JP2007091704A - IgE production inhibitor, food and drink for suppressing IgE production, antiallergic agent and food and drink for antiallergy - Google Patents

Abstract

The present invention provides an IgE production inhibitor and an IgE production-suppressing food and drink that are expected to have an excellent antiallergic action, as well as an antiallergic agent and an antiallergic food and drink.
An IgE production inhibitor comprising Bifidobacterium catenuratum as an active ingredient. A food or drink for suppressing IgE production containing the IgE production inhibitor. An antiallergic agent containing Bifidobacterium catenuratum as an active ingredient. An antiallergic food or drink containing the antiallergic agent.
[Selection figure] None

Description

  The present invention relates to an IgE production inhibitor and antiallergic agent useful for the prevention and treatment of allergic diseases, and an IgE production inhibitor food and drink and antiallergic food and drink containing them.

In recent years, patients with various allergic symptoms (allergic diseases) involving allergic reactions such as rheumatism, inflammatory bowel disease, food allergy, etc. are increasing due to changes in dietary habits and lifestyle habits. Allergic reactions are classified from type I to type IV according to their mechanism of action. Among these, diseases associated with type I allergic reactions include atopic dermatitis, food allergies, etc., and people of all ages from children to adults suffer from allergic symptoms.
Until now, prevention and treatment of allergic diseases have mainly used methods that do not allow patients to contact causative substances that cause allergies. For example, in the case of food allergies, removing causative substances from food Has been done. However, removal of the causative substance has many problems such as not only the burden on the person but also the burden on the cooking side.
A method of administering an antiallergic agent is also used. Various mechanisms of action of antiallergic agents have been proposed, for example, suppression of type I allergic reaction by suppressing production of immunoglobulin (Ig) E, which plays an important role in type I allergic reaction There are things.

On the other hand, various lactic acid bacteria such as the genus Bifidobacterium (hereinafter sometimes referred to as Bifidobacterium), Lactobacillus genus, and Enterococcus genus have various physiologically active effects (of the immune system). (Regulatory effect, bowel regulation effect, blood lipid improvement effect, etc.) have been reported. Therefore, at present, these lactic acid bacteria are generally ingested as probiotics.
Bifidobacterium is an obligately anaerobic Gram-positive pluriform gonococcus and is one of the most dominant genus of human intestinal flora. Independently from Lactobacillus in the 8th edition of Bergey's Manual published in 1974 However, systematic classification based on genetic information has been made thereafter. According to the hierarchical classification system based on the base sequence of the 16S rRNA gene proposed by Stakebrandt et al. In 1997, the genus Bifidobacterium (Genus) belongs to the class of Actinobacterium (Class), Bifidobacterium (Order), Bifidobacteriaceae (Bifidobacteriaceae) Currently, 32 subspecies of 32 species are approved.

Bifidobacteria are distributed in the digestive tract and faeces of humans and various animals, human vagina and oral cavity, honey bee digestive tract, sewage, etc., and the bacterial species include species between humans and animals, infants and adults. “Separation” has been confirmed (see Non-Patent Document 1).
At present, the bifidobacteria derived from humans include Bifidobacterium (hereinafter abbreviated as “B”), “Adrescentis”, “B. catenulatum”, and “B. pseudocatenatum”. (B. pseudocatenum), B. longum, B. infantitis, B. breve, B. bifidum, B. anguratam (B・ 10 bacterial species of angulatum, B. dentium, B. gallicum have been isolated. Among these, the dominant bifidobacteria species in the adult intestine are reported to be 4 species of B. adrescentis, B. catenulatum, B. pseudocatenuratum, and B. longum (Non-patent Document 2). reference).
In addition, it has been observed that when human stool is administered to a sterile mouse, bifidobacteria are reduced and are difficult to settle (see Non-Patent Document 3).

These facts strongly suggest that the Bifidobacteria species adapted to each environment are established because there exists a specific environment in the digestive tract depending on the species or age of the animal to which the Bifidobacterium is established. Therefore, when humans ingest bifidobacteria, the above 10 species are considered desirable.
As foods and drinks using these bifidobacteria, for example, Patent Literatures 1 and 2 disclose fermented foods using B · catenulatum or B · pseudocatenulatum.
Patent Document 3 discloses an antiallergic agent and fermented food containing four specific types of bifidobacteria (B. Infantitis, B. Breve, B. Longham, B. Bifidum) as active ingredients. It is described that these four types of bifidobacteria have an IgE production inhibitory effect. However, other than these four types of bifidobacteria, no bifidobacteria having an IgE production inhibitory effect have been reported.
Mitsuoka, Chitose, “Intestinal Bacteriology”, 87-107 (1990) Matsuki et al., Applied and Environmental Microbiology (Appl. Environ. Microbiol.), 70 (1), 167-173 (2004) Akiyama et al., Japanese Journal of Bacteriology, Vol. 46, 139 (1991) JP 2001-120178 A JP 2001-120180 A JP-A-10-309178

Nowadays, allergic diseases have become a problem for many people, and there is a strong demand for drugs and foods and drinks having an even better antiallergic action.
The present invention has been made to solve the above-described problems, and is an IgE production inhibitor and food and drink for IgE production inhibition that are expected to have an excellent antiallergic action, and an antiallergic agent and an antiallergic food and drink. The purpose is to provide.

As a result of intensive studies, the present inventors have found that B. catenuratum exhibits a strong IgE production inhibitory action and have completed the present invention.
That is, the first aspect of the present invention is an IgE production inhibitor characterized by containing B · catenulatum as an active ingredient.
The 2nd aspect of this invention is the food-drinks for IgE production suppression containing the IgE production inhibitor of said 1st aspect.
A third aspect of the present invention is an antiallergic agent characterized by containing B. catenuratum as an active ingredient.
A fourth aspect of the present invention is an antiallergic food or drink containing the antiallergic agent of the third aspect.

ADVANTAGE OF THE INVENTION According to this invention, the IgE production inhibitor by which the outstanding antiallergic action is anticipated, the food-drinks for IgE production suppression, the antiallergic agent, and the food-drinks for antiallergy can be provided.
These are useful for the prevention and treatment of allergic diseases, particularly diseases associated with type I allergic reactions (atopic dermatitis, food allergies, etc.).

  A preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention. In the present specification, the percentage is expressed by mass unless otherwise specified.

The IgE production inhibitor of the present invention contains B.catenulatum as an active ingredient.
Here, “contains B • catenulatum as an active ingredient” means that B • catenulatum has a target effect (inhibiting effect on IgE production in an IgE production inhibitor; antiallergic effect in an antiallergic agent described later). It means to contain in an effective amount that can be obtained.

B. catenulatum, which is an active ingredient of the IgE production inhibitor of the present invention, is a bacterial species isolated and identified from human adult feces and the like by Scardovi et al. In 1974 (Scardovy Y. and F. Crociani; "Int. J. Syst. Bacteriol", 24: 6-20, (1974)).
A method for separating and identifying B. catenulatum from a sample containing various bifidobacteria is not necessarily limited, and is described in a known method, for example, a DNA-DNA homology test or Japanese Patent Application Laid-Open No. 11-123093. The method can be carried out by a method using a species-specific primer targeting the 16S rRNA gene. In particular, the latter method uses primers prepared by paying attention to the bacterial species-specific gene sequences in the V2 and V3 areas out of the total of 9 variable regions of the Bifidobacterium genus 16S rRNA gene. DNA can be easily identified by extracting DNA from the colony directly or from cultured cells and examining the reactivity of the DNA with a primer specific for B. catenuratum.

The strain of B. catenuratum used in the present invention is not particularly limited, and so far, as belonging to the B. catenuratum species, American Type Culture Collection (ATCC), Japan Collection of Microorganism ( JCM), Northeast Texas Community College (NTCC), Deutsche Samlang Microorganization Men und Zerklutouren (DSM), etc. It may be an isolated strain isolated from the natural world (for example, human feces) by a known method as described above.
Examples of publicly deposited institutional strains include JCM1194 ^T strain, ATCC 27675 strain, and DSM 20224 strain. The superscript T means the reference stock.

There is no restriction | limiting in particular as a form of B * catenuratum used for this invention, What is necessary is just to contain the fungus body of B * catenuratum. In particular, it is preferable to use freeze-dried bacterial powder from the viewpoint of ease of handling such as control of the number of cells.
The freeze-dried bacterial powder can be obtained, for example, by culturing live B. catenuratum by a known method and freeze-drying.

The IgE production inhibitor of the present invention may be, for example, a pharmaceutical composition or a component added to food or drink.
In the case of a pharmaceutical composition, for example, it is preferable to produce B.catenulatum by formulating it into a desired dosage form using any additive such as a pharmaceutically acceptable excipient.
When formulated, the content of B · Katenuratamu in the formulation, depending on the dosage form, usually, is preferably in the range of ^{1 × 10 6 ~1 × 10 12} cfu / g, 1 × 10 7 More preferably, it is in the range of ˜1 × 10 ¹¹ cfu / g.

Examples of the administration route of the IgE production inhibitor of the present invention include oral administration and parenteral administration such as enteral administration.
Examples of the dosage form of the IgE production inhibitor of the present invention include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
The dose of B. catenulatum, which is an active ingredient of the IgE production inhibitor of the present invention, varies depending on the dosage form, symptoms, age, weight, etc. In order to effectively inhibit IgE production, It is preferably at least 10 ⁷ cfu / kg body weight, and more preferably in the range of 1 × 10 ⁸ to 1 × 10 ¹¹ cfu / kg body weight.
Administration of the IgE production inhibitor of the present invention can be performed once or several times a day.

  Additives that can be used for formulation are not particularly limited, and generally pharmaceutical compositions such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, solvents for injections, etc. The additive currently used for the thing can be used.

Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbit; starch derivatives such as corn starch, potato starch, α-starch, dextrin and carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, Cellulose derivatives such as hydroxypropylmethylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium magnesium magnesium silicate; phosphate derivatives such as calcium phosphate; And carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate and the like.
Examples of the binder include gelatin, polyvinyl pyrrolidone, and tuna gogol in addition to the above excipients.
Examples of the disintegrant include, in addition to the above excipients, chemically modified starch or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone.
Examples of the lubricant include talc; stearic acid; stearic acid metal salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as bee gum and geirow; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid. Carboxylic acid sodium salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and silicic acid hydrate; starch derivatives and the like; Can be mentioned.
Examples of the stabilizer include paraoxybenzoates such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid and the like.
Examples of the flavoring agent include sweeteners, acidulants, and fragrances.
Examples of the solvent for injection include water, ethanol, glycerin and the like.

B. catenuratum which is an active ingredient of the IgE production inhibitor of the present invention has an IgE production inhibitory action. Therefore, the IgE production inhibitor of this invention has the effect which suppresses the production of IgE in the living body by administering this compared with the case where it does not administer. Therefore, the IgE production inhibitor of the present invention is useful for the prevention or treatment of various diseases associated with the production of IgE.
In addition, it is known that IgE induces allergic diseases, particularly diseases associated with type I allergic reactions (type I allergic diseases) such as atopic dermatitis, food allergies, etc. Therefore, B having an IgE production inhibitory effect -Catenuratum is useful as an active ingredient of antiallergic agents. Therefore, the antiallergic agent of the present invention containing B. catenulatum as an active ingredient is useful for the prevention or treatment of allergic diseases, particularly type I allergic diseases.
Moreover, B. catenuratum is a dominant species in the intestine of humans, particularly human adults. Therefore, the IgE production inhibitor and the antiallergic agent of the present invention have no risk of disturbing the intestinal environment when administered, are highly safe, and have few side effects even when continuously administered for a long period of time. is there.
The IgE production inhibitor of the present invention may be contained in a food or drink to obtain a food or drink for suppressing IgE production. The form of the IgE production inhibitor to be contained in the food or drink is not particularly limited, and may be composed only of B. catenuratum. In addition to B. catenuratum, other ingredients allowed to be contained in the food and drink are included. You may contain.

The food / beverage products for IgE production suppression of this invention contain the IgE production inhibitor of this invention mentioned above. By ingesting the food and drink for suppressing IgE production of the present invention, the same effects as those obtained when the IgE production inhibitor of the present invention is administered can be obtained.
There is no restriction | limiting in particular in content of the IgE production inhibitor in the food-drinks for IgE production suppression of this invention. Preferably, the above-mentioned daily dose for effective suppression of IgE production preferably contains an amount of B. catenulatum that can be taken comfortably, usually 1 × 10 ⁶ to 1 × It is preferably in the range of 10 ¹² cfu / g, and more preferably in the range of 1 × 10 ⁷ to 1 × 10 ¹¹ cfu / g.

The food / beverage products for suppressing IgE production of the present invention may be manufactured using an IgE production inhibitor and other components allowed to be contained in the food / beverage products as well as the IgE of the present invention. What added the production inhibitor to the existing food-drinks containing another component may be used.
For example, when the food and drink for suppressing IgE production according to the present invention is fermented milk, the food and drink for suppressing IgE production may be one obtained by adding B. catenulatum to fermented milk, and using B. catenulatum as a starter fungus. It may be fermented milk.

  Other ingredients contained in the food and drink for suppressing IgE production are used without particular limitation as long as they are approved for use in food and drink according to food regulations such as the Food Sanitation Act. be able to. For example, sugars such as dextrin and starch; proteins such as gelatin, soybean protein and corn protein; amino acids such as alanine, glutamine and isoleucine; polysaccharides such as cellulose and gum arabic; fats and oils such as soybean oil and medium chain fatty acid triglyceride Etc. can be contained.

  The form of the food or drink for suppressing IgE production is not particularly limited. For example, beverages such as soft drinks, carbonated drinks, nutrition drinks, fruit drinks, and lactic acid drinks (including concentrated concentrates and powders for adjustment of these drinks); ice cream , Ice sherbet, shaved ice and other frozen desserts; buckwheat noodles, udon noodles, harsame, gyoza skin, sweet husk skin, Chinese noodles, instant noodles, , Jam, cream, baked confectionery, etc .; fishery and livestock processed foods such as kamaboko, ham, sausage; dairy products such as processed milk and fermented milk; salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing Oil and fat processed foods such as sauces, seasonings such as sauce, soup, stew, salad, Vegetables, pickles, bread; enteral nutrition; functional foods (foods for specified health use, and food for special dietary uses, such as food for the sick, health food), and the like.

When the food and drink for suppressing IgE production of the present invention is fermented milk using B. catenulatum as a starter fungus, B. catenulatum may be used alone for fermentation, and You may perform mixed culture.
As other starter bacteria, those generally used for the production of fermented milk can be used. For example, other bifidobacteria (B. pseudocatenuram, B. brebe, B. bifidum, B. longum, B Infantis, B. Addresscentis, etc.) and known lactic acid bacteria as starter bacteria for fermented milk can be used. Specific examples of such lactic acid bacteria include Streptococcus thermophilus, Streptococcus cremiris, Lactobacillus bulgaricus, Lactobacillus bulgaricus, Lactobacillus bulgaricus, gasseri) and the like.

Fermented milk can be manufactured by a well-known method. An example of a method for producing fermented milk is shown below.
First, a raw material mix containing raw material milk is prepared. In addition to the raw material milk, the raw material mix can contain other components such as fats and oils, sugars, and water as desired. For example, raw materials containing fats such as butter and cream can be blended as fats and oils. As the saccharide, usual sweeteners such as sucrose, maltose, glucose, fructose, dextrin, and reduced maltose can be blended. When producing hard yogurt, pre-swelled gelatin and / or pre-dissolved agar solution can be blended. When producing soft yogurt, whey protein and thickening polysaccharide can be blended. When producing a drink yogurt, 0 to 0.3% by mass of high methoxyl pectin may be added as a stabilizer, but it is preferable that no high methoxyl pectin is contained.
The method for preparing the raw material mix is not particularly limited. For example, the raw material milk can be obtained by adding other components to the raw material milk as desired and stirring and mixing.
Here, 1-15 mass% is preferable as a non-fat milk solid content, and, as for the mixture ratio of the raw material milk in a raw material mix, 8-15 mass% is more preferable.
Next, the raw mix is subjected to a heat sterilization treatment. The sterilization method and heating conditions are not particularly limited, but a heating temperature of 85 to 140 ° C. is preferable, and the heating time is preferably 5 to 15 minutes in the case of a batch type, and preferably 2 seconds to 15 minutes in the case of the HTST method.
Then, starter bacteria are inoculated to the heat-sterilized raw material mix and fermented. Fermentation conditions such as the inoculation amount of starter bacteria, fermentation temperature, and fermentation time can be appropriately set according to the type of starter bacteria and the type and properties of the fermented milk to be obtained. Depending on the product form, after inoculating the starter, the container may be filled and then fermented, or fermented in a fermentation tank.
After fermentation, it is quickly cooled to obtain fermented milk. Depending on the type and properties of the product, a step of homogenizing after cooling, or a step of adding and mixing other raw materials such as seasoning liquid after cooling may be performed.
The fermented milk obtained in this way can be made into a live-bacteria-type product that can be ingested with Bifidobacteria as a live cell without further heat sterilization. Live bacteria type products are stored, distributed and sold at a low temperature of 10 ° C. or lower, preferably 0 to 5 ° C.

It is preferable that the food / beverage products for IgE production suppression of this invention are the food / beverage products for which the use with IgE production suppression was displayed. Specifically, it is preferably sold as, for example, “a food or drink containing an IgE production inhibitor that is labeled as IgE production inhibitor and contains B. catenuratum” as an active ingredient.
Here, “display” means all actions for informing consumers of the above-mentioned use, and if the display can recall and analogize the use, the purpose of the display, the content of the display, Regardless of the object / medium to be displayed, all of them correspond to the “display” of the present invention. For example, the act of describing the above-mentioned use in the product or the product packaging related to the IgE production-suppressing food or drink of the present invention, the product or the product packaging describing the use described above, and the delivery, transfer or delivery Exhibiting, importing, or providing through telecommunications lines, displaying advertisements for products, displaying price lists or transaction documents, displaying or distributing the information, or describing the use in the information Thus, an act provided by an electromagnetic method (such as the Internet) can be exemplified.
The display is preferably a display approved by the government or the like (for example, a display that is approved based on various systems determined by the government and is performed in a mode based on such approval). Display on advertising materials at sales sites such as catalogs, pamphlets, POPs, and other documents is preferable.
In addition, for example, indications as health foods, functional foods, enteral nutrition foods, special purpose foods, functional nutrition foods, quasi-drugs, etc., and other indications approved by the Ministry of Health, Labor and Welfare, for example, , Foods for specified health use, and labeling approved under a similar system. Examples of the latter can include a display as a food for specified health use, a display as a condition specific food for specified health use, a display that affects the structure and function of the body, a display for reducing disease risk, etc. Speaking of the health promotion law enforcement regulations (April 30, 2003, Japanese Ministry of Health, Labor and Welfare Ordinance No. 86) indication as food for specified health (especially indication of health use), and Similar indications can be listed as typical examples.
Note that the wording used for the above display is not limited to the wording “for IgE production suppression”, and other words are words that express the IgE production inhibitory effect. I just need it.

The IgE production inhibitor of the present invention can also be used as an antiallergic agent for preventing or treating allergic diseases caused by IgE production in vivo.
That is, the anti-allergic agent of the present invention contains B. catenuratum as an active ingredient, and has the same configuration as the IgE production inhibitor of the present invention.
Examples of allergic diseases in which the antiallergic agent of the present invention is used include rheumatism, inflammatory bowel disease, food allergy, etc. Particularly, diseases involving type I allergic reactions such as atopic dermatitis and food allergy are preferred. .

The anti-allergic food / beverage product of the present invention contains the above-described anti-allergic agent of the present invention, and has the same configuration as the above-described food / beverage product for suppressing IgE production of the present invention.
By ingesting such an antiallergic food or drink, the same effects as when the antiallergic agent of the present invention is administered can be obtained.

The anti-allergic food / beverage product of the present invention is preferably a food / beverage product for which anti-allergic use is displayed, similar to the above-mentioned IgE production-suppressing food / beverage product. Specifically, it is preferably sold as, for example, “a food or drink containing an antiallergic agent containing B · catenulatum as an active ingredient, which is indicated as antiallergic”.
Note that the wording used for the display as described above is not limited to the word “for anti-allergy”, and other words may be used as long as they express the anti-allergic effect. Good.

EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
Test Example 1 [IgE production inhibition test on mouse spleen cells]
B. catenulatum and other dominant bifidobacteria species in the adult gut, B. adolescentens, B. pseudocatenatum and B. longum (B In the following procedure, IgE production inhibitory effect on mouse spleen cells was evaluated for a total of 4 types of bifidobacteria (longum).
1) Preparation of cell suspension Four types of bifidobacteria used four strains shown in Table 1 below as test lactic acid bacteria, respectively.
Each test lactic acid bacterium was cultured in a GAM liquid medium (manufactured by Difco), then centrifugally washed several times with a phosphate buffer (hereinafter abbreviated as PBS) and sterilized water, and lyophilized. The obtained dry bacterial powder was suspended in PBS to 5 mg / ml and sterilized at 121 ° C. for 15 minutes to obtain a bacterial cell suspension.

2) Experimental animals BALB / c mice raised in an environment (SPF) free of specific microorganisms and parasites (7 weeks old, male, purchased from Charles River Japan Co., Ltd.)

3) Preparation of cell suspension BALB / c mice shown in 2) above were used as experimental animals for preparing spleen cells. The breeding environment was a light / dark cycle environment under the control of a room temperature of 25 ± 1 ° C. with lighting from 6 am to 6 pm, and extinguishing from 6 pm to 6 am. Feeding conditions were free intake of feed CRF1 (manufactured by Oriental Yeast Co., Ltd.), and water was purified water obtained by purifying tap water with a domestic water purifier. It was acclimatized for 1 week in an aluminum cage under the above conditions.
Separately, ovalbumin (hereinafter referred to as OVA), which is a food protein, is used as an antigen, and 20 μg of OVA is an adjuvant (generic name for substances that increase antibody production and immune response by mixing or combining with antigen; immune adjuvant) As an antigen solution, 100 μl of an antigen solution was prepared by the Alum method in which 2 mg of Al (OH) ₃ was added.
100 μl of this antigen solution was injected into the abdominal cavity of the mouse after acclimatization for 1 week as described above, and was raised for 10 days under the same conditions as described above, and then the spleen cells of the mouse were collected. For the collection of spleen cells, the mice were sacrificed by cervical dislocation, disinfected with 70% ethanol, immediately opened in a clean bench, and the spleen was removed aseptically.
The spleen was suspended in a RPIM 1640 medium containing 10% fetal bovine serum (FBS) (Gilson) in a petri dish prepared in advance. This RPMI 1640 medium was prepared by adding 2- [4- (2-hydroxyethyl) -1-piperazyl] ethanesulfonic acid (HEPES) 10 mM, penicillin 100 U / ml and streptomycin 100 μg to Sigma L-glutamine-containing RPMI 1640 medium. / Ml added / prepared. Thereafter, the connective tissue attached to the spleen was removed and chopped, and then physically crushed with the back of a sterile disposable syringe to prepare a cell suspension.
The cell suspension is passed through a cell strainer (70 µm, manufactured by Falcon) set in a 50 ml conical tube (manufactured by Falcon), cell clumps and supporting tissues are removed, the cell liquid is recovered, and the amount of the liquid is measured. The culture solution was added to adjust the volume to 50 ml. This was centrifuged (1,200 rpm, 5 minutes), the supernatant was discarded, and the same centrifugation was repeated three more times to wash the cells to obtain a cell suspension.
The obtained cell suspension was used by staining cells with trypan blue (manufactured by Gibco), measuring the number of cells using a modified Neubayer hemocytometer, and adjusting the number of cells.

4) IgE production inhibition test First, the cell suspension was seeded in a 48-well plate (manufactured by Nanku Co., Ltd.) at a viable cell count of 2.5 × 10 ⁶ / well, and the cell suspension was finally added to the test well. It was added to a concentration 100 [mu] g / ml, to adjust the total amount to 1,000Myueru / well, 37 ° C., were cultured in ₂ the presence 5% CO.
After culturing for 7 days, the culture solution was collected, cell debris was removed by centrifugation (1,200 rpm, 5 minutes), and the obtained culture supernatant was stored at −80 ° C. until concentration measurement.
The IgE concentration in the culture supernatant was measured using a commercially available ELISA (enzyme-linked immunosorbent assay) kit (manufactured by Betil).
As a control (NC), a test similar to the above was performed except that phosphate buffered saline (PBS) was used instead of the cell suspension.

And from the measured density | concentration, the IgE production amount (%) was calculated | required by the following formula. The results are shown in Table 2.
Formula: IgE production amount (%) = (IgE concentration in culture supernatant of test subject / IgE concentration in NC culture supernatant) × 100
As is clear from the results, B. catenuratum was suppressed to less than 50% of the NC of IgE production in any strain.

Further, from the above results, the average IgE production amount (%) for each bacterial species was determined, and statistical processing (significant level p <0.05, by the method of Turky) was performed. The result is shown in FIG.
As is clear from FIG. 1, IgE production from mouse spleen cells was significantly suppressed by the addition of B. catenuratum. Therefore, the effect of B. catenuratum as an IgE production inhibitor and an antiallergic agent was confirmed.

Preparation Method of Strains Used in Test Example 1 All “human-derived isolates” of the bifidobacteria species used in Test Example 1 were isolated from human feces by the following separation method and identified by the following identification method.
[Separation method]
Feces collected from adults are made anaerobic with an oxygen scavenger (trade name: Aneropack Kenki, manufactured by Mitsubishi Gas Chemical Co., Ltd.), kept at a low temperature (about 5 ° C) with a cryogen, and stepwise within 24 hours Then, an anaerobic culture was performed by a steel wool method using a TOS propionic acid agar medium (Eiken Chemical Co., Ltd.) which is a bifidobacteria selective medium.
[Identification method]
Each colony formed by culturing is scraped with a platinum loop, suspended in 100 μl of PBS, added with an equal amount of Triton-X (ICN Biochemicals) and heated at 95 ° C. for 5 minutes. did. The DNA contained in the supernatant was used as a template, and the 16S rRNA gene sequence was analyzed by the method described in JP-A No. 11-123093 to identify which strain was the Bifidobacterium strain scraped from each colony.
As a result, two species identified as B. addresssentis were identified as MCC191 and MCC195 strains, respectively, and two species identified as B. catenuratum were identified as MCC76 strain, MCC78 strain, and B. pseudocatenulatum, respectively. The two species identified as MCC194 strain and MCC214 strain, and B. longum were designated as MCC124 strain and MCC133 strain, respectively.

Example 1 [Production of dry bacterial powder of B. catenuratum]
B. catenuratum JCM1194 ^T strain and MCC78 strain were inoculated into GAM bouillon liquid medium (manufactured by Nissui Pharmaceutical Co., Ltd.), respectively, followed by stationary culture at 37 ° C. for 18-24 hours. After completion of the culture, centrifugation was performed at 10,000 rpm for 5 minutes, and the supernatant was removed to obtain concentrated cells.
Subsequently, it suspended in PBS and the medium component was removed. This removal operation was performed twice each for PBS and sterilized water, frozen at −40 ° C. or lower, and then lyophilized. The number of bacteria in the obtained dry bacteria powder was 10 ¹⁰ cfu / g.
The dried B. catenuratum JCM1194 ^T strain and MCC78 strain were subjected to an IgE production suppression test in mouse spleen cells by the same test method as in Test Example 1, and the IgE production amount (%) was 42 respectively. 4% and 30.7%. From this result, it was confirmed that the dried bacterial powders of B. catenuratum JCM1194 ^T strain and MCC78 strain were effective as an IgE production inhibitor and an antiallergic agent.

Example 2 [Production of fermented milk by mixed culture]
Streptococcus thermophilus (manufactured by Hansen) and Lactobacillus bulgaricus (manufactured by Lacobacillus bulgaricus, Hansen), which are starter fungi of fermented milk, were distilled from skim milk powder 10% and yeast extract 0.1%, respectively. A culture solution obtained by inoculating a reduced skim milk medium prepared by dissolving in water and culturing at 37 ° C. for 16 hours was used as a bulk starter.
Into the raw material mix consisting of raw milk and skim milk powder, the culture solution of B. catenulatum MCC78 strain prepared in Example 1 and the bulk starter (culture solution of Streptococcus thermophilus and Lactobacillus bulgaricus) prepared in advance, 5% of each was inoculated and cultured at 37 ° C. for 16 hours to obtain fermented milk.
The obtained fermented milk contained B. catenuratum MCC78 strain at a concentration of 7.5 × 10 ⁷ cfu / mL. In addition, this fermented milk was a product having a good flavor, a delicious taste, and a high palatability.

6 is a graph showing the results of Test Example 1.

Claims (4)

  An IgE production inhibitor comprising Bifidobacterium catenatum as an active ingredient.   Food / beverage products for IgE production suppression containing the IgE production inhibitor of Claim 1.   An antiallergic agent comprising Bifidobacterium catenatum as an active ingredient. An antiallergic food or drink comprising the antiallergic agent according to claim 3.

Images