WO2023022106A1

WO2023022106A1 - Intestinal regulator and immunity enhancer

Info

Publication number: WO2023022106A1
Application number: PCT/JP2022/030747
Authority: WO
Inventors: 令古川
Original assignee: 株式会社古川リサーチオフィス
Priority date: 2021-08-18
Filing date: 2022-08-12
Publication date: 2023-02-23

Abstract

The purpose of the present invention is to provide an intestinal regulator which can be provided at low cost while having good intestinal action, is easily adopted despite generally belonging to the field of livestock, in which cost effectiveness is stringently evaluated, can be used on a daily basis as feed or food and which further allows for easy quality management in manufacturing and distribution process. According to the present invention, an intestinal regulator is provided, the active component of which is dead cells of Bacillus subtilis, which are sometimes discarded in large quantities as an industrial waste product but which at the same time can exhibit beneficial effects on the intestines.

Description

Intestinal regulator and immune enhancer

The present invention relates to an antiflatulent agent, an immunopotentiating agent, a feed and a food, characterized by containing dead cells of Bacillus subtilis as an active ingredient.

In recent years, attention has been focused on probiotics taken for the purpose of improving the balance of the intestinal flora. Probiotics are defined as live microorganisms that have beneficial effects on the host by improving the balance of the host's intestinal flora. improvement, improvement of intestinal infection diarrhea, resolution of constipation, improvement of immune function, lowering of blood pressure, etc. have been reported.

Microorganisms used in probiotics include Bacillus subtilis such as Bacillus natto, lactic acid bacteria, bifidobacteria, butyric acid bacteria, lactic acid-utilizing bacteria, and propionic acid-producing bacteria (Patent Documents 1 and 2). In particular, Bacillus subtilis is mainly provided in the form of spores, which is expected to germinate in the intestine and exert an intestinal regulation effect. provided. However, for example, regarding the Bacillus subtilis JA-ZK strain, which is a Bacillus subtilis registered as a live bacterium agent for feed additives, the number of viable bacteria in feces when its spores are administered to pigs is They do not increase in number and become undetectable soon after the final administration (Non-Patent Document 1). Considering that there is not enough oxygen for germination, division and growth in the intestinal tract, it is thought that the administered spores pass through the intestinal tract as spores without germination. Therefore, it is considered that the contribution of the viable cells germinated in the intestine is small, at least in terms of the physiological activity. In addition, regarding Bacillus natto BN strain, which is used as a raw material for pharmaceuticals, raw materials for health foods, and feed additives, even if 10 ⁸ spores/time are added to the feed, 10 spores are extracted from the feces during the administration period. Only about ³ to 10 ⁴ cells/g of bacteria have been recovered (Non-Patent Document 2). This indicates that many spores die in the body. Therefore, in probiotics ingested in the form of spores, it is considered that the physiological effect is actually not due to the viable cells germinated from the spores, but due to the spores themselves.

In addition, since probiotic agents are generally relatively expensive, the introduction of probiotic agents is becoming increasingly popular in the livestock industry, where feedstocks are subject to strict evaluation in terms of cost effectiveness. It can be difficult. For example, when calculating the reference cost per animal per day for prophylactic agents, it is possible to feed all animals for prophylactic purposes, even though they may be fed only for a few days when severe diarrhea or illness persists in livestock. It has been reported that the price will be such that there is almost no chance of such a change (Non-Patent Document 3). Therefore, livestock farms are expected to provide feed that is inexpensive and easy to use on a daily basis while exhibiting high intestinal regulation and immune enhancement effects on livestock. Furthermore, when live fungi are used in the livestock industry, they must be registered as feed additives, and the large amount of money required to apply for such registration is a major barrier to the introduction of new probiotics.

In the first place, sporulation of Bacillus subtilis for producing a viable agent requires a longer culture time and stricter control of culture conditions. Loss of the number of viable bacteria also occurs to a large extent. As a result, the production cost of probiotics generally tends to be high, and this is reflected in the market price of probiotics. It's becoming Furthermore, when manufacturing and selling a viable bacterium agent, quality control is required to guarantee the number of viable bacterium during the quality assurance period even in the process of distribution and storage.

JP 2007-131541 A JP 2010-126457 A

The problem of the present invention is that it can be provided at a low cost while having a high intestinal regulation effect or an immunopotentiating effect, and can be easily adopted even in the livestock industry where strict evaluation is generally made in terms of cost-effectiveness. The purpose of the present invention is to provide an antiflatulent agent or an immunopotentiating agent which can be used on a daily basis and for a long period of time, and whose quality control is easy in the production and distribution processes.

As a result of intensive research to solve the above problems, the present inventor surprisingly found that the number of dead cells of Bacillus subtilis, which is sometimes discarded in large quantities as industrial waste, is higher than that of Bacillus subtilis spores. The inventors have found that it exerts an intestinal regulating effect or an immunopotentiating effect, and have completed the present invention.

That is, the present invention is as specified by the following matters.
[1] An antiflatulent agent characterized by containing dead cells of Bacillus subtilis as an active ingredient.
[2] The antiflatulent agent according to [1] above, wherein the dead cells of Bacillus subtilis are dead cells of fermented waste cells of Bacillus subtilis.
[3] The antiflatulent agent of [2] above, wherein the fermented waste cells are nucleic acid fermented waste cells.
[4] The agent for controlling intestinal function according to any one of [1] to [3] above, characterized in that the intestinal regulation is caused by improvement of intestinal flora.
[5] The antiflatulent agent of [4] above, wherein the improvement of the intestinal flora is an increase in good bacteria and/or a decrease in bad bacteria in the intestine.
[6] The antiflatulent agent of [5] above, wherein the beneficial bacteria are bacteria of the genus Lactobacillus and/or the bad bacteria are bacteria of the genus Clostridium.
[7] An intestinal regulator characterized by containing dead cells of Bacillus subtilis as an active ingredient, wherein the spores of Bacillus subtilis of the same strain or the same strain as the Bacillus subtilis are used as the active ingredient, and the number of bacteria in the dead cells is The antiflatulent agent according to any one of [1] to [6] above, characterized in that it has a higher intestinal regulating activity than an agent containing the same number of .
[8] The antiflatulent agent according to any one of [1] to [7] above for the prevention or treatment of diarrhea.
[9] The antiflatulent agent according to any one of [1] to [8] above, for administration to livestock.
[10] The intestinal regulator of [9] above, wherein the livestock is a weaned pig.
[11] An immunopotentiating agent characterized by containing dead cells of Bacillus subtilis as an active ingredient.
[12] The immunopotentiating agent of [11] above, wherein the immunopotentiating results from promotion of cytokine production.
[13] The immune enhancer of [12] above, wherein the cytokine is interleukin-8 or interleukin-12.
[14] The immunopotentiating agent of any one of [11] to [13] above, wherein the immunopotentiating is caused by promotion of lymphocyte proliferation.
[15] The immunopotentiating agent of [14] above, wherein the lymphocytes are B cells and/or T cells.
[16] An immunopotentiating agent characterized by containing killed cells of Bacillus subtilis as an active ingredient, wherein the killed cells contain spores of Bacillus subtilis of the same strain or homologous strain as the Bacillus subtilis as an active ingredient. The immunopotentiating agent according to any one of [11] to [15] above, characterized in that the immunopotentiating activity is higher than that of an agent containing the same number of bacteria.
[17] The agent according to any one of [1] to [16] above, which is feed or food.

Moreover, the following can be mentioned as another aspect of implementation of the present invention.
Use of dead cells of Bacillus subtilis for producing an antiflatulent agent.
A dead body of Bacillus subtilis for use in an intestinal treatment.
A method for intestinal regulation, comprising administering an effective amount of dead cells of Bacillus subtilis to a subject in need of intestinal regulation treatment.
Use of dead cells of Bacillus subtilis for producing a preventive or therapeutic agent for diarrhea.
A killed body of Bacillus subtilis for use in the prevention or treatment of diarrhea.
A method for preventing or treating diarrhea, which comprises administering an effective amount of killed Bacillus subtilis to a subject in need of prevention or treatment of diarrhea.
Use of dead cells of Bacillus subtilis for producing an immunopotentiating agent.
A killed body of Bacillus subtilis for use in immunopotentiating treatment.
A method for enhancing immunity, comprising administering an effective amount of dead cells of Bacillus subtilis to a subject in need of treatment for enhancing immunity.

In the present invention, the dead cells of Bacillus subtilis, which is used as an active ingredient of the antiflatulent agent or immunopotentiating agent, may be discarded in large amounts as industrial waste, but on the other hand, it can exhibit a high intestinal regulating effect or immunopotentiating effect. is. Therefore, according to the present invention, it is possible to provide an inexpensive and cost-effective intestinal regulating agent or immunopotentiating agent while having a high intestinal regulating effect or immunopotentiating effect. In addition, therefore, the antiflatulent agent or immunopotentiating agent of the present invention can be easily adopted even in the field of animal husbandry, where cost-effectiveness is generally severely evaluated, and can be used routinely and for a long period of time. Furthermore, since the antiflatulent agent or immunopotentiating agent of the present invention contains dead bacteria as an active ingredient instead of viable cells or spores, quality control is easy during the manufacturing and distribution processes, and it is widely used as foods such as supplements and feeds. It is possible.

1 is a diagram showing the results of evaluation (1) of cytokine (IL-12) production promoting effect in Example 1. FIG. 1 is a diagram showing the results of evaluation (1) of cytokine (IL-8) production promoting effect in Example 1. FIG. FIG. 2 shows the results of evaluation (2) of cytokine (IL-12) production promoting effect in Example 1. FIG. FIG. 10 is a diagram showing the results of feed efficiency analysis in Example 3; FIG. 10 shows the results of peripheral blood lymphocyte subset analysis (lymphocyte count) in Example 3. FIG. FIG. 10 shows the results of peripheral blood lymphocyte subset analysis (CD8 ⁺ cell count) in Example 3. FIG. FIG. 10 shows the results of peripheral blood lymphocyte subset analysis (CD4 ⁺ CD8 ⁺ cell count) in Example 3. FIG. FIG. 10 shows the results of peripheral blood lymphocyte subset analysis (CD8 ⁺ γδTCR ⁺ cell count) in Example 3. FIG. FIG. 10 shows the results of peripheral blood lymphocyte subset analysis (MHC class II ⁺ cell count) in Example 3. FIG. FIG. 10 shows the results of organ lymphocyte subset analysis (percentage of CD8 ⁺ cells) in Example 3. FIG. FIG. 10 shows the results of organ lymphocyte subset analysis (percentage of MHC class II ⁺ cells) in Example 3. FIG. FIG. 10 shows the results of cytokine mRNA expression level analysis (IL-2, IL-4, and IL-6) in jejunal Peyer's patches in Example 3. FIG. 10 shows the results of cytokine mRNA expression level analysis (IL-7, IL-10, and IL-12) in jejunal Peyer's patch in Example 3. FIG. FIG. 10 shows the results of cytokine mRNA expression level analysis (IFN-γ and TNF-α) in jejunal Peyer's patch in Example 3. FIG. FIG. 10 is a diagram showing the results of intestinal flora evaluation in Example 3. FIG.

The antiflatulent agent or immunopotentiating agent of the present invention contains dead cells of Bacillus subtilis as an active ingredient. In the present invention, Bacillus subtilis is not particularly limited as long as it is classified as Bacillus subtilis, and may be Bacillus subtilis var. natto, for example. In the present invention, the Bacillus subtilis may have the ability to form spores or may lack the ability to form spores. Bacillus subtilis has been certified as GRAS (Generally Recognized As Safe) by the Food and Drug Administration of the United States, and there are generally no safety issues for humans and other mammals to be ingested. Bacillus subtilis is used, for example, for the production of fermented foods such as natto, as well as enzymes (nattokinase, protease, amylase, etc.), nucleic acids (orotic acid, inosinic acid, guanylic acid, etc.), amino acids (polyglutamic acid, etc.). , and other organic acids. Therefore, the dead cells of Bacillus subtilis include dead cells of food fermentation waste of Bacillus subtilis, dead cells of Bacillus subtilis of enzymatic fermentation, dead cells of Bacillus subtilis of nucleic acid fermentation, Dead cells of Bacillus subtilis fermentation waste, such as dead cells of amino acid fermentation waste and Bacillus subtilis organic acid fermentation waste, may be used.

Here, the term "fermented waste cells" refers to bacterial cells or their processed products that are left behind after the desired components are produced (i.e., fermented) by the cells and the desired components are collected. Here, the term "bacteria or processed products thereof" refers to the bacteria themselves, or the bacteria that have been physically or chemically destroyed, dried, or shaped using excipients or the like. It also includes those processed by fermenting, and may include, for example, a culture solution and its components in addition to the cells or processed products thereof.

Fermentation waste cells are usually disposed of as industrial waste after the desired components have been collected, and the disposal of fermentation waste cells as industrial waste imposes a large burden in terms of work and cost. is required. However, according to the present invention, it is possible to provide an antiflatulent agent or an immunopotentiating agent containing dead fermentation waste as an active ingredient. Work and cost burdens as well as environmental loads can be reduced. In addition, since fermented waste cells are originally industrial waste, they can be obtained at low cost. Therefore, it is possible to inexpensively manufacture and provide an intestinal regulator or an immune enhancer containing dead fermented cells as an active ingredient. can. In particular, among fermentation production, Bacillus subtilis is often used in nucleic acid fermentation, and since it is estimated that 1,000 tons of nucleic acid fermentation waste cells are generated annually, nucleic acid fermentation waste cells should be used. By doing so, it is possible to create a particularly large merit that industrial waste can be effectively used. Nucleic acid fermentation waste cells include fermentation waste cells of guanylic acid, inosinic acid, orotic acid, and the like. In addition, since the antiflatulent agent or immunopotentiator of the present invention contains dead bacteria as an active ingredient instead of viable cells and spores of fermented waste, viable cells and spores are unnecessary outside the manufacturing company or outside the laboratory. It is also possible to prevent inappropriate use by others of strains with know-how that flow out to the public.

In the present invention, the term "dead cells" refers to vegetative cells that have been sterilized by heating, pressurization, drug treatment, etc., or vegetative cells that have died without sterilization. Killed cells may be crushed products in which the structure of the bacterial cells has been destroyed by enzymatic treatment, homogenization, ultrasonic treatment, etc., or fractions of specific fractions such as cell wall fractions from the crushed products. good. Killed cells, processed products thereof, or specific fractions thereof can be recovered by techniques such as centrifugation and filter processing.

Killed bacteria can be easily obtained by collecting and sterilizing the bacteria before sporulation in the manufacturing process where live bacteria are currently produced as probiotics. In that case, it is preferable to sterilize the cells as soon as possible before the cells grow sufficiently and sporulation begins. If the sterilization process is delayed, some of the cells will sporulate and the recovery rate of the dead cells after sterilization will decrease. As shown in the examples below, spores are considered to have lower intestinal regulation activity and immunopotentiating activity than dead bacteria. It is not preferable in terms of production efficiency for producing highly active intestinal regulators or immunopotentiators. From this point of view, the antiflatulent agent or immunopotentiating agent of the present invention preferably has a ratio of dead cells to spores in the range of at least 10:0 to 5:5, preferably 10:0 to 7:3. more preferably within the range of 10:0 to 8:2, particularly preferably within the range of 10:0 to 9:1, preferably 10:0 Most preferred.

The sterilization treatment for obtaining dead cells is not particularly limited as long as it can completely kill the vegetative cells. For this purpose, for example, autoclave treatment at 121° C. for 15 minutes or more, dry heat treatment at 180° C. for 30 minutes or more or 160° C. for 1 hour or more, or the like is preferable. Sterilization can also be called inactivation.

The intestinal regulating agent or immunopotentiating agent of the present invention does not need to contain other active ingredients because it can sufficiently exhibit intestinal regulating action or immunopotentiating action by using only the dead cells of Bacillus subtilis as an active ingredient. Therefore, in one aspect, the intestinal regulator or immunopotentiating agent of the present invention substantially does not contain viable cells of Bacillus subtilis, in one aspect it does not substantially contain spores of Bacillus subtilis, and in one aspect viable Bacillus subtilis substantially free of spores and Bacillus subtilis spores. Here, "spores" may be in an active state or not (ie, alive or dead). As described above, the germination rate of Bacillus subtilis spores in the body is expected to be low (Non-Patent Document 2), so it is thought that there is almost no effect of life or death of spores when ingested by animals. be. Bacillus subtilis spores are sometimes called spores or endospores. In the present invention, the term "viable cells" refers to cells of living vegetative cells.

In the present invention, "contained as an active ingredient" means containing an effective amount. An effective amount means an amount sufficient to exhibit an intestinal regulation effect or an immunopotentiating effect in vivo. Moreover, in the present invention, the term "contained as an active ingredient" also means that other ingredients may be included in addition to the dead cells of Bacillus subtilis as long as the effects of the present invention are not impaired. The effective dose may vary depending on the severity of the disease or condition of the administration subject, the animal species, age (age in months), sex, body weight, etc. of the administration subject. In the present invention, the effective amount of dead cells of Bacillus subtilis is, for example, 10 ⁵ or more, 10 ⁶ or more, 10 ⁷ or more, or 10 ⁸ or more per 1 g or 1 mL of the antiflatulent or immunopotentiating agent. is mentioned. In the present invention, the term "substantially free" means that a small amount of Bacillus subtilis viable cells or spores may be contained, but not an effective amount. The content of viable cells or spores of Bacillus subtilis per 1 g or 1 mL is 10 ⁴ or less, 10 ³ or less, 10 ² or less, or 10 ¹ or less, or viable Bacillus subtilis cells or It contains no spores at all.

In one embodiment, the antiflatulent agent or immunopotentiating agent of the present invention, which contains dead cells of Bacillus subtilis as an active ingredient, contains spores of the same strain or homologous strain of Bacillus subtilis as an active ingredient. It has higher intestinal regulating activity or immunopotentiating activity than an agent containing the same number of bacteria. Therefore, according to the present invention, it is less expensive and easier to control quality than conventional probiotic antiflatulent agents or immunopotentiating agents that contain spores, which are widely used as viable cells of Bacillus subtilis, as active ingredients, and are highly regulated. It is possible to provide an antiflatulent agent or an immunopotentiating agent capable of obtaining an intestinal effect or an immunopotentiating effect.

Here, for the intestinal regulation activity or immunopotentiating activity, it is originally preferable to compare the activity when dead cells and spores of the same strain of Bacillus subtilis are used as active ingredients, respectively. If it is not possible to prepare killed cells and spores of the same strain, such as in the case of a strain with reduced spores, compare the activity when using killed cells and spores of the same lineage strain instead of the same strain as active ingredients. You can also Here, the term "same lineage strain" means a strain within a range in which a connection can be found in the phylogenetic tree of strain breeding, and includes, for example, parent strains.

Intestinal regulation activity or immunopotentiating activity can be evaluated by a known method, and the evaluation method is not particularly limited. A method of measuring, a method of measuring the expression level of mRNA of genes such as cytokines involved in intestinal regulation activity or immunopotentiating activity by quantitative PCR, a method of evaluating intestinal flora, and analyzing lymphocyte subsets methods and the like. Proteins involved in intestinal regulation activity or immunopotentiating activity include, for example, cytokines such as interleukin-8 (IL-8) or interleukin-12 (IL-12).

"High intestinal activity" or "high immunopotentiating activity" means that the index of intestinal regulating activity or immunopotentiating activity is statistically significantly high. Index is 1.5 times higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 6 times higher, 7 times higher, 8 times higher 9 times higher, 10 times higher, and the like.

Intestinal regulation or immune enhancement in the present invention may result from improvement of intestinal flora. Here, the improvement of the intestinal flora may be an increase in good bacteria in the intestine, a decrease in bad bacteria in the intestine, or both an increase in good bacteria and a decrease in bad bacteria in the intestine. Examples of beneficial bacteria include Lactobacillus and Brautia. Examples of bad bacteria include bacteria belonging to the genus Clostridium. Improvement of the intestinal flora can be evaluated by a known method, and the evaluation method is not particularly limited. There is a method of evaluating the ratio of bacteria to the total number of bacteria.

Intestinal regulation or immune enhancement in the present invention may result from promotion of cytokine production. Here, cytokines include, for example, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, TNF-α, IFN-γ and the like. However, in particular, intestinal regulation or immune enhancement in the present invention may result from promotion of IL-8 or IL-12 production. Here, "promotion of cytokine production" in the present invention may be either promotion of cytokine gene expression or promotion of cytokine protein expression. Promotion of production of cytokines such as IL-8 or IL-12 can be evaluated by known methods, and evaluation methods are not particularly limited. Examples thereof include a method of measuring the protein expression level and a method of measuring the expression level of IL-8 mRNA or cytokine mRNA such as IL-12 by quantitative PCR.

Intestinal regulation or immune enhancement in the present invention may be due to promotion of lymphocyte proliferation. Here, the lymphocytes may be B cells and/or T cells. Promotion of lymphocyte proliferation can be evaluated by a known method, and the evaluation method is not particularly limited. Examples thereof include a method of evaluation by subset analysis of lymphocytes in peripheral blood or organs. Here, in the subset analysis, for example, using antibodies against cell surface antigens such as CD4 (T cells), CD8 (T cells), γδ TCR (T cells), MHC class II (B cells), flow cyto The number and ratio of each antigen-positive cell may be calculated using a meter.

The intestinal regulator or immune enhancer of the present invention can be used for the prevention or treatment of indigestion, diarrhea, constipation, abdominal bloating, abdominal bloating, enteritis, or the like. In addition, it enhances the digestion and absorption of food, promotes rumen fermentation, makes the intestines healthy, improves the intestinal flora, increases the good bacteria (useful bacteria) in the intestines, It can also be used to suppress the increase of bad bacteria (harmful bacteria), promote the production of cytokines, promote the proliferation of lymphocytes, and otherwise improve the intestinal environment. That is, the intestinal regulation agent or immune enhancer of the present invention is a rumen fermentation promoter, an intestinal flora improving agent, an increase promoter for good intestinal bacteria, a decrease promoter for bad intestinal bacteria, a cytokine production promoter, and a lymphocyte proliferation promoter. , etc., can also be used.

Subjects to which the intestinal regulator or immunopotentiating agent of the present invention is administered are not particularly limited. and other avian animals, carp, goldfish, and other farmed fish. When the subject of administration is a human, the antiflatulent agent or immunopotentiating agent of the present invention can be provided in the form of food. Moreover, when the subject of administration is an animal other than humans, the intestinal regulator or immune enhancer of the present invention can be provided in the form of feed. Feed for livestock such as cattle, horses, pigs, sheep, goats, and chickens is generally strictly evaluated in terms of cost-effectiveness. is also difficult to introduce. However, since the antiflatulent agent or immunopotentiating agent of the present invention is a Bacillus subtilis agent containing as an active ingredient dead cells such as inexpensive fermented waste cells, compared with conventional Bacillus subtilis agents containing live cells as an active ingredient, It can be widely used in the livestock industry because it has an overwhelmingly high cost advantage and significantly lowers the barrier to introduction as livestock feed. Cattle, horses, pigs, chickens and the like are particularly preferred as livestock. In particular, pigs in the weaning period are particularly preferable as subjects for administration of the antiflatulent agent or immunopotentiating agent of the present invention, since they are generally susceptible to diarrhea due to disturbance of the intestinal environment due to changes in diet. Note that the weaning period is the period between the suckling period and the rearing period in which mother's milk is ingested, and refers to the period from 23 days to 40 days after birth.

The intestinal regulator or immune enhancer of the present invention contains, for example, 10 ⁵ or more dead cells of Bacillus subtilis per 1 g or 1 mL, preferably 10 ⁶ or more per 1 g or 1 mL, and 10 per 1 g or 1 mL. More preferably ⁷ or more, and even more preferably 10 ⁸ or more per 1 g or 1 mL. The number of dead cells of Bacillus subtilis per 1 g can be measured by a known general method, for example, the number of viable cells before sterilization (Colony Forming Unit: CFU). Alternatively, it may be measured by a fluorescence staining method such as the DAPI method.

The form of feed or food in the present invention may be in the form of beverage. The shape of the feed or food in the present invention is not particularly limited as long as it can be ingested by mammals and is suitable for eating. form, capsule form, cream form, and paste form. In addition, the feed or food of the present invention may contain dead cells of Bacillus subtilis alone, or may be mixed with other ingredients according to the form of each feed or food. Here, other ingredients are not particularly limited as long as they do not impair the effects of the present invention. In addition to sweeteners, acidulants such as citric acid, malic acid, and tartaric acid, excipients such as dextrin and starch, coloring agents, flavors, bittering agents, buffering agents, thickening agents, gelling agents, stabilizers, and gum bases. , binders, diluents, emulsifiers, dispersants, suspending agents, antioxidants, preservatives, preservatives, fungicides, color formers, bleaching agents, brightening agents, enzymes, seasonings, spice extracts, etc. mentioned.

The intestinal regulator or immunopotentiating agent of the present invention may be administered alone to the subject, or may be administered to other probiotics, prebiotics, antibiotics, antimicrobial agents, as long as the effects of the present invention are not impaired. It may be administered in combination with a viral agent, an anti-inflammatory agent, or the like.

Although the present invention will be described in more detail with reference to examples and the like, the technical scope of the present invention is not limited by these.

1. Preparation of Bacillus subtilis spore and dead cell samples Spore and dead cell samples for testing were prepared. Bacillus subtilis BN strain (Bacillus subtilis BN, deposit number: NITE SBD 00136) was used as Bacillus subtilis.
〔spore〕
Bacillus natto BN strain was inoculated into the sporulation medium shown in Table 1, and cultured with shaking at 37° C. for 48 hours at 120 rpm to obtain a culture solution containing spores of Bacillus natto BN strain. In this culture solution, the mother cell portion was lysed in the late stage of sporulation, and the spores were released. A centrifuge was used to remove the culture medium and the lysed mother cell portion from the culture solution, and the collected spores were washed with purified water and then centrifuged to collect the spores. The collected spores were freeze-dried to obtain a freeze-dried spore. Bacillus subtilis spores are highly durable even under harsh conditions, and can continue to survive even after freeze-drying. This spore lyophilisate was used as the "spore" sample in subsequent tests.

*) Above No. Purified water was added to 1 to 4 to make up to 1 L. After autoclave sterilization, the above No. 5-7 were added.

[Dead cells]
Bacillus natto BN strain was inoculated into LB medium (1% tryptone, 0.5% yeast extract, 1% NaOH) and aerated and stirred at 37° C. for 24 hours at 120 rpm to obtain vegetative cells of Bacillus natto BN strain. A culture solution containing viable cells was obtained. This culture solution was sterilized by heating at 121° C. for 15 minutes using an autoclave to obtain a culture solution containing dead vegetative cells. The absence of spores in this culture solution was confirmed by the absence of viable bacteria after boiling for 5 minutes. A centrifuge was used to remove the medium from this culture solution, and the collected dead cells were washed with purified water and then centrifuged to collect the dead cells. The collected dead cells were freeze-dried to obtain a freeze-dried product of dead cells. This freeze-dried product of dead cells was used as a sample of "killed cells" in subsequent tests.

2. Evaluation of cytokine production promoting effect (1)
For the measurement of IL-12p40, the mouse macrophage-derived cell line J774.1 was seeded on a cell culture plate at a concentration of 5×10 ⁵ cells/mL, and the spores and dead cells were 1 μg/mL or 10 μg/mL. (spore addition group and killed cell addition group), or without adding them (control group), 37 ° C., 48 hours, 5% CO ₂ conditions, RPMI containing 10% fetal bovine serum It was cultured using -1640 medium (manufactured by Nacalai Tesque, Inc.). Thereafter, the culture supernatant was rapidly collected, diluted 20-fold, and interleukin-12 (IL-12 ) was measured.
For the measurement of IL-8, the human intestinal epithelial cell-derived HT29 strain was seeded on a cell culture plate at a concentration of 5×10 ⁵ cells/mL, and the spores and dead cells were adjusted to 1 μg/mL or 10 μg/mL. D-MEM containing 10% fetal bovine serum was added (spore-added group and dead cell-added group) or not added (control group) at 37° C. for 48 hours under 5% CO ₂ conditions. It was cultured using a medium (manufactured by Nacalai Tesque, Inc.). Thereafter, the culture supernatant was rapidly collected, diluted 20-fold, and interleukin-8 (IL-8 ) was measured.
IL-12 and IL-8 concentrations were calculated from the absorbance at 450 nm measured with a spectrophotometer according to the kit protocol. In addition, each test group was tested with 3 wells each, and after calculating the average value and standard deviation, a statistical test was performed between the spore addition group and the dead cell addition group (n = 3 , mean±s.d., two-tailed t-test, ^* p<0.05, ^** p<0.01).

The results are shown in Figure 1 and Table 2 (IL-12 production) and Figure 2 and Table 3 (IL-8 production). Compared to the control group, the amounts of IL-12 and IL-8 produced in the spore-added group and the killed cell-added group were both larger, but when the results of the spore-added group and the killed cell-added group were compared, the results were 1 μg/ Both at the time of adding mL and adding 10 μg/mL, the amount of IL-12 produced by macrophages and IL-8 produced by intestinal epithelial cells was significantly greater in the dead cell addition group than in the spore addition group ( ^** p<0.01). For Bacillus subtilis, the usefulness of viable agents containing spores as probiotics is well known. -12 and L-8 was shown to have a production promoting effect. IL-12 is an important cytokine for the differentiation of naive T cells (Th0 cells) into Th1 cells that control innate immunity. It also induces production of interferon γ in NK cells and Th1 cells and enhances the activity of NK cells and cytotoxic T cells, mainly activating innate immunity. Furthermore, it suppresses allergic-type inflammation derived from Th2 cells. IL-8 is known as neutrophil chemoattractant, and is a cytokine that induces chemotaxis of neutrophils and granulocytes to sites of infection. Therefore, by promoting the production of IL-12 and / or IL-8, innate immune cells effective against viral and bacterial infections are activated and guided to the affected area. It is expected to have a preventive effect against disease. In addition, it is expected that the enhancement of innate immunity will improve the balance of the intestinal flora due to the restoration of the intestinal environment, and the intestinal regulation effect will be obtained.

3. Evaluation of cytokine production promoting effect (2)
When bacteria are incorporated into products such as food and feed, they are generally incorporated based on the number of bacteria rather than the mass of the bacteria, regardless of whether the form of the bacteria is spores or vegetative cells. Therefore, it is appropriate to compare the physiological activities of bacteria in the case of adding them to products such as foods and feeds, using the same number of bacteria rather than the same mass.
Therefore, in order to compare the effect per the same number of spores and dead cells, the amount of spores and dead cells to be added is both 10 ⁶ /mL, and the rest is tested in the same manner as in the above evaluation (1). was performed, and the concentration of IL-12 in the culture supernatant was measured by ELISA.

The results are shown in FIG. 3 and Table 4. Compared to the control group, the amount of IL-12 and IL-8 produced in the spore-added group and the killed cell-added group were both larger, but when comparing the spores and killed cells per the same number of bacteria, It was shown that the killed cells had a significantly and remarkably higher effect of promoting IL-12 production ( ^** p<0.01).

1. Evaluation of Rumen Fermentation Promoting Action A beef rumen liquid was provided by Professor Atsushi Tajima, T-PIRC Next Generation Agricultural Chemicals Research Division, University of Tsukuba. The bovine rumen fluid was derived from Holstein breeds, and 1 L was collected using a catheter so that the effect of saliva was diluted, and 500 mL was received except for the necessary amount for the student experiment. After filtering the bovine rumen liquid with four layers of gauze, it was stored at -65°C. Rumen fluid and McDougall buffer (Table 5) were mixed at a ratio of 5:4, and 20 mL portions were dispensed into 50 mL centrifuge tubes. In a centrifuge tube, 100 mg each of compounded feed transferred from the University of Tsukuba and 1 mg each of spores or dead cells of Bacillus natto BN strain were added (spore addition group or dead cell addition group). Only 100 mg of compound feed was added to the control group. After loosening the lid of the centrifugation tube and storing it in an aneropack anaerobic jar (manufactured by AS ONE), the tube was cultured at 39°C for 15 hours. pH, OD630 and ammonium nitrogen were analyzed after cultivation. Ammonia nitrogen was analyzed using Ammonia Test Wako (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

The pH was adjusted to 6.8 with dry ice.

The results are shown in Table 6. OD630 indicates the cell level of rumen bacteria, and the increase in ammonium nitrogen is the result of decomposition of proteins and the like by rumen bacteria. The increase in ammonia nitrogen was greater when the dead cells were added than when the spores were added, indicating that the dead cells had a stronger effect of promoting rumen fermentation than the spores. By the way, in this test, the same weight of spores and dead cells were compared, but since the number of spores is equivalent to twice the number of dead cells of the same weight, spores and dead cells can be used for the same number of bacteria. When compared, it is assumed that a larger difference will occur (see evaluation (1) and (2) of cytokine production promoting effect in Example 1).

^* pH = 7.03 before culture.

1. Addition test of dead cells to swine feed Bacillus subtilis spores and dead cells were added to feed and fed to pigs in the weaning period to evaluate the intestinal regulation action, immune enhancement action, and the like. The fattening test of the weaned pigs was conducted at Miyazaki Prefectural Livestock Experiment Station Kawaminami Branch, and the analysis was conducted at Miyazaki University Faculty of Agriculture. There were three test groups: a spore addition group, a dead cell addition group, and a non-addition group (control group), and 10 weaning pigs (4 to 5 weeks old) in each group, totaling 30 pigs, were prepared. . Bacillus subtilis spores or killed Bacillus subtilis in the spore-added group or dead cell-added group were added to the feed at 10 ⁶ spores/g feed. Feeding period was from 1 to 4 weeks after weaning. At the start of feeding, after 2 weeks, and at the end of feeding (after 4 weeks), blood was collected from all animals and their weights were measured. Three fecal samples were collected from each group at the end of feeding. After measuring the weight at the end of feeding, the average weight of each group was calculated, and 3 pigs from each group with a weight close to the average weight were selected. The selected pigs were dissected, and palatine tonsils, thymus, liver, spleen, mesenteric lymph nodes, and jejunal Peyer's patches were collected. The total length of the small intestine and Peyer's patch length of the ileum were measured. Analysis shown later was performed using peripheral blood (PB) and organs. The test was performed by Dunnet's method or Steel-Dwass method, and when P<0.05, it was determined that there was a significant difference.

2. Body Weight Measurement/Feed Efficiency Analysis The feed intake was 247.8 kg/10 animals in the control group, 235.5 kg/10 animals in the dead cell group, and 240.1 kg/10 animals in the spore addition group. There was no significant difference in weight gain between groups. Also, the feed efficiency was calculated by dividing the weight gain (Kg/10 cows) of each group by the feed intake. The results are shown in FIG. The dead cell addition group tended to have better feed efficiency. This suggested the possibility that the intestinal environment was improved by the dead bacteria.

3. Peripheral Blood Lymphocyte Subset Analysis Peripheral blood was gravity-separated using Ficoll-Paque PLUS (manufactured by GE Healthcare Bio-Sciences AB) to collect mononuclear cells. Harvested cells were stained with fluorescently labeled antibodies (Table 7). FITC labeling kit-NH2, HiLyte Fluor 555 (F555) labeling kit-NH2, and HiLyte Fluor 647 labeling (F647) kit-NH2 (manufactured by Dojindo Laboratories) were used for fluorescent labeling of the antibodies. A flow cytometer (Attune NxT Acoustic Focusing Cytometer, manufactured by ThermoFisher Scientific) was used to measure the ratio of each subset in lymphocytes, and the actual values were calculated.

The results are shown in Figures 5-9. After 4 weeks, the lymphocyte count was significantly increased in the killed cell addition group compared to the control group (Fig. 5). After 4 weeks, the number of CD8 ⁺ cells was significantly increased in the killed cell addition group compared to the control group (Fig. 6). After 4 weeks, the number of CD4 ⁺ CD8 ⁺ cells was significantly increased in the killed cell addition group compared to the control group (Fig. 7). After 4 weeks, the number of CD8 ⁺ γδTCR ⁺ cells was significantly increased in the killed cell addition group compared to the control group (Fig. 8). After 2 weeks, the number of MHC class II ⁺ cells was significantly increased in the killed cell addition group compared to the control group. In addition, the number of MHC class II ⁺ cells tended to increase in the killed cell addition group compared to the control group after 4 weeks (FIG. 9). These results suggested that the dead cells proliferate blood lymphocytes (particularly T cells and B cells) and have an effect of enhancing immunity.

4. Organ Lymphocyte Subset Analysis After creating a cell suspension of each organ, impurities were removed using a cell strainer. The collected cells are subjected to the above 3. were stained with the antibodies shown in Table 7 in the same manner as in Relative ratios were calculated based on the percentage of each cell in lymphocytes measured by a flow cytometer.

Results are shown in FIGS. The number of CD8 ⁺ cells in the liver tended to increase in the killed cell addition group compared to the control group (Fig. 10). The number of MHC class II ⁺ cells in the liver and jejunal Peyer's patch tended to increase in the killed cells addition group compared to the control group (Fig. 11). These results suggested that dead cells proliferate lymphocytes (particularly T cells and B cells) in organs and have an effect of enhancing immunity.

5. Cytokine mRNA expression level analysis in jejunal Peyer's patches RNA was extracted from the cell fluid of jejunal Peyer's patches. Using TB Green PrimeScript PLUS RT-PCR Kit (manufactured by Takara Bio), QuantStudio 3 Real-Time PCR system (manufactured by Applied Biosystems), RT-qPCR (reverse transcription at 42 ° C for 5 minutes, 95 ° C for 10 seconds was followed by 40 cycles of 95° C. for 5 seconds, 57° C. for 30 seconds, and 72° C. for 30 seconds), resulting in GAPDH, IL-2, IL-4, IL-6, Expression levels of IL-7, IL-10, IL-12, TNF-α and IFN-γ mRNA were measured and analyzed using QuantStudio software (manufactured by Applied Biosystems) (2 ^-ΔΔCt method).

The results are shown in Figures 12-14. In each group, n = 3 and the number of n was small, so no significant difference was shown, but in the killed cell addition group, IL-2, IL-4, IL-6, IL-10, IL-12, TNF- The expression levels of α and IFN-γ mRNA tended to increase. These results suggested the possibility that dead cells increase the expression level of cytokines and have the effect of enhancing immunity.

6. Intestinal flora evaluation Analysis of 16S rRNA in fecal samples was performed by H.I. U.S.A. Conducted by requesting the Group Central Research Institute, Clostridium genus (bad bacteria), Lactobacillus genus (good bacteria), or Blautia genus (good bacteria) accounting for the total number of bacteria compared the ratio of

The results are shown in FIG. In the dead cell addition group, the ratio of Clostridium spp. (bad bacteria) was significantly decreased and the ratio of Lactobacillus spp. (good bacteria) was significantly increased compared to the control group. In addition, the proportion of Brautia bacteria (good bacteria) was on the increase. These results suggest that dead bacteria reduce the number of bad bacteria in the intestine and increase the number of good bacteria in the intestine, and have an intestinal regulating effect that improves the intestinal flora.

The present invention can be effectively used in the field of manufacturing foods such as supplements and functional foods for the purpose of obtaining an intestinal regulation effect or an immune-enhancing effect, livestock feed, and the like.

Claims

An intestinal regulating agent characterized by containing dead cells of Bacillus subtilis as an active ingredient.
The antiflatulent agent according to claim 1, wherein the dead cells of Bacillus subtilis are dead cells of fermented waste cells of Bacillus subtilis.
The antiflatulent agent according to claim 2, wherein the fermented waste cells are nucleic acid fermented waste cells.
The intestinal regulation agent according to claim 1, characterized in that intestinal regulation is caused by improvement of intestinal flora.
The antiflatulent agent according to claim 4, wherein the improvement of the intestinal flora is an increase in good bacteria and/or a decrease in bad bacteria in the intestine.
The antiflatulent agent according to claim 5, wherein the beneficial bacteria are bacteria of the genus Lactobacillus and/or the bad bacteria are bacteria of the genus Clostridium.
An antiflatulent agent characterized by containing dead cells of Bacillus subtilis as an active ingredient,
2. The antiflatulent agent according to claim 1, wherein the intestinal regulation activity is higher than that of an agent containing, as an active ingredient, spores of Bacillus subtilis of the same strain or the same phylogenetic strain as the Bacillus subtilis, the same number as the number of dead cells. .
The antiflatulent agent according to claim 1 for preventing or treating diarrhea.
The intestinal regulator according to claim 1, for administration to livestock.
The intestinal regulation agent according to claim 9, wherein the livestock is a weaning pig.
An immune enhancer characterized by containing dead cells of Bacillus subtilis as an active ingredient.
The immunopotentiating agent according to claim 11, characterized in that the immunopotentiating is caused by promoting the production of cytokines.
The immunopotentiator according to claim 12, wherein the cytokine is interleukin-8 or interleukin-12.
The immunopotentiating agent according to claim 11, characterized in that the immunopotentiating is caused by promotion of lymphocyte proliferation.
The immunopotentiating agent according to claim 14, wherein the lymphocytes are B cells and/or T cells.
An immunopotentiating agent characterized by comprising dead cells of Bacillus subtilis as an active ingredient,
12. The immunity according to claim 11, characterized by having a higher immunopotentiating activity than an agent containing the same number of spores of Bacillus subtilis as the active ingredient or the same strain as the Bacillus subtilis or the same strain as the number of dead cells. Enhancer.
The agent according to any one of claims 1 to 16, which is feed or food.