JP5761679B2 - Composition for regulating activity of peripheral blood mononuclear cells, comprising fermented papaya as an active ingredient - Google Patents

Description

  The present invention relates to a composition for regulating the activity of peripheral blood mononuclear cells.

  When a pathogen, a virus, or the like enters a living body or after the invasion, various cells are involved in living body defense. Cells involved in biological defense are collectively called leukocytes. White blood cells are present in peripheral blood, and are composed of lymphocytes, granulocytes and monocytes. Cells such as lymphocytes are activated in cooperation with each other, thereby enhancing the ability of biological defense. Moreover, even when these cells are activated too much, the cells cooperate with each other to suppress the cell activity. Regulation of cell activity such as lymphocytes involves cytokines such as interleukin (IL) and interferon (IFN), and plays a role in signal transduction between cells.

  On the other hand, there were many unclear points about the activation of leukocytes by health foods, but medical evidence has been submitted since the discovery of Toll-like Receptor (TLR). A β-1.3 glucan such as a polysaccharide (lentinan) extracted from shiitake mushroom and schizophyllan (Sonifilan (registered trademark)) extracted from Suehirotake has been well known as an immune activity enhancer. Moreover, there are many reports that Gram-positive bacterial peptidoglycan (Pisibanil (registered trademark)) and LPS, which is a lipopolysaccharide derived from Gram-negative bacteria, stimulate TLR and sometimes increase immune activity (Non-patent Document 1).

  Natto, which has long been known as a health food and a fermented food, is known to have various physiological activities, and its extract has IL-12 production-inducing activity and IL-4 production. It is known to have inhibitory activity (Patent Document 1). However, as is clear from natto, which is a typical example of fermented foods, fermented foods have a characteristic odor and slime. Many. Therefore, the advent of fermented foods that have a function of regulating the activity of cells such as lymphocytes and can be easily ingested has been strongly desired.

Papaya (scientific name: Carica papaya Linn), a fruit with a rich sweetness and a faint acidity, is preferred as a favorite item for desserts and cakes. On the other hand, fermented papaya is recognized as a health food that does not have the smell and slime peculiar to fermented foods, but it is not a single ingredient like supplements, but contains various ingredients including fermented decomposition products. Food. One of its components has been reported to have an antioxidant function, which significantly suppresses free radical scavenging and lipid peroxidation in the rat brain (Non-Patent Document 2), and prevents DNA damage by H ₂ O ₂ in rat pheochromocytoma. Inhibition (Non-Patent Document 3), and the active oxygen suppression effect (Non-Patent Document 4) and the action of reducing blood glucose level (Non-Patent Document 5) in Alzheimer's disease onset model via copper ions Various effects have been reported. Many of these suggest a deep relationship with antioxidant capacity, and patent publications 2 and 3 report that they are particularly effective for hydroxy radical scavenging activity. Non-patent document 6 describes the possibility of a therapeutic ability to prevent contact allergy.

  However, research on the effectiveness of fermented papaya at the individual level is as described in Non-Patent Document 6, etc., but specific analysis at the cellular level has not been made, and defense of living bodies accompanying cellular activities such as lymphocytes The details about remained unclear.

JP 2006-001922 A Japanese Patent No. 4036385 JP 2009-102247

Naoto Ono: Dojin News. Issue: 114 Pages: 1-10 (2010) IMAO K. et al: Biochemistry and Molecular Biology International, 45 (1), PP. 11-23 (1998) Aruoma OI, et al: Biofactors. 2006; 26 (2): 147-59. Zhang et al: NEUROSCIENCE Volume 143, Number 1, November 17, (2006) Danese C, et al: Clin Ter. May-Jun; 157 (3): 195-8. (2006) Keiichi H. et al: Journal of the Science of Food and Agriculture Volume 88 Issue 7, Pages 1151-1157 (2008) Dannull J. et al: The Journal of the Clinical Investigation 2005 Dec; 115 (12): 3623-33. Epub 2005 Nov 23.

  The present invention is to provide an excellent composition for regulating the activity of peripheral blood mononuclear cells.

  As a result of intensive studies on the effectiveness of the papaya fermented product, the inventors of the present invention have found that the fermented product has an excellent effect of regulating the activity of peripheral blood mononuclear cells and completes the present invention. It came to.

  That is, the present invention provides a composition for regulating the activity of peripheral blood mononuclear cells containing a papaya fermented product as an active ingredient. Since the composition concerning this invention contains the papaya fermented material which has the activity regulation effect | action of a peripheral blood mononuclear cell, it has the effect | action which regulates the activity of a peripheral blood mononuclear cell.

  The present invention also provides food, drink, health food or feed additive (hereinafter referred to as food or drink) containing a composition for regulating the activity of peripheral blood mononuclear cells containing a fermented papaya as an active ingredient. . Since the food and drink containing the composition according to the present invention contains a papaya fermented product having an activity regulating action of peripheral blood mononuclear cells, it has an action of regulating the activity of peripheral blood mononuclear cells.

  The composition provided by the present invention regulates the activity of peripheral blood mononuclear cells, that is, promotes cell activity such as lymphocytes while suppressing cell activity such as excessive lymphocytes. Function can be demonstrated.

It is a flowchart which shows the manufacture example of the composition containing a papaya fermented material. It is a figure showing the experimental result of the flow cytometry which shows secretion of TNF- (alpha) and IL-6 from a mouse | mouth dendritic cell. It is a graph which shows the secretion of IL-6 from a human peripheral blood mononuclear cell. It is a graph which shows secretion of IL-1 ( _beta) and TNF- (alpha) from a human peripheral blood mononuclear cell. It is a graph which shows the secretion of IFN (gamma) from the human peripheral blood mononuclear cell of the atopic patient who uses a corticosteroid agent or an immunosuppressive agent. It is a graph which shows the secretion of IL-1 ( _beta) from CD8 ⁺ T cell, CD4 ⁺ T cell, and CD14 ⁺ macrophage. It is a graph which shows suppression of nonspecific cell growth by PHA stimulation. It is a graph which shows the proliferation suppression of the regulatory T cell derived from a healthy adult and an adult atopic patient.

<Regulation of peripheral blood mononuclear cell activity>
The present embodiment is a composition for regulating the activity of peripheral blood mononuclear cells containing a papaya fermented product as an active ingredient.

  Here, “activity regulation” of peripheral blood mononuclear cells means promotion of activity of peripheral blood mononuclear cells and / or suppression of activity of peripheral blood mononuclear cells.

  “Peripheral blood mononuclear cells (hereinafter referred to as PBMC)” is a generic name for cells containing “lymphocytes” and “monocytes” among leukocytes contained in blood, and that PBMC is activated. Thus, it is possible to secrete various cytokines and protect the living body from pathogens and viruses. “Lymphocytes” include T cells, B cells, natural killer cells (NK cells), and the like, and play a central role in defense against viruses and tumor cells. On the other hand, “monocytes” are supposed to differentiate into dendritic cells and macrophages, and are responsible for biological defense through phagocytosis and antigen presentation of infected cells, production of various cytokines, and the like.

In this embodiment, promotion of PBMC activity includes promotion of secretion of IL-6, IFNγ, tumor necrosis factor α (TNF-α) and IL-1 _β of PBMC. As shown in examples described later, by stimulating in composition in the present embodiment the PBMC, PBMC of IL-6, IFN [gamma], the secretion of TNF-alpha and IL-1 _beta was confirmed.

  Here, the promotion of IL-6 secretion by PBMC is not stimulated by the composition in the present embodiment, that is, compared with the amount of IL-6 secreted from non-stimulated PBMC. It means that IL-6 secretion is promoted by stimulation. Specifically, the amount of IL-6 secretion is preferably 2.0 times or more, more preferably 2.2 times or more, and even more preferably 2.5 times that of IL-6 secreted from unstimulated PBMC. That's it. The experimental conditions such as the number of cells to be stimulated and the experimental method can be referred to the examples described later.

Further, PBMC for IFN [gamma], the secretagogue of TNF-alpha and IL-1 _beta, which means that secretion as compared to stimulation by glucose negative control is facilitated. Specifically, the amount of IFNγ secretion is preferably 200 pg / ml or more, more preferably 400 pg / ml or more, and even more preferably 600 pg / ml or more, and the composition in this embodiment is further ingested. In the case of PBMC obtained from the peripheral blood of those who are, the secretion amount of IFNγ is preferably 1.5 times or more, more preferably 2.0 times or more, and even more preferably, compared to stimulation with glucose as a negative control More than 3.0 times. Moreover, the amount of TNF-α secretion is preferably 1.5 times or more, more preferably 2.0 times or more, and even more preferably 2.5 times or more, as compared with stimulation by glucose as a negative control. On the other hand, the secretion amount of IL-1 _beta is preferably 500 pg / ml or higher, more preferably 600 pg / ml or more, more preferably not less than 700 pg / ml. The experimental conditions such as the number of cells to be stimulated and the experimental method can be referred to the examples described later.

Further, PBMC for promoting the secretion of IL-1 _beta in this embodiment includes a CD8 ⁺ T cells, CD4 ⁺ T cells and CD14 ⁺ macrophages. As shown in examples described later, CD8 ⁺ T cells, secretion of IL-1 _beta was confirmed by stimulation with the compositions of this embodiment of the CD4 ⁺ T cells and CD14 ⁺ macrophages.

Here, CD8 ⁺ T cells, the CD4 ⁺ T cells and CD14 ⁺ macrophages IL-1 _beta secretion promoting means that secretion as compared to stimulation by glucose negative control is facilitated. Specifically, the secretion amount of IL-1 _beta in CD8 ⁺ T cells, and preferably from 100 pg / ml or more, more preferably 0.99 pg / ml or more, more preferably not less than 200 pg / ml. Moreover, the secretion amount of IL-1 _beta in CD4 ⁺ T cells are preferably 100 pg / ml or more, more preferably 0.99 pg / ml or more, more preferably not less than 200 pg / ml. On the other hand, the secretion amount of IL-1 _β of CD14 ⁺ macrophages is preferably 300 pg / ml or more, more preferably 400 pg / ml or more, and further preferably 500 pg / ml or more. The experimental conditions such as the number of each stimulated cell and the experimental technique can be referred to the examples described later.

Here, IFNγ is a kind of cytokine and is known to maintain Th1 cells in a dominant state among Th1 cells and Th2 cells that differentiate from CD4 ⁺ cells. It has been reported that when Th1 cells become dominant, they not only activate NK cells but also activate macrophages and induce cytotoxic T lymphocytes (CTLs), thereby improving resistance to cancer cells. Yes. On the other hand, Th2 cells become dominant when causing diseases such as hay fever, allergic rhinitis, bronchial asthma, and atopic dermatitis. Therefore, when Th1 cells are dominant, It is known to lead to treatment and prevention.

TNF-α is a cytokine that has an antitumor effect and induces cell death (apoptosis). On the other hand, IL-1 _β accounts for about 90% of IL-1 produced in vivo and is known as a lymphocyte activator.

T cells, which are one of “lymphocytes”, can be divided into CD8 ⁺ T cells and CD4 ⁺ cells according to the type of molecular marker expressed on the cell surface.

CD8 ⁺ T cells exert cytotoxic activity by recognizing viral fragments present on the surface of virus-infected cells or tumor cell-specific peptides present on the surface of tumor cells. Necrotize.

On the other hand, CD4 ⁺ T cells with undergo antigen-presenting dendritic cells are antigen-presenting cells, co-stimulatory molecules CD40 such as expressed on the cell surface, such as dendritic cells (co stimulators) of CD4 ⁺ T By binding to costimulatory molecules on cells, CD4 ⁺ T cells develop responses at the cellular level. As a result, CD4 ⁺ T cells can assist in inducing functional expression of other T cells and inducing differentiation of B cells. It is also known that cytokines such as IL-1 have costimulator activity as a humoral factor. Activated CD4 ⁺ T cells send various signals to B cells. As a result, B cells differentiate and can produce antibodies and play a role as a defense against living bodies.

  In this embodiment, the promotion of dendritic cell activity includes the promotion of TNF-α and / or IL-6 secretion. As shown in Examples described later, it was confirmed that TNF-α and IL-6 secretion were promoted by stimulating dendritic cells with the composition of the present embodiment.

  Here, the promotion of dendritic cell secretion of TNF-α and / or IL-6 means that secretion is promoted as compared with stimulation by glucose as a negative control. In addition, the amount of TNF-α secreted by dendritic cells is preferably 1.4 times or more, more preferably 1.6 times or more, and even more preferably 1.8 times or more, compared to stimulation with glucose as a negative control. On the other hand, the amount of IL-6 secreted by dendritic cells is preferably 2.0 times or more, more preferably 2.5 times or more, and even more preferably 2.8 times or more, compared to stimulation with glucose as a negative control. The experimental conditions such as the number of cells to be stimulated and the experimental method can be referred to the examples described later.

  Dendritic cells that differentiate from “monocytes” play a central role as antigen-presenting cells that present antigens by taking them in vivo. When dendritic cells are activated, co-stimulatory molecules such as CD80, CD86, and CD40 are expressed on the cell surface and used as maturation markers for dendritic cells. Dendritic cells are known to differentiate into mature dendritic cells upon stimulation with TNF-α. In addition, mature dendritic cells secrete IL-6 and play a role as a biological defense by inducing B cell proliferation and differentiation into antibody-producing cells.

  Therefore, it is preferable to use the composition in the present embodiment when the body defense ability is lowered. That is, it is preferably used after anticancer drug treatment, chemotherapy, radiotherapy or surgical invasion that causes a decrease in leukocytes including lymphocytes and monocytes. It is also suitable for use in the treatment and prevention of colds and viral diseases that cause a decrease in white blood cells including lymphocytes and monocytes.

  Moreover, even if the disease of the object which uses the composition in this embodiment is the case where the said disease or 2 or more types of diseases coexist, it is possible to use this composition. In addition to using this composition alone, immunostimulators and hematopoietic factors (macrophage colony stimulating factor, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, erythropoietin, thrombopoietin, platelet-derived growth factor, etc.), various cytokines, etc. It is also possible to use it in combination.

<Recovery from suppression of IFNγ secretion>
In this embodiment, the promotion of PBMC activity has the effect of promoting the secretion of IFNγ whose secretion is suppressed by the use of corticosteroids and immunosuppressants. As shown in the Examples described later, it was confirmed that the ingestion of the composition according to the present embodiment restored the secretion of IFNγ, the secretion of which was suppressed by the use of corticosteroids and immunosuppressants.

  Here, recovery of secretion of IFNγ, whose secretion is suppressed by the use of corticosteroids and immunosuppressants, means that those who have suppressed IFNγ secretion by the use of corticosteroids or immunosuppressants. By ingesting the composition in the embodiment, it means that the secretion of IFNγ is restored as compared with stimulation by glucose as a negative control. Specifically, the amount of IFNγ secreted when stimulating PBMC obtained from the peripheral blood of a person taking the composition in the present embodiment with the present composition is the stimulation of glucose as a negative control. In comparison, it is preferably 10 times or more, more preferably 12 times or more, and still more preferably 14 times or more. The experimental conditions such as the number of cells to be stimulated and the experimental method can be referred to the examples described later.

  Corticosteroids and immunosuppressive agents used to suppress inflammatory reactions caused by infection and allergic reactions such as bacteria and viruses, generation of abnormal metabolites, ultraviolet rays, trauma, burns, etc. are various interleukins, interferons, etc. It inhibits the transcription of cytokine genes, inhibits the production of interleukins and the like, and suppresses inflammatory reactions by reducing the activity of lymphocytes and the like. On the other hand, the action of suppressing the secretion of various cytokines causes a decrease in the activity of cells involved in biological defense such as lymphocytes, thereby increasing the risk of inducing or worsening infectious diseases. Therefore, even when an adrenocortical hormone agent or an immunosuppressive agent is used, the risk of infection or the like can be avoided by restoring the secretion of cytokines whose secretion is suppressed.

  Here, as diseases of patients who use corticosteroids or immunosuppressants, respiratory diseases (asthmatic bronchitis (including childhood asthmatic bronchitis), sarcoidosis, serum sickness bronchial asthma, drugs and allergies caused by chemical substances -Addiction, digestive diseases (ulcerative colitis, localized enteritis, etc.), liver diseases (severe hepatitis and fulminant hepatitis, cholestatic acute hepatitis, active chronic hepatitis, acute relapsed chronic hepatitis, cholestatic chronic Hepatitis, cirrhosis, etc.), kidney disease (nephrotic syndrome, etc.), neurological disease (multiple sclerosis and myotonic disorders, encephalomyelitis, myasthenia gravis, chorea, facial paralysis, spinal fistula retinitis, peripheral nerve ), Endocrine disorders (subacute thyroiditis, secondary chronic adrenocortical dysfunction primary chronic adrenocortical dysfunction, iatrogenic chronic adrenocortical dysfunction, pituitary chronic adrenocortical dysfunction, acute adrenocortical dysfunction Vice Genital syndrome, thyroid poisoning, etc.), skin diseases (acute acute eczema and chronic eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, monetary eczema, self-sensitizing dermatitis, stasis dermatitis For example, sebum-deficient eczema, progressive palmar keratoderma, female facial dermatosis, Vidar lichen, radiation dermatitis, sun dermatitis, etc.) can be mentioned as examples.

  Adrenocortical hormone means glucocorticoid, and examples include cortisol, betamethasone, prednisolone, parameterzone, diflucortron, methylprednisolone, fludrocortisone, cortisone, corticosterone, diflupredant, triamcinolone, dexamethasone. And is appropriately selected from the medical viewpoint for the above diseases.

  Further, cyclosporine, tacrolimus, mizoribine, cyclophosphamide, zafirlukast and the like can be exemplified as immunosuppressants, and are appropriately selected from the medical viewpoint for the above diseases.

  In this embodiment, the corticosteroid or immunosuppressive agent can be administered in the form of a coating agent, a swallowing agent, an injection, an inhalant, etc., as long as it follows a medically optimal administration method in the treatment of each disease. There are no restrictions.

  When using the composition in the present embodiment for recovery from IFNγ secretion suppression, there is no particular setting in the intake period of the composition, while the corticosteroid or immunosuppressive agent is used It is desirable to take.

<Tumor immunotherapy>
Suppression of PBMC activity in this embodiment includes suppression of T cell proliferation. As shown in the examples described later, it was confirmed that when PBMC undergoing non-specific growth stimulation was stimulated with the composition of this embodiment, the proliferation of T cells was suppressed.

  Here, the suppression of the proliferation of T cells means that the proliferation of T cells is suppressed as compared with the stimulation by glucose as a negative control. Specifically, the number of T cells is preferably 0.8 times or less, more preferably 0.6 times or less, compared to stimulation with glucose. The experimental conditions such as the number of cells to be stimulated and the experimental method can be referred to the examples described later.

  Inhibition of PBMC activity in this embodiment includes suppression of Treg proliferation. As shown in the Examples described later, it was confirmed that when PBMC undergoing non-specific growth stimulation was stimulated with the composition of the present embodiment, Treg proliferation was suppressed.

Here, suppression of Treg proliferation means that Treg proliferation is suppressed as compared to stimulation with glucose, which is a negative control. Specifically, the proportion of Foxp3 present in CD4 ⁺ T cells derived from healthy adults is preferably 0.7 times or less, more preferably 0.5 times or less, compared to stimulation with glucose as a negative control. On the other hand, the proportion of Foxp3 in CD4 ⁺ T cells derived from adult atopic patients is preferably 0.7 times or less, more preferably 0.5 times or less, and even more preferably 0.3 times or less, compared to stimulation with glucose as a negative control. This is the case. The experimental conditions such as the number of cells to be stimulated and the experimental method can be referred to the examples described later.

It is known that Tregs are differentiated from CD4 ⁺ T cells, and T cells that have reacted excessively by the action of Tregs are controlled to suppress the excessive reaction. It has been pointed out that Treg differentiates when Foxp3, a transcription factor, is expressed in CD4 ⁺ T cells. Therefore, Treg usually plays a role in suppressing excessive T cell responses in vivo.

  Furthermore, since the composition in the present embodiment has an effect of suppressing the proliferation of Treg, it is possible to enhance the effectiveness of tumor immunotherapy whose effect is limited by Treg.

  In cancer patients, there is an excess of Treg, and the presence of this excess Treg suppresses the anti-tumor immune response of the cancer patient, contributing to the difficulty in cancer treatment. The number of Tregs in cancer patients limits the effectiveness of tumor immunotherapy. As a means of specifically killing Treg, a therapeutic method has been devised that enhances the efficacy of tumor immunotherapy using diphtheria toxin-binding recombinant IL-2 (Non-patent Document 7). However, the above-mentioned treatment methods are expensive because they use expensive recombinant proteins such as toxin-binding IL-2. Therefore, the appearance of an alternative such as an expensive recombinant protein that is inexpensive and safely suppresses Treg proliferation has been desired.

  Here, “tumor immunotherapy” is a treatment method that suppresses cancer by enhancing immunity of immune cells using tumor cells, tissues, or tumor antigens produced in biotechnology. Tumor means colon cancer, stomach cancer, lung cancer, breast cancer and the like. Tumor immunotherapy includes LAK therapy, CTL therapy, autologous tumor vaccine therapy, dendritic cell vaccine therapy, DNA vaccine therapy and peptide vaccine therapy. Tumor antigens include tumor-specific antigens that are newly produced as cells become cancerous, and tumor-related antigens that are also produced in normal tissues and tumor tissues before canceration. (1) Virus (cervical cancer Antigens from gene products derived from human papillomavirus and human T-lymphotropic virus), (2) antigens due to mutations in oncogenes (src, ras genes, etc.) and tumor suppressor genes (p53, BRCA1, etc.), (3) canceration (4) antigens expressed only during embryogenesis (carcinoembryonic antigens, etc.).

  Any tumor immunotherapy may be employed as long as the therapeutic effect can be enhanced by suppressing the growth of Treg by using the composition in the present embodiment. Dendritic cell vaccine therapy is particularly suitable. Two or more types of tumor immunotherapy may be used in combination.

  In addition, any tumor antigen can be selected, but a protein or peptide encoded by a nucleic acid is preferable. In addition, it is possible to use tumor cells or tissues themselves, and it is also possible to use a highly antigenic part or epitope.

  In the case of performing tumor immunotherapy using the composition in the present embodiment, there is no particular setting for the period of administration, but the suppression of Treg makes the effect of tumor immunotherapy effective. It is desirable to continue ingestion during therapy.

<Method for producing composition containing fermented papaya>
A composition containing fermented papaya is prepared by adding sugar to pulverized and ground papaya, ripening or fermenting mixture 1 into filtrate 1 and residue 1 by filtration, and then adding sugar to residue 1. Then, the filtrate 2 is fractionated by filtration of the mixture 2 that has been aged or fermented, and the intermediate raw material to which saccharides are added to the mixture of the filtrate 1 and the filtrate 2 (mixed filtrate) is fermented, and further saccharides are added, It is obtained by aging for a long time after drying. It is preferable that the amount of carbohydrate added to the mixed filtrate is adjusted to 40 to 60% by mass with respect to the mixed filtrate. In the case of aging, a period of 4 to 10 days is preferable, and a more preferable aging period is 7 to 10 days. On the other hand, the long-term aging period is preferably 6 months or more.

  Papaya is `` papaya (scientific name: Carica papaya Linn) '' which is a fruit of the genus Papaya in the Papaya family, and it is not only a mature green papaya that stands in the store as a fruit, but also a green immature used for cooking as a vegetable Papaya is also included. Papaya is widely cultivated from the tropics to the subtropics, but the place of origin is not particularly limited, and any of them can be used. Moreover, although any papaya, whether mature or immature, can be used as the subject of fermentation, it is preferable to use wild immature papaya.

  In this embodiment, yeast can be used for fermentation of papaya. The yeast to be used is not particularly limited as long as it can ferment papaya, but examples include Candida, Pichia, Kluyveromyces, Saccharomyces, and Schizosaccaromyces. Etc.), but yeast of the genus Saccharomyces is preferable. Moreover, you may use the natural yeast which adheres or exists in a papaya as it is for fermentation. As long as papaya can be fermented, it may be prepared by freeze-drying by a conventional method or stored refrigerated. Optimum conditions for yeast fermentation are dependent on each yeast, and the method of use and conditions can be appropriately selected according to the conditions of the production process and the state of the target product. Furthermore, it is also possible to use 1 or 2 or more types of yeast simultaneously or sequentially.

  In this embodiment, lactic acid bacteria can be used for papaya fermentation. The lactic acid bacteria to be used are not particularly limited as long as they can ferment papaya, but examples include Streptococcus, Lactobacillus, Lactococcus, Bifidobacterium, and Leuco. The genus Nostock (Leuconostoc), Pediococcus (Pediococcus), etc. can be mentioned. Preferably, it is a lactic acid bacterium of the genus Streptococcus. Moreover, you may use the natural lactic acid bacteria adhering to or existing in papaya as it is for fermentation. Any lactic acid bacteria may be prepared by freeze-drying by a conventional method or stored refrigerated as long as papaya can be fermented. Optimum conditions for lactic acid bacteria fermentation are dependent on each lactic acid bacterium, and it is possible to appropriately select the method of use and conditions according to the conditions of the production process and the state of the target product. Furthermore, it is also possible to use 1 or 2 or more types of lactic acid bacteria simultaneously or sequentially.

  In this embodiment, amylase can be used for fermentation of papaya. Amylase is a digestive enzyme that can hydrolyze the glycosidic bonds of amylose and amylopectin contained in starch, so long as it has sufficient activity in the production process of the target product, there are special restrictions. Absent. The optimum conditions for amylase depend on the enzyme used, and the enzyme to be used can be appropriately selected according to the conditions of the production process and the state of the target product. Furthermore, it is also possible to use 1 or 2 or more types of amylases simultaneously or sequentially.

  In this embodiment, an enzyme for miso can be used for fermentation of papaya. The enzyme for miso includes secretions of Aspergillus used for producing fermented foods such as miso and soy sauce. Examples of microorganisms included in the genus Aspergillus include Aspergillus oryzae, Aspergillus niger, Aspergillus sauer and the like. Aspergillus oryzae is preferable. The enzyme for miso does not necessarily have to be purified as long as it satisfies the required quality of the target product, it may be a crudely purified enzyme, or the fungus body itself can be used. It is. The optimum conditions for the enzyme for miso are enzyme-dependent, and the method of use and conditions can be appropriately selected according to the conditions of the production process and the state of the target product. Furthermore, it is also possible to use 1 or 2 or more types of miso enzymes simultaneously or sequentially.

  Papaya is pasteurized (steam, hot water, etc.), high temperature sterilization (heated steam, hot water, etc.), microwave, high frequency, infrared, far infrared, ultraviolet, γ-ray, X-ray, It is also possible to perform sterilization treatment such as electron beam and ozone sterilization. When fermenting using mature papaya, it is preferable to perform sterilization because it is likely to cause spoilage by microorganisms. On the other hand, when wild immature papaya is used, it is preferable not to perform sterilization when microorganisms (yeast, lactic acid bacteria, etc.) attached to or present on immature papaya are beneficial for fermentation.

  Sugar, malt, dextran, sucrose (sucrose), maltose (malt sugar), glucose (dextrose), etc. can be used as sugars added for the purpose of accelerating fermentation, but the papaya enzyme is inactivated to prevent spoilage. From the viewpoint of reducing the osmotic pressure, it is preferable to use a low-molecular-weight carbohydrate that can increase the intracellular osmotic pressure and cause plasma membrane separation even with the same mass of carbohydrate. Is preferred.

<Form of composition in the present embodiment>
The form of the composition in this embodiment is milky white powder, but it can be used as it is. Further, as other forms, powders, tablets, capsules, soft capsules, liquids, syrups, pills and the like can be prepared, but the invention is not particularly limited thereto.

  The content of the composition in the present embodiment per ingestion is, for example, from 1 g (liquid mass) to 2.5 g (dry mass) of the intermediate raw material when a human subject weighing 60 kg is obtained. In terms of dry weight, it is preferably 1 to 15 g / (60 kg body weight) in terms of dry mass, and more preferably 3 to 12 g / (60 kg body weight). Further, it is preferable to increase or decrease the content of the composition based on the above criteria. Since the composition in the present embodiment is highly safe, a special upper limit value for intake is not set.

<Food and drink, health food or feed additive>
The composition in this embodiment can be used as food and drink, health food, or feed additive (hereinafter referred to as food and drink).

  When the composition in the present embodiment is used as a food or drink or health food, the composition can be used as it is, but other forms are exemplified by excipients (glucose, starch, calcium carbonate). Etc.) and additives (guar gum, glycine, etc.), sweeteners, acidulants, flavorings, vitamins or minerals etc. in combination with granules, fine granules, powders, tablets, drops, troches, gels, syrups, drinks, etc. To do.

  Other forms include cookies, cakes, bread, chocolate, sheep cake, soup flour, ice cream, pudding, crackers, chips and other sweets, tea, coffee, juice, milk, alcohol and other drinking water, spaghetti, udon, Examples include noodles such as buckwheat noodles, ramen, kneaded products such as ham, sausage, rice bran, kamaboko, seasonings such as dressing, miso, soy sauce, mayonnaise, sprinkles, and soups such as curry and stew.

  The composition in this embodiment can also be used as a feed additive. For example, it can be added to feed such as livestock (cattle, pigs, etc.), poultry (chicken, salmon, etc.), farmed fish (eg salmon, salmon, etc.), pets (dogs, cats, etc.). Even if it is added to any feed, this composition can be added as it is, and can also be added in the form of granules, fine granules, powders, tablets, capsules, etc. .

  When the composition in the present embodiment is used as a food or drink, the amount added is 2.5 g (dry weight) from the intermediate raw material as a reference when a composition of 2.5 g (dry weight) is obtained. On the other hand, when it is preferably 1.0% by mass or more, more preferably 3.0% by mass or more, and still more preferably 5.0% by mass or more, it is in a preferable state as a food or drink for sensory evaluation. Moreover, it is suitable to increase / decrease the addition amount of a composition based on the said reference | standard.

  When using the composition in the present embodiment as food or drink, for example, the content of the composition per intake of 60 kg body weight is a composition of 1 g (liquid mass) to 2.5 g (dry mass) of the intermediate raw material Is obtained, it is preferably 1 g to 15 g / (weight 60 kg) in terms of dry mass, and more preferably 3 g to 12 g / (weight 60 kg). Further, it is preferable to increase or decrease the content of the composition based on the above criteria. Since the composition in the present embodiment is highly safe, a special upper limit value for intake is not set.

  The composition in this embodiment can be used for other than humans. As non-human animals, it can also be used for pets such as dogs and cats, livestock such as cattle and pigs, poultry such as chickens and sharks, and aquaculture animals such as sharks and sharks. is there.

  Hereinafter, the present invention will be further described by way of examples. However, the following examples are for illustrative purposes only and are not intended to limit the present invention in any way.

<Example 1>
Based on the flowchart showing the production example of the composition containing the fermented papaya shown in FIG. 1, the production method will be described below.

1) Production Example of Intermediate Raw Material Used for Papaya Ferment 100 kg of wild immature papaya with white seeds was collected, washed with water to remove foreign matter, and then the loose part and the bottom of the fruit were removed. After slicing papaya with skin, it was pulverized and ground to obtain 85 kg of mash. Glucose 20 kg was added to the mash product, mixed well, and then allowed to stand for 4 days to obtain a mature mixture 1. The mixture 1 was filtered to obtain a filtrate 1 as a fruit juice and a residue 1 thereof. To 60 kg of residue 1, 10 kg of glucose was added, mixed well, and then allowed to stand for 4 days to obtain a mixture 2 that was aged. The mixture 2 was filtered to obtain a filtrate 2 as a fruit juice. To the mixed filtrate obtained by mixing the filtrate 1 and the filtrate 2, 2 kg of glucose was added and mixed well to obtain 40 kg of the desired intermediate raw material.

2) Example of production of a composition containing fermented papaya 40 kg of the above-mentioned intermediate raw material is divided into 20 kg (intermediate raw material 1), 15 kg (intermediate raw material 2), 5 kg (intermediate raw material 3). Add 0.5 kg of lactic acid bacteria and 0.1 kg of miso-enzyme to intermediate raw material 2, add 0.1 kg of amylase to intermediate raw material 3, mix well, and ferment at 40 ° C. for 48 hours. , Got 3. Add 60 kg of glucose to fermented product 1, 30 kg of glucose to fermented product 2, 10 kg of glucose to fermented product 3, mix well, dry at 18 ° C. for 1 month, grind, composition 1, 2 and 3 Got. Compositions 1, 2 and 3 were combined and homogenized, and aged for 6 months or more in a high aluminum packaging made of gas barrier to obtain a composition containing a fermented papaya (100 kg of the final composition).

<Example 2> Preparation of composition containing papaya fermented product and gram-negative bacterial lipopolysaccharide (LPS) 4 g of powder containing the composition containing papaya fermented product was suspended in 6 ml of phosphate buffered saline (PBS), 37 ° C. The mixture was sufficiently stirred for 1 hour, and further PBS was added to prepare 10 ml (40% solution). Thereafter, the mixture was centrifuged at 2000 rpm for 10 minutes, and the supernatant was filtered through a 0.45 μm filter (stock solution 400 mg / ml). LPS (cat. L-4516 derived from E. coli, Sigma) was used as a positive control. LPS was diluted to 100 μg / ml with PBS to obtain a stock solution. The composition (P) and the LPS (L) stock solution containing the papaya fermented product were each diluted with the medium used for the test (eg, 10% FCS / RPMI culture solution) to adjust the final concentration of interest. In the composition containing the fermented papaya used in this example, glucose is selected as the sugar used in the fermentation process, and the glucose concentration of the final product contains an amount that cannot be ignored for testing. Yes. Therefore, in this example, glucose (Katayama Chemical Co., Ltd.) was used as a negative control for the purpose of clarifying that the test result was not an effect due to glucose contained in the composition.

<Example 3> Induction of mouse dendritic cells and stimulation of mouse dendritic cells with a composition containing fermented papaya
Bone marrow cells were collected from BALB / c mouse femurs, and both IL-4 and GM-CSF were cultured at a concentration of 10 ng / ml for 1 week. Thereafter, LPS was stimulated with 1 μg / ml, and the floating cells after 24 hours were used as mature dendritic cells. The composition containing Papaya fermented product (P) and LPS (L) was diluted with 10% FCS / RPMI culture solution, respectively, and the final concentrations were 5 mg / ml and 1 ng / ml, respectively, to stimulate mouse dendritic cells, The concentration of glucose as a negative control was stimulated as 5 mg / ml. Stimulated the number of stimulated mouse dendritic cells as 1x10 ⁵ cells / well, and the cytokine concentration in the culture solution after 24 hours of culture was comprehensively analyzed with a flow cytometer using CBA (Cytometric Bead Array) . CBA is pre-bonded with fluorescently labeled beads (labeled with APC fluorescent dye, fluorescence concentration is changed to 6 levels) and antibodies against cytokines to be measured in advance, and reacted with 50 μl of culture test sample, then PE fluorescence It is a quantitative method that allows simultaneous measurement of multiple items by sandwiching with each labeled antibody. The amount of each cytokine was quantified using a BD CBA Human and Mouse inflammatory kit (Cat no. 551811 and 552364, BD Biosciences) and a Human Th1 / Th2 Cytokine kit (Cat no. 550749, BD Biosciences) according to the manual. Human Inflammatory kits include IL-1 _β , IL-6, IL-8, IL-10, IL-12p70, and TNF-α. Mouse Inflammatory kits include IFNγ, MCP-1, IL-6, IL-10, and IL. -12p70, TNF-α, Human Th1 / Th2 Cytokine kit can measure IFNγ, TNF-α, IL-2, IL-4, IL-5, and IL-10 simultaneously.

  The measurement results of the amount of cytokine in the culture supernatant are shown in FIG. As is clear from this result, secretion of TNF-α and IL-6 was observed from mouse dendritic cells upon stimulation with the composition (P) containing the papaya fermented product. Therefore, it was revealed that fermented papaya has the ability to activate mouse dendritic cells and promote secretion of TNF-α and IL-6.

<Example 4> Stimulation of human peripheral blood mononuclear cells (PBMC) This composition was prepared by adjusting the number of human PBMCs to 1 x 10 ⁵ cells / well in 200 µl of 10% FCS / RPMI culture medium. 1 to 5 μl of (P) stock solution (400 mg / ml) was added in 1 μl increments (final concentrations were 2, 4, 6, 8 and 10 mg / ml, respectively) to stimulate human PBMC cells. As a negative control, the stock solution of the present composition (P) is set to the condition of no addition. After culturing for 24 hours, the concentration of IL-6 in the culture was measured by ELISA. Cytokine IL-6 in the supernatant after culturing was quantified using an ELISA kit (R & D, Minneapolis, USA) according to the product manual.

  The measurement results of the amount of cytokine in the culture supernatant are shown in FIG. As is clear from this result, secretion of IL-6 was observed from human PBMC upon stimulation of the composition (P) containing the papaya fermented product. Therefore, it was revealed that fermented papaya has the ability to activate human PBMC and promote IL-6 secretion.

<Example 5> Stimulation in healthy adult (HD) and adult atopic patient (AD) in PBMC The stimulation effect in human adult PBMC of healthy adult (HD) and adult atopic patient (AD) was examined. Atopic patients are in a state where a corticosteroid agent or various immunosuppressive agents are regularly applied to the body and face. After collecting human heparinized peripheral blood from healthy adults and atopic patients, PBMCs were separated with a histopack (Sigma-Aldrich cat. 107). The number of stimulated cells of the isolated human PBMC is 1 × 10 ⁵ cells / well (IFNγ only 1 × 10 ⁶ cells / well), seeded in a flat bottom 96-well plate, LPS (L) 1 μg / ml, papaya fermented product The contained composition (P) was stimulated at a concentration of 10 mg / ml and cultured for 24 hours.

Cytokines IL-1 _β and TNF-α in the supernatant after culturing were quantified using an ELISA kit (R & D, Minneapolis, USA) according to the product manual. IFNγ is a Capture antibody using anti-human IFNγ mAb (1-D1K, MABTECH AB) diluted 500-fold with carbonate buffer (pH 9.6), and anti-human IFNγ rabbit serum is 600% with 5% FCS / PBS. The diluted antibody was used as a secondary antibody, and a goat anti-rabbit IgG-HRP (MBL) diluted 2000 times with 5% FCS / PBS was used as a secondary antibody, and measurement was performed using the sandwich method. Color development OPDA (Wako Pure Chemical, 3 mg / 10 ml citrate buffer, pH 5.0) was added 10μl of a 31% H ₂ 0 ₂ (Mitsubishi Gas Science), was measured OD values at 490 nm. L + P is a well in which both LPS (L) and a composition (P) containing a papaya fermented product coexist. As a negative control, 10 mg / ml glucose was used. Supernatants cultured for 24 hours after stimulation were collected and subjected to ELISA. Regarding the production of IFNγ, after the experiment, a composition containing a fermented papaya was continuously taken after breakfast for 3 g every day for 2 months, and then blood was collected again to conduct a similar experiment. In this experiment, blood was collected after obtaining informed consent from each subject, and the experiment was conducted.

The results of stimulation in human PBMC are shown in FIG. As apparent from FIG. 4, HD and AD both were able to confirm the secretion from human PBMC of IL-1 _beta and TNF-alpha stimulation of the composition containing the papaya fermented (P). Therefore, it became clear that the papaya fermentation product is capable of promoting the secretion of human PBMC were activated IL-1 _beta and TNF-alpha.

  The results of IFNγ secretion are shown in FIG. It was clarified that the composition (P) containing the fermented papaya stimulates the secretion of IFNγ by stimulating PBMC in HD. On the other hand, even when PBMC of AD was stimulated with a composition (P) containing a papaya fermented product, IFNγ secretion could not be promoted to a detectable level. On the other hand, when 2 days after breakfast, a composition containing 3 g of papaya fermented product (P) was ingested, the secretion of AD IFNγ was restored to the same level as HD. It was.

  From the above results, by ingesting the papaya fermented product, even in atopic patients using corticosteroids or immunosuppressants, the amount of IFNγ recovered to the same level as the amount of IFNγ secreted by healthy adults. It became clear to do.

<Example 6> Stimulation in CD8 ⁺ T cells, CD4 ⁺ T cells and CD14 ⁺ macrophages Human PBMCs were fractionated into CD8 ⁺ T cells, CD4 ⁺ T cells and CD14 ⁺ macrophages using MACS magnetic beads, respectively. Prepared to 1 × 10 ⁵ cells / well. The composition (P) containing the fermented papaya was added to the medium at a concentration of 10 mg / ml, LPS (L) at a concentration of 1 μg / ml, and glucose at a concentration of 10 mg / ml, and stimulated for 24 hours. IL-1 _beta ELISA kits in the culture supernatant after 24 h (R & D, Minneapolis, USA ) was quantified according to the product documentation used.

The secretion results of IL-1 _beta are shown in FIG. If stimulated with a composition containing papaya fermented (P), it was possible also to confirm the secretion of IL-1 _beta in either CD8 ⁺ T cells, CD4 ⁺ T cells and CD14 ⁺ macrophages. Therefore, it was revealed that the fermented papaya has the ability to activate CD8 ⁺ T cells, CD4 ⁺ T cells and CD14 ⁺ macrophages and promote IL-1 _β secretion.

<Example 7> Involvement of fermented papaya in phytohemagglutinin (PHA) stimulation of human PBMC
PHA was used to stimulate non-specific growth of human PBMC, and a composition (P) containing a papaya fermented product was added to 10% FCS / RPMI culture solution to examine its involvement. PHA is known as a reagent that proliferates only T cells. Using a 6-well plate, human PBMC 1 × 10 ⁵ cells / well was subjected to nonspecific growth stimulation under the conditions of PHA 10 μg / ml and IL-2 25 IU / ml, and a composition containing a papaya fermented product ( P) was added to a concentration of 10 mg / ml. As a negative target, glucose was added to a concentration of 10 mg / ml using glucose. The cells were cultured for 6 days and the number of cells was counted.

  The result of non-specific stimulation using PHA is shown in FIG. As is apparent from FIG. 7, the addition of the composition (P) containing the papaya fermented product suppressed the growth of human PBMC. Since PHA specifically proliferates only T cells, it was clarified that stimulation with a composition containing a papaya fermented product suppresses T cell proliferation. Therefore, it was revealed that the fermented papaya has an action of suppressing the proliferation of T cells.

<Example 8> Dynamics of Treg after PHA stimulation Human PBMC isolated from healthy adults (HD) and adult atopy patients (AD) were subjected to MACS magnetic beads (Miltenyi Biotec, USA) after the experiment described in Example 7. Then, CD4 ⁺ T cells were isolated according to the manual, and the expression of intracellular Foxp3, which is an indicator of Treg, was analyzed. Human Foxp3 was measured using an intracellular measurement kit (eBioscinece) according to the product manual. BD FACS Calibur (BD Biosciences) was used for measurement, and Cell Quest software was used for analysis.

  The results of Foxp3 expression analysis are shown in FIG. As is clear from FIG. 8, the proportion of Foxp3 in healthy adults (HD) was reduced to approximately 1/2, 0.96% with the addition of the composition containing Papaya Fermented (P), compared to 2.2% with glucose addition. It was. Therefore, it was revealed that fermented papaya has an effect of suppressing Treg growth in healthy adults (HD). In addition, the proportion of Treg in adult atopic patients (AD) decreased to about 1/4, with 1.2% addition of the composition containing Papaya fermented product (P) compared to 5.1% with glucose addition.

<Discussion>
Since the composition containing the fermented papaya used in this example contained a large amount of glucose, it was tested using glucose as a negative control. In any test, the activity of peripheral blood mononuclear cells was not regulated by glucose. LPS, a gram-negative bacterial lipopolysaccharide, is also present in the air. Therefore, various effects seen in many other health foods are often caused by LPS mixed from the outside. However, the composition containing the papaya fermented product revealed that it stimulates human PBMC via a pathway different from that of LPS (results not shown). Therefore, it has been clarified that the activity of peripheral blood mononuclear cells is controlled by the action of the papaya fermented product and not by the action of carbohydrates such as glucose contained in the composition or mixed LPS.

  The composition for regulating the activity of peripheral blood mononuclear cells containing the fermented papaya described in the present invention has safe and excellent sensory properties even when taken for a long period of time. By ingesting the composition of the present invention, it is possible not only to activate human PBMC but also to suppress the activity, thereby providing an excellent ability to regulate the activity of peripheral blood mononuclear cells. Moreover, since this composition promotes the secretion of IFNγ, it maintains Th1 cells in a dominant state and contributes to the improvement of atopy and allergic constitution. Therefore, it can be applied not only to foods and the like for the purpose of promoting health such as improvement of the body defense ability but also in the medical field such as cancer prevention.

  In addition, since IFNγ plays important roles such as activation of NK cells and macrophages, induction of CTL cells, etc., ingestion of a composition containing a fermented papaya promotes IFNγ secretion and cancer. This suggests the possibility of enhancing the immunological monitoring ability of cells.

Claims (2)

A peripheral blood mononuclear cell activity regulator containing a fermented papaya as an active ingredient ,
An activity regulator for promoting the secretion of interferon γ in peripheral blood mononuclear cells suppressed by the use of corticosteroids or immunosuppressants by ingesting the activity regulator (excluding food) . The activity regulator according to claim 1, wherein the peripheral blood mononuclear cells are derived from an atopic patient (except food) .