Novel microorganism with excellent ornithine and / or citrulline production capability

Abstract

To provide a novel strain of Tetragenococcus halophilus having superior capability for producing ornithine and citrulline, thereby enabling the efficient production of ornithine and citrulline that are beneficial amino acids, and their application in the manufacture of food compositions, supplements, pharmaceuticals and the like suitable for maintaining or promoting health, including stimulation of growth hormone secretion, regulation of sleep-wake cycles, enhancement of liver function, rejuvenation of fatigued skin, reduction of mental stress, and recovery from physical fatigue.SOLUTION: Provided is a strain of Tetragenococcus halophilus KA12022 (Deposit Number: NITE P-03886).SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a novel microorganism having an excellent ability to produce ornithine and / or citrulline, and more particularly to a novel microorganism Tetragenococcus halophilus KA12022 strain belonging to Tetragenococcus halophilus. The present invention also relates to a food composition containing the novel microorganism, a method for producing ornithine and / or citrulline using the novel microorganism, and a method for producing a food composition containing ornithine and / or citrulline. [Background technology]

[0002] Ornithine is a type of amino acid. In the body, it is one of the substances that make up the urea cycle (or ornithine cycle), which metabolizes ammonia, and is produced by the decomposition of arginine. Ornithine promotes the detoxification of ammonia, which is harmful to the body, by turning the urea cycle. Since ammonia is a substance that inhibits the production of ATP, which is the energy for vital activities, and the production of glucose, which is the main energy that drives the brain, it is thought that ornithine also contributes to smooth energy production by helping with its detoxification. In other words, it has been suggested that ornithine may be useful for maintaining or improving health by ingesting it, such as recovering from fatigue, improving sleep and awakening, improving liver function, improving tired skin, reducing mental stress, and promoting growth hormone secretion. For this reason, it is used as an ingredient in foods and supplements that claim to be high in ornithine.

[0003] Citrulline is also a type of amino acid. In the body, it is one of the substances that make up the urea cycle, and is produced together with phosphate by the reaction of ornithine and carbamoyl phosphate. Citrulline produces nitric oxide (NO) via arginine. Since NO dilates blood vessels and promotes blood circulation, it has been suggested that improving blood flow may help prevent and improve arteriosclerosis, improve sensitivity to cold, build muscle, and recover from fatigue, thereby helping to maintain or improve health. For this reason, citrulline is used as an ingredient in supplements and medicines.

[0004] On the other hand, Tetragenococcus halophilus has the arginine deiminase pathway (ADI pathway) shown in Figure 1, and is known to take up arginine into the bacterial cell, produce ornithine via citrulline, and secrete ornithine outside the bacterial cell (Non-Patent Document 1). Therefore, the present inventors have previously reported a new strain of Tetragenococcus halophilus (KA03033 strain) that highly produces citrulline (Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2022-130013 A [Non-patent literature]

[0006] [Non-Patent Document 1] Jieting Lin et. al., Appl Microbiol Biotechnol. 2020 Oct;104(20):8775-8787. doi: 10.1007 / s00253-020-10868-6.(https: / / pubmed.ncbi.nlm.nih.gov / 32880693 / ) Summary of the Invention [Problem to be solved by the invention]

[0007] However, the KA03033 strain described in Patent Document 1 has high citrulline production ability but insufficient ornithine production ability. Therefore, the present invention aims to provide Tetragenococcus halophilus with better ornithine production ability. In addition, the present invention aims to provide a method for producing ornithine and / or citrulline using the microorganism and a method for producing a food composition containing ornithine and / or citrulline. [Means for solving the problem]

[0008] As a result of intensive research, the present inventors discovered that a microbial strain isolated from a brewery in Nagano Prefecture is a novel microorganism belonging to Tetragenococcus halophilus and has a remarkably high ability to produce citrulline and arginine, and thus completed the following inventions.

[0009] (1) The novel microorganism according to the present invention is Tetragenococcus halophilus KA12022 (Accession Number: NITE P-03886) (in the present invention, may be abbreviated as "KA12022 strain").

[0010] (2) The method for producing ornithine and / or citrulline according to the present invention includes a step of causing the KA12022 strain to produce ornithine and / or citrulline.

[0011] (3) A method for producing a food composition containing ornithine and / or citrulline according to the present invention includes a step of causing the KA12022 strain to produce ornithine and / or citrulline.

[0012] (4) The food composition of the present invention contains the KA12022 strain.

[0013] (5) In the present invention, the food composition containing the KA12022 strain may be miso. Effect of the Invention

[0014] The KA12022 strain can efficiently produce the useful amino acid ornithine, which can be used to manufacture food compositions, supplements, medicines, and other products that are useful for maintaining or improving health, such as promoting growth hormone secretion, improving sleep and awakening, improving liver function, improving tired skin, reducing mental stress, and recovering from fatigue.

[0015] The KA12022 strain can efficiently produce the useful amino acid citrulline, which can be used to produce food compositions, supplements, medicines, and the like that are useful for preventing and improving arteriosclerosis, improving sensitivity to cold, building muscle, recovering from fatigue, and other unhealthy conditions, as well as for maintaining or improving health.

[0016] As shown in the examples below, the KA12022 strain is a salt-tolerant lactic acid bacterium that can grow and be active even in high salt concentrations. Therefore, for example, the KA12022 strain can be added in the production process of food compositions with high salt concentrations, such as miso, soy sauce, and pickles, to produce ornithine and citrulline in the food composition. That is, the KA12022 strain can produce, for example, miso and soy sauce that are high in ornithine and citrulline. With such food compositions, ornithine and citrulline can be ingested on a daily and habitual basis without difficulty. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram showing the arginine deiminase pathway (ADI pathway) and the arginine biosynthetic pathway (AB pathway) reported to be present in Tetragenococcus halophilus. [Diagram 2] 1 is a photograph showing the appearance of colonies that appeared on an agar medium on which PBS containing a microbial specimen was spread. [Diagram 3] This is a table showing the top 10 sequences with the highest homology to the 16S rDNA sequence of the KA12022 strain as a result of a BLAST search in the international base sequence database. [Figure 4] 1 is a bar graph showing the concentrations of arginine derivatives (I), citrulline derivatives (II), and ornithine derivatives (III) in the supernatants obtained when the KA12022 strain or the reference strain was cultured in a medium supplemented with arginine. [Diagram 5] FIG. 1 is a schematic diagram showing the homology of putative carbamoyl transferase genes (THE_06320, THE_06330) in the KA12022 strain and the type strain. [Figure 6] FIG. 1 is a schematic diagram showing the homology of the carbamate kinase genes (THE_05400, THE_05410) in the KA12022 strain and the type strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention will be described in detail below. As shown in Example 1 below, the KA12022 strain was deposited on March 27, 2015 at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under the accession number NITE P-03886, and is therefore available from this institution.

[0019] The KA12022 strain can be cultured or grown in a medium generally used for culturing lactic acid bacteria. An example of such a medium is de Man-Rogosa-Sharpe (MRS) medium. MRS medium is commercially available from a number of manufacturers, and such a commercially available product can be used. Since this strain is salt-tolerant, the medium may contain, for example, about 10 to 15% sodium chloride. Examples of culture conditions include a temperature of 0 to 40°C, preferably 10 to 35°C, stationary culture or shaking culture, a culture period of 60 minutes to several days or longer, and anaerobic or aerobic conditions.

[0020] As shown in the examples below, the KA12022 strain is excellent in producing ornithine and citrulline. Therefore, the KA12022 strain can be used to produce ornithine and citrulline. When producing ornithine and citrulline in the KA12022 strain, it is preferable to place the KA12022 strain in an environment in which arginine and citrulline are present, for example, by adding arginine and citrulline to the medium.

[0021] Ornithine and citrulline produced by the KA12022 strain can be detected by a conventional method for detecting these amino acids, such as a capillary electrophoresis-time-of-flight mass spectrometer (CE TOF / MS) or a liquid chromatography mass spectrometer (LC-MS / MS). As shown in the Examples below, these amino acids can also be derivatized and detected by high performance liquid chromatography (HPLC).

[0022] Ornithine and citrulline produced by the KA12022 strain may be used after separation and purification, or the culture or culture supernatant may be used as is, etc. The produced ornithine and citrulline may be used by being contained in a food composition, a medicine, a supplement, or a food additive.

[0023] In addition, Tetragenococcus halophilus, to which the KA12022 strain belongs, has been used in the brewing of soy sauce and miso, and is a highly safe microorganism with a long history of use in food. For this reason, the KA12022 strain can also be safely used in the production of food compositions, for example by adding it to food raw materials. By using the KA12022 strain in the production process of a food composition to produce ornithine and citrulline, a food composition containing ornithine and citrulline can be produced. In the present invention, examples of the food composition include normal food and drink forms such as health foods such as supplements, confectioneries and beverages, processed foods, and infant foods. Since the KA12022 strain has high salt tolerance, it can also be used in the production process of food compositions with high salt concentrations, such as miso, soy sauce, pickles, miso soup, and soup bases. Food compositions containing the KA12022 strain do not require sterilization or sterilization treatment, and can be safely consumed as is.

[0024] The present invention will be described below based on examples. The technical scope of the present invention is not limited to the characteristics shown by the following examples. EXAMPLES

[0025] Example 1: Isolation and identification of strains (1) Colony isolation On May 2 and 21, 2021, microbial samples were collected from breweries in Nagano Prefecture by wiping with a cotton swab, and the swab was placed in phosphate-buffered saline (PBS) and stored at 4 ° C. Next, a drop of PBS containing the microbial sample was dropped onto a medium (MRS + 10% NaCl agar medium) in which sodium chloride was added to MRS agar medium (composition shown in Table 1) to a final concentration of 10% (w / v), and the medium was streaked and cultured at 30 ° C for 3 days to allow a large number of colonies to appear. Figure 2 shows the state in which the colonies appeared. Next, a single colony was picked up using a toothpick, and a glycerol stock was prepared and stored at -80 ° C. A total of 478 colonies were obtained by this operation. [Table 1]

[0026] (2) Identification of microbial species The glycerol stock of this Example 1(1) was inoculated onto MRS+10% NaCl agar medium and cultured at 30°C for 3 days. Single colonies were picked up with a toothpick, and the full length of the 16S rRNA gene was amplified by colony direct PCR. The sequences of the primers used in PCR are shown below. Forward primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1) Reverse primer (1429R): 5'-GGCTACCTTGTTACGACTT-3' (SEQ ID NO: 2)

[0027] A band was confirmed at around 1500bp by agarose gel electrophoresis for the PCR product obtained, and the DNA was purified. The base sequence was decoded by entrusting it to Eurofins Genomics, Inc. A homology search was performed for the obtained 16S rDNA base sequence using the NCBI Basic Local Alignment Search Tool (BLAST) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to identify the affiliation. As a result, it was revealed that 133 of the 478 strains obtained from single colonies belonged to Tetragenococcus halophilus.

[0028] (3) Examination of metabolic characteristics using arginine / citrulline metabolism screening medium Tetragenococcus halophilus NBRC12172 (sometimes abbreviated as "type strain" in the present invention) was obtained from the Biotechnology Center of the National Institute of Technology and Evaluation (NITE) as a type strain of Tetragenococcus halophilus. Sodium chloride was added to the MRS liquid medium (the composition of the above MRS agar medium except for agar) to a final concentration of 10% (w / v), arginine (FUJIFILM Wako Pure Chemical Industries, Ltd.) or citrulline (FUJIFILM Wako Pure Chemical Industries, Ltd.) to a final concentration of 1% (w / v), and bromocresol purple (pH indicator) was added to a final concentration of 0.006% (w / v) to prepare a medium with a pH of 5.3, which was designated as the "arginine metabolism screening medium" or "citrulline metabolism screening medium." Bromocresol purple is a pH indicator that changes color from pH 5.2 (yellow) to 6.8 (purple). Therefore, in this medium, when the cultured strain metabolizes arginine or citrulline and the pH of the medium increases due to the production of a significant amount of ammonia, etc., the medium turns purple.

[0029] Sodium chloride was added to the MRS liquid medium to a final concentration of 10% (w / v), and this was used as the MRS+10% NaCl liquid medium. The MRS+10% NaCl liquid medium was placed in a screw-cap tube, and the glycerol stocks of the 133 strains that were found to belong to Tetragenococcus halophilus in this Example 1 (2) were inoculated and pre-cultured at 30°C for 3 days. As a comparative control, the reference strain was also inoculated into a tube containing MRS+10% NaCl liquid medium and pre-cultured under the same conditions. These media were replaced with "arginine metabolism screening medium" or "citrulline metabolism screening medium", and further cultured at 30°C for 3 days, after which the color of the medium (yellow or purple) was visually confirmed.

[0030] As a result, when the type strain was cultured in either the "arginine metabolism screening medium" or the "citrulline metabolism screening medium," the medium turned purple. In other words, it was revealed that the type strain metabolizes both arginine and citrulline, causing an increase in the pH of the medium through ammonia production, etc.

[0031] On the other hand, of the 133 strains that were identified as belonging to Tetragenococcus halophilus, 20 strains remained yellow in both the "arginine metabolism screening medium" and the "citrulline metabolism screening medium". In other words, it was revealed that these 20 strains did not cause an increase in the pH of the medium in either arginine metabolism or citrulline metabolism. There were several possible reasons for this, such as the fact that they do not produce ammonia during arginine metabolism or citrulline metabolism, or that although they do produce ammonia, they produce more lactic acid than the ammonia produced.

[0032] Therefore, the KA12022 strain, which is one of the strains that has the property of not significantly increasing the pH of the medium in either arginine-containing or citrulline-containing medium, was deposited as "Tetragenococcus halophilus KA12022" at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on April 19, 2023, under the accession number NITE P-03886.

[0033] The 16S rDNA base sequence of the KA12022 strain is shown in SEQ ID NO: 3. The BLAST search results for the KA12022 strain are shown in Figure 3. As shown in Figure 3, the 16S rDNA base sequence of the KA12022 strain (SEQ ID NO: 3) showed high homology of 99.32% to 99.44% with the 16S rDNA base sequence of Tetragenococcus halophilus at a query cover of 100%. [SEQ ID NO:3] CAAGATGAGA GTTTGATCCT GGCTCAGGAC GAACGCTGGC GGCGTGCCTA ATACATGCAA GTCGAACGCT GCTTAAGAAG AAGCTTCGGT TTTTTCTTAA GCGGAGTGGC GGACGGGTGA GTAACACGTG GGGAACCTAT CCATCAGCGG GGGATAACACAG TTGGATCATGCATGCATGG TTTTTTTCAC CTGAAAGAAA GCTCAAAGGC GCTTTACAGC GTCACTGATG GCTGGTCCCG CGGTGCATTA GCCAGTTGGT GAGGTAACGG CTCACCAAAG CAACGATGCA TAGCCGACCT GAGAGGGTGA TCGGCCACAC TGGGACTGAG ACACGGCCCA GGAGGAGGGACC TTCGGCAATG GACGCAAGTC TGACCGAGCA ACGCCGCGTG AGTGAAGAAG GTTTTCGGAT CGTAAAGCTC TGTTGTCAGC AAAGAACAGG AGAAAGAGGA AATGCTTTTT CCATGACGGT AGCTGACCAG AAAGCCACGG CTAACTACGT GCCAAGCCGAGCTAGCC GAGGTTAGTCCA GGATTTATTG GGCGTAAAGC GAGCGCAGGC GGTGATTTAA GTCTGATGTG AAAGCCCCCA GCTCAACTGG GGAGGGTCAT TGGAAACTGG ATCACTTGAG TGCAGAAGAG GAGAGTGGAA TTCCATGTGT AGCGGTGAAA TGCGTAGATA TATGGAGAGGAGGAGGAGGGGGG CTCTGGTCTG TAACTGACGC TGAGGCTCGA AAGCGTGGGT AGCAAACAGG ATTAGATACC CTGGTAGTCC ACGCCGTAAA CGATGAGTGC TAAGTGTTGG AGGGTTTCCG CCCTTCAGTG CTGCAGTTAA CGCATTAAGC ACTCCCTG GGGAGTGCCTGACCGCAAGGTT GAAACTCAAA GGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT TTAATTCGAA GCAACGCGAA GAACCTTACC AGGTCTTGAC ATCCTTTGAC CGCCCTAGAG ATAGGGTTTC CCCTTCGGGG GCAAAGTGAC AGGTGGTGCA TGGTTGTCGT CAGCTCGTGT CGTGAGATGT TGGGTTAAGT CCCGTAACGA GCGCAACCCT TATTGTTAGT TGCCAGCATT GAGTTGGGCA CTCTAGCAAG ACTGCCGGTG ACAAACCGGA GGAAGGCGGG GATGACGTCA AATCATCATG CCCCTTATGA CCTGGGCTAC ACACGTGCTA CAATGGGAAG TACAACGAGC AAGCCAAGCC GCAAGGCCTA GCGAATCTCT GAAAGCTTCT CTCAGTTCGG ATTGCAGGCT GCAACTCGCC TGCATGAAGC CGGAATCGCT AGTAATCGCG GATCAGCATG CCGCGGTGAA TCCGTTCCCG GGCCTTGTAC ACACCGCCCG TCACACCACG AGAGTTTGTA ACACCCAAAG TCGGTGCGGC AACCCTTAGG GGAGCCAGCC GCCTAAGGTG GGACGAATGA TTGGGGTGAA GTCGTAACAA GGTAGCCGTA TCGGAAGGTG CGGCTGGATC ACCTCCTTT

[0034] <Example 2> Evaluation of the ability to produce citrulline and ornithine Arginine (FUJIFILM Wako Pure Chemical Industries, Ltd.) was added to the MRS+10% NaCl liquid medium to a final concentration of 1% (w / v), and this was used as an arginine medium. The MRS+10% NaCl liquid medium was placed in a screw-cap tube, inoculated with the glycerol stock of the KA12022 strain of Example 1 (1), and precultured at 30°C for 3 days. As a comparative control, the reference strain was also inoculated into a tube containing MRS+10% NaCl liquid medium and precultured under the same conditions. These media were replaced with arginine medium, and further cultured at 30°C for 3 days. The mixture was centrifuged at 3000 x g for 20 minutes, and the culture supernatant was collected. Dabsyl chloride was added to the culture supernatant, bound to the amino acids contained in the culture supernatant, and derivatized. As a comparative control, dabsyl chloride was added to an arginine medium not used for culture in the same manner, and bound to the amino acids contained in the medium to derivatize. These were subjected to HPLC to measure the concentrations (peak areas) of the arginine derivatives, citrulline derivatives and ornithine derivatives, the results of which are shown in FIG.

[0035] As shown in FIG. 4(I), the arginine derivative concentration in the culture supernatant of the standard strain was slightly lower than that in the arginine medium, whereas it was significantly lower in the culture supernatant of the KA12022 strain. In other words, the KA12022 strain consumed almost all of the arginine. Also, as shown in FIG. 4(II), the citrulline derivative concentration was below the measurement limit in both the culture supernatant of the standard strain and the arginine medium, whereas it was significantly higher in the culture supernatant of the KA12022 strain. In other words, the KA12022 strain produced a large amount of citrulline and secreted it outside the bacterial cell. Also, as shown in FIG. 4(III), the ornithine derivative concentration was below the measurement limit in the arginine medium, and was slightly higher in the culture supernatant of the standard strain than that in the arginine medium, whereas it was significantly higher in the culture supernatant of the KA12022 strain. The ornithine derivative concentration in the culture supernatant of the KA12022 strain was about 7.5 times that of the standard strain. That is, the KA12022 strain produced a large amount of ornithine and secreted it outside the cell.

[0036] These results revealed that the KA12022 strain is a novel strain with unique properties, with an extremely high ability to produce citrulline and ornithine. In addition, a large amount of citrulline was detected in the culture supernatant of the KA12022 strain, suggesting the possibility that citrulline and arginine may be produced by a pathway other than the arginine deiminase pathway (in which citrulline is not secreted outside the bacterial cell).

[0037] <Example 3> Evaluation of arginine metabolism-related genes The whole genome sequence of the type strain has been identified and is available (Accession No.: AP012046:Genome). Therefore, the sequences of the KA12022 strain and the type strain were compared and evaluated for genes related to arginine metabolism (numbers 1 to 10 shown in Table 2 below). Specifically, first, the whole genome analysis of the DNA of the KA12022 strain using long-read sequencing by PacBio (registered trademark) Sequel was entrusted to Macrogen. Next, the base sequence and amino acid sequence of each gene of the type strain were used as a query to examine the homology with that of the KA12022 strain using the software SnapGeneR version 6.0.7 and GENETYX-MAC version 18.0.5. The results of numbers 1 to 6 are shown in Table 2, the results of numbers 7 to 8 are shown in FIG. 5, and the results of numbers 9 to 10 are shown in FIG. 6. [Table 2]

[0038] As shown in Table 2, the homology of the KA12022 strain to the type strain sequence for argininosuccinate synthase (argG, TEH_12490) was low, at 82% for the nucleotide sequence and 90% for the amino acid sequence. The homology of the KA12022 strain to the type strain sequence for argininosuccinate lyase (argH, TEH_12480) was low, at 87% for the nucleotide sequence and 90% for the amino acid sequence. The genes for carbamate kinase (arcC, THE_01270), putative transporter (THE_01260), ornithine carbamoyltransferase (THE_01280), and carbamate kinase (arcC, THE_06340) were all deleted in the KA12022 strain.

[0039] Furthermore, as shown in Figure 5, putative carbamoyl transferase no. 7 (THE_06320) and putative carbamoyl transferase no. 8 (THE_06330) were separate fragments in the type strain, whereas they existed as a single gene in the KA12022 strain.

[0040] As shown in Figure 6, carbamate kinase number 9 (THE_05400) and carbamate kinase number 10 (THE_05410) were separate fragments in the type strain, and were also cleaved in the KA12022 strain and present as two fragments, but the cleavage points were different from those of the type strain.

[0041] These results demonstrated that the KA12022 strain is a novel strain that also has a unique gene set related to arginine metabolism.

Claims

1. Tetragenococcus halophilus KA12022 strain (Tetragenococcus halophilus KA12022) (Accession number: NITE P-03886).

2. A method for producing ornithine and / or citrulline, comprising a step of causing the KA12022 strain according to claim 1 to produce ornithine and / or citrulline.

3. A method for producing a food composition containing ornithine and / or citrulline, comprising a step of causing the KA12022 strain according to claim 1 to produce ornithine and / or citrulline.

4. A food composition comprising the KA12022 strain described in claim 1.

5. The food composition of claim 4, wherein the food composition is miso.

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