Novel lactic acid bacterium belonging to genus leuconostoc capable of growing in rice porridge

Abstract

To provide a porridge product using Leuconostoc suionicum capable of growing and fermenting in rice porridge.SOLUTION: Provided is a lactic acid bacterium belonging to Leuconostoc suionicum, the lactic acid bacterium being capable of growing to 1.0×108 cfu / ml at 30°C in 4 hours by being added to rice porridge. Provided is a food or beverage comprising the lactic acid bacterium, or a fermented product or a resulting product, obtained by fermenting a food material with the lactic acid bacterium. Provided is a method for producing the fermented product, the method comprising inoculating the food material with the lactic acid bacterium and fermenting the food material.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a novel lactic acid bacterial strain belonging to Leuconostoc suionicum, which is excellent in growth and fermentation in porridge, various fruit and vegetable juices, and crushed fruits and vegetables, a food or drink containing the lactic acid bacterium, a fermented product produced by the lactic acid bacterium, a food or drink containing the fermented product, and a method for producing a fermented product using the lactic acid bacterium.

Background Art

[0002] It is said that there are more than 100 trillion bacteria in the human intestine, and the number of their types is more than 1,000. These bacteria are called the intestinal flora and have a close relationship with the host human.

[0003] The intestinal flora is known to change due to various external factors. Abnormalities in the intestinal flora not only affect bowel movements but are also involved in inflammatory bowel diseases, obesity, type 2 diabetes, and allergic diseases. As a method for improving abnormalities in the intestinal flora, there are probiotic preparations and probiotic foods that mainly use probiotics represented by lactic acid bacteria.

[0004] Lactic acid and extracellular polysaccharide (EPS) produced by lactic acid bacteria, which are probiotics, not only suppress the growth of so-called bad bacteria such as Escherichia coli and pathogenic bacteria, but also show physiological effects such as improvement of bowel movements, infection prevention by enhancing immunity, and anti-allergic effects.

[0005] As a method for ingesting such probiotics, ingestion of fermented foods that have been used since ancient times is the mainstream. Representative fermented foods are those made from dairy products such as yogurt, and bacteria such as Lactococcus lactis, Lactobacillus bulgaricus, Lactobacillus acidophilus, and Streptococcus thermophilus are used for fermentation.

[0006] On the other hand, there are people who are allergic to dairy products or who experience indigestion due to dairy products. In such cases, it is difficult for them to effectively ingest lactic acid bacteria from food. Therefore, there is a need to develop new lactic acid bacterial strains that can ferment non-dairy products and effectively ingest lactic acid bacteria.

[0007] Recently, plant-based lactic acid bacteria that can ferment plant components have attracted attention. Plant-based lactic acid bacteria can not only ferment plant components but also survive and grow in the acidic region. Therefore, it is said that they can reach the intestine alive and have a direct effect on maintaining and improving the intestinal environment. So far, lactic acid bacteria that can ferment plant components using raw materials other than milk raw materials have been isolated, and probiotic foods using plant-based lactic acid bacteria that can ferment soy milk have also been developed.

[0008] However, since there are still many people who dislike the beany smell of fermented soy milk products or are allergic to soy milk, the development of fermented products using unprecedented fermentation raw materials with plant-based lactic acid bacteria, which have a low protein content and are less likely to cause allergies, has been desired.

[0009] As fermented beverages using rice, which is the staple food of the Japanese people, there are sake and amazake, but both are alcoholic beverages and not foods. There is no report of a fermented food that can be a staple food using rice as a raw material. The inventors considered that probiotic foods could be ingested more efficiently as staple foods by the invention.

[0010] In particular, it is said that about half of the elderly patients during hospitalization and those living in nursing facilities take meals in the form of rice porridge. If probiotic foods can be ingested in the form of rice porridge, efficient intervention for elderly inpatients and those living in nursing facilities will be possible.

[0011] As a prior art, there is a high-protein functional porridge (Japanese Patent Application Laid-Open No. 2023-85194) that adds a composition derived from peas using polished rice as the main raw material, but the invention of porridge as a probiotic food is not disclosed.

Prior Art Documents

Patent Document

[0012]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0013] So far, no bacterial strain has been reported among conventional probiotics, which are so - called lactic acid bacteria, that has high growth ability in rice porridge and exhibits useful effects in vivo.

Means for Solving the Problems

[0014] The inventor has found that the plant - derived lactic acid bacterium Leuconostoc suionicum isolated from cherry blossoms has a high growth effect in rice porridge, which has not been confirmed in conventional lactic acid bacteria, and may have useful effects in vivo, and thus made the present invention.

[0015] The present invention relates to lactic acid bacteria that can grow to 1.0×10 8 cfu / ml or more in 4 hours at 30°C in rice porridge (whole rice porridge, seven - tenths rice porridge, five - tenths rice porridge, three - tenths rice porridge, thick soup). The unit cfu / ml representing the viable cell count indicates the colony - forming ability when 1 ml is inoculated on a culture plate.

[0016] The above - mentioned lactic acid bacteria may be the lactic acid bacteria deposited under Accession No. NITE AP - 04055.

[0017] The present invention is also a method for producing a food or drink containing a fermented product, which includes a fermentation step of fermenting a first food material with the above - mentioned lactic acid bacteria to obtain a fermented product, and a step of incorporating the fermented product into a second food material.

[0018] The present invention is also a fermented product obtained by fermenting a food material with the above - mentioned lactic acid bacteria.

[0019] Furthermore, the present invention includes the extracellular polysaccharide produced by the above lactic acid bacteria.

[0020] Moreover, the present invention is a method for producing a fermented product, which is characterized by inoculating the above lactic acid bacteria into a food material and fermenting it.

Advantages of the Invention

[0021] The lactic acid bacteria of the present invention can ferment rice porridge (whole rice porridge, seven-tenths rice porridge, five-tenths rice porridge, three-tenths rice porridge, thick rice soup), and can prepare a probiotic food based on rice porridge, which is the staple food for postoperative diet and nursing diet.

Brief Description of the Drawings

[0022] [Figure 1] Figure 1 shows a graph regarding the results of measuring the viable cell counts of MS009 strain and JCM6124 strain cultured with shaking at 30 °C and 200 rpm under aerobic conditions over time. [Figure 2] Figure 2 shows a graph regarding the results of the number of bacteria of MS009 strain and JCM6124 strain attached to each INT-407 cell. [Diagram 3] Figure 3 shows electron micrographs of MS009 strain and JCM6124 strain attached to NT-407 cells. [Figure 4] Figure 4 shows the results of α-diversity analysis of cecal contents in rats administered with thick rice soup cultured with MS009 strain (administered group) and non-inoculated thick rice soup (non-administered). [Diagram 5] Figure 5 shows the results of β-diversity analysis of cecal contents in rats administered with thick rice soup cultured with MS009 strain (administered group) and non-inoculated thick rice soup (non-administered). [Figure 6] Figure 6 shows the results of LEfSe analysis of cecal contents in rats administered with thick rice soup cultured with MS009 strain (administered group) and non-inoculated thick rice soup (non-administered). [Figure 7] Figure 7 shows a graph regarding the concentrations of lactic acid, acetic acid, propionic acid, butyric acid, and total short-chain fatty acids in cecal contents in rats administered with thick rice soup cultured with MS009 strain (administered group) and non-inoculated thick rice soup (non-administered). [Figure 8] FIG. 8 shows a graph of the IgA antibody concentration in the cecal contents of rats administered rice gruel cultured with the MS009 strain (treated group) and uninoculated rice gruel (non-treated group). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Hereinafter, embodiments of the present invention will be described. The following embodiments are merely examples, and the scope of the present invention is not limited to the following embodiments. Note that similar content will not be described again to avoid repetition.

[0024] definition For convenience, certain terms used in this application are collected here. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0025] Although the numerical ranges and parameters set forth in the present invention are approximate, the numerical values set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in each test measurement. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error as considered by one of ordinary skill in the art.

[0026] lactic acid bacteria The lactic acid bacteria of this embodiment can be cultured in porridge at 30°C for 4 hours at a concentration of 1.0 x 10 8 In one embodiment, the lactic acid bacterium is a lactic acid bacterium belonging to Leuconostoc sionicum, and in particular, the Leuconostoc sionicum MS009 strain deposited under accession number NITE AP-04055.

[0027] In this embodiment, the thick porridge (whole porridge, seven-tenths porridge, five-tenths porridge, three-tenths porridge, thick soup) refers to whole porridge (a mixture of water and rice at a ratio of about 5:1, heated at 95°C to 120°C for 60 minutes), seven-tenths porridge (a mixture of water and rice at a ratio of about 7:1, heated at 95°C to 120°C for 60 minutes), five-tenths porridge (a mixture of water and rice at a ratio of about 10:1, heated at 95°C to 120°C for 60 minutes), three-tenths porridge (a mixture of water and rice at a ratio of about 20:1, heated at 95°C to 120°C for 60 minutes), and thick soup (a mixture of water and rice at a ratio of about 50:1, heated at 95°C to 120°C for 60 minutes), respectively.

[0028] In this embodiment, the porridge (whole porridge, seven-tenths porridge, five-tenths porridge, three-tenths porridge, thick soup) added with MS009 strain at about 1.0×10 6 cfu / ml can increase the number of MS009 strain bacteria to about 1.0×10 8 cfu / ml or more within a fermentation time of 4 hours or less at 30°C.

[0029] The fermented product obtained by fermenting food materials with the above lactic acid bacteria, food and drink containing the fermented product, and food and drink containing the above lactic acid bacteria This embodiment includes a fermentation step of fermenting a first food material with the above lactic acid bacteria to obtain a fermented product, and a step of incorporating the fermented product into a second food material. The fermented product of this embodiment is obtained by fermenting a food material with the above lactic acid bacteria. Also, the food and drink of this embodiment contains the fermented product. The method for producing the fermented product of this embodiment is characterized by inoculating and fermenting the above lactic acid bacteria in a food material. In this embodiment, the first food material and the second food material (hereinafter, may also be simply referred to as "food material") may be any food material that can be fermented by the above lactic acid bacteria, for example, rice porridge, soy milk, fruits, and vegetables. The first food material may be the same as or different from the second food material. When producing a fermented soy milk beverage using the above lactic acid bacteria, first, the lactic acid bacteria are inoculated and cultured alone or simultaneously with other microorganisms in soy milk sterilized under conditions suitable for the soy milk to be used, and this is homogenized to obtain fermented soy milk. In the case of fruits or vegetables, fermented fruit juice or fermented vegetables can be obtained in the same manner as soy milk. It is also possible to mix these fermented products with unfermented vegetable juice, unfermented fruit juice, alcohol, etc., and further add various nutrients, vitamins, flavors, etc. to obtain a final product. These are preferable because they contain the above lactic acid bacteria in a viable state.

[0030] The food and drink of this embodiment may be in any form that can be orally ingested, such as a solution, solid form, powder, etc., and is not particularly limited. Specific examples include beverages (lactic acid beverages, fruit juice beverages, soy milk beverages, vegetable beverages, tea beverages, carbonated beverages, nutritional beverages, sports beverages, coffee beverages, soups, alcoholic beverages, etc.), dairy products (yogurt, cheese, butter, ice cream, etc.), wheat flour products (bread, noodles, cake mixes, etc.), confectioneries (chocolate, cookies, candies, caramels, jelly, gums, Japanese confectioneries, etc.), oil and fat foods (dressings, mayonnaise, creams, etc.), seasonings (sauces, tomato ketchup, vinegar, flavor seasonings, soup bases, etc.), instant foods (instant noodles, instant soups, miso soup, canned foods, retort foods, etc.), supplements (tablets, syrups, granules, capsules, rapidly disintegrating agents, etc.).

[0031] The lactic acid bacteria of this embodiment can be used for various purposes in the same way as lactic acid bacteria used in conventional probiotics, and by ingesting them, physiological effects such as intestinal regulation and antibacterial activity can be expected.

[0032] When humans or animals ingest the lactic acid bacteria of this embodiment, there is no strict limit to the amount, but the preferred amount is 10 viable bacteria count per serving. 6 cfu to 10 14 cfu is preferred. Furthermore, although it is preferred that the lactic acid bacteria of this embodiment be continuously ingested, this is not limitative, and the lactic acid bacteria may be ingested at intervals such as every other week or every other day, or for a short period of time.

[0033] The above-mentioned foods, beverages, and fermented products may contain optional ingredients such as various nutrients, various vitamins, various minerals, sweeteners, stabilizers such as emulsifiers, thickeners, dietary fiber, and flavors.

[0034] Nutrients include DHA, EPA, acetylglucosamine, catechin, turmeric, propolis, agaric, beta-cryptoxanthin, quercetin, anthocyanins, etc. Vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, folic acid, nicotinamide, biotin, vitamin K, etc. Minerals include magnesium, calcium, zinc, manganese, iron, sodium, potassium, etc. Sweeteners include sugars such as sucrose, glucose, fructose, trehalose, xylose, lactose, palatinose, high-fructose corn syrup, maltose, fructose, and honey, as well as high-intensity sweeteners such as sorbitol, xylitol, erythritol, lactitol, palatinit, aspartame, sucralose, stevia, and acesulfame K. Acidulants include citric acid, lactic acid, acetic acid, malic acid, tartaric acid, and butyric acid. Flavors include orange, citrus, berry, apple, mint, grape, apricot, shiso, lemon, grapefruit, peach, banana, tropical, herb, coffee, tea, and the like.

[0035] Furthermore, when producing the fermented product or food or drink of this embodiment, bacteria other than the lactic acid bacteria of this embodiment can also be used in combination. Examples of such bacteria include Bifidobacterium bacteria such as Bifidobacterium bifidum, Bifidobacterium lactis, and Bifidobacterium longum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus kefir, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus bulgaricus, Lactobacillus delbrueckii, and Lactobacillus pentosa. Examples of such bacteria include Lactobacillus bacteria such as Lactobacillus thermophilus and Lactobacillus plantarum, Streptococcus bacteria such as Streptococcus thermophilus and Streptococcus salivarius, Lactococcus bacteria such as Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris, Enterococcus bacteria such as Enterococcus faecalis, and yeasts belonging to the Saccharomyces cerevisiae and Candida kefir, and the Candida genus.

[0036] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0037] Isolation and culture conditions of lactic acid bacteria in the examples The novel lactic acid bacteria belonging to Leuconostoc suionicum were isolated using the sake lees filtration medium shown below. 200 g of sake lees was added to 800 mL of distilled water, heated over low heat, stirred well until boiling and dissolved. Removed from the heat and cooled to about 50 °C, filtered through a kitchen paper, adjusted to a Brix of 5.0, and autoclaved at 121 °C for 15 minutes for use as a sake lees filtration medium. The fungi attached to the flowers of Prunus jamasakura were brought into contact with the sake lees filtration medium and cultured at 30 °C for 24 hours, and the grown bacteria were isolated. After isolation, the bacteria that could be cultured under the conditions of 30 °C for 24 hours in Lactobacilli MRS Broth (manufactured by Difco) medium autoclaved at 121 °C for 15 minutes were used in subsequent experiments. Such bacteria formed circular white colonies in Lactobacilli MRS Broth (manufactured by Difco) medium. In microscopic observation, the above bacteria were cocci, non-motile, and did not form spores (not shown). The above bacteria showed positive in the Gram staining test, showed no activity in the catalase activity test, and no gas generation was observed (not shown).

[0038] Species identification using 16S rDNA nucleotide sequence The species identification of the above bacteria was performed by the following method based on the 16S rDNA nucleotide sequence. Using the DNA extracted from the cell pellet obtained by centrifuging the culture broth cultured at 30 °C for 24 hours using MRS medium as a template, the full length of the 16S rDNA sequence was amplified by the PCR method, and the nucleotide sequence of the amplification product was determined by the Dye Terminator method. The nucleotide sequence is shown in SEQ ID NO: 1. The obtained nucleotide sequence was searched in the database to identify the bacterial species. As a result, it was confirmed that the nucleotide sequence of the 16S rDNA of the above lactic acid bacteria was 99% identical to the 16S rDNA nucleotide sequences of Leuconostoc mesenteroides and Leuconostoc suionicum, but the bacterial genus could not be identified.

[0039] Whole genome analysis The strain identification of the above-mentioned bacterium was carried out by the following method based on the whole-genome nucleotide sequence. 10 ml of the bacterial culture solution cultured at 30 °C for 18 hours using MRS medium was centrifuged, and DNA was recovered from the obtained cell pellet. The purified DNA was subjected to long-read and short-read sequencing to determine the nucleotide sequence. The obtained nucleotide sequence was searched in a database to identify the bacterial species. As a result, it was confirmed that the whole-genome nucleotide sequence of the above-mentioned lactic acid bacterium was 98.8% identical to the nucleotide sequence of Leuconostoc suionicum. The above-mentioned bacterium was identified as a new strain of lactic acid bacterium belonging to Leuconostoc suionicum, named Leuconostoc suionicum MS009 strain (hereinafter referred to as MS009 strain), and was deposited on December 26, 2023, at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba, Japan. The accession number is NITE AP-04055.

[0040] The sugar assimilation properties of the MS009 strain are shown in Table 1.

[0041]

Table 1

[0042] As the lactic acid bacterium of the comparative example, Leuconostoc mesenteroides JCM6124 [purchased from the RIKEN Tsukuba Institute, BioResource Center, Microbial Materials Development Unit (JCM).] (hereinafter referred to as JCM6124 strain) was used.

[0043] Comparison of the growth properties of the MS009 strain and the JCM6124 strain in heavy water The growth properties of strains MS009 and JCM6124 were compared in 2% rice gruel supernatant (double-boiled). The double-boiled solution was prepared by adding 2% polished rice to tap water, heating at 95 °C for 60 minutes, and filtering through a sterilized metallic mesh. As pre-culture, strains MS009 and JCM6124 were inoculated into MRS liquid medium and cultured at 30 °C for 4 hours under aerobic conditions. The optical density at 600 nm (OD600) value of the pre-cultured bacterial solution was measured, and the solution was inoculated into double-boiled solution, soy milk, and MRS liquid medium respectively so that OD600 = 0.04. After inoculation, each strain was cultured with shaking at 30 °C and 200 rpm under aerobic conditions, and the viable cell count in the culture solution was measured over time. The viable cell count was calculated by serially diluting the culture solution, inoculating it onto MRS agar medium, culturing at 30 °C overnight, and counting the number of colonies grown (standard plate count method). The results are shown in Figure 1.

[0044] Evaluation of adhesion to human intestinal epithelial cells (INT-407 cells) The adhesion of strains MS009 and JCM6124 to INT-407 cells was examined in vitro. INT-407 cells were cultured at 37 °C in a 5% CO2 environment using Roswell Park Memorial Institute 1640 Medium (RPMI-1640 medium) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with fetal bovine serum (FBS) (MP Biomedicals, Ohio, USA) at a final concentration of 10% (RPMI-10%FBS). The cultured INT-407 cells were 5Dilute to a concentration of cells / ml with RPMI-10%FBS, and add 2000 μl to each well of a 6-well plate containing a collagen I-coated cover glass (12φ, 4912-010, AGC Techno Glass Co., Ltd., Shizuoka, Japan). Incubate at 37°C for 24 hours in a 5% CO2 environment. Three strains, MS009 strain and JCM6124 strain, were cultured with shaking at 37°C and 100 rpm for 18 hours in MRS liquid medium, and the cells washed with RPMI-1640 were suspended in RPMI-10%FBS to an OD600 of 1. After 24 hours of preculture, it was confirmed that the INT-407 cells had reached 80% confluence. The supernatant was discarded, and after washing once with PBS, 1800 μl of RPMI-10%FBS was added to each well of the 6-well plate. 200 μl of the bacterial solution was added to each well of the 6-well plate, and the cells were cultured at 37°C for 2 hours in a 5% CO2 environment. After removing the collagen I-coated cover glass from the 6-well plate and washing it three times with PBS, it was fixed with a 2.5% glutaraldehyde solution for 12 hours and post-treated with a 2% osmium tetroxide solution for 30 minutes. A JSM-6610LV scanning electron microscope (JEOL, Tokyo, Japan) was used for imaging. To determine the viable cell count after culture, the same treatment was performed on another plate excluding the collagen I-coated cover glass, and the number of bacteria in the culture solution was calculated by the standard plate count method. The results of the number of bacteria attached per cell are shown in Figure 2. An electron microscope image of the bacteria attached to the cells is shown in Figure 3.

[0045] Experiment on administration of MS009 strain using heavy water to rats Heavy water prepared by the method described above (containing 4.0x10 8 cfu / ml of MS009 strain) was administered to rats, and feces were analyzed. Four-week-old SD rats (n = 14) were divided into an MS009 administration group (n = 7) and a non-administration group (n = 7). Heavy water cultured with the MS009 strain (administration group) and non-inoculated heavy water (non-administration) were each administered for 14 days. After 14 days of administration, samples of the cecal contents of the administration group and the non-administration group were taken and analyzed.

[0046] Analysis of the Bacterial Flora in Rat Cecal Contents Total DNA was extracted from 200 mg of cecal contents collected from rats in the administration group and the non - administration group. The NucleoSpin (registered trademark) DNA Stool kit (MACHEREY - NAGEL, Duren, Germany) was used for the extraction. For the extraction method, according to the manual of the same kit, the sample was placed in a tube containing the attached crushing beads, and shaken at 4,200 rpm for 30 seconds three times with a bead crusher (Yasui Kikai, Tokyo, Japan) to disrupt the bacterial cell wall. The disrupted sample was purified using a spin column to a DNA with a final concentration of 60 - 80 ng / μl. The purified DNA was used as a universal primer targeting the V3 - V4 region (460 bp) of 16S rDNA, Forward Primer (5’TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 3’)(SEQ ID NO: 2) and It was amplified by the Polymerase Chain Reaction method (PCR method) using the Reverse Primer (5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC 3’) (SEQ ID NO: 3). After initial heating (94°C, 60 seconds), PCR was performed with 25 cycles of denaturation (94°C, 15 seconds), annealing (50°C, 20 seconds), and extension (72°C, 30 seconds), followed by a final extension (72°C, 420 seconds) to completion. The amplified DNA was subjected to PCR for tagging and then sequenced using a next-generation sequencer (MiSeq) (Illumina Inc, San Diego, USA) to obtain read data of the nucleotide sequences for each sample. The obtained read data was analyzed using Quantitative Insights Into Microbial Ecology version 2 (QIIME2). After removing primer sequences, quality checking, correcting error sequences, and removing noise using QIIME2, it was classified into clusters (Operational Taxonomic Units: OTUs) with a similarity of 97% or more. The OTUs were matched with the SILVA database (Ver 2021.11) for species attribution, and the composition ratios were calculated for each classification level (Level 1-7) of the bacterial flora. Next, based on the number of OTUs and the phylogenetic tree constructed from the OTUs, alpha diversity analysis (Shannon Index (Shannon), Chao1, Observed OTUs, Faith’s Phylogenetic Diversity (Faith’s PD), Pielou’s Evenness Index (Pielou’s Evenness), Simpson Index (Simpson)) and beta diversity analysis (Jaccard distance, Bray-Curtis distance, Weighted unifrac distance, Unweighted unifrac distance) of the bacterial flora were performed. Furthermore, Linear discriminant analysis effect size (LEfSe) was performed based on the analysis data by Qiime2.The results of the α-diversity of cecal contents in the administration group and the non-administration group are shown in Figure 4. The results of the β-diversity of cecal contents in the administration group and the non-administration group are shown in Figure 5. The results of the LEfSe analysis of cecal contents in the administration group and the non-administration group are shown in Figure 6.

[0047] Measurement of short-chain fatty acid concentration in rat cecal contents Lactic acid, acetic acid, propionic acid, butyric acid, and total short-chain fatty acids in cecal contents collected from rats in the administration group and the non-administration group were measured by high-performance liquid chromatography (HPLC). The cecal contents sampled from rats were weighed to 100 mg, 900 μl of purified water was added and homogenized, and then centrifuged (4 °C, 15,000 rpm, 10 minutes) to obtain the supernatant. The separated supernatant was filtered through a 0.45 μm filter (Merck KGaA, Darmstadt, Germany) to obtain a sample. A volatile fatty acid standard Mix (Sigma-Aldrich Japan KK, Tokyo, Japan) was measured simultaneously as a standard solution. Chromaster (registered trademark) (Hitachi High-Tech Corporation, Tokyo, Japan) was used for the measurement. A Gelpack (registered trademark) GL-C601H-S (Hitachi High-Tech Corporation, Tokyo, Japan) was used as the column. 3 mmol / l perchloric acid water was used as the eluent at a flow rate of 0.5 ml / min, and the absorbance at 440 nm was measured by the post-column method using a BTB solution. The concentrations of lactic acid, acetic acid, propionic acid, butyric acid, and total short-chain fatty acids in the sample were calculated from the measurement curve of the standard solution. The measured lactic acid, acetic acid, propionic acid, butyric acid, and total short-chain fatty acids are shown in Figure 7.

[0048] Measurement of IgA antibody concentration in rat cecal contents The cecal contents of the rats in the administration group and non - administration group were weighed to 100 mg, and 900 μl of PBS containing 50 mM EDTA and 0.1 mg / ml trypsin inhibitor (NACALAI TESQUE, INC., Kyoto, Japan) was added and suspended. After centrifugation (4°C, 10,000 rpm, 10 minutes), the supernatant was collected. The supernatant obtained by centrifugation again (4°C, 10,000 rpm, 10 minutes) was stored at - 80°C until measurement as the fecal extract. 50 μl of the primary antibody (Anti - Rat IgA [α - chain specific] antibody produced in goat) (Merck KGaA, Darmstadt, Germany) diluted to 2 μg / ml with 0.1 M disodium hydrogen phosphate solution was added to each well of a 96 - well microplate (MICROLON half - Area) (Greiner Bio - One, Kremsmunster, Austria) and left standing at 4°C overnight. After the microplate was washed three times with PBS added with 0.05% polyoxyethylene (20) sorbitan monolaurate (PBS / Tween), 50 μl of PBS added with 2% skim milk was added to each well of the microplate and blocked at room temperature for 2 hours. The sample was diluted 10,000 - fold by adding PBS / Tween (2% skim milk - PBS / Tween) added with 2% skim milk to the stored fecal extract (cecal contents·rectal feces). After the blocked microplate was washed three times with 2% skim milk - PBS / Tween, the sample and the standard solution (Rat IgA Isotype Control) (Thermo Fisher Scientific, Waltham, USA) diluted in 8 steps from 0 - 100 ng / ml were added to the microplate at 50 μl / well each and left standing at 37°C for 2 hours.After washing the microplate three times with 2% skim milk-PBS / Tween, 50 μl / well of a secondary antibody (Anti-Rat IgA (α-chain specific)-Alkaline Phosphatase antibody produced in goat) (Merck KGaA, Darmstadt, Germany) diluted to 0.5 μg / ml with 2% skim milk-PBS / Tween was added, and the mixture was allowed to stand at room temperature for 2 hours. The microplate was washed three times with 2% skim milk-PBS / Tween, and 50 μl / well of 4-Nitrophenyl Phosphate (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was spread as a chromogenic solution. After color development at room temperature for 30 minutes, 50 μl / well of 1N NaOH was added to stop the reaction, and then the absorbance at 405 nm was measured with a microplate reader (Multiskan FC) (Thermo Fisher Scientific, Waltham, USA). The measured IgA antibody concentrations in the cecal contents of the administered and non-administered groups of rats are shown in Fig. 8.

[0049] Fig. 1 shows the growth properties of the MS009 strain and the JCM6124 strain in heavy water. The MS009 strain showed good growth properties in heavy water and reached approximately 1.0×10 8 cfu / ml in 4 hours, reaching a maximum of 1.3×10 9 cfu / ml in 24 hours. The related strain JCM6124 could not grow to 1.0×10 8 cfu / ml in 4 hours, and the maximum number of colonies thereafter was 5.8×10 7 cfu / ml at 24 hours and did not grow to 1.0×10 8 cfu / ml or more. It was revealed that the MS009 strain had approximately 20-fold higher growth properties in heavy water compared to the related strain JCM6124.

[0050] Figure 2 shows the adhesiveness of MS009 strain and JCM6124 strain to INT-407 cells. It was revealed that the MS009 strain has significantly higher adhesiveness compared to the related JCM6124 strain. In the electron microscope image of Figure 3, an image with a large number of bacteria attached to the INT-407 cells themselves was confirmed.

[0051] Figures 4, 5, and 6 show the analysis results of the intestinal flora of cecal contents collected in the MS009 strain administration experiment to rats. An increase in α-diversity (Figure 4) was observed in the administration group, and in the β-diversity analysis (Figure 5), the administration group and the non-administration group formed different clusters, revealing that the bacterial composition of the intestinal flora had changed. The LEfSe analysis (Figure 6) revealed that the bacterial group of the class Clostridia containing butyrate-producing bacteria had increased.

[0052] Figure 7 shows the results of the lactic acid and short-chain fatty acid analysis of cecal contents collected in the MS009 strain administration experiment to rats. It was found that the administration of the MS009 strain increased the butyric acid concentration and the total short-chain fatty acid concentration in the cecal contents.

[0053] Figure 8 shows the results of the measurement of the IgA antibody concentration in cecal contents collected in the MS009 strain administration experiment to rats. It was found that the administration of the MS009 strain increased the IgA antibody concentration in the cecal contents.

[0054] The MS009 strain showed high growth ability (about 20 times) in heavy water compared to the related JCM6124 strain and high adhesiveness to INT-407 cells, which are small intestinal epithelial cells. The good growth ability in porridge and high adhesiveness to small intestinal epithelial cells may exhibit high effects as a probiotic food.

[0055] In the administration experiment to rats, it was found that the intestinal flora was changed, and it was revealed that the bacterial group of the class Clostridia containing butyrate-producing bacteria had increased, and it was found that the butyric acid concentration in the cecal contents had increased.

[0056] In addition, it was revealed that the IgA antibody concentration in the cecal contents was increased and that it had an effect of enhancing intestinal immunity.

Claims

1. Lactic acid bacteria that can grow to 1.0×10 8 cfu / ml or more in rice porridge at 30°C for 4 hours.

2. The lactic acid bacterium according to Claim 1, which is deposited under accession number NITE AP-04055.

3. A method for producing a food or drink containing a fermented product, comprising a fermentation step of fermenting a first food material with the lactic acid bacterium according to Claim 1 to obtain a fermented product, and a step of incorporating the fermented product into a second food material.

4. A fermented product obtained by fermenting a food material with the lactic acid bacterium according to Claim 1.

5. A food or drink containing the lactic acid bacterium according to Claim 1.

6. A method for producing a fermented product, characterized by inoculating a food material with the lactic acid bacterium according to Claim 1 and fermenting it.

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