JP7670255B2 - Putrescine-producing lactic acid bacteria - Google Patents

Description

NPMD NITE P-03449 NPMD NITE P-03450

The present invention relates to putrescine-producing lactic acid bacteria, foods and beverages containing said lactic acid bacteria, and a method for increasing putrescine production.

Putrescine is a type of polyamine produced in living organisms from the amino acid ornithine, and has the chemical name "1,4-diaminobutane." Its functions include physiological effects such as stabilization and structural changes of nucleic acids through interaction with nucleic acids, promotion of nucleic acid synthesis, activation of protein synthesis, acetylation of histones, promotion of phosphorylation of non-histone chromatin proteins, stabilization of cell membranes and enhancement of permeability to substances, and activation of enzymes affected by divalent metals, as well as antioxidant effects on fats and oils.
It has been reported that putrescine is also produced in human somatic cells and in intestinal bacteria, but the amount produced decreases with age.
Due to these circumstances, in recent years, putrescine has begun to be used as a health food.
Putrescine can also be synthesized by chemical synthesis, but since highly toxic hydrogen cyanide is used, there are problems not only with the environmental impact but also with safety.
Other methods for producing putrescine have been reported, including a method for extracting and purifying putrescine from fish milt, dairy materials, yeast, plants, or the like that contain a large amount of polyamines, and a method for producing putrescine using a microorganism (e.g., Patent Documents 1 and 2, etc.). However, from the viewpoints of production yield and safety for humans, a new method for producing putrescine is desired.

On the other hand, lactic acid bacteria are used in the production of dairy products such as yogurt and cheese, and fermented foods such as pickles, miso, and soy sauce. In recent years, they have been known as probiotics, having various health-maintaining effects such as intestinal regulation by improving the bacterial flora in the digestive tract, immune enhancement, and antitumor effects, and are therefore known as a useful microbial resource that is deeply involved in human health. However, when lactic acid bacteria are ingested as food, they are exposed to various stresses such as gastric acid and bile acid, which reduces their survival rate in the intestine and reduces the beneficial effects that lactic acid bacteria inherently possess.

JP 2006-191808 A JP 2010-057396 A

The present invention aims to provide a novel lactic acid bacterium that has high putrescine production capacity and can be used as a probiotic, and to provide a method for increasing putrescine production using the lactic acid bacterium.

As a result of intensive research conducted by the present inventors to solve the above problems, the present inventors discovered that a specific strain of Leviractobacillus paucivorans has the ability to produce putrescine and is also resistant to bile acids, thereby solving the above problems.

Specifically, the present invention relates to the following items.
1. A lactic acid bacterium which is Reviractobacillus paucivorans strain N36 (accession number: NITE P-03449) or strain N50 (accession number: NITE P-03450).
2.1. A food or drink containing the lactic acid bacteria described above.
3.1. A method for increasing putrescine production, comprising adding glucose to a skim milk medium in which the lactic acid bacterium described above is cultured.

The specific lactic acid bacteria belonging to the genus Leviractobacillus according to the present invention have the ability to produce putrescine and are resistant to bile acids, and therefore can be used as probiotics.
The lactic acid bacteria of the present invention can be cultured in milk such as skim milk and used together with the putrescine produced as a highly safe food or drink, making them extremely useful.
Furthermore, when the lactic acid bacterium is cultured in a skim milk medium, the amount of putrescine produced can be increased by adding glucose.

FIG. 2 is a diagram showing the 16S rDNA base sequence of the lactic acid bacteria N36 strain of the present invention. FIG. 2 is a diagram showing the 16S rDNA base sequence of the lactic acid bacteria N50 strain of the present invention. 1 is a chart showing the analysis results by high performance liquid chromatography in the putrescine production confirmation test in Example 2. 1 is a graph showing the bile acid resistance (%) of the GG strain, the N36 strain of the lactic acid bacteria of the present invention, and the N50 strain in the probiotic compatibility test of Example 3. 1 is a graph showing the amounts of putrescine produced by the lactic acid bacteria strains N36 and N50 of the present invention when no sugar is added and when sugar is added in Test 1 for Confirmation of Putrescine Production Amount During Skim Milk Cultivation in Example 4. 1 is a graph showing the amount of putrescine produced when the lactic acid bacteria strains N36 and N50 of the present invention are co-cultured with starter lactic acid bacteria in Test 2 for Confirmation of Putrescine Production in Skim Milk Cultivation in Example 5.

The present invention relates to a lactic acid bacterium of Leviractobacillus paucivorans strain N36 (accession number: NITE P-03449) or strain N50 (accession number: NITE P-03450).
The genus Leviractobacillus in the present invention is one of the genera that were reclassified into a total of 25 genera in 2020 as a result of reevaluating the genus classification at the genome level using genome data of the type strains of all species previously classified into the genus Lactobacillus (261 species, as of August 19, 2019). The genus Leviractobacillus included approximately 9.2% (24 species) of all species that were reclassified.
The present invention will be described in detail below.

<Reviractobacillus paucivorans strain N36, strain N50>
Levilactobacillus paucivorans N36 and N50 strains are lactic acid bacteria isolated from "rice bran pickles" and are strains stored in the lactic acid bacteria library of the Livestock Product Function Unit, Livestock Product Research Area, Livestock Research Division, National Agriculture and Food Research Organization. As a result of a homology search using 16S rDNA base sequence, it was identified as Levilactobacillus paucivorans. Since it is a species belonging to the genus Lactobacillus, which has been certified as "GRAS (Generally Recognized As Safe)" as a food generally recognized as safe in the United States, its safety to humans is extremely high.
Both the N36 strain and the N50 strain of the present invention have been deposited at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation, an independent administrative institution, with the accession numbers NITE P-03449 (deposit date: March 19, 2021) for the N36 strain and NITE P-03450 (deposit date: March 19, 2021) for the N50 strain.
The strain characteristics of each of the Leviractobacillus paucivorans strains N36 and N50 are as follows.
(N36 strain)
Cell shape: Bacillus Gram staining: Positive Growth temperature: Grows at 20-35°C. Does not grow at 40°C.
Motility: None Sugar assimilation: Ribose, glucose, fructose, maltose Gas production using glucose as a carbon source: +
Salt tolerance: 6%
Bile acid resistance: 0.6%
(N50 stock)
Cell shape: Bacillus Gram staining: Positive Growth temperature: Grows at 20-35°C. Does not grow at 40°C.
Motility: None Sugar assimilation: Ribose, glucose, fructose, maltose Gas production using glucose as a carbon source: +
Salt tolerance: 6%
Bile acid resistance: 0.6%

<Method of producing putrescine>
Putrescine can be produced using the lactic acid bacteria strains N36 and N50 of the present invention. The method for producing putrescine relates to a method for culturing the lactic acid bacteria strains N36 and N50 of the present invention in a medium containing a putrescine-producing substrate.

(Cultivation process)
The culture medium for producing putrescine using the lactic acid bacteria strains N36 and N50 of the present invention may include a medium containing a putrescine-producing substrate in addition to necessary nutrient sources such as carbon sources, nitrogen sources, or inorganic salts that can be assimilated by lactic acid bacteria belonging to the genus Leviractobacillus. A preferred example of the putrescine-producing substrate is ornithine.
The concentration of the putrescine-producing substrate contained in the medium is not particularly limited, but may usually be about 0.01 to 2% by weight/volume, preferably about 0.1 to 1% by weight/volume.
Any medium, whether natural or synthetic, may be used as long as it allows lactic acid bacteria to grow, and those skilled in the art can select an appropriate known medium to be used. As the carbon source, glucose, fructose, etc. may be used, and as the nitrogen source, meat extract, peptone, yeast extract, casein hydrolysate, whey protein hydrolysate, soy protein hydrolysate, etc. may be used. As the inorganic salt, phosphate, sodium, potassium, magnesium, etc. may be used. Examples of media suitable for culturing lactic acid bacteria include M17 medium (with the above carbon source added) and MRS liquid medium.
It has been confirmed by the experimental examples described below that the lactic acid bacteria strains N36 and N50 of the present invention increase the amount of putrescine produced by adding glucose to a skim milk medium.

The culture conditions are not particularly limited as long as they allow the growth of lactic acid bacteria.
Although there are no particular limitations on the pH of the medium, it is usually in the range of pH 5 to 8, preferably pH 6 to 7. The medium and culture device can be sterilized at 105 to 121° C. for 15 minutes before use.
The culture temperature may be set within the range of growth temperatures for the lactic acid bacteria N36 and N50 strains, preferably within the range of the optimal growth temperatures, for example, 20° C. to 35° C., preferably about 25° C. to 30° C. The culture time is not limited, but may be, for example, about 1 to 6 days.
There is no need to block or supply oxygen during cultivation. Therefore, putrescine can be produced by simply covering the culture vessel with a lid. Cultivation methods include stationary culture, shaking culture, and tank culture.

<Food and beverages>
The lactic acid bacteria strains N36 and N50 of the present invention can be used as probiotics by taking advantage of their putrescine-producing ability, and one embodiment of this is their use in foods and beverages.
Examples of foods and beverages in the present invention include beverages such as milk drinks (including concentrated original solutions and powders for adjusting these beverages), dairy products such as processed milk, fermented milk, yogurt, butter, and cheese, and lactic acid bacteria fermented foods such as kimchi and pickled vegetables.
They can also be made into medicines or quasi-drugs (supplements), and can be used as formulations such as powders, granules, tablets, capsules, suspensions, liquids, emulsions, and injections.

The present invention will be described below with reference to examples, but the technical scope of the present invention is not limited to these examples.
Example 1: Lactic acid bacteria strain selection test
(1) Test samples A food-derived lactic acid bacteria library of 360 strains, covering 44 species, was maintained by the Livestock Product Functional Unit, Livestock Research Division, Livestock Research Department, National Agriculture and Food Research Organization (NARO). First, a test was conducted to confirm whether or not the bacteria produced amines, in order to select lactic acid bacteria capable of producing putrescine.
(2) Test method: After culturing in a lactic acid bacteria growth liquid medium (MRS medium) at 30° C. for 24 hours, the cells were centrifuged to discard the culture supernatant, and the cells were washed with physiological saline. After washing, the following amine production test medium containing ornithine as a putrescine-producing substrate was added, and the cells were cultured at 30° C. for 24 hours.
[Amine production test medium, pH 5.0]
Tryptone 5 g/L, yeast extract 5 g/L, sodium chloride 5 g/L, glucose 1 g/L, surfactant (Tween 80) 0.5 g/L, magnesium sulfate heptahydrate 0.2 g/L, calcium carbonate 0.1 g/L, bromocresol purple 0.06 g/L, manganese sulfate tetrahydrate 0.05 g/L, ferrous sulfate heptahydrate 0.04 g/L, and ornithine 10 g/L were dissolved in purified water, the pH was adjusted to 5.0 with 1 N hydrochloric acid, and the solution was heat sterilized at 121° C. for 15 minutes.
(3) Determination of the ability to produce amines This can be confirmed by the presence of bromocresol purple, which will turn the medium purple if the bacterium is producing an amine, or yellow if the bacterium is producing only an acid.
(4) Results From the results of the above test, it was confirmed that, among the 360 strains of lactic acid bacteria tested, two strains of the lactic acid bacteria of the present invention, N36 and N50, produced amines.

<Example 2: Putrescine production confirmation test>
(1) Test specimens Amine-producing media of the lactic acid bacteria strains N36 and N50, which had turned purple in the above "Example 1: Lactic acid bacteria strain selection test", were used.
(2) Test method: The test sample in (1) above was diluted with 0.1N hydrochloric acid, and then 1,7-diaminoheptane was used as an internal standard substance, and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) was added to dansylate the amino groups. This was extracted with ether, and the extract was dissolved in acetonitrile and subjected to high performance liquid chromatography (HPLC) analysis using the following analytical equipment and conditions.
(Analysis equipment and conditions)
Equipment: High performance liquid chromatography (LC-VP series) manufactured by Shimadzu Corporation
Pump: LC-10ADvp
Column oven: CTO-10ASvp
Detector: SPD-M10vp
Column: Kromasil C18 (250 x 4.6 mm)
Oven temperature: 30°C
Flow rate: 0.8mL/min
Mobile phase: acetonitrile Sample injection volume: 10 μL
(3) Results As shown in FIG. 3 below, a putrescine peak was detected in the amine production medium of the lactic acid bacteria strains N36 and N50 of the present invention, confirming that they produce putrescine.

Example 3: Probiotic compatibility test
The lactic acid bacteria strains N36 and N50 of the present invention, which were confirmed to have putrescine production ability, were tested for resistance to bile acids in order to clarify their suitability as probiotics. The lactic acid bacteria used as a control was Lacticaceibacillus rhamnosus GG strain, which is widely used as a probiotic lactic acid bacterium. The GG strain was registered with the American Type Culture Collection (ATCC) (Manassas, VA, USA) under the accession number ATCC 53103. Each strain was pre-cultured overnight in MRS medium at the optimum growth temperature, and then inoculated in 20 μL each into MRS medium without bile and MRS medium with 0.3 and 0.6% bile (4 mL each). The GG strain was cultured at the optimum growth temperature of 37° C., and the N36 and N50 strains were cultured at 30° C. for 48 hours. OD620 after the culture was measured, and the OD620 value after the culture in the MRS medium to which bile had been added was set as 100, and the value after the culture in the MRS medium to which bile had been added is shown in FIG.
Both the N36 strain and the N50 strain showed better growth in bile-supplemented medium than the GG strain, confirming that they have bile acid resistance and are expected to be effective as probiotics.

<Example 4: Test 1 for confirming the amount of putrescine produced during skim milk culture>
The lactic acid bacteria strains N36 and N50 of the present invention, which were confirmed to have the ability to produce putrescine, were tested to confirm the amount of putrescine produced during skim milk culture. The amount of putrescine produced by adding sugar during skim milk culture was also confirmed.
Each strain was pre-cultured overnight in MRS medium (30°C), and the cells were washed, and the washed cells were resuspended in 10% skim milk medium (sugar-free) or 10% skim milk medium (sweetened) to which 0.5% glucose was added, so that the cell concentration was 10 x 10 ⁹ cfu/mL. cfu stands for colony forming unit. The skim milk medium inoculated with each lactic acid bacterium was cultured at 30, 35, and 40°C for 24 to 48 hours. The culture solution was thoroughly stirred with a vortex and diluted with 1N hydrochloric acid, and then 1,7-diaminoheptane was used as an internal standard substance, and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) was added to dansylate the amino group. This was extracted with ether, and the extract was dissolved in acetonitrile and analyzed by high performance liquid chromatography (HPLC), and the results are shown in Figure 5.
Both strains N36 and N50 were shown to produce putrescine in 10% skim milk medium at a culture temperature of 30 to 40° C. Furthermore, the addition of 0.5% glucose increased the putrescine concentration in the medium by 1.5 times.

<Example 5: Test 2 to confirm the amount of putrescine produced during skim milk culture>
A test was carried out to confirm the amount of putrescine produced by culturing the lactic acid bacteria strains N36 and N50 of the present invention in skim milk in the presence of a commercially available yogurt starter strain.
Each strain was pre-cultured overnight in MRS medium (30°C), the cells were washed, and the washed cells were resuspended in 10% skim milk medium to a concentration of 10 x 10 ⁷ cfu/mL. Commercially available yogurt starter bacteria were inoculated into 10% skim milk medium, cultured at 37°C for 16 hours, and added at 1%. The skim milk medium inoculated with each lactic acid bacteria and yogurt starter bacteria was cultured at 37°C for 6 to 168 hours. The culture solution was thoroughly stirred with a vortex and diluted with 1N hydrochloric acid, and then 1,7-diaminoheptane was used as an internal standard substance, and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) was added to dansylate the amino group. This was extracted with ether, and the extract was dissolved in acetonitrile and analyzed by high performance liquid chromatography (HPLC), with the results shown in Figure 6.
The commercially available yogurt starter did not produce putrescine in the skim milk medium. Both strains N36 and N50 were shown to produce putrescine even in the presence of yogurt starter bacteria.

The specific lactic acid bacteria belonging to the genus Leviractobacillus of the present invention can be used as a probiotic because it has the ability to produce putrescine and is resistant to bile acids. In addition, when the lactic acid bacteria is cultured in a skim milk medium, the amount of putrescine produced can be increased by adding glucose.
Furthermore, the lactic acid bacteria of the present invention can be cultured in milk such as skim milk and used together with the produced putrescine as a highly safe food or drink, making them extremely useful.

Claims (3)

A lactic acid bacterium that is the Rebilactobacillus paucivorans strain N36 (accession number: NITE P-03449) or N50 strain (accession number: NITE P-03450). A food or drink containing the lactic acid bacteria according to claim 1. A method for increasing putrescine production, comprising adding glucose to a skim milk medium in which the lactic acid bacteria according to claim 1 is cultured.