JP2632657B2 - Method for selecting a strain capable of inducing IgA - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a secretory Ig having an action of inhibiting the binding of microorganisms and allergen substances to mucous membranes.
The present invention relates to a method for confirming that the IgA-inducing ability of A has a large difference between different strains of the same genus or the same species, and using this IgA-inducing ability as an index, a method capable of selecting a strain having a high IgA-inducing ability.

[0002]

2. Description of the Related Art Antibodies and immunoglobulins, which are proteins having structural and functional relationships with the antibodies, are classified into five classes according to the functional properties of many different classes of immunoglobulins. IgA therein has two subclasses (IgA1 and IgA2). IgA2 is different from IgA1 in that L chains are linked to each other by an S--S bond instead of H chains (IgA1).
In 1, the light chain is covalently linked to the heavy chain). Ig
A1 accounts for 90% of all serum IgA, whereas IgA2 accounts for 60% of the secreted IgA.

[0003] IgA production sites are present in submucosal plasma cells such as the lamina propria of the gastrointestinal tract, salivary glands, and mammary glands. In the lamina propria of the human gastrointestinal tract, about 2 IgA-producing cells are present.
The number of IgG-producing cells was reduced by 0: 1, and 1: 3 (IgG
A: Contrast with the lymph nodes and spleen in the proportion of IgG). IgA found in mucus secretions is made as a dimer with one J-chain component and has an accessory secretion fragment found only in small amounts in serum IgA. It is added to dimeric IgA molecules as they emerge from the plasma cells under the mucous membranes of the intestine and airways into the mucus secretion.

[0004] Secretory IgA in mucus secretions inhibits the binding of strongly pathogenic microorganisms and allergens to mucous membranes, thus preventing not only infection but also food components that can act as allergens. It binds and does not pass through the digestive tract wall. For example, when exotoxins are secreted, protection by the antibody depends on the direct action of the antibody bound to the surface of the microorganism. Antibodies can exert various effects by directly binding to microorganisms.

[0005] On the other hand, there is a substance having the ability to non-specifically stimulate cells of an antibody-producing system so that an antigen can be more effectively processed, and this property is often called an adjuvant. For example, Vibrio cholera
Cholera toxin, the causative toxin of diarrhea produced by ae), is known to act on the small intestinal mucosa and alter its ion permeability, causing a large amount of electrolytes and water to be excreted from the small intestine, causing diarrhea. . In addition, cholera toxin B subunit, which is a non-toxic component excluding the body of the toxin, penetrates into the small intestinal mucosa and has an immune response (IgA inducing ability) to promote the production of IgA antibody, and is known to act as an adjuvant. Have been.

On the other hand, Bifidobacterium, an intestinal bacterium that does not show any pathogenicity to humans and animals, has 99% of its intestinal flora during breastfeeding within the first few weeks of birth. It was thought to play a role in host defense. One of these is Bifidobacterium longum (B
Certain strains of S. ifidobacterium longum) have been reported to increase total fecal IgA levels by oral administration.

[0007]

However, focusing on the secretory IgA, it has not been performed to compare the IgA inducing ability of different strains of the same genus or species with each other. A simple selection method has not been established.

[0008] For example, the usefulness of intestinal bacteria has not yet been fully elucidated, and in particular, the species and bifidobacterium species of Bifidobacterium known to have no pathogenicity to humans and animals are known. It was not yet clear whether the difference in IgA-inducing ability was observed.

Therefore, in the present invention, the ability to induce IgA is used as one index to select from different strains of the same genus or the same species.
A simple selection method using IgA-producing cells for selecting strains having high gA inducibility was established, and the operation was easy and in large quantities.
In a short period of time, intensive studies were conducted for the purpose of selecting strains having high IgA-inducing ability from different test strains of the same genus or the same species. As a result, between strains of the same genus or species,
A great difference was recognized, and a strain having a stronger IgA-inducing ability than the known bacteria could be selected.

[0010]

According to the method for selecting a strain having the ability to induce IgA according to the present invention, a plurality of culture solutions obtained by aseptically culturing Peyer's patch cells containing a large amount of IgA-producing cells are prepared, and individual cultures are prepared. A different test strain of the same genus or the same species is added to the solution, and cultured for a predetermined period of time.The amount of IgA secreted from the IgA-producing cells in each culture after the cultivation for the predetermined period is measured, and the same or different species of the same or different species A strain having high IgA-inducing ability is selected from different test strains of the same genus or the same species depending on the difference in the amount of IgA in the culture solution to which the test strain has been added.

[0011]

In the present invention, a plurality of culture solutions prepared by aseptically culturing Peyer's patch cells containing a large amount of IgA-producing cells are prepared, and different test strains of the same genus or the same species are added to the respective culture solutions for a predetermined period. After culturing, the amount of IgA secreted from the IgA-producing cells in each of the cultures after the cultivation for a predetermined period was measured, and the difference in the amount of IgA in the culture medium to which the different test strains of the same genus or the same species were added, Alternatively, to select a strain having a high IgA inducing ability from different test strains of the same species, the operation is easy, a large amount, in a short time, a strain having a high IgA inducing ability from a different test strain of the same genus or the same species. Can be sorted out.

[0012]

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the finding that three out of 120 Bifidobacterium isolates isolated from the stool of newborns, babies, infants, adults, and the elderly are more sensitive to Peyer's strain than known Bifidobacterium strains. It is intended to strongly induce IgA production of plate cells.

[0013] The known bacteria used this time are the American
Type Culture Collection (ATCC) and Japan Collection Microorganisms (JC
M).

Next, selection of a Bifidobacterium strain having a strong IgA-inducing ability of the present invention will be described with reference to examples. In addition, the identification, preparation method, and experimental method of the strain used in the experiment are as follows. (1) Identification of Bacteria Isolated The bacteria were identified in accordance with the Mitsuoka et al.'S sugar fermentation test method (World of Intestinal Bacteria- Isolation and Identification of Anaerobic Bacteria- , Bunsosha).

(2) Bacterial Cell Preparation Various strains of Bifidobacterium isolates (120 strains) isolated from feces of newborns, infants, infants, adults and the elderly
Was inoculated into a GAM medium and cultured at 37 ° C. for 48 hours under anaerobic conditions. These bacteria were washed twice with a phosphate buffer, and
Heat treatment was performed at 00 ° C. for 30 minutes before use.

(3) Separation of Peyer's patch cells Peyer's patches are aseptically removed from the small intestine of a mouse, and a dispase solution (1.5 mg Dispase / ml Joklik-m) is used.
odified MEM), and the mixture was 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.

Example 1 The Bifidobacterium isolate (120 strain) obtained by performing single colony isolation three times was examined for its ability to induce IgA. The method for measuring the amount of IgA produced was as follows. Using a 96-hole flat bottom plate, 5 × 10 ⁵ per hole
Of Peyer's patch cells and suspensions of various strains at a concentration of OD ₆₆₀ = 0.275, Eagle medium containing 5% fetal calf serum (Eagle MEM (Nippon Pharmaceutical) 9.4 g / l, 200 mM glutamine 10
ml / l (2mM), MEM nonessential amins acid (× 100G1BC
O) 10ml / l, 100mM sodium pyruvate (Sigma) 10ml /
1 (1 mM) pH 7.2 (prepared with 1N NaOH)) at 37 ° C. in an atmosphere of 5% CO ₂ / Air.

During the culture, nutrient mixture (nutrient mixture) (MEMessential amino acid
d (× 50G1BCO) 5ml, MEM non essential amino acid (× 1
0G1BCO) 2.5ml, 200mM glutamine 2.5ml, Dextrose 500mg,
Eagle MEM (-NaHCO ₃ ) (manufactured by Nissui Pharmaceutical Co., Ltd.) was adjusted to 35 ml, pH 7.2 (1N NaOH), and 7.5% NaHCO ₃ was added at 7.5 ml) to 0.02 (ml /
(Hole) Dispensed. IgA secreted into culture supernatant for 7 days
The amount was measured over time by ELISA.

The ELISA method uses a sodium carbonate buffer (pH 9.
Using 6), 100 μl of goat anti-mouse IgA (Cappel) was added to each well of the immunoplate,
The mixture was allowed to react overnight at 0 ° C. and adsorbed. Wash solution (0.05% Triton
(X-100, PBS) 3 times, then to cover unadsorbed part,
A sodium carbonate buffer containing 1% bovine serum albumin (BSA) was added and reacted at 37 ° C. for 1.5 hours. After washing again with the washing solution, each sample (culture supernatant) was diluted, added at 90 μl / well, and allowed to stand at 37 ° C. for 1.5 hours to react. After washing, peroxidase-conjugated goat anti-mouse I
gS (IgA + IgG + IgM) (Cappel) was added at 100 μl / well and left at 37 ° C. for 1.5 hours. After washing again with the washing solution, the substrate solution (20 mg of o-phenylenediamine dissolved in 50 ml of citrate buffer pH 5.0 and 10 μl of hydrogen peroxide added immediately before use) was added.
The mixture was added at 100 μl / well and reacted at 37 ° C. for 10 minutes. The reaction was stopped immediately by adding 50 μl / well of 2.5 M sulfuric acid, and the absorbance (OD _{492 nm} ) of each well was measured using Titertec Multiscan (Flow).

Standard IgA is mouse IgA (myelo
na) (ICN Immuno Biolog
ical)) was used. IgA values were expressed in μg / ml culture supernatant. The rate of increase in IgA was determined by the following equation (1).

[0021]

(Equation 1)

As shown in the following Table 1, three strains having stronger IgA-inducing ability (Index value of 12 or more) were detected as compared to the seven known strains (YIT4062, 4063, 406).
4). YIT4062, YIT4063, YIT4
064 has an overwhelming ability to induce IgA as compared with a known strain of Bifidobacterium deposited at a public organization, and the IgA content of the test substance-added group relative to the IgA amount of the test substance-free group The ratio of the amount (increase rate) is 12 or more, which strongly induces IgA production of IgA-producing cells.

The YIT4062 and YIT406
3, 3 strains of YIT4064 are available from
No. (The first microbial laboratory deposited on April 19, 1989)
No. 0670 was transferred on March 19, 1990), and No. 2823, a microfabrication laboratory, and No. 10671, deposited on April 19, 1989, on March 19, 1990. Has been deposited as Microtechnical Research Article No. 2824 (Microtechnical Research Microorganism No. 10672 deposited on April 19, 1989 and transferred on March 19, 1990).

[0024]

[Table 1]

(Example 2) The three strains having strong IgA induction were identified by a sugar fermentation test and a DNA homology test method. The results of the sugar fermentability test are shown in Table 2 below. YIT
4062 is B. longu (B. longu)
m), YIT4063, YIT4063, 4064 was found to be Bifidobacterium breve.

[0026]

[Table 2]

As described above, the three strains according to the present invention are Bifidobacterium longum and Bifidobacterium breve, strains of the genus Bifidobacterium having very low antigenicity (H.YASUI). , A.MIKE, and M.OHWAK
I, J. Dairy Sci. 72: 30-35), and furthermore, it was confirmed to strongly enhance IgA production of Peyer's patch cells, and thus acts as an adjuvant.

As described above, according to the present invention, there is a possibility that a substance or a strain having IgA-inducing ability that can be easily operated, can be searched in a large amount and in a short time can be searched.

Since the Bifidobacterium strain searched by the selection method of the present invention is known to have no pathogenicity to humans and animals, its IgA-inducing ability is considered as its own foreign substance. Is very likely to be IgA-inducing as an adjuvant effect, rather than antigenic. The above 3 obtained by the screening method according to the present invention.
All experiments with bacterial strains were performed with heat-killed cells, and it was confirmed that IgA production of Peyer's patch cells was strongly enhanced, suggesting that some cells activate IgA-inducing ability in the cell wall. For this reason, since it is presumed that even living bacteria have the same effect, they can be formulated into powders, drinks, tablets and the like, and can also be used as functional food materials.

These three strains strongly enhance the IgA production of Peyer's patch cells, which are the main tissues of the gastrointestinal mucosal immune system.
It is also expected to enhance gA production and luminal endocrine IgA, and thus strongly enhance IgA production in mucous membranes other than Peyer's patches, so that infection protection in the intestine, and
It is effective in inhibiting absorption of allergic sources, further preventing cold,
It may also have a sufficient effect on prevention of allergic rhinitis and the like. In addition, these cells can be administered orally,
It is extremely easy to use without worrying about safety. Furthermore, the microbial cells are used as an active ingredient,
It is easy to formulate, store and use because it can be a dead cell. Therefore, it can be used as a preventive agent for intestinal infections and allergies, and can also be widely used as a nutritional supplement or as a compounding component in so-called health foods and functional foods.

[0031]

According to the present invention, a plurality of culture solutions prepared by aseptically culturing Peyer's patch cells containing a large amount of IgA-producing cells are prepared, and different test strains of the same genus or the same species are added to each culture solution. After culturing for a predetermined period, the amount of IgA secreted from the IgA-producing cells in each culture solution after culturing for the predetermined period is measured, and the difference in the IgA amount of the culture solution to which the different test strains of the same genus or the same species are added is determined. To select a strain having a high IgA-inducing ability from different test strains of the same genus or the same species, it is easy to operate, and has a high IgA-inducing ability from a different test strain of the same genus or the same species in a large amount in a short time. The strain can be selected.

 ────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tatsuhiko Suga 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (56) References J Dairy Sci, 72 (1), P. 30-35, 1989 Metabolism, 21 (Special Issue, Immunity '84), p. 93-102, 1984 Bifidus, 2 (Supplement, Special Issue of the 8th Annual Scientific Meeting), p. 37-38, 1988 J Immunol Methods, 48, p. 33-44, 1982 Gastrointestinal tract and immunity, no. 20, p. 204-207, 1988 Austrian Journal of Experimental Biology and Medical Science, 63 (Pt. 2) P. 177-182, 1985 Infection and Immunity, 55 (5), P. 1085-1089, 1987

Claims (1)

(57) [Claims] 1. A plurality of culture solutions prepared by aseptically culturing Peyer's patch cells containing a large amount of IgA-producing cells are prepared, and different test strains of the same genus or the same species are respectively added to the individual culture solutions and cultured for a predetermined period of time. The amount of IgA secreted from the IgA-producing cells in each culture after the cultivation for a predetermined period is measured, and the difference in the amount of IgA of the culture to which the different test strains of the same genus or the same species are added, A method for selecting a strain having IgA-inducing ability, comprising selecting a strain having high IgA-inducing ability as an adjuvant effect from different test strains.