Composition for promoting antimicrobial peptide production

Abstract

To provide a novel material capable of increasing antimicrobial peptide production from epithelial cells, thereby exhibiting an infection-preventing effect.SOLUTION: The present invention provides a composition for promoting antimicrobial peptide production, containing lactic acid bacteria of the genus Lactobacillus as an active ingredient. The present invention also provides a composition for promoting antimicrobial peptide production, containing a component derived from Lactobacillus bacterial cells as an active ingredient. The present invention further provides a composition for promoting antimicrobial peptide production in which the bacterial cell-derived component is one or more selected from the group consisting of S-layer protein and degradation products of the S-layer protein.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a composition for promoting the production of an antimicrobial peptide, and in particular to a composition for promoting the production of an antimicrobial peptide, comprising lactic acid bacteria of the genus Lactobacillus. [Background technology]

[0002] Antimicrobial peptides are one of the defense systems of living organisms and have a wide range of antimicrobial activity against bacteria, fungi, viruses, etc. Inducing the expression of antimicrobial peptides is expected to have preventive and therapeutic effects against infections, and thus several solutions for increasing the production of antimicrobial peptides have been disclosed. Patent Document 1 aims to stimulate the production of beta-defensin in skin cells using non-pathogenic bacteria, and discloses, as a solution to this problem, an extract containing heat-treated Lactobacillus plantarum cells, and a composition containing its active fraction. Patent Document 2 aims to provide an antimicrobial peptide inducer that is safe and has no side effects, and discloses an antimicrobial peptide inducer that contains lactic acid bacteria as an active ingredient as a means to solve this problem. Patent Document 3 aims to provide a substance that induces the secretion of antimicrobial peptides and to provide a therapeutic agent for diseases caused by bacteria or viruses, and discloses, as a means to achieve this, an antimicrobial peptide secretion inducer that contains branched-chain amino acids as active ingredients. However, the solution provided by the present application is neither disclosed nor suggested in any of the documents. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 5224807 [Patent Document 2] WO2015 / 087919 Brochure [Patent Document 3] JP 2003-95938 A Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a novel material that enhances the production of antimicrobial peptides from epithelial cells and has an infection prevention effect. [Means for solving the problem]

[0005] In order to solve the above problems, the present invention includes the following configurations. [1] A composition for promoting the production of antimicrobial peptides, comprising as an active ingredient one or more selected from the group consisting of S-layer proteins, which are components derived from Lactobacillus bacteria, and degradation products of the S-layer proteins. [2] A composition for promoting the production of antimicrobial peptides, comprising as an active ingredient Lactobacillus helveticus cells or a culture thereof, which contains an S-layer protein, a component derived from Lactobacillus cells, and a decomposition product of the S-layer protein. [3] The composition for promoting antimicrobial peptide production described in [1] or [2], wherein the lactic acid bacterium of the Lactobacillus genus is Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus amylovorus or Lactobacillus buchneri. [4] A composition for promoting antimicrobial peptide production according to any one of [1] to [3], wherein the antimicrobial peptide is a β-defensin. [5] The composition for promoting antimicrobial peptide production according to any one of [1] to [4], wherein the promotion of antimicrobial peptide production is promotion of antimicrobial peptide production in epithelial cells. [6] A food or drink for promoting antimicrobial peptide production, comprising the composition for promoting antimicrobial peptide production described in any one of [1] to [5]. [7] A food or drink for preventing infection, comprising a composition for promoting antimicrobial peptide production described in any one of [1] to [5]. Effect of the Invention

[0006] By ingesting materials that induce the production of antimicrobial peptides, such as S-layer protein (hereinafter sometimes referred to as SLP) derived from Lactobacillus lactic acid bacteria and its degradation products, or Lactobacillus lactic acid bacteria cells containing SLP and their cultures, it is expected that the body's defense against various infections will be improved. [Brief description of the drawings]

[0007] [Figure 1] 1 is a photograph showing the results of SDS-PAGE of purified SLPs. [Diagram 2] Photographs showing the results of SDS-PAGE of SLP, heated SLP, and SLP decomposition products. [Diagram 3] 1 is a graph showing the expression level of the hBD2 gene when Lactobacillus helveticus lactic acid bacteria cells or SLPs of the lactic acid bacteria were added to Caco-2 cells. [Figure 4] 1 is a graph showing the expression level of the hBD2 gene when Lactobacillus helveticus SLP, heated SLP, and SLP digests were added to Caco-2 cells. [Diagram 5] 1 is a graph showing the expression level of the hBD2 gene when SLPs prepared from Lactobacillus helveticus SBT2171, Lactobacillus acidophilus JCM1132, Lactobacillus amylovorus JCM1126, and Lactobacillus buchneri JCM1115 were added to Caco-2 cells. [Figure 6] 1 is a graph showing the expression level of the hBD2 gene when Lactobacillus helveticus lactic acid bacteria cells or SLPs of the lactic acid bacteria were added to HSC-4 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The present invention provides a novel composition for promoting the production of antimicrobial peptides. The composition for promoting antimicrobial peptide production of the present invention will be described in detail below. (Composition for promoting antimicrobial peptide production) S-layer proteins (hereinafter sometimes simply referred to as SLPs) are found in the outer layer of the cell walls of various bacteria and are characterized by their regular crystalline structure. Since SLPs are non-covalently bound to cell wall components, they can be extracted from bacterial cells using chaotropic agents such as lithium chloride. Furthermore, the extracted SLPs reform into a regular crystalline structure when the chaotropic agent is removed. This characteristic makes it possible to purify them from bacterial cells. It has been found that S-layer is expressed in the genus Lactobacillus among lactic acid bacteria, and in a report by Mukai et al. (Japanese Journal of Lactic Acid Bacteria, 19(1):21-29, 2008), the expression of SLP has been confirmed or estimated in 13 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. The SLP used in the composition for promoting antimicrobial peptide production of the present invention is derived from the genus Lactobacillus and can be any one that has the relevant action, but Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus amylovorus, or Lactobacillus buchneri is preferred. Among Lactobacillus helveticus, SBT2171 (FERM BP-5445) is most preferred. In addition, the SLPs do not need to be purified from the bacterial cells, and the bacterial cells themselves or cultures containing the SLPs can be used, but purified SLPs are more preferable. The bacterial cells themselves may be live or killed. Antibacterial peptides include the defensin family (such as α-defensin and β-defensin), cathelicidin (LL-37), dermcidin, LEAP-1 (hepcidin), LEAP-2, lactoferrin, and Reg peptides, of which the defensin family is preferred, and β-defensin is even more preferred.

[0009] (Method of producing a composition for promoting antimicrobial peptide production) SLP is purified from lactic acid bacteria cells according to the following method. After the target Lactobacillus lactic acid bacteria are sufficiently cultured in a liquid medium such as MRS liquid medium or skim milk, the cells are collected and washed as necessary. The cells may be collected from colonies grown on an agar medium or the like. The obtained cells are suspended and stirred in a chaotropic reagent solution such as lithium chloride, urea, or guanidine hydrochloride as is or after freeze-drying to solubilize the SLP on the cell surface. After removing solid matter from the solution containing the solubilized SLP, the chaotropic reagent is removed by dialysis or the like to precipitate the SLP. The precipitated SLP is collected and washed as necessary to obtain purified SLP. The SLP used in the composition for promoting antimicrobial peptide production of the present invention may be heated since the antimicrobial peptide promoting effect is maintained even when heated.

[0010] (SLP decomposition product) The SLP degradation product is prepared according to the following method. Various proteases are added to a suspension of SLP at appropriate concentrations and reacted appropriately. The type of protease and the degree of protease degradation are not limited. As an example, the conditions for degradation treatment with proteinase K are preferably 37°C for 2 to 24 hours, more preferably 3 to 20 hours.

[0011] (Intake amount) Since the antimicrobial peptide production promoting effect of SLP is exerted even in the form of decomposition products, it is considered that the effect is small even if it is decomposed by gastric acid after oral ingestion. For this reason, it is preferable to add SLP to food and take it orally. There is no particular limit on the intake amount, but in the case of Lactobacillus helveticus SBT2171, it is expected that antimicrobial peptides can be induced by continuously taking approximately 1 mg of SLP per day.

[0012] (Incorporation into food, medicine, and feed) Since the purified SLPs do not lose their antimicrobial peptide production-promoting activity even when heated, they can be incorporated into foods, medicines, and feed in the usual manner. The food and drink containing the composition for promoting antimicrobial peptide production of the present invention is useful as a food and drink for promoting antimicrobial peptide production, and by ingesting the food and drink, it promotes antimicrobial peptide production from epithelial cells, making it possible to prevent various infections, and even if an infection occurs, symptoms can be kept mild and a quick recovery can be expected. When the food and drink is contained in animal feed, the same effect can be expected for animals that ingest it. In addition, a medicine containing the composition for promoting antimicrobial peptide production of the present invention can be used for diseases that can be treated or prevented by promoting the production of antimicrobial peptides, such as various infectious diseases caused by pathogenic bacteria.

[0013] (Evaluation method) The antimicrobial peptide production-promoting action refers to the action of promoting the production of an antimicrobial peptide in a subject, and refers to the expression of an antimicrobial peptide by inducing the expression of a gene for expressing the antimicrobial peptide. Therefore, in this specification, the antimicrobial peptide production-promoting action and the antimicrobial peptide-inducing action are used synonymously. The antimicrobial peptide induction effect of the SLP of the present invention is evaluated by the following method. SLP or its degradation product is added to cultured cells, and the expression level of the antimicrobial peptide gene is measured after a predetermined time. Various cells can be used for the evaluation, but epithelial cells, which are expected to come into contact with the outside of the body, are particularly preferred. The antimicrobial peptides to be measured are not limited, but the defensin family is preferred, and β-defensin secreted from epithelial cells is particularly preferred. EXAMPLES

[0014] Examples of the present invention will be described in detail below, but the present invention is not limited to these examples.

[0015] The preparation examples of Lactobacillus bacteria, SLPs, and SLP degradation products used in this test are shown below. (Example of preparation of Lactobacillus lactic acid bacteria cells) Lactobacillus helveticus SBT2171 was cultured in 100 mL of MRS liquid medium at 37°C for 16 hours, after which the cells were harvested by centrifugation (8,000 × g, 4°C, 10 minutes), washed twice with saline and once with sterilized MilliQ water, and then freeze-dried (freeze-dried cells). In the following tests, freeze-dried cells were used.

[0016] (Example of SLP preparation) Lactobacillus helveticus SBT2171 was cultured in 100 mL of MRS liquid medium at 37°C for 16 hours, after which the cells were harvested 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 after stirring was centrifuged (10,000 × g, 4 °C, 20 minutes) and the supernatant was collected. The precipitate was TM 5M LiCl supplemented with Protease Inhibitor Cocktail (Roche) The mixture was resuspended in 10 mL of solution and stirred again at room temperature for 30 minutes. The suspension after stirring was centrifuged (10,000 × g, 4 ° C, 20 minutes) and the supernatant was collected. The collected supernatants were pooled and 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) to collect the precipitated SLP. 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. After purification, it was confirmed that the band of the desired size, 43 kDa, was observed by SDS-PAGE (Figure 1). The amount of SLP obtained was 3 mg.

[0017] (Example of preparation of SLP decomposition product) Proteinase K (from Tritirachium album, P6556: Sigma) (10 mg / mL) was added to SLP suspended in PBS at 5 mg / mL to a final concentration of 100 μg / mL, and the mixture was allowed to react overnight at 37°C. After the reaction, the SLP was heated at 96°C for 10 minutes to inactivate proteinase K, and the resulting SLP was digested. For comparison, the bands of SLP suspended in PBS, SLP suspended in PBS and heated at 96°C for 10 minutes, and SLP digested with proteinase K were confirmed by SDS-PAGE (Figure 2). In addition to the bands at around 43 kDa in the heated SLP, bands at around 37 kDa and 5 kDa were observed. In addition, no clear bands of 5 kDa or more were observed in the SLP digests.

[0018] [Test Example 1] Evaluation of antimicrobial peptide induction activity (1) Comparison with bacterial cells 1. Test Method Subcultured Caco-2 cells (human intestinal epithelial cells) were added to 2.0 × 10 5The cells were seeded in a 12-well plate at 100 cells / well and cultured overnight at 37°C in a 5% CO2 incubator. For the culture, DMEM-high glucose (D5796: Sigma) was supplemented with FBS (final conc. 10%) (Lot. 11D264: Sigma), NEAA (final conc. 1×) (M7145: Sigma), and Penicillin-Streptomycin (final conc. 100U-100μg / mL) (15140-122: Life technologies). After confirming that the cells were 70-80% confluent, the medium was removed and replaced with medium (DMEM-high glucose + 1× NEAA) without FBS or antibiotics, and cultured at 37°C for another 24 hours. After the incubation, the medium was removed from the 12-well plate, and a medium containing 10 or 50 μg / mL of Lactobacillus helveticus SBT2171 cells or SLP purified from Lactobacillus helveticus SBT2171 was added and incubated for 6 hours. After the incubation, the medium was removed from the 12-well plate, washed with PBS, and total RNA was extracted from the cells using an RNeasy Mini Kit (Qiagen). The concentration of the extracted total RNA was measured using a NanoDrop2000 (Thermo Fisher Scientific). cDNA was synthesized from the extracted total RNA solution using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). For reverse transcription, a TaKaRa PCR Thermal Cycler Dice (registered trademark) Gradient (Takara Bio) was used. The obtained reverse transcription reaction solution was used as template cDNA for real-time PCR. For real-time PCR, TaqMan® Fast Advanced Master Mix (Life Technologies, Cat#4444556) and TaqMan Gene Expression Assay (hBD2: Hs00175474_m1, GAPDH: Hs03929097_g1) (Life Technologies) were used. Reactions were performed in 384-well plates (Life Technologies, Cat#4309849) using the ViiA TM 7 (Life Technologies) was used. LPS was added at a concentration of 10 μg / mL as a positive control, and gene expression at each level was evaluated as the relative expression level to the positive control by the ΔΔCt method. The test was performed with n=3, and the level including the sample in which no amplification was observed was recorded as not detected (nd).

[0019] 2. Test Results As a result of the evaluation, increased expression of the hBD2 gene was observed in both SBT2171 cells and SLP (Figure 3). In SBT2171 cells, hBD2 gene expression was approximately 3 times higher than the positive control at an added concentration of 10 μg / mL, and approximately 28 times higher at an added concentration of 50 μg / mL. On the other hand, in SLP, gene expression was approximately 19 times higher at an added concentration of 10 μg / mL, and approximately 45 times higher at 50 μg / mL. From these results, it is considered that SLP purified from SBT2171 has a stronger antimicrobial peptide induction effect than SBT2171 cells. In the figure, the symbols a, b, c, and d indicate that there is a significant difference (P<0.05) between the different symbols. (The same applies to the following Figures 4 to 6).

[0020] [Test Example 2] Evaluation of antimicrobial peptide-inducing activity (2) Evaluation of SLP degradation products 1. Test Method As in the above "Test Example 1 (1) Comparison with bacterial cells", Caco-2 cells were seeded and the medium was replaced, and then SLP of Lactobacillus helveticus SBT2171 was added to a concentration of 50μg / mL either as is, heated at 96℃ for 10 minutes, or decomposed by proteinase K treatment, and cultured for 6 hours. Then, total RNA extraction, cDNA synthesis, and hBD2 gene expression analysis by real-time PCR were performed as described above.

[0021] 2. Test Results As a result of the evaluation, not only untreated SLP, but also heat-treated SLP and SLP decomposed with proteinase K showed significantly higher hBD2 gene expression than the positive control (Figure 4). From this result, it is considered that purified SLP has antimicrobial peptide induction activity even in the decomposition state.

[0022] [Test Example 3] Evaluation of antimicrobial peptide induction activity (3) Comparison of bacterial strains 1. Test Method As in the above "Test Example 1 (1) Comparison with Bacteria", Caco-2 cells were seeded and the medium was replaced, and then SLP purified from Lactobacillus helveticus SBT2171, Lactobacillus acidophilus JCM1132, Lactobacillus amylovorus JCM1126, and Lactobacillus buchneri JCM1115 was added to a concentration of 10 μg / mL and cultured for 6 hours. Then, total RNA extraction, cDNA synthesis, and hBD2 gene expression analysis by real-time PCR were performed as described above. The case without SLP was used as a negative control and evaluated as the relative expression level to the positive control.

[0023] 2. Test Results As a result of the evaluation, higher hBD2 gene expression was observed in the SLPs purified from all strains compared to the negative control, and hBD2 gene expression was observed to be equal to or higher than that of the positive control (Figure 5). From these results, it was considered that the antimicrobial peptide induction effect of the purified SLPs is widely common to the Lactobacillus genus. The molecular weight of SLPs produced by lactic acid bacteria varies greatly between bacterial species, and it has been shown that their genetic structures are highly diverse even within the same bacterial species. Therefore, it was surprising that the antibacterial peptide-inducing effect of the SLPs of the present invention was found to be widespread among the Lactobacillus genus.

[0024] [Test Example 4] Evaluation of antimicrobial peptide induction activity (4) Evaluation in oral epithelial cells 1. Test Method Subcultured HSC-4 cells (human tongue epithelial cells) were added to 1.0 × 10 5 The cells were seeded onto a 24-well plate at 100 cells / well and cultured overnight at 37°C in a 5% CO2 incubator. The cells were cultured in DMEM-high glucose (D5796: Sigma) supplemented with FBS (final conc. 10%) (Lot. 42F4160K: Gibco) and Penicillin-Streptomycin (final conc. 100U-100μg / mL) (15140-122: Life technologies). ) was added to the wells. After the culture, it was confirmed that the cells had reached 70-80% confluence, and the medium was removed and replaced with medium (DMEM-high glucose) without FBS or antibiotics, and the cells were further cultured at 37°C for 24 hours. After the culture, the medium was removed from the 24-well plate, and medium containing Lactobacillus helveticus SBT2171 cells or SLP purified from Lactobacillus helveticus SBT2171 at 10 or 50 μg / mL was added, and the cells were cultured for 6 hours. After that, total RNA extraction, cDNA synthesis, and hBD2 gene expression analysis by real-time PCR were performed as described above.

[0025] 2. Test Results As a result of the evaluation, it was found that both the bacterial cells and SLP increased hBD2 gene expression, as in the case of Caco-2 cells. Furthermore, higher hBD2 gene expression was observed with the addition of SLP compared to the bacterial cells (Figure 6). This result was similar to that of Caco-2 cells, and it was considered that SLP has a stronger antimicrobial peptide induction effect than the bacterial cells.

[0026] (Examples of food additives) 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 bag to produce the stick-shaped health food for promoting antimicrobial peptide production of the present invention.

[0027] (Example of mixing into feed) 2 g of SLP purified from Lactobacillus helveticus SBT2171 was suspended in 3998 g of deionized water, heated to 40°C, and then stirred and mixed for 20 minutes at 3,600 rpm in a TK homomixer (MARK II 160 type; manufactured by Tokushu Kika Kogyo Co., Ltd.) to obtain a 2 g / 4 kg SLP solution. 2 kg of this SLP solution was mixed with 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, 0.5 kg of vitamin mixture, 0.3 kg of cellulose, and 0.2 kg of mineral mixture, and sterilized at 120°C for 4 minutes to produce 10 kg of the feed for promoting antimicrobial peptide production of the present invention.

[0028] (Example of drug formulation) The liquid culture of Lactobacillus helveticus SBT2171 was centrifuged at 4°C and 7,000 rpm for 15 minutes, and then washed with sterilized water and centrifuged three times to obtain washed cells. The washed cells were freeze-dried to obtain a cell powder. One part of the cell powder was mixed with four parts of skim milk powder, and the mixed powder was tableted in 1g portions using a tableting machine in a conventional manner to prepare the tablets for promoting antimicrobial peptide production of the present invention. [Industrial Applicability]

[0029] By ingesting materials that induce the production of antimicrobial peptides, such as SLPs and SLP hydrolysates derived from Lactobacillus lactic acid bacteria, or Lactobacillus lactic acid bacteria cells containing SLPs and their cultures, it is expected that the body's defense against various infections will be improved. [Accession number]

[0030] <Reference to deposited biological material> (1) SBT2171 (a) The name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation, Patent Microorganism Depository Center Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818) Date of deposit of biological material in a depository institution in the Republic of Korea March 6, 1996 The accession number given to the deposit by the depository institution in question FERM BP-5445

Claims

1. A composition for promoting β-defensin production, comprising as an active ingredient an S-layer protein, which is a component derived from the bacterial cells of Lactobacillus helveticus.

2. The composition for promoting β-defensin production according to claim 1, wherein the promotion of β-defensin production is promotion of β-defensin production in epithelial cells.

3. A food or drink for promoting β-defensin production, comprising the composition for promoting β-defensin production described in claim 1 or 2.

4. The food or beverage for promoting β-defensin production according to claim 3, which is for preventing infection.

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