Composition for promoting IgA production

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]

[0001] The present invention relates to a composition for promoting IgA production, and in particular to a composition for promoting IgA production that contains an S-layer protein, which is a component derived from the cells of Lactobacillus lactic acid bacteria. [Background technology]

[0002] IgA is one of the major antibodies that function in innate immunity. Secretory IgA (SIgA), in particular, plays a key role in mucosal immunity, functioning as the front line of the immune system in the gastrointestinal and respiratory tracts. In the intestinal tract, SIgA is secreted from plasma cells in the muscularis mucosae, penetrates the epithelium, and is secreted onto the epithelial surface, where it captures antigens and pathogenic bacteria, preventing them from adhering to the epithelium. For these reasons, IgA is expected to play a role in the body's defense against infection, and several solutions for increasing IgA production have been disclosed.

[0003] Patent Document 1 aims to provide new lactic acid bacteria that are useful as probiotics and have excellent mucosal immune stimulating effects and improve the body's defense mechanisms, as well as final products containing them (food and beverage products such as fermented milk and lactic acid bacteria drinks).As a solution to this problem, it discloses at least one lactic acid bacterium selected from the group consisting of Lactobacillus plantarum ONRIC b0239 and Lactobacillus plantarum ONRIC b0240, which have the ability to promote IgA production.

[0004] Patent Document 2 aims to provide an intestinal immunity enhancer containing as an active ingredient a component derived from lactic acid bacteria that has a high intestinal immunity enhancing effect, and as a means of solving this problem, it discloses an intestinal immunity enhancer containing as an active ingredient a cell wall component of Lactobacillus plantarum that has the effect of improving IgA antibody production.

[0005] Patent Document 3 aims to provide pharmaceuticals, nutritional compositions, foods, beverages, and feeds that can prevent both infectious diseases and autoimmune diseases by promoting the production of IgA antibodies and suppressing the production of IgG antibodies, which are autoantibodies, and discloses lactic acid bacteria belonging to the lactic acid bacterium Lactobacillus helveticus, particularly the Lactobacillus helveticus SBT2171 strain, as a means of solving this problem.

[0006] Patent Document 4 aims to provide a novel material that activates JNK, and as a means of solving this problem, it discloses a JNK-activating composition containing, as an active ingredient, S-layer protein (hereinafter sometimes referred to as SLP), a component derived from Lactobacillus bacteria, or a hydrolysate of the SLP, as well as foods and drinks for JNK activation, antioxidant foods and drinks, and cell defense foods and drinks containing the same. Patent Document 5 aims to provide a novel material that increases the production of antimicrobial peptides from epithelial cells and has an infection-preventing effect, and as a means of solving this problem, it discloses a composition for promoting antimicrobial peptide production, containing, as an active ingredient, SLP, a component derived from Lactobacillus bacteria, or a hydrolysate of the SLP, as well as foods and drinks for promoting antimicrobial peptide production and infection-preventing foods and drinks containing the same.

[0007] However, since the Lactobacillus plantarum described in Patent Documents 1 and 2 does not contain SLP, it is clear that the IgA antibody production promoting effect described in these documents is an effect of the Lactobacillus plantarum cells or cell wall components as active ingredients, and the IgA antibody production promoting effect of SLP is not disclosed. Furthermore, the IgA antibody production-promoting effect and IgG antibody production-inhibiting effect described in Patent Document 3 are the effects of using Lactobacillus helveticus cells or a culture of cells as the active ingredient, and the IgA antibody production-promoting effect of SLP is not disclosed. Furthermore, Patent Documents 4 and 5 do not disclose the IgA production promoting activity of SLPs 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. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 3818319 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-125446 [Patent Document 3] Patent No. 6739155 [Patent Document 4] Japanese Patent Publication No. 2020-152653 [Patent Document 5] International Publication No. 2020 / 111172 Brochure Summary of the Invention [Problem to be solved by the invention]

[0009] 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. [Means for solving the problem]

[0010] In order to solve the above problems, the present invention includes the following configurations. [1] A composition for promoting IgA production, characterized by containing, as an active ingredient, an S-layer protein, which is a component derived from bacteria of the genus Lactobacillus. [2] The composition for promoting IgA production according to [1], wherein the lactic acid bacterium of the genus Lactobacillus is Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus buchneri, Lactobacillus brevis, or Lactobacillus helveticus. [3] The composition for promoting IgA production according to [1] or [2], wherein the S-layer protein is purified. [4] A food or drink for promoting IgA production, comprising the composition for promoting IgA production according to any one of [1] to [3]. [5] A pharmaceutical 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]. [Effects of the Invention]

[0011] By ingesting materials that induce IgA production, such as S-layer proteins (hereinafter simply referred to as SLPs) derived from Lactobacillus lactic acid bacteria, or Lactobacillus lactic acid bacteria containing SLPs and their cultures, it is expected that the body's defenses against various infections will be improved through the promotion of IgA production. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a photograph showing the results of SDS-PAGE of purified SLPs of Lactobacillus helveticus. [Figure 2] 1 is a graph showing the IgA concentration in the culture supernatant when SLPs were added to PBMCs (peripheral blood mononuclear cells) and the cells were cultured. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention provides a novel composition for promoting IgA production. The composition for promoting IgA production of the present invention will be described in detail below. (Composition for promoting IgA production) The S-layer proteins of the present invention are found in the outer layer of the cell walls of various bacteria and are characterized by their regular crystalline structure. Because SLPs are noncovalently bound to cell wall components, they can be extracted from bacterial cells using chaotropic agents such as lithium chloride. Furthermore, the extracted SLPs re-form their regular crystalline structure upon removal of the chaotropic agent. This characteristic allows for their purification from bacterial cells. Among lactic acid bacteria, the Lactobacillus genus has been found to express S-layer proteins. Mukai et al. (Japanese Journal of Lactic Acid Bacteria, 19(1):21-29, 2008) reported that SLP expression was confirmed or predicted in 13 species, including Lactobacillus acidophilus. The SLPs expressed by these lactic acid bacteria share common features, such as being basic proteins with an isoelectric point of 9-10 and possessing a signal peptide sequence of 23-30 amino acid residues at the N-terminus. However, the molecular weight of SLPs produced by lactic acid bacteria varies greatly between species, and their genetic structure has been shown to vary significantly even within the same species. The SLP used in the composition for promoting IgA production of the present invention is derived from the genus Lactobacillus, and any bacterial species that expresses an S-layer and has the relevant effect can be used. Bacterial species that express SLP or SLP-like proteins include Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus kitasatonis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, and Lactobacillus helveticus. Examples of the bacterium include Lactobacillus helveticus, Lactobacillus kefiri, and Lactobacillus parakefiri, and among these, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus buchneri, Lactobacillus brevis, and Lactobacillus helveticus are more preferred. The SLPs of the present invention do not necessarily have to be purified from bacterial cells, and bacterial cells themselves or cultures containing SLPs can be used, but purified SLPs are more preferred. The bacterial cells themselves may be live or killed.

[0014] (Method of producing a 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. SLP can be purified using various known purification methods, and a typical method is shown below. After thoroughly culturing the target Lactobacillus lactic acid bacteria in a liquid medium such as MRS liquid medium or skim milk, the cells are collected and washed as necessary. The cells may also be collected from colonies grown on an agar medium or the like. The obtained cells, either directly or after lyophilization, are suspended and stirred in a chaotropic reagent solution such as lithium chloride, urea, or guanidine hydrochloride to solubilize the SLP on the cell surface. After removing solids from the solution containing the solubilized SLP, the chaotropic reagent is removed by dialysis or the like, and the SLP is precipitated. The precipitated SLP is collected and washed as necessary to obtain the purified SLP used in the present invention. For further purity, further purification by chromatography or the like is preferred. If one wishes to obtain a fraction with a high SLP content rather than purified SLP, the cells can be disrupted and only the insoluble fraction collected to obtain an SLP fraction with a higher concentration than the cells. 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.

[0015] (Food, beverages, medicines, and feed containing SLP) The SLP obtained by the above-mentioned production method of the present invention can be used as a raw material or ingredient for food and drink products as it is, and can be produced according to the standard method for each food product, except for adding a composition containing SLP. Therefore, an effective amount of SLP of the present invention may be incorporated into any food or beverage, or may be added to raw materials during the manufacturing process of the food or beverage. Examples of foods and beverages include, but are not limited to, dairy products such as cheese, fermented milk, dairy lactic acid bacteria drinks, lactic acid bacteria drinks, butter, margarine, etc., milk drinks, fruit juice drinks, soft drinks, etc., egg products such as jelly, candy, pudding, mayonnaise, etc., sweets and breads such as butter cake, various types of powdered milk, infant foods, nutritional compositions, etc. The food and drink containing an effective amount of SLP produced in this manner is provided as a food and drink for promoting IgA production.

[0016] The SLP obtained by the above-described production method of the present invention can be used as a raw material for pharmaceuticals as it is, and tablets, capsules, powders, syrups, etc. can be produced by conventional methods except for adding the SLP. Therefore, when formulating a pharmaceutical containing the SLP of the present invention as an active ingredient, it can be formulated by appropriately mixing it with excipients, stabilizers, flavoring agents, etc. that are approved for pharmaceutical use, or it can be dried directly and used as a powder or sachets. Furthermore, it can also be formulated by mixing it with excipients, binders, disintegrants, lubricants, flavoring agents, suspending agents, coating agents, and any other drugs within a range that does not interfere with the IgA production-promoting effect. Possible dosage forms include tablets, capsules, granules, powders, dusts, and syrups. A pharmaceutical containing an effective amount of SLP produced in this manner is provided as a pharmaceutical for promoting IgA production.

[0017] The SLP obtained by the above-described production method of the present invention can be used as a raw material for feed as it is, and the feed may be produced according to a standard method for producing feed, except for adding the SLP. Therefore, an effective amount of SLP of the present invention may be blended into any feed, similar to the above-mentioned foods and drinks, or may be added to the raw materials during the production process of the feed. The feed containing an effective amount of SLP produced in this manner is provided as a feed for promoting IgA production.

[0018] (Intake amount) 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 or animals that take it, but a rough estimate would be 0.1 mg / day to 10 mg / day of SLP, with 0.5 mg / day to 5 mg / day being preferred, 0.7 mg / day to 1.2 mg / day being even more preferred, and 1 mg / day being most preferred.

[0019] The subjects for taking the composition for promoting IgA production of the present invention are humans or animals in need of enhanced IgA production in the body, and for example, the effects of the present invention can be expected by taking the composition in subjects whose IgA production is lower than the reference value as determined by the following evaluation method. Also, the subject is a human or animal in need of protection against various infections through the promotion of IgA production. The effect of the SLPs of the Lactobacillus lactic acid bacteria of the present invention as active ingredients is to promote IgA production, which is different from the antimicrobial peptide production promotion effect that has already been reported for the SLPs. Antimicrobial peptides such as defensins act directly on bacterial cell membranes, destroying them and thereby exerting a bactericidal effect on bacteria. On the other hand, IgA can bind not only to bacteria but also to toxins produced by bacteria, and during infection, it binds to pathogens and pathogenic toxins to prevent them from entering the body. In addition, IgA not only works to eliminate pathogens, but also to maintain the symbiotic relationship between the host and resident bacteria (Chemistry and Biology, 55(9):596-601, 2017). For this reason, it has been suggested that increasing IgA production improves the intestinal flora (Immunity, 41,152-165, 2014). Disturbances in the intestinal flora are known to lead to various ailments such as constipation and rough skin, and promoting IgA production and improving the intestinal flora can improve these ailments.

[0020] (Evaluation method) The IgA production-promoting effect of the SLPs of the present invention can be evaluated by comparing the amount of IgA in the saliva, intestinal tract, intestinal contents, or feces of humans or animals when SLPs are administered to them and when they are not, and if the amount of IgA is higher when SLPs are administered than when they are not administered, the IgA production-promoting effect of the present invention can be evaluated. Furthermore, in an ex vivo IgA measurement test, for example, immune cells containing IgA-producing cells are plated on a plate, and the IgA concentration in the medium is compared between when SLP is added to the medium and when it is not added. If the IgA concentration is higher when SLP is added than when it is not added, the IgA production-promoting effect of the present invention can be evaluated. [Example]

[0021] Examples of the present invention will be described in detail below, but the present invention is not limited to these examples. (Example of SLP preparation) Lactobacillus helveticus SBT2171 was cultured in 100 mL of MRS liquid medium at 37°C for 16 hours, and then the cells were collected by centrifugation (8,000 × g, 4°C, 10 minutes) and washed once with sterilized MilliQ water. TM The mixture was suspended in 10 mL of 1 M LiCl solution containing Protease Inhibitor Cocktail (Roche) and stirred at room temperature for 30 minutes. The suspension was centrifuged (10,000 × g, 4 °C, 20 minutes) and the supernatant was collected. The precipitate was then collected by centrifuging at 10,000 × g for 20 minutes at 4 °C. TM The cells were resuspended in 10 mL of 5 M LiCl solution containing Protease Inhibitor Cocktail (Roche) and stirred again at room temperature for 30 minutes. The stirred suspension was centrifuged (10,000 × g, 4°C, 20 minutes), and the supernatant was collected. The collected supernatants were combined, passed through a 0.2 μm filter, and then dialyzed against sterile MilliQ water using a Slide-A-Lyzer G2 10K (Pierce) to precipitate SLPs. The dialyzed solution was centrifuged (12,000 × g, 4°C, 20 minutes), and the precipitated SLPs were collected. The recovered SLPs were washed with 1 mL of sterile 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 SLPs.

[0022] After purification, SDS-PAGE confirmed that a band of the desired size, approximately 43 kDa, was observed (Figure 1). The yield of SLP obtained was 3 mg. Furthermore, SLPs prepared by a similar method from Lactobacillus acidophilus SBT2062, Lactobacillus brevis SBT10966, Lactobacillus helveticus SBT11380, Lactobacillus helveticus JCM1120T, Lactobacillus amylovorus JCM1126T, and Lactobacillus buchneri JCM1115T showed bands of the desired size of 43 to 55 kDa by SDS-PAGE (not shown).

[0023] [Test Example 1] Examination of the IgA production promoting effect of SLP 1. Test Method Cryopreserved human PBMCs (peripheral blood mononuclear cells) (Lot. 4661MA20: ASTRATE Biologics) were thawed in a water bath at 37°C and resuspended in RPMI 1640 (11875-093: Gibco) supplemented with FBS (final conc. 10%) (Lot. 42Q3780K: Gibco), MEM Vitamin Solution (final conc. 1x) (11120052: Gibco), MEM Non-Essential Amino Acids Solution (final conc. 1x) (11140050: Gibco), Penicillin-Streptomycin (final conc. 100U-100μg / mL) (15140-122: Life Technologies), and Sodium Pyruvate (final conc. 100μg / mL) (15140-122: Life Technologies). After washing with a medium supplemented with StemSure® 2-Mercaptoethanol Solution (final concentration 0.05 mM) (198-15781: Wako), 5.0 × 10 5Cells were seeded onto a 96-well plate at 100 μL / well. Culture medium containing 20 μg / mL of SLP purified from each lactic acid bacteria strain was added to the seeded cells at 100 μL / well (final concentration: 10 μg / mL), and the cells were cultured at 37°C in a 5% CO2 incubator for 7 days. After culture, the medium was collected from the 96-well plate, and the cells were removed by centrifugation (1,500 × g, 4°C, 5 minutes) to prepare a sample for IgA concentration measurement. A control without sample was used as a negative control, and a control with recombinant human IL-6 (AF-200-06: Peprotech) added at a concentration of 10 ng / mL was used as a positive control. The test was performed with n=6.

[0024] IgA concentrations were measured by ELISA. AffiniPure Goat Anti-Human Serum IgA, α Chain Specific (Jackson Immuno Research Laboratories) was diluted with 0.05M Carbonate-Bicarbonate Buffer (pH 9.6) (C3041: Sigma) to 20 μg / mL, and 50 μL / well of the solution was dispensed into a 96-well ELISA plate (Corning® 96-well EIA / RIA plate) (3590: Corning) and allowed to stand overnight at 4°C. After incubation, the plate was drained and 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 the plate was incubated at room temperature for 1 hour. The liquid was then removed from the plate, and the plate was washed four times with 300 μL / well of wash buffer. After washing, 50 μL / well of 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 the plate was incubated at room temperature for 2 hours. The liquid was then removed from the plate, and the plate was washed four times with 300 μL / well of wash buffer. After washing, 50 μL / well of Peroxidase-AffiniPure Goat Anti-Human Serum IgA, α Chain Specific (Jackson Immuno Research Laboratories) diluted to 0.16 μg / mL with diluent was added and the plate was incubated at room temperature for 2 hours. After standing, the liquid was removed from the plate, and the plate was washed four times with 300 μL / well of wash buffer.After washing, 100 μL / well of eBioscience™ TMB Solution (00-4201-56: Invitrogen) was added and the plate was left to stand at room temperature until sufficient color development was observed. 100 μL / well of 1N HCl was then added, and the absorbance at 450 nm (OD450) was measured using a VARIOSKAN FLASH (Thermo Scientific). Measurement results were corrected by subtracting the OD620 value from the OD450 value. Measurements were performed in two wells per sample, and the average of the two well values ​​was used as the measured value. In addition, a calibration curve was created using IgA from human serum as a standard, and the IgA concentration was calculated from the measured values ​​for each sample.

[0025] 2. Test Results As a result of the evaluation, a significant increase in IgA concentration was observed at most levels where SLP derived from Lactobacillus lactic acid bacteria was added at a concentration of 10 μg / mL compared to the control level (Figure 2). Compared to the control level (14.5 ng / mL), the Lactobacillus acidophilus SBT2062 added level (54.2 ng / mL) increased IgA levels by approximately 3.7 times, the Lactobacillus brevis SBT10966 added level (58.8 ng / mL) increased IgA levels by approximately 4.1 times, the Lactobacillus helveticus SBT11380 added level (46.1 ng / mL) increased IgA levels by approximately 3.2 times, and the Lactobacillus helveticus SBT11380 added level (46.1 ng / mL) increased IgA levels by approximately 3.2 times. The IgA concentration increased approximately 2.5-fold with the addition of BT2171 strain (36.7 ng / mL), approximately 2.7-fold with the addition of Lactobacillus helveticus JCM1120T strain (39.0 ng / mL), approximately 3.3-fold with the addition of Lactobacillus amylovorus JCM1126T strain (47.1 ng / mL), and approximately 3.8-fold with the addition of Lactobacillus buchneri JCM1115T strain (54.8 ng / mL). These results suggest that SLP derived from Lactobacillus lactic acid bacteria promotes IgA production. In the figure, *, **, and *** indicate significant differences from the control level (*: P<0.05, **: P<0.01, ***: P<0.001).

[0026] (Example of supplement manufacturing) 10 mg of SLP purified from Lactobacillus helveticus SBT2171 was mixed with 30 g of skim 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. The mixture was packed into a stick-shaped bag to produce the supplement for promoting IgA production of the present invention.

[0027] (Example of feed production) Two grams of purified SLP from Lactobacillus helveticus SBT2171 was suspended in 3998 g of deionized water and heated to 40°C. The suspension was then mixed in a TK homogenizer (Model MARK II 160; Tokushu Kika Kogyo Co., Ltd.) at 3,600 rpm for 20 minutes to obtain a 2 g / 4 kg SLP solution. This SLP solution (2 kg) was then combined with 1 kg soybean meal, 1 kg skim milk powder, 0.4 kg soybean oil, 0.2 kg corn oil, 2.3 kg palm oil, 1 kg corn starch, 0.9 kg wheat flour, 0.2 kg bran, 0.5 kg vitamin mixture, 0.3 kg cellulose, and 0.2 kg mineral mixture. The mixture was then sterilized at 120°C for 4 minutes to produce 10 kg of the feed for promoting IgA production.

[0028] (Example of pharmaceutical manufacturing) 10 mg of SLP purified from Lactobacillus helveticus SBT2171 was mixed with 40 g of skim milk powder. One part of this mixture was mixed with 4 parts of skim milk powder, and the mixed powder was compressed into 1 g tablets using a tablet press in a conventional manner to prepare the tablets for promoting IgA production of the present invention. [Industrial Applicability]

[0029] According to the present invention, it is now possible to provide a new composition for promoting IgA production, which contains SLP derived from Lactobacillus lactic acid bacteria as an active ingredient, as well as foods, beverages, pharmaceuticals, and feed for promoting IgA production, which contain SLP derived from Lactobacillus lactic acid bacteria as an active ingredient.

Claims

1. A composition for promoting IgA production, comprising an S-layer protein, which is a component derived from Lactobacillus bacteria, as an active ingredient, The composition for promoting IgA production, wherein the lactic acid bacterium of the genus Lactobacillus is Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus buchneri, or Lactobacillus brevis.

2. 2. The composition for promoting IgA production according to claim 1, wherein the S-layer protein is purified.

3. A food or drink for promoting IgA production, comprising the composition for promoting IgA production according to claim 1 or 2.

4. A pharmaceutical for promoting IgA production, comprising the composition for promoting IgA production according to claim 1 or 2.

5. A feed for promoting IgA production, comprising the composition for promoting IgA production according to claim 1 or 2.

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