TWI418356B - Lactobacillus paracasei strain lt12 as immunity regulatory agent - Google Patents

Description

Lactobacillus paracasei LT12, which acts as an immunomodulator

The present invention relates to a novel Lactobacillus paracasei and its use for modulating an immune response and treating an allergy-related disease.

Allergies are an over-immune reaction of the immune system to foreign substances, such as bacteria, pollen or dust. The foreign body is commonly known as an "allergen." These allergies may be considered harmful or dangerous by the human body to activate part of the immune system, but in fact the allergen does not cause infection or toxicity. When the human body interacts with allergens to cause an immune response, histamine and prostaglandins may then be released to cause allergic symptoms, including respiratory inflammatory reactions, atopic dermatitis, allergic conjunctivitis, urticaria, eczema, specific Gastrointestinal disease or asthma.

It has been reported that children in developed countries are more susceptible to allergies because they are over-emphasizing public health and often vaccinated or use antibiotics, thus reducing the chance of infection, which usually fights allergies or asthma. In medicine, this phenomenon is called the "hygiene hypothesis", which mainly discusses the lack of contact with infectious agents in childhood and tends to an immune response to Th-2, such as overproduction of IL-4 (interleukin-4). ), IL-5 and IL-13. These Th-2 cytokines attract eosinophils, basophils, and mast cells to the site of inflammation. These cells can be used alone or in combination with immunoglobulin E (IgE). An allergic reaction occurs. In addition, IL-4 and IL-13 can promote B cells to perform an isotype switch of antibodies to increase the amount of IgE in the blood. According to the "hygiene hypothesis", immune responses induced by Th-1 (T helper 1) cells, such as promoting the expression of IFN-γ, may be effective in slowing allergic reactions.

Many studies have pointed out that Lactobacillus sp. has the ability to regulate immune responses in organisms, including promoting the secretion of cytokines, activating macrophages and natural killer cells and antibody production (Madsen et al. , 2001, Gastroenterology 121). :580-591). Collins et al. (US Pat. No. 7,390,519) have shown that strains of Lactobacillus salivarius AH102, AH103, AH105, AH109 or AH110 can be used as an anti-inflammatory biotherapeutic agent to effectively prevent and/or treat inflammatory reactions. Hsu et al. (US Pat. No. 6,994,848) also discloses that Lactobacillus paracasei GM-080 promotes the secretion of IFN-[gamma] and can be used to treat allergy-related diseases.

However, a variety of lactic acid bacteria present in nature have not yet been fully discovered and fully studied. The present invention provides a composition comprising a novel Lactobacillus paracasei and its immunomodulatory application, which differs from the lines disclosed in the related prior literature.

One aspect of the present invention provides a novel Lactobacillus paracasei LT12 having an immunomodulatory function.

Another aspect of the present invention provides a composition comprising the above-described Lactobacillus paracasei LT12 and a carrier. The composition may be a food (eg, yogurt, biscuits, cereals, chocolates, and snack bars), a beverage (eg, tea, non-alcoholic beverages, milk, and juice), or a drug containing a medically acceptable carrier. .

Yet another aspect of the present invention provides a method of modulating an immune system (e.g., innate immunity or adaptive immunity) or treating allergy, and a composition thereof, which is administered to an individual in need thereof An effective amount of any combination of the above. The individual (eg, a human patient) can be a patient with an IgE-mediated immediate hypersensitivity or a T cell-mediated delayed hypersensitivity.

Still another aspect of the present invention also encompasses the use of the aforementioned Lactobacillus paracasei LT12 for the preparation of a medicament for modulating an immune system or treating allergy.

One or more specific embodiments of the invention are described below. This hair Other features and advantages will be apparent from the following figures, examples, and claims.

The present invention provides a novel Lactobacillus paracasei strain, designated LT12, which can be used to modulate immune responses and treat allergies. The strain LT12 was deposited on September 21, 2009 at the American Agricultural Stem Breeding Center (1815N. University Street, Peoria, Illinois, 61601, U.S.A.) under the accession number NRRL-B50327. The strain LT12 was deposited with the Food Industry Development Research Institute of the Republic of China on February 10, 2010. The deposit number is BCRC910461.

The LT12 strain of Lactobacillus paracasei is isolated from human feces and must be separated within 12 hours of sample sampling. A sample weighing (about one gram) was placed in a homogenizer (Stomacher 400, Seward, UK) and homogenized for 30 seconds, followed by a pre-reduced brain heart infusion broth. Diluted and then cultured in a suitable selective medium. Appropriate dilutions were applied to lactic acid bacteria acetate medium (Rogosa Acetate agar, Difco, USA) or lactic acid bacteria acetate medium (Rogosa Acetate agar, Difco, USA) containing 12 μg/ml vancomycin (vancomycin, Sigma, USA). to cultivate all Lactobacillus and vancomycin-resistant lactic acid bacteria, for example: Lactobacillus paracasei group (L.paracasei group), comprising Lactobacillus paracasei (L. paracasei), Lactobacillus casein (of L.casei) Leiman Shi and Lactobacillus (L.rhamnosus) and so on. The API 50 CHL system (bioMérieux, Inc. USA) was used to identify strains in vancomycin-resistant Lactobacillus paracasei. The identification results are shown in Table 1.

The microbiological characteristics of LT12 are as follows:

(1) Morphological characteristics

(a) Cell shape and size: After the cells were cultured overnight at 37 ° C in MRS medium, the shape observed under a microscope was rod-shaped and the edges were round.

(b) Activity: Yes

(c) Flagella: none

(d) Spore formation: none

(e) Gram stain: positive

(2) Cultivation characteristics:

(a) Medium: MRS medium (Difco, USA), final pH 6.2-6.5.

(b) Culture conditions: 37 ° C facultative anaerobic

(3) Physiological characteristics:

(a) Catalase: positive

(b) Oxidase: negative

(c) API 50 CHL test: test results are shown in Table 1.

(4) Genetic characteristics:

As shown in Example 2, the Random Amplification Polymorphic DNA (RAPD) method was applied to LT12, Lactobacillus paracasei (Cell biotech Co., Ltd., Korea) (hereinafter referred to as LP-CBT), Lactobacillus paracasei GMNL32. (hereinafter referred to as: GMNL32), Lactobacillus paracasei subsp. paracasei BCRC 14023 (hereinafter referred to as: BCRC 14023) and Lactobacillus paracasei subsp. paracasei BCRC 12188 (hereinafter referred to as BCRC 12188) were analyzed for RAPD. The 12 random nucleic acid primers are listed in Table 2. Electropherogram results show that LT12 has different strip modes (as shown in Table 3). Based on the above identification results, LT12 is a novel strain of Lactobacillus.

According to a particular embodiment of the invention, LT12 or a functional variant thereof in the form of a living cell can be used. According to another embodiment of the invention, a form of non-viable cells, such as a heat lethal cell, comprising a beneficial factor produced by strain LT12 can be used. The term "functional variant strain" as used herein refers to a strain having the same characteristics as LT12, which achieves the same efficacy.

The present invention provides an LT12 strain and a functional variant thereof having an activity of modulating an immunological activity and/or an antiallergic reaction. The term "modulating immune activity" as used herein refers to an immune system that is self-steady or low. Plaque activity (immunostimulation activity), or suppression of excess or excessive immune response to restore to the appropriate level (inhibition of immune activity). In another aspect, this is used to regulate the balance of immune cells (such as T cells, B cells, or macrophages) to stabilize the overall immune response. For example, but not limited to, stimulating or inhibiting the production of cytokines, activating lymphocytes, balancing Th1/Th2 activity, and suppressing allergic reactions.

The present invention also provides a LT12 strain or a functional variant thereof which can be used for the treatment of allergies. The term "allergy" as used herein refers to an immune response caused by physiological protective immunity that harms the host. The two most common allergies are immediate allergic reactions mediated by IgE and T-cell-mediated delayed allergic reactions. Clinical symptoms of allergies include allergic rhinitis, allergic asthma, food allergies, allergic dermatitis, eye allergies, and systemic allergic reactions (anaphylaxis). The term "treatment of allergy" as used herein refers to a series of immune responses that reverse the aforementioned immune symptoms, such as, but not limited to, blocking a Th-2 mediated immune response and reducing Th-2. Cytokines (eg, IL-4, IL-5, IL-13) increase Th-1 type cytokines (such as INF-γ) and inhibit the activation or differentiation of IgE ⁺ memory B cells and eosinophils.

According to a specific embodiment of the present invention, it was confirmed by ELISA analysis that LT12 can stimulate peripheral blood mononuclear cells (PBMCs) isolated from donor blood to secrete INF-γ (see Table 4). INF-γ is a cytokine that is important for innate and adaptive immunity against viral and bacterial infections. It is a representative of Th-1 type cytokines, which can inhibit the differentiation of Th-2 type cells, thus reducing the production of L-4, IL-5, IL-13, which stimulates the conversion of immunoglobulins into IgE. , as well as the number of original and IgE ⁺ memory B cells amplified. Therefore, promoting the performance of INF-γ may effectively treat allergies.

According to another embodiment of the present invention, LT12 also stimulates peripheral blood mononuclear cells to secrete other cytokines, including IL-1α, IL-6, which are analyzed by a human cytokine antibody array. , IL-10, IFN-γ, MCP-2, and TNF-α (see Figures 1A-1E and Table 6).

Accordingly, the present invention provides a method and composition for modulating immunity comprising administering to a subject an effective amount of Lactobacillus paracasei LT12 in combination with a pharmaceutically acceptable carrier.

The invention also provides a method and composition for treating allergy comprising administering to a subject an effective amount of Lactobacillus paracasei LT12 in combination with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes, but is not limited to, physiological saline, buffer, dextrose, water, glycerol, ethanol, and related compositions.

Surprisingly, the present inventors have found that a composition comprising LT12 and a second Lactobacillus paracasei also has immunomodulatory and/or antiallergic activity. In one embodiment, the second strain of Lactobacillus paracasei is LP-CBT, and the ratio of LT12 to its bacteria is 100:1. Preferably, the ratio of bacteria to LT12 and the second strain is 2:1. As shown by the results of Example 4, the composition also stimulates peripheral blood mononuclear cells to secrete other cytokines, including IL-1α, IL-6, which are analyzed by a human cytokine antibody array. IL-10, IFN-γ, MCP-2, and TNF-α. In addition, MCP-3 is produced only by peripheral blood mononuclear cells stimulated by the LT12 composition.

According to the present invention, LT12 provides excellent immunomodulatory and antiallergic activity. According to one embodiment of the invention, LT 12 or a combination thereof can be used as an active ingredient of the composition. The composition can be formulated as a food, beverage or medicament. In addition, various additives may be included, and the types of the additives are, for example, but not limited to, artificial colors (for example, β-carotene, annatto red, turmeric, paprika, pharmaceutical, and cosmetic dyes). Etc.), seasonings, flavors, sweeteners, emulsifiers and/or thickeners, preservatives, vitamins and antioxidants (eg vitamins A, C, D, E, B-1, B-5, B-) 6, zinc, selenium, calcium, α-tocopherol, glutathione, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and cysteine ( Cyceine)).

The invention will be further illustrated by the following examples, but the actual invention is not limited to the examples set forth in the specification.

Example 1: Separation of Lactobacillus lacticis strain LT12

Lactobacillus paracasei LT12 is isolated from human feces and must be separated within 12 hours of sample sampling. A sample weighing (about one gram) was placed in a homogenizer (Stomacher 400, Seward, UK) and homogenized for 30 seconds, followed by a pre-reduced brain heart infusion broth. Diluted and then cultured in a suitable selective medium. Appropriate dilutions were applied to lactic acid bacteria acetate medium (Rogosa Acetate agar, Difco, USA) or lactic acid bacteria acetate medium (Rogosa Acetate agar, Difco, USA) containing 12 μg/ml vancomycin (vancomycin, Sigma, USA). in all Lactobacillus culture, and vancomycin resistant lactic acid bacteria, for example: Lactobacillus paracasei group (L.paracasei group), comprising Lactobacillus paracasei (L. paracasei), Lactobacillus casein (of L.casei) Leiman Shi and Lactobacillus (L.rhamnosus) and so on. The API 50 CHL system (bioMérieux, Inc. USA) was used to identify vancomycin-resistant strains of Lactobacillus paracasei.

Example 2: Identification of Lactobacillus paracasei

The API 50 CHL system is used to identify lactic acid bacteria. The properties of the lactic acid bacteria strain are established by a series of enzyme reactions. The results of the API 50 CHL system identification of LT12 are shown in Table 1.

Random amplified polymorphic DNA (RAPD analysis) identification:

The following strains were cultured for 40 hours and then subjected to DNA extraction: LT12, Lactobacillus paracasei (Cell biotech Co., Ltd., Korea) (abbreviated as LP-CBT), Lactobacillus paracasei GMNL32 (abbreviation: GMNL32), Lactobacillus paracasei subsp. paracasei BCRC 14023 (abbreviation: BCRC 14023), Lactobacillus paracasei subsp paracasei BCRC 12188 ( abbreviation: BCRC 12188)., after extraction RAPD analysis to chromosome 12 polymorphic nucleic acid random primers (table 2). The results of RAPD analysis are shown in Table 3. It can be observed by electrophoresis that LT12 has different band patterns from the other four strains.

Comprehensive API 50 CHL identification and RAPD analysis, LT12 is different from the typical Lactobacillus paracasei strain. Therefore, LT12 is a novel strain of Lactobacillus.

Example 3: Allergy regulation function of LT12 and other lactic acid bacteria

Preparation of experimental cells

LT12, LP-CBT and Lactobacillus rhamnosus GG (hereinafter referred to as LGG) were cultured in a lactic acid bacteria MRS culture solution at 37 ° C for 24 hours, and then the bacterial solution was centrifuged at 3000 rpm for 15 minutes. The precipitate was washed twice with 1 ml of sterile buffered saline, and then heated to 95 ° C for 30 minutes to prepare a heat-killing liquid.

Preparation of peripheral blood mononuclear cells (PBMC)

Fresh blood was obtained from healthy donors (two men and three women, ages 24 to 40 years), centrifuged at 2000 rpm for 10 minutes at room temperature, and then a sample of the buffy coat layer was collected. The collected samples were added to an equal amount of dilution (Dilute medium containing RPMI-1640, 2% fetal bovine serum) and slowly transferred to a 4 ml solution of Histopaque-1077 ficoll plus. After centrifugation at 1200 rpm for 30 minutes at room temperature, 8 ml of a washing solution (Wash medium containing RPMI-1640, 2% fetal bovine serum, 1% PBS) was added and uniformly washed twice and PMBC was collected from the interface of the sample. The collected PMBC was resuspended in complete medium (containing RPMI-1640, 10% fetal bovine serum, 1% PBS), and cultured in a 24-well plate at a concentration of 10 ⁶ cells/ml.

Stimulate IFN-γ secretion and cytokine IFN-γ quantification

PBMCs were co-cultured with each of the three strains (LT12, LP-CBT, and LGG) at a given concentration (5 x 10 ⁶ , 5 x 10 ⁷ or 5 x 10 ⁸ CFU/ml). After 48 hours of co-cultivation, the cell supernatant of each well was collected, and the cytokine IFN-γ was quantitatively analyzed by an enzyme immunoassay kit (ELISA kit) as follows. Among them, 5 μg/ml phytohemagglutinin and 10 μg/ml lipopolysaccharides (LPS) were used as positive control groups.

100 μL/well of 4 μg/ml of anti-IFN-γ antibody (RayBio® Human IFN-γ ELISA, RayBiotech Inc., USA) was added to a 96-well plate and allowed to stand overnight at 4 °C. The liquid in the well was removed, washed with 250 μL/well wash buffer, and the liquid in the well was removed, and the above washing steps were repeated 5 times. 200 μL/well of assay diluent was added and allowed to stand at room temperature for 1 hour. The washing was repeated by adding 250 μL/well of the washing buffer, and the above washing steps were repeated 5 times. Then, 100 μL/well of PMBCs sample was added and reacted at room temperature for two hours. After washing five times with 250 μL/well of washing buffer, 100 μL/well of secondary antibody was added to each well for one hour. After washing five times with 250 μL/well of washing buffer, 100 μL/well of horseradish peroxidase (Avidin-HRP) (1:250) was added for 30 minutes. The cells were washed 7 times with 250 μL/well of washing buffer, followed by 100 μL/well of a substance solution. After standing at room temperature for 3 to 15 minutes, the reaction was stopped by adding 50 μL/well of 2N H ₂ SO ₄ . The wells of each well were measured for absorbance at a wavelength of 450 nm, and the concentration of cytokine (IFN-γ) in the sample was converted according to a standard curve. The test results are shown in Table 4.

Among the strains, LT12 has the strongest ability to stimulate IFN-γ production at different cell concentrations.

Example 4: Functional evaluation of LT12 and its compositions for production of cytokines by peripheral blood mononuclear cells

The heat-killing LT12, and its composition, including LT12 and LP-CBT with a ratio of 2:1, were prepared as described above. PBMCs (source C and source E) were incubated with LT12 (5 x 10 ⁷ /ml) and LT12 composition (5 x 10 ⁷ /ml) for 48 hours, followed by human cytokine antibody array (RayBio ^® Human cytokine antibody array , Cat. AAH-CYT-1, RayBiotech Inc., USA) evaluated the induced cytokine secretion profile.

The analysis steps are shown in the description of the analysis kit ( http://www.raybiotech.com/cytokine_antibody_array.asp ). To put it simply, cut the membrane package, carefully pry the membrane into the eight-hole plate attached to the kit, and inject 2 mL/well of blocking buffer along the wall to place it on the shaker. Shake and incubate for 30 minutes at room temperature. The blocking buffer was removed and a 1 mL/well sample was injected. After gently shaking for 2 hours on a shaker, the cells were washed twice with 2 mL/well of Wash Buffer I, and then washed 3 times with 2 mL/well of Wash Buffer II. A 1 mL/well biotin-antibody solution was added and shaken on a shaker for 2 hours. After removing the antibody solution, it was washed twice with 2 mL/well of Wash Buffer I, and then washed 3 times with 2 mL/well of Wash Buffer II. 2 mL/well of horseradish peroxidase avidin (HRP-Streptavidin) was injected and shaken for 2 hours on a shaker. Carefully pry the membrane out of the membrane and discard the remaining liquid, then return to the well plate from which the liquid has been removed, and inject 0.5 mL/well of the detection buffer to avoid the light reaction for 2 minutes. Use a pair of tweezers to clamp the film into the two plastic sheets attached to the kit or a plastic wrap. Place the film together with the plastic sheet in a dark box for 40 seconds in the darkroom and develop. . The positional map of the cytokine antibody array on the membrane is shown in Table 5.

The results of the analysis are shown in Table 6 and Figures 1A-E. There were 5 groups in the experimental analysis group, which were PBMC source C blank group (Fig. 1A), PBMC source C and LT12 co-culture group (Fig. 1B), PBMC source C and LT12 composition co-culture group (Fig. 1C). The blank group of PBMC source E (Fig. 1D) and the PBMC source E and LT12 co-culture group (Fig. 1E). An increase in cytokine secretion was observed in each co-culture test group, including IL- 1α , IL-6, IL-10, INF-γ, MCP-2, and TNF- α , among which the cytokines IL-1 α and INF- γ, MCP-2 and TNF- α were only induced by PBMCs stimulated by the LT12 and LT12 compositions. Comparing the groups of the same PBMC source C, it was found that the ability of the LT12 composition to induce GRO- α , IL-10 and MCP-2 production was higher than that of LT12. In addition, only LT12 compositions can induce PMBCs to exhibit MCP-3.

Those skilled in the art will be able to implement them without departing from the spirit of the invention. Examples of changes and modifications. It is to be understood that the invention is not to be limited by the scope of the embodiments disclosed herein,

The foregoing description and the embodiments may achieve better illustrative effects by the additional drawings. To enhance the description of the invention, the drawings of the appropriate embodiments are set forth herein. It is to be noted that the present invention is not limited to the descriptions set forth herein.

Figure 1 is a photograph showing the analysis of the blots of each group using a human cytokine antibody array (Human cytokine antibody Array): peripheral blood mononuclear cell-derived C blank group (Fig. 1A), stimulator peripherally with LT12 alone Blood mononuclear cell source C (Fig. 1B), LT12 and second Lactobacillus paracasei stimulate peripheral blood monocyte source C (Fig. 1C), peripheral blood mononuclear cell source E blank group (Fig. 1D) and LT12 Stimulate peripheral blood mononuclear cell source E (Fig. 1E). The positional map of the cytokine antibody array on the membrane is shown in Table 5.

<110> Litong Co., Ltd.

<120> Lactobacillus paracasei LT12, which acts as an immunomodulator

<140> 098144461

<141> 2009-12-23

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Claims (8)

An isolated Lactobacillus paracasei strain LT12, wherein the Lactobacillus paracasei strain LT12 is deposited in the American Agricultural Stem Breeding Center, registered as NRRL-B50327, and deposited in the Food Industry Development of the Republic of China Institute, registration number is BCRC910461. A composition comprising Lactobacillus paracasei LT12 as described in claim 1 of the patent application. The composition of claim 2, wherein the Lactobacillus paracasei LT12 is a living body. The composition of claim 2, wherein the Lactobacillus paracasei LT12 is non-activated. The composition of claim 2, which may be a food, a drink, or a drug. The composition of claim 2, which may further comprise a second Lactobacillus paracasei strain. The composition of claim 6, wherein the ratio of the bacteria of the Lactobacillus paracasei to the second Lactobacillus paracasei strain is between 2:1 and 100:1. The composition of claim 6, which may be a food, a drink, or a drug.