JP2020025506A - Novel lactic acid bacterium and method for producing soy sauce using the same - Google Patents

Abstract

To provide novel soy sauce lactic acid bacteria improving the flavor of soy sauce without producing histamine in soy sauce brewed moromi (unrefined soy sauce), and to provide methods for producing soy sauce using the lactic acid bacteria.SOLUTION: A novel lactic acid bacterium belonging to Tetragenococcus halophilus is used as a soy sauce lactic acid bacterium to produce soy sauce, the novel lactic acid bacterium having no histidine decarboxylase gene (HDC gene), not degrading arginine, degrading aspartic acid, producing alanine, having cohesiveness, and highly producing diacetyl.SELECTED DRAWING: None

Description

  The present invention relates to a novel lactic acid bacterium belonging to Tetragenococcus halophilus. In particular, the present invention relates to a novel soy sauce lactic acid bacterium suitable for soy sauce production. The present invention also relates to a method for producing soy sauce using the lactic acid bacterium and a method for suppressing histamine production in soy sauce production.

  Soy sauce and miso are fermented foods that are indispensable for Japanese meals. In the production thereof, three kinds of microorganisms, koji mold, lactic acid bacteria and yeast, are involved, and high-quality and good-tasting soy sauce and miso are brewed while coexisting with each other. Especially during soy sauce production, sugar and amino acids are produced by enzymes derived from koji mold, fermentation of lactic acid is produced by lactic acid bacteria, and scent is formed by yeast during ripening and moromi. Is brewed. Lactic acid bacteria, among others, maintain the growth environment of soy sauce yeast by accumulating lactic acid in moromi and lowering the pH, and show various assimilation properties for sugars and amino acids, soy sauce other than sour sauce derived from lactic acid It is thought to impart various flavors and flavors to soy sauce, and is an important microorganism for soy sauce production.

  Among these three microorganisms, aspergillus and yeast have been developed and provided as excellent bacteria, but lactic acid bacteria, especially in small and medium-sized breweries, still rely on wild bacteria with storage. The problem is that histamine, which may cause allergic symptoms, is accumulated in products by converting histidine into histamine by wild lactic acid bacteria in soy sauce mash. For this reason, a highly safe lactic acid bacterium (excellent lactic acid bacterium) that does not generate unnecessary substances such as histamine is also required for lactic acid bacteria.

  In addition, as an example of the prior literature regarding the manufacturing method of soy sauce lactic acid bacteria and soy sauce, the following Patent Document 1 and Non-Patent Documents 1 to 3 can be cited.

JP-A-2000-245443

Toru Kaneko, "Precautions for reducing histamine and tyramine in soy sauce brewing" Japan Soy Sauce Association 2015 By Setuya Hanwaka, "Notes for small factories on amine reduction," Japan Soy Sauce Association 2016 Tatsuroro Tochikura, "Enhanced Soy Sauce Science and Technology" Japan Brewing Association 1994

  As described above, in recent years, in soy sauce production, histamine has been accumulated in soy sauce due to conversion of histidine into histamine by lactic acid bacteria in moromi. There is an urgent need to clean and use good lactic acid bacteria. In particular, in small- and medium-scale sites where soy sauce production is conventionally performed using wild storage lactic acid bacteria as soy sauce lactic acid bacteria, the use of excellent lactic acid bacteria that do not produce histamine is required.

  The present invention has been developed to solve such a problem, and is a novel lactic acid bacterium belonging to Tetragenococcus halophilus suitable for soy sauce production, which is not capable of converting histidine in soy sauce mash into histamine. The task is to provide Another object of the present invention is to provide a lactic acid bacterium capable of imparting good flavor to soy sauce.

  Another object of the present invention is to provide a fermented food using the lactic acid bacterium and a method for producing soy sauce. Another object of the present invention is to provide a method for suppressing histamine production that may occur in soy sauce production.

  The present inventors have been diligently studying soy sauce moromi, which is owned by many soy sauce factories in Miyazaki Prefecture, and have no ability to convert histidine to histamine (having histamine production) without a histidine decarboxylase gene. No lactobacillus belonging to Tetragenococcus halophilus which is suitable for the production of soy sauce having no good ability and good flavor, and the present lactic acid bacterium is confirmed to be a novel lactic acid bacterium which has not been known before, thereby completing the present invention. Reached.

The invention includes the following embodiments:
(I) Novel lactic acid bacteria (I-1) Lactic acid bacteria belonging to Tetragenococcus halophilus having the following characteristics (a) to (d):
(A) having no histidine decarboxylase gene;
(B) assimilate erythritol, D-xylose, and methyl-α-D-mannopyranoside and do not assimilate D-ribose;
(C) does not decompose arginine,
(D) Decompose asparagine to produce alanine.
(I-2) The lactic acid bacterium according to (I-1), further having the following characteristics (e) and (f):
(E) cohesiveness,
(F) Diacetyl productivity.
(I-3) "MS0204" (Accession No. NITE P-02727), which is a lactic acid bacterium collected from soy sauce moromi and belonging to Tetragenococcus halophilus, or a subculture thereof.
(I-4) The lactic acid bacterium according to any one of (I-1) to (I-3), which is a soy sauce lactic acid bacterium.

(II) Fermented foods (II-1) Fermented foods using the lactic acid bacteria described in any of (I-1) to (I-4).
(II-2) The fermented food according to (II-1), wherein the fermented food is soy sauce or miso.
(II-3) The fermented food according to (II-2), wherein the soy sauce is fried soy sauce.

(III) A method for producing soy sauce (III-1) A method for producing soy sauce, wherein the lactic acid bacteria described in any of (I-1) to (I-4) are used as lactic acid bacteria for soy sauce in the process for producing soy sauce. .

(IV) Method for suppressing histamine production in soy sauce production (IV-1) In the soy sauce production process, lactic acid bacteria described in any of (I-1) to (I-4) are used as soy sauce lactic acid bacteria, A method for suppressing histamine production in soy sauce production.

  The lactic acid bacterium of the present invention is a lactic acid bacterium belonging to Tetragenococcus halophilus characterized by having no histidine decarboxylase gene, and has characteristic saccharide assimilation properties and amino acid assimilation properties. Therefore, the production of histamine can be suppressed, and the histamine can be suitably used for production of highly flavored soy sauce.

A region specific to the histidine decarboxylase (HDC) gene by PCR method for various soy sauce lactic acid bacteria (the soy sauce lactic acid bacteria of the present invention [MS0204], existing strains [NBRC12172, NBRC100498, NBRC100726, NBRC100727, 3737, NBRC100499]) Figure 3 shows a microchip electrophoresis pattern of the amplified product. In the figure, from the left lane, Lane1-3: ladder, Lane4: MS0204 , Lane5: NBRC12171, Lane6: NBRC100498, Lane7: NBRC100726, Lane8: NBRC100727, Lane9: 3737, Lane10: NBRC100499, Lane11: negative control (dH 2 0) , Lane12: shows the result of positive control. The band observed in the positive control is the band corresponding to the HDC gene. It is a figure which shows the arginine metabolism ability (Arg → Orn) in various soy sauce lactic acid bacteria (the soy sauce lactic acid bacteria [MS0204] of the present invention, existing strains [NBRC12172, NBRC100498, NBRC100726, NBRC100727, 3737]). In the figure, “Arg medium” means an arginine-containing MRS medium cultured without inoculating soy sauce lactic acid bacteria. The vertical axis indicates the total amino acid concentration (mg / 100 mL) of arginine (Arg) and ornithine (Orn) in the culture. In each bar graph, the light color indicates the arginine concentration, and the dark color indicates the ornithine concentration. It is a figure which shows the aspartic acid metabolism ability (Asp → Ala) in various soy sauce lactic acid bacteria (the soy sauce lactic acid bacteria [MS0204] of the present invention, existing strains [NBRC12172, NBRC100498, NBRC100726, NBRC100727, 3737]). In the figure, “Asp medium” means an aspartic acid-containing MRS medium cultured without inoculating soy sauce lactic acid bacteria. The vertical axis shows the total concentration (mg / 100 mL) of the amino acids of aspartic acid (Asp) and alanine (Ala) in the culture. In each bar graph, the light color indicates aspartic acid concentration, and the dark color indicates alanine concentration. It is a graph which shows the distribution of filter paper transmittance | permeability of 218 strains of soy sauce lactic acid bacteria isolate | separated from soy sauce moromi. For lactic acid bacteria isolated from soy sauce moromi (MS0204, selected strains MS0304, MS0305, MS1308, MS6501, MS6504), diacetyl in a medium without citric acid (light-colored bar graph) and a medium with 0.3% citric acid (dark-colored bar graph) It is a graph which shows productivity. The vertical axis indicates the diacetyl concentration (μg / L) in each medium. The pH change in the brewing process of the soy sauce moromi produced using each soy sauce lactic acid bacterium (the soy sauce lactic acid bacterium [MS0204] of the present invention, a commercially available soy sauce lactic acid bacterium) is shown. The change in pH of soy sauce moromi produced without using soy lactic acid bacteria is also shown (no addition). The change of the amino acid content in the brewing process of the soy sauce moromi produced using each soy sauce lactic acid bacterium (the soy sauce lactic acid bacterium [MS0204], a commercially available soy sauce lactic acid bacterium) is shown. The changes in the amino acid content of soy sauce moromi produced without using soy sauce lactic acid bacteria are also shown (no addition). (A) shows a change in the content of arginine. (B) Changes in the content of ornithine are shown. The change of the amino acid content in the brewing process of the soy sauce moromi produced using each soy sauce lactic acid bacterium (the soy sauce lactic acid bacterium [MS0204], a commercially available soy sauce lactic acid bacterium) is shown. The changes in the amino acid content of soy sauce moromi produced without using soy sauce lactic acid bacteria are also shown (no addition). (A) shows a change in the content of aspartic acid. (B) shows the change in the content of alanine. (A) LC / MS / MS chromatogram of soy sauce moromi after fermentation with soy sauce lactic acid bacteria of the present invention for 90 days, (B) LC / MS / MS of soy sauce moromi fermentation without soy sauce lactic acid bacteria for 90 days Each MS / MS chromatogram is shown. The peak near the retention time of 8 minutes observed in (B) corresponds to the histamine peak.

(I) New Lactic Acid Bacteria The lactic acid bacteria of the present invention are lactic acid bacteria belonging to Tetragenococcus halophilus isolated from soy sauce moromi using MRS agar plate medium adjusted to pH 7 by adding 10% sodium chloride according to a conventional method. And at least the following characteristics (a) to (d). The lactic acid bacterium can further have the following characteristics (e) and (f).
(A) having no histidine decarboxylase gene;
(B) assimilate erythritol, D-xylose, and methyl-α-D-mannopyranoside and do not assimilate D-ribose;
(C) does not decompose arginine,
(D) decomposing aspartic acid to produce alanine;
(E) cohesiveness,
(F) Diacetyl productivity.

These characteristics can be evaluated and confirmed by the following tests.
(A) Confirmation of the presence or absence of the histidine decarboxylase gene (HDC gene) in the lactic acid bacterium of the present invention having no histidine decarboxylase gene was performed using hdcA (GenBank: Accession No .: HDC gene of Tetragenococcus halophilus). AB076394.1) can be carried out by preparing primers specific to AB076394.1), performing PCR, and subjecting the resultant to electrophoresis to confirm the presence or absence of an amplification product caused by hdcA. The details can be performed according to the description in Experimental Example 1 (2). The lactic acid bacterium of the present invention has no HDC gene. For this reason, the lactic acid bacteria of the present invention cannot produce histamine using histidine as a substrate.

(B) Evaluation and confirmation of the saccharide assimilation of the lactic acid bacterium of the present invention, which assimilates erythritol, D-xylose, and methyl-α-D-mannopyranoside, and does not assimilate D-ribose, examines carbohydrate metabolism of bacteria. The test can be performed using a commercially available test kit (for example, API50CH [Sysmex BioMerieux Co., Ltd.]). Table 4 of Experimental Example 2 shows the results of evaluating the saccharide assimilation properties of the lactic acid bacterium of the present invention using “MS0204” contained in the lactic acid bacterium of the present invention as an example. As shown in Table 4, it differs from the existing lactic acid bacteria belonging to Tetragenococcus halophilus in that it assimilates erythritol, D-xylose, and methyl-α-D-mannopyranoside, and does not assimilate D-ribose. However, the lactic acid bacteria of the present invention were unique. Therefore, the lactic acid bacteria of the present invention are characterized in that they utilize erythritol, D-xylose, and methyl-α-D-mannopyranoside, and do not use D-ribose, and at least have new properties in these respects. Is recognized as a new lactic acid bacterium.

(C) Confirmation of the presence or absence of arginine metabolizing ability in the lactic acid bacterium of the present invention which does not decompose arginine can be performed by an assimilation test using a medium supplemented with L-arginine. The details can be performed according to the description in Experimental Example 3 (1). The lactic acid bacterium of the present invention does not have arginine metabolism ability (arginine utilization). That is, arginine in a medium (eg, soy sauce moromi) is not used as a substrate to produce ornithine.

(D) Confirmation of the presence or absence of aspartic acid metabolizing ability in the lactic acid bacterium of the present invention, which degrades aspartic acid to produce alanine, can be carried out by an assimilation test using a medium supplemented with L-aspartic acid. The details can be performed in accordance with the description in Experimental Example 3 (2). The lactic acid bacterium of the present invention has aspartic acid metabolizing ability (aspartic acid assimilation). That is, alanine is produced using aspartic acid in a medium (eg, soy sauce moromi) as a substrate. Aspartic acid is a sour component, while alanine is a sweet component. For this reason, the lactic acid bacteria of the present invention are useful as soy sauce lactic acid bacteria for producing soy sauce having a mild taste.

(E) Aggregability The presence or absence of agglutination of the lactic acid bacteria of the present invention is confirmed by determining the filter paper transmittance of a culture obtained by culturing the lactic acid bacteria. The details can be performed as described in Experimental Example 4. The lactic acid bacterium of the present invention has a low filtration permeability of 30% or less, preferably 20% or less, and has high cohesiveness (cohesive lactic acid bacterium). For this reason, the lactic acid bacteria of the present invention are useful as soy sauce lactic acid bacteria capable of producing raw soy sauce with high clarity, that is, high-quality soy sauce.

(F) Diacetyl productivity The confirmation of the presence or absence of diacetyl productivity of the lactic acid bacteria of the present invention is carried out by determining the presence or absence of diacetyl production (diacetyl concentration in the medium) of the culture obtained by culturing the lactic acid bacteria. The details can be performed as described in Experimental Example 5. The lactic acid bacteria of the present invention have the ability to produce diacetyl (aggregating lactic acid bacteria). For this reason, the lactic acid bacteria of the present invention are useful as soy sauce lactic acid bacteria for producing flavored soy sauce due to the diacetyl flavor (butter flavor, cheese flavor).

Table 1 shows the results of comparing the characteristics of the lactic acid bacteria of the present invention with those of the existing soy sauce lactic acid bacteria. Each of these existing lactic acid bacteria is a lactic acid bacterium belonging to Tetragenococcus halophilus suitable for brewing soy sauce that can grow on a medium having a salt concentration of 18%.
As can be seen from Table 1, the lactic acid bacterium of the present invention is a novel lactic acid bacterium belonging to Tetragenococcus halophilus having characteristics different from those of existing lactic acid bacteria.

  Table 3 of Experimental Example 2 shows the bacteriological properties of the lactic acid bacteria of the present invention having such characteristics. That is, the lactic acid bacterium of the present invention is a lactic acid bacterium belonging to Tetragenococcus halophilus having the mycological properties shown in Table 3, and has the above-mentioned characteristics (a) to (d). A lactic acid bacterium having such a property should be subjected to a screening operation using soy sauce moromi that does not produce histamine as a raw material, referring to the descriptions of the above (a) to (d), specifically, the descriptions of Experimental Examples 1 to 3. Can be isolated. Furthermore, the lactic acid bacteria having the above-mentioned characteristics (e) to (f) are subjected to a screening operation by referring to the descriptions of (e) to (f), specifically, the descriptions of Experimental Examples 4 to 5, in addition to the screening. Can be isolated.

  One of the lactic acid bacteria of the present invention thus separated and identified from soy sauce moromi was designated as “MS0204” for the designation of microorganism (indication of identification), and on June 13, 2018, Kazusa Kamatari, Kisarazu-shi, Chiba, Japan Deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center, which has an address at 2-5-8 (accession number NITE P-02727, notification date June 13, 2018 / notification number 2018-0186) . In the present specification, lactic acid bacteria belonging to Tetragenococcus halophilus having the above-mentioned properties, including such deposited bacteria, are collectively referred to as "lactic acid bacteria of the present invention" or "lactic acid bacteria of soy sauce of the present invention". As shown in Experimental Example 5 (4) described below, soy sauce prepared using the lactic acid bacterium of the present invention has a soft aroma, aroma, well-balanced, good aftertaste, and both taste and aroma in a sensory test. Since good evaluation was obtained, the lactic acid bacteria of the present invention are considered to be excellent soy sauce lactic acid bacteria.

(II) Fermented food and its production method The food targeted by the present invention is the fermented food using the lactic acid bacteria of the present invention described above. As shown in Experimental Example 1 described later, the lactic acid bacteria of the present invention have a property of growing in an environment having a salt concentration of 18% (salt tolerance), and are preferably fermented foods containing salt. Here, the fermented food containing salt may be a fermented food containing salt, and examples thereof include a salt concentration in the range of 3 to 25%, and a fermented food having a salt concentration in the range of 3 to 25%. It is. Examples of fermented foods containing salt in such a range include soy sauce, miso, and pickles. Preferred are soy sauce and miso, and particularly preferred is soy sauce.

  The soy sauce includes dark soy sauce, light soy sauce, tame soy sauce, recharged soy sauce and white soy sauce, all of which are included in the soy sauce of the present invention. The miso includes rice miso, barley miso, bean miso, and combined miso, depending on the raw materials used, and these are all included in the miso of the present invention. The pickles to which the present invention is directed include pickles using lactic acid fermentation, such as pickled pickles, Chinese cabbage kimchi, and sauerkraut.

  Such a salt-containing fermented food can be produced by a method including a step of fermenting a raw material mixture of a salt-containing fermented food using the lactic acid bacterium of the present invention described above according to a standard method.

  The salt concentration in the raw material mixture of the salt-containing fermented food is adjusted to be in the range of 3 to 25% depending on the type of the salt-containing fermented food. For example, when the salt-containing fermented food is soy sauce, it is in the range of 15 to 25% by weight, preferably 15 to 20% by weight / volume. When it is miso, the range is 10 to 15% by weight / weight. In the case of, a range of 3 to 10% by weight /% by weight can be mentioned, but it is not limited thereto.

  Hereinafter, as an example, a method for producing the salt-containing fermented food will be briefly described for each case of soy sauce, miso, and pickles.

(II-1) Method for Producing Soy Sauce The soy sauce of the present invention can be produced according to a conventional method except that the above-mentioned lactic acid bacterium of the present invention is used as a soy sauce lactic acid bacterium (seed fungus). For example, there is a method in which the lactic acid bacteria of the present invention are added together with soy sauce koji and salt water at the time of preparation, and fermentation and aging are performed. The brewing method, the mixed brewing method, and the mixed method may be used, but the brewing method should be adopted in that it has a taste and aroma utilizing the saccharide-utilizing properties and amino acid-utilizing properties inherent to the lactic acid bacteria of the present invention. Is preferred. When soy sauce is produced using the soy sauce lactic acid bacteria of the present invention, the soy sauce lactic acid bacteria are preferably pre-cultured. For example, it is preferable that lyophilized soy sauce lactic acid bacteria be cultured in a liquid medium before use and used as a starter. The liquid medium in this case includes, for example, a medium having a composition of fried soy sauce 25%, sodium chloride 7.5%, and glucose 2% adjusted to pH7.

  The soy sauce moromi obtained by the fermentation aging process is squeezed using a nylon filter cloth and the like, and the obtained squeezed liquid is separated and clarified, if necessary, as an oil and the like, and is then prepared as raw fried soy sauce. . In addition, according to the mixing method, the soy sauce moromi is squeezed and then squeezed and mixed with an amino acid solution to prepare soy sauce. Then, it can be prepared as a soy sauce by standard preparation, burning, and squeezing (filtration).

  As shown in Experimental Example 5 (4) described below, soy sauce prepared using the lactic acid bacterium of the present invention has a good taste and aroma such as a soft aroma, aroma, well-balanced, and good aftertaste, according to a sensory evaluation test. Good evaluations have been obtained on both sides. In addition, as shown in Experimental Example 6, the production of histamine in soy sauce can be suppressed by brewing soy sauce using the lactic acid bacteria of the present invention. In other words, it is considered that soy sauce produced using the lactic acid bacteria of the present invention does not contain histamine, or even if it does contain histamine, the concentration is suppressed to an extremely low concentration.

(II-2) Method for Producing Miso The miso of the present invention can be produced by a method having a step of fermenting a raw material mixture (prepared for miso) for producing miso using the lactic acid bacteria of the present invention described above. . The salt concentration of this miso preparation varies depending on the type and taste of the miso, but is usually in the range of 10 to 15% by weight. This is kept at about 25 to 30 ° C. for 1 to 12 months and fermented and aged. During the fermentation, it is preferable that the miso is dug up once or twice to homogenize the miso (turning back). After the fermentation and ripening, the miso is dug up, blended with two or more types for quality adjustment as necessary, and then commercialized as granulated miso or strained with a chopper to produce commercialized miso.

(II-3) Method for Producing Pickles The pickles of the present invention have a process of fermenting pickle raw materials (for example, in the case of bran pickles, a mixture of rice bran, vegetables and seasonings) using the above-described lactic acid bacteria of the present invention. Can be manufactured. The salt concentration of the pickled raw material varies depending on the type and taste of the pickled food, but is usually in the range of 3 to 10% by weight. This is kept at about 10 to 30 ° C. for 5 to 30 days and fermented to obtain pickles.

(III) Method for suppressing histamine production in soy sauce production As described above, histamine production in soy sauce production can be suppressed by using the lactic acid bacteria of the present invention as soy sauce lactic acid bacteria in the soy sauce production process. Therefore, the present invention provides a method for suppressing the production of histamine that may occur in soy sauce production by using the lactic acid bacterium of the present invention as a soy sauce lactic acid bacterium in a soy sauce production process. This method can be carried out by treating according to a conventional soy sauce manufacturing method, except that the lactic acid bacterium of the present invention is used as a soy sauce lactic acid bacterium (seed bacterium).

  Hereinafter, the configuration and effects of the present invention will be described using experimental examples. However, the present invention is not affected by these experimental examples at all. Unless otherwise specified, the following experiments were performed under atmospheric pressure and room temperature (25 ± 5 ° C.). Unless otherwise specified, “%” means “% by mass”.

Experimental example 1
Among soy sauce moromi obtained from a soy sauce plant in Miyazaki Prefecture, a lactic acid bacterium having no histidine decarboxylase gene (HDC gene) was found from soy sauce moromi not producing histamine, and the lactic acid bacterium was isolated and identified.

(1) Isolation and Identification of Lactic Acid Bacteria Among soy sauce mashes, soy sauce mash in which histamine was not produced was spread on a sterilized plate medium (MRS medium, pH 7) for lactic acid bacteria culture containing 10% sodium chloride (see Table 2).

  This was cultured under anaerobic conditions at 30 ° C. for 4 days, and the white colony that formed a halo was subjected to Gram staining and microscopic examination to confirm that it was a lactic acid bacterium having a gram positive and quadrangular shape. Furthermore, catalase activity, presence or absence of gas generation, fermentation format, growth at 15 ° C. and 45 ° C., growth under acidic and weakly alkaline conditions, and growth under 18% high salt concentration conditions were evaluated. And the mycological properties described in Table 3.

  A homology search was performed on the base sequence of the 16S rRNA gene of the lactic acid bacterium using Micro SEQ microorganism identification software v3.0 (Applied Biosystems, U.S.), which is a gene database. As a result, the homology with the known Tetragenococcus halophilus registered in the database was 100%. As another identification method, primers (193f: 5'-AGCTCAAAGGCGCTTTAC-3 '[SEQ ID NO: 1], 480r: 5'-TTCTGGTCAGCTACCGTC which amplify a region specific to 16S rDNA of Tetragenococcus halophilus were used as another identification method. -3 '[SEQ ID NO: 2]) (A. Juste', B. Lievens, M. Klingeberg, CW Michiels, TL Marsh, KA Willems: Food Microbiology, 25, 413). -421 (2008)). From these results, the bacteria isolated from the soy sauce moromi were identified as lactic acid bacteria belonging to Tetragenococcus halophilus (lactic acid bacteria of soy sauce). These fungi are stored in a state that they can be distributed at the Miyazaki Prefectural Food Development Center located at 16500-2 Higashigami Naka, Sadohara-cho, Miyazaki City, Miyazaki Prefecture, and one of them is transferred to Kazusa, Kisarazu City, Chiba Prefecture, Japan. Entered into the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (NPMD), which has an address at 2-5-8, Kamatari, on June 13, 2018 (Date of Deposit) under the name "Indication of Microbial Identification: MS0204". Deposited in Japan (Accession number: NITE P-02727).

(2) Confirmation of the histidine decarboxylase gene (HDC gene) It is known that a lactic acid bacterium belonging to Tetragenococcus halophilus (soy sauce lactic acid bacterium) that produces histamine has a pyruvyl-type HDC gene (hdcA) in a plasmid. I have. From this, a primer specific to hdcA (GenBank: Accession No .: AB076394.1) of soy sauce lactic acid bacteria was prepared (2745f: 5'-TTGAACACACTTGGGGTTGA-3 '[SEQ ID NO: 3], 3566r: 5'-AATTGAGCCACCTGGAATTG -3 '[SEQ ID NO: 4]), the lactic acid bacteria isolated in (1) above were subjected to PCR according to a standard method, and the resulting amplified product was subjected to electrophoresis. As a comparative test, in addition to the lactic acid bacteria isolated in (1), PCR was performed in the same manner for the six existing soy sauce lactic acid bacteria (NBRC12171, NBRC100498, NBRC100726, NBRC100727, 3737, NBRC100499), and the resulting amplified product was subjected to electrophoresis. Was served. The results are shown in FIG. In FIG. 1, the results shown in lane 4 are the results for the lactic acid bacteria isolated in (1). As can be seen, no PCR-amplified product was detected from the results of the isolated bacteria, which confirmed that the lactic acid bacteria isolated in (1) did not have the HDC gene. That is, the lactic acid bacterium cannot convert histidine present in the soy sauce mash into histamine (no histamine generating ability).

Experimental Example 2 Evaluation of saccharide assimilation of soy sauce lactic acid bacteria In the soy sauce lactic acid bacteria of Example 1 for which the presence or absence of the HDC gene was confirmed, the assimilation ability to saccharides was measured. Specifically, various soy sauce lactic acid bacteria cultured on a plate medium (MRS medium, pH 7) containing 10% sodium chloride were transferred to API50CH (manufactured by Sysmex BioMerieux, Inc.), a test kit for testing carbohydrate metabolism of bacteria. Then, the reactivity (positive (+), negative (-), undeterminable (±)) for each sugar substrate was evaluated according to the manual. Table 4 shows the results.

  As can be seen from the results, the soy sauce lactic acid bacterium of the present invention has assimilation properties for erythritol, D-xylose and methyl-α-D-mannopyranoside, but has no assimilation property for D-ribose. , It was confirmed that the soy sauce lactic acid bacteria had a saccharide assimilation property clearly different from that of the existing soy sauce lactic acid bacteria. In particular, as shown in the experimental example 1 (2), among the existing soy sauce lactic acid bacteria, NBRC12172, NBRC100498, NBRC100726, NBRC100727, and 3737 are common to the soy sauce lactic acid bacteria of the present invention in having no HDC gene, It was confirmed that they had different characteristics in saccharide assimilation. From this, the soy sauce lactic acid bacteria of the present invention are novel soy sauce lactic acid bacteria different from existing soy sauce lactic acid bacteria.

Experimental Example 3 Evaluation of Amino Acid Metabolizing Ability of Soy Sauce Lactic Acid Bacteria Soy Sauce Lactic Acid Bacteria whose saccharide utilization was measured in Example 2 (Soy Sauce Lactic Acid Bacteria [MS0204], Existing Soy Sauce Lactic Acid Bacteria [NBRC12172, NBRC100498, NBRC100726, NBRC100727, and 3737]) The metabolic ability of amino acids (arginine and aspartic acid) was measured for the target.

(1) Evaluation of arginine metabolism ability The arginine metabolism ability of soy sauce lactic acid bacteria can be confirmed by an assimilation test using a medium supplemented with L-arginine. Specifically, first, a culture solution of each soy sauce lactic acid bacterium was inoculated into an MRS medium (pH 7.5) containing 0.3% L-arginine and 10% sodium chloride, and cultured at 30 ° C. for 7 days. . Next, the obtained culture was subjected to amino acid analysis to determine the concentration of arginine and ornithine in the culture, and the arginine assimilation of each soy sauce lactic acid bacterium, that is, the arginine metabolizing ability was confirmed from the amounts.

  The results are shown in FIG. As can be seen from FIG. 2, almost no ornithine was detected in the culture of the soy sauce lactic acid bacterium of the present invention, indicating that the soy sauce lactic acid bacterium of the present invention had arginine assimilation, that is, it did not have arginine metabolizing ability. On the other hand, existing soy sauce lactic acid bacteria (NBRC12172, NBRC100498, NBRC100726) which are common to the soy sauce lactic acid bacteria of the present invention in that they do not have an HDC gene have arginine assimilation (arginine metabolizing ability) of the present invention. It was confirmed that it had characteristics different from those of soy sauce lactic acid bacteria.

(2) Evaluation of aspartic acid metabolizing ability The aspartic acid metabolizing ability of soy sauce lactic acid bacteria can be confirmed by an assimilation test using a medium supplemented with L-aspartic acid. Specifically, first, an MRS medium (pH 7.5) containing 0.3% L-aspartic acid and 10% sodium chloride was inoculated with a culture solution of each soy sauce lactic acid bacterium, and allowed to stand still at 30 ° C. for 7 days. did. Next, the obtained culture was subjected to amino acid analysis, the aspartic acid and alanine concentrations in the culture were determined, and the aspartic acid assimilation of each soy sauce lactic acid bacterium, that is, the aspartic acid metabolizing ability was confirmed from the amounts.

  The results are shown in FIG. As can be seen from FIG. 3, alanine was detected in the culture of the soy sauce lactic acid bacterium of the present invention, indicating that the soy sauce lactic acid bacterium of the present invention has aspartic acid assimilation, that is, aspartic acid metabolizing ability. In addition, this property is a property that existing soy sauce lactic acid bacteria also have in common, but by having this property, aspartic acid in soy sauce moromi can be assimilated and converted to alanine with a mellow taste, and as a result, The flavor of soy sauce can be mild.

Experimental Example 4 Evaluation of Coagulability of Soy Sauce Lactic Acid Bacteria The cohesion of the soy sauce lactic acid bacteria (MS0204) of the present invention was measured by the cohesion test "Tatsuro Ueki, Yoshifumi Izawa, Kazunori Oba, Yoshiharu Noda: Research on Soy Sauce Technique, 26, 197-207 (2000) ", which can be carried out by determining the filter paper permeability of a culture solution of lactic acid bacteria of soy sauce. Specifically, first, lactic acid bacteria are converted into MRS containing 16.5% sodium chloride. 10 mL of a culture solution cultured at 30 ° C. for 4 days in a medium (pH 7) was filtered using filter paper (No. 2, ADVANTEC), and the turbidity (absorbance at 660 nm: OD ₆₆₀ ) of the culture solution before and after filtration was measured. Measured with a spectrophotometer (UV2100; Shimadzu Corporation) The percentage of the turbidity (OD ₆₆₀ ) of the culture solution after filtration to the turbidity (OD ₆₆₀ ) of the culture solution before filtration is defined as “filter paper transmittance”. The calculated lactic acid bacteria with soy sauce having a filter paper transmittance of 30% or less were determined to be "cohesive It is determined that the Ri ".

  As a result, the soy sauce lactic acid bacterium of the present invention had a filter paper transmittance of 16%, and was confirmed to have cohesiveness. FIG. 4 shows the results of similarly measuring the filter paper transmittance of the soy sauce lactic acid bacteria 221 contained in the soy sauce moromi. From these results, soy sauce lactic acid bacteria having no cohesion accounted for 95.9%, and soy sauce lactic acid bacteria had a small number of 4.1%. Among them, it was confirmed that the soy sauce lactic acid bacteria having a high cohesiveness were only 0.9% of the whole, having a filter paper transmittance of 20% or less, and were rare.

Experimental Example 5 Evaluation of Diacetyl Productivity of Soy Sauce Lactic Acid Bacteria The presence or absence of diacetyl productivity was evaluated for the soy sauce lactic acid bacteria (MS0204) of the present invention. Specifically, a medium (citrate-supplemented medium) adjusted to pH 7 by adding 0.3% citric acid monohydrate to an MRS liquid medium containing 10% sodium chloride, and citric acid monohydrate The soy sauce lactic acid bacteria (MS0204) of the present invention were cultured at 30 ° C. for 7 days using each of the media (medium without citric acid) adjusted to pH 7 without addition, and the diacetyl concentration of the culture after fermentation was measured. As a result, the presence or absence of diacetyl productivity of the soy sauce lactic acid bacteria was confirmed.

The diacetyl concentration of the culture was measured by head space solid phase microextraction (SPME) using a gas chromatograph mass spectrometer. The analysis conditions are as follows.
Equipment: GCMS-QP2010 Plus, AOC-5000 Auto Injector Shimadzu Corporation (SPME)
SPME fiber: 60 μm Polyethylene Glycol
Sample: 3 mL / 5 mL vial
SPME conditions: vial temperature 40 ° C, extraction time 30 minutes (agitation ON), headspace method
(GC conditions)
Inlet temperature: 230 ° C
Column: DB-WAX 30 m, Diam. 0.25 mm, Film 0.25 μm
Oven: 40 ℃ (10min) -4 ℃ / min -230 ℃ (5min)
Interface temperature: 230 ℃
(MS condition)
Ionization mode: EI
Ion source temperature: 200 ℃
Measurement mode: Selected ion detection method (SIM) m / z = 86.

  Like the soy sauce lactic acid bacteria (MS0204) of the present invention, other soy sauce lactic acid bacteria (MS0304, MS0305, MS1308, MS6501, MS6504) isolated from soy sauce moromi were cultured in a citric acid-added medium or a citric acid-free medium, respectively. The result of measuring the diacetyl concentration in the culture is shown in FIG. As a result, the diacetyl concentration of the culture in the medium added with citric acid by the soy sauce lactic acid bacterium of the present invention was 1140 μg / L, and the diacetyl concentration in the culture in the medium without citric acid was 326 μg / L. It was confirmed that it was. The diacetyl concentration is significantly higher than the diacetyl concentration in the culture of other soy sauce lactic acid bacteria similarly isolated from soy sauce moromi, which confirms that the soy sauce lactic acid bacteria of the present invention have high diacetyl productivity. Was done. Since diacyl is a compound having a butter-like or cheese-like scent, it is thought that the soy sauce lactic acid bacterium of the present invention can impart a complex and high-quality scent to soy sauce.

Experimental Example 5 Production of Soy Sauce and Its Evaluation (1) Production of Fresh Fried Soy Sauce Soy sauce was produced using the above-described soy sauce lactic acid bacteria (MS0204) of the present invention. Specifically, soy sauce koji obtained by koji-making soybean and wheat having a mass ratio of 1: 1 by a conventional method was mixed with 2065 mL of 23.2% salt water and mixed, and the mixture was mashed to obtain a liter volume of 3000 mL (3630 g) and a 4 L capacity. No. 5 fruit liquor bottle. Simultaneously with the charging, soy sauce lactic acid bacteria were added thereto at a volume ratio of 1/500 (6 mL with respect to the 3000 mL) so that the viable bacteria would be 10 ⁶ CFU / mL. After the preparation, the soy sauce moromi was manufactured by fermenting in a dark place at 15 ° C. for one month and fermenting in a dark place at 30 ° C. for 90 days after the first month. At the time of preparation and after 5, 15, 30, 45, 60, and 90 days from the start of cultivation (fermentation) at 30 ° C., the sap was carefully taken so that various bacteria were not mixed into the soy sauce mash. Collected and measured for pH and free amino acid concentration.

  The soy sauce mash aged by fermentation for three months was pressed according to a standard method, and the obtained sap was prepared as freshly fried soy sauce. If necessary, components such as sodium chloride may be added to the sap and the components may be adjusted, followed by heating (burning) to produce soy sauce (burned soy sauce).

(2) Change in pH of Soy Sauce Moromi At the time of preparation, the pH of the soy sauce moromi on days 5, 15, 30, 45, 60, and 90 days after culture is shown in FIG. FIG. 6 shows, in addition to the pH change of soy sauce moromi by the soy sauce lactic acid bacterium of the present invention, the pH change of soy sauce moromi by a commercially available soy sauce lactic acid bacterium (comparative example) and a system without the soy sauce lactic acid bacterium (control). As can be seen from the results, the lactic acid bacterium of the present invention, similarly to the commercially available lactic acid lactic acid bacterium, rapidly reduced the pH of the soy sauce mash from 30 days after lactic acid fermentation to 45 days from the lactic acid fermentation. (Alcohol fermentation environment) was confirmed.

(3) Amino acid content of soy sauce moromi At the time of preparation, the concentrations (mg / 100 mL) of arginine and ornithine in soy sauce moromi at 5, 15, 30, 45, 60, and 90 days after cultivation are shown in FIG. FIG. 8 shows the concentrations of aspartic acid and alanine (mg / 100 mL). 7 and 8, in addition to the change in the amino acid concentration in soy sauce mash by the soy sauce lactic acid bacteria of the present invention, commercially available soy sauce lactic acid bacteria (comparative example: arginine-decomposed / asparagine-non-degraded strain) and a system without the soy sauce lactic acid bacteria (control) 2) shows the change in pH of the soy sauce mash. The concentration of each free amino acid in the soy sauce moromi was determined by diluting the soy sauce moromi sap 200-fold with 0.02 mol / L hydrochloric acid, filtering through a 0.45 μm membrane filter, and then using a high-speed amino acid analyzer (L- 8900, Hitachi, Ltd.) and measured by the ninhydrin coloring method.

  As can be seen from FIG. 7, the soy sauce moromi prepared by a commercially available soy sauce lactic acid bacterium, which is an arginine-degrading strain, had a low arginine / ornithine content as a result of arginine being degraded to produce ornithine. On the other hand, the soy sauce moromi prepared by the lactic acid bacteria of the present invention, which is a non-degraded arginine strain, had a high arginine content / no ornithine content because arginine was not degraded. Also, as can be seen from FIG. 8, as with the commercially available soy sauce lactic acid bacterium, which is an aspartic acid-decomposing strain, the soy sauce moromi prepared by the soy sauce lactic acid bacterium of the present invention, which is an aspartic acid-decomposing strain, is degraded of aspartic acid to produce alanine. It was confirmed that it was generated. As shown in FIG. 8, the aspartic acid degradability is higher in the order of the commercially available strain> the lactic acid bacterium of the present invention> the strain existing in the additive-free system.

(4) Sensory evaluation of fried soy sauce In (1) above, for fried soy sauce produced using the soy sauce lactic acid bacteria of the present invention (soy sauce of the present invention), and fried soy sauce produced using commercially available soy sauce lactic acid bacteria (standard soy sauce), The taste and aroma were evaluated by 12 panelists who had experienced sensory evaluation of soy sauce. Specifically, the taste, aroma, and overall evaluation of the standard soy sauce are used as a reference (a score of 3), and the taste, aroma, and overall performance of the soy sauce of the present invention are compared with the standard soy in five stages (1: low to 5: high). Evaluation (scoring method).
Table 5 shows the average of the scores of the 12 panelists.
As can be seen from the above table, the freshly fried soy sauce produced using the soy sauce lactic acid bacteria of the present invention gave favorable evaluations in both taste and aroma, such as soft aroma, aroma, well-balanced, and good aftertaste.

Experimental Example 6 Histamine Concentration of Brewed Soy Sauce In Experimental Example 5 (1), the amount of histamine contained in the fermented soy sauce mash was measured. Specifically, the sap collected by filtration from soy sauce moromi after culturing and fermenting for 90 days was diluted 1000-fold with water, filtered through a 0.45 μm membrane filter, and set under the conditions described in Table 4. Histamine was analyzed by subjecting it to a liquid chromatograph mass spectrometer (LCMSMS) (API3200, Ab Sciex). The analysis of the amount of histamine was carried out for each of soy sauce (soy sauce moromi sap) produced using the lactic acid bacteria of the present invention and soy sauce with no soy sauce lactic acid bacteria-added system (control).

FIG. 9 shows the results. As shown in FIG. 9, the production of histamine was observed in the soy sauce produced without the addition of lactic acid bacteria, whereas the production of histamine was not observed in the soy sauce produced using the lactic acid bacteria of the present invention. . The reason why the production of histamine was observed in the soy sauce produced without the addition of lactic acid bacteria may be that a small amount of lactic acid bacteria attached during koji making were histamine-producing bacteria. On the other hand, the reason that no histamine was produced in the soy sauce produced using the lactic acid bacteria of the present invention was speculated, but by adding the lactic acid bacteria of the present invention, the growth of lactic acid bacteria mixed in a trace amount was suppressed. It is considered that the lactic acid bacteria of the present invention worked predominantly, so that histamine production by the mixed lactic acid bacteria was suppressed. From this fact, it is considered that the use of the soy sauce lactic acid bacterium of the present invention in the production of soy sauce makes it possible to suppress histamine production that may occur in soy sauce brewing.

  By using the novel lactic acid bacterium of the present invention for soy sauce brewing, the amount of histamine in soy sauce can be reduced. In addition, soy sauce with good taste and aroma can be stably produced. That is, by distributing the lactic acid bacterium, it is possible to reduce the histamine and improve the quality of the soy sauce of the recipient (for example, a soy sauce manufacturer in Miyazaki Prefecture).

NITE P-02727

  SEQ ID NOs: 1 and 2 show the nucleotide sequences of a forward primer (193f) and a reverse primer (480r) for amplifying a region specific to 16S rDNA of Tetragenococcus halophilus, respectively. SEQ ID NOS: 3 and 4 show the nucleotide sequences of a forward primer (2745f) and a reverse primer (3566r) for amplifying a region specific to hdcA of soy sauce lactic acid bacteria, respectively.

Claims (6)

A lactic acid bacterium belonging to Tetragenococcus halophilus having the following characteristics (a) to (d):
(A) having no histidine decarboxylase gene;
(B) assimilate erythritol, D-xylose, and methyl-α-D-mannopyranoside and do not assimilate D-ribose;
(C) does not decompose arginine,
(D) Decompose aspartic acid to produce alanine. The lactic acid bacterium according to claim 1, further having the following characteristics (e) and (f):
(E) cohesiveness,
(F) Diacetyl productivity.   The lactic acid bacterium according to claim 1 or 2, which is a soy sauce lactic acid bacterium.   A fermented food using the lactic acid bacterium according to claim 1.   A method for producing soy sauce, wherein the lactic acid bacterium according to any one of claims 1 to 3 is used as a soy sauce lactic acid bacterium in a soy sauce production process.   A method for suppressing histamine production in soy sauce production, comprising using the lactic acid bacterium according to any one of claims 1 to 3 as a soy sauce lactic acid bacterium in a soy sauce production process.