JP7430312B2 - Composition for promoting intestinal short-chain fatty acid production and food and beverages containing the same - Google Patents

Description

NPMD NITE BP-03259

The present invention relates to a functional material that has the effect of promoting the production of short-chain fatty acids, particularly acetic acid , propionic acid, butyric acid, etc., in the intestine.

The villi of the large intestine are covered with epithelial cells, and crypts are present at the base of the villi. Inside the crypts are mucosal secretory cells, which constantly produce mucus that coats the epithelial cells, protects the mucous membrane, and facilitates the movement of feces. Recent studies have revealed that the presence of butyric acid is essential for the formation of the mucosal layer of the large intestine.

Short-chain fatty acids produced by intestinal bacteria include acetic acid, propionic acid, and butyric acid, and these organic acids are absorbed in the large intestine and serve as an important energy source for the host. In the case of pigs, 30% of the energy absorbed is due to these organic acids. Substances absorbed from the large intestine are transported to the liver through the portal vein, but when organic acids in portal blood are analyzed, only acetic acid and propionic acid are detected. Since only acetic acid is detected in peripheral blood, it is thought that propionic acid is used for gluconeogenesis in the liver. On the other hand, butyric acid is immediately utilized as an energy source for the regeneration of the colonic mucosa. In addition to being used as an energy source for the proliferation of mucosal epithelial cells, it has also been reported to promote the division of epithelial cells, contribute to mucosal thickening, and further promote colonic motility (Non-patent Document 1, (See 2, 3).

On the other hand, focusing on the important role of such short chain fatty acids, Patent Document 1 discloses that bacterial cells of Lactobacillus acidophilus and/or Bifidobacterium longum are used as an active ingredient. The invention of an agent for promoting increase in intestinal butyrate concentration is disclosed.

Moreover, Patent Document 2 discloses an invention of an intestinal butyric acid production promoter characterized by containing as an active ingredient killed bacterial cells isolated from the intestines of animals.

Shimotoyodome, A. , Meguro,S. , Hase, T. , Tokimitsu, I. , Sakata, T. (2000) Short chain fatty acids but not lactate or succinate stimulate mucus release in the rat colon. Comp. Biochem. Physiol. 125(4):pp. 525-531. Sakata, T. (1997) Influence of short-chain fatty acids on experimental growth and functions. In: Dietary Fiber in Health and disease (Kritchevsky, D. and Bonfield, C. eds.), pp. 191-199. Plenum Press, New York, NY. Yajima, T. (1995) Sensory mechanisms for short-chain fatty acids in the colon. pp. 209-221. In: Physiological and clinical aspects of short-chain fatty acids. (Cummings, J.H., Rombeau, J.L. and Skata, T. eds.), Cambridge University Press, Cambridge, UK.

Japanese Patent Application Publication No. 10-84909 Japanese Patent Application Publication No. 2004-346043

However, in Patent Document 1, it is described that when rats are fed feed containing bacterial cells of Lactobacillus acidophilus and Bifidobacterium longum, the concentration of butyrate in the caecum of rats increases; It is not clear about the short chain fatty acids of

In addition, Patent Document 2 describes that when pigs are fed Enterococcus faecalis bacteria in feed, the concentration of butyric acid in the caecum increases compared to the control; It is stated that no significant change was obtained with respect to acids, and no change over time in the total short-chain fatty acid concentration was observed (paragraph 0046 of Patent Document 2).

In view of the prior art, an object of the present invention is to provide a new functional material that has the effect of promoting the production of short chain fatty acids, particularly acetic acid , propionic acid, butyric acid, etc. in the intestine.

In order to achieve the above object, the present inventors conducted various studies and discovered that Cremolys bacteria, known as lactic acid bacteria derived from Caspian Sea yogurt, have the effect of promoting the production of short chain fatty acids in the intestine. The invention was completed.

That is, the present invention provides a composition for promoting intestinal short-chain fatty acid production, which is characterized by containing Cremoris bacteria.

As the cremoris bacteria, Lactococcus lactis subsp. Cremoris Flora aid strain (receipt number: NITE B P-03259) is preferred.

The composition for promoting intestinal short-chain fatty acid production according to the present invention can be used to increase the amount of intestinal production of at least one or more selected from the group consisting of acetic acid, propionic acid, and butyric acid. I can do it.

The composition for promoting intestinal short-chain fatty acid production according to the present invention can be used as a food or drink, a material for adding food or drink, a pharmaceutical, a material for adding to medicine, an animal feed, or a material for adding to animal feed.

In another aspect, the present invention can provide a food or drink containing the composition for promoting intestinal short chain fatty acid production.

According to the present invention, it is possible to provide a new functional material that has the effect of promoting the production of short-chain fatty acids, particularly acetic acid , propionic acid, butyric acid, etc. in the intestines, using Cremoris bacteria.

1 is a chart showing the results of measuring the short chain fatty acid concentration in a rat cecal content sample treated with Cremoris bacteria in Test Example 1. 2 is a chart showing the results of measuring the concentration of short chain fatty acids in a rat cecal content sample treated with a bacterial composition commonly used in yogurt in Test Example 2.

The Cremoris bacterium used in the present invention is known as a lactic acid bacterium derived from Caspian Sea yogurt, and its scientific name is Lactococcus lactis subsp. cremoris. For example, Lactococcus lactis subsp., which has been deposited at the National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary (NPMD). cremoris Flora aid strain (receipt number: NITE B P-03259).

Cultivation of cremoris bacteria, maintenance of bacterial cells, etc. can be performed by various techniques. For example, a medium suitable for culturing lactic acid bacteria includes a skim milk powder medium, or a liquid medium containing yeast extract, peptone, meat extract, salts, minerals, and the like. Commercially available media include "MRS Broth MERCK" (trade name, Chemicals Inc.), "Difco Lactobacilli MRS Broth" (trade name, Nippon Becton Dickinson Co., Ltd.), and such commercially available media may be used.

In general, lactic acid bacteria can be cultured by, for example, standing still, although there are no restrictions. Furthermore, culture (neutralized culture) may be performed while adjusting the pH by adding an alkaline agent to the medium so as to suppress a decrease in pH due to metabolic products (lactic acid, etc.) of lactic acid bacteria. In that case, as the alkaline agent to be added, for example, an aqueous solution of sodium hydroxide, potassium hydroxide, calcium hydroxide, etc., ammonia, etc. can be used. The pH during culture may be in the range of pH 5.0 to 7.5, or may be in the range of pH 6.0 to 7.0, and is preferably adjusted and maintained at that pH. Although pH adjustment may be performed manually, it is easier and more accurate to use an automatic pH controller (pH stat).

In the present invention, cremoris bacteria may be prepared from a culture solution to prepare a cell concentrate in which cremoris bacteria is concentrated, or preferably, an excipient may be added thereto and then freeze-dried. A bacterial cell concentrate can be prepared by concentrating the culture solution as it is, but it is preferable to collect the bacteria by means such as centrifugation or filtration, and then wash the bacterial cells with purified water or the like to obtain the desired bacteria. It can be prepared by suspending it in purified water etc. so that it has a body concentration. The content of Cremoris bacteria in 100% by mass of the bacterial cell concentrate may be in the range of 0.1 to 30% by mass in terms of dry bacterial cells, and may be in the range of 0.5 to 20% by mass. It may well range from 1 to 10% by weight.

Further, the bacterial cell concentrate may contain an excipient. According to this, even after freezing or freeze-drying, the properties as a living bacterium are easily maintained after being reconstituted with water. Furthermore, in another embodiment, when the bacterial cells are crushed and dispersed, reagglomeration of the obtained lactic acid bacteria powder can be prevented. The content of the excipient may be in the range of 10 to 99% by weight, may be in the range of 20 to 95% by weight, or may be in the range of 50 to 90% by weight in terms of dry matter.

Excipients are not particularly limited and include, for example, dextrin; maltodextrin; xanthan gum; sugar alcohols such as lactitol, maltitol, mannitol, sorbitol, xylitol; sugars such as dextrose, fructose, glucose, lactose, and sucrose; Examples include organic acids such as adipic acid, citric acid, fumaric acid, glutaric acid, malic acid, succinic acid, and tartaric acid.

In the present invention, cremoris bacteria may be subjected to pulverization and dispersion treatment. For example, the above-mentioned bacterial cell concentrate can be pulverized and dispersed using means such as stirring, a mixer, a homogenizer, a ball mill, a bead mill, a jet mill, and a generator.

As the cremoris bacteria used in the present invention, living bacteria may be used. In that case, it is preferable to use a powder prepared by adding the above-mentioned excipients and freeze-drying the mixture after culturing. According to this, it is convenient to provide the cremoris bacteria alive in the form of capsules, granules, yogurt, etc.

On the other hand, killed cells of Cremoris bacteria may be used. For example, the bacterial cell concentrate can be subjected to heat treatment before or after the above-described process of crushing and dispersing the bacterial cell concentrate. Furthermore, after the above-described process of crushing and dispersing the bacterial cell concentrate, it can be made into a dry powder. Examples of dry powdering methods include freeze drying, reduced pressure spray drying, and spray drying using hot air. Note that by performing spray drying using hot air, lactic acid bacteria are usually destroyed and dead bacteria can be obtained.

The composition for promoting intestinal short-chain fatty acid production according to the present invention uses Cremoris bacterium or its processed product, which can be prepared as described above, as an active ingredient, and is administered to humans or non-human animals. This promotes the production of short-chain fatty acids in the intestines. The administration method is not particularly limited, but for example, oral administration is preferable. Specifically, short-chain fatty acids in the intestine include acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, lactic acid, and succinic acid. The composition for promoting intestinal short chain fatty acid production of the present invention particularly promotes the production of acetic acid, propionic acid, and/or butyric acid by bacterial flora in the large intestine. It is thought to increase the concentration.

The composition for promoting intestinal short-chain fatty acid production according to the present invention increases short-chain fatty acids in the intestine, and specifically promotes absorption of minerals by acetic acid and propionic acid, and increases colonic absorption by butyric acid. The effect of suppressing the onset of cancer can be obtained (see Reference 1 below). In addition, for example, by improving the intestinal barrier function and acting on regulatory T cells (Treg) in the intestinal mucosa, it is possible to suppress excessive immunity and alleviate allergic symptoms and infectious enteritis symptoms. (See Reference 2 below). In addition, energy consumption can be increased, for example, through GPR41, which is expressed in intestinal L cells and is known as a short-chain fatty acid receptor (see Reference 2 below). In addition, fat accumulation can be suppressed, for example, through GPR43, which is also expressed in intestinal L cells and is known as a receptor for short chain fatty acids (see Reference 2 below).

・Reference 1: Hiroshi Hara, "Physiological effects of short chain fatty acids produced in the large intestine from prebiotics", Journal of Enteric Bacteriology (2002) 16: pp. 35-42
・Reference 2: Hidenori Shimizu, Ryuji Ohue-Kitano, Ikuo Kimura “Regulation of host energy metabolism by gut microbiota-derived short-chain fatty acids” Glycative Stress Research 2019; 6 (3): 181-191

In another aspect of the present invention, the composition for promoting intestinal short chain fatty acid production according to the present invention also suppresses the proportion of microorganisms belonging to the genus Firmicutes among intestinal bacteria, for example. I can do it. Alternatively, for example, the proportion of microorganisms belonging to the genus Bacteroidetes among intestinal bacteria can be increased. This can reduce the Firmicutes/Bacteroidetes ratio and balance the intestinal flora.

The dosage of the composition for promoting intestinal short-chain fatty acid production according to the present invention is determined depending on the subject's health condition and age, or how much of the effect of promoting intestinal short-chain fatty acid production is required. , may be set appropriately. Typically, the intake amount in terms of dry matter of the cells of the Cremoris bacterium or its processed product may be in the range of 0.001 mg to 100 mg/day/kg body weight, and 0.01 mg to 20 mg/day. /kg body weight and may range from 0.1 mg to 10 mg/day/kg body weight.

The composition for promoting intestinal short-chain fatty acid production according to the present invention may contain cells of microorganisms other than those derived from Cremoris bacteria or processed products thereof, as long as the effects of the present invention are not inhibited. You can stay there. Examples include lactic acid bacteria other than cremolys bacteria, bifidobacteria, Bacillus subtilis, butyric acid bacteria, yeast, Aspergillus aspergillus, actinomycetes, and the like.

The composition for promoting intestinal short chain fatty acid production according to the present invention may be provided as a food or drink for promoting intestinal short chain fatty acid production. That is, the cells of the Cremoris bacterium or the processed product thereof may be used as is or in combination with other raw materials for food and drink to form a food and drink. Examples of raw materials for foods and drinks to be combined with the cremoris cells or processed products thereof include various carbohydrates, emulsifiers, sweeteners, acidulants, fruit juices, flavors, and the like. More specifically, examples include sugars such as glucose, sucrose, fructose, and honey, sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, and palatinit, emulsifiers such as sucrose fatty acid ester, glycerin sugar fatty acid ester, and lecithin. Other examples include various vitamins such as vitamin A, vitamin B, vitamin C, and vitamin E, herbal extracts, grain components, vegetable components, milk components, and the like.

Examples of the food and drink include, but are not limited to, cookies, rice crackers, jelly, candies, yogurt, confectionery such as steamed buns, soft drinks, nutritional drinks, soups, and the like. Other examples of food and beverages include health foods for promoting intestinal short-chain fatty acid production, supplements, foods for specified health uses, and foods with functional claims, such as tablets, granules, powders, capsules, and drinks. , may be provided in the form of jelly, etc.

On the other hand, the composition for promoting intestinal short-chain fatty acid production according to the present invention can be used as an additive to foods and drinks for promoting intestinal short-chain fatty acid production, which is used as an additive to foods and drinks as described above. may be done.

The composition for promoting intestinal short chain fatty acid production according to the present invention may be provided as a pharmaceutical for promoting intestinal short chain fatty acid production. That is, the cells of the Cremoris bacterium or a processed product thereof may be used as is or in combination with other pharmaceutical raw materials to form a pharmaceutical composition. There are no particular restrictions on other pharmaceutical raw materials to be combined with the Cremoris cells or the processed product thereof, and if necessary, pharmaceutically acceptable base materials and carriers may be added and the preparation may be carried out by a known formulation method, for example. It can be used as an oral preparation in the form of a tablet, granule, capsule, pill, powder, liquid, powder, jelly, lozenge, etc.

On the other hand, the composition for promoting intestinal short chain fatty acid production according to the present invention may be used as a pharmaceutical additive material for promoting intestinal short chain fatty acid production, which is used as an additive to such pharmaceuticals. .

The composition for promoting intestinal short chain fatty acid production according to the present invention may be an animal feed for promoting intestinal short chain fatty acid production. That is, for example, the cells of the Cremoris bacterium or a processed product thereof may be used as is or in combination with other animal feed materials to form a composition for animal feed. For example, it may be used as feed for animals such as livestock, racehorses, ornamental animals, and pets.

On the other hand, the composition for promoting intestinal short chain fatty acid production according to the present invention is an animal feed additive material for promoting intestinal short chain fatty acid production, which is used as an additive to such animal feed. Good too.

In the composition for promoting intestinal short-chain fatty acid production according to the present invention, the content of the above-mentioned microbial cells or the processed product thereof determines the relationship between the amount used and the effective dose when it is in various forms. It may be decided as appropriate after consideration. Typically, the content of the above-mentioned bacterial cells or their processed product on a dry matter basis may be in the range of 0.1 to 100% by mass, may be in the range of 1 to 50% by mass, and may be in the range of 10 to 100% by mass. It may be in the range of 30% by weight. Further, the content converted to the number of bacterial cells may be in the range of 1×10 ⁵ to 1×10 ⁸ cfu/g, and may be in the range of 1×10 ⁶ to 5×10 ⁷ cfu/g. , 1×10 ⁷ to 3.3×10 ⁷ cfu/g.

The present invention will be specifically described below with reference to Examples, but these Examples do not limit the scope of the present invention.

<Test Example 1>
After diluting the contents of the rat cecum 5 times with PBS (-), Lactococcus lactis subsp. cremoris Flora aid strain (receipt number: NITE B P-03259) was added to a final concentration of 1 x 10 ⁷ cfu/mL, and the concentration of short chain fatty acids (acetic acid, propionic acid, butyric acid) was measured after culturing in an anaerobic environment for 10 hours. Short-chain fatty acids were measured by pre-treating the sample and then performing HPLC analysis.

(Preprocessing)
0.3 mL of the contents of the cecum was collected, suspended in 0.6 mL of distilled water, 0.09 mL of 12% perchloric acid was added, and the suspension was thoroughly suspended and allowed to stand on ice for 10 minutes. After precipitating the protein by centrifugation, the supernatant was filtered through a 0.45 μm filter and degassed under reduced pressure to remove carbon dioxide gas, and 5 μL of the sample was used for analysis.

(HPLC analysis)
・Shim-Pack SCR-102(H) (H-type sulfo group, 7 μm, 300 mm x 8 mm, Shimadzu Corporation)
・Column temperature: 45℃
・Mobile phase: Distilled water for HPLC containing 5mM p-toluenesulfonic acid ・Post-column reaction phase: Distilled water for HPLC containing 5mM p-toluenesulfonic acid, 20mM Bis-tris, 100 μM EDTA ・Detector: Waters 431 conductivity meter ( Detection base voltage: 2000mV, detection sensitivity: 0.01)
・System controller: CBM-20A (Shimadzu Corporation)
・Component identification: CBM-20A built-in data module

As a result, as shown in Table 1 and FIG. 1, the amount of short-chain fatty acids was increased in the rat cecal contents sample treated with Cremolys bacteria compared to the control without addition.

<Test Example 2>
Regarding a complex bacterial composition (containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) commonly used in yogurt, a 5-fold dilution of rat cecal contents with PBS (-) has a final concentration of 1 x 10 ⁷ cfu/mL. The test was carried out in the same manner as in Test Example 1.

As a result, as shown in Table 2 and Figure 2, there was no increase in the amount of short chain fatty acids in rat cecal contents samples treated with the bacterial composition used in regular yogurt compared to the control without the addition. .

Claims (3)

A composition for promoting intestinal short-chain fatty acid production characterized by containing cremoris bacteria as an active ingredient ( excluding those containing galactomannan as an active ingredient),
The cremoris bacterium is Lactococcus lactis subsp. Cremoris Flora aid strain (receipt number: NITE BP-03259), a composition for promoting intestinal short chain fatty acid production . The composition for promoting intestinal short-chain fatty acid production according to claim 1 , which is used to increase the amount of intestinal production of at least one or more selected from the group consisting of acetic acid, propionic acid, and butyric acid. thing. The composition for promoting intestinal short-chain fatty acid production according to claim 1 or 2 , which is a food or drink, a food or drink additive material, a pharmaceutical, a drug additive material, an animal feed, or an animal feed additive material.