JP2968374B2 - IgA production promoter - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an IgA having an activity of promoting secretory IgA production which has an action of inhibiting the binding of pathogenic microorganisms to the mucosa of the digestive tract and inhibiting the passage of allergen substances to the digestive tract wall. It relates to a production promoter.

[0002]

2. Description of the Related Art It is known that milk which is a complete diet for the growth and development of infants and young animals contains protective substances such as immunoglobulin, complement, lysozyme and lactoferrin.

[0003] Secretory IgA, which is an immunoglobulin secreted into milk, inhibits the binding of microorganisms having strong pathogenicity to the intestinal mucosa and inactivates the action by binding specifically to bacterial toxins. It is known that it binds to a dietary antigen that acts as an allergen, prevents it from passing through the digestive tract wall, and suppresses allergic reactions.

[0004] In addition, IgG antibodies against dietary antigens appear frequently in artificially fed infants and patients with IgA deficiency raised in infant formula without secretory IgA, causing allergic diseases and autoimmune diseases. It is known that the frequency is high.

[0005] As described above, as the excellent infection-protecting action of IgA has been found, it has been desired to develop a food or drink to which IgA is added, which has an effect of protecting infection and suppressing allergic reactions.

[0006]

However, the IgA
Is difficult to supply, not only expensive, but also physically unstable, making it almost impossible to use it industrially. For this reason, foods to which IgA has been added as a component for preventing infection or preventing allergic reactions have not yet been provided.

[0007] IgA production sites are present in mucosal plate plasma cells such as the lamina propria of the gastrointestinal tract, salivary glands, and mammary glands, and the IgA production promoting activity nonspecifically stimulates these production cells. (IgA-inducing ability) has been confirmed.

The present inventors have disclosed in Japanese Patent Application Laid-Open No. 2-280059 that IgA can be easily operated in a large amount in a short time.
As a method of searching for a substance having an inducing ability, a Bayer plate cell containing a large amount of IgA-producing cells is cultivated aseptically, and the ratio of I.
A method of selecting a substance having an ability to induce IgA according to the ratio of the amount of gA has already been proposed, and further, it has been found that a specific Bifidobacterium bacterium has a strong ability to induce IgA using this method. ing.

The present invention has found a more useful IgA inducer, and has no effect on the protection against infection with pathogenic microorganisms and the addition of IgA having an antiallergic effect to foods and the like. An object of the present invention is to provide an IgA production promoting agent which stimulates a production site, promotes IgA production activity to increase the amount of IgA production, inhibits excellent infection protection and inhibits allergic reactions, and a food or drink having an ability to promote IgA production. And

[0010]

The IgA production promoter according to the present invention comprises, as an active ingredient, a protoplast of Bifidobacterium (hereinafter referred to as B) or a cytoplasmic membrane of Bifidobacterium. Things.

[0011]

In the present invention, the presence or absence of the ability to induce the production of IgA was examined for the cell component fraction of B bacterium using the method disclosed in the above-mentioned JP-A-2-280059. as a result,
The present inventors have now found that they show strong IgA-inducing ability in protoplasts of B bacillus or cytoplasmic membrane fraction of B bacillus, and have reached the present invention. By adding the present protoplasts or the cytoplasmic membrane fraction of B bacterium according to the present invention to foods and drinks, it is possible to provide foods and drinks useful for protection against infections that promote IgA production activity in the intestinal tract.

Bacterial cell fractions of B bacterium are obtained by treating B bacterium with a cell wall lysing enzyme (M1 enzyme) to remove a cell wall portion, a protoplast fraction, a M1 enzyme soluble fraction, and the protoplast with an organic solvent. The cytoplasmic membrane fraction obtained by the treatment, the lipoteichoic acid fraction, the cell wall fraction obtained by crushing B cells and treating with a nuclease / proteolytic enzyme are examined, and a protoplast fraction,
IgA stronger than the cells themselves derived from the cytoplasmic membrane fraction
It was confirmed that it exhibited inducibility. The fact that it shows stronger IgA-inducing ability than the cells itself derived from the protoplast fraction and the cytoplasmic membrane fraction was confirmed not for a specific B bacterium but for a general B bacterium.

That is, in the examples described later, the B bacterium used in the present invention was Bifidobacterium bacterium having a high IgA-inducing ability disclosed in the above-mentioned JP-A-2-280059.
Breve ( Bifidobacterium breave ) YIT4063
B. Breve YIT4064 and newborn, infant,
B. isolated from infant, adult and elderly stool and identified as B. Breve Ka-6, B.I. Breve 9-7,
B. Breve KN-6, B.I. Longum ( Bifidobacter
ium longum ) 04-6 was used, and it was confirmed that any of them exhibited stronger IgA inducing ability than the cells themselves derived from the protoplast fraction and the cytoplasmic membrane fraction.

Incidentally, the measurement of the IgA production promoting activity in the examples described later was carried out according to the method described in JP-A-2-280059. That is, Bayer plate cells, which are one of the intestinal immunity tissues containing a large amount of IgA-producing cells, are aseptically cultured, and the above-mentioned nuclear component is added to the culture solution, cultured for a predetermined period, and cultured after the predetermined period of culture. Ig secreted from IgA producing cells in solution
The amount of IgA secreted from A-producing cells was measured, and the IgA inducing ability (IgA production promoting activity) was measured by the ratio of the amount of IgA in the group without the various dairy components to the amount of IgA in the group without the various dairy components. It is.

[0015]

EXAMPLES The present invention is based on the finding that the protoplast or cytoplasmic membrane of the B strain is higher in Ig than the cells before fractionation.
It provides that A production is more strongly induced.

The B strain used this time was YIT4063 (Microcosms No. 10671) and YIT4064 (Microcosms No. 10672), which have already been deposited as strains having high IgA inducing ability, and newborn babies. Several strains isolated from the stool of infants, infants, adults and the elderly and identified as Bifidobacterium were used.

Next, the IgA-inducing ability of the protoplasts or the cytoplasmic membrane fractionated from the B strain of the present invention will be described with reference to experimental examples. The method for fractionating the cells used in the experiment and the method for measuring the ability to induce IgA are as follows.

(1) Bacterial Cell Fraction Each B strain was inoculated into a GAM medium (Nissui Pharmaceutical) and cultured at 37 ° C. for 48 hours under anaerobic conditions. These bacteria were washed twice with a phosphate buffer and heat-treated at 100 ° C. for 30 minutes to obtain whole cells, which were used as starting materials for fractionation.

All cells were treated with 5 mM Tris-malate buffer (T
ris-malate buffer) (pH 6.4, 2 × 10 ^-3 M Mg
Cl _2, was suspended in 0.15 M NaCl), it was added N- acetylmuramidase (N-acethylmuramidase) (M1 enzymes, Seikagaku Corporation). After treatment at 37 ° C. overnight, the mixture was centrifuged to separate a supernatant and a sediment. The precipitate was washed with distilled water and freeze-dried. The fraction was used in experiments as a fraction insoluble in M1 enzyme, so-called protoplast.

Chloroform, methanol and water were added to the above protoplasts at a ratio of 2: 1: 1 and stirred at 50 ° C. for 1 hour. The aqueous layer was separated into a centrifuged aqueous layer, a chloroform: methanol layer, and a fluff layer existing between the two.
The Fluff layer was washed with distilled water, freeze-dried, and used as a cytoplasmic membrane fraction in the experiment.

(2) Method for measuring IgA inducibility The Peyer's patch was aseptically removed from the small intestine of a mouse, and a dispase (Dispase) solution (1.5 mg Dispase / ml Joklik) was used.
-modified MEM), and stirred at 37 ° C. for 30 to 40 minutes to collect single cells that came out of the solution. This operation was repeated 4 to 5 times, followed by centrifugal washing to obtain Peyer's patch cells. However, in the Peyer's patch cell culture system, the whole cells and the cell fraction components were contained in one.
It was added so as to be 00 μg / ml.

( Experimental Example 1: For protoplast fractionation of B bacterium)
Enhancement of IgA-inducing ability by B) Several strains of B bacterium were examined for IgA-inducing ability of the protoplast fraction and all cells before fractionation. As a result, although the B strains examined as shown in Table 1 had different IgA-inducing abilities, all were made into protoplast fractions, so that the IgA-inducing ability was about 2 times higher than that of all cells before fractionation. It was confirmed that the concentration was increased from 2-fold to 3-fold.

[0023]

[Table 1]

(Experimental Example 2: IgA induction of cytoplasmic membrane fraction
NohFor protoplasts, all cells are treated with cell wall lysing enzyme.
This is the fraction from which the cell wall has been removed.
It is composed of components within the substance. Here we have a protoplast
Of the cytoplasmic membrane treated with organic solvent to induce IgA
I examined it. As a result, they were examined as shown in Table 2.
B. Breve 4064; With Breve Ka-6
For the plasma membrane fractionation, stronger IgA induction than whole cells before fractionation
Noh was recognized. The same is true when compared to protoplasts.
Or more IgA induction.

[0025]

[Table 2]

[0026]

As described above, according to the present invention, the B strain can be obtained by converting the cells into protoplasts or cytoplasmic membranes.
It was shown that the ability to induce IgA was further enhanced. Therefore, it strongly indicates that the action main body of the IgA-inducing ability of the B strain exists in a cell component different from the cell wall. From this point, it can be said that there is a high possibility that a substance having a novel immunostimulating effect is found from the protoplast of the B strain or the cytoplasmic membrane. In the future, when developing materials such as foods that emphasize functions (so-called functional foods), it is essential to clarify the active substance. In particular, bacterial cell components are known to have various physiological activities, and the method of fractionating the cells into their constituent components and comparing and examining the activity present in each fraction is described above. It is effective in elucidating the active substance.

As described above, the ability to induce IgA of the protoplast or the B strain mainly composed of the cytoplasmic membrane was clearly stronger than that of the whole cells before fractionation. Therefore, the protoplast or the B strain having a cytoplasmic membrane as a main component can be a more effective material for enhancing IgA, and can be a food or beverage having an ability to promote IgA production.

Continued on the front page (72) Inventor Kazuhito Hayakawa 1-1-1 Higashi-Shimbashi, Minato-ku, Tokyo Co., Ltd. Yakult Honsha Co., Ltd. (72) Inventor Makoto Owaki 1-1-1-1 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Co., Ltd. In-house (56) References JP-A-2-280059 (JP, A) JP-A-1-242532 (JP, A) JP-A-4-335885 (JP, A) JP-A-3-244367 (JP, A) (58) Fields surveyed (Int.Cl. ⁶ , DB name) A61K 35/74

Claims (1)

(57) [Claims] (1) Bifidobacterium (Bifidobacteriu
m) Genus protoplasts or Bifidobacterium
An IgA production promoter containing a bacterial cytoplasmic membrane as an active ingredient.