HK40080024B - Composition containing lactic acid bacteria - Google Patents

Description

Compositions containing lactic acid bacteria

Technical Field

This invention relates to a composition containing lactic acid bacteria that has anti-allergic effects.

Background Technology

Allergic diseases are controlled by the interaction of various factors, such as genetic factors and environmental factors (e.g., the birth environment during infancy and early childhood, gut microbiota, etc.).

It is believed that gut bacteria influence the development of the host's immune system (Non-Patent Literature 1). Lactic acid bacteria are cited as an example of gut bacteria associated with allergic diseases. Furthermore, reports indicate that the environment in which infants and young children live with pets is an important environmental factor in allergic diseases. For instance, human-pet microbial crosstalk resulting from contact with pets (dogs, cats, etc.) during infancy can suppress allergic reactions (Non-Patent Literature 2 and 3). Fujimura et al. reported a significant increase in the proportion of *Lactobacillus Johnsonii* in the gut microbiota of mice exposed to house dust in a dog-raising environment. *Lactobacillus Johnsonii* plays an important role in suppressing allergic respiratory diseases (Non-Patent Literature 4).

On the other hand, Taylor et al. administered Lactobacillus acidophilus to infants born to mothers with allergic diseases until 6 months of age to analyze the effect of lactic acid bacteria on allergic attacks. The results showed that at 1 year of age, there was no significant difference in the incidence of atopic dermatitis between the administration group and the placebo group, and the positive rate of IgE antibodies was higher in the Lactobacillus acidophilus administration group (non-patent literature 5).

The above indicates that lactic acid bacteria have a certain impact on allergic diseases, but the results show the opposite (Non-patent literature 5 shows a negative result, while non-patent literature 4 shows a positive result), and further analysis is needed to determine which bacterial species are more effective and practical in inhibiting allergies.

Existing technical documents

Non-patent literature

Non-patent literature 1: Macphers and Harris, Nat Rev Immunol. 4:478-485 2004.

Non-patent literature 2: Hesselmar et al., Clin Exp Allergy. 29:611-617 1999.

Non-patent literature 3: Fall et al., JAMA Pediatr. 169e153219 2015

Non-patent literature 4: Fujimura et al., Proc Natl Aca Sci USA 14:805-810 2014.

Non-patent literature 5: Tylor et al., J Allergy Clin Immuno. 119:184-191 2007.

Summary of the Invention

In view of the above, the objective of the present invention is to provide a composition containing lactic acid bacteria that is effective in suppressing allergic reactions.

The inventors believe that canine gut bacteria have certain effects on the host's immune system and compared the gut microbiota of healthy dogs and atopic dogs, selecting bacteria that are dominant in healthy dogs. Specifically, among 240 strains isolated from the feces of healthy dogs, 184 strains belonged to the selected bacteria, and these bacteria were systematically classified and analyzed based on 16S rRNA genes. The results identified 13 species from 4 genera: Bacteroides, Enterococcus, Lactobacillus, and Streptococcus. Among these, Lactobacillus animalis, a Lactobacillus species that has not been previously reported to be associated with allergic reactions, was selected as the subject of analysis.

When the inventors orally administered *Lactobacillus animalis* to mouse models of atopic dermatitis and allergic asthma, they observed its effect in suppressing various symptoms induced by allergies. Furthermore, they found that *Lactobacillus animalis* exhibited a higher anti-allergic effect than *Lactobacillus johnsonii*, which had previously been reported to have anti-allergic effects.

This invention is based on the above insights.

That is, the present invention relates to the following (1) to (7).

(1) A composition comprising lactic acid bacteria belonging to animal lactobacillus or a bacterial cell treatment thereof.

(2) The composition according to (1) above, wherein the lactic acid bacteria are animal lactobacillus strains preserved with accession numbers NITE BP-03137, NITE BP-03138, NITE BP-03139 and/or NITE BP-03140.

(3) The lactic acid bacteria or composition according to (1) or (2) above have anti-allergic activity.

(4) The composition according to any one of (1) to (3) above, wherein the composition is a pharmaceutical composition.

(5) The composition according to any one of (1) to (3) above, wherein the composition is a feed composition.

(6) The composition according to any one of (1) to (3) above, wherein the composition is a sanitary composition.

According to the present invention, a lactic acid bacteria strain can be provided that exhibits a higher anti-allergic effect than previously reported lactic acid bacteria strains.

Attached Figure Description

Figure 1 shows the analysis results using the atopic dermatitis model (1). The number of scratching behaviors per hour in the model mice was measured (n=8) 4 weeks (A) and 7 weeks (B) after the start of transdermal administration of the mite antigen. Data represent the mean, and error bars represent the standard error. **P<0.01.

Figure 2 shows the analytical results using an atopic dermatitis model (2). The results are obtained by scoring skin symptoms weekly from the start of transdermal administration of the mite antigen up to week 10 (left panel) (n=8). Data represent the mean, and error bars represent the standard error. Also shown are photographs of the skin symptoms of the model mice after mite antigen administration (right panel).

Figure 3 shows the analytical results using the atopic dermatitis model (3). The results are obtained by measuring the thickness of the back skin weekly from the start of transdermal administration of the mite antigen until week 10 (n=8). Data represent the mean, and error bars represent the standard error. **P<0.01, *P<0.05.

Figure 4 shows the analytical results using the atopic dermatitis model (4). The results are obtained by counting the number of CD3 ⁺ CD4 ⁺ T cells (A), CD19 ⁺ IgE ⁺ B cells (B), and CD11c ⁺ CD40 ⁺ dendritic cells (C) present in auricular lymph nodes collected from model mice the day after final administration of the mite antigen (n=8). Data represent the mean, and error bars represent standard errors. **P<0.01, *P<0.05. Intact represents results using mice not treated with mites, and Control represents results using model mice not treated with lactic acid bacteria (the same applies in the following figures).

Figure 5 shows the analysis results using the atopic dermatitis model (5). The amounts of various cytokines produced in auricular lymph nodes collected from model mice the day after final administration of the mite antigen were measured (A: IL-4, B: IL-9, C: IL-13, D: IL-17, E: TNFα) (n=8). ND is below the detection limit. Data represent the mean, and error bars represent the standard error. **P<0.01, *P<0.05.

Figure 6 shows the analytical results using the atopic dermatitis model (6). The amounts of various cytokines produced in auricular skin tissue collected from model mice the day after final administration of the mite antigen were measured (A: IL-1α, B: IL-4, C: IL-5, D: IL-9, E: IL-13, F: IL-17) (n=8). Data represent the mean, and error bars represent the standard error. **P<0.01, *P<0.05.

Figure 7 shows the analytical results using the atopic dermatitis model (7). The amounts of various cytokines produced in auricular skin tissue collected from model mice the day after final administration of mite antigen were measured (A: IL-33, B: TNFα, C: TSLP) (n=8). Data represent the mean, and error bars represent the standard error. *P<0.05.

Figure 8 shows the analytical results using the atopic dermatitis model (8). Serum was prepared from blood collected from model mice the day after the final administration of the mite antigen, and the total IgE content in the serum was measured (n=8). Error bars represent standard errors.

Figure 9 shows the analytical results using an allergic asthma model (1). The results are obtained by counting the number of CD3 ⁺ CD4 ⁺ T cells (A), CD19 ⁺ IgE ⁺ B cells (B), and CD11c ⁺ CD40 ⁺ dendritic cells (C) present in hilar lymph nodes collected from model mice the day after final induction of the mite antigen (n=8). Data represent the mean, and error bars represent the standard error. **P<0.01, *P<0.05.

Figure 10 shows the analysis results using an allergic asthma model (2). The amounts of various cytokines produced in hilar lymph nodes collected from model mice the day after final induction with mite antigen were measured (A: IL-4, B: IL-5, C: IL-9, D: IL-13, E: IL-17) (n=8). ND is below the detection limit. Data represent mean values, and error bars represent standard errors. **P<0.01, *P<0.05.

Figure 11 shows the analytical results using an allergic asthma model (3). The results are obtained by counting eosinophils (A) and neutrophils (B) in bronchoalveolar lavage fluid collected from model mice the day after final induction with mite antigen (n=8). Data represent the mean, and error bars represent the standard error. **P<0.01, *P<0.05.

Detailed Implementation

The first embodiment of the present invention is a composition containing lactic acid bacteria belonging to Lactobacillus animalis or a processed product thereof (hereinafter also referred to as "the composition of the present invention"). As used in embodiments of the present invention, *Lactobacillus animalis*, for example, strains whose gene sequences have been registered with NCBI accession number NZ_AYYW00000000.1 are preferred, in addition to strains whose gene sequences have been registered with NCBI accession number NZ_AYYW00000000.1. These strains are those specifically deposited on February 21, 2020 (the original deposit date) at the Patent Microbial Collection Center (NPMD) of the Biotechnology Center of the Technical Base for Product Evaluation, Independent Administrative Institution (Postal Code 292-0818, 2-5-8 Kazusa Kamata, Kisarazu City, Chiba Prefecture, Japan, with accession numbers NITE P-03137 (identification name: L11-2), NITE P-03138 (identification name: L13-1), NITE P-03139 (identification name: L41-1), and NITE P-03140 (identification name: M08-1). It should be noted that the specific strains with accession numbers NITE P-03137, NITE P-03138, NITE P-03139, and NITE P-03140 were subsequently transferred from their original depositary institutions to international depositary institutions based on the Budapest Treaty (date of issue of the "Certificate of Accession" and "Certificate of Viability" of the original deposit: March 4, 2021), with accession numbers NITE BP-03137, NITE BP-03138, NITE BP-03139, and NITE BP-03140, respectively. These deposited strains can be obtained from the aforementioned depositary institutions.

There are no particular limitations on the culture method for *Lactobacillus animalis*. As a method for culturing lactic acid bacteria, any method that is commonly chosen by those skilled in the art is acceptable. For example, it can be cultured at a temperature of 20–50°C, preferably 25–40°C, under anaerobic conditions.

There are no particular limitations on the culture medium used for the propagation of *Lactobacillus animalis*, and any culture medium commonly selected by those skilled in the art can be used. For example, any culture medium containing the following components in a composition suitable for the strain is acceptable: carbon source (glucose, galactose, mannose, lactose, sucrose, cellobiose, trehalose, etc.), nitrogen source (ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, etc.), inorganic salts (sodium chloride, potassium chloride, potassium sulfate, magnesium sulfate, calcium chloride, calcium nitrate, etc.), and organic components (peptone, yeast extract, meat extract, soybean flour, etc.). MRS medium, LBS medium, etc., are preferred.

Examples of bacterial cell treatment products of the present invention include bacterial cultures and fermentation products, in which the contained lactic acid bacteria can be either live or dead. Furthermore, examples of bacterial cell treatment products include substances obtained by subjecting lactic acid bacteria to processes such as heating, paste formation, drying, freezing, lysis, disruption, and extraction, as well as supernatants obtained by removing the solid components of bacterial cell fragments, bacterial cultures, and fermentation products, but are not limited to these.

In addition, the compositions of the present invention may contain other substances besides *Lactobacillus animalis* or its cell-processed form. There are no particular limitations on these other substances; for example, they may contain lactose, glucose, mannitol, sucrose, dextrin, cyclodextrin, starch, cellulose, collagen, citric acid, acetic acid, salt, vitamins, etc.

The compositions of the present invention have the effect of inhibiting allergic reactions and can be provided, for example, as pharmaceutical compositions, feed compositions, and hygiene compositions, but are not limited to these compositions, depending on the intended use.

When the composition of the present invention is a pharmaceutical composition, its dosage form is not particularly limited. Examples include tablets, capsules, granules, powders, syrups, liquid preparations, suppositories, or injections. These preparations can be prepared according to conventional methods. Furthermore, in the case of liquid preparations, they can be dissolved or suspended in water or other suitable solvents at the time of use. Additionally, tablets and granules can be coated using known methods. In the case of injections, the antibodies or functional fragments of the present invention can be prepared by dissolving them in water, and can be dissolved in physiological saline or glucose solution as needed. Buffers and preservatives can also be added.

When the compositions of the present invention are provided as hygiene compositions, their form is not particularly limited. For example, they can be toothpaste, skin cream, soap, shampoo, cosmetic, aerosol, spray, coating, etc., and can also be hygiene compositions for non-human or non-animal use.

Furthermore, the compositions of the present invention can be feed compositions that can be ingested by non-human animals and pharmaceutical compositions for animals. Animal feed mentioned herein includes not only food consumed as a source of nutrition for animals, but also animal supplements given as animal sustenance.

The second embodiment of the present invention is a method for preventing allergic reactions (hereinafter also referred to as "the prevention method of the present invention"), which includes administering the pharmaceutical composition of the present invention to a subject.

In this context, "prevention" refers to measures taken to prevent allergic reactions from occurring in advance.

The object of the prevention method of the present invention is not particularly limited, and can be any animal classified as a mammal, such as pets such as dogs, cats, and rabbits, and livestock such as cattle, pigs, sheep, and horses, in addition to humans. Particularly preferred "mammals" are humans and dogs.

In cases where this instruction manual is translated into English and includes the singular forms of the words “a,” “an,” and “the,” both singular and plural forms are included unless explicitly stated in the text.

The following embodiments are shown to further illustrate the present invention, but these embodiments are merely illustrative examples of implementation of the present invention and do not limit the scope of the present invention.

Example

1. Experimental Methods

1-1. Isolation of lactic acid bacteria from feces and systematic classification

Of the 240 bacterial strains isolated from the feces of 16 healthy dogs, 184 strains that were dominant in the gut microbiota of healthy dogs compared to those of atopic dogs were selected for systematic classification and analysis. Specifically, feces collected from dogs were cultured overnight at 37°C under anaerobic conditions using MRS or LBS liquid medium. After culture, bacteria were isolated using MRS or LBS plates. Purification was performed at least three times. The systematic classification of bacteria was based on 16S rRNA sequencing. As a result, 13 species from 4 genera (Bacteroides, Enterococcus, Lactobacillus, and Streptococcus) were identified. Among these, *Lactobacillus animalis*, a *Lactobacillus* species that has not been previously reported to be associated with allergic reactions, was selected as the target for analysis. It should be noted that *Lactobacillus johnsonii*, considered to be associated with allergic reactions, was used as a positive control in this example. The animal lactobacillus used in this embodiment was prepared by mixing strains preserved with accession numbers NITE BP-03137, NITE BP-03138, NITE BP-03139 and NITE BP-03140 in a ratio of 1:1:1:1.

1-2. Analysis using disease model mice

All animal experiments used in this embodiment were conducted after being approved by the Animal Experiment Special Committee of Azabu University.

1-2-1. Atopic dermatitis model

Six-week-old female NC/Nga mice (Charles River Laboratories Japan Co., Ltd.) were used in the experiment. After one week of acclimatization, each mouse was orally administered 0.2 ml ( ^10⁹ ) of *Lactobacillus animalis* or *Lactobacillus johnsonii*. Two weeks later, sensitization with mite antigen (*Dermatophagoides farinae*) was initiated. Before sensitization with mite antigen, the skin on the back of the neck was shaved. After 10 tape removals on the back of the neck, 30 μl (0.25 mg/ml) of mite suspension (10 μl) was transdermally administered to each ear using a pipette. The mite antigen was administered twice a week for 12 weeks, with weekly monitoring of pruritus behavior, scoring of skin symptoms, and measurement of back skin thickness.

Monitoring of pruritus behavior involved recording mouse behavior using a camera, defining each instance of pruritus behavior as biting or scratching the mite antigen-applied area (ear and back skin) with the hind limbs, forelimbs, or tongue. The number of pruritus behaviors was recorded over a 60-minute period.

Skin observation involves scoring the crusting, ulceration, and redness of the skin on the auricle and back (scores are based on a scale of 0 to 4), and the cumulative scores are used as a total score for evaluation.

The thickness of the skin on the back is measured once a week using calipers.

The day after final administration of the mite antigen, blood was collected from mice under isoflurane inhalation anesthesia, followed by euthanasia, and sampling of auricular lymph nodes and auricular skin was performed.

After separating serum from the blood, the total amount of IgE in the blood was quantified using the ELISA method.

Auricular lymph nodes were obtained by separating cells using flow cytometry based on cell surface antigens after single-cell isolation. Helper T cells (CD3 ⁺ CD4 ⁺ T cells) from the obtained cells were cultured for 24–96 hours in the presence of anti-CD3 and anti-CD28 antibodies (VERITAS Co., Ltd.), and the production of various cytokines in the culture supernatant was measured using ELISA.

The collected auricular skin was frozen in liquid nitrogen, and the amounts of various cytokines in the supernatant obtained by homogenization in 500 μl of PBS using an electric homogenizer were measured by ELISA.

1-2-2. Allergic Asthma Model

Six-week-old female BALB/c mice (Charles River Laboratories Japan Co., Ltd.) were used in the experiment. After one week of acclimatization, each mouse was orally administered 0.2 ml ( ^10⁹ ) of *Lactobacillus animalis* or *Lactobacillus johnsonii*. Two weeks later, sensitization to the mite antigen (durine mite, *Dermatophagoides farinae*) was initiated. Sensitization to the mite antigen was performed under isoflurane inhalation anesthesia by pipetting 30 μl (1 mg/ml) of mite suspension intranasally. The mite antigen was administered once a week for three consecutive weeks. In the fourth week, the same method was used to induce sensitization in the fourth week by pipetting 5 μl (0.2 mg/ml) of mite suspension intranasally for three consecutive days under isoflurane inhalation anesthesia. The day after the final induction of the mite antigen, blood was collected from the mice under isoflurane inhalation anesthesia, followed by euthanasia, and samples were taken from the hilar lymph nodes and bronchoalveolar lavage fluid.

Bronchoalveolar lavage fluid is used to count the number of eosinophils and neutrophils in the lavage fluid.

Hilar lymph nodes were obtained by separating cells using flow cytometry based on cell surface antigens after single-cell isolation. Helper T cells (CD3 ⁺ CD4 ⁺ T cells) from the obtained cells were cultured for 24–96 hours in the presence of anti-CD3 and anti-CD28 antibodies, and the production of various cytokines in the culture supernatant was measured using ELISA.

1-3. Statistical Analysis

For the obtained data, the mean and standard error of each group were calculated, and multiple comparison tests were performed for each experiment across all groups. In the multiple comparison tests, Bartlett's method was used to test variance, and Dunnett's test was employed when variance was considered. For each test item, statistically significant differences between groups were analyzed at significance levels of 5% and 1%.

2. Results

2-1. Atopic dermatitis model

Atopic dermatitis is a pathological condition characterized by alternating episodes of itching and skin inflammation that exacerbate symptoms. Therefore, this study investigated the effects of lactic acid bacteria on pruritus by measuring pruritus behavior in a mouse model every hour immediately following weekly administration of a mite antigen.

Based on the analysis results of a mouse model of atopic dermatitis induced by administration of mite antigen, it was found that the mice given Lactobacillus animalis and the mice given Lactobacillus johnsonii showed significantly fewer scratching behaviors compared with the control mice (without lactobacillus). (Figures 1A and 1B)

Furthermore, Figure 2 shows the results of scoring atopic skin symptoms during mite antigen administration. Atopic symptom presentation varied considerably among individuals; although no statistically significant differences were found, the *Lactobacillus animalis* and *Lactobacillus johnsonii* groups showed greater symptom relief compared to the control group. Moreover, when comparing the *Lactobacillus animalis* and *Lactobacillus johnsonii* groups, the *Lactobacillus animalis* group showed a greater degree of symptom relief.

As the symptoms of atopic dermatitis worsen, epidermal thickening and cellular infiltration occur, leading to an increase in skin thickness. Therefore, the thickness of the skin on the back of model mice was measured. The results showed a significant reduction in skin thickness in both the *Lactobacillus animalis* and *Lactobacillus johnsonii* treatment groups compared to the control group. The reduction in thickness observed in the *Lactobacillus animalis* treatment group was statistically significant, even compared to the *Lactobacillus johnsonii* treatment group (Figure 3).

Following the final dissection, auricular lymph nodes were collected, and the number of allergy-related immune cells (helper T cells (CD3 ⁺ CD4 ⁺ T cells), IgE-positive B cells (CD19 ⁺ IgE ⁺ B cells), and activated dendritic cells (CD11c ⁺ CD40 ⁺ dendritic cells)) in the lymph nodes was counted using flow cytometry. The results showed a decrease in the number of cells in both the *Lactobacillus animalis* and *Lactobacillus johnsonii* treatment groups. In particular, a significant reduction in helper T cells and IgE-positive B cells was observed compared to the control group, with the reduction being most pronounced in the *Lactobacillus animalis* treatment group (Figure 4).

Next, helper T cells collected from auricular lymph nodes were cultured in the presence of CD3 and CD28 antibodies, and the production levels of inflammatory cytokines (IL-4, IL-9, IL-13, IL-17, and TNFα) were measured. The results showed that, compared with the control group, the production levels of any inflammatory cytokine were significantly reduced in both the *Lactobacillus animalis* and *Lactobacillus johnsonii* treatment groups (Figure 5). In particular, a significant reduction in the production levels of inflammatory cytokines was observed in the *Lactobacillus animalis* treatment group compared to the *Lactobacillus johnsonii* treatment group.

The levels of inflammatory cytokines (IL-1α, IL-4, IL-5, IL-9, IL-13, and IL-17) in auricular skin tissue were measured. Similar to the results in auricular lymph nodes, a significant reduction in cytokine levels was observed in both the *Lactobacillus animalis* and *Lactobacillus johnsonii* treatment groups (Figure 6). Furthermore, the results of quantifying pruritus-related cytokines (IL-33, TNFα, and TSLP (thymic stromal lymphopoietin)) in auricular skin tissue are shown in Figure 7. Although no significant reduction in TSLP was observed, a significant reduction in the production of keratinocyte-derived cytokines such as IL-33 and TNFα was observed in both the *Lactobacillus animalis* and *Lactobacillus johnsonii* treatment groups compared to the control.

Next, the total IgE level in serum prepared from model mice was measured. The results showed a significant reduction in total IgE levels in both the *Lactobacillus johnsonii* treatment group and the *Lactobacillus johnsonii* treatment group (Figure 8).

2-2. Allergic Asthma Model

After final dissection of the allergic asthma model mice, hilar lymph nodes were collected. Similar to the atopic dermatitis model, the number of allergy-related immune cells (helper T cells, IgE-positive B cells, and activated dendritic cells) in the lymph nodes was analyzed using flow cytometry. The results showed a significant reduction in the number of all allergy-related immune cells in the hilar lymph nodes of both the *Lactobacillus johnsonii* and *Lactobacillus johnsonii* treatment groups. The reduction was particularly significant in the *Lactobacillus johnsonii* treatment group (Figure 9).

Next, helper T cells collected from hilar lymph nodes were cultured in the presence of CD3 and CD28 antibodies, and the production of inflammatory cytokines (IL-4, IL-5, IL-9, IL-13, and IL-17) was measured. The results showed that, compared with the control group, the production of any inflammatory cytokine was significantly reduced in both the *Lactobacillus animalis* and *Lactobacillus johnsonii* treatment groups (Figure 10). In particular, a significant reduction in the production of inflammatory cytokines was observed in the *Lactobacillus animalis* treatment group compared to the *Lactobacillus johnsonii* treatment group.

At the final dissection of the allergic asthma model mice, the lungs were washed with PBS, and the number of eosinophils and neutrophils in the washing fluid (bronchoalveolar lavage fluid, BALF) was counted using flow cytometry (Figure 11). For both cell species, a decrease in cell number was observed in both the *Lactobacillus animalis* and *Lactobacillus johnsonii* groups, with a particularly significant decrease observed in the *Lactobacillus animalis* group.

Industrial availability

This invention provides a composition that can suppress allergic reactions, and its potential applications are expected not only in the medical and veterinary fields, but also in the manufacture of hygiene products.

Collection Number

Accession number NITE BP-03137

Accession number NITE BP-03138

Accession number NITE BP-03139

Accession number NITE BP-03140.

Claims (4)

1. A composition with anti-allergic activity, comprising lactic acid bacteria belonging to *Lactobacillus animalis* or a processed form thereof. The lactic acid bacteria mentioned are *Lactobacillus animalis* strains preserved with accession numbers NITE BP-03137, NITE BP-03138, NITE BP-03139, and NITE BP-03140. The bacterial culture product is a bacterial culture or fermentation product. 2. The composition according to claim 1, wherein the composition is a pharmaceutical composition. 3. The composition according to claim 1, wherein the composition is a feed composition. 4. The composition according to claim 1, wherein the composition is a sanitary composition.