Short-chain fatty acid production promoter

Abstract

A short-chain fatty acid production promoter containing gluconic acid derivatives that promotes the production of short-chain fatty acids by intestinal bacteria in the intestinal tract. [Solution] To provide a novel prebiotic material that promotes the production of short-chain fatty acids by intestinal bacteria.

Description

Technical Field

[0001] The present invention relates to a short-chain fatty acid production promoter that promotes the production of short-chain fatty acids by intestinal bacteria in the intestinal tract.

Background Art

[0002] It is widely known that improving the intestinal environment contributes to the health of humans and animals, and many foods containing probiotics (bacterial cells) and prebiotics (the "food" for probiotics) are on the market.

[0003] Prebiotics are defined as "indigestible food ingredients that selectively change the growth and activity of specific bacteria in the large intestine, thereby having a beneficial effect on the host and improving the host's health" (Gibson, G. R., and Roberfroid, M. B.: Dietary modulation of the human colonic microbiota: introducing the concept of pebiotics. J Nutr, 125: 1401-1412, 1995.). Representative examples of prebiotics include polysaccharides and oligosaccharides.

[0004] The function of this prebiotic is expected to maintain health from all angles through the host's intestinal flora, not only having an intestinal regulating effect but also suppressing allergies and enhancing intestinal immunity.

[0005] In recent years, the production of short-chain fatty acids by intestinal bacteria has attracted attention as a mechanism by which prebiotics exert various health functions. Short-chain fatty acids are a type of organic acid and refer to fatty acids with 6 or fewer carbon atoms. Specifically, formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and 2-methylbutyric acid are applicable.

[0006] Of these, acetic acid, propionic acid, and butyric acid in particular have been reported to improve glucose tolerance, suppress obesity, and exhibit anti-inflammatory effects (Chambers et al., 2019, Gut, 68(8): 1430-1438., Kimura et al., 2013, Nat. Commun., 4: 1829., Thorburn et al., 2015, Nat. Commun., 6: 7320., etc.). [Overview of the project] [Problems that the invention aims to solve]

[0007] However, the changes in the gut microbiota and short-chain fatty acids differ depending on the prebiotics ingested. Therefore, it is necessary to first identify prebiotic materials that are expected to promote the production of short-chain fatty acids.

[0008] Therefore, the present invention aims to provide a novel prebiotic material that promotes the production of short-chain fatty acids by intestinal bacteria. [Means for solving the problem]

[0009] The above problems are solved by the present invention as described below. In other words, the present invention (1) provides a short-chain fatty acid production promoter that contains gluconic acid and promotes the production of short-chain fatty acids by intestinal bacteria in the intestinal tract.

[0010] Furthermore, the present invention (2) provides a short-chain fatty acid production promoter according to (1), characterized in that the gluconic acids are one or more selected from gluconic acid, sodium gluconate, potassium gluconate, calcium gluconate, zinc gluconate, copper gluconate, and glucono-δ-lactone. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide a novel prebiotic material that promotes the production of short-chain fatty acids by intestinal bacteria. [Brief explanation of the drawing]

[0012] [Figure 1] This shows the increase or decrease in the abundance of Bifidobacterium bacteria in Experiment 1. [Figure 2] This shows the increase or decrease in the abundance of Fecalibacterium bacteria in Experiment 1. [Figure 3] This shows the increase or decrease in the abundance of Blautia bacteria in Experiment 1. [Figure 4] This result shows the increase or decrease in the abundance of Bilophila bacteria in Test 1. [Modes for carrying out the invention]

[0013] The short-chain fatty acid production promoter of the present invention contains gluconic acid and is a short-chain fatty acid production promoter that promotes the production of short-chain fatty acids by intestinal bacteria in the intestinal tract.

[0014] In other words, the short-chain fatty acid production promoter of the present invention is a short-chain fatty acid production promoter that contains gluconic acid as an active ingredient and promotes the production of short-chain fatty acids by intestinal bacteria in the intestinal tract. To put it another way, the short-chain fatty acid production promoter of the present invention is a prebiotic aimed at promoting the production of short-chain fatty acids by intestinal bacteria in the intestinal tract.

[0015] When the short-chain fatty acid production promoter of the present invention is used as a prebiotic, the short-chain fatty acids whose production is promoted by intestinal bacteria in the intestinal tract are fatty acids with 6 or fewer carbon atoms, and examples include formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and 2-methylbutyric acid.

[0016] Examples of gluconic acids related to the short-chain fatty acid production promoter of the present invention include gluconic acid, non-toxic salts of gluconic acid such as sodium gluconate, potassium gluconate, calcium gluconate, zinc gluconate, and copper gluconate, and glucono-δ-lactone.

[0017] There are no particular restrictions on the methods for producing gluconic acid, non-toxic salts of gluconic acid, and glucono-δ-lactone; as long as they are used as food products, there are no particular restrictions. For example, a method for producing gluconic acid, a non-toxic salt of gluconic acid, and glucono-δ-lactone is a fermentation method using raw materials containing sugars such as corn. Furthermore, gluconic acid, sodium gluconate, non-toxic salts of gluconic acid, and glucono-δ-lactone may be commercially available products.

[0018] The short-chain fatty acid production promoter of the present invention may be in the form of a powder, an aqueous solution, or granules. Since sodium gluconate, potassium gluconate, calcium gluconate, zinc gluconate, copper gluconate, and glucono-δ-lactone can exist in powder form, the short-chain fatty acid production promoter of the present invention can be a powder containing sodium gluconate, potassium gluconate, calcium gluconate, zinc gluconate, copper gluconate, glucono-δ-lactone, etc. The short-chain fatty acid production promoter of the present invention can also be an aqueous solution containing sodium gluconate, potassium gluconate, calcium gluconate, zinc gluconate, copper gluconate, glucono-δ-lactone, etc. The short-chain fatty acid production promoter of the present invention can also be a granule containing sodium gluconate, potassium gluconate, calcium gluconate, zinc gluconate, copper gluconate, glucono-δ-lactone, etc., which is granulated using an excipient. Furthermore, since gluconic acid dehydrates and changes into glucono-δ-lactone when isolated from an aqueous solution, it is difficult to maintain in powder form. Therefore, an aqueous solution containing gluconic acid is used as the short-chain fatty acid production promoter of the present invention. Note that gluconic acid and glucono-δ-lactone are in equilibrium in water, so the aqueous solution contains both gluconic acid and glucono-δ-lactone.

[0019] When the short-chain fatty acid production promoter of the present invention is an aqueous solution, the concentration of the short-chain fatty acid production promoter is preferably 0.01 to 60% by mass.

[0020] In the intestinal tract, bacteria that produce short-chain fatty acids include Bifidobacterium (bacteria belonging to the genus Bifidobacterium), Blautia (bacteria belonging to the genus Blautia), Faecalibacterium (bacteria belonging to the genus Faecalibacterium), Propionibacterium (bacteria belonging to the genus Propionibacterium), and some Clostridium bacteria (Clostridium cluster I, IV, XIVa, etc.). Among these, the short-chain fatty acid production promoter of the present invention promotes the growth of Bifidobacterium and Blautia, and increases the metabolic amount of short-chain fatty acids.

[0021] The inventors of the present invention studied the production of short-chain fatty acids by intestinal bacteria in the intestinal tract and found the following. · Gluconic acids have a high effect of promoting the growth of Bifidobacterium and Blautia. · Therefore, like glucose and fructooligosaccharides that have been conventionally used as prebiotics, gluconic acids promote the production of short-chain fatty acids.

[0022] That is, the short-chain fatty acid production promoter of the present invention is a short-chain fatty acid production promoter that particularly effectively promotes the production of short-chain fatty acids by Bifidobacterium and Blautia.

[0023] In addition to gluconic acids, the short-chain fatty acid production promoter of the present invention can contain, as necessary, acidulants containing gluconic acids, pH adjusters, antioxidants, nutritional fortifiers, seasonings, etc.

[0024] As an example of the method of using the short-chain fatty acid production promoter of the present invention, it is mixed with food, food additives or excipients and processed into supplements, foods, beverages, etc.

[0025] Since short-chain fatty acids exhibit effects such as improving glucose tolerance, suppressing obesity, and having an anti-inflammatory effect, the short-chain fatty acid production promoter of the present invention that promotes the production of short-chain fatty acids by intestinal bacteria in the intestinal tract is expected to be applied to glucose tolerance improvers, obesity suppressants, and anti-inflammatory agents.

[0026] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples shown below. [Examples]

[0027] (Example 1, Comparative Examples 1 and 2, Reference Examples 1 and 2) A culture test was conducted based on the method described in Japanese Patent Publication No. 7051175. First, human fecal suspension was cultured under anaerobic conditions after adding sodium gluconate (Example 1), sodium malate (Comparative Example 2), disodium succinate (Comparative Example 3), glucose (Reference Example 1), or fructooligosaccharide (Reference Example 2). As a control, the sample was cultured under anaerobic conditions without any additives (Comparative Example 1). Next, for each cultured sample, the bacterial flora profile was identified by metagenomic analysis using 16S rRNA gene sequences. Furthermore, organic acids and short-chain fatty acids after culturing were quantified by LC-TOF / MS of the culture supernatant. For human fecal suspensions, six subjects with different gut microbiota were selected, and all fecal samples were mixed with anaerobic culture medium under anaerobic conditions to achieve the same concentration.

[0028] <Test 1 (Metagenomic Analysis)> Fresh stool samples from six subjects were used to prepare the stool suspensions. After homogenization, the stool was added to anaerobic culture medium to achieve the same final concentration (w / v) to create the stool suspensions. Subsequently, each test compound was added to the stool suspension to achieve the same final concentration (w / v). Next, the stool-test compound mixture was dispensed into a 96-well plate and incubated at 37°C under anaerobic conditions for the specified time. The cultured stool-test compound mixture was centrifuged, and the pellet was used for nucleic acid extraction for microbiome analysis, while the supernatant was used for metabolite extraction for metabolome analysis. In this study, three cultures were performed (n=3), and after confirming that there were no problems with the culture by turbidity measurement (OD600), nucleic acid extraction and metabolite extraction were carried out, and an averaged sample obtained by uniformly mixing the three samples was used for measurement.

[0029] Next, metagenomic analysis using the 16S rRNA gene sequence was performed on each sample. The metagenomic analysis followed the method of Murakami et al. (Murakami, S., et al. (2015) The Consumption of Bicarbonate-Rich Mineral Water Improves Glycemic Control. Evidence-Based Complementary and Alternative Medicine, 2015, Article ID: 824395.). First, using DNA extracted from the centrifuged pellet of the sample as a template, DNA fragments of the V1-V2 region of the 16S rRNA gene were amplified by PCR, and then the sequences of the PCR products were analyzed using the paired-end method with Illumina MiSeq. The average number of read pairs obtained per sample was 29,780 (maximum: 42,245, minimum: 18,216). This indicates that a sufficient amount of base sequences were obtained for all 66 samples. The obtained nucleotide sequences were subjected to pretreatment steps including paired-end sequence merging, PhiX-derived sequence removal, and quality filtering to obtain high-quality nucleotide sequences. These nucleotide sequences were mapped to a 16S rRNA gene database using bowtie2. The 16S rRNA gene database used was the OTU obtained by clustering the SILVA SSU Ref provided by silva (https: / / www.arb-silva.de) with a 99% threshold (hereinafter referred to as the SILVA database). Through the above mapping, each nucleotide sequence is assigned to the most similar OTU in the SILVA database. By counting the number of nucleotide sequences assigned to (mapped) each OTU, the bacterial phylogenetic composition of the stool culture microbiota is quantified. In practice, 10,000 sequences were randomly selected to unify the detection sensitivity between samples before calculating the bacterial phylogenetic composition. The resulting increases and decreases in the abundance of specific microorganisms are shown in Figures 1 to 4. In the figures, significant increases or decreases are indicated by (*) (P<0.05; Wilcoxon signed-rank test).

[0030] (Test compound) Example 1: Sodium gluconate Comparative Example 1: Additive-free Comparative Example 2: Sodium Malate Comparative Example 3: Disodium succinate Example 1: Glucose Example 2: Fructooligosaccharides

[0031] (Fecal sample after culture) In Figures 1 to 4, FK01 to FK06 show the results of stool samples (after addition of the test compound and incubation) obtained from six subjects.

[0032] <Test 2 (Quantitative Analysis of Short-Chain Fatty Acids)> To evaluate the effect of adding the test compound on metabolic reactions originating from the gut microbiota, a quantitative evaluation of short-chain fatty acids was performed on the metabolites contained in the supernatant of the sample from Test 1. First, the supernatant obtained by centrifugation of the sample was filtered using a tube equipped with a filter unit. The sample solution was subjected to liquid-liquid extraction using an organic solvent, followed by derivatization treatment, and an internal standard substance for elution time correction was added. Metabolites were measured using LC-TOF / MS, and the column retention time, mass-to-charge ratio (m / z), and peak area of ​​the detected peaks were obtained. By comparing this information with the measurement results of standard samples, the metabolites corresponding to each peak were identified. These peaks were corrected so that the area ratio with the internal standard substance was constant for each sample, and converted into values ​​that allow for relative quantification between samples (relative area ratio). Absolute quantification was performed on 13 of the metabolites by comparing them to a calibration curve created using standard samples of known concentration. Table 1 shows the short-chain fatty acids that showed a significant difference in abundance compared to Comparative Example 1 (no additive). A significant increase is indicated by an upward arrow (↑), a significant decrease by a downward arrow (↓), and an equal or equal amount by a horizontal bar (-) (P<0.05; Wilcoxon signed-rank test). Compounds that showed a significant difference in the Friedman-Nemenyi test are marked with (Fr).

[0033] [Table 1]

[0034] The results of the above tests showed that sodium gluconate (Example 1) was highly effective in promoting the growth of Bifidobacterium and Blautia bacteria. On the other hand, other organic acid salts besides gluconate, such as sodium malate (Comparative Example 2) and disodium succinate (Comparative Example 3), did not have any effect on promoting the growth of Bifidobacterium and Blautia bacteria. Furthermore, sodium gluconate (Example 1), like glucose (Reference Example 1) and fructooligosaccharides (Reference Example 2), was found to significantly promote the production of short-chain fatty acids compared to the case without additives (Comparative Example 1). On the other hand, sodium malate (Comparative Example 2) and disodium succinate (Comparative Example 3), which are organic acid salts other than gluconates, did not promote the production of short-chain fatty acids.

[0035] Based on these findings, it can be inferred that gluconic acids, which are highly effective in promoting the growth of Bifidobacterium and Blautia under anaerobic conditions compared to other intestinal bacteria, also promote the production of short-chain fatty acids in the human intestinal tract.

Claims

1. A short-chain fatty acid production promoter containing gluconic acid derivatives that promotes the production of short-chain fatty acids by intestinal bacteria in the intestinal tract.

2. The short-chain fatty acid production promoter according to claim 1, characterized in that the gluconic acid is one or more selected from gluconic acid, sodium gluconate, potassium gluconate, calcium gluconate, zinc gluconate, copper gluconate, and glucono-δ-lactone.

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