JP7792086B2 - Food composition for improving intestinal environment - Google Patents

Description

IPOD FERM BP-22254

This disclosure relates to a food composition for intestinal regulation.

The so-called intestinal regulating effect is achieved in the following way: When dietary fiber ferments in the large intestine, short-chain fatty acids (SCFA) such as butyric acid and lactic acid increase in the large intestine, creating an acidic environment within the large intestine. An acidic environment inhibits the growth of harmful bacteria. Butyric acid also stimulates peristalsis and water absorption in the large intestine. As a result, the intestinal environment improves, bringing about an intestinal regulating effect.

In addition, some beneficial bacteria present in the large intestine act directly on the intestinal immune system of the large intestine to improve immunity. Beneficial bacteria include lactic acid bacteria and some clostridium bacteria.
Dietary fiber has an intestinal regulating effect. Enzymes such as lactic acid bacteria are believed to promote the fermentation of dietary fiber. Products that allow the intake of dietary fiber and enzymes have been developed (Patent Document 1). In addition, products that allow the intake of bacteria that act on SCFA production and intestinal immunity have been developed (Patent Document 2).

Japanese Patent Application Laid-Open No. 2001-299276 Special Publication No. 2018-532758

The ability to break down dietary fiber is determined by the intestinal bacteria present in the large intestine. Therefore, once the intake of dietary fiber exceeds a certain level, the intestinal regulating effect will plateau even if the intake is further increased. Furthermore, when bacteria that affect SCFA production or intestinal immunity are orally administered, most of the bacteria are broken down in the stomach or small intestine before reaching the large intestine. Therefore, even if bacteria that affect SCFA production or intestinal immunity are orally administered, it is difficult for them to have an intestinal regulating effect. Furthermore, intestinal flora varies greatly from person to person, and the effects expected from measures described in conventional technology also vary greatly from person to person, making them limited. In one aspect of the present disclosure, it is preferable to provide a food composition for intestinal regulation that has a high intestinal regulating effect.

One aspect of the present disclosure is a food composition for intestinal regulation containing a substance derived from the microalga Coccomyxa as an active ingredient. The food composition for intestinal regulation that is one aspect of the present disclosure has a high intestinal regulation effect.

1 is a graph showing the results of a principal component analysis of all data on the intestinal bacteria of pigs obtained from the experimental and control groups before and after the feeding test. 1 is a graph showing the average value of principal component 1 obtained by principal component analysis of the intestinal bacteria of pigs obtained from the experimental group and the control group before and after the feeding test. FIG. 1 is an explanatory diagram showing a network of 53 genera of bacteria that decompose or ferment dietary fiber. This is a graph obtained by plotting the value of principal component 1 and the 16S rDNA copy number ratio of Fibrobacter . 1 is a graph showing the results of analyzing short-chain fatty acids in samples obtained from the experimental group and the control group after the feeding test. FIG. 1 is an explanatory diagram showing bacterial groups with a correlation coefficient of 0.6 or higher with the content of each short-chain fatty acid in feces. 1 is a graph showing the results of analyzing decay products in samples obtained from the experimental group and the control group after the feeding test. FIG. 1 is an explanatory diagram showing bacterial groups that are negatively correlated with the content of putrefactive products in feces. This is a graph showing the results of the 16S rDNA copy number ratio of bacteria of the family Veillonellaceae in samples obtained from the experimental and control groups after the feeding test. 1 is a graph showing the results of the 16S rDNA copy number ratio of Clostridium XIVa bacteria in samples obtained from the experimental group and the control group after the feeding test. This is a graph showing the results of the 16S rDNA copy number ratio of Veillonellaceae bacteria contained in feces before and after ingestion of the Coccomyxa KJ strain in humans in a human ingestion test. This is a graph showing the results of the 16S rDNA copy number ratio of Clostridium XIVa bacteria contained in feces before and after ingestion of the Coccomyxa KJ strain in humans in a feeding test.

Exemplary embodiments of the present disclosure will be described with reference to the drawings.
1. Food Composition for Intestinal Regulation The food composition for intestinal regulation of the present disclosure contains a substance derived from the microalga Coccomyxa as an active ingredient. The substance derived from the microalga Coccomyxa is all or part of the components of the microalga Coccomyxa. The substance derived from the microalga Coccomyxa is, for example, a substance derived from the Coccomyxa KJ strain. The Coccomyxa KJ strain was deposited on June 4, 2013, with the National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under accession number FERM P-22254. The Coccomyxa KJ strain was also transferred to an international deposit under the Budapest Treaty on June 2, 2015, under accession number FERM BP-22254.

Substances derived from the microalga Coccomyxa include, for example, a cell wall and a substance encapsulated in the cell wall. The cell wall is the cell wall of the microalga Coccomyxa. The substance encapsulated in the cell wall is a part of the microalga Coccomyxa. The substance encapsulated in the cell wall is, for example, an active ingredient that has an intestinal regulating effect. The cell wall includes, for example, alginane.

Substances derived from the microalga Coccomyxa activate, for example, a group of bacteria (hereinafter referred to as a specific group of bacteria) involved in the decomposition or fermentation of dietary fiber.
For example, at least a portion of the specific bacterial group is a bacterial group capable of producing short-chain fatty acids. Examples of bacterial groups capable of producing short-chain fatty acids include the family Ruminococcaceae, Lachnospiraceae, the genus Sedimentibacter, the genus Phascolarctobacterium, and the genus Allobaculum. Examples of bacterial groups from the family Ruminococcaceae include the genus Oscillibacter, the genus Faecalisbacterium, and the genus Subdoligranurum. Examples of bacterial groups from the family Lachnospiraceae include the genus Parasporobacterium, the genus Malvinbrianchia, and the genus Syntrophococcus. Examples of short-chain fatty acids produced by the specific bacterial group include butyric acid, propionic acid, and acetic acid.

For example, at least a portion of the specific bacterial group is a bacterial group that has the ability to suppress the production of putrefactive products that cause fecal odor. Examples of bacterial groups that have the ability to suppress the production of putrefactive products that cause fecal odor include the Prevotellaceae family, the Coriobacteriaceae family, and the Akkermansia genus. Examples of bacterial groups in the Prevotellaceae family include the Prevotella genus and the Paraprevotella genus. Examples of bacterial groups in the Coriobacteriaceae family include the Adrecrautia genus and the Enterohabdus genus.

The substance derived from the microalga Coccomyxa activates, for example, bacteria of the family Veillonellaceae and Clostridium XIVa . Clostridium XIVa produces butyric acid. The food composition for intestinal regulation of the present disclosure further contains, for example, at least one of a hardly degradable carbohydrate and a dietary fiber.

The food composition for intestinal regulation of the present disclosure preferably further contains, for example, at least one of a persistent carbohydrate and dietary fiber. Ingestion of a persistent carbohydrate or dietary fiber can enhance the effect of improving the activity of the bacterial network involved in the decomposition and fermentation of dietary fiber by substances derived from the microalga Coccomyxa.

The bacterial groups that make up an individual's intestinal flora form an interaction network based on antagonistic, symbiotic, or complementary relationships, and even if a specific intestinal bacterium is ingested, it is unlikely to change due to the buffering action of other bacterial groups. Therefore, we sought to create an intestinal regulating food composition that acts on the entire bacterial group that forms the interaction network in each individual's unique intestinal flora, increasing its activity. In particular, many of the bacterial groups that work near the large intestine and are involved in the breakdown and fermentation of dietary fiber produce short-chain fatty acids such as butyric acid and propionic acid in the large intestine, which have a significant impact on intestinal regulating effects. Therefore, we have created an intestinal regulating food composition that increases the activity of the bacterial network involved in the breakdown and fermentation of dietary fiber.

Because most nutrients are broken down and absorbed in the stomach and small intestine, which are digestive organs between the mouth and the large intestine, encapsulation or other methods were necessary to deliver nutrients to the network of bacteria involved in the breakdown and fermentation of dietary fiber near the large intestine. Meanwhile, dietary fiber contained in vegetables and other foods cannot be broken down in the human stomach or small intestine and therefore reaches the large intestine, where it becomes food for the bacteria that break it down and ferment it, producing lactic acid and short-chain fatty acids. Microalgae are rich in proteins, vitamins, minerals, and other nutrients contained within their cell walls, the main component of which is dietary fiber. In other words, the abundant nutrients contained inside the microalgae shells are released by cell wall breakdown—that is, by the action of bacteria present in the large intestine that break down dietary fiber—making them the perfect agents for reaching the large intestine. However, the cell walls of many microalgae, such as chlorella, are easily destroyed during food processing, such as high-pressure sterilization and heat sterilization, resulting in a high percentage of nutrients being absorbed before reaching the large intestine. Therefore, they focused on Coccomyxa, which contains a hard component called alginane in its cell walls, and thought that by using this microalgae, it would be possible to deliver nutrients to the large intestine even after sterilization.

2. Manufacturing Methods of Food Compositions for Intestinal Regulation (2-1) First Manufacturing Method A cultured Coccomyxa KJ strain is prepared. The algae density of the Coccomyxa KJ strain is, for example, 0.2 to 0.5 g/L. A concentrate is obtained by concentrating the culture using a centrifuge until the algae density reaches 10 to 20 g/L. A dried powder of the Coccomyxa KJ strain is obtained by drying the concentrate using a drum dryer heated to 120 to 150°C. The dried powder of the Coccomyxa KJ strain corresponds to a substance derived from the microalga Coccomyxa and a food composition for intestinal regulation. The food composition for intestinal regulation manufactured by the first manufacturing method can also be used, for example, as livestock feed. Microalgae Coccomyxa other than the Coccomyxa KJ strain may also be used as a raw material.

(2-2) Second Manufacturing Method A concentrate is obtained in the same manner as in the first manufacturing method. The concentrate is heat-treated to suppress the pheophorbide-producing activity. A dried powder of the Coccomyxa KJ strain is obtained from the concentrate by a drying process such as spray drying or freeze-drying. The dried powder of the Coccomyxa KJ strain corresponds to a substance derived from the microalga Coccomyxa and a food composition for intestinal regulation. Microalgae Coccomyxa other than the Coccomyxa KJ strain may also be used as a raw material.

3. Effects of the Food Composition for Intestinal Regulation (1A) The food composition for intestinal regulation of the present disclosure has a high intestinal regulation effect.
(1B) When the food composition for intestinal regulation of the present disclosure further contains at least one of a persistent carbohydrate and dietary fiber, the intestinal regulation effect is even higher.

(1C) The substance derived from the microalga Coccomyxa includes, for example, a cell wall and a substance encapsulated in the cell wall. In this case, when the food composition for intestinal regulation is orally administered, the cell wall prevents the encapsulated substance from being broken down or absorbed in the stomach or small intestine. Therefore, the substance encapsulated in the cell wall easily reaches the large intestine. As a result, the intestinal regulating effect of the food composition for intestinal regulation is even greater.

(1D) The cell wall contains, for example, alginane. In this case, the cell wall further prevents the encapsulated substance from being broken down or absorbed in the stomach or small intestine. Therefore, the substance encapsulated in the cell wall is more likely to reach the large intestine. As a result, the intestinal regulating effect of the food composition for intestinal regulation is even greater.

(1E) For example, a substance derived from the microalga Coccomyxa activates a specific bacterial group, which further enhances the intestinal regulating effect of the food composition for intestinal regulation.
(1F) For example, at least a portion of the specific bacterial group activated by a substance derived from the microalga Coccomyxa has the ability to produce short-chain fatty acids. In this case, the intestinal regulating effect of the food composition for intestinal regulation is further enhanced.

(1G) For example, at least a portion of the specific bacterial group activated by a substance derived from the microalga Coccomyxa has the ability to suppress the production of putrefactive products that cause fecal odor. In this case, the intestinal regulating effect of the intestinal regulating food composition is even greater.

(1H) For example, substances derived from the microalga Coccomyxa activate bacteria of the Veillonellaceae family and Clostridium XIVa. In this case, the intestinal regulating effect of the food composition for intestinal regulation is further enhanced.
4. Examples (4-1) Production of Food Composition for Intestinal Regulation A food composition for intestinal regulation was produced using the Coccomyxa KJ strain as a raw material by the first production method. The food composition for intestinal regulation contains a dry powder of the Coccomyxa KJ strain. The dry powder of the Coccomyxa KJ strain corresponds to a substance derived from the microalga Coccomyxa. The dry powder of the Coccomyxa KJ strain contains a cell wall and a substance encapsulated within the cell wall. The cell wall contains alginane. The produced food composition for intestinal regulation was used in the feeding test described below.

(4-2) Conducting the Feeding Test Ten piglets weighing approximately 30 kg were divided into two groups: an experimental group and a control group. Each group consisted of five pigs. A feeding test was conducted for each of the experimental and control groups from December 18, 2020 to March 12, 2021. The feed given to the experimental group during the feeding test was a mixed feed consisting of a base feed mixed with 0.5% w/w of a food composition for intestinal regulation. The base feed was a concentrated feed containing grains, specifically Super Hawk. The feed given to the control group during the feeding test was only the base feed.

(4-3) Analysis of intestinal flora Fecal samples were obtained from five rats in the experimental group and five rats in the control group on December 10, 2020, before the feeding test, and on March 12, 2021, after the feeding test. The samples were collected in dedicated containers within 5 minutes after defecation and frozen to prevent changes in the contents.

Next, the bacteria in the collected feces were disrupted using a shaking tissue homogenizer (Precellys Evolution, Bertin), and DNA was extracted using a GENE PREP STAR PI-48 (Kurabo). The V3-V4 region (approximately 430 bp) of the 16S rDNA of the extracted DNA was amplified by PCR. Next, amplicon sequencing analysis was performed using Miseq, and the amplified genes were identified using the RDP microbial identification database.

(4-4) Analysis Results of Intestinal Flora Based on the results of "(4-3) Analysis of Intestinal Flora" above, all data on intestinal bacteria for the eight animals, excluding the two animals that were in poor health, was subjected to principal component analysis. The results are shown in Figure 1. In Figure 1, "Before test" refers to samples obtained before the feeding test. "After test" refers to samples obtained after the feeding test.

As shown in Figure 1, in samples taken before the feeding trial, principal component 1 fell between -1 and 0 in both the experimental and control groups. In samples taken after the feeding trial, principal component 1 in the experimental group shifted in the positive direction compared to principal component 1 in the control group.

Figure 2 shows the average values of principal component 1 obtained by principal component analysis of the intestinal bacteria of pigs obtained from the experimental and control groups before and after the feeding trial. Principal component 1 in the experimental group showed greater mobility in the positive direction than in the control group. Positive mobility refers to the degree to which principal component 1 in the sample obtained after the feeding trial has moved in the positive direction compared to principal component 1 in the sample obtained before the feeding trial.

From the bacterial groups that influence the positive mobility of principal component 1, we selected the top 18 genera with the highest factor loadings, as well as bacterial groups with a correlation coefficient of 0.7 or higher with these 18 genera. We constructed a network of bacterial groups with a correlation coefficient of 0.8 or higher from the selected bacterial groups, and discovered a bacterial group network of 53 genera that decompose or ferment dietary fiber, as shown in Figure 3.

Principal component analysis was again performed on these 53 genera of bacteria. The values of principal component 1 were then plotted against the 16S rDNA copy number ratio of Fibrobacter . The results are shown in Figure 4. Fibrobacter is a bacterium that exists in the bacterial community network shown in Figure 3 and decomposes dietary fiber. Figure 4 shows that as the piglets grow, the number of Fibrobacter in the large intestine increases, the number of bacteria in the positive direction of principal component 1 increases, and the mobility of principal component 1 in the positive direction is significantly greater in the experimental group. Therefore, Figure 4 indicates that the Coccomyxa KJ strain contains a component (hereinafter referred to as the active component) that activates the bacteria that decompose or ferment dietary fiber.

In order to deliver the active ingredient to the large intestine, it is preferable to prevent the active ingredient from being broken down or absorbed in the stomach or small intestine. For example, if the active ingredient is encapsulated in the cell wall, the active ingredient is less likely to be broken down or absorbed in the stomach or small intestine. If the cell wall contains alginane, the active ingredient is even less likely to be broken down or absorbed in the stomach or small intestine. Alginane is contained in the cell wall of the microalga Coccomyxa. The food composition for intestinal regulation produced in "(4-1) Production of food composition for intestinal regulation" above contains cell walls containing alginane and a substance encapsulated in the cell wall.

Based on the results of intestinal flora analysis of samples obtained on December 10, 2020, before the feeding test, and on March 12, 2021, after the feeding test, the 16S rDNA copy ratios of the bacterial groups contained in the bacterial network shown in Figure 3 were calculated. The results are shown in Tables 1 and 2.

Of the 53 genera included in the bacterial community network, 51 genera showed a tendency for the 16S rDNA copy ratio of the bacterial community in the experimental area to be higher.
(4-5) Analysis of Fecal Components After the feeding trial, on March 12, 2021, fecal samples were collected from five rats in the experimental group and five rats in the control group. The samples were collected in dedicated containers within five minutes of defecation and frozen to prevent changes in the contents. SCFA and decay products in the feces were analyzed by HPLC. SCFAs were acetic acid, propionic acid, and butyric acid. Decay products were phenol, 4-ethylphenol, indole, p-cresol, and skatole.

Among the results of the above analysis, the results of analyzing the short-chain fatty acids in the samples are shown in Figure 5. A tendency for the contents of all short-chain fatty acids to be higher in the experimental group was observed.
Figure 6 shows bacterial groups with a correlation coefficient of 0.6 or higher with the content of each short-chain fatty acid in the feces. The "*" mark in Figure 6 indicates bacteria included in the bacterial group network of principal component 1. It is presumed that the reason for the high content of short-chain fatty acids in the feces in the experimental group is that the intake of Coccomyxa strain KJ activated the bacterial groups that break down or ferment dietary fiber.

Next, Figure 7 shows the results of the analysis of putrefactive products in the samples from the above analysis. A tendency for the content of all putrefactive products to be lower in the experimental group was observed. Figure 8 shows the bacterial groups that have a negative correlation with the content of putrefactive products in the feces.

Of the bacterial groups shown in Figure 8, all bacterial groups other than the Alloprovetella genus are bacteria included in the bacterial group network of main component 1. The reason for the lower content of putrefactive products in the feces in the experimental group is presumed to be that the intake of Coccomyxa KJ strain activated the bacterial groups that decompose or ferment dietary fiber. Putrefactive products are substances that cause fecal odor. The above analysis results confirmed that the intake of a food composition for intestinal regulation can suppress fecal odor.

In addition to the 53 genera included in the bacterial community network, bacterial groups with higher 16S rDNA copy ratios were also observed in the experimental group. The results are shown in Figures 9 and 10. Among the Veillonellaceae bacteria in Figure 9, there are many that utilize lactic acid to produce SCFA such as propionic acid. Clostridium XIVa in Figure 10 is known to include a bacterial group capable of producing butyrate, and it is thought that an increase in these bacterial groups also influenced the increase in SCFA in the feces.

In addition to the pig tests, a human ingestion test of the Coccomyxa KJ strain was also conducted. 31 subjects were asked to ingest 1.5 g of Coccomyxa KJ strain tablets daily, and changes in the intestinal flora in the feces were analyzed before and after 30 days of ingestion. The results showed that ingestion of the Coccomyxa KJ strain increased the number of Veillonellaceae bacteria and Clostridium XIVa. The results are shown in Figures 11 and 12.

The reason why tests using pigs were conducted in the examples is as follows: The human intestinal flora is affected by diet, lifestyle, and other factors, making it prone to change and variation, making statistical analysis difficult.

On the other hand, for pigs, the feed they receive and their living environment can be controlled in common, which reduces changes and variations in their intestinal flora, making them suitable for verifying effects through statistical analysis. In particular, for intestinal food compositions used as supplements to activate the bacteria involved in the breakdown and fermentation of dietary fiber near the large intestine, the intake rate is limited to less than 1%, so testing in pigs, where changes and variations in their intestinal flora can be reduced, is appropriate.

The intestinal microbiota of humans and pigs share a common feature in that three bacteria - Firmicutes, Bacteroidetes, and Proteobacteria - predominate (see: Development of alternatives to antimicrobial growth promoters (AGPs) in pigs based on research into the porcine intestinal microbiome, Tohoku Society of Livestock Science Bulletin 68(1):1-7, 2018), and Nature has also reported that the roles of each intestinal bacterium are very similar (see: Dirty pigs are healthy pigs. Study finds link between outdoor living and immune health. Nature 2009).

Therefore, we investigated the effects of a food composition for intestinal regulation containing a substance derived from the microalga Coccomyxa as an active ingredient, primarily using pigs, which have an intestinal flora similar to that of humans.
5. Other Embodiments Although the embodiments of the present disclosure have been described above, the present disclosure is not limited to the above-described embodiments and can be implemented in various modifications.

(5-1) Multiple functions possessed by one component in the above embodiments may be realized by multiple components, or one function possessed by one component may be realized by multiple components. Furthermore, multiple functions possessed by multiple components may be realized by one component, or one function realized by multiple components may be realized by one component. Furthermore, part of the configuration of the above embodiments may be omitted. Furthermore, at least part of the configuration of the above embodiments may be added to or substituted for the configuration of another of the above embodiments.

(5-2) In addition to the intestinal regulating food composition described above, the present disclosure can also be realized in various forms, such as a system that includes the intestinal regulating food composition as a component, or a method for producing the intestinal regulating food composition.

Claims (5)

The active ingredient is a substance derived from the microalga Coccomyxa.
The substance derived from the microalga Coccomyxa is a substance derived from the Coccomyxa KJ strain,
The cell wall contains alginane, and a substance encapsulated in the cell wall,
It has the effect of improving the activity of the bacterial network involved in the decomposition and fermentation of dietary fiber .
Food composition for improving intestinal environment . The food composition for improving intestinal environment according to claim 1,
the encapsulated substances include proteins, vitamins, and minerals;
The cell walls are not decomposed in the stomach or small intestine, but after reaching the large intestine, the cell walls are decomposed by bacteria that decompose dietary fiber, thereby releasing the encapsulated substances,
The encapsulated substance is released, thereby activating the bacterial network that produces short-chain fatty acids, and suppressing putrefactive products in feces.
Food composition for improving intestinal environment . The food composition for improving intestinal environment according to claim 1 or 2,
The bacterial network is a food composition for improving the intestinal environment, comprising a group of bacteria of the Veillonellaceae family and Clostridium XIVa. The food composition for improving intestinal environment according to any one of claims 1 to 3 ,
A food composition for improving the intestinal environment, further comprising at least one of a persistent carbohydrate and dietary fiber. The food composition for improving intestinal environment according to any one of claims 1 to 4,
A food composition for improving the intestinal environment, which is livestock feed.