JP2012087102A - IgA PRODUCTION PROMOTER AND IMMUNOPOTENTIATOR - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an IgA production promoter which has excellent action, and has excellent safety, environmental properties and productivity, an immunopotentiator, a method for extracting IgA, and IgA obtained by the extraction method.SOLUTION: The IgA production promoter comprises tempeh as an active ingredient, and the immunopotentiator comprises tempeh as an active ingredient. In the method for extracting IgA, feces is extracted from an object to which tempeh has been administrated, a solvent and a protease inhibitor are added to the feces, suspension is caused to prepare a suspension, the suspension is subjected to centrifuging to obtain a supernatant, and IgA is extracted from the supernatant. The IgA is obtained by the extraction method.

Description

  The present invention relates to an IgA production promoter and an immunostimulant, an IgA extraction method, and IgA obtained by the extraction method.

  The modern diet is becoming more westernized, and the tendency for high-fat, low-fiber diets is growing. Accordingly, an increase in lifestyle-related diseases such as obesity has become a problem. In recent years, with the westernization of such meals, the incidence of colon diseases such as colorectal cancer and inflammatory bowel disease has been increasing year by year, and a high fat diet has been pointed out as one of the causes.

  IgA is known as a factor that affects the intestinal environment of the large intestine. Skin and mucous membranes, which are the boundaries between which the living body comes into contact with the outside world, and particularly gastrointestinal mucosa such as intestinal mucosa, are in contact with many substances such as viruses, bacteria, parasites, pathogenic antigens, and food antigens. IgA is secreted not only in the gastrointestinal tract but also in the tracheal mucosa and protects the respiratory tract, and is also secreted into milk and protects the intestinal tract of the newborn baby. At the forefront of the immune system in the gastrointestinal tract and respiratory tract, it has the action of adhering, fixing, and preventing invasion of pathogens that invade the mucosal surface, and neutralizing toxins and bacteria via the mucosal surface To do. When the IgA secretion amount increases, the intestinal tract immune function is enhanced and various diseases such as infectious diseases, inflammatory bowel diseases, and allergic diseases are prevented (see Non-Patent Document 1). Furthermore, the relationship between IgA and colorectal cancer has also been reported (see Non-Patent Document 2).

  Under such circumstances, Okazaki et al. Of Hiroshima University recently revealed that ingestion of curcumin, a polyphenol, under the condition of a high-fat diet increases IgA in feces. It points out the involvement of IgA increase in the prevention mechanism (see Non-Patent Document 3).

  Therefore, there is a demand for various natural preparations that are excellent in safety, environment and productivity, can be ingested on a daily basis, and are inexpensive, yet have at least one of an excellent IgA production promoting action and an immunostimulating action. Is extremely strong.

Huang MT, et al. , Cancer Research, vol. 54, p. 5841-5847, 1994 Lambert JD, et al. , Am J Clin Nutr, vol. 81, p. 284-291, 2005 Okazaki Y, et al. , J Nutr Sci Vitamol, vol. 56, p. 68-71, 2010

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, an object of the present invention is to provide an IgA production promoter that has an excellent IgA production promoting action and is highly safe. Another object of the present invention is to provide an immunostimulator having an excellent immunostimulatory action and high safety. Another object of the present invention is to provide an IgA extraction method and IgA obtained by the extraction method.

  In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following findings. That is, it has been found that tempe has an excellent IgA production promoting action and an excellent immunostimulatory action.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> An IgA production promoter comprising tempe as an active ingredient.
<2> An immunostimulator comprising tempe as an active ingredient.
<3> A food and drink comprising at least one of the IgA production promoter according to <1> and the immunostimulator according to <2>.
<4> A feces was collected from a subject administered with tempeh, and a suspension was prepared by adding a solvent and a proteolytic enzyme inhibitor to the feces to prepare a suspension, which was obtained by centrifuging the suspension. It is an IgA extraction method characterized by extracting IgA from a supernatant.
<5> IgA obtained by the IgA extraction method according to <4>.

According to the IgA production promoter of the present invention, conventional problems can be solved, and an IgA production promoter having excellent IgA production promotion activity and high safety can be provided.
According to the immunostimulant of the present invention, conventional problems can be solved, and an immunostimulant having excellent immunostimulatory action and high safety can be provided.
According to the IgA extraction method and IgA of the present invention, conventional problems can be solved, and an excellent IgA extraction method and IgA obtained by the extraction method can be provided.

(IgA production promoter and immunostimulator)
The IgA production promoter of the present invention contains tempe as an active ingredient, and further contains other ingredients as necessary.
The immunostimulant of the present invention contains tempe as an active ingredient, and further contains other ingredients as necessary.

<Tempe>
The tempe is a composition obtained by fermenting cereals such as soybeans with Tempe fungus ( Rhizopus oligosporus ).
The tempeh is known as an Indonesian traditional food and is also called Indonesian natto, but it does not produce viscous substances or unique odors that pull the system like natto. Tempe is said to have been made in Java from 400 to 500 years ago and spread throughout Indonesia in the early 19th century. A clear record is the start of the 1895 Dutch Scientific Research Team's report on the analysis of components of tempeh in Java and the search for microorganisms.
However, there are very few studies on the health effects of taking tempeh itself.
There is no restriction | limiting in particular as said grain, According to the objective, it can select suitably, For example, a soybean, peanut, rice, wheat, etc. are mentioned. Among these, soybean is preferable.
The method for producing the tempeh is not particularly limited, and a known method can be selected according to the purpose. For example, after absorbing grains such as soybeans, boiled and cooled, acidified by adding vinegar and the like. And koji containing Rhizopus oligosporus , which is a kind of spider fungus belonging to the zygomycete called Tempe fungus, is mixed, thinned and fermented.
There is no restriction | limiting in particular as temperature of the said fermentation, Although it can select suitably according to the objective, 30 to 40 degreeC is preferable and 32 to 35 degreeC is more preferable. If the temperature is less than 30 ° C, the fermentation may not proceed, and if it exceeds 40 ° C, it may be rancid.
There is no restriction | limiting in particular as time of the said fermentation, Although it can select suitably according to the objective, 20 hours-30 hours are preferable, and 22 hours-26 hours are more preferable. When the temperature is less than 20 hours, the fermentation may not proceed sufficiently, and when it exceeds 30 hours, it may be rancid.
There is no restriction | limiting in particular as said tempe, According to the objective, it can select suitably, A commercial item can be used. As this commercial item, Tempe (made by Kuninobu) etc. are mentioned, for example.

  Since the tempe has an IgA production promoting action and an immunostimulating action, it can be used as an IgA production promoting agent and an immunostimulating agent using these actions.

  The IgA production promoter of the present invention may be composed only of the tempe or may contain other components. There is no restriction | limiting in particular as content of the said tempe in the said IgA production promoter, According to the objective, it can select suitably.

  The immunostimulant of this invention may consist only of the said tempe, and may contain another component. There is no restriction | limiting in particular as content of the said tempe in the said immunostimulant, According to the objective, it can select suitably.

<Other ingredients>
The tempe is formulated into an arbitrary dosage form such as powder, granule, tablet, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin and any other auxiliary agent. It can be provided and used in other compositions (for example, oral pharmaceuticals), and can also be used as an ointment, a liquid for external use, a patch, and the like. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a corrigent, a corrigent and the like can be used.

  Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include , Purified talc, stearate, borax, polyethylene glycol and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, Oh lactate and the like, as the flavoring agent include sucrose, bitter orange peel, citric acid, and tartaric acid.

  In addition, the IgA production promoter and immunostimulant of this invention can mix | blend and use the other natural extract etc. which have either an IgA production promotion effect | action and an immunostimulation effect | action as needed.

The IgA production promoter exerts an IgA production promoting action by the action of tempe contained as an active ingredient.
The immunostimulant exerts an immunostimulatory action by the action of tempe contained as an active ingredient.

  According to the IgA production promoter of the present invention, through an excellent IgA production promoting action, for example, the intestinal tract immune function is enhanced to prevent or treat various diseases such as infectious diseases, inflammatory bowel diseases, allergic diseases, and colon cancer. It is possible to use it suitably. However, the IgA production promoter of the present invention can be used for all purposes that are meaningful for exerting an IgA production promoting effect in addition to these uses.

  According to the immunostimulant of the present invention, it is suitable for the prevention or treatment of various diseases such as infectious diseases, inflammatory bowel diseases, allergic diseases, colorectal cancer, etc., through enhanced immunostimulatory action, for example, enhancing intestinal immunity function. It becomes possible to use it. However, the immunostimulant of the present invention can be used for all uses other than these uses that are meaningful for exerting an immunostimulatory action.

  Since the IgA production promoter and immunostimulant of the present invention have an excellent action and have edible tempeh as an active ingredient and are excellent in safety, they are suitable for cosmetic foods and drinks or oral medicines. Suitable for blending.

  Moreover, since the IgA production promoter and immunostimulant of the present invention have an excellent action, they can be suitably used as reagents for research of diseases related to IgA and immunostimulation.

  The IgA production promoter and immunostimulant of the present invention are preferably applied to humans, but should be applied to animals other than humans as long as their respective effects are exhibited. You can also.

(Food)
The food / beverage products of this invention contain at least any one of the IgA production promoter of this invention, and an immunostimulant, and also contain another component as needed.

The foods and drinks are those that are less likely to harm human health and are taken by oral or gastrointestinal administration in normal social life, such as foods, pharmaceuticals, and quasi drugs in administrative divisions. It is not limited to a category, and means, for example, a wide range of foods that are taken orally, such as general foods, health foods, health functional foods, beauty foods, quasi drugs, and pharmaceuticals.
Moreover, the food / beverage products of this invention can be simply eaten / drinked by processing into shapes, such as a drink, a hard capsule, a soft capsule, a granule, and can be utilized widely.

  The food or drink is not particularly limited as long as it contains at least one of the IgA production promoter and the immunostimulator, and can be appropriately selected according to the purpose. For example, soft drinks, carbonated drinks, nutrition Beverages such as beverages, fruit beverages and lactic acid beverages (including concentrates and powders for preparation of these beverages); frozen desserts such as ice cream, ice sherbet and shaved ice; buckwheat, udon, harsame, gyoza skin, scented skin, Chinese noodles, instant noodles and other noodles; sweets such as crab, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, baked confectionery, bread; crab, salmon, clams, tuna, sardines, shrimp Seafood such as bonito, mackerel, whale, oyster, saury, squid, red scallop, scallop, abalone, sea urchin, salmon roe, tokobushi; -Fish and processed fishery products such as sage; Dairy products such as processed milk and fermented milk; Processed fats and oils such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing; seasonings such as sauce and sauce; Retort pouches such as curry, stew, oyakodon, rice bowl, miscellaneous rice, Chinese rice bowl, tempura, eel rice bowl, hayashi rice, oden, marbodorf, beef bowl, meat sauce, egg soup, omelet rice, dumplings, shumai, hamburg, meatballs, etc. Foods; Salads, pickles and other prepared foods; various forms of health, beauty and nutritional supplements; tablets, granules, capsules, drinks, troches and other pharmaceuticals, quasi drugs, and the like. The tempe may be used as a substitute for meat. In addition, the said food / beverage products are not limited to the said illustration.

  The content of the IgA production promoter and immunostimulant in the food or drink varies depending on the food or drink to be added, and cannot be specified unconditionally, but in the case of tablets or capsules, 1% to 90% by weight. In other foods and beverages, 1% by mass to 90% by mass is preferable. Moreover, it is preferable to prepare so that the intake amount of at least one of an IgA production promoter and an immunostimulant per day for an adult may be about 5 to 15 g in consideration of the general intake amount of the food and drink to be added.

Examples of the other components include auxiliary raw materials and additives that are usually used in manufacturing the food and drink.
The auxiliary raw material or additive is not particularly limited and may be appropriately selected depending on the intended purpose. For example, glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, lactose, citrus Acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, Examples include gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamin B, nicotinic acid amide, calcium pantothenate, amino acids, calcium salts, pigments, fragrances and preservatives.

  The food and drink of the present invention can be taken orally on a daily basis, and at least one of IgA production promoting action and immunostimulating action can be achieved very effectively by the action of tempe which is an active ingredient. it can.

  In addition, although the food / beverage products of this invention are suitably applied with respect to a human, as long as each effect is show | played, it can also apply with respect to animals other than a human.

(IgA extraction method and IgA)
In the IgA extraction method of the present invention, feces are collected from a subject administered with the tempe, a suspension is prepared by adding a solvent and a protease inhibitor to the feces, and the suspension is centrifuged. It is necessary to extract IgA from the supernatant obtained by separation.
The IgA of the present invention needs to be obtained by the above IgA extraction method.
There is no restriction | limiting in particular as said solvent, According to the objective, it can select suitably, For example, phosphate buffered saline, physiological saline, Tris buffered saline, etc. are mentioned.
The proteolytic enzyme inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include trypsin inhibitor, serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and aprotinin; leupeptin and the like Serine / cysteine protease inhibitors; Cysteine protease inhibitors such as E-64; Metalloprotease inhibitors such as ethylenediaminetetraacetic acid (EDTA) and glycol etherdiaminetetraacetic acid (EGTA); Lactacystine, MG-115, MG-132, etc. And proteasome inhibitors. These may be used individually by 1 type and may use 2 or more types together.
The IgA extraction method is not particularly limited as long as the above conditions are satisfied, and can be appropriately selected according to the purpose. For example, proteolysis such as trypsin inhibitor, EDTA, PMSF and the like is performed on feces collected from subjects administered with the tempe. Add phosphate buffered saline (PBS, pH 7.2) containing an enzyme inhibitor, stir with a test tube mixer for 1 minute, and then let stand at 4 ° C. overnight, then at 4 ° C., 9,000 × g. A method of extracting or purifying IgA from the supernatant obtained by centrifugation for 10 minutes by a known method can be mentioned.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not restrict | limited to a following example.

[Animal rearing]
SD (Sprague-Dawley) male rats (3 weeks old, initial weight 40-50 g, manufactured by Charles River Japan Inc.) were used as experimental animals. Laboratory animals were raised according to the regulations for handling laboratory animals at Hiroshima University. Specifically, each animal is placed in a stainless steel cage and reared in a constant temperature environment (24 ° C. ± 1 ° C.) of 12 hours light-dark alternation (8: 00-20: 00 light, 20: 00: 8:00 dark the next morning). did. The pre-breeding period was 7 days, and commercially available solid feed (MF, manufactured by Oriental Yeast Co., Ltd.) and tap water were freely ingested.
After preliminary breeding, the rats were divided into 3 groups (8 animals / 1 group), respectively, a control group, a tempeh group, and a separated soy protein (SPI) group, and a restricted diet (experimental diet) shown in Table 1 below, The animals were bred for 21 days with free access to deionized water. The restricted diet is a high-fat diet of 30% by weight beef tallow, the protein source being 20% by mass of casein in the control group, 25% by mass of tempe and 7.1% by mass of casein in the tempe group, and isolated soybeans. The protein (SPI) group contains 12.3% by mass of SPI and 7.1% by mass of casein. Table 2 shows the amino acid composition of Tempe and SPI. As the tempe, tempe made from soybeans (Tempe, manufactured by Kuninobu Co., Ltd.) and Solby 4000 (produced by Nisshin Oillio Group) as the SPI were used.
The experimental meal is 9 g on the first day, 10 g on the second to fourth days, 12 g on the fifth to seventh days, 14 g on the eighth to thirteenth days, and 15 g on the twenty-first to twenty-first days (19:00) To rats. Both gave enough to eat by the next morning. Body weight was measured every morning (10:00). Feces were collected during the last 3 days of breeding. The wet weight of feces was measured, lyophilized for 12 hours, and then ground using a mortar. The dried feces was stored frozen at −30 ° C. and analyzed for fecal IgA.

<Analysis method>
<< Method for measuring IgA in feces >>
Fecal IgA was measured in accordance with the method of Sharma et al. (Sharma A. et al., Infection and Immunity. Vol. 69: p. 2928-2934, 2001) as follows.

(IgA extraction)
Phosphoric acid containing 40-fold trypsin inhibitor (0.1 mg / ml, derived from soybeans, manufactured by Wako Pure Chemical Industries, Ltd.), EDTA (50 mM), phenylmethylsulfonyl fluoride (PMSF, 1 mM) in dry feces (0.1 g) Buffered saline (PBS, pH 7.2) was added. The mixture was stirred for 1 minute with a test tube mixer and allowed to stand overnight at 4 ° C. Thereafter, the mixture was centrifuged at 9,000 × g for 10 minutes at 4 ° C., and the supernatant was collected and used as a sample for IgA measurement. The obtained sample was stored frozen at −30 ° C. until measurement.

(Measurement of IgA amount)
The IgA amount was measured by an ELISA method using a Rat IgA ELISA Quantification Kit (manufactured by BETHYL). A goat anti-rat IgA antibody was used as an antigen and diluted with 0.05 M calcium carbonate buffer (pH 9.6) to a concentration of 1 μl antigen stock / 0.1 ml. 100 μl of this diluted solution was placed in each well of a 96-well plate and allowed to stand overnight at 4 ° C. for antigen treatment. The next day, each well was washed three times with a washing solution (0.05% Tween 20, 50 mM Tris buffer, 0.14 M NaCl, pH 8.0), and each well was treated with 50 mM Tris buffer containing 1% BSA, 0.14 M. 200 μl of NaCl solution (pH 8.0) was added, and after stirring with a microplate mixer, blocking was performed at room temperature (20 ° C. to 25 ° C.) for 30 minutes. Sample or rat IgA antibody standard was diluted with diluent (1% BSA, 0.05% Tween 20, 50 mM Tris buffer-0.14 M NaCl, pH 8.0). After washing each well three times with a washing solution, 100 μl of a sample and a rat IgA antibody standard were added, stirred with a microplate mixer, and incubated at room temperature (20 ° C. to 25 ° C.) for 1 hour. Each well was washed 5 times with a washing solution, 100 μl of diluted HRP-labeled goat anti-rat IgA antibody solution was added as a secondary antibody, stirred with a microplate mixer, and incubated at room temperature (20 ° C. to 25 ° C.) for 1 hour. Each well was washed five times with a washing solution, and then 0.1 mg citrate phosphate buffer (pH 5.0) containing 0.4 mg / ml OPD (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.026% H ₂ O ₂ was used. The mixture was reacted at room temperature (20 ° C. to 25 ° C.) for 15 minutes while being shielded from light with aluminum foil. After the reaction, 100 μl of 2MH ₂ SO ₄ was added to stop the reaction, and the absorbance at 450 nm was measured with a microplate reader.

<< Statistical processing method >>
The data of each group obtained by the experiment is shown as mean ± standard error, and after performing a one-way analysis of variance, the significance of each group was determined using Fisher's multiple-range test (p <0.05).

<Result>
Table 3 shows the results of measuring the amount of IgA in feces from the intake of restricted food in each group. The amount of IgA per dry feces in the tempe group was significantly increased compared to the control group (p <0.05). In addition, although the dry weight of feces decreased in the tempeh group, the water content in the feces increased (data not shown; p <0.05). When the fecal IgA was expressed as the total excretion amount for 3 days, the tempeh group significantly increased 3.87 times compared to the control group (p <0.05). Even in the SPI group, an increase in the amount of IgA per dry feces and the total excretion amount of IgA in feces was observed for 3 days, but was not as significant as in the Tempe group (p <0.05).

  From the results in Table 3, it was confirmed that tempe had an excellent IgA production promoting action. The SPI group was also found to have an IgA production promoting effect, but the effect was more pronounced in the Tempe group. Moreover, since tempe has an excellent IgA production promoting effect, it was considered that tempe had an excellent immunostimulatory effect.

  Since the IgA production promoter and the immunostimulant of the present invention have excellent IgA production promoting effect and immunostimulating effect, the intestinal tract immune function is enhanced, and infection, inflammatory bowel disease, allergic disease, colon cancer It can be suitably used for the prevention or treatment of various diseases such as.

Claims (5)

  An IgA production promoter comprising tempe as an active ingredient.   An immunostimulant comprising tempe as an active ingredient.   A food / beverage product comprising at least one of the IgA production promoter according to claim 1 and the immunostimulator according to claim 2.   A feces is collected from a subject to which tempe has been administered, and a suspension is prepared by adding a solvent and a protease inhibitor to the feces to prepare a suspension. From the supernatant obtained by centrifuging the suspension. An IgA extraction method characterized by extracting IgA.   IgA obtained by the IgA extraction method according to claim 4.