WO2015182530A1

WO2015182530A1 - Indigestible substance and method for manufacturing same

Info

Publication number: WO2015182530A1
Application number: PCT/JP2015/064835
Authority: WO
Inventors: 一典三輪; 公子南田
Original assignee: アテリオ・バイオ株式会社
Priority date: 2014-05-24
Filing date: 2015-05-24
Publication date: 2015-12-03
Also published as: JPWO2015182530A1; JP6306170B2

Abstract

[Problem] The purpose of the present invention is to provide an indigestible substance that makes it possible to deliver probiotics to the distal colon and that has an exceptional effect for improving the intestinal environment, and a method for manufacturing the indigestible substance. [Solution] The present invention provides a method for manufacturing an indigestible substance, the method including: a step for preparing a mixture that contains dietary fiber, spore-forming bacteria and/or spores thereof, a sugar, and a protein or an amino acid; and a step for heating the mixture to form melanoidin. This makes it possible to provide an indigestible substance that makes it possible to improve bowel movement and promote proper defecation, and to prevent repeated diarrhea and constipation seen in irritable bowel syndrome through continual ingestion.

Description

Indigestible substance and method for producing the same

The present invention relates to an indigestible substance excellent in improving the intestinal environment and a method for producing the same.

In order for microorganisms effective for improving the intestinal environment, called probiotics, to reach the lower gastrointestinal tract, especially the large intestine, after ingestion, gastric acid with a strong bactericidal action and food in the small intestine Must be resistant to exposure to various substances involved in digestion and absorption.

However, not many microorganisms originally have the above resistance. Bacillus coagulans , a spore-forming lactic acid bacterium, is highly resistant to gastric acid and has the ability to reach the intestine and produce lactic acid. It is not satisfactory. Spores of spore-forming lactic acid bacteria show high resistance to the surrounding environment, but the spore that has reached the environment suitable for growth, for example, the digestive tract below the stomach, germinates and changes into vegetative cells. Loses resistance to Butyric acid bacteria have similar problems. Therefore, microorganisms that are used as probiotics, including spore bacteria useful for humans such as spore-forming lactic acid bacteria and butyric acid bacteria, as well as microorganisms that do not have various resistances or are insufficient. However, there is a great need for formulation-like devices that can provide the desired resistance exogenously.

In addition to the probiotics that are microorganisms, similar needs exist for nutrients that affect the growth of enteric bacteria, particularly prebiotics. Specifically, so that prebiotics are not assimilated by microorganisms other than probiotics before reaching the desired site, or are not altered by exposure to various substances involved in digestion and absorption of food, There is a great need for formulation-like devices that can provide the desired resistance exogenously to prebiotics.

Furthermore, it is preferable that the probiotics have a characteristic of proliferating predominately over other intestinal bacteria in the intestinal environment where a very wide variety of microorganisms are mixed to form a flora. A formulation ingenuity that can provide such growth characteristics exogenously, that is, can provide a favorable growth environment for the desired probiotics, can also be effective for improving the intestinal environment using probiotics.

¡Preparative measures for selectively bringing an active ingredient to a desired site have been made. For example, Patent Document 1 discloses an intracolon pressure collapse type colon delivery capsule using ethyl cellulose. However, capsule technology using such a polymer film is mainly for the purpose of drug delivery, and it is disadvantageous in terms of cost in providing useful substances such as probiotics and prebiotics in the form of food. There are also problems such as reducing the stability of probiotics and prebiotics, which are objects, in the formulation process.

As an example of food containing spores of spore-forming lactic acid bacteria, Patent Document 2 discloses frozen bread containing lactic acid bacteria. Moreover, the stabilization method of the spore using a specific emulsifier and thickening polysaccharide is disclosed. However, even though these techniques can maintain the spore state before ingestion orally, the spore germinates in the small intestine after ingestion and changes to vegetative cells, resulting in the necessary amount in the large intestine, etc. There remains a risk that the spores or sporic lactic acid bacteria could not be delivered. Furthermore, these techniques do not improve the growth characteristics of spore-forming lactic acid bacteria in the large intestine.

US Pat. No. 5,637,319 Japanese Patent Laid-Open No. 10-84845

The present invention improves the intestinal environment suitable for use in the form of food, allowing probiotics to reach a desired location in the intestine, especially the distal large intestine, and promote its growth at such location. An object of the present invention is to provide an indigestible substance having excellent effects and a method for producing the same.

The present inventors have completed the following inventions while variously examining probiotic protection means using dietary fiber.

(1) preparing a mixture containing dietary fiber, spore fungus and / or spore, saccharide and protein or amino acid;
And a step of forming melanoidin by heating the mixture.
(2) The method according to (1), wherein the dietary fiber is fine powdered okara having a particle diameter of 200 μm or less, cellulose, or a derivative thereof.
(3) The method according to any one of (1) and (2), wherein the saccharide and protein or amino acid are cereal grains or a processed product thereof.
(4) The method according to (3), wherein the processed grain product is soybean flour, rice flour or barley extract.
(5) The method according to any one of (1) to (4), wherein the mixture further comprises prebiotics.
(6) An indigestible substance produced by any one of the methods (1) to (5).
(7) An indigestible substance in which spores and melanoidin are fixed to dietary fiber.
(8) The indigestible substance according to (7), wherein the dietary fiber is fine powdered okara having a particle diameter of 200 μm or less, cellulose, or a derivative thereof.
(9) The indigestible substance according to (7) or (8), further comprising prebiotics.
(10) A therapeutic agent for irritable bowel syndrome comprising the indigestible substance according to any one of (6) to (9) as an active ingredient.

According to the present invention, a useful substance having an action to improve the intestinal environment represented by probiotics is efficiently delivered to the distal part of the large intestine and proliferated to improve the intestinal environment in the distal part of the large intestine. can do. As a result, bowel movements can be improved to promote proper defecation, and continuous ingestion can prevent repeated diarrhea and constipation seen in irritable bowel syndrome.

It is a microscope picture which shows the external appearance of the water-insoluble dietary fiber before heat processing. It is a microscope picture which shows the external appearance of the water-insoluble dietary fiber after heat processing. It is a graph which shows the proliferation in the pseudo proximal colon environment of the spore-forming lactic acid bacteria in an indigestible substance. It is a graph which shows the proliferation in the pseudo-distal colon environment of the spore-forming lactic acid bacteria in an indigestible substance. It is a graph which shows the proliferation in the pseudo proximal colon environment of the indigestible substance after artificial digestion treatment, and the spore-forming lactic acid bacteria in the non-heat-treated control goods. It is a graph which shows the proliferation in the pseudo-distant large intestine environment of the spore-forming lactic acid bacteria in the indigestible substance after artificial digestion treatment, and the non-heat-treated control goods. 6 is a graph showing the growth of the indigestible substance and spore-forming lactic acid bacteria produced in Examples 3 to 5 in a pseudo-distal colon environment. The cecum excised from the rat fed with the indigestible substance produced in Example 2 and the indigestible substance reaching the cecum (location indicated by → in the figure) are shown.

The present invention relates to an indigestible substance comprising a step of preparing a mixture comprising dietary fiber, spore fungus and / or spore thereof, sugar, and protein or amino acid, and a step of forming melanoidin by heating the mixture. A manufacturing method is provided.

Examples of dietary fiber in the present invention include pure dietary fiber, water-soluble dietary fiber such as indigestible dextrin, water-insoluble dietary fiber such as cellulose, fruit peel, cereal bran, bran, okara, or vegetable ( A food material containing the above-mentioned dietary fiber obtained by refining or grinding a water-insoluble solid content after squeezing fruit (including moss and beans) or fruits can also be used. Many of these dietary fiber-containing food materials contain a small amount of saccharides and / or proteins derived from raw materials, and therefore are advantageous dietary fibers in the present invention in forming melanoidins described later.

The dietary fiber-containing food material that can be used in the present invention includes “Beat fiber” (http://www.nitten.co.jp/product/fiber.html, water-insoluble dietary fiber of beet) manufactured by Nippon Sugar Sugar Co., Ltd. “PURE FIBER” from Fi Nutrition Co., Ltd. (http://www.fi-nutrition.jp/product/product01_01.html, water-insoluble dietary fibers such as wheat, oats, and sugarcane) And so on "(http://3maru.co.jp/sanchemifa/q.seni.htm, water-insoluble dietary fiber of corn).

Particularly preferable dietary fiber-containing materials in the present invention include fine powdered okara containing water-insoluble dietary fiber, for example, having a particle diameter of 200 μm or less, preferably 150 μm or less. The pulverized okara having various particle sizes including the fine okara having the above particle size is already on the market, and in the present invention, a commercially available fine okara may be used. Alternatively, a normal okara that has not been pulverized is used as a raw material, a pulverization process or a crushing process known to those skilled in the art is performed, and a product prepared by sieving using a sieve of 100 mesh size or more is referred to in the present invention. You may use as fine powder Okara.

In addition, another example of the preferred dietary fiber in the present invention is cellulose or a derivative thereof. Cellulose can be used as long as it is taken orally, and cellulose powder, particularly microcrystalline cellulose, which is widely used as a food additive or a pharmaceutical excipient, is preferred. Microcrystalline cellulose is an example of a water-insoluble dietary fiber, and examples include Theolas (registered trademark) UF, ST or FD grade of Asahi Kasei Corporation, KC Flock (registered trademark) of Nippon Paper Group. Moreover, the cellulose derivative should just be ingestible orally, and hydroxypropyl methylcellulose, carboxymethylcellulose, etc. can be utilized.

Spores are generally bacteria that can form durable spores when placed in an unfavorable environment for growth, but in the present invention, they are resistant to digestion and have excellent effects on improving the intestinal environment. Because it aims to provide a substance, spore bacteria are limited to those useful for the intestinal environment. Such spore bacteria beneficial to the human intestinal environment include sporic lactic acid bacteria (reference: Yuichi Makiura, “FOOD Style 21”, 2002, 6, 9, pp. 77-80) and butyric acid bacteria. Is mentioned.

Examples of sporic lactic acid bacteria include Bacillus coagulans, Sporolactobacillus inulinus, and the like. B. Coagulance is commercially available under names such as Lacris (registered trademark) -S. In the present invention, B.I. The use of a coagulance strain is preferred. The use of the coagulance lilac-01 strain is preferred. The lilac-01 strain is described in Japanese Patent No. 5006986, and is deposited at the National Institute of Technology and Evaluation Microorganisms Deposit Number: NITE P-1102.

Moreover, Clostridium butyricum ( Clostridium butyricum ) can be mentioned as an example of a butyric acid bacterium. C. Butyricum is marketed under a name such as Miyabae.

Spores of spore bacteria useful in the intestinal environment are prepared by performing known treatments that can form spores on vegetative cells of spore bacteria, such as heating, drying, and addition of chemicals. be able to. Conveniently, it can be prepared by heating or drying a medium in which spore bacteria are grown.

Examples of the saccharide in the present invention include monosaccharides, disaccharides, oligosaccharides, and polysaccharides having a reducing end, combinations thereof, or mixtures containing them. The saccharide may be added to the mixture separately from other raw materials, or may be a saccharide derived from a dietary fiber-containing food material or a protein raw material described below.

Examples of proteins (including peptides and polypeptides) include plant proteins and / or animal proteins. In the present invention, it is particularly preferable to use cereal grains containing both saccharides and proteins or processed products thereof. Examples thereof include soybean powder, oryza protein (registered trademark, reference URL: http://www.oryza.co.jp/product/detail/oryza_protein_igai.html) manufactured by Oriza Oil Co., Ltd., or Ozeki Co., Ltd. Indigestible rice proteins such as profiber (reference URL: http://www.ozeki.co.jp/food_bio/jyozo2.html), fermented barley extract from Sanwa Sake Co., Ltd. (http: //www.b- barley extract such as fermentation.com/product/index.php?action=story & story_id = 1), "Potein" of Toyo Shinyaku Co., Ltd. (http://www.toyoshinaku.co.jp/gn It is preferred to use such -material / potein01.html) potato protein, such as the use of soybean flour or indigestible rice protein is more preferable.

The amino acid is not particularly limited as long as it is an amino acid capable of forming melanodine, or may be an amino acid contained in the dietary fiber-containing food material.

The method of the present invention includes a step of preparing a mixture by mixing the dietary fiber, spore fungus and / or spore thereof, saccharide, protein or amino acid, respectively described above. In such a process, saccharides may be prepared separately from other ingredients, but originally included when using dietary fiber-containing food materials or when using grains or processed products thereof as proteins. It may be a saccharide derived from a raw material.

Mixing is preferably performed with an appropriate amount of moisture. The moisture may be added to the mixture independently of dietary fiber, spore fungus and / or its spore, saccharide and protein or amino acid, or may contain dietary fiber, spore fungus and / or its spore, saccharide and protein or amino acid. It may be brought into the mixture as water used for wetting or a solution or suspension containing these. The amount ranges from the amount that gives moisture enough to knead the mixture of dietary fiber, spore fungus and / or spore, saccharide, and protein or amino acid without scattering the powder, and the amount from which the mixture becomes pasty. It is preferable to adjust as appropriate.

The mixing amount ratio between the saccharide and the protein or amino acid in the mixture can be set according to the number of functional groups contributing to the Maillard reaction contained therein, that is, the reducing end of the saccharide and the amino group of the protein or amino acid, For example, the ratio of sugar: protein or amino acid is appropriately adjusted within a range of about 0.1: 10 to 10: 0.1, or 1: 5 to 5: 1, or 0.5: 1 to 1: 0.5. It is preferable to do. The mixing ratio of dietary fiber to saccharide and protein or amino acid is 500: 1 to 1: 5, preferably 500: 1 to 1, with the ratio of dietary fiber: saccharide and protein or amino acid being the weight ratio excluding moisture. It is preferable to adjust appropriately within the range of: 1.

In addition, when dietary fiber-containing materials used as dietary fiber, for example, fine okara itself or cereals or processed products thereof originally contain sugars and / or proteins or amino acids, the content of sugars, proteins or amino acids for each raw material The amount of saccharide and protein or amino acid contained in the mixture may be adjusted within the above range by specifying the amount and taking these into consideration.

Difficulty in the indigestible substance finally produced by adjusting the amount of saccharide and protein or amino acid in the mixture in consideration of the content of saccharide and / or protein or amino acid contained in dietary fiber as raw material The content of digestible carbohydrates (resistant carbohydrate, abbreviated as RC) and / or indigestible proteins (resistant protein, abbreviated as RP) can be adjusted.

Probiotics, which are microorganisms that contribute to the improvement of the intestinal environment, each have a content ratio of carbohydrate (C) and protein (P) suitable for their dominant growth. It is not considered to adjust or control the content ratio (C / P ratio) of quality (C) and protein (P), particularly the ratio of RC and RP (RC / RP ratio). The method of the present invention produces an indigestible substance having a C / P ratio suitable for the growth of spore bacteria and further an RC / RP ratio by appropriately adjusting or controlling the C / P ratio in the mixture. Can do.

In addition, the number of spores and / or the number of spores contained in the mixture is determined in consideration of the preferred daily intake per adult for each spore fungus (generally said to be about 1 million to 1 billion). You just have to decide. For example, approximately 10,000 to 10 billion, preferably 100,000 to 1 billion spores and / or spores of the same number of spores per unit weight (gram) of the indigestible substance. Spores or vegetative cells of 10 to 100 times these spores may be contained in the mixture. However, the number of spores and / or spores contained in the mixture is not limited to the above range. It is possible within the scope of the methods disclosed herein to prepare a lesser or greater amount of spore bacteria and / or an indigestible material comprising the spores.

In a preferred embodiment of the step of preparing the mixture of the present invention, spore bacteria are prepared by suspending fine powdered okara and peptone or cereal flour such as soybean flour, resistant rice protein or barley extract in an appropriate amount of water. This is a step of mixing a culture solution obtained by culturing for an appropriate time.

The present invention further includes a step of forming melanoidin by heating the mixture. Melanoidin is a brown product produced by a reaction between a protein or amino acid and a reducing sugar, that is, a Maillard reaction (also referred to as an aminocarbonyl reaction), and is a reaction product that is commonly found in cooking and the like. The heating in the present invention may be performed until the mixture turns brown over an appropriate temperature and time, and special methods and conditions, and no special equipment are required for the temperature and time. Specifically, depending on the amount of the mixture and the water content, the degree of browning and the dry state are appropriately confirmed at a temperature in the range of about 80 ° C. to 150 ° C., preferably 90 ° C. to 120 ° C. for 10 minutes to 120 minutes. The mixture may be heated while heating.

Examples of the method of heating the mixture in the present invention include a method of heating the mixture while winding it with high-temperature hot air, a method of heating the mixture by exposure to infrared or radiant heat while stirring, and as necessary. Examples of the method include frying the mixture while stirring, but are not limited thereto. A preferable heat treatment method in the present invention is a method in which the mixture is fried while stirring the mixture on or in a suitable container. In particular, in the present invention, it is preferable to further perform a drying treatment with or after heating.

The melanoidin formed by heating is melanoidin formed between sugars and / or proteins or amino acids contained in dietary fiber and other components in the mixture, and when the dietary fiber itself has a reducing end. Mention may be made of melanoidins formed between the reducing end and the protein or amino acid in the mixture. In the case of this melanoidin, the dietary fiber itself is converted into merinosin. Melanoidin can also be formed between components other than dietary fiber. In the case of this melanoidin, it adheres to the dietary fiber so as to burn, and does not easily leave the dietary fiber. Thus, in the present invention, the state in which the dietary fiber itself is melanoidized or the melanoidin adheres to the dietary fiber and does not leave easily is expressed as being adhered to the dietary fiber.

In addition, the spore bacteria after heating and / or the spores are sandwiched between dietary fiber and melanoidin adhering thereto, embedded in melanoidin adhering to dietary fiber, spores and food Melanoidin is formed between the fiber and integrated with dietary fiber, or is fixed to dietary fiber as melanoidin formed between spore and saccharide and / or protein or amino acid It is guessed. In such a state, the state in which the spore bacteria and / or the spores thereof are not easily separated from the dietary fiber is expressed in the present invention as the spore bacteria and / or the spores are fixed to the dietary fiber. In addition, when the mixture before a heating contains the vegetative cell of a spore bacteria, it is guessed that many form a durable spore in the middle of a heating, and this spore adheres to a dietary fiber.

Thus, the indigestible substance produced by the method of the present invention is such that the dietary fiber itself is melanoidized by including other components and spore bacteria, or the spores and melanoidins of spore bacteria cannot be easily separated. A distinction is made between adhering to dietary fiber, i.e., spore and melanodine fixed to dietary fiber, and in a mere mixture of dietary fiber, spores of spore fungi and melanoidin.

The mixture in the production method of the present invention may contain dietary fiber, spore fungus and / or spore thereof, saccharide, and any useful substance other than protein or amino acid as long as it does not interfere with the formation of melanoidin by heating. In particular, the useful substance can be fixed to the dietary fiber by adding the useful substance to the mixture.

A typical example of a useful substance in the present invention is a so-called prebiotic that selectively promotes the growth of useful intestinal bacteria including spore bacteria. Indigestible substances produced via a mixture further containing prebiotics are useful as synbiotics containing probiotics and prebiotics simultaneously.

Prebiotics include disaccharides such as turanose, cellobiose and lactulose, panose, gentianose, melezitose, stachyose, galactooligosaccharide, fructooligosaccharide, soybean oligosaccharide, dairy oligosaccharide, xylooligosaccharide, isomaltoligosaccharide, raffinose, mannan oligo Sugar, nigerooligosaccharide, cellooligosaccharide, maltotriose, cyclodextrin, oligosaccharide such as coffee bean manno-oligosaccharide, organic acid such as gluconic acid, polydextrose, inulin, guar gum degradation product, soluble dietary fiber such as psyllium, rice starch, There may be mentioned starches such as shinshin flour, shiratama flour, corn starch, potato starch, rice flour and brown rice flour.

In addition, in place of or in addition to the prebiotics, nutritional aspects such as amino acids, vitamins, minerals, etc., provided that the formation of melanoidin and the growth of spore bacteria in the distal large intestine are not hindered. An effective substance or an effective substance generally regarded as a food functional ingredient may be added to the mixture and fixed to the dietary fiber.

The present invention further provides an indigestible substance obtained by adhering spores of spore bacteria and melanoidin to dietary fiber.

Here, dietary fiber and its particle diameter, spores of spore bacteria and the term “adherence” are as described above. Moreover, the content of dietary fiber, saccharides, protein, and spores of spore bacteria contained in the indigestible substance may be within a range determined by the use amount described in the above production method. In addition, the indigestible substance of the present invention contains various substances such as pharmacologically effective substances and nutritionally effective substances in addition to the prebiotics described above in dietary fiber in the same form as spores. As described above, it may be fixed. That is, a typical or suitable example of the indigestible substance of the present invention is an indigestible substance produced by the method of the present invention.

The indigestible substance of the present invention can be in any form such as block, granule or powder. For example, the mixture in the present invention described above may be put into a suitable mold and heated to form a lump, or after forming melanoidin, a suitable binder or binder is added and processed into a suitable mold. It is good also as a lump. Furthermore, the mixture can be heated to a granular or powdery indigestible substance by heating with stirring, or a product processed into a lump shape may be pulverized or crushed into a granular or powdery indigestible substance.

The indigestible substance of the present invention is effective in improving the intestinal environment, particularly in the distal part of the large intestine. The dietary fiber, spore-forming spore and melanoidin contained in the indigestible substance of the present invention are all resistant to degradation in the stomach internal environment and the small intestine internal environment, and thus ingested the indigestible substance. It is expected to be protected from digestion and absorption by the host itself. At the same time, the protective action by dietary fiber and melanoidin is expected to prevent the spores of spore bacteria from transforming into vegetative cells in the stomach and small intestine.

On the other hand, the decomposition of the indigestible substance of the present invention is finally started in earnest under the internal environmental conditions of the large intestine. Most of the spores of the spore bacteria fixed by this decomposition are released. In addition, the dietary fiber to which the spore has adhered and the melanoidin itself or its degradation product, which has adhered to the spore, become a germination inducer of spore bacteria and / or a nutrient source for cell growth. And the growth of spore bacteria begins.

If reasonably inferred based on the above fact, the indigestible substance of the present invention passes through the stomach and intestine of the host ingesting it almost without being decomposed and reaches the proximal part of the large intestine. As a result of continuing to undergo degradation gradually until reaching the distal end of the large intestine, the spores of spore bacteria are released throughout the large intestine, especially at the distal end where degradation proceeds, and melanodine or its degradation products are supplied as nutrients. It is expected to change the spore to a vegetative cell and promote the dominant growth of spore bacteria in the distal large intestine.

Generally, Westernized foods are low in dietary fiber and high in lipids, so that the amount of bile secretion from the host is increased, and it is said that the balance between lactic acid production and lactic acid utilization in the intestine fermentation, especially the large intestine, is likely to be lost. Especially in the distal part of the large intestine, when the growth of lactic acid bacteria is weak and the amount of lactic acid is low, the pH rises and the water content becomes low, so it becomes black and hard stool. So it tends to cause constipation. On the other hand, if lactic acid is present in excess, the pH decreases and the activity of lactic acid-assimilating bacteria decreases, leading to further accumulation of lactic acid. As a result, colonic atrophy tends to occur and diarrhea tends to occur. It is thought to cause colorectal disease.

As mentioned above, lactic acid bacteria and butyric acid bacteria are known as probiotics effective for improving the intestinal environment. However, the conventional intestinal preparations using these probiotics regard the entire large intestine as one gastrointestinal tract having the same environment, and it is not considered to maintain an optimal fermentation state for each large intestine region. In addition, butyric acid bacteria are formulated in the above-mentioned intestinal preparation in a so-called naked state, and it is not sufficient to consider the ability to reach the large intestine, particularly the distal part of the large intestine. For these reasons, the bowel movement improving effect of conventional intestinal preparations, specifically the effect of improving constipation, constipation-like or diarrhea-prone, or soft stool-like bowel movements, is not always satisfactory.

On the other hand, the indigestible substance of the present invention promotes the production of an appropriate amount of lactic acid in the intestine, particularly in the distal part of the large intestine, based on the above characteristics, and further by converting butyric acid from this lactic acid, or butyric acid itself. It is expected that peristaltic movement of the intestine is induced by promoting the generation, and appropriate defecation can be promoted. An appropriate amount of butyric acid also favors normal renewal of colonic epithelial cells and prevention of colorectal cancer.

In addition to improving the intestinal environment at the distal part of the large intestine, the indigestible substance of the present invention can stably maintain the improved environment by continuous ingestion, thereby allowing the irritable intestine to remain stable. It is expected to prevent repeated diarrhea and constipation seen in the syndrome.

The indigestible substance of the present invention may be ingested as it is, suspended in a suitable beverage such as water, fruit juice, milk, soy milk or mixed with bread, white rice, cereal or other foods, or cookies, cakes, You may take in the form of processed foods such as bread. In addition, the indigestible substance of the present invention may be used in the form of health supplements, supplements or pharmaceuticals produced as one of raw materials, and these are various raw materials and additives usually used by those skilled in the art. Other substances and the indigestible substance of the present invention can be combined in an appropriate form and produced according to a usual method.

The intake of the indigestible substance of the present invention can be determined as appropriate according to the spore bacteria and / or the number of spores contained per unit weight of the indigestible substance. In general, the preferred intake of spore bacteria is said to be 1 million to 1 billion per day for adults, and it is sufficient to consume an indigestible substance in an amount that can provide a number in this range. Preferably, considering not only the number of spores and / or the number of spores but also the effective intake of dietary fiber and melanoidin, 1 mg to 1000 g per day, preferably 0.1 g to 1000 g, more preferably 1 g to The intake amount of the indigestible substance of the present invention may be adjusted in the range of 100 g, more preferably 1 to 10 g.

The following examples further illustrate the present invention.

<Example 1>
B. Coagulance lilac-01 strain was inoculated into 100 mL (pH 7.0) of a medium containing 0.5% peptone SE 50M, 0.5% yeast extract, 0.5% glucose, 0.1% magnesium sulfate and 5 ppm manganese sulfate. Spore culture was carried out at 40 ° C. for 2 days to form spores. After centrifugation, the cells were washed once with sterile physiological saline and then suspended in sterile physiological saline. The spore suspension (200 μL) was added to a soybean powder suspension obtained by adding 100 mL of distilled water to 5 g of soybean powder (Minami Sangyo Co., Ltd.), and 200 g of fine powder Okara (Kikkoman Beverage Co., Ltd.) was added and mixed. The indigestible substance of the present invention was produced by heating and drying 100 g of this mixture in a frying pan for 8 minutes. The number of spores of the lilac-01 strain per 1 g of the indigestible substance was 4.0 × 10 ² .

<Example 2>
B. The coagulance lilac-01 strain was inoculated into 4 L of soy milk (Kikkoman Beverage Co., Ltd.) and cultured with shaking at 55 ° C. for 24 hours. After the cultivation, 4 kg of fine powder Okara (Kikkoman Beverage Co., Ltd.) was added and mixed. 10 g of this mixture was taken as a control product, and the remaining 4 kg was transferred to a kneader (manufactured by Samsung Corp.), followed by heating and drying at 120 ° C. for 20 minutes. After drying, it is passed through a sieve of analytical sieve (mesh No. 24, stitch size is 0.71 mm), and the portion not passed (grain size is 1-2 mm) is recovered to make the indigestible of the present invention It was considered as a sex substance. The number of spores of the lilac-01 strain per gram of the indigestible substance was 1.9 × 10 ⁸ . Moreover, the micrographs of the fine powder okara which is a part of the raw material and the indigestible substance produced in Example 2 are shown in FIG. 1 and FIG. It can be seen that the appearance of fine powdered okara, which is a water-insoluble dietary fiber, is changed by heat treatment, and browned to produce melanoidin.

<Test Example 1>
1) Test in simulated proximal colon environment model The indigestible substance produced in Example 1 was crushed with a lab miller (LAB CAT) and processed into a powder form. Spores of lilac-01 strain (1 ml of medium) suspended in physiological saline as a control in medium (pH 6.5) containing peptone SE 50M 0.25%, yeast extract 0.1% and magnesium sulfate 0.1% And 20 g of the above powdery indigestible substance (containing 20 spores per 1 ml of medium), and anaerobically cultivated at 37 ° C. Number was measured. The result is shown in FIG.

In the control, the growth of the lilac-01 strain reaches the logarithmic growth phase 15 hours after the start of the culture and reaches the equilibrium phase after 24 hours, whereas in the examples, it germinates and grows later than the control. confirmed.

2) Test in simulated distal colon environment model Spores of lilac-01 strain suspended in 10 mL of 0.85% sterile physiological saline (pH 7.0) and physiological saline as a control (20 per 1 mL of physiological saline) ) And 0.5 g of the above powdery indigestible substance (containing 20 spores per 1 ml of physiological saline), anaerobically cultured at 37 ° C., and the number of living bacteria of lilac-01 over time Was measured. The result is shown in FIG.

In this medium, the control spore did not germinate, and the growth of the fungus was not confirmed. On the other hand, in the examples, a growth curve as shown in FIG. 4 was shown. From this, it was confirmed that the spores of spore-forming lactic acid bacteria included in the Examples germinate in the pseudo-distal colon environment and can grow using the components contained in the indigestible substance as a nutrient source.

3) Resistance test against artificial digestion treatment About the indigestible substance produced in Example 1 and Example 2 and the control product not subjected to the heat treatment of each Example, Honma et al. (Shokugaku, 2008, Volume 55, pages 18-24), the resistance to artificial digestion was evaluated.

0.88 g of a control product having the same solid content in 10 mL / conical tube of 20 mM phosphate buffer (pH 6.9) containing 10 mM sodium chloride (the water contents of Examples 1 and 2 are 49.3% and 46.46, respectively). 8%) and 0.5 g of indigestible substance (the water contents of Examples 1 and 2 were 10.7% and 6.2%, respectively), pre-warmed at 37 ° C. for 15 minutes, and then 25 μL of α Amylase (Sigma catalog number A-0521, 1 mg / mL) was added and allowed to react at 37 ° C. for 15 minutes. Next, 1M hydrochloric acid was added to adjust the pH to 2.5, 62.5 μL of pepsin (Sigma catalog number P-7012, 0.5 mg / mL) was further added, and the mixture was reacted at 37 ° C. for 30 minutes. Furthermore, after adjusting to pH 7.1 by adding 1 M sodium hydroxide, 125 μL of pancreatin (Sigma catalog number P-7545, 20 mM phosphate buffer containing 0.5 mg / mL of 10 mM sodium chloride, pH 6.9) ) Was added and reacted at 37 ° C. for 90 minutes.

A control product obtained by artificial digestion treatment and further washed once with physiological saline in 10 mL of anaerobic culture medium (medium obtained by diluting GAM medium of Nissui Pharmaceutical Co., Ltd. 4 times and excluding agar) and the indigestibility of Example 1 The substances were added so that the number of spores was 20 per 1 ml of the medium, respectively, and anaerobically cultured at 37 ° C., and the number of living bacteria of the lilac-01 strain was examined over time. This culture is for confirming the proliferation ability in the pseudo proximal colon environment. The result is shown in FIG.

In addition, the control product and the digestible substance of Example 2 which were further washed with physiological saline once after artificial digestion treatment in 5 mL of 0.85% sterilized physiological saline, each had 3 × 10 spores per 1 ml of physiological saline. ^In addition to ⁶ cells, anaerobic culture was performed at 37 ° C., and the viable count of lilac-01 strain was examined over time. This culture is for confirming the proliferative ability in the pseudo-distal colon environment. The result is shown in FIG.

From the results of the above resistance test against artificial digestion treatment, the indigestible substance of the present invention reaches the large intestine and germinates later in the pseudo proximal colon environment than in the control product, and in the pseudo distal colon environment than in the control product. It was confirmed that the growth rate was large.

<Example 3>
B. The coagulance lilac-01 strain was inoculated into 100 mL (pH 7.0) of a medium containing 0.5% peptone, 0.5% yeast extract, 0.5% glucose, 0.1% magnesium sulfate and 5 ppm manganese sulfate. And cultured for 2 days with shaking to form spores. After centrifugation, the cells were washed once with sterile physiological saline and then suspended in sterile physiological saline. The previous spore suspension (20 μL) was added to 30 mL of 5% sterilized soybean powder suspension, and 15 g of finely divided cellulose (Asahi Kasei Chemicals Corporation) was added and mixed. This mixture was heated and dried in a frying pan for 5 minutes, passed through an analytical sieve (mesh No. 24, the size of the stitch was 0.71 mm), and the portion that did not pass through (the size of the grain was 1-2 mm). By recovering, the indigestible substance of the present invention was produced. The number of spores of lilac-01 strain per gram of the indigestible substance was 2.1 × 10 ² .

<Example 4>
B. The coagulance lilac-01 strain was inoculated into 100 mL (pH 7.0) of a medium containing 0.5% peptone, 0.5% yeast extract, 0.5% glucose, 0.1% magnesium sulfate and 5 ppm manganese sulfate. And cultured for 2 days with shaking to form spores. After centrifugation, the cells were washed once with sterile physiological saline and then suspended in sterile physiological saline. The spore suspension (20 μL) was added to a solution obtained by adding 20 mL of distilled water to 2.0 g of peptone and 11.0 g of honey, and further 15 g of finely divided cellulose (Asahi Kasei Chemicals Corporation) was added and mixed. This mixture was heated and dried in a frying pan for 5 minutes, passed through an analytical sieve (mesh No. 24, the size of the stitch was 0.71 mm), and the portion that did not pass through (the size of the grain was 1-2 mm). By recovering, the indigestible substance of the present invention was produced. The number of spores of lilac-01 strain per gram of the indigestible substance was 1.3 × 10 ² .

<Example 5>
B. The coagulance lilac-01 strain was inoculated into 100 mL (pH 7.0) of a medium containing 0.5% peptone, 0.5% yeast extract, 0.5% glucose, 0.1% magnesium sulfate and 5 ppm manganese sulfate. And cultured for 2 days with shaking to form spores. After centrifugation, the cells were washed once with sterile physiological saline and then suspended in sterile physiological saline. The spore suspension (20 μL) was added to 30 mL of a solution containing 0.5% peptone and 0.5% glucose, and 15 g of sodium carboxymethyl cellulose (Nippon Paper Industries Co., Ltd.) was added and mixed. This mixture was heated and dried in a frying pan for 5 minutes, passed through an analytical sieve (mesh No. 24, the size of the stitch was 0.71 mm), and the portion that did not pass through (the size of the grain was 1-2 mm). By recovering, the indigestible substance of the present invention was produced. The number of spores of lilac-01 strain per gram of the indigestible substance was 2.1 × 10 ² .

<Test Example 2>
2 of Test Example 1) 0.5 g of indigestible substance produced in Examples 3 to 5 (including 13 to 20 spores per 1 ml of physiological saline) according to the test in the pseudo-distal colon environment model As a control, spore of lilac-01 strain (400 per 1 ml of physiological saline) suspended in physiological saline was added to 5 mL of 0.85% sterile physiological saline (pH 7.0), followed by anaerobic culture at 37 ° C. The viable count of lilac-01 strain was measured over time. The result is shown in FIG.

In the physiological saline, the control spore did not germinate and the growth of lilac-01 strain was not confirmed. On the other hand, regarding Examples 3 to 5, the lilac-01 strain exhibited a growth curve as shown in FIG. Therefore, spores of spore-forming lactic acid bacteria contained in the indigestible substances produced in Examples 3 to 5 germinate in the pseudo-distal colon environment and proliferate using the ingredients contained in the indigestible substances as nutrients. Confirmed that you can.

<Test Example 3>
WKAH / HkmSlc rats (5-week-old male, approximately 120 g body weight) were preliminarily raised, and then a feed mixed with the indigestible substance produced in Example 2 (5 × 10 ⁶ spore bacteria per gram of feed) was freely ingested. However, it was raised for 2 weeks. After breeding, the animals were dissected and the cecum was removed and the contents were observed (FIG. 8). As a result, it was confirmed that a number of indigestible substances (locations indicated by → in the figure) reached the cecum without being digested. In addition, after diluting a lump of indigestible substance collected from the cecum with sterile physiological saline, it is mixed-cultured on a standard agar medium (55 ° C. for 2 days), and the number of viable bacteria and spores contained therein As a result, the number of viable bacteria was 1.5 × 10 ⁴ /0.01 g of indigestible substance, and the number of spores was 1.1 × 10 ⁴ /0.01 indigestible substance. From these results, it was confirmed that many of the lilac-01 strains germinated and changed into viable bacteria in the rat cecum.

<Test Example 4>
A domestic cat (16 years old, male, weighing about 4 kg) was administered the indigestible substance produced in Example 2 (1 × 10 ⁷ spores per day). After feeding for one week, the color of feces was compared before and after ingestion of indigestible substances. Feces before ingestion were dark brown, whereas feces after ingestion turned yellow. From this, it was confirmed that the indigestible substance of the present invention improves the intestinal environment of the domestic cat.

<Test Example 5>
SD-type Slc rats (5-week-old male, approximately 120 g body weight) were preliminarily raised and divided into 2 groups. The control group contained only sporic lactic acid bacteria (1 × 10 ⁶ spore bacteria per gram of feed). Were reared for 2 weeks while freely ingesting the feed containing the indigestible substance produced in Example 2 (1 × 10 ⁶ spores per gram of feed). The cecum was dissected after breeding and the number of viable spore-forming lactic acid bacteria in the cecal contents was smeared on a standard agar medium (Nissui Pharmaceutical) after dilution and measured after aerobic culture at 55 ° C for 1 day. . The number of spores of spore-forming lactic acid bacteria was measured by taking a 10-fold diluted solution in a microtube, heating at 90 ° C. for 10 minutes, and then culturing in the same manner. When the germination rate was calculated from these values, it was confirmed that the germination rate of the control group was 6% and that of the test group was 29%.

<Test Example 6>
WKAH / HkmSlc rats (5-week-old male, body weight: about 120 g) were preliminarily raised, and then freely fed with a diet containing indigestible substances produced in Example 2 (5 × 10 ⁶ spores per gram of feed). Reared for 2 weeks. The cecum was dissected after breeding and the cecal contents were taken out, and the concentration of short-chain fatty acids in the cecal contents was measured with an organic acid measurement HPLC (LC-10ADvp; Shimadzu Corporation). As a result, as shown in Table 1, in the test group, acetic acid, propionic acid and butyric acid increased significantly compared to the control group which did not ingest the indigestible substance or ingested only the spore-forming lactic acid bacteria. It was shown that.

In general, short-chain fatty acids are known to improve the intestinal environment and have therapeutic effects on inflammatory bowel diseases such as ulcerative colitis. Butyrate is particularly useful for the treatment of irritable bowel syndrome. It is known to be effective (eg, Zateski et al., Przegrad Gastroenterology, 2013, Vol. 8, pages 350-353). Therefore, it is considered that the indigestible substance of the present invention is also effective as a therapeutic agent for intestinal regulating agent, inflammatory bowel disease or irritable bowel syndrome.

INDUSTRIAL APPLICABILITY The present invention is useful for improving the intestinal environment, particularly the intestinal environment in the distal part of the large intestine, and can promote proper defecation, as well as prevent repeated diarrhea and constipation seen in irritable bowel syndrome. As an intestinal regulating agent and a method for producing the same, it has industrial applicability.

Claims

Preparing a mixture comprising dietary fiber, spore fungi and / or spores, sugars and proteins or amino acids;
And a step of forming melanoidin by heating the mixture.
The method according to claim 1, wherein the dietary fiber is fine powdered okara having a particle diameter of 200 µm or less, cellulose, or a derivative thereof.
The method according to claim 1 or 2, wherein the saccharide and the protein or amino acid are cereal grains or a processed product thereof.
4. The method according to claim 3, wherein the processed grain product is soybean flour, rice flour or barley extract.
The method according to any one of claims 1 to 4, wherein the mixture further comprises prebiotics.
Indigestible substance produced by the method according to any one of claims 1 to 5.
Indigestible substance in which spores of spore bacteria and melanoidin adhere to dietary fiber.
The indigestible substance according to claim 7, wherein the dietary fiber is fine powdered okara having a particle diameter of 200 μm or less, cellulose, or a derivative thereof.
The indigestible substance according to claim 7 or 8, further comprising prebiotics.
A therapeutic agent for irritable bowel syndrome comprising the indigestible substance according to any one of claims 6 to 9 as an active ingredient.