JP2023096453A - Iga production promotion composition - Google Patents

Abstract

To provide a new material which promotes IgA production from an immune cell and has an infection prevention effect.SOLUTION: An IgA production promotion composition includes as an active ingredient an S-layer protein which is a component derived from a bacterial body of a bacterium of the genus Lactobacillus. A lactic acid bacterium of the genus Lactobacillus is Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus buchneri, Lactobacillus brevis, or Lactobacillus helveticus.SELECTED DRAWING: Figure 2

Description

TECHNICAL FIELD The present invention relates to a composition for promoting IgA production. In particular, it relates to a composition for promoting IgA production containing an S-layer protein, which is a component derived from lactic acid bacteria of the genus Lactobacillus.

IgA is one of the major antibodies that act in innate immunity. In particular, secretory IgA (SIgA) plays a major role in mucosal immunity and functions as the forefront of the immune system in the digestive tract and respiratory system. In the intestinal tract, SIgA is secreted from plasma cells of the muscularis mucosae, penetrates the epithelium, and is secreted to the surface of the epithelium to trap antigens and pathogenic bacteria, thereby preventing them from adhering to the epithelium. Based on these facts, IgA is expected to work for defense against infection in living organisms, and several solutions for increasing IgA production have been disclosed so far.

Patent Document 1 discloses a new lactic acid bacterium that can exhibit excellent mucosal immunostimulatory action, improvement action of biological defense mechanism, etc., and is useful as probiotics, and final products containing the same (fermented milk, food and drink such as lactic acid bacteria beverages). As a solution thereof, at least one species selected from the group consisting of Lactobacillus plantarum ONRIC b0239 and Lactobacillus plantarum ONRIC b0240 having the ability to promote IgA production, Lactic acid bacteria are disclosed.

Patent Document 2 aims to provide an intestinal immunity enhancer containing, as an active ingredient, a component derived from lactic acid bacteria having a high intestinal immunity enhancing effect. It discloses an intestinal immunity enhancer containing a cell wall component of plantarum as an active ingredient.

Patent Document 3 discloses pharmaceuticals and nutritional compositions that can provide both the effects of preventing infectious diseases and preventing autoimmune diseases by promoting the production of IgA antibodies and suppressing the production of IgG antibodies, which are autoantibodies. The problem is to provide products, food and drink, and feed, and as a means for solving the problem, a lactic acid bacterium belonging to the lactic acid bacterium Lactobacillus helveticus, particularly the Lactobacillus helveticus SBT2171 strain is disclosed.

Patent Document 4 aims to provide a novel material that activates JNK. or a JNK activating composition containing the SLP degradation product as an active ingredient, as well as a JNK activating food and drink, an antioxidant food and drink, and a cell defense food and drink containing the same. Patent Document 5 aims to increase the production of antibacterial peptides from epithelial cells and to provide a novel material having an infection-preventing effect. Disclosed are an antimicrobial peptide production-enhancing composition containing a degradation product of SLP as an active ingredient, an antimicrobial peptide production-enhancing food and drink, and an infection-preventing food and drink containing the composition.

However, since the Lactobacillus plantarum described in Patent Documents 1 and 2 does not have SLP, the IgA antibody production promoting action described in this document is effective for the cells and cell wall components of Lactobacillus plantarum. It is clear that the IgA antibody production-promoting action of SLP, which is a component action, is not disclosed.
Further, the effect of promoting the production of IgA antibodies and the effect of suppressing the production of IgG antibodies described in Patent Document 3 are the effects of using Lactobacillus helveticus cells or cell cultures as active ingredients, and promoting the production of IgA antibodies by SLP. No action is disclosed.
Moreover, Patent Documents 4 and 5 do not disclose the IgA production-enhancing effect of SLP of the genus Lactobacillus.
As described above, the solution provided by the present application is neither disclosed nor suggested in any of the above documents.

Japanese Patent No. 3818319 JP 2014-125446 A Japanese Patent No. 6739155 JP 2020-152653 A International publication 2020/111172 pamphlet

An object of the present invention is to provide a novel material that promotes IgA production from immune cells and has an infection-preventing effect.

In order to solve the above problems, the present invention includes the following configurations.
[1] A composition for promoting IgA production, comprising as an active ingredient an S-layer protein, which is a component derived from the cells of the genus Lactobacillus.
[2] The lactic acid bacteria of the genus Lactobacillus are Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus buchneri, Lactobacillus brevis obacillus brevis), or Lactobacillus The composition for promoting IgA production according to [1], which is Lactobacillus helveticus.
[3] The composition for promoting IgA production according to [1] or [2], wherein the S-layer protein is purified.
[4] Food and drink for promoting IgA production, comprising the composition for promoting IgA production according to any one of [1] to [3].
[5] A drug for promoting IgA production, comprising the composition for promoting IgA production according to any one of [1] to [3].
[6] A feed for promoting IgA production, comprising the composition for promoting IgA production according to any one of [1] to [3].

Material that induces IgA production such as S-layer protein (hereinafter sometimes simply referred to as SLP) derived from Lactobacillus lactic acid bacteria, or Lactobacillus lactic acid bacteria cells containing SLP and cultures thereof By ingesting into the body, it can be expected to improve the defense ability against various infections through the IgA production promoting effect.

Fig. 3 is a photograph showing the results of SDS-PAGE of purified SLP of Lactobacillus helveticus. 1 is a graph showing the IgA concentration in the culture supernatant when SLP was added to PBMC (peripheral blood mononuclear cells) and cultured.

The present invention provides a novel composition for promoting IgA production.
The composition for promoting IgA production of the present invention is described in detail below.
(Composition for promoting IgA production)
The S-layer protein of the present invention is a protein characterized by being found in the cell wall outer layer of various bacteria and exhibiting a regular crystal structure. Since SLP is non-covalently bound to cell wall components, it can be extracted from the cells with a chaotropic reagent such as lithium chloride. Also, the extracted SLP reforms a regular crystal structure by removing the chaotropic reagent. Utilizing this characteristic, purification from bacterial cells becomes possible.
Among lactic acid bacteria, it has been found that S-layer is expressed in the genus Lactobacillus, and Mukai et al. reported (Japanese Journal of Lactic Acid Bacteria, 19 (1): 21-29, 2008) that - Expression of SLP has been confirmed or presumed in 13 bacterial species including Lactobacillus acidophilus group lactic acid bacteria. The SLPs expressed by these lactic acid bacteria have in common that they are basic proteins with an isoelectric point of 9 to 10 and have a signal peptide sequence consisting of 23 to 30 amino acid residues in the N-terminal region. . On the other hand, it has been shown that the molecular weights of SLPs produced by lactic acid bacteria vary greatly between bacterial species, and that their gene structures are highly diverse even within the same bacterial species.
The SLP used in the composition for promoting IgA production of the present invention is derived from the genus Lactobacillus, and among bacterial species expressing S-layer, any one having the effect can be used. can. Bacterial strains expressing SLP or SLP-like proteins include Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum (La ctobacillus gallinarum), Lactobacillus kitasatonis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus brevis, Lactobacillus buchne Li (Lactobacillus buchneri), Lactobacillus fur Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus kefiri, Lactobacillus parakefiri, among which lactobacillus cilus acidophilus, lactobacillus amylovorus, lacto Bacillus buchneri, Lactobacillus brevis, or Lactobacillus helveticus are more preferred.
In addition, the SLP of the present invention does not necessarily have to be purified from bacterial cells, and can be used as bacterial cells themselves containing SLP or cultures, but purified SLP is more preferable. The cells themselves may be viable cells or dead cells.

(Method for producing composition for promoting IgA production)
SLP, the active ingredient of the present invention, can be purified from lactic acid bacteria cells according to the following method. Various known purification methods may be used for purification of SLP, and typical methods are shown below. After sufficiently culturing the target Lactobacillus lactic acid bacteria in a liquid medium such as MRS liquid medium or in skim milk, the cells are collected and washed as necessary. Cells may be recovered from colonies grown on an agar medium or the like. The obtained cells are directly or after freeze-drying, suspended in a chaotropic reagent solution such as lithium chloride, urea or guanidine hydrochloride, and stirred to solubilize the SLP on the surface of the cells. After removing the solid matter from the solution containing the solubilized SLP, the chaotropic reagent is removed by dialysis or the like to precipitate the SLP. After collecting the precipitated SLP, it can be washed as necessary to obtain the purified SLP used in the present invention. If it is desired to further increase the purity, further purification by chromatography or the like is preferred.
In addition, when obtaining a fraction with a high SLP content instead of purified SLP, it is possible to obtain an SLP fraction with a higher concentration than the cells by crushing the cells and collecting only the insoluble fraction. .
The SLP used in the composition for promoting IgA production of the present invention may be heated since the IgA promoting effect is maintained even when heated.

(Food and drink containing SLP, pharmaceuticals, feed)
The SLP obtained by the above-described production method of the present invention can be used as it is as a raw material for food and drink, and can be produced according to the usual method for each food except that the composition containing SLP is added.
Therefore, an effective amount of SLP of the present invention may be incorporated into any food or drink, and may be added to raw materials during the manufacturing process of the food or drink. Examples of food and drink include cheese, fermented milk, dairy lactic acid drink, lactic acid drink, butter, margarine and other dairy products, milk drinks, fruit juice drinks, soft drinks and other beverages, jellies, candies, puddings, mayonnaise and other eggs. Processed products, sweets and breads such as butter cakes, various milk powders, infant foods, nutritional compositions, etc. may be mentioned, but are not particularly limited.
Food and drink products containing an effective amount of SLP produced in this manner are provided as food and drink products for promoting IgA production.

The SLP obtained by the above production method of the present invention can be used as it is as a raw material for pharmaceuticals, and tablets, capsules, powders, syrups and the like may be produced by standard methods except for the addition of SLP.
Therefore, when formulating a drug containing the SLP of the present invention as an active ingredient, the excipients, stabilizers, corrigents, etc. that are permitted in the formulation are appropriately mixed and formulated, and the SLP is dried as it is. It can also be used as powders and powders. In addition, excipients, binders, disintegrants, lubricants, flavoring agents, suspending agents, coating agents, and other optional agents can be mixed and formulated as long as they do not interfere with the IgA production promoting action. can. Dosage forms include tablets, capsules, granules, powders, powders, syrups, and the like.
A pharmaceutical containing an effective amount of SLP produced in this manner is provided as an IgA production-promoting pharmaceutical.

The SLP obtained by the above production method of the present invention can be used as it is as a raw material for feed, and can be produced by a conventional method for feed except that SLP is added.
Therefore, an effective amount of SLP of the present invention may be blended into any kind of feed as in the case of the above food and drink, and may be added to raw materials during the production process of the feed.
A feed containing an effective amount of SLP produced in this manner is provided as a feed for promoting IgA production.

(Intake)
The intake amount of the composition for promoting IgA production of the present invention is not particularly limited as long as it is an effective amount that can be expected to promote IgA production in humans and animals to be ingested. 0.5 mg/day to 5 mg/day is preferred, 0.7 mg/day to 1.2 mg/day is even more preferred, and 1 mg/day is most preferred.

Subjects for ingestion of the composition for promoting IgA production of the present invention are humans or animals that require promotion of IgA production in vivo. The effect of the present invention can be expected by ingesting it. It is also a human or animal that requires prevention against various infections through IgA production promoting action.
The action of the SLP of the Lactobacillus lactic acid bacterium of the present invention as an active ingredient is an IgA production promoting action, which is different from the antibacterial peptide production promoting action already reported for the SLP. Antibacterial peptides such as defensins act directly on the cell membrane of bacteria and show bactericidal action against bacteria by destroying the cell membrane. On the other hand, IgA can bind not only bacteria but also toxins and the like produced by bacteria, and during infection, it binds to pathogens and pathogenic toxins to prevent them from entering the body.
In addition, IgA works not only to eliminate pathogens, but also to maintain symbiotic relationships between hosts and indigenous bacteria (Kagaku to Biology, 55(9):596-601, 2017). Therefore, it has been suggested that an increase in IgA production improves the intestinal flora (Immunity, 41, 152-165, 2014). Disturbance of the intestinal flora is known to lead to various disorders such as constipation and rough skin, and it is possible to improve such disorders by promoting IgA production and improving the intestinal flora.

(Evaluation method)
The IgA production promoting effect of SLP of the present invention is obtained by comparing the amount of IgA in saliva, intestinal tract, intestinal contents, or feces when SLP is administered to humans or animals and when SLP is not administered. It can be evaluated that the IgA production promoting effect of the present invention is present when the administration is increased.
In addition, in the ex vivo IgA measurement test, for example, immune cells containing IgA-producing cells are seeded on a plate, and the IgA concentration in the medium is compared when SLP is added to the medium and when it is not added. When the IgA concentration is higher with SLP addition than with addition, it can be evaluated that the IgA production promoting effect of the present invention is present.

Examples of the present invention will be described in detail below, but the present invention is not limited to these.
(Preparation example of SLP)
After culturing Lactobacillus helveticus SBT2171 in 100 mL of MRS liquid medium at 37° C. for 16 hours, the cells were collected by centrifugation (8,000×g, 4° C., 10 minutes) and washed once with sterilized MilliQ water. . The obtained cells were suspended in 10 mL of 1 M LiCl solution containing cOmplete ^™ Protease Inhibitor Cocktail (Roche) and stirred at room temperature for 30 minutes. After stirring, the suspension was centrifuged (10,000×g, 4° C., 20 minutes) to collect the supernatant. The precipitate was resuspended in 10 mL of 5 M LiCl solution with cOmplete ^™ Protease Inhibitor Cocktail (Roche) and stirred again for 30 minutes at room temperature. After stirring, the suspension was centrifuged (10,000×g, 4° C., 20 minutes) to collect the supernatant. Collected supernatants were pooled, passed through a 0.2 μm filter, and then dialyzed against sterile MilliQ water on a Slide-A-Lyzer G2 10K (Pierce) to precipitate SLP. The dialyzed solution was centrifuged (12,000×g, 4° C., 20 minutes) to collect the precipitated SLP. The recovered SLP was washed with 1 mL of sterilized MilliQ water, suspended in 500 μL of 1 M LiCl solution, and kept on ice for 15 minutes with appropriate stirring. The LiCl solution was removed by centrifugation, washed again with 1 mL of sterile MilliQ water, resuspended in sterile MilliQ water, and lyophilized to obtain purified SLP.

It was confirmed by the SDS-PAGE method that the purified SLP had a band around the target size of 43 kDa (Fig. 1). Moreover, the obtained SLP was 3 mg.
In addition, SLPs prepared from Lactobacillus acidophilus SBT2062, Lactobacillus brevis SBT10966, Lactobacillus helveticus SBT11380, Lactobacillus helveticus JCM1120T, Lactobacillus amylovorus JCM1126T, and Lactobacillus buchneri JCM1115T in a similar manner are SD S-PAGE A band was observed around the target size of 43-55 kDa by the method (not shown).

[Test Example 1] Examination of IgA production promoting effect of SLP 1. Test method Cryopreserved Human PBMC (Peripheral Blood Mononuclear Cell: Peripheral Blood Mononuclear Cell) (Lot.4661MA20: ASTRATE Biologics) was dissolved in a water bath at 37°C, and FBS (final conc.) was added to RPMI1640 (11875-093: Gibco). .10%) (Lot.42Q3780K: Gibco), MEM Vitamin Solution (final conc.1×) (11120052: Gibco), MEM Non-Essential Amino Acids Solution (final conc.1×) (11140050: G ibco), Penicillin- Streptomycin (final conc. 100 U-100 μg/mL) (15140-122: Life technologies), Sodium Pyruvate (final conc. 1 mM) (11360070: Gibco), StemSure (registered trademark) 2-Mercaptoeth Anol Solution (final cone. 0.05 mM ) (198-15781: Wako) and then seeded in a 96-well plate at 5.0×10 ⁵ cells/100 μL/well. 100 μL/well of a medium containing 20 μg/mL of SLP purified from each lactic acid bacteria strain was added to the seeded cells (final conc. 10 μg/mL) and cultured at 37° C. for 7 days in a 5% CO ₂ incubator. After culturing, the medium was collected from the 96-well plate, and the cells were removed by centrifugation (1,500×g, 4° C., 5 minutes) to obtain a sample for IgA concentration measurement. Control (Negative Control) was defined as the level without addition of the sample, and Positive Control was defined as the level with addition of Recombinant Human IL-6 (AF-200-06: Peprotech) at a concentration of 10 ng/mL. The test was performed with n=6.

IgA concentration was measured by ELISA method. AffiniPure Goat Anti-Human Serum IgA, α Chain Specific (Jackson Immuno Research Laboratories) diluted with 0.05 M Carbonate-Bicarbonate Buffer (pH 9.6) (C3041: Sigma) 20 μg / mL, 96 well ELISA plate (Corning ( 50 μL/well was dispensed into a 96-well EIA/RIA plate (registered trademark) (3590: Corning) and allowed to stand at 4° C. overnight. The liquid was removed from the plate after standing, and the plate was washed four times with 300 μL/well of wash buffer (50 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 0.05% Tween 20). After washing, 300 μL/well of blocking buffer (50 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 1% BSA) was added and allowed to stand at room temperature for 1 hour. The liquid was removed from the plate after standing, and the plate was washed 4 times with 300 μL/well of wash buffer. After washing, 50 μL/well of a sample diluted 10-fold with diluent (50 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 1% BSA, 0.05% Tween 20) was added and allowed to stand at room temperature for 2 hours. The liquid was removed from the plate after standing, and the plate was washed 4 times with 300 μL/well of wash buffer. After cleaning, DILOXIDASE -AFFINIPUAT ANTI -HUMAN SERUM IGA, αCHAIN SPECIFIC (JACKSON IMMUNO RESEARCHL) Aboratories) 50 μL / WELL was added and kept at room temperature for 2 hours. The liquid was removed from the plate after standing, and the plate was washed 4 times with 300 μL/well of wash buffer. After washing, 100 μL/well of eBioscience (TM) TMB Solution (00-4201-56: Invitrogen) was added and allowed to stand until sufficient color development was observed at room temperature. ) was used to measure the absorbance (OD450) at 450 nm. The measurement results were corrected by subtracting the OD620 value from the OD450 value. Two wells were measured per sample, and the average of the values of the two wells was adopted as the measured value. Also, a calibration curve was prepared using IgA from human serum as a standard, and the IgA concentration was calculated from the measured values of each sample.

2. Test Results As a result of the evaluation, a significant increase in IgA concentration was observed in most of the levels to which SLP derived from Lactobacillus lactic acid bacteria was added at a concentration of 10 μg/mL compared to the Control level (FIG. 2). Compared to the Control level (14.5 ng/mL), the addition level of Lactobacillus acidophilus SBT2062 strain (54.2 ng/mL) is about 3.7 times, and the addition level of Lactobacillus brevis SBT10966 strain (58.8 ng/mL) ) is about 4.1 times, Lactobacillus helveticus SBT11380 strain addition level (46.1 ng / mL) is about 3.2 times, Lactobacillus helveticus SBT2171 strain addition level (36.7 ng / mL) is about 2.5 times about 2.7 times at the addition level of Lactobacillus helveticus JCM1120T strain (39.0 ng/mL), about 3.3 times at the addition level of Lactobacillus amylovorus JCM1126T strain (47.1 ng/mL), and Lactobacillus buchneri At the JCM1115T strain addition level (54.8 ng/mL), an approximately 3.8-fold higher IgA concentration was observed. From this result, it was considered that SLP derived from Lactobacillus lactic acid bacteria exhibits an IgA production promoting effect. In the figure, the symbols *, **, and *** indicate significant differences from the control level (*: P<0.05, **: P<0.01, ***: P<0. 001).

(Production example of supplement)
To 10 mg of SLP purified from Lactobacillus helveticus SBT2171, 30 g of skimmed milk powder, 40 g of an equal mixture of vitamin C and citric acid, 100 g of granulated sugar, and 60 g of an equal mixture of cornstarch and lactose were added and mixed. The mixture was packed in a stick-like bag to produce the supplement for promoting IgA production of the present invention.

(Example of production of feed)
2 g of SLP purified from Lactobacillus helveticus SBT2171 was suspended in 3998 g of deionized water, heated to 40° C., and stirred at 3,600 rpm for 20 minutes with a TK homomixer (MARK II 160 type; manufactured by Tokushu Kika Kogyo Co., Ltd.). A 2 g/4 kg SLP solution was obtained by mixing. To 2 kg of this SLP solution, 1 kg of soybean meal, 1 kg of skimmed milk powder, 0.4 kg of soybean oil, 0.2 kg of corn oil, 2.3 kg of palm oil, 1 kg of corn starch, 0.9 kg of wheat flour, 0.2 kg of bran, and 0.5 kg of vitamin mixture. , 0.3 kg of cellulose, and 0.2 kg of a mineral mixture, and heat-sterilized at 120° C. for 4 minutes to produce 10 kg of the feed for promoting IgA production of the present invention.

(Example of pharmaceutical production)
To 10 mg of SLP purified from Lactobacillus helveticus SBT2171, 40 g of skimmed milk powder was added and mixed. 1 part of this mixture was mixed with 4 parts of powdered skim milk, and 1 g of this mixed powder was tableted by a tableting machine in a conventional manner to prepare tablets for promoting IgA production of the present invention.

According to the present invention, a new IgA production-promoting composition containing SLP derived from Lactobacillus lactic acid bacteria as an active ingredient, and an IgA production-promoting food, drink, and pharmaceutical product containing SLP derived from Lactobacillus lactic acid bacteria as an active ingredient , it became possible to provide feed.

Claims (6)

A composition for promoting IgA production, comprising as an active ingredient an S-layer protein, which is a component derived from a bacterium belonging to the genus Lactobacillus. The lactic acid bacteria of the genus Lactobacillus include Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus buchneri, and Lactobacillus brevis. illus brevis), or Lactobacillus helveticus helveticus). 3. The composition for promoting IgA production according to claim 1 or 2, wherein the S-layer protein is purified. Food and drink for promoting IgA production, comprising the composition for promoting IgA production according to any one of claims 1 to 3. A pharmaceutical product for promoting IgA production, comprising the composition for promoting IgA production according to any one of claims 1 to 3. A feed for promoting IgA production comprising the composition for promoting IgA production according to any one of claims 1 to 3.

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