Novel halophilic lactic acid bacteria, and method for producing recovery amino acids and food compositions using the same

Abstract

To provide a strain that allows mass-production of recovering amino acids.SOLUTION: The present invention discloses the halophilic lactic acid bacteria, Tetragenococcus halophilus KA03033 strain (NITE P-03264).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a novel Tetragenococcus halophilus strain that produces recovery-based amino acids at high concentrations and a method for producing food compositions such as miso using the same.

Background Art

[0002] Miso is one of the fermented foods and is produced by mixing grains such as soybeans, rice, and wheat with salt and koji and fermenting them. The fermentation of miso is closely related to microorganisms, and it is known that the growth environment of microorganisms varies depending on the climate and water of the land.

[0003] First, koji mold is propagated and fermented on steamed rice, wheat, soybeans, etc. to decompose proteins and starches and produce amino acids and sugars. Although koji mold is inactivated by salt, lactic acid fermentation and alcohol fermentation occur due to the action of the enzymes of koji mold and lactic acid bacteria and yeast added for aging, adding flavor and aroma to miso.

[0004] Since a high concentration of salt is added to miso, most microorganisms cannot be active. For example, halotolerant yeast Zygosaccharomyces rouxii and halotolerant (halophilic) lactic acid bacterium Tetragenococcus halophilus ( Tetragenococcus halophilus ) are known to be used for the aging of miso.

[0005] Patent Document 1 discloses a method for producing miso containing ornithine, and miso containing ornithine is produced using Lactobacillus brevis ( Lactobacillus brevis ) 9E53 found from the leaves of cabbage. However, in Patent Document 1, the salt concentration is 7% by weight or less, that is, the production of reduced-salt miso is described, and Lactobacillus brevis 9E53 cannot grow at the normal salt concentration of miso (11 to 12% by weight), and there is a problem that it is limited to use at the stage before adding salt and yeast. Also, nothing is described regarding citrulline.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a strain capable of producing a large amount of recovery amino acids even in miso with normal salt concentration.

Means for Solving the Problems

[0008] In one aspect of the present invention, a new halophilic bacterium capable of converting arginine to citrulline under normal salt concentration is provided. In another aspect of the present invention, a method for producing a food composition such as miso having a high content of citrulline, which uses a new halophilic bacterium capable of converting arginine to citrulline, is provided.

[0009] In another aspect of the present invention, a method for producing citrulline in the presence of arginine, which is characterized by using a strain capable of converting arginine to citrulline, is provided. In still another aspect of the present invention, a food composition such as miso containing citrulline produced by the above halophilic bacterium is provided.

[0010] That is, the present invention relates to the following. (1) Halophilic lactic acid bacterium Tetragenococcus halophilus ( Tetragenococcus halophilus ) Strain KA03033 (NITE P-03264). (2) A method for producing a food composition containing recovery amino acids, which comprises performing lactic acid fermentation using a lactic acid bacterium capable of converting arginine to citrulline during the food production process. (3) The method according to (2), wherein the food composition is miso. (4) The method according to (2) or (3), wherein the lactic acid bacterium is Tetragenococcus halophilus KA03033 strain. (5) A food composition produced by the method according to any one of (2) to (4). (6) A method for producing recovery amino acids, which comprises growing a halophilic lactic acid bacterium, Tetragenococcus halophilus KA03033 strain, in the presence of arginine. (7) The method according to (6), wherein the recovery amino acid is citrulline. (8) A food composition comprising citrulline produced by the method according to (7) as an active ingredient. (9) The food composition according to (8), wherein the food composition is miso.

Advantages of the Invention

[0011] The novel halophilic Tetragenococcus halophilus strain of the present invention can produce recovery amino acids, particularly citrulline, at a high concentration.

Brief Description of the Drawings

[0012]

Figure 1

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Embodiments for Carrying Out the Invention

[0013] The halophilic Tetragenococcus halophilus strain of the present invention can produce recovery amino acids, particularly citrulline, at high concentrations. In addition to citrulline, it can also produce ornithine.

[0014] The halophilic Tetragenococcus halophilus strain of the present invention is, for example, strain KA03033, which has been deposited with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NPMD), Biotechnology Center, at the deposit number of NITE P-03264 on September 11, 2020. The KA03033 strain of the present invention is a Gram-positive coccus and has microbiological characteristics such that it can be cultured in a medium containing about 10 to 20% NaCl.

[0015] In the present invention, the recovery amino acids refer to citrulline or ornithine. Citrulline is a type of amino acid and is expected to maintain the body temperature of hands when feeling cold as a functional food. It is sold as a supplement or pharmaceutical for the purpose of improving blood flow, alleviating arteriosclerosis, enhancing muscles, and recovering from fatigue. Ornithine is also a type of amino acid and is one of the substances constituting the urea cycle (or ornithine cycle). It is known that the intake of ornithine improves liver function, and as a functional food, it is also expected to relieve fatigue caused by mental stress and reduce fatigue and drowsiness when waking up.

[0016] Tetragenococcus halophilus has an arginine deiminase pathway. As follows, it is known that arginine is converted to citrulline by the arcA enzyme and then to ornithine by the arcB enzyme (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 / )).

Chemical formula

[0017] The KA03033 strain of the present invention is considered to have a high ability to convert arginine to citrulline, for example, due to the high growth rate of the strain and the high expression of the arcA enzyme expressed by the KA03033 strain. On the other hand, the expression of the arcB enzyme is suppressed or deficient, and it is considered to have a high ability to accumulate citrulline in the cells.

[0018] In the present invention, the "normal salt concentration" refers to the concentration of salt in miso production. In the present invention, the normal salt concentration in miso production is, for example, 10 to 13% by weight, 11 to 12% by weight, and the low salt concentration in miso production refers to, for example, 7% by weight or less.

[0019] In the present invention, "production at a high concentration" and "high production" refer to high productivity and production amount for recovery amino acids such as citrulline or ornithine, and "high content" refers to a high content. "Production at a high concentration" and "high production" mean that, for example, in the production of miso, the production of recovery amino acids is such that the amount of recovery amino acids per 20 g of miso (one cup of miso soup) is, for example, 20 mg or more, 30 to 40 mg. The produced recovery amino acids can be detected, for example, by capillary electrophoresis-time of flight mass spectrometer, high performance liquid chromatography mass spectrometer.

[0020] The method for producing miso of the present invention comprises a koji-making step of adding koji mold to steamed raw materials such as soybeans, rice, and wheat and culturing to obtain koji; a charging step of mixing the koji obtained in the koji-making step with steamed soybeans, rice, wheat, etc., salt, water, and other optional materials in a desired ratio to obtain a koji-mixed material; and a fermentation and aging step of fermenting and aging the koji-mixed material to obtain a fermented product.

[0021] The method for producing miso of the present invention is characterized in that a halophilic Tetragenococcus halophilus strain, for example, the KA03033 strain is added in the charging step or the fermentation and aging step. In the method for producing miso of the present invention, the salt concentration in the charging step may be a low concentration or a normal concentration. Since the halophilic Tetragenococcus halophilus strain can grow at the normal salt concentration of miso (for example, 11 to 12% by weight), the method for producing miso of the present invention can be carried out regardless of whether it is for producing reduced-salt miso or miso with a normal salt concentration, and miso containing a high content of recovery amino acids can be produced. The production method of the present invention also includes a method for producing a food composition other than miso. For example, using a halophilic Tetragenococcus halophilus strain such as strain KA03033, a food composition can be produced by lactic acid fermentation under anaerobic conditions or aerobic conditions. The food composition is not limited as long as it is produced through the lactic acid fermentation process, and examples include soy sauce and pickles.

[0022] In addition, the halophilic Tetragenococcus halophilus strain of the present invention can be used for the production of recovery amino acids in the presence of arginine. The culture conditions of the halophilic Tetragenococcus halophilus strain of the present invention are not particularly limited, but it is preferably cultured at 0°C to 40°C under anaerobic conditions or aerobic conditions, and more preferably cultured at 10°C to 30°C. As described above, since strain KA03033 can produce citrulline at a high concentration in the presence of arginine, the obtained citrulline can be added to the food composition. The KA03033 strain of the present invention is considered to have a high ability to convert arginine into citrulline due to the high growth rate of the strain itself or the high expression of the arcA enzyme expressed by the KA03033 strain. The addition amount of the recovery amino acids in the food composition can be, for example, 100 mg to 500 mg. In addition, the halophilic Tetragenococcus halophilus strain of the present invention can also be provided as a production kit for food compositions such as miso, which includes grains such as soybeans, salt, and koji mold.

[0023] The recovered amino acids produced by the Tetragenococcus halophilus strain of the present invention can be incorporated into food compositions after separation and purification, or provided as supplements. The food compositions of the present invention can be used, for example, as foods that support sound sleep and foods that maintain the body temperature of the hands when feeling cold. Foods include health foods, functional foods, dietary supplements, health functional foods (foods for specified health uses, foods with functional claims, and foods with nutritional functions) and supplements. Examples of foods include processed foods such as miso soup and soup, and foods containing high concentrations of salt such as miso, soy sauce, and pickles.

Example

[0024] The present invention will be further specifically described by the following examples, but the technical scope of the present invention shall not be limited to these examples.

[0025] Example 1: Metabolome analysis of miso In order to comprehensively analyze the metabolites present in miso, metabolome analysis was performed. The metabolites contained in the sample were extracted using methanol and comprehensively measured and quantified using a capillary electrophoresis-time of flight mass spectrometer. The results are shown in Table 1 below.

Table 1

[0026] Example 2: Analysis of bacterial flora before and after ripening of miso from KA Co., Ltd. Next, for the miso of Company KA containing a high content of ornithine, a bacterial flora analysis was performed. Using the DNA extracted from the sample as a template, the V3-V4 region in the 16S rRNA gene of bacteria was amplified by PCR, and the sequences of the amplified products were comprehensively decoded using a next-generation sequencer. The obtained sequence data was analyzed using a program for microbial community analysis (QIIME, Caporaso JG. et al. QIIME allows analysis of high-throughput community sequencing data. Nature methods. 2010; 7(5): 335-336.), and the types and proportions of bacteria contained in the sample were estimated. The samples used were as follows.

Table 2

[0027] Example 3: Isolation of microorganisms from ornithine-rich miso The isolation of Tetragenococcus halophilus was carried out by culturing on MRS agar medium (medium for lactic acid bacteria) with varying salt concentrations (0%, 5%, 10% NaCl) at 30 °C for 3 to 4 days. 192 strains were isolated and preserved. Subsequently, as described in Int J Food Microbiol, 153:269-274, 2012, the strain was inoculated into an arginine metabolism screening medium, and strains capable of metabolizing arginine were selected (Figure 3). After 7 days of culture, strain KA03033 was found as the strain with the highest arginine metabolism rate (Figure 4).

[0028] Example 4: Analysis of the growth rate of strain KA03033 The growth rate of strain KA03033 was compared with that of the lactic acid bacterium reference strain NBRC12172 as the absorbance at a predetermined time. Strain KA03033 shifted to the stationary phase in 48 hours, while strain NBR12172 shifted to the stationary phase in 72 hours. The results are shown in Figure 6. Therefore, the growth rate of strain KA03033 was faster than that of strain NBR12172, suggesting that the arginine metabolism ability was due to the growth rate.

[0029] Example 5: Identification of the bacterial species of strain KA03033 For strain KA03033, amplification and sequence analysis of the 16S rRNA gene were performed. A partial sequence of the 16S rRNA gene sequence of strain KA03033 is shown in SEQ ID NO: 1. When compared with the Nucleotide BLAST database, it was shown to be highly likely to be Tetragenococcus halophilus.

[0030] Example 6: Microbial metabolome analysis Next, strain KA03033, Tetragenococcus halophilus reference strain NBRC12172, and probiotic lactic acid bacteria ( Lactobacillus rhamnosus GG, LGG strains) were subjected to microbial metabolome analysis. The results are shown in Table 3 below.

Table 3

Claims

Claim 1 Halophilic lactic acid bacterium Tetragenococcus halophilus strain KA03033 (NITE P-03264). Claim 2 A method for producing a food composition containing citrulline or ornithine, which comprises performing lactic acid fermentation using Halophilic lactic acid bacterium Tetragenococcus halophilus strain KA03033 (NITE P-03264) that converts arginine to citrulline during the food production process. Claim 3 The method according to claim 2, wherein the food composition is miso. Claim 4 A method for producing citrulline or ornithine, which comprises growing Halophilic lactic acid bacterium Tetragenococcus halophilus strain KA03033 (NITE P-03264) in the presence of arginine. Claim 5 The method according to claim 4, which is a method for producing citrulline.

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