TWI667031B - Lactic acid bacterium having immunomodulatory and anti-allergic effects and pharmaceutical composition containing the same - Google Patents

Abstract

The present invention provides a strain of Lactobacillus plantarum subsp. plantarum K37, which is deposited in the DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN under the accession number DSM 27445. ), and is deposited with the Food Industry Development Research Institute, under the number BCRC 910619. The present invention further provides a pharmaceutical composition comprising the isolated lactic acid bacteria (Lactobacillus plantarum subsp. K37). Further, the present invention provides a use of an isolated lactic acid bacterium (Lactobacillus plantarum subsp. K37) for the preparation of a pharmaceutical composition for preventing or treating a disease in an individual.

Description

Lactic acid bacteria having immunomodulating and anti-allergic effects and pharmaceutical compositions containing the same

The present invention relates to a lactic acid bacterium, in particular to a novel lactic acid bacteria strain having immunomodulatory and anti-allergic effects in an individual.

Allergic diseases such as allergic rhinitis, atopic dermatitis, allergic asthma and food allergies have become more common in many countries. These diseases not only affect the quality of life of individuals, but also become a social medical burden. The allergic system reacts with T helper cell type 2 (Th2 cells) associated with both T cells and B cells. The Th2 response is characterized by the production of certain cytokines, including interleukin (IL) IL-4, IL-5, IL-13, and the production of total immunoglobulin (Ig) E, antigen-specific IgE, and IgG1. Th2 cytokines produced by Th2 cells enhance IgE production and eosinophilic aggregation. Among Th2 cytokines, IL-5 is known to be important for the differentiation, maturation and recruitment of eosinophils, while IL-4 and IL-13 have great potential for the treatment of asthma and other Th2 cell-associated diseases. Furthermore, IL-13 directly enhances excessive mucus secretion and gas Airway hyperresponsiveness (AHR). The production of cytokines is thought to be a T cell response, and the production of immunoglobulins is thought to be a B cell response. Th1 cells can inhibit the Th2 response by secreting interferon (IFN)-γ, IgG2a, IL-2, and IL-3. Therefore, by suppressing the Th2 cell response while enhancing the Th1 response to regulate the immune response, it is expected to help treat allergic and other Th2-responsive diseases, such as asthma, which is caused by multiple airway obstructions, airway eosinophilic inflammation, and bronchial hyperreactivity. Chronic complex respiratory diseases caused by sex.

Many studies have suggested that lactic acid bacteria (LAB), whether live or heat-killing, can alleviate allergy symptoms by regulating the Th1/Th2 response toward Th1. For example, Lactobacillus paracasei KW3110 live bacteria were orally administered to allergic mice, and the results showed anti-allergic effects including induction of the Th1 cytokine IL-12 and inhibition of the Th2 cytokine IL-4. Lactobacillus casei strain Shirota (LcS) stimulates the secretion of IL-12, which shifts the production pattern of cytokines, which shifts from Th2 cells to Th1 cells, thereby inhibiting IgE production and IgG1 response. Systemic allergic reactions with humans. Lactobacillus brevis SBC8803 inhibits the production of IgE and histamine secretion by improving the balance of Th1/Th2 and tending to Th1. Oral administration of Lactobacillus acidophilus L-55 to BALB/c mice sensitized with ovalbumin (OVA) showed that it can inhibit nasal symptoms induced by OVA, such as sneezing and sneezing. nose. Therefore, both live bacteria or heat-killed LAB can exhibit the ability to improve allergic reactions in rodents or humans.

In the present invention, the Lactobacillus plantarum subsp. K37 (hereinafter referred to as K37) is isolated from the traditional Chinese fermented mustard product, which is selected because it can induce human peripheral blood mononuclear cells ( Human peripheral blood mononuclear cells (hPBMCs) produce large amounts of IFN-γ, and have strong immunomodulatory effects in vitro. Different levels of K37, such as 10 ⁵ , 10 ⁷ , and 10 ⁹ CFU (colony forming units) were orally administered to OBA-sensitized and induced BALB/c mice by measuring the amount of immunoglobulin and cytokines in serum. Explore the effect of K37 on systemic allergy. Airway hyperreactivity to methacholine was assessed using a non-invasive whole volume descriptor. In addition, histological analysis is also evaluated.

The present invention provides a lactic acid bacterium for immunomodulation, which is a Lactobacillus plantarum subsp. plantarum K37, and is deposited with the DSMZ German Microbiology and Cell Culture Collection Center (DSMZ-DEUTSCHE) under the accession number DSM 27445. SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH), and is deposited with the Food Industry Development Institute under the number BCRC 910619.

The present invention further provides a pharmaceutical composition for preventing or treating a disease in an individual, which comprises a subspecies K37 and an excipient of Lactobacillus plantarum for immunomodulation.

In a specific embodiment of the present invention, the lactic acid bacteria are subjected to heat-killing bacteria, and the lactic acid bacteria are orally administered to the individual.

In a specific embodiment of the invention, the pharmaceutical composition is used for pre- Prevent or treat individual diseases. In a specific embodiment of the invention, the number of cells in the individual is reduced, and the cell line is selected from the group consisting of macrophages, eosinophils, neutrophils, and lymphocytes.

In a specific embodiment of the invention, the disease is related to an allergic disease. In a specific embodiment of the present invention, the allergic disease is selected from the group consisting of allergic rhinitis, atopic dermatitis, allergic asthma, food allergy, and high respiratory responsiveness.

In a specific embodiment of the present invention, the allergic disease is related to selected from the group consisting of IgG1, IgG2a, IgE, IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL. -12. Protein expression of a group consisting of IL-13 and chemokine (eotaxin). In a specific embodiment of the invention, the expression of IL-2, IL-12 and IFN-γ is increased. In a specific embodiment of the invention, the expression of IL-4, IL-5, IL-13, TNF-α, chemokines and IgE is decreased.

The present invention further provides a use of the lactic acid bacterium of the present invention for the preparation of a pharmaceutical composition for preventing or treating a disease in an individual.

Fig. 1 to Fig. 1C show electrophoresis patterns of PCR fingerprints of Lactobacillus plantarum plant subspecies, wherein SEQ ID NO: 3/SEQ ID NO: 4 (Fig. 1A) and SEQ ID NO: 5 (Fig. 1B) are used. And the primer of SEQ ID NO: 6 (Fig. 1C). Lane M is a DNA ladder (250-10000 base pairs); Lane 1 is Lactobacillus plantarum subspecies K37; Lane 2 is Lactobacillus plantarum subsp. ATCC 14917 ^T ; Lane 3 is Lactobacillus plantarum Anshia ATCC 17638 ^T ; Figure 2 shows the experimental schedule of the BALB/c mouse model sensitized with ovalbumin (OVA). Six-week-old female BALB/c mice were fed with live or heat-killing Lactobacillus plantarum subsp. K37 for 4 weeks, and 50 μg of OVA dissolved in 100 μl of Al(OH) _{3 was} intraperitoneally injected on days 1 and 14 three times. . On days 28, 29, and 30, mice were induced by intranasal administration with OVA (100 μl per mouse per day in 1% PBS) or PBS. Serum was collected weekly for immunoglobulin assays. On day 34, the mice were sacrificed and the spleen was removed for spleen cell preparation; Figure 3 to Figure 3D shows the effect of oral administration of K37 on the production of immunoglobulin in the serum of OVA-sensitized mice. From day 1 to day 34, heat-killing K37 with 10 ⁵ (K37-L), 10 ⁷ (K37-M) and 10 ⁹ (K37-H) CFU and 10 ⁹ CFU of K37 live bacteria (K37-A) BALB/c mice were fed, and 50 μg of OVA dissolved in 100 μl of Al(OH) _{3 was} intraperitoneally injected on days 1 and 7. The healthy control group (CON) and the allergy control group (OVA) were given PBS during the experiment, while the healthy control group was not sensitized with OVA. Total IgE (Fig. 3A), OVA-specific IgE (Fig. 3B), OVA-specific IgG1 (Fig. 3C) and OVA-specific IgG2a (Fig. 3D) in serum were determined by ELISA. Each value represents the mean ± standard deviation (N = 8). When P <0.05(*) and P <0.01(**), there was a statistically significant difference between the K37 group and the OVA group; Figure 4 shows the respiratory resistance value (Penh) showing that K37 was used in OVA-sensitized mice. After the last OVA induction for 24 hours, the airway response to aerosolized acetylcholine. Allergy control mice (OVA group) were fed with 200 μl PBS and sensitized and induced with OVA. The healthy control group (CON) was fed with 200 μl PBS and sensitized and induced with PBS. MK mice were fed 10 ⁵ (K37-L), 10 ⁷ (K37-M) and 10 ⁹ (K37-H) CFU heat-killing K37 and 10 ⁹ CFU K37 live bacteria (K37-A). When P <0.05(*) and P <0.01(**), it was confirmed that there was a statistically significant difference between the K37 group and the OVA group; Figure 5 and Figure 5B show that K37 was induced by OVA after OVA induction. The effect of infiltration of inflammatory cells in the lung tissue of sensitive mice. Figure 5A shows healthy control (CON), allergy test group (OVA), K37-L (10 ⁵ CFU, heat lethal), K37-M group (10 ⁷ CFU, heat lethal), K37-H group (10) ⁹ CFU, heat lethal) and the total number of cells in the lung lavage fluid of mice in the K37-A group (10 ⁹ CFU, live). Figure 5B shows the ratio of macrophages, eosinophils, neutrophils and lymphocytes in the lung lavage fluid, in groups of six mice, expressed as mean ± standard deviation. When P <0.05(*) and P <0.01(**), it was confirmed that there was a statistically significant difference between the K37 group and the OVA group; Figure 6 shows that K37 was used to induce the lung tissue of OVA-sensitized mice after OVA induction. The effect of inflamed cell infiltration and airway remodeling. Representative hematoxylin and Yi in healthy control group (CON), allergy control group (OVA), K37-H group (10 ⁹ CFU, heat lethal) and K37-A group (10 ⁹ CFU, live bacteria) Red (hematoxylin and eosin, H & E) lung tissue section staining (magnification 40 times); Figure 7 to Figure 7 F shows oral administration of K37 to ovarian lavage fluid (BALF) in OVA-sensitized mice The impact. Determination of IL-2 (Fig. 7A), IL-4 (Fig. 7B), IL-12 (Fig. 7C), IL-5 (Fig. 7D), IFN- in lung lavage fluid by ELISA Concentrations of γ (Fig. 7E) and IL-13 (Fig. 7F). Each value represents the mean ± standard deviation (n = 8). When P < 0.05 (*) and P < 0.01 (**), it was determined that there was a statistically significant difference between the K37 group and the OVA group; Figure 8 to Figure 8 F shows that K37 was orally administered to OVA-sensitized mice. The spleen produces a cytokine effect. Determination of IL-2 (Fig. 8A), IL-4 (Fig. 8B), IL-12 (Fig. 8C), IL-5 (Fig. 8D), IFN- in lung lavage fluid by ELISA Concentrations of γ (Fig. 8E) and IL-13 (Fig. 8F). Each value represents the mean ± standard deviation (n = 8). When P <0.05(*) and P <0.01(**), there was a statistically significant difference between the K37 group and the OVA group; and Figure 9 shows that the Lactobacillus plantarum subsp. plantarum K37 was orally administered to the lung lavage fluid. Inflammatory cytokines such as TNF-α, IL-6 and chemokines. When P <0.01 (**), it was determined that there was a statistically significant difference between the K37 group and the OVA group.

The invention is illustrated by the following specific examples, which are readily understood by those skilled in the art. The present invention may be embodied or applied by other different embodiments, and the various details of the present invention may be variously modified and changed without departing from the spirit and scope of the invention.

The invention has been described in terms of numerous embodiments. The following examples are not to be construed as limiting the scope of the invention.

Example

Example 1 Identification of the isolation and genotyping of Lactobacillus plantarum subsp. K37.

Lactobacillus plantarum subsp. plantarum K37 (hereinafter referred to as K37) is derived from the menu of traditional fermented mustard products in Taiwan. The 16S rDNA and phe S sequences were used in combination to identify the type of K37. The 16S rDNA (SEQ ID NO: 1) and phe S (SEQ ID NO: 2) of K37 were analyzed by direct sequencing PCR amplification of the product by about 500 nucleotides. Therefore, genomic DNA extraction, PCR amplification, purification of PCR products, and purification of PCR product sequencing are performed.

The sequence obtained is placed in the sequence alignment software provided on the Internet by the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), and the artificial sequence is performed. Align and compare the representative 16S rDNA or phe S sequences of the organisms belonging to the genus Lactobacillus. For comparison, the 16S rDNA and phe S sequences were also obtained from the database provided by NCBI on the Internet.

Based on the results of this analysis, Table 1 below lists the organisms whose 16S rDNA sequence (Table 1A) and phe S (Table 1B) sequences show the highest similarity values compared to the K37 sequence.

The combined results of KS 16S rDNA and pheS sequence analysis showed the highest similarity with Lactobacillus plantarum subspecies. Therefore, K37 represents a strain which is a subspecies of Lactobacillus plantarum plants.

Lactobacillus plantarum subsp. K37 has been deposited with the German Treaty on Microbiology and Cell Culture (DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH) on June 27, 2013 (Inhoffenstr. 7 B, D-38124 Braunschweig, Germany) And the accession number DSM 27445 was given by the International Depositary Authority. In addition, the Lactobacillus plantarum subspecies K37 was deposited with the Food Industry Development Institute on April 8, 2014 under the number BCRC 910619. This biomaterial has been tested and passed the viability test.

Example 2 Identification of bacterial strains using PCR-fingerprint

The PCR-fingerprint of K37 and two other Lactobacillus plantarum strains were compared. PCR was carried out under the conditions shown in Table 2. DNA was extracted from these strains as a template, and the resulting amplification products were subjected to electrophoresis and comparison, as shown in Fig. 1 to Fig. 1C, in which SEQ ID NO: 3/NO: 4 was used (Fig. 1 A), NO: 5 (Fig. 1B) and NO: 6 (Fig. 1C).

PCR reaction conditions:

94 ° C, 2 minutes; 5 cycles (94 ° C, 30 seconds; 36 ° C, 1 minute; 72 ° C, 1.5 minutes); 30 cycles (94 ° C, 20 seconds; 36 ° C, 30 seconds; 72 ° C, 1.5 minutes); 72 ° C, 3 minutes.

As shown in Fig. 1A to Fig. 1C, lane M represents a DNA ladder (250-10000 base pairs); lane 1 represents a Lactobacillus plantarum subspecies K37; and lane 2 represents a Lactobacillus plantarum subspecies. ATCC 14917 ^T ; Lane 3 represents Lactobacillus plantarum subsp. ATCC 17638 ^T . The results showed that the form of the amplified product of K37 was different from the other two strains of Lactobacillus plantarum.

Example 3 Analytical Profile Index (API) typing

The present invention uses the API50CHL kit (bioMerieux, France) The sugar utilization rate of K37 was estimated, and the results are shown in Table 3. Fermentation experiments indicated that K37 carries similar biochemical properties to plant subspecies of Lactobacillus plantarum.

^a. +, positive; -, negative

Example 4 Evaluation of anti-allergic effects of K37 in vivo

1. Materials and methods:

(1) Chemicals and reagents

De Man, Rogosa, and Sharpe (MRS) broth was purchased from Difco (Sparks, MD). Acetylcholine and OVA were purchased from Sigma-Aldrich (St. Louise, MO). RPMI-1640 medium, fetal bovine serum (FBS), L-glutamate, antibiotics (penicillin, streptomycin, and amphotericin) were purchased from Gibco (BRL, NY). All other chemicals were purchased from Merck (Darmstadt, Germany).

(2) Preparation of K37

The Lactobacillus plantarum subsp. K37 (hereinafter referred to as K37) is isolated from the traditional fermented mustard product of Taiwan and preserved in the preservation tube of the strain. K37 was inoculated into a culture medium of MRS (de Man, Rogosa and Sharpe; pH 5.4; Difco, USA), cultured at 30 ° C for 21 hours, collected by centrifugation (1500 g, 10 minutes), and washed twice with sterile PBS. K37 was then resuspended in PBS to a final concentration of 10 ¹⁰ CFU/ml and stored at -20 °C until use. For the preparation of the heat-killed K37, 10 ¹⁰ CFU/ml of K37 was thermally killed at 100 ° C for 20 minutes and stored at -20 ° C until use.

(3) Animal experiments and breeding

Four-week-old female BALB/c strains were purchased from the National Experimental Animal Center of Taiwan and kept in the animal room of the National Yangming University. The animal house was maintained at a 12:12 hour light-dark cycle with a temperature of 25 ± 2 °C. And humidity 55 ± 15%. Mice were fed a standard laboratory diet (LabDiet Autoclavable Rodent Diet 5010, PMI International Nutrition, Brentwood, USA) and adapted for two weeks for OVA sensitization and bacterial feeding. All animal testing procedures were reviewed and approved by the National Yangming University Animal Management Committee.

To assess the antiallergic effect of K37, 6-week-old mice were sensitized and induced with OVA to establish a BALB/c mouse model of respiratory allergy induced by OVA. Figure 2 summarizes the experimental procedure for OVA immunization, administration of K37, and sample collection in the OBA-sensitized BALB/c mouse model. Group 5 (n=8 per group) mice were assigned for different treatments for 34 days. The healthy control group (CON) and the allergy control group (OVA) were orally administered with PBS using a stainless steel feeding tube. The K37 group was given 10 ⁵ (K37-L), 10 ⁷ (K37-M) and 10 ⁹ (K37-H) CFU heat-killing K37 and 10 ⁹ CFU K37 live bacteria (K37-A), respectively. All groups, except the normal control group, were intraperitoneally injected on day 1 and day 14 with 100 μl of Al(OH) ₃ containing 50 μg of OVA. The healthy control mice received only Al(OH) ₃ . On days 28, 29 and 30, mice were induced by intranasal administration and in OVA (1% in PBS, 100 μl/mouse, daily) or PBS. On day 32, the AHR of the mice was determined. At the end of the assessment, all mice were sacrificed and a study of lung lavage fluid was performed. The spleen was aseptically removed for further cultivation. The lungs were removed for histological analysis.

Mouse body weight was measured daily during the study period. There were no significant differences in food intake, feeding efficiency or body weight between groups. On the designated date (Fig. 2), blood was collected using blood sampling from the orbital vein, and serum was prepared by centrifugation (centrifugation at 2,000 rpm for 10 minutes). Serum was stored at -20 °C prior to immunoglobulin analysis.

(4) Measurement of airway overreaction (AHR)

The measurement of AHR was performed by a whole body plethysmography. The pressure difference between the main chambers of the volume descriptor containing the animal and the reference chamber (box pressure signal) was determined. Mice were induced with aerosol saline (for baseline measurements) or methotrexate (6.25, 12.5, 25, and 50 mg/mL) for three minutes and recorded three minutes after nebulization and averaged readings. The enhanced pause (Penh) ratio per minute was recorded, and after the third recorded value, the average Penh value was divided by the Penh of saline and expressed as the relative percentage increase of Penh.

(5) Analysis of the cellular composition of the lung lavage fluid

Immediately after the sacrifice of the mice, the lungs were lavaged 3 times with 1 ml of Hanks' balanced salt solution (HBSS). Bronchoalveolar lavage fluid (BALF) was cooled on ice and centrifuged (1200 rpm, 4 ° C, 10 min), the supernatant was collected for determination of cytokines, and the cell pellet was resuspended in 1 ml of HBSS. The total number of cells in the lung lavage fluid was counted using a standard haemocytometer. Differentiating at least 200 cells in a cytospin preparation stained with Liu's stain solution (Chi I Pao, Taipei, Taiwan) by standard morphological criteria for macrophages, eosinophils, neutrophils , cell count of lymphocytes.

(6) Preparation of spleen cells

Briefly, spleen cells were adjusted to 1 with RPMI 1640 medium containing 10% FBS, 1% L-glutamate, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 μg/ml amphotericin. × 10 ⁶ cells/ml. The cells were cultured in a humidified incubator at 37 ° C, 5% CO ₂ for 48 hours. After the incubation, the supernatant was collected and stored at -20 ° C for further analysis of cytokines.

(7) Determination of immunoglobulins and cytokines by enzyme-linked immunosorbent assay (ELISA)

Total IgE and OVA-specific IgE, IgG1 and IgG2a levels were determined using a commercial ELISA kit (total IgE kits were purchased from Bethyl Laboratory Inc., Montgomery, TX, OVA-specific Ig kits were purchased from Alpha Diagnostic International Inc., San Antonio, TX). According to the manufacturer The manual uses the ELISA program to determine the concentrations of IL-2, IL-4, IL-5, IL-6, IL-12, IL-13, TNF-α and IFN-γ (IL-2, IL-4, IL). -10, TNF-[alpha] and IFN-[gamma] kits were purchased from eBioscience, Boston, MA; sets of IL-5, IL-6, IL-13 and chemokines were purchased from R&D Systems, Minneapolis, MN).

(8) Tissue identification of rat lung tissue

Immediately after lavage, the lungs were removed and fixed in 10% v/v (v/v) buffered formalin (in PBS, pH 7.4) for 24 hours and then embedded in paraffin. Further staining with hematoxylin and eosin (Sigma, St. Louis, MO) was performed using an optical microscope (Leica DM750) for tissue evaluation.

(9) Statistical analysis

Data are expressed as mean ± standard deviation (SD). Differences between means were tested for statistical differences using one-way ANOVA and Tukey's post hoc test. When P < 0.05 (*) or < 0.01 (**) represents a statistically significant difference between the control group and the other groups.

2. Results

(1) Effect of oral administration of K37 on immunoglobulin expression in OVA-sensitized mice

The serum immunoglobulin content was first studied to understand the effect of LAB on OVA-sensitized mice. Certain strains of lactic acid bacteria having a Th1 dominant reaction have been proposed to effectively modulate OVA-induced immunoglobulin production. In this study, respectively ^{10 5 (K37-L),} 10 7 (K37-M) , and 10 ⁹ (K37-H) CFU of lethal heat K37 viable and 10 ⁹ CFU K37 (K37-A) for oral administration of small The rats were 34 days old and intraperitoneally injected with OVA/Al(OH) _{3 on} days 1 and 14 (Fig. 2). As shown in Figure 3, on day 7, serum total IgE in OVA-sensitized mice increased and continued to increase to day 34. On day 34, compared with the OVA sensitization group (OVA), the high-dose heat-killed K37 group (10 ⁹ CFU, K37-H) and the K37 live bacteria group (10 ⁹ CFU, K37-A) showed total serum. IgE (Fig. 3A) and OVA-specific IgE (Fig. 3B) were significantly decreased ( P < 0.01). Serum OVA-specific IgG1 and Th2 immunoglobulin levels were significantly lower in the K37-M, K37-H, and K37-A groups than in the OVA sensitization group (OVA) (Fig. 3C; P <0.01). The K37 group increased serum OVA-specific IgG2a and Th1 type immunoglobulin. The content of OVA-specific IgG2a was significantly different in the K37-H and K37-A groups compared to the OVA sensitization group (OVA) ( P <0.05; Figure 3 D).

(2) airway overreaction

To assess the effect of K37 on AHR, a non-invasive overall volume descriptor was used for evaluation 1 day after the last induction. As shown in Fig. 4, compared with PBS-sensitized and PBS-induced mice (CON group), BABA/c mice sensitized with OVA and nasally induced showed Penh values after inhalation of methotrexate Increase (OVA and K37 groups). In the OVA group, the AHR for inhaled methotrexate was significantly increased compared to the CON group. However, oral administration of K37 relieved the development of AHR compared to the OVA group. The Penh values of the K37-H and K37-A groups were similar to those of the CON group, and significantly lower than the AHR of the OVA group (amylcholine 25 mg/mL, P <0.05; 50 mg/ml, P < 0.01). The Penh value of the K37-M group was significantly lower than that of the OVA group at 50 mg/ml of methotrexate.

(3) Cell differentiation of lung washing fluid

The number of cells of macrophages, eosinophils, neutrophils, and lymphocytes was counted to obtain cell differentiation of lung lavage fluid to evaluate the effect of K37 on lung inflammation. Check for the influx of inflammatory cells into the lungs. As shown in Figure 5, the total number of lung lavage fluid cells in the OVA group was significantly increased. The total number of cells in the K37-M, K37-H and K37-A groups was significantly lower than that in the OVA group ( P < 0.05). The cellular composition of BALF was further analyzed (Fig. 5B). In the OVA group, the percentage of eosinophils and neutrophils increased significantly compared to the CON group, while the percentage of macrophages decreased. As for the K37-H and K37-A groups, the number of cells of macrophages increased, whereas eosinophilic infiltration was relieved compared with the OVA group.

(4) Tissue examination of rodent lung tissue

The effect of K37 on OVA sensitization and OVA-induced lung inflammation in mice was further evaluated by histological examination. As shown in Fig. 6, according to H & E staining, inflammatory changes such as cell infiltration and increase in epithelial cell thickness were observed in the OVA group. Compared to the OVA group, mice given K37 orally had significantly reduced inflammation around the bronchi and perivascular areas based on less cell infiltration and a thinner epithelial cell layer (Fig. 6).

(5) Effects of oral administration of K37 on cytokines and spleen cells in lung washing fluid of OVA-sensitized mice

The production of cytokines was used to assess the effect of K37 on T cell responses. Lung lavage fluid (Fig. 7A to Fig. 7F) was assayed by ELISA using spleen cell culture (Fig. 8 to Fig. 8F), Th1 cytokines such as IL-2, IL-12 and IFN. - Concentrations of gamma and Th2 cytokines such as IL-4, IL-5 and IL-13. As shown in Fig. 7A, Fig. 7C and Fig. 7E, Th1 cytokines such as IL-2, IL-12 and IFN-γ in the lung lavage fluid were compared with the OVA group in the K37 group. The median dose-dependent increase. The levels of IL-2 and IL-12 in the K37-H and K37-A groups were significantly higher than those in the OVA group (Fig. 7A and Fig. 7C, P < 0.05). The content of IFN-γ was significantly increased in K37-M ( P < 0.05), K37-H ( P < 0.01), and K37-A groups ( P < 0.01). The content of Th2 cytokines including IL-4, IL-5 and IL-13 in the lung lavage fluid was also determined. Compared with the OVA group (K37-M, P <0.05), K37-H and K37-A ( P <0.01), the IL-4 content in the lung wash solution of the K37 group (Fig. 7B) was significantly decreased. In the K37-M, K37-H and K37-A groups, the IL-5 content was significantly lower than that in the OVA group (Fig. 7; K37-M, P < 0.05, K37-H and K37-A, P <0.01). . However, only in the K37-H and K37-A groups, the IL-13 content was significantly decreased (Fig. 7F).

As for the cytokines in the spleen cells, the content of IL-2 (Fig. 8A) in the K37 group was higher than that in the OVA group, however, there was a significant difference only in the K37-H and K37-A groups ( P <0.05). In all OVA sensitization groups (OVA and K37 groups), the IL-12 content was lower than that of the non-sensitized group (CON group) (Fig. 8C). In the K37-M, K37-H and K37-A groups, the IFN-γ content was higher than that in the OVA group, showing significant differences ( P < 0.05 and P < 0.01, respectively). In the K37 group (K37-H and K37-A, P < 0.05), a decrease in IL-4 content was observed. The levels of IL-5 and IL-13 were increased in a dose-dependent manner in the K37 group (for K37-M, P < 0.05 and for K37-H and K37-A, P < 0.01). However, the content of IL-4 in all groups (Fig. 8B) was comparable. Overall, cytokine assays showed that treatment with K37 induced the production of Th-1 cytokines IL-2 and IFN-γ in BALF and spleen cell cultures. In the K37 group, a decrease in the secretion of Th2 cytokines IL-4, IL-5 and IL-13 was observed. Cytokines such as TNF-α, IL-6 and chemokines in proinflammatory cytokines in BALF were also measured to assess the effect of K37 on the inflammatory state of the lungs. As shown in Fig. 9, the content of TNF-α was comparable in the CON and OVA groups, but decreased in a dose-dependent manner in the K37 group. Compared with the CON group, the levels of IL-6 and chemokines in the OVA group were significantly increased. However, in the K37-H and K37-A groups, the levels of IL-6 and chemokines were similar to those of the CON group. The levels of TNF-α and IL-6 in spleen cell culture showed the same trend as BALF (data not shown). Overall, treatment with K37 reduced the production of TNF-α, IL-6 and chemokines in BALF in OVA-sensitized mice.

The present invention investigates the effect of oral administration of K37 on an OVA-induced allergic asthma BALB/c mouse model. The current results show that K37 inhibits allergic parameters, including AHR, airway inflammation, total IgE, and OVA-specific IgE. The distribution of cytokine production in BALF and spleen cell culture showed that K37 made the immune response prone to Th-1 immune response, increased the content of Th-1 cytokines, and decreased the content of Th-2 cytokines. The levels of inflammatory mediators such as TNF-[alpha], IL-6 and chemokines in BALF were dose-dependently decreased by oral administration of K37. A decrease in the number of eosinophils and neutrophils in BALF means that K37 can improve inflammatory conditions. Histological observations of lung sections also showed less cellular infiltration.

K37 is a traditional fermented mustard product from Taiwan. The cytokine production was previously evaluated by hPBMCs in vitro. Therefore, it can be speculated that the specific lactic acid bacteria having a Th1-polarizing potential may exhibit an anti-allergic effect in vivo.

Ovalbumin (OVA) is the most commonly used allergen in animal models of allergy. The lower the content of IgE and OVA-specific IgE and IgG1, the milder the allergic reaction. In the present invention, the anti-allergic effect of K37 is investigated by intraperitoneal OVA sensitization and intranasal OVA-induced BALB/c mouse model (Fig. 2). As shown in Fig. 3, the levels of serum IgE, OVA-specific IgE, and IgG1 in the OVA group increased, representing the establishment of an allergic animal model and a B-cell type exhibiting a Th-2 response. At the end of the assessment, the total IgE, OVA-specific IgE, and OVA-specific IgG1 levels in the K37 group were significantly lower than in the OVA group (K37-M, P <0.05; and K37-A, P < 0.01) (Fig. 3). Furthermore, in the K37 group (Fig. 3D), a significant increase in OVA-specific IgG2a was observed. Analysis of serum immunoglobulin showed a systemic antiallergic effect of K37. Furthermore, the effect of K37 on OVA-induced immunoglobulin secretion was shown to be dose-dependent.

The results showed that K37 showed systemic and respiratory anti-allergic effects, and K37-H (10 ⁹ CFU, heat lethal) and K37-A (10 ⁹ CFU, live bacteria) showed the best activity, this recommendation K37 resistance Allergic effects are dose dependent.

Airway hyperresponsiveness is defined as an increased sensitivity to certain bile basic drugs, such as methotrexate, which causes smooth muscle contraction and increases airway resistance by reducing the airway. Asthma is a kind of airway hyperresponsiveness The disease is characterized by the accumulation of inflammatory cells, the increase in mucus production, and the release of certain Th2 cytokines such as IL-4, IL-5, IL-13 and IgE. In the present invention, changes in airway remodeling by K37 treatment have been explored. K37 significantly attenuated OVA-induced AHR inhalation of methotrexate (Fig. 4). According to the histopathological study of the lungs stained with H & E, the inflammatory cell infiltration was inhibited in the K37 group compared to the OVA group.

The expression of cytokines, such as IL-4, IL-5, IL-13 associated with T cell responses, and the production of immunoglobulin G1 (IgG1) associated with B cell responses, are thought to be related to Th2 type immunity. Th2 cytokines play an important role in asthma. Among the Th 2 cytokines, IL-4 chemises native T helper cells into a Th2 cell phenotype and induces B cells to switch isotype antibodies to IgE. IL-5 produced by Th2 cells is responsible for the growth, differentiation, movement, complementation, activation and survival of eosinophils. IL-13 plays an important role in the pathogenesis of asthma. Excessive production of IL-4, IL-5, and IL-13 is closely related to the development of asthma. The results show that T-cell-reactive Th2 cytokines are increased from BALF and spleen cell cultures in the OVA group, such as IL-4, IL-5 and IL-13 (as shown in Figures 6 and 7, respectively). This evidence indicates that the regulatory effect of Th1/Th2 in lactic acid bacteria is used in animal models of allergies. In the present invention, AHR is reduced by oral administration of K37, and it is proved that its antiallergic effect can be attributed to a decrease in Th2 cytokines and chemokines, which is known to be important in the development of AHR. It is speculated that the inhibitory effect on IL-13 contributes to the antiallergic activity of K37 (Fig. 7F and Fig. 8F). In addition to inhibiting Th2 cytokines, K37 also promotes the production of Th1 cytokines. K37 increased IFN-γ production in hPBMCs (data not shown). K37 The levels of IL-2, IL-12 and IFN-γ in BALF and spleen cell cultures can also be increased (Fig. 7 and Fig. 8, respectively). Taken together, the results of the present invention show that K37 has a promising effect on the regulation of T cell responses in OVA-sensitized mice towards the Th1 response line.

The present invention observes inflammatory responses induced by OVA and induced lung tissue and BALF (Figs. 5 and 6). Many types of inflammatory cells are involved in airway inflammation processes such as mast cells, eosinophils, and T lymphocytes. Among these cells, eosinophils play a key role in the pathogenesis of allergic diseases. Eosinophils are attracted to chemoattractants by CC chemokine receptor 3 (CCR3), such as chemokines released in the airways. Clinical and experimental studies have established eosinophils as indicators of allergic diseases. K37 reduced the content of OVA-specific IgE and total IgE. Furthermore, in the K37 group, the OVA-specific IgG1 line was decreased (Fig. 3).

Chemokines are thought to play an important role in allergy because they induce supplementation of eosinophils, basophils, and Th2 lymphocytes in the lungs. Inflammatory infiltration around the bronchi and around the blood vessels usually involves eosinophils and mast cells. As shown in Fig. 5B, the number of eosinophils was significantly reduced in the K37-H and K37-A groups compared to the OVA group. In addition, infiltration of eosinophils was observed in the periphery of the bronchi of the lung sections of the OVA group. However, in the K37 group, there was less cell infiltration (Fig. 6). In addition, the chemokine content was similar to the CON group in the K37-M, K37-H, and K37-A groups. A decrease in TNF-α and IL-6 was also observed in the K37-H group and the K37-A group (Fig. 9). In summary, these results indicate that the inflammation of the lungs is attributed to Anti-allergic effect of K37.

In summary, the present invention shows that K37 can ameliorate asthmatic responses such as OVA-sensitized BALB/c mice. AHR was assessed by the Enhanced Breath Interval (Penh) method using the Whole Volume Descriptor and it was found that administration of K37 significantly reduced AHR in OVA-sensitized BALB/c mice. K37 elicits significant immunomodulatory effects on most of the parameters tested. Therefore, K37 is a promising candidate for the protection and prevention of allergic diseases.

The above-described embodiments are merely illustrative of the principles of the invention and its effects, and those skilled in the art can make modifications and changes to the embodiments described above without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes that may be made without departing from the spirit and scope of the invention are to be covered by the appended claims.

Claims (1)

A method for identifying a lactic acid bacterium comprising: comparing a 16S rDNA sequence or a phe S gene sequence of the lactic acid bacterium to the Lactobacillus, wherein the lactic acid bacterium and the 16S rDNA sequence Accession No. D79210 of the subspecies of Lactobacillus plantarum have 99.6% sequence similarity And the lactic acid bacterium has a sequence similarity to the phe S gene sequence Accession No. AM087714 of the subspecies of the plant Lactobacillus plantarum , wherein the lactic acid bacteria Lactobacillus plantarum subsp. plantarum K37, Registered at DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH under the accession number DSM 27445 and deposited with the Food Industry Development Institute under the number BCRC 910619; and subject to the lactic acid bacteria and sugars Fermentation test, in which the following sugars were positive in the fermentation test: L-arabinose, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, L- Rhamnose, D-mannitol, D-sorbitol, methyl-α-D-mannopyranoside, N-acetylglucosamine, amygdalin, bearberry , Qiyelu ferric citrate, glucoside, D-fiber disaccharide, D-maltose, bovine source D-lactose, D-melose, D-sucrose, D-trehalose, inulin, D-sabitriose , D-raffinose, bitter fructose, D-pineose and potassium gluconate; and the following sugars were negative in the fermentation test: glycerol, butyl alcohol, D-arabinose, D-wood Sugar, L-xylose, D-ribitol, methyl-β-D-xylopyranoside, L-sorbose, galactitol, cyclohexanol, methyl-α-D-glucopyranoside, Starch, glycogen, xylitol, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluconic acid Potassium and potassium 5-ketogluconate.