WO2008001769A1

WO2008001769A1 - Intestinal immunity-activating substance or agent, and food, beverage and animal feed containing the same

Info

Publication number: WO2008001769A1
Application number: PCT/JP2007/062799
Authority: WO
Inventors: Futoshi Yokomizo; Masahisa Ibuki; Yoshinori Mine; Shigeru Katayama
Original assignee: Fuji Oil Company, Limited
Priority date: 2006-06-26
Filing date: 2007-06-26
Publication date: 2008-01-03
Also published as: JPWO2008001769A1

Abstract

Disclosed is an intestinal immunity-activating substance or agent containing β-1,4-mannobiose.

Description

Specification

Intestinal immunity stimulating substance and agent, food and drink containing these, and technical field to which the invention belongs

[0001] The present invention is characterized in that the production of immunoglobulin A (IgA), an antibody important for intestinal immunity, is increased, characterized by comprising a plant mannan degradation product containing β_1,4_mannobiose as a main component. The present invention relates to a composition having an intestinal immunity-stimulating action and a disease-preventing function caused by pathogenic bacteria and viruses, or a food or drink containing them.

Conventional invention

In recent years, the intestinal tract has been found to be the most important immune organ in the animal body.

Foods are carried by loca and enter the body. With food, pathogenic bacteria and viruses enter the body at the same time, and if they reach the intestine, they enter the body along with food ingredients. If you allow this invasion, it may lead to a life crisis. The intestinal tract that avoids this crisis is considered more immune functions. The intestinal immune system distinguishes between safe and non-safe. Safe things are foods and useful microorganisms that are absorbed to help your health. However, even safe items exhibit some immune response, and in extreme cases allergies can occur. This is called oral tolerance, but I'm immune, but I'm immune. Care should be taken as it will disrupt the balance of oral tolerance. In other words, it is not all good to be said to be an immunostimulant, but it is important to use it after understanding the power and what is being activated. It is very dangerous academically to judge that everything is healthy with the word “immunity activation”.

[0003] When pathogenic bacteria and viruses enter the body, immune globulin A (IgA) is produced in an attempt to eliminate it. This IgA is produced by the cells that make IgA in the Peyer's patch in the intestinal tract, and moves to the lower layer of the epithelial cells covering the intestinal lumen, and begins to make IgA. This IgA enters the intestinal lumen and prevents the invasion of pathogenic bacteria and viruses. Therefore, This IgA is very important for disease prevention against these pathogenic bacteria and viruses, and if it has a composition or food power that can enhance IgA without compromising oral immune tolerance, It will greatly contribute to the food safety that has been said recently, and can be very effective. In addition, this IgA is transported intracellularly and secreted to other mucosal surfaces, which can prevent not only resistance to pathogenic bacteria and viruses, but also the entry of allergens from the small intestine and nose. It is also important for prevention of onset.

[0004] Various foods focusing on this IgA have been proposed. For example, JP-A-2005-75740 and JP-A-2005-97133 disclose that a mushroom extract power ¾A production promoting function has been recently discussed. Further, JP-A-2005-330213 discloses that uridinoleic acid has its function. These are considered to have certain functions, and are required to be cheaper and easier to use.

[0005] In response to such demand, Japanese Patent Application Laid-Open No. 2002-27922 proposes a simple hydrolyzed cobra meal or palm kernel meal as an immunostimulator with excellent safety and economy. . However, this patent document only shows that macrophage stimulation is equivalent to that of lipopolysaccharide (LPS), which is usually used as a control, in view of the production of interleukin-1 (IL-1) in immunization experiments. The proposed product itself stimulates the body's immune system, and there is concern about the problem of macrophage inflammation due to increased IL-1. There is also concern that these will place an extra burden on the body.

[0006] In addition, JP-A-2005-35896 proposes that α-type mannobiose, which can be removed relatively easily from the cell wall of yeast, has an effect of enhancing production of interleukin-12 (IL-12). However, this IL-12 is a cell-mediated immune function that activates sputum cells and the like, and is not intended to promote the production of IgA. Furthermore, in Japanese Patent Application Laid-Open No. 2004-5 1582, vegetable β-type mannose oligosaccharide extracted from coffee is preferably useful as an immunostimulant, and stimulates the lymphocytes to promote their cell proliferation. It is exemplified. This is because the proposed product itself does not stimulate the immune system, and in a test using a force test or ConA stimulation, which is a simulated test, it is proposed that immunostimulation occurs only when a bacterial infection situation is reached. It can be said that it is an immunostimulant with no additional power. Because the PHA irritation is stronger, this disclosure stimulates T lymphocytes. It is disclosed that it is growing. Certainly, the ability to proliferate T lymphocytes is immunostimulatory. This invention is not a proposal to promote IgA production. Based on the above situation, an inexpensive intestinal immunity stimulating substance that can promote the production of IgA that directly prevents the invasion of pathogenic bacteria, viruses, and allergens that cause allergies, without inflicting load on the body was invented. The current situation is that.

Disclosure of the invention

Problems to be solved by the invention

[0007] The problems to be solved by the present invention are excellent in safety and economy, reliably promote IgA production without imposing a heavy load on the body, and prevent infection with external pathogenic bacteria and viruses. Another object of the present invention is to provide an intestinal immunity stimulating composition, food and drink, and feed that can prevent allergies.

Means for solving the problem

[0008] As a result of intensive studies to solve such problems, the present inventors have made mannan-degrading enzymes act on mannan-containing natural products, particularly cobra meal and palm kernel meal, and simply hydrolyze them. However, those containing at least 10% by weight or more of i3 1,4-mannobiose relative to mannan before decomposition are not affected by the site of IgA without the involvement of site force-in such as interleukin-6 (IL-6). It was found that the production was promoted, the increase in T lymphocytes, the inability to pass through the urinary pathway, and the discovery of a completely novel immune system stimulating ability, which led to the present invention.

That is, the present invention is based on a mannan-degrading product containing a mannan-degrading enzyme acting on a mannan-containing natural product and containing at least 10% by weight of / 3-1, 4_mannobiose based on mannan before decomposition. It is a composition having an immunostimulatory action characterized in that it relates to an immunostimulatory composition having an effect of promoting production of IgA, foods and drinks and feeds containing them.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

In the present invention, β-1,4-mannobiose is composed of two D mannose molecules, 1, 4- It is a glycoside bond. In the present invention, β -1,4 mannobiose can be obtained by, for example, a method of synthesizing from mannose or a method of decomposing β-1,4 mannan (hereinafter also simply referred to as mannan).

The method for decomposing β-1, 4 monomannan is more preferable in terms of raw material resources and reaction efficiency, and can provide / 3-1,4_mannobiose. In this method, for example, mannan-degrading enzyme is added to mannan-containing natural products such as coconut cake, palm kernel meal, cobra meal, gua gum, locust bean gum, etc. that are rich in mannan, or mannan extracted from these natural products. By acting, you can obtain / 3-1— 4 _ Mannobiose.

[0010] The coconut cake is used as an edible solid product obtained by crushing the endosperm in coconut fruit into a slurry and solid-liquid decomposition. Cobra meal generally refers to a residue obtained when palm oil is extracted from cobra obtained by drying core meat in coconut pulp by sun drying or hot air drying. In the present invention, the sun or hot air drying process is performed. Also includes oil extraction residue extracted without passing through. The method for extracting coconut oil is not particularly limited, such as extraction using a solvent, etastruder or a combination of these. Palm kernel meal is a residue obtained by extracting palm kernel oil from palm kernels, which are seeds of oil palm, and can also be extracted by solvent extraction, etastruder extraction or a combination thereof, but is particularly limited. It is not a thing. Among these, coconut cake is used for food, and is more preferably used in that the cost can be reduced by omitting the extraction and purification of mannobiose described later.

In the present invention, the decomposition of mannan must be enzymatic hydrolysis. Typical examples of the hydrolysis method include acid decomposition. However, hydrolysis by these methods causes acid modification of the resulting composition, and it is difficult to obtain the expected effect immediately. As the enzyme used, mannanase is not particularly limited as long as it decomposes mannan and produces at least 10% by weight of / 3-1, 4_mannobiose to mannan before decomposition. Examples thereof include hemicellulases such as mannosidase. For example, commercially available preparations, culture solutions obtained by culturing bacterial cells, or those obtained after cell strength separation can be used. For example, hemicellulase GM “AMANO” (Amano Pharmaceutical Co., Ltd.) Sumiteam ACH (manufactured by Shin Nippon Chemical Industry Co., Ltd.), Singuchi Shin GM5 (Hankyu Bio Industry Co., Ltd.) and the like can be preferably used. In addition to these, even those commercially available as xylanase and cellulase, those having the hydrolysis activity can be used. For example, cellulase Y-NC (manufactured by Yakult Pharmaceutical Co., Ltd.) can be used. it can. In particular, mannosidase (exo type) activity is low and mannanase (endo type) activity is high. Hemicellulase GM “AMANO” (manufactured by Amano Pharmaceutical Co., Ltd.) and Sumiteam ACH (manufactured by Shin Nippon Chemical Industry Co., Ltd.) are responsible for the production of mannose. It is preferable in that it can be suppressed and a large amount of mannobiose can be generated.

[0012] Furthermore, the enzyme used in the present invention acts on mannan-containing natural products or extracted mannan as an enzyme solution dissolved or dispersed in water. In order to perform an efficient reaction in the case of using a mannan-containing natural product, it is important to adjust moisture in a reaction system composed of the mannan-containing natural product, a mannan-degrading enzyme, and water. The amount of water added for water adjustment is preferably 50 to 10000 parts by weight with respect to 100 parts by weight of mannan, more preferably 50 to 1500 parts by weight. By making the amount of water added in such a range, the mannan fiber can be sufficiently swollen in the presence of sufficient moisture, and the enzyme solution can be easily contacted. However, an excessive amount of water results in an increase in the drying cost in the drying process when the enzyme concentration is diluted and, as a result, if the reaction efficiency is lowered and drying is performed by force, drying is performed. Therefore, considering the above, it is appropriate to add 50 to 500 parts by weight of water to 100 parts by weight of mannans.

[0013] The amount of the enzyme and the reaction time are at least 10% by weight of mannobiose produced with respect to mannan before decomposition, preferably 10 to 80% by weight of mannobiose produced by hydrolysis with respect to mannan before decomposition. %, So long as it is about%, a wet enzyme-treated product can be obtained under such conditions. However, since an enzyme having a high mannanase (endo type) activity usually also has a mannosidase (exo type) activity, if the enzyme reaction time is too long, mannobiose is decomposed and the amount of mannose increases. Therefore, the reaction time is preferably not longer than necessary. These enzyme reaction conditions are appropriately set so that the amount of mannobiose produced is as large as possible. in this case, beta-1, 4 if mannobiose is example preferred tool embodiment to set to include more than mannose, beta-1, 4 more preferable that the Ru der proportion force 60 weight ^_0/0 following mannose for mannobiose instrument 20 It is particularly preferred that it is not more than wt%.

As described above, for example, when palm kernel meal (mannan content is approximately 36%) is used as a raw material for 3 to 36 hours, the amount of mannobiose depends on the type, amount, and time of the enzyme used. Due to 100 parts by weight of raw material, 6 to 17 parts by weight can be generated.

[0014] The obtained decomposed product may be an aqueous solution composition as it is, preferably with a drying force. The drying method is not particularly limited, and examples thereof include freeze-drying, spray drying, fluidized bed drying and the like, which are free from excipients such as dextrin. If necessary, ethanol is used to remove impurities from the obtained enzyme degradation product. In addition, as a solvent that may remove impurities, ethanol is preferable from the viewpoint of power safety that can include methanol, isopropanol, hexane, and the like. After removing impurities with ethanol, water extraction may be performed. In this case, since 1,4 mannobiose, which is considered as a main component, is water-soluble, a concentration operation may be performed. The drying method of the obtained water-soluble component is not particularly limited. In particular, the aqueous composition may be used as it is without drying. Examples of the drying method include freeze-drying, spray drying and fluidized bed drying with an excipient such as dextrin.

[0015] The β-1, 4 mannobiose content is preferably hydrolyzed so as to be 10% by weight or more based on the weight of mannan before hydrolysis. There is no promotion of IgA production that is expected to be less than 10. IgA production promoting ability seen in the present invention Whether or not it is caused only by this β −1, 4_mannobiose is not certain, but it is expected to act as at least a main component. However, since it is limited to coconut cake, cobra meal and palm kernel meal as a mannan-containing composition in order to obtain the expected effect of promoting IgA production, trace amounts of components present in these raw materials And β _ 1, 4_ mannobiose are considered to exhibit a unique effect. Therefore, it is considered that the effect of the present invention can be obtained even when the amount of β _ 1, 4_ mannobiose is 100%, but it is economical to extract β _ 1 '4 _ mannobiose with high purity. Because it is burdensome and no synergistic effect with the estimated trace components can be expected, the content of β-1, 4 mannobiose is preferably 10% by weight or more and less than 90% by weight 15% by weight or more, 40% by weight Less than.

[0016] The intestinal immunity stimulating substance for promoting IgA production of the present invention may be appropriately formulated by a public method and used in the form of an intestinal immunity activator. It can be added to breads, confectionery, vitamins and other health foods and consumed as food and drink, and is not particularly limited. Similarly, it can be used as feed by adding it to fishery and terrestrial animal feeds.

Example

Hereinafter, the present invention will be specifically described by way of examples. The present invention is not limited to these examples.

Cobrameal, 30% by weight of mannan containing 30% by weight of mannanase and coprameal A (the drying process) with 0.25 parts by weight of hemicellulase (hemicellulase GM “AMANO” manufactured by Amano Pharmaceutical Co., Ltd.) in water (Free dried oil), mixed with 100 parts by weight of palm kernel meal or coconut cake, and after 12 hours at 60 ° C, fluid bed drying is performed until the water content is less than 10%. Got.

Add twice the amount of ethanol to 100 parts by weight of the dried product, filter after stirring, repeat this operation three times, and then remove residual ethanol. Then, 500 parts by weight of 60 ° C hot water was added, and the filtrate obtained after filtration was lyophilized to obtain the intestinal immunity stimulating substance of the present invention. Its sugar composition was as follows

[0018] [Table 1] Table 1 Sugar composition of invention

Example 1 Example 2 Example 3 Copramysole raw material Palm kernel meal raw material Two pramille lees raw material arabinose 0.. 1 6 (%) 0.1 8 (%) 0.2 0 (%) Galactose 0 1 4 0. 1 3 0. 2 1 Guzore course 2.. 5 4 2. 3 1 1 0. 0 3 Mannose 1.. 3 8 1. 5 0 1 .5 4 Fructose 1 .. 3 8 1. 4 1 2. 7 1 β-1, 4-N-Nobiose 2 1,. 7 4 1 9. 8 0 3 6. 0 7

Next, the ability as an IgA production promoting activator according to Examples 1 and 2 was evaluated by measuring IgA in mouse spleen cell culture supernatant. Mouse spleen cells were collected from 6-week-old BALB females (Charles River).

The collected spleen cells were loosened in RPMI1640 (Gibco BRL) containing 50 U / ml penicillin and 50 _β g / ml streptomycin, and a cell suspension was obtained by centrifugation.

LPS (5 μ / ml) or concanaparin A (ConA) (0.5 z / ml each) in this cell suspension (1 X 10 ⁶ cell / ml) and the IgA production promotion obtained in Examples 1 and 2 Activator is added to a concentration of 50 μg / ml, cultured at 37 ° C for 2 days for cyto force-in measurement, and 5 days for immunoglobulin measurement. The IgA content was measured by sandwich ELISA. Similarly, IgG was also measured.

[0020] Similarly, in order to confirm the cell proliferation of T cells or B cells, the absorbance at 450 nm was evaluated using a WST-1 assay kit (Roche Molecular Biochemimcal).

In addition, interleukin 6 (IL-6), which is important for IgA production, and interferon _γ (IFN-γ), which is an indicator of B cell and macrophage activation, were also measured using this ELISA using a sandwich ELISA method. . For IFN γ, add the above supernatant to an ELISA microtiter plate coated with anti-mouse IFN γ monoclonal antibody, bind INF γ in the supernatant, and use anti-mouse IN F-γ stained with piotin. The mixture was made into a sandwich, reacted with a mixed enzyme solution of avidin and horseradish peroxidase, the color developed by the substrate TMB was measured with a microplate reader at 450 nm, and the content was calculated with a calibration curve. For IL-6, ELISA kit (OptEIA set, The content was measured by BD Bioscience).

Table 2 shows the results of the effect of promoting production of IgA, IgG, INF-γ, and IL 6 in the examples.

[Table 2]

Table 2 Effects of Examples 1 and 2 on Ig A, Ig G, I N F—γ, I L

As can be seen from the above results, it is recognized that the product of the present invention has the effect of stimulating the immune system to promote the production of IgA and IgG. However, the production of INF-γ and IL-6, which are site forces, is not promoted, indicating that it is a stimulant for a completely new immune system.

Table 3 shows the presence or absence of sputum cells or sputum cell activation promoting effects.

[Table 3]

Table 3 Effect of Examples on Sputum Cells and Sputum Cells

From the above results, the present invention was found to have low T cell and B cell proliferation promoting effects. Therefore, the product of the present invention stimulates and proliferates the conventionally called T lymphocytes and B cells, and directly produces IgA and IgG, which does not prevent bacterial invasion. It turns out that it is a new one that promotes and protects against bacteria and the like. Industrial applicability

According to the present invention, it is possible to increase IgA production, an antibody important for intestinal immunity, characterized by comprising a plant mannan degradation product containing / 3_1,4_mannobiose as a main component. It has an intestinal tract immunostimulatory effect, and can be used for a composition having a disease-preventing function due to pathogenic bacteria or viruses, or a food or drink containing them.

Claims

The scope of the claims

[1] An intestinal immunity stimulating substance or agent containing β -1, 4_mannobiose.

[2] per weight of mannan contained in coconut cake, cobra meal or palm kernel meal

The water-soluble component of a composition containing j3-1,4 mannobiose obtained by using a mannan degrading enzyme so that 10% by weight or more of j3_1,4_mannobiose is produced is an active ingredient. Intestinal immunity stimulating substance or agent as described in 1.

[3] Per weight of mannan contained in coconut cake, cobra meal or palm kernel meal

10 weight ^_0/0 or more 1, 4 mannobiose dried is i3 1 obtained by a work mannan degrading enzyme to generate, 4 mannobiose-containing composition, ethanol and water was added to the SC Ete extracted residue component The intestinal immunity stimulating substance or agent according to claim 1, wherein the extracted ingredient is an active ingredient.

[4] The intestinal immunity stimulating substance or agent according to any one of claims 1 to 3, which has an action of increasing the production amount of immunoglobulin A (IgA).

[5] A food or drink or feed containing the intestinal immunity stimulating substance according to any one of claims 1 to 4, which has a disease-preventing effect against pathogenic bacteria and viruses.

[6] A food or drink or feed comprising the intestinal immunity stimulating substance according to any one of claims 1 to 4, which has an allergic effect.