Subcritical extract of microbial cell preparation and method for producing subcritical extract of microbial cell preparation

Abstract

To provide a subcritical extract of a bacterial preparation having an M2 macrophage differentiation induction effect that can correct M1 / M2 balance and a method of producing the same.SOLUTION: The present invention provides a subcritical extract of at least one bacterial preparation selected from the group consisting of a bacterial cell, a treated bacterial cell, and a cultured bacterial, and a method for producing a subcritical extract of at least one bacterial preparation selected from the group consisting of a bacterial cell, a treated bacterial cell, and a cultured bacterial.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a subcritical extract of a microbial preparation and a method for producing the subcritical extract of a microbial preparation. The present invention also relates to an immunomodulator, a food or drink, an additive for beverages or foods, a cosmetic composition, an anti-inflammatory agent, and an M2 macrophage differentiation inducer containing the subcritical extract of a microbial preparation, and methods for producing them.

Background Art

[0002] Macrophages (Mφ) have long been known to be one type of immune cell responsible for immune function. In recent years, it has been found that macrophages not only have the function of simply removing infectious microorganisms and foreign substances, but also play an important role in controlling the inflammatory state of the living body. Among them, tissue-resident macrophages, which have attracted much attention, are constantly present in almost all tissues and are thought to be involved in maintaining the homeostasis of the living body in tissues, such as regulating the intensity of the immune response and regulating energy metabolism.

[0003] Macrophages are roughly classified into two phenotypes, inflammatory macrophages (M1Mφ) and anti-inflammatory macrophages (M2Mφ), according to the mode of activation. And it has been found that the balance of these Mφ (M1 / M2 balance) is very important in maintaining the homeostasis of the living body. For example, obesity, which is a type of lifestyle-related disease, is thought to contribute to the fact that the Mφ balance (M1 / M2 balance) in subcutaneous adipose tissue is tilted toward the inflammatory (M1) side, and the function of macrophages has become pro-inflammatory. In order to normalize the M1 / M2 balance tilted toward the M1Mφ side in this way, the function of M2Mφ, which converges inflammation, is important. Control of the inflammatory action and control of the localization of macrophages based on the function of such M2Mφ are also in increasing demand in the field of regenerative medicine.

[0004] Patent Document 1 describes lactic acid bacteria that exhibit inducibility to M2 macrophages. In Patent Document 1, it has been shown that a heat-treated product (75°C, 1 hour) of a specific lactic acid bacterium (Lactobacillus rhamnosus OLL2838) has an IL-10 production promoting effect and an effect of inducing differentiation from undifferentiated (M0) Mφ to M2Mφ.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, the IL-10 production promoting effect and the differentiation inducing effect to M2Mφ described in Patent Document 1 are only effects possessed by specific bacterial species. It cannot be said that any lactic acid bacterium can adapt, and therefore it has low versatility and is difficult to widely penetrate. Also, in the treated product described in Patent Document 1, the induction of M0Mφ to M1Mφ has not disappeared. That is, it is considered that the induction effect of macrophages that have already become M1Mφ to M2Mφ cannot be expected.

[0007] Therefore, it is hard to say that the M2 macrophage differentiation inducer described in Patent Document 1 can obtain a complete anti-inflammatory effect. That is, even if the M2 macrophage differentiation inducer described in Patent Document 1 was applicable for the prevention of inflammation, it was insufficient for the purpose of curing inflammation.

[0008] The present invention has been made in view of the above problems, and in order to treat already-occurring inflammation, an object of the present invention is to provide a highly versatile, safe, and simple method for inducing macrophages induced to M1Mφ to M2Mφ in order to correct the M1 / M2 balance.

Means for Solving the Problems

[0009] The present invention relates to a subcritical extract of at least one bacterial cell preparation selected from the group consisting of bacterial cells, processed bacterial cell products, and bacterial cell cultures.

[0010] Preferably, the subcritical extract is of a bacterial cell culture which is a suspension of the bacterial cells, a fermentation broth containing the bacterial cells, or a fermentation residue containing the bacterial cells.

[0011] Preferably, the subcritical extract is of a bacterial cell which is at least one selected from the group consisting of lactic acid bacteria, yeast, Bacillus natto, Aspergillus oryzae, and acetic acid bacteria.

[0012] Preferably, the subcritical extract is of a lactic acid bacterium belonging to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium.

[0013] Preferably, the subcritical extract is of a fermentation residue containing the bacterial cells which is brewing lees.

[0014] Preferably, the subcritical extract is of brewing lees which is at least one selected from the group consisting of sake lees, shochu lees, and mirin lees.

[0015] The present invention also relates to a method for producing a subcritical extract, characterized by subjecting at least one bacterial cell preparation selected from the group consisting of bacterial cells, processed bacterial cell products, and bacterial cell cultures to extraction treatment with subcritical water adjusted to a temperature of 155°C or higher and 220°C or lower and a pressure of 0.3 MPa or higher and 2.0 MPa or lower.

[0016] Preferably, the method for producing a subcritical extract is such that the bacterial cell culture is a suspension of the bacterial cells, a fermentation broth containing the bacterial cells, or a fermentation residue containing the bacterial cells.

[0017] Preferably, the method for producing a subcritical extract is such that the bacterial cell is at least one selected from the group consisting of lactic acid bacteria, yeast, Bacillus natto, Aspergillus oryzae, and acetic acid bacteria.

[0018] A method for producing a subcritical extract, wherein the lactic acid bacterium belongs to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium, is preferable.

[0019] A method for producing a subcritical extract, wherein the fermentation residue containing the cells is brewing lees, is preferable.

[0020] A method for producing a subcritical extract, wherein the brewing lees is at least one kind of brewing lees selected from the group consisting of sake lees, shochu lees, and mirin lees, is preferable.

[0021] The present invention also relates to an immunomodulator containing a subcritical extract of at least one kind of cell preparation selected from the group consisting of cells, cell-treated products, and cell cultures.

[0022] An immunomodulator, wherein the cell culture is a suspension of the cells, a fermentation broth containing the cells, or a fermentation residue containing the cells, is preferable.

[0023] An immunomodulator, wherein the cells are at least one kind of cells selected from lactic acid bacteria, yeast, Bacillus natto, Aspergillus oryzae, and acetic acid bacteria, is preferable.

[0024] An immunomodulator, wherein the lactic acid bacterium belongs to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium, is preferable.

[0025] An immunomodulator, wherein the fermentation residue containing the cells is brewing lees, is preferable.

[0026] An immunomodulator, wherein the brewing lees is at least one kind of brewing lees selected from the group consisting of sake lees, shochu lees, and mirin lees, is preferable.

[0027] It is preferable that the immunomodulator is an anti-inflammatory cytokine production promoter.

[0028] It is preferable that the immunomodulator is an inflammatory cytokine production inhibitor.

[0029] It is preferable that the immunomodulator is an immunomodulator for the treatment or prevention of a disease in which a Th1-predominant immune response is involved in the exacerbation of the disease state.

[0030] It is preferable that the immunomodulator is an immunomodulator for the treatment or prevention of a disease related to the imbalance of the M1 / M2 balance of macrophages.

[0031] The present invention also relates to a method for producing an immunomodulator, which comprises subjecting at least one bacterial cell preparation selected from the group consisting of bacterial cells, processed bacterial cell products, and bacterial cell cultures to extraction treatment with subcritical water adjusted to a temperature of 155°C or higher and 220°C or lower and a pressure of 0.3 MPa or higher and 2.0 MPa or lower.

[0032] A method for producing an immunomodulator is preferable, wherein the bacterial cell culture is a suspension of the bacterial cells, a fermentation broth containing the bacterial cells, or a fermentation residue containing the bacterial cells.

[0033] A method for producing an immunomodulator is preferable, wherein the bacterial cells are at least one selected from lactic acid bacteria, yeast, Bacillus natto, Aspergillus, and acetic acid bacteria.

[0034] A method for producing an immunomodulator is preferable, wherein the lactic acid bacteria belong to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium.

[0035] A method for producing an immunomodulator is preferable, wherein the fermentation residue containing the bacterial cells is brewing lees.

[0036] A method for producing an immunomodulator in which the brewing residue is at least one selected from the group consisting of sake lees, shochu lees, and mirin lees is preferable.

[0037] The present invention also relates to a food or drink containing a subcritical extract of at least one cell preparation selected from the group consisting of cells, cell-treated products, and cell cultures.

[0038] A food or drink in which the cell culture is a suspension of the cells, a fermentation broth containing the cells, or a fermentation residue containing the cells is preferable.

[0039] A food or drink in which the cells are at least one selected from lactic acid bacteria, yeast, natto bacteria, koji mold, and acetic acid bacteria is preferable.

[0040] A food or drink in which the lactic acid bacteria belong to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium is preferable.

[0041] A food or drink in which the fermentation residue containing the cells is brewing residue is preferable.

[0042] A food or drink in which the brewing residue is at least one selected from the group consisting of sake lees, shochu lees, and mirin lees is preferable.

[0043] It is preferable that the food or drink has an anti-inflammatory effect.

[0044] The present invention also relates to a method for producing a food or drink, characterized in that at least one cell preparation selected from the group consisting of cells, cell-treated products, and cell cultures is subjected to extraction treatment with subcritical water adjusted to a temperature of 155°C or higher and 220°C or lower and a pressure of 0.3 MPa or higher and 2.0 MPa or lower.

[0045] A method for producing a food or drink in which the cell culture is a suspension of the cells, a fermentation broth containing the cells, or a fermentation residue containing the cells is preferable.

[0046] A method for producing a food or beverage product is preferred, wherein the microbial cells are at least one selected from lactic acid bacteria, yeast, Bacillus natto, Aspergillus oryzae, and acetic acid bacteria.

[0047] A method for producing a food or beverage product is preferred, wherein the lactic acid bacteria belong to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium.

[0048] A method for producing a food or beverage product is preferred, wherein the fermentation residue containing the microbial cells is brewing lees.

[0049] A method for producing a food or beverage product is preferred, wherein the brewing lees is at least one selected from the group consisting of sake lees, shochu lees, and mirin lees.

[0050] The present invention also relates to an additive for beverages or foods containing a subcritical extract of at least one microbial cell preparation selected from the group consisting of microbial cells, microbial cell-treated products, and microbial cell cultures.

[0051] An additive for beverages or foods is preferred, wherein the microbial cell culture is a suspension of the microbial cells, a fermentation broth containing the microbial cells, or a fermentation residue containing the microbial cells.

[0052] An additive for beverages or foods is preferred, wherein the microbial cells are at least one selected from lactic acid bacteria, yeast, Bacillus natto, Aspergillus oryzae, and acetic acid bacteria.

[0053] An additive for beverages or foods is preferred, wherein the lactic acid bacteria belong to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium.

[0054] An additive for beverages or foods is preferred, wherein the fermentation residue containing the microbial cells is brewing lees.

[0055] The additive for beverages or foods, wherein the brewing residue is at least one selected from the group consisting of sake lees, shochu lees, and mirin lees, is preferred.

[0056] It is preferable that the additive for beverages or foods has an anti-inflammatory effect.

[0057] The present invention also relates to a method for producing an additive for beverages or foods, characterized by subjecting at least one kind of microbial cell preparation selected from the group consisting of microbial cells, processed microbial cells, and microbial cell cultures to extraction treatment with subcritical water adjusted to a temperature of 155°C or higher and 220°C or lower and a pressure of 0.3 MPa or higher and 2.0 MPa or lower.

[0058] The method for producing an additive for beverages or foods, wherein the microbial cell culture is a suspension of the microbial cells, a fermentation broth containing the microbial cells, or a fermentation residue containing the microbial cells, is preferred.

[0059] The method for producing an additive for beverages or foods, wherein the microbial cells are at least one selected from lactic acid bacteria, yeast, Bacillus natto, Aspergillus oryzae, and acetic acid bacteria, is preferred.

[0060] The method for producing an additive for beverages or foods, wherein the lactic acid bacteria belong to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium, is preferred.

[0061] The method for producing an additive for beverages or foods, wherein the fermentation residue containing the microbial cells is brewing residue, is preferred.

[0062] The method for producing an additive for beverages or foods, wherein the brewing residue is at least one selected from the group consisting of sake lees, shochu lees, and mirin lees, is preferred.

[0063] The present invention also relates to a cosmetic composition containing a subcritical extract of at least one bacterial cell preparation selected from the group consisting of bacterial cells, processed bacterial cell products, and bacterial cell cultures.

[0064] A cosmetic composition is preferred in which the bacterial cell culture is a suspension of the bacterial cells, a fermentation broth containing the bacterial cells, or a fermentation residue containing the bacterial cells.

[0065] A cosmetic composition is preferred in which the bacterial cells are at least one selected from the group consisting of lactic acid bacteria, yeast, Bacillus natto, Aspergillus oryzae, and acetic acid bacteria.

[0066] A cosmetic composition is preferred in which the lactic acid bacteria belong to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium.

[0067] A cosmetic composition is preferred in which the fermentation residue containing the bacterial cells is brewing lees.

[0068] A cosmetic composition is preferred in which the brewing lees is at least one selected from the group consisting of sake lees, shochu lees, and mirin lees.

[0069] The cosmetic composition is preferably a cosmetic composition having an anti-inflammatory effect.

[0070] The present invention also relates to a method for producing a cosmetic composition, characterized in that at least one bacterial cell preparation selected from the group consisting of bacterial cells, processed bacterial cell products, and bacterial cell cultures is subjected to extraction treatment with subcritical water adjusted to a temperature of 155°C or higher and 220°C or lower and a pressure of 0.3 MPa or higher and 2.0 MPa or lower.

[0071] A method for producing a cosmetic composition is preferred in which the bacterial cell culture is a suspension of the bacterial cells, a fermentation broth containing the bacterial cells, or a fermentation residue containing the bacterial cells.

[0072] A method for manufacturing a cosmetic composition in which the microbial cells are at least one selected from lactic acid bacteria, yeast, Bacillus natto, Aspergillus oryzae, and acetic acid bacteria is preferable.

[0073] A method for manufacturing a cosmetic composition in which the lactic acid bacteria are lactic acid bacteria belonging to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium is preferable.

[0074] A method for manufacturing a cosmetic composition in which the fermentation residue containing the microbial cells is brewing lees is preferable.

[0075] A method for manufacturing a cosmetic composition in which the brewing lees are at least one selected from the group consisting of sake lees, shochu lees, and mirin lees is preferable.

[0076] The present invention also relates to an anti-inflammatory agent containing a subcritical extract of at least one microbial cell preparation selected from the group consisting of microbial cells, microbial cell-treated products, and microbial cell cultures.

[0077] An anti-inflammatory agent in which the microbial cell culture is a suspension of the microbial cells, a fermentation broth containing the microbial cells, or a fermentation residue containing the microbial cells is preferable.

[0078] An anti-inflammatory agent in which the microbial cells are at least one selected from lactic acid bacteria, yeast, Bacillus natto, Aspergillus oryzae, and acetic acid bacteria is preferable.

[0079] An anti-inflammatory agent in which the lactic acid bacteria are lactic acid bacteria belonging to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium is preferable.

[0080] An anti-inflammatory agent in which the fermentation residue containing the microbial cells is brewing lees is preferable.

[0081] An anti-inflammatory agent in which the brewing residue is at least one selected from the group consisting of sake lees, shochu lees, and mirin lees is preferred.

[0082] The present invention also relates to a method for producing an anti-inflammatory agent, which comprises subjecting at least one kind of microbial cell preparation selected from the group consisting of microbial cells, processed microbial cell products, and microbial cell cultures to extraction treatment with subcritical water adjusted to a temperature of 155°C or higher and 220°C or lower and a pressure of 0.3 MPa or higher and 2.0 MPa or lower.

[0083] A method for producing an anti-inflammatory agent in which the microbial cell culture is a suspension of the microbial cells, a fermentation broth containing the microbial cells, or a fermentation residue containing the microbial cells is preferred.

[0084] A method for producing an anti-inflammatory agent in which the microbial cells are at least one selected from lactic acid bacteria, yeast, natto bacteria, koji mold, and acetic acid bacteria is preferred.

[0085] A method for producing an anti-inflammatory agent in which the lactic acid bacteria belong to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium is preferred.

[0086] A method for producing an anti-inflammatory agent in which the fermentation residue containing the microbial cells is brewing residue is preferred.

[0087] A method for producing an anti-inflammatory agent in which the brewing residue is at least one selected from the group consisting of sake lees, shochu lees, and mirin lees is preferred.

[0088] The present invention also relates to an M2 macrophage differentiation inducer containing a subcritical extract of at least one kind of microbial cell preparation selected from the group consisting of microbial cells, processed microbial cell products, and microbial cell cultures.

[0089] An M2 macrophage differentiation inducer in which the microbial cell culture is a suspension of the microbial cells, a fermentation broth containing the microbial cells, or a fermentation residue containing the microbial cells is preferred.

[0090] The M2 macrophage differentiation inducer in which the microbial cells are at least one selected from lactic acid bacteria, yeast, Bacillus natto, Aspergillus oryzae, and acetic acid bacteria is preferable.

[0091] The M2 macrophage differentiation inducer in which the lactic acid bacteria belong to the lactic acid bacteria selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium is preferable.

[0092] The M2 macrophage differentiation inducer in which the fermentation residue containing the microbial cells is brewing lees is preferable.

[0093] The M2 macrophage differentiation inducer in which the brewing lees is at least one selected from the group consisting of sake lees, shochu lees, and mirin lees is preferable.

[0094] The M2 macrophage differentiation inducer in which the differentiation induction includes induction from M1 macrophages to M2 macrophages is preferable.

[0095] The M2 macrophage differentiation inducer is preferably an M2 macrophage differentiation inducer having an anti-inflammatory effect.

[0096] The M2 macrophage differentiation inducer is preferably an M2 macrophage differentiation inducer that promotes regeneration induction.

[0097] The present invention also relates to a method for producing an M2 macrophage differentiation inducer, which comprises subjecting at least one microbial cell preparation selected from the group consisting of microbial cells, microbial cell-treated products, and microbial cell cultures to extraction treatment with subcritical water adjusted to a temperature of 155°C or higher and 220°C or lower and a pressure of 0.3 MPa or higher and 2.0 MPa or lower.

[0098] The method for producing an M2 macrophage differentiation inducer in which the microbial cell culture is a suspension of the microbial cells, a fermentation broth containing the microbial cells, or a fermentation residue containing the microbial cells is preferable.

[0099] A method for producing an M2 macrophage differentiation inducer in which the microbial cells are at least one selected from lactic acid bacteria, yeast, Bacillus natto, Aspergillus oryzae, and acetic acid bacteria is preferable.

[0100] A method for producing an M2 macrophage differentiation inducer in which the lactic acid bacteria belong to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium is preferable.

[0101] A method for producing an M2 macrophage differentiation inducer in which the fermentation residue containing the microbial cells is brewing lees is preferable.

[0102] A method for producing an M2 macrophage differentiation inducer in which the brewing lees is at least one selected from the group consisting of sake lees, shochu lees, and mirin lees is preferable.

[0103] The present invention also relates to the use of a subcritical extract of at least one microbial cell preparation selected from the group consisting of microbial cells, microbial cell-treated products, and microbial cell cultures in the production of a medicament for preventing and / or treating inflammation.

[0104] According to the present invention, it can act on M1Mφ that has already differentiated into M1 macrophages and induce it into M2Mφ. Furthermore, the induction from undifferentiated (M0) Mφ to M1Mφ can be eliminated. Therefore, according to the induction of M2 macrophage differentiation by the present invention, it is expected that inflammatory cells can be induced into anti-inflammatory cells and the M1 / M2 balance can be corrected.

Effects of the Invention

[0105] The subcritical extract of the present invention, as well as the immunomodulator, food and beverage, additive for beverage or food, cosmetic composition, anti-inflammatory agent and M2 macrophage differentiation inducer, can induce M0Mφ to M2Mφ and eliminate the induction from M0Mφ to M1Mφ. Furthermore, macrophages pre-induced to M1Mφ can be induced to M2Mφ, which is inhibitory to inflammation. Therefore, not only can inflammation be prevented, but also the healing of existing inflammation that could not be achieved conventionally can be carried out.

[0106] Also, according to the method for producing the subcritical extract of the present invention, as well as the immunomodulator, food and beverage, additive for beverage or food, cosmetic composition, anti-inflammatory agent and M2 macrophage differentiation inducer, M0Mφ can be induced to M2Mφ and the induction from M0Mφ to M1Mφ can be eliminated, and macrophages already induced to M1Mφ can also be induced to M2Mφ. Thus, the subcritical extract, as well as the immunomodulator, food and beverage, additive for beverage or food, cosmetic composition, anti-inflammatory agent and M2 macrophage differentiation inducer can be produced.

Brief Description of the Drawings

[0107]

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Mode for Carrying Out the Invention

[0108] The inventors of the present invention have intensively studied the action of various extracts of the bacterial cells with respect to the inflammation / anti-inflammation cycle brought about by the bacterial cells. As a result, surprisingly, when the subcritical extract obtained by extracting the bacterial cells by the subcritical treatment described below is allowed to act on macrophages, the induction from M0Mφ to M1Mφ, which has been confirmed when using a hot water extract or the like, disappears, and it has been found that the induction effect from M1Mφ to M2Mφ is expressed.

[0109] Subcritical extract The subcritical extract of the bacterial cell preparation of the present invention (hereinafter also referred to as the subcritical extract of the present invention) is an extract obtained by extracting at least one bacterial cell preparation selected from the group consisting of bacterial cells, bacterial cell-treated products, and bacterial cell cultures by subcritical treatment.

[0110] When the subcritical extract of the bacterial cell preparation is allowed to act on macrophages, as described above, the induction from M0Mφ to M1Mφ, which has been confirmed when a hot water extract of lactic acid bacteria or the like is allowed to act on macrophages and when untreated bacterial cells are allowed to act on macrophages, disappears, and the induction from M1Mφ to M2Mφ can be expressed. In addition, the induction from M0Mφ to M2Mφ is also expressed. Therefore, the subcritical extract of the present invention can enhance the M2Mφ activity.

[0111] Subcritical treatment of the bacterial cells can cause the reduction of their molecular weight. Therefore, for example, the subcritical extract of the present invention obtained by subjecting bacterial cells such as lactic acid bacteria to subcritical treatment contains substances in which functional biopolymers such as the cell components of bacterial cells such as lactic acid bacteria and various functional components contained within the cell membrane are extracted and / or decomposed by subcritical treatment. Since the subcritical extract of the present invention exhibits the effect of inducing disappearance from M0Mφ to M1Mφ and the effect of inducing expression from M1Mφ to M2Mφ, which cannot be achieved by extracts prepared by extraction methods other than subcritical extraction or untreated raw materials, these effects are considered to be due to the low-molecular-weight functional biopolymers contained in the subcritical extract of the present invention.

[0112] In the present invention, as the bacterial cells of the bacterial cell preparation subjected to subcritical treatment, any bacterial cells such as wild bacterial cells or commercially available bacterial cells can be used. For example, fermented foods such as yogurt, natto, and sake lees, foods such as vegetables and fruits, or other natural products may be used as the bacterial cells as they are, or those isolated and / or cultured from these raw materials may be used as the bacterial cells. Considering that those isolated from products used for edible and / or medicinal purposes such as foods can be safely ingested. However, the bacterial cells used in the present invention are not limited to these. The bacterial cells may be those isolated from humans. In addition, mutants of the above bacterial cells or genetically modified microorganisms (such as bacterial cells) prepared using genetic recombination techniques can also be used.

[0113] The bacterial cells used in the present invention may be live bacteria or dead bacteria. Dead bacteria can be obtained, for example, by subjecting live bacteria to sterilization treatments such as heat treatment, UV irradiation treatment, and cell disruption.

[0114] As the bacterial cell treatment product used in the present invention, dried products of bacterial cells (such as spray-dried products, freeze-dried products, vacuum-dried products, drum-dried products, etc.), frozen products, pasted products, aqueous dispersions, etc. can be used. Further, ground products or crushed products obtained by performing treatments such as grinding or crushing on the obtained live bacteria or dead bacteria may be used.

[0115] As the microbial cell culture used in the present invention, a suspension of microbial cells, a mixture of microbial cells (fermentation broth containing microbial cells), fermentation residue containing microbial cells, etc. after culturing the microbial cells can be used. Further, cytoplasm or cell wall fractions obtained by treating the microbial cell culture may also be used. The medium for culturing the microbial cells is not particularly limited, and the microbial cells may be cultured in a medium containing a normal carbon source, nitrogen source, inorganic salts, organic nutrients, etc. Normal culture conditions (culture temperature, pH during culture, culture period, etc.) for culturing the microbial cells can also be used.

[0116] The microbial cells used in the present invention are not particularly limited as long as the effects of the present invention are not impaired. Suitable examples include, for example, lactic acid bacteria, yeasts, Bacillus natto, Aspergillus oryzae, acetic acid bacteria, and the like. Such microbial cells can be included in the microbial cell preparation of the present invention in the form of live microbial cells, dead microbial cells, or disrupted microbial cells.

[0117] Examples of lactic acid bacteria include lactic acid bacteria belonging to a genus selected from the group consisting of the genus Lactobacillus, the genus Lactococcus, the genus Leuconostoc, the genus Pediococcus, the genus Streptococcus, the genus Weissella, the genus Tetragenococcus, the genus Oenococcus, the genus Enterococcus, the genus Vagococcus, the genus Carnobacterium, the genus Melissococcus, the genus Trichococcus, the genus Atopobium, the genus Bifidobacterium, and the like. More preferable lactic acid bacteria include lactic acid bacteria belonging to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium. However, the lactic acid bacteria of the present invention are not limited thereto.

[0118] Examples of yeasts include yeasts belonging to a genus selected from the group consisting of, for example, the genus Saccharomyces, the genus Candida, the genus Zygosaccharomyces, the genus Debaryomyces, the genus Hansenula, the genus Sterigmatomyces, the genus Rhodotorula, the genus Sirobasidium, etc. However, it is not limited to these.

[0119] Examples of Bacillus natto include Bacillus natto belonging to the genus Bacillus, but it is not limited to these.

[0120] Examples of Aspergillus oryzae include Aspergillus oryzae belonging to the genus Aspergillus. As examples, Aspergillus niger, Aspergillus kawachii, or Aspergillus flavus can be preferably used. However, it is not limited to these.

[0121] Examples of acetic acid bacteria include acetic acid bacteria belonging to the genus Acetobacter or the genus Gluconacetobacter. However, it is not limited to these.

[0122] Preferable examples of the fermentation residue used in the present invention include brewing lees. Here, brewing lees means by-products generated in the production process of fermented foods, such as residues after alcohol brewing. Examples of brewing lees in the present invention include sake lees (residues after sake production), shochu lees (residues after shochu distillation), and mirin lees (residues after mirin production), etc.

[0123] Method for producing subcritical extract The subcritical extract of the present invention is characterized in that it is a bacterial extract obtained by subcritically treating a bacterial preparation. In the present invention, the bacterial extract is an extraction product obtained by subjecting a raw material, which is a bacterial preparation, to an extraction treatment, and specifically, it is obtained by an extraction step of performing subcritical treatment using a solvent.

[0124] The subcritical treatment in the extraction step of the present invention refers to a treatment of bringing a subcritical fluid as an extraction solvent in a subcritical state under conditions of a predetermined temperature and a predetermined pressure into contact with a raw material to be extracted (in the present invention, a bacterial preparation), thereby extracting a desired component from the raw material to be extracted. For example, when water is heated to a pressure of 22.12 MPa or more and a temperature of 374.15 °C or more, it shows a state that is neither a liquid nor a gas. This point is called the critical point of water, and hot water at a temperature and pressure in the vicinity lower than the critical point is called subcritical water. This subcritical water has excellent component extraction action and hydrolysis action due to a decrease in dielectric constant and an improvement in ionic product.

[0125] Examples of the extraction solvent used for the subcritical treatment may include water, or a solvent other than water, such as ethylene, ethane, propane, carbon dioxide, methanol, ethanol, and mixtures thereof. Among these solvents, it is most preferable to use water from the viewpoint of safety. Also, it is considered that the extraction efficiency of water-soluble components is improved by using water.

[0126] When water is used as the extraction solvent for subcritical treatment, whether it is high-temperature water treatment, water in a liquid state or water in a gaseous state can be used without problems. That is, steam may be supplied to the treatment tank for subcritical treatment, or water may be supplied, or both of them may be supplied. The temperature of water or steam is preferably 100°C or higher. Also, since the reaction proceeds more easily in a liquid state than in a gaseous state as the reaction field, it is desirable to use water in a so-called subcritical state, which is forcibly made into a liquid state in a sealed container, as a suitable reaction field. As a more specific example of the reaction field, it can be mentioned that raw materials and water as the extraction solvent are put into a pressure-resistant container such as metal or ceramics, sealed, and the raw materials and water are brought into contact for a certain period of time. That is, in such a reaction field, an extract obtained by bringing subcritical water and raw materials into contact for a certain period of time or longer can be used as the subcritical-treated product of the present invention.

[0127] Therefore, as a method for producing the subcritical extract of the present invention, it is preferable to include an extraction step of subjecting the cell preparation to subcritical treatment using water. By subcritical treatment having a strong hydrolysis action, the cell preparation can be dissolved, decomposed, and made into low molecular weight. Furthermore, since extraction by subcritical treatment can be performed in a short time and with a high extraction rate, an extract can be efficiently obtained from a small amount of raw material, that is, the cell preparation.

[0128] The extraction step using subcritical water can be performed at a temperature of 155°C or higher and 220°C or lower. Furthermore, for the reason of enhancing the extraction effect, the extraction step is preferably performed at a temperature between 165 and 220°C, and more preferably at a temperature between 180 and 200°C. When the subcritical treatment is performed at a temperature lower than 155°C, even if the obtained subcritical extract is allowed to act on macrophages, the inducing activity from M0Mφ to M1Mφ does not disappear and remains. Also, when the subcritical treatment is performed at a temperature of 220°C or higher, the active ingredients contained in the cell preparation are over-decomposed, and the inducing activity from M1Mφ to M2Mφ disappears.

[0129] In the extraction process using subcritical water, the subcritical treatment pressure is preferably equal to or higher than the saturated vapor pressure at each temperature. The saturated vapor pressure at each temperature can be determined by referring to, for example, the steam tables of the Japan Society of Mechanical Engineers (1968). Exemplifying the saturated vapor pressures at 10°C intervals between 160 and 210°C, they are: 160°C: 0.62 MPa (6.30 at), 170°C: 0.79 MPa (8.08 at), 180°C: 1.00 MPa (10.22 at), 190°C: 1.25 MPa (12.80 at), 200°C: 1.55 MPa (15.68 at), 210°C: 1.90 MPa (19.45 at). By setting the subcritical treatment pressure to such pressures, it becomes easier to efficiently obtain the bacterial extract in the extraction process. Note that although the upper limit of the subcritical treatment pressure is not particularly defined, it may be preferably suppressed to around 20 to 30 MPa due to the specifications of the high-pressure device.

[0130] In the present invention, from the perspective of the inducibility to M2Mφ, which is a characteristic of the subcritical extract of the present invention, the subcritical treatment pressure in the extraction process is preferably 0.3 MPa or more and 2.0 MPa or less. If the subcritical treatment pressure is less than 0.3 MPa, the inducing activity of the obtained subcritical extract from M0Mφ to M1Mφ will not disappear and will remain. Also, if the subcritical treatment pressure is 2.0 MPa or more, the active ingredients contained in the bacterial cell preparation will be over-decomposed, and the inducing activity from M1Mφ to M2Mφ will disappear.

[0131] The subcritical treatment in the extraction process of the bacterial cell preparation of the present invention is preferably carried out with a treatment time between 5 and 150 minutes, more preferably between 5 and 60 minutes. Among these, it is more preferably carried out with a treatment time between 5 and 30 minutes. By performing the subcritical treatment within such a treatment time range, the functional components of the bacterial cell preparation can be efficiently obtained in the extraction process.

[0132] Therefore, the subcritical extract of the microbial cell preparation of the present invention is produced by subjecting at least one microbial cell preparation selected from the group consisting of, for example, microbial cells, processed microbial cells, and microbial cell cultures to extraction treatment with subcritical water adjusted to a temperature of 155°C or higher and 220°C or lower and a pressure of 0.3 MPa or higher and 2.0 MPa or lower. Further, preferably, the subcritical treatment of the microbial cell preparation using water as the extraction solvent can be carried out under the conditions that the treatment temperature is 180 to 200°C, the treatment pressure is equal to or higher than the saturated vapor pressure at each temperature, and the treatment time is 5 to 30 minutes. By performing the extraction step under such conditions, the induction from M0Mφ to M2Mφ and the induction from M1Mφ to M2Mφ of the subcritical extract of the obtained microbial cell preparation are significantly improved, and the induction from M0Mφ to M1Mφ disappears.

[0133] The subcritical extract of the present invention thus obtained can be directly put to practical use, but if necessary, the subcritical extract can be subjected to a solid-liquid separation step, and the obtained liquid portion can also be used.

[0134] Here, the solid-liquid separation step means a step of separating the subcritical extract of the microbial cell preparation into an extract and a raw material residue (solid matter). Specific solid-liquid separation steps include filtration using filter paper, centrifugation, decantation, screw press, roller press, rotary drum screen, belt screen, vibrating screen, multi-plate vibrating filter, vacuum dehydration, pressure dehydration, belt press, centrifugal concentration dehydration, multi-disc dehydration, and the like. Among them, filtration is preferred because of its simple operation and excellent separation efficiency.

[0135] Furthermore, the extract obtained by solid-liquid separation in the solid-liquid separation step, or the subcritical extract obtained from the extraction step of the cell preparation, may be diluted, concentrated, or may be prepared into a paste, a solid form with excellent preservability, a powder form, etc. through an additional drying step. As such a drying step, general drying methods can be used, including natural drying, heat transfer drying such as box drying and spray drying in a heating system, internal heat generation drying such as microwave drying, freeze drying, vacuum drying, suction drying, pressure drying, ultrasonic drying, etc. in a non-heating system. It is of course also possible to use a general and convenient oven or constant temperature bath for drying. Also, the extract or the subcritical extract may be appropriately purified by a known method.

[0136] The subcritical extract of the present invention can be used for various applications such as, but not limited to, food, cosmetics, pharmaceuticals, and the reagent clinical field. Also, the subcritical extract of the present invention may be for oral ingestion or for percutaneous ingestion. Preferably, the uses of the subcritical extract of the present invention include food and drink products, additives for beverages or foods, cosmetic compositions, and pharmaceuticals such as immunomodulators, anti-inflammatory agents, and M2 macrophage differentiation inducers.

[0137] Although not intended to be bound by theory, the subcritical extract of the present invention is excellent in the inducibility from M0Mφ to anti-inflammatory M2Mφ and the inducibility from M1Mφ to anti-inflammatory M2Mφ, and eliminates the inducibility from M0Mφ to inflammatory M1Mφ. This is because when the cell components and functional components are reduced in molecular weight by the subcritical treatment, the M1Mφ-inducing substances originally possessed by the cells are decomposed, while the M2Mφ-inducing substances retained by the cells are extracted from the cells by the subcritical treatment and further dissolved in water, making it possible to produce an extract having high M2Mφ-inducing activity.

[0138] Therefore, the subcritical extract of the present invention can be provided as, for example, a food or drink product, a supplement, or an additive for beverages or foods, expecting an anti-inflammatory effect. In this case, it can be in any form that can be used orally. In the present invention, the food or drink product includes processed foods added with the subcritical extract of the present invention and so-called foods for specified health uses. Also, the dosage unit form when used as a supplement is not particularly limited, and examples include tablets, capsules, granules, liquids, powders, and the like.

[0139] Further, the subcritical extract of the present invention may be provided as a cosmetic composition, expecting an anti-inflammatory effect. In this case, it can be in any form that can be used transdermally, for example. The cosmetic composition of the present invention can be applied to the skin, mucous membranes, body hair, hair, scalp, nails, teeth, facial skin, lips, and the like. Also, the cosmetic composition may contain one or more other components other than the subcritical extract of the present invention. As the other components, there is no particular limitation as long as the effects of the present invention are not impaired, and components usually used in cosmetics, for example, whitening agents, moisturizing agents, antioxidants, oily components, ultraviolet absorbers, surfactants, thickeners, alcohols, powder components, coloring materials, aqueous components, water, various skin nutrients, and the like can be appropriately blended as needed. Also, the dosage form of the cosmetic composition is not limited, and for example, an aqueous solution system, a solubilized system, an emulsion system, a powder system, an oil solution system, a gel system, an ointment system, an aerosol system, a water-oil two-layer system, a water-oil-powder three-layer system, etc. can be appropriately selected.

[0140] In addition, the use of the subcritical extract of the present invention may be a medicine. The medicine containing the subcritical extract of the present invention can be administered by various administration routes. Either an oral or a parenteral administration route can be used, and either systemic administration or local administration is also acceptable. For example, the medicine of the present invention can be administered via routes such as oral, intravenous, intramuscular, intraperitoneal, intradermal, transdermal, nasal, inhalation, intralesional, etc. The dosage form of the medicine of the present invention is not particularly limited, and various dosage forms can be applied. For example, tablets, powders, granules, dry syrups, syrups, capsules, ointments, creams, injections, solutions, nasal drops, suppositories, patches, etc. can be mentioned. Further, the medicine of the present invention can optionally contain other pharmaceutically acceptable carriers, excipients, diluents, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, etc. within the range that does not impair the effects of the present invention.

[0141] The medicine of the present invention is, for example, an immunomodulator. The immunomodulator of the present invention has a high induction activity from M0Mφ and M1Mφ to M2Mφ. Macrophages are activated to secrete immunomodulatory cytokines and are known to respond to various signals via the secreted cytokines. When macrophages are activated by stimulation with TLR ligands and IFN-γ, they are induced into M1 macrophages, which are inflammatory macrophages. M1 macrophages secrete inflammatory cytokines such as TNF-α and IL-12, promote Th1 responses, and play a role in resistance to intracellular parasites. When macrophages are activated by stimulation with IL-4 / IL-13, they are induced into M2 macrophages, which are anti-inflammatory macrophages. M2 macrophages secrete anti-inflammatory cytokines such as IL-10, promote Th2 responses, and are involved in reducing inflammation and promoting tissue repair. Therefore, the immunomodulator of the present invention can regulate the balance of M1 macrophages / M2 macrophages to adjust the immune function.

[0142] Although not intended to be limited to a specific mechanism of action, the immunomodulating agent of the present invention can be used as a medicament for the treatment or prevention of diseases related to the imbalance of the M1 / M2 balance of macrophages. The M1 / M2 balance of macrophages is considered to be involved in the pathogenesis of various diseases. Here, the imbalance of the M1 / M2 balance in the present invention means a state in which M1 macrophages are present in excess compared to M2 macrophages, or M2 macrophages are smaller in number compared to M1 macrophages. Examples of diseases related to such an imbalance of the M1 / M2 balance of macrophages include diabetes, arteriosclerosis, multiple sclerosis, inflammatory bowel disease, and the like. The immunomodulating agent of the present invention can correct the imbalance of the M1 / M2 balance by inducing differentiation into M2 macrophages, and as a result, can treat or prevent diseases related to the imbalance of the M1 / M2 balance of macrophages. Therefore, the present invention also relates to a method for preventing or treating such diseases, which method comprises administering an effective amount of the subcritical extract of the present invention.

[0143] The immunomodulating agent of the present invention is particularly preferably used for diseases in which a Th1-dominant immune response is known to be involved in the exacerbation of the disease state. For example, arteriosclerosis is one of such diseases. According to the immunomodulating agent of the present invention, since macrophages can be induced from M1 to M2, it is considered that diseases in which a Th1-dominant immune response is known to be involved in the exacerbation of the disease state can be advantageously treated or prevented.

[0144] In addition, since the immunomodulating agent of the present invention can induce M0Mφ and M1Mφ into M2Mφ that secretes anti-inflammatory cytokines, it can act as an anti-inflammatory cytokine production promoter that promotes the secretion of anti-inflammatory cytokines. Furthermore, since the immunomodulating agent of the present invention eliminates the induction of M1Mφ that secretes inflammatory cytokines and also induces existing M1Mφ into M2Mφ, it can act as an inflammatory cytokine production inhibitor.

[0145] In the present invention, since inflammatory cells can be induced to become anti-inflammatory, it is possible to treat already-occurring inflammation. Therefore, the subcritical extract of the bacterial cell preparation of the present invention and the subcritical extract of the bacterial cell preparation obtained by the production method of the present invention can be used in the production of a medicament for preventing and / or treating inflammation. Further, the medicament of the present invention can be provided as an anti-inflammatory agent having a therapeutic effect on inflammation.

[0146] The present invention provides an M2 macrophage differentiation inducer effective for inducing the differentiation of macrophages into M2 macrophages. The M2 macrophage differentiation inducer of the present invention contains a subcritical extract of a bacterial cell preparation, and as described above, in addition to the inducibility from M0Mφ to M2Mφ, it also has the inducibility from M1Mφ to M2Mφ. Therefore, the M2 macrophage differentiation inducer of the present invention includes induction of differentiation from M1 macrophages to M2 macrophages. It is considered that the induction to M2 macrophages is further enhanced. Although not intended to be bound by theory, it has become clear that M2 macrophages have effects such as parasite removal, immune regulatory functions, reduction of inflammation, and promotion of induction of regeneration of the living body. Therefore, increasing the induction to M2 macrophages is an effective means for preventing and treating inflammation and promoting induction of regeneration of the living body, for example, promoting tissue repair and angiogenesis. However, the M2 macrophage differentiation inducer of the present invention is not limited to use as a medicament, and may be used in vitro, for example, as a research reagent.

Examples

[0147] The present invention will be specifically described based on examples, but the present invention is not limited thereto.

[0148] Bacterial cell preparation for testing <Lactic acid bacteria> As the raw material bacterial cells, Lactobacillus brevis, Lactobacillus plantarum OLL2712 (FERM BP-11262), Lactobacillus rhamnosus OLL2838 (NITE P-313), Lactobacillus casei, Streptococcus equi subsp. Zooepidemicus HA-116 (ATCC 39920), and Bifidobacterium bifidum NBRC 100015 were used. Among these, the ATCC strains are available from the American Type Culture Collection. Also, the FERM strains, NITE strains, and NBRC strains are available from the Biotechnology Center of the National Institute of Technology and Evaluation.

[0149] Note that the Lactobacillus brevis strain and Lactobacillus casei strain were isolated from commercially available yogurt. After analyzing their respective 16s ribosomal DNA sequences and performing a search in the NCBI database, they were found to match the registered sequences of Lactobacillus brevis and Lactobacillus casei with 99% and 99% identity, respectively.

[0150] Each strain was statically cultured at 37°C for 18 hours or more. Bifidobacterium bifidum was statically cultured at 37°C for 18 hours or more after being replaced with nitrogen gas. All strains were cultured in de Man, Rogosa, and Shape (MRS) medium (Becton, Dickinson Co, Sparks, Md, USA).

[0151] These cultures were centrifuged at 3,000 rpm for 10 minutes to collect only the bacterial cells. After repeating washing and centrifugation twice with sterilized distilled water, sterilized distilled water was added to adjust the solid content concentration to 0.65% to prepare a bacterial cell preparation for testing. The solid content concentration was converted from the weights of the sample before and after treatment, which was obtained by placing the bacterial suspension in a dried container and allowing it to stand at 105 °C for 4 hours or more.

[0152] <Brewing residue> The shochu lees obtained from a shochu manufacturer in Kagoshima Prefecture (distillation residue generated from the distillation process during shochu production; solid content 6.2 - 6.4%) was used as it was as a bacterial cell preparation for testing in the experiment.

[0153] <Preparation of test samples> · Example 1 15 g of lactic acid bacteria (strain: Lactobacillus brevis), which was a bacterial cell preparation for testing adjusted to a solid content concentration of 0.65% as described above, was placed in a 2 L pressure-resistant container, and subcritical treatment was performed at a treatment temperature of 195 °C, a treatment pressure of 1.40 MPa, and a treatment time of 10 minutes. After the subcritical treatment was completed, the treated product (subcritical treatment liquid + solid matter) in the treatment container was collected in a 500 mL container. The treated product was adjusted to a solid content concentration of 0.1% to obtain a subcritical extract for testing. The obtained subcritical extract for testing was stored frozen at -25 °C. When used for each evaluation described below, it was thawed at room temperature and then used as a test sample.

[0154] · Example 2 The same treatment as in Example 1 was performed except that lactic acid bacteria (strain: Lactobacillus casei) was used.

[0155] · Example 3 The same treatment as in Example 1 was performed except that streptococcus (strain: Streptococcus equi subsp. Zooepidemicus) was used.

[0156] · Example 4 The same procedures as in Example 1 were performed, except that lactic acid bacteria (species: Lactobacillus plantarum) were used.

[0157] · Example 5 The same procedures as in Example 1 were performed, except that lactic acid bacteria (species: Lactobacillus rhamnosus) were used.

[0158] · Example 6 The same procedures as in Example 1 were performed, except that Bifidobacterium (species: Bifidobacterium bifidum) were used.

[0159] · Examples 7 to 12 Each of the subcritical extracts for testing obtained in Examples 1 to 6 was subjected to solid-liquid separation and sterilization using a 0.22 μm membrane filter (manufactured by Millipore) through filtration. Only the obtained filtrate was used as the subcritical treatment filtrate for testing and was used as a test sample for each evaluation described below.

[0160] · Example 13 The same procedures as in Example 7 were performed, except that lactic acid bacteria (species: Lactobacillus brevis) were used and subcritical treatment was carried out at a treatment temperature of 165°C, a treatment pressure of 0.70 MPa, and a treatment time of 20 minutes.

[0161] · Example 14 The same procedures as in Example 7 were performed, except that lactic acid bacteria (species: Lactobacillus brevis) were used and subcritical treatment was carried out at a treatment temperature of 175°C, a treatment pressure of 0.89 MPa, and a treatment time of 10 minutes.

[0162] · Example 15 The same procedures as in Example 7 were performed, except that lactic acid bacteria (species: Lactobacillus brevis) were used and subcritical treatment was carried out at a treatment temperature of 175°C, a treatment pressure of 0.89 MPa, and a treatment time of 30 minutes.

[0163] · Example 16 The same treatment as in Example 7 was performed, except that lactic acid bacteria (strain: Lactobacillus brevis) were used and subcritical treatment was carried out at a treatment temperature of 195°C, a treatment pressure of 1.40 MPa, and a treatment time of 30 minutes.

[0164] · Example 17 200 g of shochu lees was placed in a 2L pressure-resistant container, and subcritical treatment was carried out at a treatment temperature of 135°C, a treatment pressure of 0.31 MPa, and a treatment time of 20 minutes. After the subcritical treatment was completed, the treated product (subcritical treatment liquid + solid matter) in the treatment container was collected in a 1L container. The treated product was stored frozen at -25°C. When used for each evaluation, the frozen product was thawed at room temperature, centrifuged (3,000 rpm, 5 minutes), and the obtained supernatant was adjusted to a solid content concentration of 1%, filtered through a 0.22 μm membrane filter (manufactured by Millipore), and only the obtained filtrate was used as the test subcritical treatment filtrate and subjected to each evaluation described below as a test sample.

[0165] · Example 18 The same treatment as in Example 17 was performed, except that subcritical treatment was carried out at a treatment temperature of 155°C, a treatment pressure of 0.54 MPa, and a treatment time of 20 minutes.

[0166] · Example 19 The same treatment as in Example 17 was performed, except that subcritical treatment was carried out at a treatment temperature of 175°C, a treatment pressure of 0.89 MPa, and a treatment time of 20 minutes.

[0167] · Example 20 The same treatment as in Example 17 was performed, except that subcritical treatment was carried out at a treatment temperature of 195°C, a treatment pressure of 1.40 MPa, and a treatment time of 20 minutes.

[0168] · Comparative Example 1 50 g of lactic acid bacteria (Lactobacillus brevis) adjusted to a solid content concentration of 0.65% were placed in a 200 mL beaker, and heat treatment was performed at a treatment temperature of 95 °C for 30 minutes. After the heat treatment was completed, the treated material in the beaker was collected in a 50 mL centrifuge tube container. The obtained heat-treated extract was adjusted to a solid content concentration of 0.1% and stored frozen at -25 °C. When used for each evaluation described below, it was thawed at room temperature and then used.

[0169] · Comparative Example 2 The same treatment as in Comparative Example 1 was performed except that lactic acid bacteria (species: Lactobacillus casei) were used.

[0170] · Comparative Example 3 The same treatment as in Comparative Example 1 was performed except that streptococci (species: Streptococcus equi subsp. Zooepidemicus) were used.

[0171] · Comparative Example 4 30 g of lactic acid bacteria (Lactobacillus rhamnosus) adjusted to a cell count of 1.0×10 8 cells / mL (solid content concentration 0.045%) were placed in a 200 mL beaker, and heat treatment was performed at a treatment temperature of 70 °C for 60 minutes. After the heat treatment was completed, the treated material in the beaker was collected in a 50 mL centrifuge tube container. The obtained heat-treated extract was adjusted to a solid content concentration of 0.1% and stored frozen at -25 °C. When used for each evaluation, it was thawed at room temperature and then used.

[0172] · Comparative Example 5 The same treatment as in Comparative Example 1 was performed except that lactic acid bacteria (species: Lactobacillus rhamnosus) were used.

[0173] · Comparative Example 6 The supernatant obtained by centrifuging sake lees was adjusted to a solid content concentration of 1%. The adjusted product was filtered through a 0.22 μm membrane filter (manufactured by Millipore), and only the resulting filtrate was used for each evaluation described below.

[0174] · Comparative Example 7 200 g of sake lees was placed in a 500 mL beaker, and heat treatment was performed at a treatment temperature of 95 °C for 30 minutes. After the heat treatment, the treated product in the beaker was collected in a 500 mL container and stored frozen at -25 °C. When used for each evaluation, the frozen product was thawed at room temperature, centrifuged (3,000 rpm, 5 minutes), the resulting supernatant was adjusted to a solid content concentration of 1%, filtered through a 0.22 μm membrane filter (manufactured by Millipore), and only the resulting filtrate was used.

[0175] <Induction of Macrophage Differentiation and Its Evaluation> (1) Macrophage Cells M0 macrophages are prepared by a method of differentiating monocytes present in bone marrow and blood, which are generally known, into macrophages by stimulating them with M-CSF, or a method of obtaining macrophage-like cell lines, for example, a method of obtaining a macrophage-like cell line by differentiating THP-1 (human monocyte-like cells) by stimulation with PMA, or a method of using RAW264 cells, which are mouse-derived macrophage-like cell lines. In the present invention, RAW264 cells, which are mouse-derived macrophage-like cell lines, were used. The RAW264 cells were obtained from the RIKEN.

[0176] (2) Induction of Macrophage Differentiation M0 macrophages were induced to differentiate by adding a predetermined amount of the test samples prepared in the examples and comparative examples to RAW264 cells. The detailed method will be described later.

[0177] When evaluating the inductivity described in the following (3) to (5), when evaluating the test sample itself, the experiment was conducted without filtering the test sample through a membrane filter. However, since this increases the risk of contamination by other bacteria and other impurities during the test, after evaluating the properties of each test sample itself, each test sample that had been filtered through a membrane filter was used for subsequent evaluation.

[0178] (3) Inductivity to M1 macrophages To confirm the inductivity to inflammatory M1 macrophages, a method of quantifying the production amount of NO is generally known. M1 macrophages express iNOS and enhance bactericidal properties by producing NO from arginine (Fernando Oneissi Martinez et al., Frontiers in Bioscience 13, 453 - 461, January 1, 2008.). The produced NO is oxidized to NO 2 and further reacts with water to form nitrous acid or nitric acid. In the method using Griess reagent, the production amount of NO can be indirectly evaluated by quantifying the generated nitrous acid.

[0179] (4) Inductivity to M2 macrophages To confirm the inductivity to anti - inflammatory M2 macrophages, a method of evaluating the activity of arginase is known. M2 macrophages express arginase 1 and produce ornithine, and polyamines and proline downstream from arginine. This suppresses NO production at the repair site and promotes tissue repair via polyamines and proline (J Immunol 2001, 167 (11): 6533 - 6544.). That is, arginase 1 deprives and depletes the substrate (arginine) used for the production of inflammatory cytokines at the initial stage of inflammation. Therefore, arginase 1 is considered to have the function of switching from inflammation to inflammation suppression.

[0180] Both iNOS and arginase 1 use the same arginine as a substrate. That is, M2 macrophages negatively regulate NO production by expressing high levels of arginase 1, biasing the NO metabolic pathway towards proline production. The activity of arginase can be calculated by quantifying the amount of urea synthesized in the production of ornithine from arginine.

[0181] In addition, anti-inflammatory M2 macrophages are known to express IL-10, which is known as an anti-inflammatory cytokine. Therefore, the inducibility of M2 macrophages can be evaluated by quantifying the amount of IL-10 produced.

[0182] (5) Inducibility from M1 macrophages to M2 macrophages Macrophages are induced into M1 macrophages by adding lipopolysaccharide (LPS) and interferon gamma (IFN-γ) (Adams, DO, Immunol Today, 1989, 10, 33-5). The evaluation of the inducibility from M1 macrophages to M2 macrophages was performed by adding a test sample to the M1 macrophages induced in this way and evaluating the inducibility to M2 macrophages.

[0183] <Evaluation of NO production amount> After RAW264 cells were subcultured more than 4 times in a petri dish, only the medium was removed from the petri dish using an aspirator. Then, 10 mL of medium was added, and the cells remaining on the bottom of the petri dish were detached from the petri dish using a cell scraper. This was collected in a 15 mL centrifuge tube and centrifuged at 800 rpm for 3 minutes to collect the cells. After collection, the cells were dispersed in the medium to a concentration of 1.0×10 5 cells / mL to prepare a cell suspension, and 500 μL of the cell suspension was added to each well of a 24-well plate. Then, the cells were grown by leaving them overnight in an incubator at 37°C in the presence of 5% CO 2 After confirming that the cells had grown sufficiently, the medium was removed using an aspirator.

[0184] When evaluating the induction effect of the test sample on pre-induced M1 Mφ, after changing the medium here, INF-γ and LPS are added to the medium so that the final concentration becomes 10 ng / mL, and 5% CO 2 is left standing overnight in an incubator at 37°C in the presence to induce differentiation into M1 Mφ.

[0185] After adding 450 μL of medium to each well containing M0 macrophages or M1 macrophages, 50 μL of the test subcritical extract or test subcritical treatment filtrate with a solid content concentration of 0.1% as the test sample is added, and 5% CO 2 is left standing overnight in an incubator at 37°C in the presence for culturing. By adding Greiss reagent to the collected culture solution, as described above, nitrous acid in the culture solution can be quantified, and the NO production amount can be indirectly evaluated. Sodium nitrate is used as a standard for the quantification of nitrous acid, and a calibration curve is prepared using a 10-fold dilution series as the nitrous acid standard solution.

[0186] As positive control (PC), LPS and INF-γ, as negative control (NC), sterilized water, and the cell culture solutions obtained by adding the test subcritical extract or test subcritical treatment filtrate respectively were each sampled 100 μL into 24 wells. 100 μL of 100 mg / mL Greiss-Romijn nitrous acid reagent (manufactured by Wako Pure Chemical Industries, Ltd.) was added and left standing at room temperature for 20 minutes for reaction. After the reaction was completed, the absorbance at 540 nm was measured using a microplate reader. The nitrous acid concentration in the culture supernatant was calculated from the calibration curve prepared in advance. The results are shown in Figure 1.

[0187] Figure 1 shows the evaluation results of NO production (that is, the inducibility from M0 Mφ to M1 Mφ) in Examples 1 to 6 and Comparative Examples 1 to 5. From Figure 1, it can be seen that in Examples 1 to 6 using the test samples subjected to subcritical treatment, NO production disappeared under all conditions. On the other hand, in Comparative Examples 1 to 5 using the test samples subjected to hot water treatment, NO production was confirmed under all conditions.

[0188] From the above, it was found that the cell preparation subjected to subcritical treatment can suppress the induction from M0 macrophages to M1 macrophages (M0M1φ→M1Mφ) regardless of the bacterial species of the raw material. However, in Comparative Examples 1 to 5 subjected to heat treatment, the inducibility to M1Mφ was confirmed. Therefore, Comparative Examples 1 to 5 of heat treatment were excluded from the subsequent examination.

[0189] <Evaluation of Arginase Activity> After RAW264 cells were subcultured in a petri dish four or more times, only the medium was removed from the petri dish using an aspirator, and then 10 mL of medium was added. The cells remaining on the bottom of the petri dish were detached from the petri dish using a scraper. This was collected in a 15 mL centrifuge tube and centrifuged at 800 rpm for 3 minutes to collect the cells. After collection, 5 The cells were dispersed in the medium to a concentration of 1.0×10 2 cells / mL to prepare a cell suspension, and 500 μL of the cell suspension was added to each well of a 24-well plate. Then, the cells were grown by leaving them standing overnight in an incubator at 37°C in the presence of 5% CO

[0190] After changing the medium, INF-γ and LPS were added to the medium to a final concentration of 10 ng / mL, and the cells were allowed to stand overnight in an incubator at 37°C in the presence of 5% CO 2 to induce differentiation into M1Mφ.

[0191] For the evaluation of arginase activity, a dedicated kit (QuantiChrom TM Arginase Assay kit, manufactured by BioAssay Systems) was used. After adding 40 μL of the test subcritical treatment filtrate, 10 μL of the substrate buffer of the kit was added to each well, and the mixture was left standing in an incubator at 37°C for 2 hours to react. 200 μL of the urea reagent attached to the kit was added to each well. Then, the mixture was left standing at room temperature for 60 minutes, and according to the procedure manual attached to the kit, the absorbance at 430 nm was measured to calculate the urea concentration. The results are shown in Figure 2.

[0192] Figure 2 shows the evaluation results of arginase activity (i.e., the inducibility from M1Mφ to M2Mφ) when the test samples of Examples 7 to 12 (subcritical-treated products, which are the test samples of Examples 1 to 6 where the effect of suppressing M0M1φ→M1Mφ was discovered, filtered through a filter to obtain test samples) were applied to pre-induced M1Mφ. As shown in Figure 2, in Examples 7 to 12 with subcritical treatment, regardless of the raw material bacterial species, a high amount of urea was produced under all conditions, indicating high arginase activity.

[0193] From the above, it was found that the subcritical-treated cell preparation has a remarkable effect that it can induce anti-inflammation for macrophages pre-induced to M1Mφ regardless of the raw material bacterial species.

[0194] <Evaluation of IL-10 production> After RAW264 cells were subcultured in a petri dish more than 4 times, only the medium was removed from the petri dish using an aspirator, then 10 mL of medium was added, and the cells remaining on the bottom of the petri dish were detached from the petri dish using a scraper. This was collected into a 15 mL centrifuge tube and centrifuged at 800 rpm for 3 minutes to collect the cells. After collection, the cells were dispersed in the medium to a concentration of 1.0×10 5 cells / mL to prepare a cell suspension, and 500 μL of the cell suspension was added to each well of a 24-well plate. Then, the cells were grown by leaving them standing overnight in an incubator at 37°C in the presence of 5% CO 2 After confirming that the cells had grown sufficiently, the medium was removed using an aspirator.

[0195] After changing the medium, INF-γ and LPS were added to the medium to a final concentration of 10 ng / mL, and the cells were induced to differentiate into M1Mφ by leaving them standing overnight in an incubator at 37°C in the presence of 5% CO 2 After that, the cells were left standing overnight in an incubator at 37°C in the presence of 5% CO

[0196] For the evaluation of IL-10 production, a dedicated kit (Quantikine® ELISA mouse IL-10, manufactured by R&D) was used. The evaluation was carried out using the test subcritical treatment filtrate obtained by filtering the test subcritical extract with a solid content concentration of 0.1%. The measurement was performed according to the procedure manual attached to the kit. The results are shown in Figure 3.

[0197] Figure 3 shows the evaluation results of the IL-10 production amount (i.e., the inducibility from M1Mφ to M2Mφ) when the test samples of Examples 7 to 11 (subcritical treatment products which are the test samples of Examples 1 to 5 where the effect of suppressing M0M1φ → M1Mφ was discovered, and were filtered to obtain the test samples) were applied to the pre-induced M1Mφ. As shown in Figure 3, in Examples 7 to 11 with subcritical treatment, regardless of the raw material bacterial species, a significant increase in IL-10 production was confirmed, where the production amount of IL-10 was higher in the presence of the test sample compared to the absence under all conditions.

[0198] From the above, it was found that the subcritically treated cell preparation has a remarkable effect of being able to induce anti-inflammation even for macrophages pre-induced to M1Mφ, regardless of the raw material bacterial species.

[0199] From Figure 1, it was found that when subcritical treatment was performed, regardless of the raw material bacterial species, the induction from M0 macrophages to M1 macrophages could be eliminated. Also, from Figures 2 and 3, it was found that for macrophages pre-induced to M1 macrophages, by adding the subcritical extract of the cell preparation, regardless of the raw material bacterial species, they could be induced to M2 macrophages at a high level.

[0200] Therefore, in subsequent tests, the bacterial species was narrowed down to Lactobacillus brevis, and further verification was carried out on the effectiveness of subcritical treatment.

[0201] <Evaluation of NO production amount using Lactobacillus brevis> Using the same method as the evaluation of the NO production amount shown in Fig. 1, the NO production amount was examined when a subcritical extract using Lactobacillus brevis as the cell body was used. However, in this method, the induction effect of the test sample on macrophages pre-induced into M1 macrophages was evaluated. As the test samples, the samples of Examples 7 and 14 were used. The results are shown in Fig. 4.

[0202] From Fig. 4, it was found that by using the test sample subjected to subcritical treatment, regardless of the subcritical treatment conditions, the NO production that should have been originally produced from macrophages pre-induced into M1 macrophages was suppressed. From this result, it is considered that once-induced M1 macrophages also disappeared by using the test sample subjected to subcritical treatment.

[0203] <Evaluation of arginase activity using Lactobacillus brevis> Using the same method as the evaluation of the arginase activity shown in Fig. 2, the arginase activity was examined when a subcritical extract using Lactobacillus brevis as the cell body was used. As the test samples, the samples of Examples 7, 13, 15 and 16 were used. The results are shown in Fig. 5.

[0204] From Fig. 5, high arginase activity was confirmed in all of the test samples subjected to subcritical treatment. Regardless of the subcritical treatment conditions, it was found that by using the test sample subjected to subcritical treatment, macrophages pre-induced into M1Mφ can also be induced to be anti-inflammatory.

[0205] From the results shown in Figs. 4 and 5, it was found that the cell preparation subjected to subcritical treatment can transdifferentiate inflammatory M1 macrophages into anti-inflammatory M2 macrophages by applying them to inflammatory M1 macrophages regardless of the subcritical treatment conditions.

[0206] <Evaluation of NO production amount using shochu lees> Using the same method as the evaluation of the NO production amount shown in Fig. 4, that is, adding a test sample to macrophages pre-induced into M1 macrophages, the NO production amount when using a subcritical extract with shochu lees as the raw material of the cell preparation was examined. As the test samples, the samples of Example 20 and Comparative Examples 6 and 7 were used. The results are shown in Fig. 6.

[0207] As shown in Fig. 6, it was found that in Example 20, which is a test sample subjected to subcritical treatment, the NO production that should have been originally produced from macrophages pre-induced into M1 macrophages was suppressed. Therefore, it is considered that once induced M1 macrophages also disappeared by using a test sample derived from shochu lees subjected to subcritical treatment. On the other hand, in Comparative Example 6 where untreated shochu lees was the test sample, and Comparative Example 7 where shochu lees subjected to hot water treatment was the test sample, it was found that NO production was not suppressed and remained as it was. That is, when using the test samples of the comparative examples, it is considered that inflammatory M1 macrophages remain inflammatory without being induced even after the test sample is applied.

[0208] <Evaluation of arginase activity using shochu lees> Using the same method as the evaluation of arginase activity shown in Fig. 5, the arginase activity when using a subcritical extract with shochu lees as the raw material of the cell preparation was examined. As the test samples, the samples of Example 20 and Comparative Examples 6 and 7 were used. The results are shown in Fig. 7.

[0209] As shown in Fig. 7, high arginase activity was confirmed in Example 20, which is a test sample subjected to subcritical treatment. Therefore, it was found that once induced M1 macrophages can be induced into anti-inflammatory by using a test sample derived from shochu lees subjected to subcritical treatment. On the other hand, in Comparative Example 6 where untreated shochu lees was the test sample, and Comparative Example 7 where shochu lees subjected to hot water treatment was the test sample, although arginase activity about the positive control was observed, these activities were only low compared with the activity confirmed in Example 20.

[0210] <Evaluation of IL-10 production using sake lees Using the same method as the evaluation of IL-10 production shown in Fig. 3, the IL-10 production when using a subcritical extract with sake lees as the raw material of the cell preparation was examined. As test samples, the samples of Examples 17 to 20 and Comparative Examples 6 and 7 were used. The results are shown in Fig. 8.

[0211] As shown in Fig. 8, in Examples 17 to 20 which are test samples subjected to subcritical treatment, high IL-10 production was confirmed in all test samples regardless of the conditions of subcritical treatment. Therefore, it was found that once induced M1 macrophages can also be induced to be anti-inflammatory by using test samples derived from sake lees subjected to subcritical treatment. On the other hand, in Comparative Example 6 where untreated sake lees was the test sample and Comparative Example 7 where sake lees subjected to hot water treatment was the test sample, although IL-10 production was confirmed, the production amount was only much lower compared to the production amount confirmed in Example 20.

[0212] From the above results, it can be seen that the subcritical extract of the present invention and the filtrate obtained by filtering the subcritical extract, regardless of the bacterial species of the cell preparation as the raw material and regardless of the conditions of subcritical treatment, have a remarkable effect of eliminating the induction from M0 macrophages to M1 macrophages and inducing M1 macrophages to M2 macrophages in the same manner as M0 macrophages. In the untreated cell preparation and the hot water extract, since the induction to M1 macrophages remains, this effect is considered to be an effect peculiar to subcritical treatment, that is, due to the low molecular weight of the functional biopolymer by subcritical treatment.

Claims

1. A method for producing an M2 macrophage differentiation inducer, comprising subjecting at least one kind of bacterial cells or bacterial cell cultures selected from the group consisting of lactic acid bacteria, streptococci, bifidobacteria, and shochu lees to extraction treatment with subcritical water adjusted to a temperature of 155°C or higher and 220°C or lower and a pressure of 0.3 MPa or higher and 2.0 MPa or lower.

2. The method for producing an M2 macrophage differentiation inducer according to Claim 1, wherein the lactic acid bacteria belong to a genus selected from the group consisting of the genus Lactobacillus, the genus Streptococcus, and the genus Bifidobacterium.

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