JP7798564B2 - Composition for promoting IgA production - Google Patents

Description

The present invention relates to a composition for promoting IgA production. In particular, the present invention relates 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.

IgA is one of the main 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 digestive 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.

Patent Document 1 aims to provide new lactic acid bacteria useful as probiotics that exhibit excellent mucosal immunostimulatory effects and enhance the body's defense mechanisms, as well as final products containing the bacteria (food and beverage products such as fermented milk and lactic acid bacteria drinks). As a means to achieve this goal, 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.

Patent Document 2 addresses the issue of providing an intestinal immunity enhancer that contains, as an active ingredient, a component derived from lactic acid bacteria that has a strong intestinal immunity enhancing effect, and discloses, as a means of resolving this issue, an intestinal immunity enhancer that contains, as an active ingredient, a cell wall component of Lactobacillus plantarum that has the effect of improving IgA antibody production.

Patent Document 3 addresses the issue of providing 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. It discloses lactic acid bacteria belonging to the genus Lactobacillus helveticus, particularly the Lactobacillus helveticus SBT2171 strain, as a means of resolving this issue.

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 said SLP, as well as foods and beverages for JNK activation, antioxidant foods and beverages, and cell defense foods and beverages containing the same. Patent Document 5 aims to provide a novel material that increases the production of antimicrobial peptides from epithelial cells and has infection prevention effects, 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 said SLP, as well as foods and beverages for promoting antimicrobial peptide production and infection prevention containing the same.

However, since the Lactobacillus plantarum described in Patent Documents 1 and 2 does not have SLP, it is clear that the IgA antibody production promoting action described in these documents is an action using the bacterial body or cell wall components of Lactobacillus plantarum as active ingredients, and the IgA antibody production promoting action of SLP is not disclosed.
Furthermore, the IgA antibody production-promoting effect and IgG antibody production-inhibiting effect described in Patent Document 3 are effects of 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.

Patent No. 3818319 JP 2014-125446 A Patent No. 6739155 Japanese Patent Application Laid-Open No. 2020-152653 International Publication No. 2020/111172 Brochure

The objective of the present invention is to provide a novel material that promotes IgA production from immune cells and has infection prevention effects.

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 Lactobacillus bacteria.
[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].

By ingesting materials that induce IgA production, such as S-layer protein (hereinafter simply referred to as SLP) derived from Lactobacillus lactic acid bacteria, or Lactobacillus lactic acid bacteria cells containing SLP and their cultures, it is expected that the body's defenses against various infections will be improved by promoting IgA production.

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

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 protein of the present invention is found in the outer layer of the cell wall of various bacteria and is characterized by its regular crystalline structure. Since SLP is non-covalently bound to cell wall components, it can be extracted from bacterial cells using a chaotropic agent such as lithium chloride. Furthermore, the extracted SLP reforms its regular crystalline structure upon removal of the chaotropic agent. Utilizing this characteristic, it can be purified from bacterial cells.
Among lactic acid bacteria, the S-layer has been found to be expressed in the Lactobacillus genus, and a report by Mukai et al. (Japanese Journal of Lactic Acid Bacteria, 19(1):21-29, 2008) confirmed or predicted the expression of SLP in 13 bacterial species, including Lactobacillus acidophilus group lactic acid bacteria. The SLPs expressed by these lactic acid bacteria share common features, such as being basic proteins with an isoelectric point of 9-10 and having a signal peptide sequence consisting of 23-30 amino acid residues in the N-terminal region. On the other hand, the molecular weight of SLP produced by lactic acid bacteria varies greatly between bacterial species, and its gene structure has also been shown to be 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 any bacterial species that expresses the 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, and Lactobacillus johnsonii. johnsonii), Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus kefiri, Lactobacillus parakeefiri, Lactobacillus Among these, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus buchneri, Lactobacillus brevis, or Lactobacillus helveticus is more preferred.
Furthermore, the SLP of the present invention does not necessarily have to be purified from bacterial cells, and bacterial cells themselves or a culture containing SLP can be used, but purified SLP is more preferred. The bacterial cells themselves may be live or dead.

(Method for 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. The target Lactobacillus lactic acid bacteria are thoroughly cultured in a liquid medium such as MRS liquid medium or skim milk, and then 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. If further purity is desired, further purification by chromatography or the like is preferred.
If a fraction with a high SLP content is to be obtained 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 activity is maintained even when heated.

(Food, beverages, medicines, and feed containing SLP)
The SLP obtained by the above-mentioned production method of the present invention can be used as it is as a material or ingredient for food and drink, 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 drink, or may be added to raw materials during the manufacturing process of the food or drink. Examples of food and drink 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., confectioneries and breads such as butter cake, various types of milk powder, 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.

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 excipients, stabilizers, flavoring agents, etc. that are approved for pharmaceutical use, or it can be dried as is and used as a powder or sachets. Furthermore, it can also be formulated by mixing 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, syrups, etc.
A pharmaceutical containing an effective amount of SLP thus produced can be provided as a pharmaceutical for promoting IgA production.

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 the addition of 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 thus produced is provided as a feed for promoting IgA production.

(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 roughly speaking, the amount is 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.

The subjects for taking the composition for promoting IgA production of the present invention are humans or animals in need of promoting 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 level 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 SLP of the Lactobacillus lactic acid bacteria of the present invention as an active ingredient is to promote IgA production, which is different from the antimicrobial peptide production promotion effect that has already been reported for the SLP. 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 maintains the symbiotic relationship between the host and resident bacteria (Chemistry and Biology, 55(9):596-601, 2017). Therefore, 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.

(Evaluation method)
The IgA production-promoting effect of the SLP of the present invention can be evaluated by comparing the amount of IgA in the saliva, intestinal tract, intestinal contents, or feces when SLP is administered to humans or animals with and without administration, and if the amount of IgA is higher when SLP is administered than when it is 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, it can be evaluated that the present invention has an IgA production-promoting effect.

Examples of the present invention will be described in detail below, but the present invention is not limited to these examples.
(Example of preparation of SLP)
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. 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. The stirred suspension was centrifuged (10,000 × g, 4 ° C, 20 minutes) and the supernatant was collected. The precipitate was resuspended in 10 mL of 5 M LiCl solution containing cOmplete ^™ Protease Inhibitor Cocktail (Roche) and stirred again at room temperature for 30 minutes. After stirring, the 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 sterilized MilliQ water using a Slide-A-Lyzer G2 10K (Pierce) to precipitate SLP. The dialyzed solution was centrifuged (12,000 × g, 4 ° C, 20 minutes), and the precipitated SLP was collected. The collected 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 sterilized MilliQ water, resuspended in sterilized MilliQ water, and lyophilized to obtain purified SLP.

It was confirmed that the purified SLP gave a band of approximately 43 kDa, the target size, by SDS-PAGE (Figure 1). The yield of the purified SLP 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 at the target size of 43 to 55 kDa by SDS-PAGE (not shown).

[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 the cells were diluted in RPMI 1640 (11875-093: Gibco) supplemented with FBS (final conc. 10%) (Lot. 42Q3780K: Gibco), MEM Vitamin Solution (final conc. 1x) (11120052: Gibco), and MEM Non-Essential Amino Acids Solution (final conc. 1x) (11120052: Gibco). After washing with medium supplemented with 1× (11140050: Gibco), Penicillin-Streptomycin (final conc. 1×) (15140-122: Life technologies), Sodium Pyruvate (final conc. 1 mM) (11360070: Gibco), and StemSure® 2-Mercaptoethanol Solution (final conc. 0.05 mM) (198-15781: Wako), the cells were seeded onto a 96-well plate at 5.0×10 ⁵ cells/100 μL/well. 100 μL/well of 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 for 7 days at 37 ° C in a 5% CO ₂ incubator. 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 level without sample addition was used as a control (negative control), and a level 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.

IgA concentrations were measured using ELISA. AffiniPure Goat Anti-Human Serum IgA, α Chain Specific (Jackson ImmunoResearch Laboratories) was diluted with 0.05M Carbonate-Bicarbonate Buffer (pH 9.6) (C3041: Sigma) to 20 μg/mL, and dispensed at 50 μL/well into a 96-well ELISA plate (Corning® 96-well EIA/RIA plate) (3590: Corning) and left overnight at 4°C. After standing, the liquid was removed from the plate, 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 the plate was left to stand at room temperature for 1 hour. 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, 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. 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, 50 μL/well of Peroxidase-AffiniPure Goat Anti-Human Serum IgA, α Chain Specific (Jackson ImmunoResearch Laboratories) adjusted to 0.16 μg/mL with Diluent was added, and the plate was allowed to stand at room temperature for 2 hours. The liquid was removed from the plate after standing, 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). The 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 wells 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.

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 strain added level (54.2 ng/mL) was approximately 3.7 times higher, the Lactobacillus brevis SBT10966 strain added level (58.8 ng/mL) was approximately 4.1 times higher, the Lactobacillus helveticus SBT11380 strain added level (46.1 ng/mL) was approximately 3.2 times higher, and the Lactobacillus helveticus SBT11380 strain added level (46.1 ng/mL) was approximately 3.2 times higher. At the BT2171 strain addition level (36.7ng/mL), the IgA concentration was approximately 2.5 times higher, at the Lactobacillus helveticus JCM1120T strain addition level (39.0ng/mL), the IgA concentration was approximately 2.7 times higher, at the Lactobacillus amylovorus JCM1126T strain addition level (47.1ng/mL), the IgA concentration was approximately 3.3 times higher, and at the Lactobacillus buchneri JCM1115T strain addition level (54.8ng/mL), the IgA concentration was approximately 3.8 times higher. From these results, it was considered that SLP derived from Lactobacillus lactic acid bacteria exhibits an IgA production promoting effect. In the figure, *, **, *** symbols indicate that there is a significant difference (*: P<0.05, **: P<0.01, ***: P<0.001) between the control level and the SLP.

(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.

(Example of feed production)
SLP2g purified from Lactobacillus helveticus SBT2171 was suspended in 3998g of deionized water, heated to 40 ℃, then TK homomixer (MARK II 160 type; manufactured by Tokushu Kika Kogyo Co., Ltd.), stirred and mixed at 3,600 rpm for 20 minutes to obtain a 2g/4kg SLP solution. This SLP solution 2kg was mixed with soybean meal 1kg, skim milk powder 1kg, soybean oil 0.4kg, corn oil 0.2kg, palm oil 2.3kg, corn starch 1kg, wheat flour 0.9kg, bran 0.2kg, vitamin mixture 0.5kg, cellulose 0.3kg, mineral mixture 0.2kg, 120 ℃, 4 minutes of heat sterilization, to produce 10kg of the feed for promoting IgA production of the present invention.

(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 in a tablet press by a conventional method to prepare tablets for promoting IgA production of the present invention.

The present invention makes it 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 (5)

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. The composition for promoting IgA production according to claim 1, wherein the S-layer protein is purified. A food or beverage for promoting IgA production, comprising the composition for promoting IgA production described in claim 1 or claim 2. A pharmaceutical for promoting IgA production, comprising the composition for promoting IgA production described in claim 1 or claim 2. A feed for promoting IgA production comprising the composition for promoting IgA production described in claim 1 or claim 2.