JP6479396B2 - Novel microorganism having ability to produce putrescine and method for producing putrescine using the same - Google Patents

Description

  The present invention relates to a microorganism having the ability to produce putrescine and a method for producing putrescine using the same. The present invention also relates to a method for producing a salt-containing fermented food containing putrescine.

  Putrescine is a kind of polyamine contained in the living body. Functions include nucleic acid stabilization and structural changes through interaction with nucleic acids, nucleic acid synthesis system activation, protein synthesis system activation, histone acetylation, non-histone chromatin protein phosphorylation, cell membrane stability Physiological effects such as oxidization and enhancement of substance permeability, activation of enzymes affected by divalent metals, and antioxidant effects on fats and oils are known. In addition, it is known that mice fed with a high polyamine diet live significantly longer than mice not fed, so that polyamines act as an anti-aging factor (Non-patent Document 1). For these reasons, putrescine has recently been used in health foods and other foods and drinks, cosmetics, pharmaceuticals, and quasi drugs.

  Putrescine has been conventionally used as a raw material for nylon 46, which is a plastic material, but its chemical synthesis uses a highly toxic hydrogen cyanide, and therefore has a large environmental burden and high production cost.

  On the other hand, as a method for producing putrescine, a method of extracting and purifying putrescine from fish larvae, dairy materials, yeasts, plants or the like that contain a large amount of polyamine; since there are many microorganisms that produce polyamine, There are also reports on how to produce. Specifically, microorganisms belonging to Escherichia coli (Non-patent document 2), lactic acid bacteria (Non-patent document 3), Enterobacteriaceae (Enterobacter genus (Patent document 1), Citrobacter genus (Patent document 2)) are used. A method for producing putrescine by using it is known.

Ali, M.A., et al., Food and Nutrition Research, 55,5572 (2011) Donald T. Dubin et al., “The Acetulation of Polyamines in Escherichia coli.” The Journal of Biological Chemistry Vol. 235, No. 3, March 1960 M.E.Arena, et al., Journal of Applied Microbiology 2001, 90 (2), 158-162 Coton M. etc: Food Microbiology, 27, 1078-1085 (2010) (see Fig. 1) Yasuei Hamana: Microbiol. Cult. Coll., June, 17-43 (2002) Listed in Table 3 Okamoto A, et al., Biosci Biotechnol Biochem; 61: 1582_4. (1997)

JP 2006-191808 A JP 2010-57396 A

  As described above, there are many microorganisms that produce polyamines, and microorganisms with higher productivity are being searched, but microorganisms with higher production efficiency are desired. As one method, if putrescine can be produced using microorganisms at a high salt concentration, the risk of growth of other microorganisms during cultivation is greatly reduced. It is thought that putrescine can be produced.

  Based on this idea, the present invention provides a microorganism (putrescine-producing salt-tolerant microorganism) having the ability to produce putrescine in the presence of sodium chloride, particularly in the presence of high salt concentration of 3% or more. The main object is to provide a method for producing putrescine using the same. Another object of the present invention is to provide a salt-containing food containing putrescine, particularly a food containing a high concentration of 3% or more of sodium chloride (a high-concentration salt-containing food), and a method for producing the same.

  Incidentally, a microorganism belonging to the genus Tetragenococcus, known as soy sauce lactic acid bacteria, is known to grow in the presence of a high concentration of sodium chloride, but no report has been made to produce putrescine. In addition, the gene for ornithine decarboxylase (ODC), an enzyme for producing putrescine, has not been found from Tetragenococcus (Non-patent Documents 4 and 5). Moreover, although there is a report that soy sauce contains about 500 mg / L when putrescine is high (Non-patent Documents 6 and 1), soy sauce containing more is not known.

  As a result of intensive studies to solve the above problems, the present inventors have found that among microorganisms belonging to Tetragenococcus, which is a salt-resistant lactic acid bacterium, microorganisms having the ability to produce putrescine under a high salt concentration (tetra The present invention was completed with the discovery of Tetragenococcus halophilus.

  The present invention has the following embodiments.

(I) Microorganism having putrescine production ability (I-1) Tetrageno having a nucleotide sequence of 16S rRNA gene (16S rDNA) 90% or more homologous to the nucleotide sequence shown in SEQ ID NO: 1 and having putrescine production ability A microorganism belonging to the genus Coccus.
(I-2) The microorganism according to (I-1), wherein the microorganism belonging to the genus Tetragenococcus is Tetragenococcus halophilus.

(I-3) The microorganism described in (I-1) or (I-2) having the following mycological and physiological properties:
(A) Mycological properties (6% w / v salt-containing sweetened broth medium, initial pH 7, culture at 30 ° C for 5 days)
(A) Morphological properties
(1) Cell shape and size: double or quadruple streptococci with a diameter of 0.8-1 μm
(2) Presence or absence of cell polymorphism: None
(3) Existence of mobility: None
(4) Presence or absence of spores: None
(b) Culture properties
(1) Plate culture Colony morphology Diameter: 0.5-1.5mm
Color tone: White
Shape: Circular Protrusion: Lens shape Perimeter: Smooth Surface shape: Smooth Gloss: None
(2) Liquid culture: cloudiness (B) physiological properties
(1) Gram staining: Positive
(2) MR test: positive
(3) VP test: negative
(4) Indole production: negative
(5) Reduction of nitrate: negative
(6) Starch hydrolysis: None
(7) Dye generation: None
(8) Oxidase: Negative
(9) Catalase: negative
(10) Generate L-lactic acid from glucose
(11) Gas productivity from glucose: negative
(12) has ornithine decarboxylase
(13) Range of growth:
Temperature: grows under 15-40 ° C conditions, does not grow under 45 ° C conditions
pH: Does not grow under pH 5 conditions, grows under pH 5.7-9 conditions
NaCl: grows later than 5 days under 0% condition, grows under 5-20 wt / vol% condition
(14) Attitude toward oxygen Aerobic growth: positive Anaerobic growth: positive
(15) OF test: F.

  (I-4) Described in (I-1) or (I-3) having the physiological properties (sugar utilization) listed in Table 1 in addition to the properties described in (I-3) above. Microorganisms:

(I-5) The above-mentioned microorganism belonging to the genus Tetragenococcus is Tetragenococcus halophilus PAM-26 strain (deposit number: NITE P-01873), or its passage strain (I-1) To the microorganism described in any one of (I-4).

(II) Putrescine production method (II-1) A culture according to any one of (I-1) to (I-5) is cultured in a medium containing a putrescine-producing substrate, and a culture containing putrescine A process for producing putrescine, comprising the step of obtaining
(II-2) A method for producing putrescine, comprising a step of collecting putrescine from the culture obtained by the production method according to (II-1).
(II-3) The production method according to (II-1) or (II-2), wherein the putrescine-producing substrate is ornithine.
(II-4) The production method according to any one of (II-1) to (II-3), wherein the salt concentration of the medium is 3 to 20% by weight / volume.

(III) Method for producing salt-containing fermented food containing putrescine (III-1) A raw material mixture of salt-containing fermented food is any one of (I-1) to (I-5) in the presence of a putrescine-producing substrate. A method for producing a salt-containing fermented food containing putrescine, comprising a step of fermenting using the microorganism described.
(III-2) The manufacturing method as described in (III-1) whose salt concentration of the raw material mixture of salt containing fermented food is 3-20 weight / volume% (or weight / weight%).
(III-3) The production method according to (III-1) or (III-2), wherein the high-salt fermented food is any one selected from the group consisting of soy sauce, miso, and pickles.

(IV) Salt-containing fermented food containing putrescine (IV-1) manufactured using the microorganism described in any one of (I-1) to (I-5), containing the microorganism and putrescine-containing salt Fermented food.
(IV-2) The salt-containing fermented food according to (IV-1), wherein the salt concentration is 3 to 20% by weight / volume% (or weight / weight%).
(IV-3) The salt-containing fermented food described in (IV-1) or (IV-2), which is any one selected from the group consisting of soy sauce, miso, and pickles.
(IV-4) The salt-containing fermented food according to any one of (IV-1) to (IV-3), wherein the salt-containing fermented food is soy sauce containing 1000 mg / L or more of putrescine.

  According to the present invention, it is possible to provide a salt-resistant lactic acid bacterium that is a novel microorganism having the ability to produce putrescine in a salt-containing medium. By using the microorganism, putrescine can be produced by a fermentation process. In addition, since the microorganism is salt-tolerant, it is possible to suppress contamination by other microorganisms by culturing using a medium with a high salt concentration, so the target putrescine can be efficiently and inexpensively performed with simple equipment. Can be manufactured.

  Moreover, according to the microorganism of the present invention (salt-resistant lactic acid bacteria), putrescine can be produced even in the presence of 3% or more of sodium chloride to produce a fermented food. For example, soy sauce, miso and pickles A fermented food containing a relatively high concentration (fermented food containing high-concentration salt) can also be provided as a putrescine-containing fermented food.

The results of measuring the amount of putrescine produced over time when the microorganism of the present invention (PAM-26 strain) was cultured in the presence of 3 to 20% by weight / volume sodium chloride (NaCl) are shown (Example 2).

I. Microorganism having ability to produce putrescine The microorganism of the present invention is a lactic acid bacterium belonging to the genus Tetragenococcus, and is characterized by having salt tolerance and ability to produce putrescine.

  Specifically, the microorganism is a microorganism having a base sequence of 16Sr DNA that is 90% or more homologous to the base sequence shown in SEQ ID NO: 1 and having the ability to produce putrescine from ornithine. Preferably, it is a microorganism having a 16S rRNA gene that is homologous to 95% or more, more preferably 98% or more, and still more preferably 99% or more of the base sequence shown in SEQ ID NO: 1.

  Here, “homologous” means identity to the base sequence shown in SEQ ID NO: 1. In general, identity (%) is the step of comparing the sequences between two molecules to be compared (an oligonucleotide having a reference sequence and an oligonucleotide having a sequence whose identity to the reference sequence is unknown). It can be determined by counting the number of fits between, dividing by the length of the reference sequence, and multiplying the result by 100. Conveniently, the identity of the nucleotide sequence is determined by the Carlin and Arthur mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7). Programs called BLASTN and BLASTX based on the BLAST algorithm have also been developed (Altschul, SF., Et al., J Mol Biol, 1990, 215, 403). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.Nih.gov/).

  Preferred examples of the microorganism include Tetragenococcus halophilus. When the nucleotide sequence represented by SEQ ID NO: 1 is subjected to homology search against the GenBank database (Non-reducdant nucleotide database) using BLAST, 99% of the nucleotide sequence matches the nucleotide sequence of Tetragenococcus halophilus 16Sγ DNA (99 % Homology).

Furthermore, the microorganism of the present invention preferably has the following mycological and physiological properties.
(A) Mycological properties (6% w / v salt-containing sweetened broth medium, initial pH 7, culture at 30 ° C for 5 days)
(A) Morphological properties
(1) Cell shape and size: double or quadruple streptococci with a diameter of 0.8-1 μm
(2) Presence or absence of cell polymorphism: None
(3) Existence of mobility: None
(4) Presence or absence of spores: None
(b) Culture properties
(1) Plate culture Colony morphology Diameter: 0.5-1.5mm
Color tone: White
Shape: Circular Protrusion: Lens shape Perimeter: Smooth Surface shape: Smooth Gloss: None
(2) Liquid culture: cloudiness (B) physiological properties
(1) Gram staining: Positive
(2) MR test: positive
(3) VP test: negative
(4) Indole production: negative
(5) Reduction of nitrate: negative
(6) Starch hydrolysis: None
(7) Dye generation: None
(8) Oxidase: Negative
(9) Catalase: negative
(10) Generate L-lactic acid from glucose
(11) Does not produce gas from glucose
(12) has ornithine decarboxylase
(13) Range of growth:
Temperature: grows at 15-40 ° C, does not grow at 45 ° C pH: does not grow at pH 5, grows at pH 5.7-9
NaCl: grows later than 5 days at 0%, grows at 5-20% w / v
(14) Attitude toward oxygen Aerobic growth: positive Anaerobic growth: positive
(15) OF test: F
(16) Utilization of sugar

  Preferred examples of Tetragenococcus halophilus having the above salt tolerance, putrescine production ability, and bacteriological and physiological properties include PAM-26 strain. The PAM-26 strain is a microorganism contained in the strain preserved by the present applicant (Higashimaru Soy Sauce Co., Ltd.). As of June 30, 2014, Kazusa Kamashita, Kisarazu City, Chiba Prefecture, Japan 2-5-8 Deposited in Japan as “Accessory Number: NITE P-01873” at the Patent Microorganism Depositary Center, National Institute of Technology and Evaluation, which has an address in Room 122.

  In addition to the PAM-26 strain, the microorganism of the present invention has a 16S rRNA gene (16S rDNA) having a nucleotide sequence of 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 1, as described above, In addition, as long as it has the above-mentioned salt tolerance and ability to produce putrescine, a related microorganism of the PAM-26 strain is also included. The related microorganisms include microorganisms having the same or equivalent properties as the above-mentioned mycological and physiological properties of the PAM-26 strain. A related microorganism of the PAM-26 strain can be isolated using the base sequence described in SEQ ID NO: 1 (16S rDNA sequence), putrescine production ability, and salt tolerance as indicators. For example, a test sample is added and cultured in a medium containing ornithine, preferably a salt concentration of 6 to 15% by weight / volume, and the culture is subjected to HPLC analysis to select a sample in which high production of putrescine is observed. (Primary screening). Next, the culture solution of the selected test sample is cultured on a plate having a salt concentration of 6 to 15% by weight / volume, and colonies are formed to isolate microorganisms, and then the isolated microorganisms are cultured and subjected to HPLC analysis. Select microorganisms with high putrescine production (secondary screening). The base sequence of 16Sγ DNA of the obtained microbial strain is subjected to homology search against the BLAST base sequence database, and is 90% or more, preferably 95% or more, more preferably 98% or more with the base sequence shown in SEQ ID NO: 1. Particularly preferably, a microorganism having 99% or more of 16S rDNA is selected.

  The microorganism of the present invention can be cultured (growth, proliferated) by a method similar to that used for culturing known microorganisms belonging to the genus Tetragenococcus, preferably Tetragenococcus halophilus, which is a salt-resistant lactic acid bacterium. it can.

  Examples of the medium include a medium containing a necessary nutrient source such as a carbon source, a nitrogen source, or an inorganic salt that can be assimilated by a microorganism belonging to the genus Tetragenococcus. As long as this is the case, the medium is not particularly limited, and either a natural medium or a synthetic medium may be used. The carbon source used in the medium is not particularly limited as long as microorganisms belonging to the genus Tetragenococcus can be assimilated, and carbohydrates such as glucose, fructose and maltose are preferably used. Any nitrogen source may be used as long as the microorganism can assimilate, and nitrogen-containing compounds such as peptone, yeast extract, malt extract, and meat extract are preferably used. For convenience, a commercially available medium for lactic acid bacteria can be used, but if salt is added thereto, it can be used as a suitable medium. Although a medium for general bacteria can be used, it is preferable to add a carbon source such as glucose and salt to this medium. More desirably, the addition of a soy sauce cake extract to the medium can promote the growth, and a medium mainly composed of the soy sauce cake extract can also be used. The salt concentration of the medium is preferably 5 to 15%, more preferably 10 to 15%.

  What is necessary is just to set the culture temperature to the range of the growth temperature of the microorganisms of this invention, Preferably the range of the optimal growth temperature can mention the range of 20-40 degreeC, for example, Preferably it is about 25-30 degreeC. . The culture time is usually about 3 to 6 days. Moreover, it is preferable to maintain the pH of the medium at about 6 to 7 during the culture period. The pH of the medium can be adjusted using an inorganic acid or organic acid aqueous solution, an alkaline aqueous solution, or the like.

(II) Method for producing putrescine Putrescine can be produced by using the microorganism of the present invention described above. The putrescine production method of the present invention is characterized in that the microorganism of the present invention is cultured in a medium containing a putrescine-producing substrate. Specifically, the putrescine production method of the present invention includes a step of culturing the microorganism in a medium containing a putrescine-producing substrate, and a step of collecting putrescine from the culture.

(II-1) Cultivation process In addition to the necessary nutrient sources such as carbon sources, nitrogen sources, or inorganic salts that can be assimilated by microorganisms belonging to the genus Tetragenococcus, the culture medium used for cultivation includes a putrescine-producing substrate, And a medium containing sodium chloride (salt). Here, the putrescine production substrate is preferably ornithine. As long as this is the case, the medium is not particularly limited, and either a natural medium or a synthetic medium may be used.

  Although it does not restrict | limit especially as a density | concentration of the putrescine production | generation substrate contained in a culture medium, Usually, 0.01-5 weight / volume%, Preferably about 0.2-2 weight / volume% can be mentioned. The salt concentration in the medium is not particularly limited as long as it is within a range in which the microorganism of the present invention can grow, and examples thereof include a range of about 5 to 20% by weight / volume. If it is 5% by weight or more, the growth of thermotolerant bacteria is suppressed, and if it is 15% by weight or more, the number of microorganisms that can grow is greatly limited. Accordingly, the salt concentration is preferably in the range of 5 to 20% by weight / volume, and within this range, the concentration can be appropriately set such as 10 to 20% by weight / volume%, 15 to 20% by weight / volume. .

  The carbon source, nitrogen source, inorganic salts and the like to be blended in the medium are as described above in (I). Although pH of a culture medium is not restrict | limited in particular, Usually pH 6-8, Preferably the range of pH 6-7 can be mentioned. The medium and the culture apparatus can be sterilized at a low temperature of 100 ° C. or lower.

  What is necessary is just to set the culture temperature to the range of the growth temperature of the microorganisms of this invention, Preferably the range of the optimal growth temperature can mention the range of 20-40 degreeC, for example, Preferably it is about 25-30 degreeC. . Although culture | cultivation time is not restrict | limited, For example, about 3 to 6 days can be mentioned. However, in order to make putrescine highly produced in the medium, it can be cultured (brewed) for more than half a year.

  It should be noted that oxygen is not particularly required to be cut off or supplied during the culture. For this reason, it is possible to produce putrescine by covering the incubator with a simple lid. In addition, suppressing the sedimentation of the bacterial cells by performing gentle stirring during the cultivation leads to an improvement in productivity.

  Culturing can also be performed by immobilizing the microorganism of the present invention. Examples of the immobilization method include known methods such as a carrier binding method, a crosslinking method, and a comprehensive method.

(II-2) Putrescine collection step Putrescine accumulated in the culture solution by the above-described culture step can be collected from the culture solution by a conventional method. Moreover, it can also refine | purify further as needed. As the purification method, commonly used means such as filtration, centrifugation, ion exchange chromatography, adsorption chromatography and the like may be appropriately combined as necessary.

(III) Salt-containing fermented food containing putrescine and method for producing the same The food targeted by the present invention is a salt-containing fermented food containing putrescine.

  Here, the salt-containing fermented food may be any fermented food containing salt, but is preferably a fermented food having a salt concentration of 3 to 20%. Examples of fermented foods containing salt within this range include soy sauce, miso, and pickles. Soy sauce and miso are preferred, and soy sauce is particularly preferred.

  The soy sauce includes concentrated soy sauce, light soy sauce, tame soy sauce, re-prepared soy sauce, white soy sauce, fresh soy sauce, reduced salt soy sauce, and powdered soy sauce, all of which are included in the soy sauce of the present invention. In addition, miso includes rice miso, barley miso, bean miso, and combined miso depending on the raw materials used, all of which are included in the miso of the present invention. Examples of the pickles targeted by the present invention include pickles using lactic acid fermentation such as quick pickles, Chinese cabbage kimchi and sauerkraut.

  These fermented foods of the present invention are characterized by containing putrescine. Although the content of putrescine varies depending on the fermented food, it can be 500 mg / L (mg / Kg) or more, preferably 600 mg / L or more for soy sauce and pickles, and 200 mg / Kg or more for miso. In particular, the soy sauce preferably contains putrescine at a rate of 1000 mg / L or more.

  Such a salt-containing fermented food containing putrescine can be produced by a method comprising a step of fermenting a raw material mixture of salt-containing fermented food using the microorganism of the present invention described above in the presence of a putrescine-producing substrate. .

  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% according to the type of the salt-containing fermented food. For example, when the salt-containing fermented food is soy sauce, the range is 15 to 25% by weight / volume%, when it is miso, the range is 10 to 15% by weight / weight%, and when it is pickled, it is 3 to 10% by weight / weight%. However, it is not limited to these ranges.

  The putrescine-producing substrate is as described above, and ornithine is preferable. The concentration of the putrescine-producing substrate to be blended in the salt-containing fermented food material mixture is not particularly limited, but is usually 0.01 to 2% by weight / volume% (or weight / weight%), preferably 0.2 to 1% by weight / About volume% (or weight / weight%) can be mentioned.

  Below, the manufacturing method is demonstrated easily about each case where salt containing fermented food is soy sauce, miso, and pickles, for example.

(III-1) Method for Producing Soy Sauce The soy sauce of the present invention is prepared by blending a putrescine-producing substrate with a raw material mixture (soy sauce charged) for producing soy sauce, and fermenting and ripening it using the microorganism of the present invention described above. It can manufacture by the method which has a process to make. The salt concentration in this soy sauce charge varies depending on the type of soy sauce, but is usually in the range of 15 to 25% by weight / volume. The content of the putrescine production substrate is as described above. This is fermented and matured at 15 ° C. to 30 ° C. over several months. The soy sauce moromi obtained by the fermentation and ripening process is squeezed using a nylon filter cloth or the like, and the resulting squeezed liquid is prepared as soy sauce after oil separation, standard preparation, burning, orientation pulling and the like.

(III-2) Miso manufacturing method The miso of the present invention is a process in which a putrescine-producing substrate is blended with a raw material mixture (miso charging) for manufacturing miso and fermented with the aforementioned microorganism of the present invention. It can manufacture by the method which has this. The salt concentration of the miso preparation varies depending on the type of miso, but is usually in the range of 10-15 wt / wt%. The content of the putrescine production substrate is as described above. This is maintained at about 25-30 ° C. for 1-12 months and fermented. During the fermentation, it is preferable to dig up the miso 1-2 times to homogenize the miso (returning). The miso that has been fermented and matured is dug up, and two or more types are blended for quality adjustment as necessary, and then commercialized as a granulated miso, or kneaded with a chopper and commercialized as a miso miso.

(III-3) Method for producing pickles The pickles according to the present invention are prepared by blending a putrescine-producing substrate with a raw material for pickles (for example, a mixture of rice bran, vegetables and seasonings) It can manufacture by the method which has the process fermented using. The salt concentration of the raw material for pickles is usually in the range of 3 to 10% by weight / weight%, although it varies depending on the kind and preference of the pickles. The content of the putrescine production substrate is as described above. This is maintained at about 10-30 ° C. for 5-30 days and fermented to obtain pickles.

  Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to these examples.

Example 1
1) Acquisition of microorganisms Among the strains collected from various commercially available bran pickles, soy sauce moromi, and high-concentration salt-containing foods such as soy sauce moromi possessed by the present applicant, a strain that highly produces putrescine was screened.

  Specifically, first, strains (including Applicant-preserved strains) collected from various high-concentration salt-containing foods were sterilized media (salt concentration: 10 wt / volume%, pH 6) each having the composition described in Table 3. And adjusted to 0.5) and cultured at 30 ° C. for 1 week. In Table 3, the soy sauce cake extract was prepared by adding 1.5 L of 20 wt / vol% saline to 1 kg of soy sauce cake, placing it at room temperature, and filtering after 3 weeks.

  After the cultivation, the amount of putrescine contained in each culture solution was quantitatively analyzed by HPLC under the following conditions. The sample used for the analysis was prepared by diluting the culture solution 100-fold with 0.1N hydrochloric acid and filtering with a 0.45 μm filter.

(HPLC conditions)
Column: Shodex Asahipak ODP-50 4D (4.6mm × 150mm)
Mobile phase: 50 mM borate buffer (pH 9.9): acetonitrile (containing 2 mM o-phthalaldehyde and 2 mM N-acetyl-L-cysteine) = 77: 23 (volume ratio)
Column temperature: 40 ° C
Flow rate: 0.5ml / min
Detector: Fluorescence detector (excitation wavelength 330 nm, fluorescence wavelength 430 nm).

  The putrescine concentration in each culture solution was calculated from a standard calibration curve prepared using a 0.1N hydrochloric acid solution containing putrescine in the range of 0 to 25 mg / L. When the measured value exceeded the upper limit of the calibration curve, it was further diluted with 0.1N hydrochloric acid.

  As a result, a salt-tolerant strain having the ability to produce putrescine was found out of the strains stored by the applicant, and this was named PAM-26. On June 30, 2014, the PAM-26 shares were entrusted to the Patent Microbiology Depositary Center of the National Institute of Technology and Evaluation, which has an address in Room No. 2-5-8 Kazusa Kamashi, Kisarazu, Chiba, Japan. No .: NITE P-01873 ”.

2) Identification of PAM-26 strain
The nucleotide sequence of the 16S ribosomal RNA (rRNA) gene (16S rDNA) of the PAM-26 strain was determined, and homology search was performed. Specifically, the PAM-26 strain was inoculated into a sterilized sweetened broth medium (Table 4) (salt concentration: 10%, ornithine hydrochloride 0.5%, adjusted to pH 6.5), and cultured at 30 ° C. for 5 days. Subsequently, the cells were collected by centrifugation, and genomic DNA was extracted. Marmur method was used for extraction of genomic DNA. Using the extracted genomic DNA as a template, a 500 bp region on the 5 ′ end side of the 16S rRNA gene (16S rDNA) was amplified by PCR according to the method described in the Japanese Pharmacopoeia. Thereafter, the amplified base sequence was sequenced using a DNA sequencer (ABI3730xl: manufactured by Applied Biosystems), and the base sequence of 16S rDNA of PAM-26 strain was determined. The determined base sequence is shown in SEQ ID NO: 1.

  The base sequence of the obtained 16S rDNA was searched for homology with the GenBank database (Non-redundant nucleotide database) using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) However, it showed 99% homology with the 16S rDNA base sequence of Tetragenococcus halophilus species. This revealed that the PAM-26 strain is a salt-tolerant lactic acid bacterium belonging to the genus Tetragenococcus halophilus.

3) Mycological and physiological / biochemical properties of PAM-26
The bacteriological and physiological properties of PAM-26 were performed according to Bergey's Manual of Systematic Bacteriology, Volume 2, Williams & Wilkins, 1986. Unless otherwise specified in the mycological and physiological properties tests, the medium was used based on a sweetened broth medium (Table 4). Unless otherwise specified in each test, the salt concentration of the medium was 6% w / v, the initial pH was 7, the culture temperature was 30 ° C., and the culture period was 5 days. The bacteriological properties and physiological / biochemical properties obtained as a result are as follows and are consistent with Tetragenococcus halophyllus.

(A) Mycological properties (a) Morphological properties
(1) Cell shape and size: double or quadruple streptococci with a diameter of 0.8-1 μm
(2) Presence or absence of cell polymorphism: None
(3) Existence of mobility: None
(4) Presence or absence of spores: None.

(b) Culture properties
(1) Plate culture Colony morphology Diameter: 0.5-1.5mm
Color tone: White Shape: Circular Raised state: Lens shape Edge: Smooth Surface shape: Smooth Gloss: None
(2) Liquid culture: cloudiness (B) physiological properties
(1) Gram staining: Positive
(2) MR test: positive
(3) VP test: negative
(4) Indole production: negative
(5) Reduction of nitrate: negative
(6) Starch hydrolysis: None
(7) Dye generation: None
(8) Oxidase: Negative
(9) Catalase: negative
(10) Generate L-lactic acid from glucose
(11) Does not produce gas from glucose
(12) has ornithine decarboxylase
(13) Range of growth:
Temperature: grows under 15-40 ° C conditions, does not grow under 45 ° C conditions
pH: Does not grow under pH 5 conditions, grows under pH 5.7-9 conditions
NaCl: grows later than 5 days under 0% condition, grows under 5-20 wt / vol% condition
(14) Attitude toward oxygen Aerobic growth: positive Anaerobic growth: positive
(15) OF test: F.

(C) Physiological properties (sugar utilization)
Sugar assimilation was performed using API API50CHL (Sysmex Biomelieu Co., Ltd.). The medium was supplemented with 6% salt / volume by volume.

  Table 5 shows the results. Observations were made after 3 days and 7 days, and were indicated as “+” when positive after 3 days and “+ s” when positive after 7 days. There was no gas production from any sugar. This result is consistent with Tetragenococcus halophilus.

Example 2 Production of putrescine using PAM-26 strain The sodium chloride concentration was adjusted to 5 types: 3 wt / vol%, 6 wt / vol%, 10 wt / vol%, 15 wt / vol% and 20 wt / vol%. Each of the media shown in Table 3 was inoculated with the PAM-26 strain and cultured at 30 ° C. for 30 days. During the culture period, the concentration of putrescine contained in the culture medium was measured by sampling over time. As a control, three lactic acid bacterial strains belonging to the genus Tetragenococcus halophilus used by the applicant as a lactic acid fermentation starter were cultured under the same conditions as described above, and the putrescine concentration in the culture solution was measured.

  FIG. 1 shows changes with time of the putrescine concentration in the culture medium by the PAM-26 strain culture. As shown in FIG. 1, it was confirmed that the PAM-26 strain produced putrescine at any salt concentration. In particular, the results at a salt concentration of 6 to 15% by weight / volume were good. On the other hand, the putrescine concentration in the culture solution (saline concentration of 3 to 20% by weight / volume%) of other Tetragenococcus cultures used as a control was below the detection level (20 ppm), and no putrescine production ability was observed. It was.

Example 3 Production of soy sauce containing putrescine An equivalent amount of defatted soybeans and wheat produced as usual in the applicant's factory, soy sauce cake using Aspergillus oryzae, and lactic acid bacteria of the present invention, putresin was used. A soy sauce containing was produced.

That is, 1.8 kg of soy sauce cake, 3 L of saline (salt concentration 22.5%) and 19 g of ornithine hydrochloride were charged into a 5 L glass container. The temperature in the charging chamber was 15 ° C. for 2 weeks, 25 ° C. for 4 weeks, 30 ° C. for 2 weeks, and then maintained at 25 ° C. One week after the preparation, lactic acid bacteria PAM26 strain previously cultured using the soy sauce cake extract as a medium was added to 10 ⁵ cells / ml. In addition, 6 weeks after preparation, yeast was added at 10 ⁵ pieces / ml. As a control group, instead of the lactic acid bacteria PAM26 strain, Tetragenococcus halophilus H10 strain used by the applicant as a lactic acid fermentation starter was added and the same soy sauce brewing was performed. During the preparation period, the agitation was performed approximately once every two weeks, and after 20 weeks, the mixture was pressed and filtered to obtain freshly fried soy sauce, which was analyzed.

  The general components of this soy sauce are as shown in Table 6. There was no difference between the test group and the control group, and the flavor was good.

  Putrescine in the test section using the milk pasteurized PAM26 strain was 1,550 ppm, and other amines such as histamine, tyramine, spermidine and spermine were all less than 20 ppm. The putrescine in the control group using normal lactic acid bacteria was less than 20 ppm.

Example 4 Base mass and putrescine production amount The relationship between the amount of ornithine as a substrate and the production amount of putrescine was observed. Ornithine hydrochloride was added to the medium of Table 3 adjusted to a salt concentration of 10% w / v to prepare a medium (pH 7) with ornithine hydrochloride 0.5, 1.0, 2.0% w / v. . This was heated in a 95 ° C water bath for 10 minutes and then cooled. Each strain was inoculated with PAM26 at 10 ⁵ / ml, cultured at 30 ° C for 5 or 15 days, and centrifuged to contain putrescine. A culture was obtained.

  As the results shown in Table 7, the putrescine production amount increased in direct proportion to the added amount of the substrate. The conversion rate of ornithine to putrescine was constant at approximately 75%.

    NITE P-01873

Claims (8)

Is a microorganism belonging to Tetragenococcus genus with putrescine-producing ability, Tetragenococcus-halophilus (Tetragenococcus halophilus) PAM-26 strain (accession number: NITE P-01873), or a their passage strain, Tsugidaikabu Said microorganism having the following mycological and physiological properties:
(A) Mycological properties (6% w / v salt-containing sweetened broth medium, initial pH 7, culture at 30 ° C for 5 days)
(A) Morphological properties
(1) Cell shape and size: double or quadruple streptococci with a diameter of 0.8-1 μm
(2) Presence or absence of cell polymorphism: None
(3) Existence of mobility: None
(4) Presence or absence of spores: None
(b) Culture properties
(1) Plate culture
Colony morphology
Diameter: 0.5-1.5mm
Color tone: White
Shape: Circular
Raised state: lenticular
Perimeter: Smooth
Surface shape: smooth
Gloss: None
(2) Liquid culture: cloudiness
(B) Physiological properties
(1) Gram staining: Positive
(2) MR test: positive
(3) VP test: negative
(4) Indole production: negative
(5) Reduction of nitrate: negative
(6) Starch hydrolysis: None
(7) Dye generation: None
(8) Oxidase: Negative
(9) Catalase: negative
(10) Generate L-lactic acid from glucose
(11) Gas productivity from glucose: negative
(12) has ornithine decarboxylase
(13) Range of growth:
Temperature: grows under 15-40 ° C conditions, does not grow under 45 ° C conditions
pH: does not grow under pH 5 conditions, grows under pH 5.7-9 conditions
NaCl: grows later than 5 days under 0% condition, grows under 5-20 wt / vol% condition
(14) Attitude toward oxygen
Aerobic growth: positive
Anaerobic growth: positive
(15) OF test: F
(16) Putrescine production ability: positive.   A method for producing putrescine, comprising culturing the microorganism according to claim 1 in a medium containing a putrescine-producing substrate to obtain a culture containing putrescine.   The production method according to claim 2, wherein the putrescine-producing substrate is ornithine.   The manufacturing method of the salt containing fermented food containing putrescine which has the process of fermenting the raw material mixture of salt containing fermented food using the microorganisms of Claim 1 in presence of a putrescine production substrate.   The manufacturing method according to claim 4, wherein the salt-containing fermented food is any one selected from the group consisting of soy sauce, miso, and pickles.   A salt-containing fermented food containing the microorganism according to claim 1 and putrescine derived from the microorganism.   The salt-containing fermented food according to claim 6, which is any one selected from the group consisting of soy sauce, miso, and pickles.   The salt-containing fermented food according to claim 6 or 7, wherein the salt-containing fermented food is soy sauce containing 1000 mg / L or more of putrescine.