TWI398259B - Composition and use of lactobacillus paracasei strain gmnl-133 in treating atopic dermatitis or other allergic diseases - Google Patents

Description

Composition of lactic acid bacteria strain GMNL-133 for improving atopic dermatitis or other allergic diseases and use thereof

The present invention relates to a strain of lactate and its use, and more particularly to a strain of Lactobacillus paracasei and a composition and use thereof for improving allergy diseases.

Allergy refers to an acquired immunological adverse reaction to normal harmless substances. Symptoms caused by allergic reactions include itching, coughing, wheezing, sneezing, tears, inflammation and fatigue. Allergic reactions are thought to include early specific immune responses and advanced inflammatory responses. Allergens (such as pollen and dust mites) have been reported to mediate early allergic reactions by stimulating high-affinity immunoglobulin E (IgE). Specifically, when mast cells and basophils are stimulated by allergens, histamine and cytokines are released. Cytokines then mediate late allergic reactions by aggregating inflammatory cells. It has also been reported that the collection of eosinophils, macrophages, lymphocytes, neutrophils and platelets opens the cycle of advanced malignant inflammatory response. The late allergic reaction enhances the initial immune response, which in turn promotes the release of more inflammatory cells.

A variety of therapies have been used to treat allergy symptoms, including the use of anti-allergic agents and histamine H-receptor antagonists (antihistamines). Antihistamine can alleviate symptoms such as congestion, itching and swelling caused by histamine acting on target tissues, and can prevent or alleviate many symptoms caused by degranulation of mast cells. However, it has also been reported that antihistamines have some side effects such as reduced alertness, unresponsiveness and lethargy.

Atopic dermatitis is one of the most common allergies in children. The traditional method for treating atopic dermatitis is topical steroid therapy, but in fact, atopic dermatitis symptoms are difficult to eradicate, and often relapse after stopping the drug. Furthermore, long-term use of steroid therapy is prone to side effects such as skin atrophy and thinning, skin pigmentation, long hair, and long acne. Therefore, the side effects are small and the safety is high, and the medicine that can be used with confidence can be regarded as a patient and a clinical one.

Probiotics have been recognized by the medical and scientific communities in recent years to help human health and have no side effects. The Food and Agriculture organization of the United Nations (FAO) and the World Health Organization (WHO) define probiotics as: living microorganisms, when it is administered in an appropriate amount, The host can be given a health benefit. There are many types of microorganisms currently available as probiotics, such as Lactobacillus, Bifidobacterium, Lactococcus, Enterococcus, Yeasts, and Chains. Streptococcus and the like.

Lactobacillus is a group of Gram-positive facultative anaerobic bacteria commonly found in the human gastrointestinal tract and vagina, capable of fermenting sugars and using lactic acid as the main metabolite. There are many studies and clinical trials that have confirmed the important correlation between lactobacilli and health. At present, the efficacy of Lactobacillus has been discovered: (1) to improve the bacterial phase balance of the intestinal microbiota; (2) to prevent diarrhea; (3) to reduce the risk of colon cancer; (4) Stimulates the normal development and function of the gastrointestinal epithelial mucosal system; (5) produces various vitamins and nutrients; and (6) prevents and treats vaginosis.

In summary, one aspect of the present invention provides an isolated lactic acid strain GMNL-133, which is deposited at the Food Industry Research and Development Institute (FIRDI) in Taiwan. The number is BCRC 910520.

Another aspect of the present invention is to provide a use of a strain of Lactobacillus paracasei GMNL-133 for the manufacture of a composition for treating atopic dermatitis.

According to the above aspect of the present invention, there is provided a composition for treating atopic dermatitis, comprising the lactic acid strain GMNL-133, and the content of the lactic acid strain GMNL-133 is effective for improving ectopicity. Dermatitis.

Still another aspect of the present invention provides a use of a strain of Lactobacillus paracasei GMNL-133 for use in the manufacture of a composition for treating an allergic disease.

According to the above aspect of the present invention, a composition for treating an allergic disease, comprising a strain of Lactobacillus paracasei GMNL-133, and a content of a strain of Lactobacillus paracasei GMNL-133 is effective for improving an allergic disease.

According to an embodiment of the present invention, the lactic acid bacteria strain GMNL-133 may be a live bacteria or a heat-treated dead bacteria. The composition for treating atopic dermatitis or an allergic disease may be a pharmaceutical composition or a food composition.

According to another embodiment of the present invention, the composition for treating atopic dermatitis or an allergic disease may further comprise at least one other strain selected from the group consisting of Lactobacillus acidophilus and Lactobacillus. Plantarum ), Bifidobacterium longum , Lactobacillus fermentum , Lactobacillus bulgaricus , Streptococcus thermophilus , Lactobacillus cremors , Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus reuteri and any combination of the above.

It has been reported that allergies can be treated by regulating cytokines. Among them, γ-interferon (interferon-γ, IFN-γ) can inhibit the excessive expression of cytokines in type 2 helper T cells (Th2), especially inhibiting interleukin-4 (IL-4). ) secretion, which in turn reduces the proliferation of B lymphocytes. In addition, IFN-γ can also stimulate the immune response of type 1 helper T cells (Th1) and inhibit the synthesis of IgE. Therefore, Xianxin IFN-γ is effective for treating allergies.

Accordingly, the present invention provides a novel Lactobacillus casei GMNL-133 having the ability to stimulate secretion of IFN-γ by spleen cells of a mouse, and can be used for the manufacture of a composition for treating allergic diseases. Lactobacillus paracasei GMNL-133 was deposited on July 5, 2011 at the Food Industry Research and Development Institute (FIRDI) in Taiwan under the registration number BCRC 910520. Also, according to the Budapest Treaty, the Phytolactin strain GMNL-133 was also deposited on the China Center for Type Culture Collection (CCTCC) on September 26, 2011 (Wuhan, Wuhan). University, 430072, People's Republic of China), the deposit number is CCTCC M 2011331. The Lactobacillus paracasei GMNL-133 (BCRC 910520) may comprise at least one pharmaceutically acceptable excipient and/or diluent such as other ingredients such as glucose, maltodextrin, infant formula, fructooligosaccharide, hard Magnesium oleate, eucalyptus, other difficult to separate ingredients, or any combination thereof, and such other ingredients and methods of formulation are well known to those skilled in the art. Further, the Lactobacillus paracasei GMNL-133 (BCRC 910520) may be used in an amount of from about 1 x 10 ⁶ to about 1 x 10 ¹¹ colony-forming unit (CFU) per gram (CFU/g).

The term "allergic disease" refers to atopic dermatitis, allergic rhinitis and asthma, and allergies caused by certain foods and insect bites. These diseases can cause a considerable degree of inflammatory response, resulting in chronic inflammation of the skin, mucosal tissue or blood vessels.

According to an embodiment of the present invention, the composition for treating an allergic disease comprises Lactobacillus paracasei GMNL-133, and the content of Lactobacillus paracasei GMNL-133 is effective for improving allergy diseases.

The above-mentioned lactic acid strain GMNL-133 may be a live bacteria or a heat-treated dead bacteria. The composition for treating an allergic disease can be an oral composition such as a pharmaceutical composition or a food composition.

According to another embodiment of the present invention, the composition for treating an allergic disease may further comprise at least one other strain. Other strains are selected from Lactobacillus acidophilus , Lactobacillus plantarum , Bifidobacterium longum , Lactobacillus fermentum , Lactobacillus bulgaricus , and A group consisting of Streptococcus thermophilus , Lactobacillus cremors , Lactobacillus rhamnosus , Lactobacillus reuteri , and any combination of the above. In general, the amount of bacteria of other strains may be about 1 x 10 ⁷ CFU/g or more.

Example 1. Isolation, screening and identification of strains

The inventor of the present invention isolated more than 100 strains of lactobacilli from healthy human gastrointestinal tract samples and established a library of isolated strains. In order to find the Lactobacillus with the potential to alleviate the treatment of allergic diseases, the Lactobacillus in the strain bank was co-cultured with the mouse spleen cells, and the IFN-γ secretion of the mouse spleen cells was analyzed, and the isolates were screened out. GMNL-133 is a lactobacillus that stimulates the secretion of large amounts of IFN-γ from mouse spleen cells.

The isolate GMNL-133 was subjected to various strain identification analysis. Figure 1 is a diagram showing the morphology of the isolate GMNL-133 under a microscope. The preliminary test results of the isolate GMNL-133 showed Gram-positive bacilli, which did not have enzymes, oxidases and motility, and grew in both aerobic and anaerobic environments.

Further analysis was performed with the 16S rDNA sequence and the API 50 CHL identification system. The 16S rDNA partial sequence of the isolate GMNL-133 is shown in SEQ ID No: 1. Table 1 shows the results of the analysis of the isolate 50nL-133 by the API 50CHL identification system.

The above identification results showed that the isolate GMNL-133 was Lactobacillus paracasei.

Example 2. Difference analysis between Lactobacillus paracasei GMNL-133 and other commercially available products of Lactobacillus paracasei

The broiler lactic acid strain GMNL-133 and the commercially available yoghurt (for example, uniform LP33 yogurt, containing the L. lactis strain LP33), Yogurt (for example, the Japanese unicorn company's vitality yoghurt, containing the cheese Differential analysis of strains in products containing Lactobacillus paracasei, such as Lactobacillus strain KW3110), probiotic health foods (eg, Probiotics Probiotics, BRAP-01) Analysis of the amplified DNA of RAPD (Random Amplification of Polymorphic DNA) and API 50 CHL identification system.

RAPD electrophoresis analysis using the primer sequence 5'-CCGCGACGTT-3' (SEQ ID No: 2) for polymerase chain reaction to analyze the strain of lactic acid bacteria GMNL-133 and the commercially available product of Lactobacillus paracasei The difference between polymorphic DNA was randomly amplified. The polymerase chain reaction reaction conditions were 93 ° C, and the double-stranded DNA was denatured into single strands in 10 minutes. The following cycle was followed 35 times: 93 ° C, 1 minute; 36 ° C, 1 minute; 74 ° C, 1 minute. Finally, the DNA fragment was amplified by performing at 75 ° C for 5 minutes. After the reaction was completed, DNA electrophoresis analysis was carried out with 1.5% acacia.

Figure 2 is a RAPD electropherogram of Lactobacillus paracasei GMNL-133 and other commercially available products. Lanes M are molecular weight markers, and lanes 1 to 4 are RAPD patterns of LP33, KW3110, GMNL-133 and BRAP-01, respectively. From the results of Fig. 2, the RAPD spectra of GMNL-133 and KW3110, LP33, and BRAP-01 are different.

The results of the analysis of the API 50 CHL identification system of the paratuberculosis strain GMNL-133 and other commercially available products of Lactobacillus paracasei are shown in Table 2.

It can be seen from Table 2 that the biochemical reactions of the lactic acid bacteria strains GMNL-133, LP33 and BRAP-01 are different. Among them, the biochemical reaction of L-sorbose, methyl-αD-glucoside, amygdalin and D-containing pine triose was completely different for L. paramedis strain GMNL-133 and LP33. The lactic acid bacteria strains GMNL-133 and BRAP-01 for L-arabinose, D-ribose, methyl-βD-xyloside, L-rhamnose, inositol, D-sorbitol, D-lactose The biochemical reactions of D-melose, inulin, D-containing pine triose, D-pine disaccharide, D-lyxose, D-tagatose and gluconic acid are completely different.

In summary, it can be seen that the lactic acid strains GMNL-133, KW3110, LP33 and BRAP-01 are different in gene sequence and biochemical reaction, and the four strains of lactic acid strains are indeed different strains, confirming the cheese of the present invention. Lactobacillus strain GMNL-133 is a novel strain.

Example 3. Comparison of the secretion of IFN-γ produced by lactic acid strain GMNL-133 and other strains of Lactobacillus paracasei in stimulating mouse spleen cells

The spleen cells of the mouse spleen cells were compared with the spleen cells of normal mice by GM3-133, KK3110, LP33 and BRAP-01, respectively, in the various commercially available products. - γ secretion amount.

RPMI 1640 medium (GIBCO/BRL) containing 10% fetal bovine serum , Gaithersburg, MD, USA) Cultured spleen cells from BALB/c mice. ⁴ ×10 ⁵ mouse spleen cells were added to a 96-well plate, and 10 ^{5 of} each of the above-mentioned Lactobacillus strains were separately added, and cultured at 37 ° C for 48 hours in an environment containing 5% carbon dioxide. Thereafter, the cell culture supernatant was used as a commercially available ELISA kit (OptEIA Mouse IFN-γ Set, BD Biosciences Pharmingen) , San Diego, CA, USA) Determination of IFN-γ content therein.

Fig. 3 is a graph showing the amount of IFN-γ secreted by spleen cells of mouse lactic acid strains GMNL-133 and other commercially available products. From the results of Fig. 3, it can be seen that the lactic acid strain GMNL-133 can stimulate the secretion of a large amount of IFN-γ, and the secretion amount is at least 3 times higher than that of the other strains of the commercially available products, showing the present invention. The lactic acid strain GMNL-133 has a better effect on treating allergies than other Lactobacillus paracasei strains.

Example 4. Effect of lactic acid strain GMNL-133 on cytokine secretion in sensitized mice

The sensitized mouse model mice were female BALB/c mice purchased from Taiwan Lesco Biotechnology. Breeding in animal rooms that control light and temperature. One week after the BALB/c mice entered the chamber, the test was started after the animals were stabilized.

Mice were divided into normal group, experimental group and placebo group for experiment. In the experimental group and the placebo group, 50 μg of ovalbumin (OVA) and 4 mg of aluminum hydroxide adjuvant (alum) were intraperitoneally injected into the mice on the 0th day after the start of the experiment, and the experiment was performed. At 14 and 28 days, mice were repeatedly injected with 25 μg of OVA and 4 mg of alum.

During the experiment, 0.5 ml of RO water was fed daily from the first day and the placebo group, while the experimental group was fed 2×10 ⁷ of the live strain of the honeysuckle strain GMNL-133. The experiment was carried out for 35 days, the mice were sacrificed on the 35th day, and blood was collected from the tail of the mouse before sacrifice, and the serum was isolated for subsequent analysis of IgE and IgG2a.

After each group of BALB/c mice was sacrificed by the CO ₂ asphyxiation method, the spleen was taken out in a petri dish by a aseptic technique, 6 ml of PBS was added, and the spleen was crushed and ground to a suspension state with a glass rod. The aspirated suspension was slowly added to a 15 ml centrifuge tube of 6 ml of Ficoll-Hypaque (17-1400-02, Pharmacia), and subjected to gradient centrifugation to separate the spleen cells (720 g, 20 min), and the precipitate was red blood cells. The spleen cells were taken out from the interface, washed twice with PBS, adjusted to a cell concentration of 4 × 10 ⁶ cells/ml in RPMI-1640 medium, and spleen cells were injected into a 96-well plate at 100 μl per well.

The spleen cells were cultured for 48 hours after adding OVA (culture concentration: 30 μg/mL), and the supernatant was taken, and the concentrations of TGF-β, IFN-γ, IL-10, and IL-17 were detected by ELISA.

Figures 4A to 4B are bar graphs showing the amount of IgE and IgG2a in the serum of each group normalized by the amount of placebo in the sensitized mouse model test. The * mark indicates statistically significant compared with the placebo group. Significant difference (p < 0.05). From the results of Figs. 4A to 4B, it was found that the OVA-specific IgE in the serum of the BALB/c mice of the experimental group (feeding the Lactobacillus paracasei GMNL-133) was significantly decreased as compared with the placebo group. However, the OVA-specific IgG2a in the serum of the BALB/c mice of the experimental group (feeding the Lactobacillus paracasei GMNL-133) was significantly increased compared with the placebo group. According to the literature, IgG2a antibodies are secreted by Th1 cells. Therefore, feeding the Lactobacillus paracasei GMNL-133 can activate Th1 cells in BALB/c mice, thereby reducing the production of OVA-specific IgE in serum.

In Figures 5A to 5D, respectively, in the sensitized mouse model experiment, the expression levels of TGF-β, IFN-γ, IL-10 and IL-17 in the spleen cell culture medium of each group were normalized by the performance of the placebo group. Bar graph, where the * mark indicates a statistically significant difference (p < 0.05) compared to the placebo group. From the results of the 5A to 5D results, it was found that the TGF-β, IFN-γ, IL-10 in the spleen cell culture solution of the experimental group were compared with the placebo group after the feeding of the Lactobacillus paracasei strain GMNL-133. There was a significant increase in IL-17 concentration.

Many allergic diseases are caused by an imbalance in the immune response between Th1 cells and Th2 cells, and the response to allergens is more likely to be biased toward the immune response of Th2 cells. However, it has been pointed out that by improving the balance of the Th1/Th2 immune system, the Th2 cell response can be shifted to the Th1 immune response, which can alleviate allergic diseases. When the immune response is biased toward the immune pathway of Th1 cells, Th1 cells secrete IFN-γ, help the production of IgG2a antibodies, activate macrophages, natural killer cells and poisonous T cells, and strengthen cellular immune responses. In addition, Th17 cells secrete IL-17, which can serve as a bridge between innate immunity and acquired immunity, which means that Th17 cells are located at the pivotal position between cellular immunity and humoral immunity.

Furthermore, another group of regulatory T cells can inhibit the development of allergic diseases by inhibiting the immune response of Th2 cells. Regulatory T cells are characterized by secretion of IL-10 or TGF-β, which in turn inhibits activation and proliferation of the target cells.

In summary, the lactic acid strain GMNL-133 of the present invention can stimulate Th-17 cells of the immune system in vivo, thereby transferring the Th1/Th2 immune response to the Th1 pathway. Furthermore, the lactic acid strain GMNL-133 of the present invention can also stimulate regulatory T cells, thereby inhibiting the immune response of Th2 cells and alleviating allergic reactions.

Example 5. Evaluation of the improved efficacy of lactic acid bacteria strain GMNL-133 in mice with atopic dermatitis

A 6-week-old normal male BALB/c mouse will be purchased from Taiwan Lesco Biotech. Breeding in animal rooms that control light and temperature. One week after the BALB/c mice entered the chamber, the test was started after the animals were stabilized.

Mice were divided into normal group, experimental group and placebo group for experiment. In the experimental group and the placebo group, 10 μl of 0.3% trinitrochlorobenzene solution was applied to the inner side of the right ear of BALB/c mice on the 0th day after the start of the experiment, and then three times a week during the experiment. The same portion was coated with the same dose of trinitrochlorobenzene solution to induce atopic dermatitis in mice, forming a mouse model of atopic dermatitis. At the same time, during the experiment, 0.5 ml of RO water was fed to the normal group and the placebo group from the first day, while the experimental group was fed with 2×10 ⁷ of the live strain of the honeysuckle strain GMNL-133.

The experiment was carried out for 21 days. The mice were sacrificed on the 22nd day, and the thickness of the right ear of the mouse was measured by thickness gauge before sacrifice, the ear tissue of the mouse was collected, and blood was collected from the tail of the mouse, and the serum was isolated for immunoglobulin analysis. The mouse ear tissue was placed in a 50 mM phosphate buffer solution containing protease inhibitors, homogenized, and then centrifuged at 18,870 g for 30 minutes. The supernatant was taken to measure the tumor necrosis factor (Tumor necrosis) in the supernatant by enzyme immunoassay. The content of factor-α, TNF-α).

Fig. 6A is a bar graph showing the results of the thickness of the right ear of each group of mice in a model test of atopic dermatitis. Figures 6B to 6C are bar graphs of the amount of IgE and IgG1 in the serum of each group of mice standardized in the placebo group in the model test of atopic dermatitis. Fig. 6D is a bar graph of the amount of TNF-α expression in the ear tissue of each group of rats in a model group of atopic dermatitis in the placebo group. The results of Figures 6B to 6D are expressed as 100% of the placebo group, and the normal and experimental groups were compared with the placebo group, respectively. The * mark indicates that compared with the placebo group. There was a statistically significant difference (p < 0.05).

According to the results of Figures 6A to 6D, the ear thickness, serum IgE, IgG1 and ear tissue of the mice fed the GMNL-133 live bacteria (experimental group) in the atopic dermatitis mouse model were shown. The amount of TNF-α secreted was significantly decreased compared with the mice that were not fed the live strain of the honeysuckle strain GMNL-133 (placebo group). Therefore, the strain of the honeysuckle strain GMNL-133 of the present invention can significantly improve the symptoms of atopic dermatitis and also has an inhibitory effect on allergic reactions in the living body.

<110>Jingyue Biotechnology Co., Ltd.

<120> Composition of Lactobacillus paracasei GMNL-133 for improving atopic dermatitis or other allergic diseases and use thereof

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<210>SEQ ID NO: 1

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<212>DNA

<213> Lactobacillus paracasei

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<210>SEQ ID NO: 2

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<212>DNA

<213>Artificial sequence

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<223>Chemical synthesis sequence

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Figure 1 is a pattern of the isolate GMNL-133 under the microscope.

Figure 2 is a RAPD electropherogram of Lactobacillus paracasei GMNL-133 and other commercially available products.

Fig. 3 is a bar graph showing the amount of IFN-γ produced by the spleen cells of the lactic acid bacteria strain GMNL-133 and other commercially available products, respectively.

Figures 4A to 4B are bar graphs showing the amount of IgE and IgG2a in the serum of each group normalized by the amount of placebo in the sensitized mouse model test. The * mark indicates statistically significant compared with the placebo group. Significant difference (p < 0.05).

Figures 5A to 5D are the sensitized mouse model experiments. The expression levels of TGF-β, IFN-γ, IL-10 and IL-17 in the spleen cell culture medium of each group were standardized by the performance of the placebo group. Bar graphs, * markers indicate statistically significant differences (p < 0.05) compared to placebo groups.

Fig. 6A is a bar graph showing the results of right ear thickness in each group of mice in a model of atopic dermatitis. The * mark indicates a statistically significant difference (p < 0.05) compared with the placebo group.

Figures 6B to 6C are bar graphs of the IgE and IgG1 expression levels in the serum of each group of rats in the atopic dermatitis model, which were normalized by the amount of the placebo group, and the * mark indicates that compared with the placebo group. There was a statistically significant difference (p < 0.05).

Fig. 6D is a bar graph of the expression of TNF-α in the ear tissue of each group of mice in a model of atopic dermatitis. The * mark indicates that compared with the placebo group. There was a statistically significant difference (p < 0.05).

Claims (10)

An isolated lactic acid strain GMNL-133, which is deposited in the Food Industry Research and Development Institute (FIRDI), Taiwan, under the registration number BCRC 910520. A use of the Lactobacillus paracasei GMNL-133 as claimed in claim 1 for the manufacture of a composition for the treatment of atopic dermatitis. A use of the Lactobacillus paracasei GMNL-133 as claimed in claim 1 for the manufacture of a composition for the treatment of allergic diseases. The use according to the second or third aspect of the patent application, wherein the lactic acid strain GMNL-133 is a living bacteria. The use of claim 2, wherein the composition is a pharmaceutical composition or a food composition. A composition for treating atopic dermatitis, comprising the lactic acid bacteria strain GMNL-133 according to claim 1 of the patent scope, and the content of the lactic acid strain GMNL-133 is effective for improving atopic skin inflammation. A composition for treating allergy symptoms, comprising the lactic acid bacteria strain GMNL-133 according to claim 1 of the patent application, and the content of the lactic acid bacteria strain GMNL-133 is effective for improving allergy diseases. The composition of claim 6 or 7, wherein the lactic acid strain GMNL-133 is a live bacterium. The composition of claim 6 or claim 7, wherein the composition is a pharmaceutical composition or a food composition. The composition according to claim 6 or 7, further comprising: at least one other strain selected from Lactobacillus acidophilus , Lactobacillus plantarum , Bifidobacterium longum , Lactobacillus fermentum , Lactobacillus bulgaricus , Streptococcus thermophilus , Lactobacillus cremors , Lactobacillus rhamnosus ), a group consisting of Lactobacillus reuteri and any combination of the above.