for 7 days (5 mice), 14 days (5 mice) or 21 days (5 mice) at a dose of 109CFU/250 μ L/mouse/day each morning, respectively. Similarly, the lactic acid bacteria (non-viable cells) of the present invention prepared in (2) were orally administered to 15 mice of the group to which the lactic acid bacteria (non-viable cells) of the present invention were administered, for 7 days (5 mice), 14 days (5 mice) or 21 days (5 mice), respectively. After the respective dosing period, mice of each group were decapitated and their blood was collected in tubes and centrifuged at 3000 rpm for 10 minutes at 4 ℃ to obtain serum. Peyer's patch cells were prepared by the following method. After each group of mice was sacrificed, the small intestine was removed and peyer's patches were removed from the outer surface of the small intestine with ophthalmic scissors. Peyer's patches were placed in 24-well microplates containing incomplete medium (RPMI 1640 containing 10mg gentamicin) and cooled with ice. The resulting culture broth was screened to prepare a single cell suspension, and the wells were washed with 5mL of incomplete medium. The resulting suspension was filtered and centrifuged at 1000 rpm for 10 minutes at 4 ℃. After centrifugation, the culture supernatant was aspirated, and the pellet was suspended in 5mL of incomplete medium. After repeating the above-described steps including washing, filtration, centrifugation and aspiration once, the obtained pellet was used as peyer's patch cells.

Control mice (15 mice) of the saline-administered group were not fed with the lactic acid bacteria (live and non-live cells) of the present invention, and their sera and peyer's patch cells were prepared in the same manner as described above at 7 days (5 mice), 14 days (5 mice) or 21 days (5 mice) after the start of the test.

IgA production test

The peyer's patches cells (pellets) thus prepared were suspended in 0.5mL of complete medium (RPMI 1640 containing 2mM L-glutamine, 50. mu.M mercaptoethanol, 100U/mL penicillin, 100mg/mL streptomycin, and 10% FBS) and adjusted to a cell concentration of 2X 10⁶cells/mL. After counting the number of viable cells, 100 μ L of the cell suspension was seeded into each well of a 96-well cell culture plate.

The amount of IgA produced by peyer's patch cells was evaluated by two methods, i.e., one method comprising culturing the peyer's patch cells as above and then measuring the amount of IgA produced, and the other method comprising culturing the peyer's patch cells in cultured cells containing the lactic acid bacterium (non-living cell) of the present invention as a stimulator of the peyer's patch cells and then measuring the amount of IgA produced. The conditions used in the latter method are considered to be closer to the real in vivo environment. In particular, when the lactic acid bacteria of the invention (live and non-live cells) were taken orally in this test, it was expected that the digested lactic acid bacteria would provide some stimulation to the peyer's patch cells.

The lactic acid bacteria (non-viable cells) of the present invention, which are stimulators of peyer's lymph node cells, were prepared according to the following method.

Lactic acid bacteria (non-viable cells) of the invention for peyer's patch stimulation

The lactic acid bacteria (living cells) suspension of the present invention prepared previously for oral administration was further diluted with phosphate buffer to a concentration of 10⁷CFU/mL (turbidity at 660nm of 0.275), the resulting cell suspension was autoclaved (heating at 121 ℃ for 15 minutes) and then sonicated using a sonicator (BRANSON 2510) for 45 minutes.

In the method using the peyer's patch cell stimulator, 10 μ L of the lactic acid bacteria (non-viable cells) of the present invention for the stimulation of peyer's patch cells was added to each well, and then 100mL of RPMI1640 without FCS was added to each well, and the peyer's patch cells were cultured at 37 ℃ for 7 days in the presence of 5% CO 2. In the method without using the peyer's patch cell stimulant, 10. mu.L of physiological saline was added to each well instead of the lactic acid bacteria (non-viable cells) of the present invention, and then the peyer's patch cells were cultured by the same procedure as above.

(4) Measuring

The culture supernatants were separated from the cell culture solution by centrifugation and they were stored frozen at-80 ℃ until they were used to measure the total concentration of IgA produced in the culture supernatants.

The total IgA concentration of the culture supernatant and the total IgG concentration of the serum were determined by ELISA using a commercial kit.

(5) Results

The results (IgA concentration and IgG concentration, respectively) are shown in fig. 1 and 2.

FIG. 1 is a bar graph showing IgA concentration (. mu.g/mL) of culture supernatants. In fig. 1, white bars show the results of the saline administration group of the control (labeled "saline"). The results (labeled "b 0240 viable cells") of the group administered with lactic acid bacteria (viable b0240 cells) of the present invention are shown by the hatched bars. The black bars show the results (labeled "b 0240 non-viable cells") for the lactic acid bacteria (non-viable b0240 cells) administration group of the present invention. "non-stimulated" means the case where peyer's patch cells from mice of each group were cultured in a culture system without the lactic acid bacteria (non-viable cells) of the present invention. "cell stimulation" means the case where the peyer's patch cells from the mice of each group were cultured by adding lactic acid bacteria (non-viable cells) to the culture system under the stimulation of the lactic acid bacteria of the present invention. Results obtained using 5 mice per group are shown as mean ± standard deviation (mean ± SD). The p-value shown above the results represents the level of significance in the student's t-test relative to the control.

The results shown in fig. 1 clearly show that:

(1) administration for 7 days

In the case of cell stimulation, the group administered with lactic acid bacteria (non-viable cells) of the present invention had a significantly higher value (p: 0.01.0) than the group administered with physiological saline.

(2) Administration for 14 days

The group administered with lactic acid bacteria (non-viable cells) of the present invention had a significantly higher value (p ═ 0.048) than the control group (no stimulation after administration of physiological saline) (non-stimulated black bars) without stimulation.

In the case of cell stimulation, both groups (non-viable cells and viable cells) of the lactic acid bacteria-administered group of the present invention had significantly higher values (p ═ 0.034 and p ═ 0.002, respectively) than the control group (physiological saline-administered group).

(3) Administration for 21 days

The group administered with lactic acid bacteria (non-viable cells) according to the invention had a significantly higher value (p ═ 0.047) than the control group without stimulation.

In the case of cell stimulation, both groups administered with the lactic acid bacteria of the present invention (non-viable cells and viable cells) had significantly higher values (p ═ 0.015 and p ═ 0.005, respectively) than the control group.

FIG. 2 is a bar graph showing the effect of administration of lactic acid bacteria (non-viable cells) of the present invention for 21 days on IgG production. Serum IgG concentrations (. mu.g/mL) are plotted on the vertical axis.

The results shown in fig. 2 clearly show that the lactic acid bacteria (non-viable cells) administered group of the present invention had a significantly higher serum IgG concentration (p ═ 0.0064) than the control group (physiological saline administered group); the lactic acid bacteria (live cells) administered group of the present invention also had a significantly higher serum IgG concentration than the control group (normal saline administered group).

The above results are considered to occur as follows: the lactic acid bacteria of the present invention induce mucosal immune response by stimulating the immunocompetent cells in the peyer's lymph nodes or small intestine epithelial cells and the surrounding immunocompetent cells, ultimately increasing the total IgA production of the peyer's lymph node cells. The results also clearly show that administration of the lactic acid bacteria of the present invention can increase not only IgA but also serum IgG. These conditions indicate that ingestion of the lactic acid bacteria of the present invention stimulates not only mucosal immunity but also systemic immunity, so that the immune response in vivo is stimulated doubly, thereby enabling the host organism to defend internally and externally. Since not only living cells but also non-living cells show such activity, the lactic acid bacteria of the present invention are expected to be useful in new probiotic methods, such as oral vaccines.

Example 4

This example will demonstrate the effectiveness of the lactic acid bacteria of the present invention in preventing lower respiratory tract influenza infection.

Mucosal immunity is the first step in the infection defense mechanism when pathogens contact the mucosa (Brandtzaeg, p., curr. top. microbiol. immunol. 146: 131989). Mucosally secreted IgA (S-IgA) has defensive properties against pathogens such as bacteria and viruses (Czinn, S.J. et al, Vaccine 11: 637, 1993; Renegar, K. et al, J.Immunol.146: 1972, 1991) and also plays an important role in neutralizing toxins produced by microorganisms (Brandtzaeg, P., APMIS 103: 1, 1995; Kilian, M. et al, Microbiol.Rev.52: 2961988). In recent years, many studies and developments have been made on drugs for infectious diseases against infection protective effects by the mucosal immune system. Influenza infection has a high mortality rate in children with underdeveloped immune systems and in elderly people whose immune function has decreased, and there is a need to develop more effective vaccines to replace existing vaccines. In particular, since the types of influenza viruses that are circulating change every year, various attempts have been made to develop mucosal vaccines based on moderate-specific IgA produced by mucosal immunity at the site of viral infection instead of highly specific IgG produced by transdermal administration. Foods using lactic acid bacteria, such as fermented milk, have been reported to have IgA-based infection protective effects. For example, Yasui et al performed an experiment in which rotavirus, which is a virus causing a major cause of infantile diarrhea, infected a mouse, in which b.breve YIT4064 was administered to a mother mouse, and then a child mouse was fed with milk of the mother mouse, and reported that the diarrhea of the child mouse was suppressed (h.yasui et al, j.infect.dis., 172: 403, 1995). Yasui et al also reported that administration of b.breve YIT4064 increased influenza virus-specific IgG in serum, thereby protecting mice against influenza infection, since the degree of protection against influenza virus infection was correlated with levels of humoral and cellular immunity, such as mucosal immunoglobulin a (iga) and serum IgG in the respiratory tract (h.yasui et al, clin.diagn.lab.immunol.6: 186, 1999).

In order to investigate the IgA-based infection protective effect of lactic acid bacteria, the infection protective effect of taking the composition of the present invention (fermented milk prepared using lactic acid bacteria of the present invention) was evaluated using influenza virus (IFV) to lower respiratory tract infection model mice, using the number of days surviving after infection as an index. The test was carried out in the following manner.

(1) Laboratory animal

5-week-old female mice of SPF/VA/VAF pure line (BALB/cAnNCrj strain) were purchased from Charles river Japan, quarantined for 4 days under the conditions shown below, and then divided into 3 groups (distilled water group, milk group and fermented milk group containing the lactic acid bacteria of the present invention) such that the average body weight of each group was substantially the same.

And (3) feed supply: MF solid diet (product of Oriental Yeast Co., Ltd.)/free-feed

Water supply: tap water/free feed from bottle

Environment: the temperature is 23 +/-2 ℃; humidity is 60 +/-10%.

Illumination time: illumination period, 7:00 to 19: 00; dark phase, 19:00 to 7: 00.

(2) Test method

The test substance ((1) distilled water, (2) cow's milk or (3) fermented milk containing lactic acid bacteria of the present invention) was administered to each group of mice (n ═ 45) for 2 weeks with MF solid diet (product of Oriental Yeast Co., Ltd.).

The milk used for the test was prepared by diluting LL milk (Oaso milk; product of Rakunous mothers (Kumamoto Dairy Co., Ltd.)) to 75% with distilled water. Fermented milk for testing containing the lactic acid bacteria of the present invention was prepared using lactobacillus plantarum (l.plantarum) ONRIC b0240 suspended in a 10% skim milk aqueous solution and stored frozen at-80 ℃ as a starting material. Starting Material (viable cell number: 10)⁸Cells) were added to 1 liter of milk and fermented at 33 ℃ for 16 hours to a concentration of 5 x10⁷cells/mL, diluted to 75% with distilled water.

The test subjects were fed ad libitum via water bottles. The feeding intake was calculated from the weight loss of the test article by comparing the initial weight of the test article with the weight after feeding.

Two weeks after the start of feeding, mice in each group were anesthetized with "Ketalar" (ketamine hydrochloride), nasally inoculated with IFV through one of the nasal cavities, and administered with 50. mu.L of 10, 10 concentration²Or 10³Mice were infected with IFV solution of pfu/50. mu.L PBS/mouse (15 mice per group). Mice in each group were examined daily for survival or death. Mice had free access to the test article from the start of infection until death was confirmed.

IFV stored at Otsuka pharmaceutical research institute for microbiology: the A/PR/8/34/H1N1 strain was used as IFV strain. The strain was suspended in MEM containing 0.1% BSA and 10mM HEPES, and diluted with PBS (+) to a concentration of 10-10³pfu/50. mu.L, thus providing a viral solution for IFV vaccination. PBS (+) was prepared by dissolving 9.55 g of PBS (-) powder (product of Kojin-Bio Inc.), 100.00mg of anhydrous calcium chloride and 46.90mg of anhydrous magnesium chloride in distilled water to a volume of 1000 mL.

Results

The number of days that the mice in each group survived nasal inoculation with IFV was examined by observation, i.e., twice daily, in the morning (8:30-9:00) and in the evening (17:30-18: 00).

When the virus takes 10²pfu/concentration of mice upon inoculation, all mice in the control group (distilled water-administered group) and the comparative group (milk-administered group) died within 7 days. When the virus takes 10³pfu/concentration of mice upon inoculation, all mice in both groups died by night on day 6. In contrast, the group administered with fermented milk containing the lactic acid bacteria of the present invention showed a tendency that the survival time of the mice exceeded that of the control group.

When the virus was inoculated at a concentration of 10 pfu/mouse, 70% or more of the mice survived in all groups at 14 days; 86.7% of the mice survived in the group administered fermented milk containing the lactic acid bacteria of the present invention, and thus showed a tendency to increase the survival rate compared to the control group (80%).

The weight of mice in each group was measured every two days from the start of intake of the test article to the time of infection, and every morning (8:30-9:00) after infection. The measurement was performed on the surviving mice on each measurement day, and the average of all the measurement values of the mice in the same group was shown as the obtained value.

In all groups, a slight weight loss was observed from day 2 onwards. The trend of weight change was similar in all groups and no clear difference was observed.

Experience is good

From the results of this test and the results in examples 2 and 3, it was deduced that the lactic acid bacteria of the present invention and the fermented milk containing the lactic acid bacteria had a protective effect against IFV infection.

Industrial applicability

The present invention provides lactic acid bacteria capable of stimulating mucosal immunity and promoting IgA production, and a composition comprising the same. The lactic acid bacteria and the composition can inhibit invasion of infectious microorganisms through mucosa, thereby providing host protective effect.

Claims (13)

1. A composition comprising a lactic acid bacterium selected from the group consisting of Lactobacillus (Lactobacillus) ONRIC b0239 having a deposit number FERM BP-10064 and Lactobacillus (Lactobacillus) ONRIC b0240 having a deposit number FERM BP-10065, and an edible carrier, which composition is capable of stimulating mucosal immunity and is in the form of a food or beverage.

2. The composition of claim 1, wherein the composition is a fermented milk, a lactic acid bacteria beverage, a fermented vegetable beverage, a fermented fruit beverage, or a fermented soy milk beverage.

3. Use of a composition according to claim 1 or 2 for the preparation of a food or beverage composition for stimulating mucosal immunity in a human subject in need thereof.

4. Use of a composition according to claim 1 or 2 for the preparation of a food or beverage composition for promoting IgA production in a human subject in need of such treatment.

5. The composition of claim 1, which is in the form of a granule, powder, tablet, effervescent product, or pudding.

6. Use of a lactic acid bacterium selected from the group consisting of lactobacillus ONRIC b0239 having a deposit number FERM BP-10064 and lactobacillus ONRIC b0240 having a deposit number FERM BP-10065 for the preparation of a food or beverage composition for stimulating mucosal immunity in a human subject in need thereof.

7. Use of a lactic acid bacterium selected from the group consisting of lactobacillus ONRIC b0239 having a deposit number FERM BP-10064 and lactobacillus ONRIC b0240 having a deposit number FERM BP-10065 for the preparation of a food or beverage composition for promoting IgA production in a human subject in need of a treatment for promoting IgA production.

8. A pharmaceutical composition for the immunostimulation of human mucosa comprises a lactic acid bacterium selected from Lactobacillus onRIC b0239 having a deposit number FERM BP-10064 and Lactobacillus onRIC b0240 having a deposit number FERM BP-10065, and a pharmaceutically acceptable excipient or diluent.

9. A pharmaceutical composition for promoting IgA production in human comprises a lactic acid bacterium selected from Lactobacillus onRIC b0239 having a deposit number FERM BP-10064 and Lactobacillus onRIC b0240 having a deposit number FERM BP-10065, and a pharmaceutically acceptable excipient or diluent.

10. Use of the composition of claim 8 for the manufacture of a medicament for stimulating mucosal immunity in a human subject in need thereof.

11. Use of a composition according to claim 9 in the manufacture of a medicament for promoting IgA production in a human subject in need of such treatment.

12. Use of a lactic acid bacterium selected from the group consisting of lactobacillus ONRIC b0239 having a deposit number FERM BP-10064 and lactobacillus ONRIC b0240 having a deposit number FERM BP-10065 for the manufacture of a medicament for stimulating mucosal immunity in a human subject in need thereof.

13. Use of a lactic acid bacterium selected from the group consisting of lactobacillus ONRIC b0239 having a deposit number FERM BP-10064 and lactobacillus ONRIC b0240 having a deposit number FERM BP-10065 for the manufacture of a medicament for promoting IgA production in a human subject in need of a treatment for promoting IgA production.