JP2023023981A - LACTIC ACID BACTERIA FORMULATION FOR PROMOTING IN VIVO IgA AND IFN-γ PRODUCTION AND ORAL COMPOSITION - Google Patents

Abstract

To provide a probiotic formulation that has high immunostimulation activity, has very high stability as a viable cell, and also has excellent productivity.SOLUTION: The present invention provides a lactic acid bacteria formulation for promoting in vivo IgA and IFN-γ production, comprising a strain belonging to the spore-forming lactic acid bacteria Weizmannia coagulans, which promote IgA and IFN-γ production, a mutant of the strain or a crushed, cultured, extracted, or processed product thereof. The spore-forming lactic acid bacteria Weizmannia coagulansas are preferably Weizmannia coagulans SANK70258.SELECTED DRAWING: Figure 1

Description

The present invention relates to a lactic acid bacterium preparation having a high production-enhancing effect on IgA and IFN-γ, and an oral composition containing this lactic acid bacterium preparation.

The immune system is roughly divided into mucosal immunity and systemic immunity, and the immune response is roughly classified into cell-mediated immunity and humoral immunity. Appropriate maintenance and enhancement of each of these immune systems and immune responses is an important issue not only for maintaining human health but also for improving breeding efficiency in the livestock field.

A living body constantly takes in foreign foreign substances (antigenic substances) through physiological actions essential for maintaining life activities such as respiration and eating and drinking. Therefore, the surfaces of the respiratory and digestive organs are constantly exposed to antigenic substances such as pathogenic bacteria, viruses, pollen, and food-derived allergens. The body is equipped with an immune defense mechanism to eliminate this, and the immune system at the forefront is mucosal immunity. Immunoglobulin A (IgA), which is a type of immunoglobulin, plays a central role. IgA is known to have low antigen specificity and high affinity to various antigens and high neutralizing activity due to its ability to form multimeric structures (Non-Patent Document 1). In other words, IgA can be said to be an important primary defense mechanism for suppressing invasion of pathogenic viruses and allergens into the body.

When antigenic substances break through mucosal immunity and pathogens invade the body, there is innate immunity that functions immediately after invasion, and leukocytes play a central role. Among leukocytes, dendritic cells and macrophages not only have the function of phagocytosing invading pathogens, but also participate in the induction of acquired immunity by presenting antigens to T cells (Non-Patent Document 2). In addition, natural killer cells (NK cells), a type of lymphocyte, have cytotoxic activity and cytokine-producing ability, and play a central role in eliminating infections including viruses and tumor cells. It has been shown that NK cells function on mucosal surfaces such as the digestive tract (Non-Patent Document 3). Dendritic cells and macrophages that sense pathogen-derived components secrete inflammatory cytokines to promote the activation of NK cells. On the other hand, these macrophages and NK cells are known to be activated by IFN-γ (interferon-gamma), and NK cells themselves also produce IFN-γ and activate dendritic cells and T cells. It has been reported that it contributes to the improvement of

It is known that infants, the elderly, and people with reduced physical strength have weakened immunity and low resistance to external foreign substances such as pathogenic bacteria. Furthermore, it has been reported that modern people have weakened intestinal immunity due to factors such as disordered eating habits, reduced opportunities for exposure to bacteria, and use of antibiotics, leading to an increase in allergic diseases (Non-Patent Document 4). ), the development of immunostimulatory agents and immune system regulating agents has become an even more important issue. Improving immune function is also an important issue in the livestock industry. In other words, in recent years, from the viewpoint of economic efficiency, livestock farming has been shifting to a large-scale management form that takes advantage of the efficiency of mechanization and the scale merits of multi-head breeding. In such an overcrowded environment, infection with pathogenic viruses and bacteria leads to a serious loss of productivity. Young livestock are particularly susceptible to disease, and the economic loss caused by contracting the disease is large. Therefore, it is important to prevent disease outbreaks as much as possible. Due to concerns about the emergence of the disease, it is being restricted worldwide, and livestock farmers are currently struggling to deal with the frequent disease (Non-Patent Document 5).

Lactic acid bacteria have long been known as probiotics, and one of their effects is immunostimulatory action (Non-Patent Document 6). As lactic acid bacteria that induce IgA production, several strains belonging to the genus Lactobacillus, Leuconostoc, and Enterococcus have been reported (Patent Documents 1, 2, 3, and 4), and Bifidobacterium has also been reported. There is (Patent Document 5). In addition, strains belonging to the genus Lactobacillus and Streptococcus are exemplified as those that induce IFN-γ production (Patent Document 6).

However, since most of these lactic acid bacteria and bifidobacteria are obligate anaerobes, production under special anaerobic conditions is required. Some of the lactic acid bacteria are facultative anaerobes, so they can be produced with relatively simple equipment. Cost burden is high. In addition, it is difficult to stably maintain these lactic acid bacteria and bifidobacteria as viable bacteria for a long period of time, and distribution as viable bacteria in food is limited to product forms that have undergone special processing, such as encapsulation. . Also, in the livestock industry, probiotics are expected to be added to the feed, but in the summer, the feed is exposed to high temperatures and humidity, so feeding live bacteria as live bacteria to livestock is more stable. Desired. However, the above special processing is not suitable in terms of cost.
For these reasons, there has been a demand for the development of probiotic preparations that have higher immunostimulatory capacity than conventional probiotics, high productivity, and high stability as viable bacteria.

T.Suzukietal.ProcNatl Acad Sci USA.2015 Jun. 23;112(25):7809-14. Chemistry and Biology Vol. 53, No. 9, 2015 E. Vivier et al Nature Immunology volume 9, p.503-510 (2008) Chemistry and Biology Vol. 44, No. 1, 2006 Journal of Japanese Society of Chemotherapy Vol. 67, No. 1 Holistic Medicine 2019 Vol. 17 No. 1 p. 8-19

Japanese Unexamined Patent Application Publication No. 2008-201708 Japanese Patent Application Laid-Open No. 2008-179630 JP 2007-308419 A WO2010/001509 WO2006/087913 Japanese Patent Application Laid-Open No. 2007-302628

An object of the present invention is to provide a probiotic preparation that has high immunostimulatory activity, extremely high stability as viable bacteria, and excellent productivity.

The present inventors focused on Weizmannia coagulans (synonym Bacillus coagulans), a spore-bearing lactic acid bacterium that is known as a bacterium with very high environmental resistance and can be mass-produced by aerobic culture, and are keenly interested in its immunostimulatory activity. proceeded with the research. As a result, it was found that this bacterium has the effect of promoting IgA secretion from Peyer's patch cells in the intestinal tract, and that this effect was also exhibited in the mouse oral ingestion test. It was found to promote the secretion of γ. In addition, the present invention was completed based on the finding that the respective production-inducing abilities are higher than those of the genus Lactobacillus and the genus Leuconostoc, which have been previously reported to have high induction abilities.

That is, the gist of the present invention is as follows.

[1] In vivo IgA comprising a strain belonging to the spore-forming lactic acid bacterium Weizmannia coagulans that promotes IgA and IFN-γ production, a mutant strain of the strain, a homogenate, a culture, an extract, or a processed product thereof A lactic acid bacteria preparation that promotes IFN-γ production.

[2] The lactic acid bacteria preparation according to [1], wherein the spore-bearing lactic acid bacterium Weizmannia coagulans is Weizmannia coagulans SANK70258.

[3] An oral composition containing the lactic acid bacteria preparation of [1] or [2].

[4] The oral composition according to [3], which is an additive for food and drink, a food and drink, a supplement, or an additive for feed.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a probiotic preparation that has high immunostimulatory activity, extremely high stability as viable bacteria, and excellent productivity.
That is, the spore-forming lactic acid bacterium Weizmannia coagulans has a high ability to induce the secretion of IgA and IFN-γ. Animal feed, pharmaceuticals, and the like can be provided. In addition, since the spore-bearing lactic acid bacterium Weizmannia coagulans has high heat resistance, it can be used in a wide range of applications such as processed foods and molded feeds that require heat processing.

1 is a graph showing the evaluation results of the IgA production-promoting action of various lactic acid bacteria measured using mouse small intestine Peyer's patch-derived cells in Example 1. FIG. 4 is a graph showing the difference in the amount of IgA in mouse feces depending on whether Weizmannia coagulans strain SANK70258 was administered or not, measured in Example 2. FIG. 4 is a graph showing the difference in the number of mouse IgA-producing cells depending on whether or not Weizmannia coagulans strain SANK70258 was administered, measured in Example 3. FIG. 2 is a graph showing the evaluation results of the IFN-γ production-promoting action of various lactic acid bacteria in mouse spleen-derived cells, measured in Example 4. FIG. 10 is a graph showing the difference in abundance ratio of IFN-γ-expressing NK cells, helper T cells, and cytotoxic T cells depending on whether or not Weizmannia coagulans strain SANK70258 was administered, as measured in Example 5. FIG.

Preferred embodiments for carrying out the present invention will be described below. It should be noted that the embodiments described below are examples of representative embodiments of the present invention, and the scope of the present invention should not be construed narrowly.

[Lactic acid bacteria preparation]
A lactic acid bacteria preparation that promotes in vivo IgA and IFN-γ production (hereinafter sometimes referred to as "IgA/IFN-γ production promoter"), which is an embodiment of the present invention, belongs to the spore-bearing lactic acid bacterium Weizmannia coagulans. It is characterized by containing a strain, a mutant strain of the strain, a homogenate, a culture, an extract, or a processed product thereof.

Weizmannia coagulans (synonym Bacillus coagulans) is a lactic acid bacterium belonging to spore-bearing lactic acid bacteria. Germinate and become vegetative cells, which then proliferate and produce lactic acid. Due to these characteristics, Weizmannia coagulans is highly resistant to bacteria of the genus Lactobacillus, and it can be expected that even a relatively small amount of administration will produce a sufficient effect.
In addition, since Weizmannia coagulans is known to have an effect of improving intestinal function and skin condition, ingestion of the IgA/IFN-γ production promoter according to the embodiment of the present invention can improve the production of IgA and IFN-γ. In addition to the promotion effect, an intestinal regulation effect and a skin condition improving effect can also be expected.

As Weizmannia coagulans, Weizmannia coagulans SANK70258 strain, P-22 strain, lilac-01 strain, SIM-7 DSM14043 strain, C101 strain, NBRC12583 strain, GBI-1 strain, GBI-20 strain, GBI-30 strain, GBI- 40 strains, among others, Weizmannia coagulans strain SANK70258 is preferable from the viewpoint of stable supply and easy availability. Mutants derived from these strains may also be used as long as they have the effect of promoting the production of IgA and IFN-γ. In addition, crushed products, cultures, extracts, and processed products (for example, heat-dried dead cells) of these strains may also be used.
These may be used alone or in combination of two or more.

Commercially available Weizmannia coagulans can be used. Commercial products of Weizmannia coagulans include, for example, Mitsubishi Chemical Corporation "Lacris (registered trademark)-S", "Lacris (registered trademark)-S granules", "Lacris (registered trademark)-15", and "Feed Lacris ®-10”, and Kerry Inc. , SABINSA, AterioBio, UNIQUEBIOTECH, Asahi Biocycle, and the like.
These commercially available products may be appropriately cultured in an appropriate medium and used. Moreover, these can be used in the state of spores, in the state of a mixture of spores and vegetative cells, or in the state of disrupted cells or a culture medium containing them.
Moreover, as the form, it can be used in any form such as powder, granule or liquid.

The IgA/IFN-γ production promoter of the embodiment of the present invention may contain oligosaccharides that can be utilized by Weizmannia coagulans. Specific examples of such oligosaccharides include galacto-oligosaccharides, fructo-oligosaccharides, xylooligosaccharides, isomalto-oligosaccharides, mannooligosaccharides, cyclodextrin, inulin, guar gum decomposition products, pectin, glucomannan, sodium alginate, resistant starch, Acacia dietary fiber and the like.

The IgA/IFN-γ production promoter of the embodiment of the present invention may contain bacteria other than Weizmannia coagulans. Examples of other bacteria include lactic acid bacteria other than Weizmannia coagulans, butyric acid-producing bacteria, Bifidobacterium, Bacillus natto, Aspergillus oryzae, and yeast. Examples of lactic acid bacteria other than Weizmannia coagulans include bacteria belonging to the genus Lactobacillus and Bacillus.

Examples of bacteria belonging to the genus Lactobacillus include Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus bulgaricus, and Lactobacillus bulgaricus. Gasseri (Lactobaillus gaceli), Lactobacillus plantarum (Lactobacillus plantrum) and the like. Examples of bacteria belonging to the genus Bacillus include Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus cereus.

When the IgA/IFN-γ production promoter of the embodiment of the present invention contains bacteria other than Weizmannia coagulans, the ratio of Weizmannia coagulans to the total amount of bacteria is usually 1% by weight or more, preferably 10% by weight or more. , is preferably 50% by weight or more, and the upper limit is usually 100% by weight.

Further, the IgA/IFN-γ production promoter of the embodiment of the present invention includes salts such as sodium salt, potassium salt, calcium salt and magnesium salt; amino acids such as lysine, methionine, threonine, tryptophan and valine; Organic acids; Vitamins such as vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, pantothenic acid, biotin, folic acid, and ascorbic acid may be included.

[Oral composition]
Another embodiment of the present invention is an oral composition containing the IgA/IFN-γ production promoter, and the oral composition may be specifically a food, a beverage, or a It may be an additive for food, a supplement, or an additive for feed.

As used herein, "food" includes health food, functional food, food with health claims (food for specified health use, food with nutrient function claims, food with function claims, etc.), health supplement, and dietary supplement. Moreover, the shape of the food can be appropriately selected from solid, liquid, paste, and the like.

"Beverage" as used herein includes soft drinks, milk drinks, and alcoholic beverages.

The "supplement" according to the present invention may be in any form, such as tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, solutions, emulsions, and the like. Also, in order to protect the IgA/IFN-γ production promoter from gastric acid and allow it to act in the intestine, it may be an enteric agent having different solubility in different pH.

The additive for food and drink according to the present invention can be used as an additive for food and drink. In addition, feed additives can be used as feed additives for livestock and pets. Moreover, these may be in any form, such as tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, solutions, emulsions, and the like.

The oral composition of the present invention may further contain stabilizers, excipients, pH adjusters and the like, if necessary. Stabilizers include silicic anhydride and the like. Excipients include grains such as corn, wheat, soybeans, and milo, sugars such as lactose, sucrose, and glucose, plants such as soybean meal, corn gluten meal, sesame oil meal, corn farm meal, rapeseed oil meal, and distilled grain residue. Oil cakes, wheat bran, rice bran, defatted rice bran, bran such as corn gluten feed, animal materials such as fishmeal, vegetable oils such as coconut oil, lard, corn oil, starches such as cornstarch and potato starch, carbonic acid Minerals such as calcium, vitamins such as vitamin E, dextrin, cornstew liquor, paprika extract, and alfalfa meal. Examples of pH adjusters include citric acid and lactic acid.

[IgA and IFN-γ production promotion effect]
The mechanism of the IgA/IFN-γ production promoting action of the IgA/IFN-γ production promoting agent of the present invention is not limited, and as a result, the production amounts of IgA and IFN-γ are improved, and the effect of enhancing immunity is exhibited. Anything is fine. Here, the "IgA and IFN-γ production promoting action" preferably refers to the amount of IgA and IFN-γ antibody production at a statistically significant level of 5% before and after ingestion of the IgA/IFN-γ production promoting agent. is improved.

In the present invention, the IgA and IFN-γ production-promoting effects are preferably evaluated by the following methods.
For IgA, Weizmannia coagulans is suspended in physiological saline or the like, added to mouse small intestine Peyer's patch cells and cultured for a certain period of time, and the IgA antibody production in the culture supernatant is measured by ELISA. Alternatively, mice are fed with Weizmannia coagulans at 0.2% (10 ⁹ per day) of the weight of the diet, and fecal IgA is measured by ELISA after feeding for 7 days.
For IFN-γ, a suspension of Weizmannia coagulans is added to mouse spleen-derived cells, cultured for 48 hours, and IFN-γ released into the medium is measured by ELISA.

The intake of Weizmannia coagulans by the IgA/IFN-γ production promoter or oral composition of the present invention is preferably 1×10 ⁷ cfu or more, more preferably 1×10 ⁸ cfu or more per day. Also, the upper limit is preferably 5×10 ¹⁰ cfu or less per day.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to the following examples as long as it does not exceed the gist of the invention.

[Example 1]
Regarding the IgA production-inducing activity of the Weizmannia coagulans SANK70258 strain, the ability to promote secretion was evaluated using small intestinal Peyer's patch cells of mice. As a control, the strains shown in Table 1 were purchased from the National Institute of Physical and Chemical Research Bioresource Research Center Microbial Material Development Office and used. Lactic acid bacteria used as a control group were collected after culturing in MRS medium for one day, washed with physiological saline, and freeze-dried. All strains were tested after being sterilized with gamma rays.

Peyer's patches collected from the small intestine of 6-week-old female BALB/C mice were used. After treating the collected tissue with the enzyme solution for 15 minutes at 37° C., a cell suspension was prepared through a cell strainer, and the cell concentration was adjusted to 5×10 ⁵ cells/500 μL/well and seeded on a plate. bottom. The sterilized various lactic acid bacteria strains described above were added to the final concentration of 5×10 ⁶ cells/mL, and cultured in RPMI1640 medium (Sigma-Aldrich, USA) containing 10% FBS (fetal bovine serum). °C for 7 days. After that, the culture supernatant was collected, and the IgA concentration in the culture supernatant was measured by ELISA.

The results are shown in FIG. The numerical values shown in FIG. 1 are the average values of the measured values obtained by repeating the test four times. Error bars indicate standard error.
From FIG. 1, it was confirmed that among the tested lactic acid bacteria, Weizmannia coagulans SANK70258 strain (referred to as "W. coagulans" in FIG. 1 and FIGS. 2 to 5 below) has a strong IgA production promoting effect.

[Example 2]
The ability of Weizmannia coagulans SANK70258 strain to induce IgA production was evaluated by a mouse oral administration test. Gamma sterilized Weizmannia coagulans strain SANK70258 was administered to 6-week-old female BALB/C mice in the diet at 0.2% of the diet weight (10 ⁹ per day). Seven days after the start of administration, fresh feces were collected and the amount of IgA contained was measured by ELISA in the same manner as in Example 1.
At the same time, a test group was prepared to which the bacterial cells were not administered, and the amount of IgA in feces was compared with this group.
For the test, 18 mice were prepared for each group.

The results are shown in FIG. In addition, (-) in FIG. 2 indicates a non-administered group, and (+) indicates a group administered Weizmannia coagulans strain SANK70258.
As shown in Figure 2, the fecal IgA of 18 mice in each group was measured. An increasing trend was confirmed.

[Example 3]
Changes in the abundance ratio of IgA-producing cells due to administration of Weizmannia coagulans strain SANK70258 were investigated. Gamma sterilized Weizmannia coagulans strain SANK70258 was administered to 6-week-old female BALB/C mice in the diet at 0.2% of the diet weight (10 ⁹ per day). Seven days after the start of administration, Peyer's patches were collected from the small intestine, treated with an enzyme solution for 15 minutes at 37° C., and passed through a cell strainer to prepare a cell suspension. The obtained cells were stained with anti-CD16/32, biotin-anti-IgA, streptavidin-PE and anti-B220-FITC (Biolegend) for cell surface antigens and analyzed by flow cytometry.
A test group was prepared without administering the bacterial cells at the same time, and the test was conducted using 6 mice in each group. For comparison of the mean values of each group, the presence or absence of homogeneity of variance was confirmed by F-test, and then unpaired t-test (two-tailed test) was used.

The results are shown in FIG. (-) in FIG. 3 indicates the non-administered group and (+) indicates the administered group. * indicates that the P value was <0.05.
A comparison of the abundance ratio of IgA-producing cells in each of the non-administration group and six mice in the administration group revealed that the abundance ratio of IgA-producing cells was significantly increased in the administration group.

[Example 4]
The IFN-γ inducing activity of Weizmannia coagulans strain SANK70258 was evaluated using cells derived from mouse spleen. As a control group, the lactic acid bacteria strains shown in Table 1 were used. Spleens were harvested from 6-week-old C57BL/6 male mice and cell suspensions were prepared. The cell suspension was passed through a cell strainer to prepare a spleen-derived single cell suspension, which was seeded on a plate at 5×10 ⁵ cells/500 μL/well. Various strains of lactic acid bacteria sterilized with gamma rays were added thereto to a final concentration of 5×10 ⁵ cells/mL, and cultured in the following medium for 48 hours.

<Culture medium>
RPMI-1640: SIGMA, 10% FCS: SIGMA, 100 U/mL penicillin, 100 μg/mL streptomycin: Meiji, 10 mM HEPES pH 7.4, 1% MEM NEAA: WAKO, 1 mM sodium lactate, 100 μM β-mercaptoethanol with the addition of

After that, the culture supernatant was collected, and the amount of IFN-γ protein in the culture supernatant was measured by ELISA.

The results are shown in FIG. The numerical values shown in FIG. 4 are the average values of the measured values obtained by repeating the test three times.
From FIG. 4, it was confirmed that among the tested lactic acid bacteria, the Weizmannia coagulans SANK70258 strain in particular has a strong IFN-γ production-inducing activity.

[Example 5]
In order to know the action point of the IFN-γ-inducing ability of Weizmannia coagulans strain SANK70258, the abundance of NK cells, helper T cells and cytotoxic T cells expressing IFN-γ was evaluated. Spleens were harvested from 6-week-old C57BL/6 male mice and cell suspensions were prepared. The cell suspension was passed through a cell strainer to prepare a spleen-derived single cell suspension, which was seeded on a plate at 2×10 ⁶ cells/mL. Weizmannia coagulans SANK70258 strain sterilized by gamma rays was added thereto to 10 μg/mL and cultured for 12 hours. After that, 1 μL of Monensin and Breferdin A (both manufactured by BioLegend) were added and cultured for another 5 hours. Cells were collected, fixed using Fixation Buffer and Intracellular Staining Perm Wash Buffer (manufactured by BioLegend), and permeabilized. The cells were stained with anti-CD49b-APC and anti-IFN-γ-PECy7 (all manufactured by BioLegend) and analyzed by flow cytometry.
A test group was prepared without administering the bacterial cells at the same time and tested in the same manner. For comparison of the mean values of each group, the presence or absence of homogeneity of variance was confirmed by F-test, and then unpaired t-test (two-tailed test) was used.

The results are shown in FIG. The numerical value shown in FIG. 5 is the average of the measured values obtained by repeating the test four times. (-) in FIG. 5 indicates the non-administered group, and (+) indicates the administered group. * indicates that the P value was <0.05.
When comparing the abundance of helper T cells, cytotoxic T cells, and NK cells expressing IFN-γ, the number of NK cells expressing IFN-γ was lower in the group to which Weizmannia coagulans SANK70258 strain was added. It was confirmed that the abundance was significantly increased.
From this, it was found that the Weizmannia coagulans SANK70258 strain promotes the production of IFN-γ by NK cells.

Claims (4)

In vivo IgA and IFN-γ, comprising a strain belonging to Weizmannia coagulans, a spore-bearing lactic acid bacterium that promotes IgA and IFN-γ production, a mutant strain of the strain, a homogenate, a culture, an extract, or a processed product thereof A lactic acid bacteria preparation that promotes production. 2. The lactic acid bacterium preparation according to claim 1, wherein the spore-forming lactic acid bacterium Weizmannia coagulans is Weizmannia coagulans SANK70258. An oral composition comprising the lactic acid bacteria preparation according to claim 1 or 2. The oral composition according to claim 3, which is an additive for food and drink, a food and drink, a supplement, or an additive for feed.

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