Acquisition method of arginine non-degradable lactic acid bacterium strains

Abstract

To provide an acquisition method of arginine non-degradable lactic acid bacterium strains capable of establishing a method for dominantly growing, arginine non-degradable lactic acid bacterium strains where arginine deiminase path function is short of, for selecting arginine non-degradable lactic acid bacterium strains from various lactic acid bacterium strains, or selecting arginine non-degradable mutation bacterium strains from specific bacterium strains, easily, for efficiently acquiring a fermentation starter which can be used for producing fermented food products such as soy sauce.SOLUTION: The invention is configured to culture lactic acid bacteria in a medium including analogous compound of arginine, for dominantly growing arginine non-degradable lactic acid bacterium strains, for selecting efficiently, lactic acid bacterium strains suitable for producing fermented food products, or acquiring efficiently, arginine non-degradable lactic acid bacteria fermentation starter.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for obtaining an arginine non-decomposing lactic acid bacteria strain. [Background technology]

[0002] Lactic acid bacteria such as Tetragenococcus halophilus are indispensable and useful bacteria in the production of fermented foods such as soy sauce, pickles, and cheese, and they play a role in lowering the pH of the food and imparting its characteristic flavor by producing various components including organic acids. In the production of fermented foods, such as the brewing of soy sauce, it is possible to obtain high-quality soy sauce by appropriately adding a superior strain of these lactic acid bacteria that has favorable properties as a fermentation starter.

[0003] It is known that some lactic acid bacteria have an arginine deiminase pathway, which includes a reaction that converts arginine in the bacterial cell into citrulline or ornithine. Citrulline, a metabolic product of the arginine deiminase pathway, is a precursor of ethyl carbamate, which is known to be carcinogenic, so strains that do not have the arginine deiminase pathway are generally preferred as fermentation starters for the production of fermented foods (Patent Document 1).

[0004] However, many lactic acid bacteria isolated from nature have the arginine deiminase pathway (Non-Patent Document 1). Therefore, when using a strain that does not have the arginine deiminase pathway as a fermentation starter, it was necessary to select a strain that does not have the arginine deiminase pathway and is suitable as a fermentation starter from wild lactic acid bacteria.

[0005] In recent years, it has been reported that it is possible to breed mutant strains that do not have the arginine deiminase pathway using lactic acid bacteria that have the arginine deiminase pathway as parent strains (Non-Patent Document 2). However, with this method, only five strains were successfully bred from 20,000 parent strains, and since it requires a great deal of effort to select the desired strain, it is difficult to say that this method is practical.

[0006] On the other hand, a method is known in which canavanine, an analogue of arginine, is added to an agar medium, yeast is inoculated on the medium, and a canavanine-resistant strain is obtained from the colonies that grow, thereby obtaining a useful strain that has lost the ability to take up arginine into cells (Non-Patent Documents 3 and 4). However, there have been no examples of this method being used for lactic acid bacteria used in the production of fermented foods. [Prior art documents] [Non-patent literature]

[0007] [Patent Document 1] Patent Publication No. 5-227914 [Patent Document 2] Patent Publication 2013-202008 [Non-Patent Document 1] Koichi Iizuka, Masao Ushirosu. Seasoning Science, 1973, 20, 17. [Non-Patent Document 2] Wakinaka & Watanabe, Applied and Environmental Microbiology, 2019, 85, e00208-19. [Non-Patent Document 3] Akita, Journal of the Brewing Society of Japan, 1989, Vol. 84, No. 2, pp. 96-99 [Non-Patent Document 4] Takamine, Shika Kogyo News, April 1996, No. 33, pp. 8-9 Summary of the Invention [Problem to be solved by the invention]

[0008] In other words, the object of the present invention is to establish a method for preferentially growing arginine-non-decomposing lactic acid bacteria strains, such as those that have a functional deficiency in the arginine deiminase pathway, thereby making it easy to select arginine-non-decomposing lactic acid bacteria strains from among various lactic acid bacteria strains, or to obtain arginine-non-decomposing mutant strains from specific strains, thereby obtaining a method for efficiently obtaining a fermentation starter that can be used in the production of fermented foods such as soy sauce. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above problems, and have found that lactic acid bacteria that do not have an arginine synthesis system cannot survive if they lose the ability to take up arginine, and that arginine analogues such as canavanine inhibit the growth of lactic acid bacteria strains that have the arginine deiminase pathway.In addition, the growth of arginine-decomposing lactic acid bacteria strains is not easily inhibited by arginine analogues, so that arginine-decomposing lactic acid bacteria strains can be grown predominantly and efficiently selected by culturing lactic acid bacteria in a liquid medium containing arginine analogues.

[0010] In other words, the present invention relates to an efficient method for selecting lactic acid bacteria strains suitable for producing fermented foods, characterized in that lactic acid bacteria are cultured in a medium containing an arginine-related compound, thereby allowing arginine-non-decomposing lactic acid bacteria strains to grow predominantly, and an efficient method for obtaining an arginine-non-decomposing lactic acid bacteria fermentation starter. Effect of the Invention

[0011] The present invention provides a method for preferentially growing arginine-nondegrading lactic acid bacteria strains, such as those with a functional deficiency in the arginine deiminase pathway, by culturing lactic acid bacteria in a medium containing an arginine analogue. The arginine-nondegrading lactic acid bacteria strains selected using this method are excellent strains suitable for the production of fermented foods and can be used as fermentation starters, etc. [Brief description of the drawings]

[0012] [Figure 1] Figure 1 shows the results of measuring the absorbance of the culture solution after culturing the NBRC12172 strain and the mutant strain obtained in Example 2 and confirmed to have an insertion sequence transferred to arcD. The amounts of L-arginine and canavanine sulfate added to the medium are shown below the figure. The results are shown as the average value of triplicate experiments, and the error bars indicate ± standard deviation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention relates to a method for obtaining a non-arginine-decomposing lactic acid bacteria strain, comprising: (1) a step of culturing lactic acid bacteria in a liquid medium containing an arginine analog; and (2) a step of selecting an arginine non-decomposing lactic acid bacteria strain from the cultured lactic acid bacteria. The present invention also relates to a method for producing a lactic acid bacteria starter containing an arginine non-decomposing lactic acid bacteria strain by culturing lactic acid bacteria in a liquid medium containing an arginine analog. Regarding.

[0014] The lactic acid bacteria referred to in the present invention are not particularly limited as long as they are a species that can be used to brew fermented foods, but are preferably lactic acid bacteria that can be used to produce fermented seasonings such as soy sauce and miso, and more preferably Tetragenococcus halophilus.

[0015] In the present invention, the inability of a lactic acid bacteria strain to decompose arginine means that the lactic acid bacteria strain has lost the ability to metabolize arginine due to reasons such as a deficiency in the function of a gene involved in the arginine deiminase pathway or the arginine uptake pathway.

[0016] The arginine analogue in the present invention is not particularly limited as long as it is a compound that can be recognized as a substrate by the arginine / ornithine exchanger of lactic acid bacteria, and among them, canavanine ((2S)-2-amino-4-{[(diaminomethylidene)amino]oxy}butanoic acid) is preferable. The arginine analogue may be used as a salt such as a sulfate salt.

[0017] In the method of the present invention, lactic acid bacteria are cultured in a liquid medium containing an analogue of arginine. This process allows arginine non-decomposing lactic acid bacteria to grow predominantly. At this time, the lactic acid bacteria may be subjected to mutagenic treatment in advance by known methods such as physical methods such as ultraviolet irradiation or chemical methods using various mutagens, while some species or strains, such as those known to have active transposons, may not require mutagenic treatment.

[0018] Furthermore, when an attempt is made to obtain a strain resistant to an arginine analogue by preparing an agar medium or the like containing an arginine analogue and seeding a lactic acid bacteria strain thereon as in the conventional method, the overwhelming majority of strains having the arginine deiminase pathway form colonies, making it impossible to obtain a strain that does not decompose arginine.

[0019] In the method of the present invention, lactic acid bacteria are cultured in a liquid medium containing an analogue of arginine. The composition of the liquid medium is not particularly limited as long as it can culture a non-arginine decomposing strain of the target lactic acid bacteria species and contains an analogue of arginine. For example, when Tetragenococcus halophilus is used as the lactic acid bacteria, a liquid medium containing 0.5% beef extract, 0.5% hypopeptone, 0.5% yeast extract, 0.1% thioglycolic acid, 10% sodium chloride, 0.1% glucose, and 0.1% arginine analogue (e.g., canavanine sulfate) can be used.

[0020] In preparing a liquid medium, for example, the raw materials for the medium are mixed, the pH is adjusted to 7.5 using sodium hydroxide, and the medium is sterilized in an autoclave. The medium composition and preparation method are not limited to those described above, and can be appropriately determined taking into consideration the characteristics of the target lactic acid bacteria.

[0021] By culturing lactic acid bacteria in a liquid medium containing an arginine analogue such as those exemplified above, it is possible to grow predominantly arginine non-decomposing strains.

[0022] In the culture of lactic acid bacteria, the above liquid medium was inoculated with lactic acid bacteria, and the number of bacteria was increased to 10 6 ~10 10 The culture may be continued until the number of cfu / mL of medium is reached. The culture conditions are appropriately adjusted depending on the strain used, the composition of the medium, and the like, but the culture temperature can be set to, for example, 18 to 45° C. The culture time can be selected from, for example, 3, 6, 12, 18, 24, 36, 48, 60, 72, 96, 120, 150, 180, or 240 hours, and any culture time between two of the above values ​​may be set.

[0023] The culture medium in which the arginine non-decomposing strains have grown predominantly by the above-mentioned method can be used as a fermentation starter as it is. Alternatively, the arginine non-decomposing strains can be selected and isolated from the cultured lactic acid bacteria in a conventional manner, and then cultured in pure form to be used as a fermentation starter.

[0024] A known method can be used to select and isolate arginine non-degrading strains from the culture medium, for example, a method in which each strain is cultured in arginine indicator broth containing bromocresol purple, and a strain in which the color of the broth changes from purple to yellow is isolated as an arginine non-degrading strain (Non-Patent Document 2), or a method in which the base sequence of a gene region including an arginine deiminase gene is analyzed to check for the presence or absence of gene disruption due to transposon insertion or the like can be used.

[0025] The lactic acid bacteria strains and fermentation starters obtained by the method of the present invention can be used in the production of various fermented foods, including soy sauce and miso, depending on the species and characteristics of the lactic acid bacteria. For example, when used in the production of soy sauce, the fermentation starter can be added at the start of the preparation of moromi mash or at any stage during the fermentation process of the moromi mash in a process of producing soy sauce according to a conventional method. EXAMPLES

[0026] The present invention will be described below with reference to examples, but the present invention is not limited to these in any way.

[0027] (Example 1) Attempt to obtain arginine non-degrading strain by conventional method

[0028] Agar medium was prepared by adding 10% salt and 0.2% canavanine sulfate to the minimal medium composed of the composition shown in Table 1 below, and dispensed into petri dishes. Tetragenococcus halophilus NBRC12172 strain was inoculated onto the agar medium plate by a conventional method, and then cultured at 30°C for 5 days. As a result, the NBRC12172 strain having the arginine deiminase pathway formed many colonies, and it was not possible to select lactic acid bacteria strains not having the arginine deiminase pathway.

[0029] [Table 1]

[0030] (Example 2) Attempt to obtain arginine non-degrading strain by the method of the present invention A liquid medium was prepared by adjusting the pH of an aqueous solution of 0.5% beef extract, 0.5% polypeptone, 0.5% yeast extract, 0.1% thioglycolic acid, 10% sodium chloride, 0.1% glucose, and 0.1% canavanine sulfate to 7.5 and sterilizing it in an autoclave. Tetragenococcus halophilus NBRC12172 was inoculated into this liquid medium and cultured for 3 days.

[0031] The cultured lactic acid bacteria were appropriately diluted and inoculated onto an LA13 agar plate (1% hypopeptone (Nihon Seiyaku), 0.4% yeast extract, 1% potassium dihydrogen phosphate, 12% salt, 0.5% glucose, 5% dark soy sauce (Yamasa Shoyu), 1.5% agar) to form colonies. 48 of these colonies were isolated and inoculated into arginine indicator broth, and the arginine decomposition ability of each isolated colony was examined using the color of the medium containing bromocresol purple as an indicator according to the method described in Non-Patent Document 2.

[0032] As a result, 41 of the 48 colonies were mutant strains that could not decompose arginine.

[0033] One of these was selected and the DNA sequence of the gene region containing arginine deiminase was analyzed. It was found that the insertion sequence ISTeha4 had been transferred onto the ORF of the arginine-ornithine exchanger gene (arcD), and the DNA sequence also confirmed that a mutation had occurred that caused a loss of arginine hydrolysis.

[0034] Therefore, it was found that the method of the present invention makes it possible to obtain a lactic acid bacteria strain that does not have arginine deiminase with a high probability.

[0035] (Example 3) Attempt to obtain arginine non-degrading strain by conventional method 2 To the minimal medium having the composition shown in Table 1, salt equivalent to 10% (w / v) was further added, and the concentrations of L-arginine and canavanine sulfate were adjusted to the four concentrations shown in Table 2, and each medium was prepared by autoclaving.

[0036] The Tetragenococcus halophilus NBRC12172 strain and the mutant strain obtained in Example 2 and confirmed to have an insertion sequence transferred to arcD (hereinafter, sometimes referred to as the "arcD mutant strain") were inoculated into these four types of media, and after culturing for 3 days, the absorbance at a wavelength of 600 nm was measured. The results are shown in Figure 1.

[0037] [Table 2]

[0038] The NBRC12172 and arcD mutant strains could not grow in a medium that did not contain arginine, suggesting that they do not have an arginine synthesis system. Therefore, it was inferred that they could not survive if they lost the ability to take up arginine. On the other hand, both the NBRC12172 and the arcD mutant strains could grow in the presence of 0.2% L-arginine, and the presence or absence of canavanine sulfate did not affect the growth of the NBRC12172 and arcD mutant strains. The fact that Tetragenococcus halophilus cannot grow in the absence of arginine, but can grow in the presence of arginine, even in the presence of canavanine, shows that attempts to obtain an arginine-incompetent strain using the conventional method for obtaining a canavanine-resistant strain have not been successful.

[0039] (Consideration) The reason why the non-arginine degrading strain grew preferentially in the presence of canavanine was presumably because the arginine degrading strain has a strong activity to metabolize arginine, so that arginine in the bacterial cell is converted to other metabolic products, while canavanine accumulates in the bacterial cell, increasing the ratio of canavanine to arginine.

[0040] It is known that the toxicity of canavanine is exerted by the fact that canavanine is mistakenly incorporated into an amino acid residue that should be composed of arginine during protein synthesis, resulting in a loss of protein function. Therefore, as in the case of arginine-decomposing strains, when the amount of arginine metabolized in the bacterial cell increases and the ratio of canavanine to arginine increases, the toxicity is more pronounced. On the other hand, since arginine-decomposing strains also have the ability to take up arginine, they take up a certain amount of canavanine, but unlike arginine-decomposing strains, the ratio of canavanine does not increase in the bacterial cell, so they do not exert strong toxicity. It was thought that this was the reason why arginine-decomposing strains preferentially grew in the presence of canavanine.

Claims

1. (1) culturing lactic acid bacteria in a liquid medium containing an arginine analogue; (2) selecting a non-arginine decomposing lactic acid bacteria strain from the cultured lactic acid bacteria; A method for obtaining an arginine non-degrading lactic acid bacteria strain.

2. A method for producing a fermentation starter comprising an arginine non-decomposing lactic acid bacteria strain by culturing lactic acid bacteria in a liquid medium containing an arginine analogue.

3. The method according to claim 1 or 2, wherein the arginine analogue is canavanine.

4. The culture in liquid medium was carried out at a bacterial count of 10 ６ ~10 １０ The method according to claim 1 or 2, wherein the method is carried out until cfu / mL of medium is obtained.

5. The method according to claim 1 or 2, wherein the lactic acid bacteria is a lactic acid bacterium belonging to the genus Tetragenococcus.

6. A method for producing a fermented food, comprising using the arginine non-degrading lactic acid bacteria strain obtained according to claim 1 as a fermentation starter.

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